Peptide self-assembling scaffolds have been widely used in tissue engineering. Much work has been focused on modifying the self-assembling scaffolds with functional motifs for desired biological activities. We report here the development of a biological material designed specifically for neural tissue engineering (NTE). Using RADA-16 (AcN–RADARADARADARADA–CONH2) as a base scaffold, we synthesized a 31 amino acid peptide RADA-FRM (AcN–RADARADARADARADAGGSIDRVEPYSSTAQ–CONH2) containing the neural cell adhesion molecule (NCAM)-derived mimetic peptide FRM (SIDRVEPYSSTAQ), which could undergo self-assembly into a nanofiber scaffold. We tested the characterization of the nanofiber scaffold using atomic force microscopy (AFM) and accessed the rheological properties of FRM-containing nanofiber scaffold (FRM-NS). Then we examined its biocompatibility on neural stem cells (NSCs) from neonatal rats. Regrettably, we found that FRM-NS had no effect on differentiation of NSCs. However, we tested that FRM-NS was non-cytotoxic. Furthermore, compared to pure RADA-16 scaffold, we found that the designer self-assembling peptide scaffold containing FRM motif could significantly promote NSCs proliferation and stimulate NSCs migration into the 3-D scaffold. Our results indicate that the novel designer peptide scaffold containing FRM had excellent biocompatibility with NSCs and may be useful for central nervous tissue repair.

Polysaccharide hydrogels are good candidates for skin scaffolds due to their inherent biocompatibility and water transport properties. In the current study, hydrogels were made from a mixture of four polysaccharides: xanthan gum, konjac gum, iota-carrageenan, and kappa-carrageenan. Gel formation, strength, and structure of these polysaccharides were studied using rheological and thermal techniques. All gel samples studied were strong gels at all times due to the gradual water loss. However, after 12 hours of storage, elastic (G') and loss (G“) moduli of hydrogel mixture containing all the ingredients is of one to two orders of magnitude greater than that of mixtures not containing either xanthan gum or iota-carrageenan, which confirmed the varied levels of gel strength. This is mainly due to the rate of water loss in each of these mixtures, resulting in gels of varying structures and dynamic moduli over a period of time. Iota carrageenan and xanthan gum differ in their effect on gel strength and stability in combination with konjac gum and kappa carrageenan.

Compared with early bare-metal stents, drug-eluting stents (DESs) are more effective in treating coronary artery diseases, especially in inhibiting restenosis. However, in-stent restenosis still clinically occurs at a non-negligible rate. More importantly, delayed endothelialization, inflammation, and hypersensitivity trigger subacute or late adverse events, particularly stent thrombosis, and thereby raise more concerns over the long-term safety of DESs. These problems are mostly associated with the permanent polymeric materials, non-optimal therapeutic drugs, and/or metallic stent platforms used in current DES design. It is critically important to further improve and optimize DES design and apply newer strategies for developing next generation DES. These new generation DESs should maintain their clinical efficacy and meanwhile eliminate the long-term safety concerns. In this review article, the current information on the optimization of DES design was critically reviewed based on DES's basic components, namely, stent platform, restenotic drug, and polymer coating. The available strategies for designing next-generation DESs were also summarized, ranging from degradable polymer DESs, to polymer-free DESs, to fully biodegradable DESs.

Bone morphogenetic proteins (BMPs)-immobilized polydioxanone (PDO)/Pluronic F127 porous particles were prepared as a bone graft using a melt-molding particulate-leaching method, and the sequential binding of heparin and BMPs (BMP-2 and BMP-7, single or dual) onto the porous particles. The prepared PDO/Pluronic F127 porous particles gradually degraded with time, with approximately 30 % of the initial particle weight remaining after 16 wks. The degradation rate of the PDO/Pluronic F127 porous particles may parallel the bone healing rate. The BMPs were easily immobilized onto the pore surfaces of PDO/Pluronic F127 particles via heparin binding and were released in a sustained manner for up to 21 days, regardless of BMP type. The BMPs (single BMP-2 or dual BMP-2/BMP-7)-immobilized porous particles were effective for in vitro osteogenesis of bone marrow stem cells (BMSCs), as analyzed by alkaline phosphatase activity, calcium content, time polymerase chain reaction using specific markers for osteogenesis (Type I collagen, osteocalcin, osteopotin, and RunX2), and immunohistochemical staining. The BMPs (single BMP-2 or dual BMP-2/BMP-7)-immobilized porous particles were also effective in promoting new bone formation, as analyzed by the preliminary animal study using a full-thickness skull defect model of Sprague–Dawley rats (micro-computed tomography). The synergistic effect of dual BMPs on the osteogenesis of BMSCs and bone regeneration was not significant in our system. The BMP-2 or dual BMPs (BMP-2/BMP-7)-immobilized PDO/Pluronic F127 porous particles may be a promising candidate as a bone graft for the delayed and insufficient bone healing in clinical fields.

The main problem of percutaneous osseointegrated implants is poor skin-implant integration, which may cause infection. This study investigated the effects of pore size (Small, 40-100 microns and Large, 100-160 microns), nanotubular surface treatment (Nano), and duration of implantation (3 and 6 weeks) on skin ingrowth into porous titanium. Each implant type was percutaneously inserted in the back of 35 rats randomly assigned to 7 groups. Implant extrusion rate was measured weekly and skin ingrowth into implants was determined histologically after harvesting implants. It was found that all 3 types of implants demonstrated skin tissue ingrowth of over 30% (at week 3) and 50% (at weeks 4-6) of total implant porous area under the skin; longer implantation resulted in greater skin ingrowth (p<0.05). Only one case of infection was observed (infection rate 2.9%). Small and Nano groups showed the same implant extrusion rate which was lower than the Large group rate (0.06±0.01 vs. 0.16 ± 0.02 cm/week; p<0.05). Ingrowth area was comparable in the Small, Large and Nano implants. However, qualitatively, the Nano implants showed greatest cellular inhabitation within first three weeks. We concluded that percutaneous porous titanium implants allow for skin integration with the potential for a safe seal.

As a dynamic and hierarchically organized composite, native extracellular matrix (ECM) not only supplies mechanical support which the embedded cells needs but also regulates the functions of various cellular through interaction with them. Based on the ECM-mimetic principle, good biocompatibility and appropriate mechanical properties are the two basic requirements that the ideal scaffolds for the tissue engineering or regenerative medicine need. Some fibers and tubes have been shown effective to reinforce scaffolds for tissue engineering or regenerative medicine. In this review, three parts, namely properties affected by the addition of fibers or tubes, scaffolds reinforced by fibers or tubes for soft tissue repair and scaffolds reinforced by fibers or tubes for hard tissue repair, are stated, which shows that tissue repair or regeneration efficacy was enhanced significantly by fiber or tube reinforcement. Also, it indicates that these reinforcing agents can improve the biocompatibility and biodegradation of the scaffolds in most cases. However, there are still some concerns, such as the homogeneousness in structure or composition throughout the reinforced scaffolds, the adhesive strength between the matrix and the fibers or tubes, cytotoxicity of nanoscaled reinforcing agents, etc., which were also discussed in the conclusion and perspectives part.

Heparin conjugation of poly(l-lactide) fibrous scaffolds fabricated by electrospinning was accomplished by surface functionalization with amine (–NH₂) groups using a sequential treatment with Ar-NH₃ and H₂ plasmas. The density of the incorporated –NH₂ group was determined by combining a chemical derivatization method with X-ray photoelectron spectroscopy. The time of Ar-NH₃ plasma treatment significantly affected the N/C, –NH₂/N, and –NH₂/C fractions, whereas the plasma power, Ar-NH₃ gas composition, and time of H₂ plasma treatment only influenced the –NH₂/N and –NH₂/C fractions. Scaffold surface functionalization by –NH₂ groups significantly increased the amount of covalently bonded heparin compared to a hydrolysis method. The function of immobilized heparin was confirmed by the decrease of platelet attachment during the exposure of the scaffolds to blood from Sprague-Dawley rats. In vitro experiments with bovine aorta endothelial cells demonstrated that heparin conjugation enhanced cell infiltration through the fibrous scaffolds, regardless of the amount of covalently immobilized heparin.

An injectable biodegradable hydrogel was prepared for temperature responsive pulsatile release of insulin. Triblock copolymer of poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) was prepared by ring opening bulk copolymerization and characterized using FTIR, ¹HNMR and GPC. Aqueous solution of PECE formed an injectable hydrogel which was solution at room temperature and transformed into gel at 37 °C. The temperature responsive sol-gel transition and crystallinity of PECE hydrogel was studied and compared with Pluronic, a well-studied non-biodegradable injectable hydrogel. In vitro release study revealed that insulin release profile of PECE was similar to pluronic and its viscosity was 1/30^th of Pluronic sol at 10000 s^-1 shear rate. Release behavior of insulin from PECE hydrogels followed Fickian diffusion of first order. Insulin retained its secondary structure after release as confirmed by CD spectrum. A three-fold increase in Fickian diffusion coefficient was evidenced when temperature was increased from 34 °C to 40 °C due to crystalline melting of PCL part of PECE. Pulsatile release of insulin showed a correlation coefficient of 0.90 with the change of temperature.

A novel polystyrene (PS) substrate with microscale porous structure was facilely fabricated by crystalline-controlled casting method using mixed solvent (N, N-dimethylformamide (DMF) and ethyl alcohol (v/v)) based on the non-solvent induced phase separation process. The substrate surfaces exhibited a bi-continuous microscale porous morphology with high porosity, large pore size and pore–pore connection structure. Moreover, behaviors of the normal human liver cell line (HL-7702) seeded on this substrate surface were carefully investigated. The results indicated that the cell adhesion, spread and cell-cell connection on the surface with subcellular pore size (~10μm) were similar to the cells proliferated on the flat PS surface. However, the number of HL-7702 cells proliferated on the PS microscale porous surface was higher than cells on the conventional PS flat surface, suggesting that the pore-pore structure was conducive to HL-7702 cell proliferation. Furthermore, hematoxylin and eosin (H&E) staining and micronucleus test (MNT) were performed. The results showed that fewer damages for nuclear and cytoplasm and less cell genotoxicity were caused by the microscale porous structure within the scope of pore size (~10μm) than that of the flat surface.

We have described an approach to fabricate 3-D cell-based structures using functionalized super paramagnetic iron oxide nanoparticles (SPIONs) as patterning agents to guide the assembly of endothelial cell spheroids into 3-D patterns using the magnetic forces generated by a pre-fabricated magnetic template. SPIONs were first uptaken by endothelial cells before they were assembled into uniform-sized spheroids through a home-made robotic spheroid maker. In order to guide the magnetic spheroids, a unique magnetic template was fabricated using computer-aided design (CAD) and cut from a magnetic sheet. The spheroids were then guided to the pre-fabricated magnetic template through the attractive magnetic forces between the SPIONs inside the endothelial cells and the magnetic template. Fusion of endothelial cell spheroids over time while adhered to the magnetic template allowed for the formation of 3-D cell-based structures. Subsequent removal of the pre-fabricated magnetic template left 3-D endothelial cell sheets, which may be stacked to fabricate complicated 3-D multi-cellular tissue structures. To enhance the cytocompatibility, SPIONs were silica-coated before use. At low concentrations, the SPIONs did not adversely affect cell viability, proliferation, and phenotype stability. Light and confocal microscopy showed that endothelial cell spheroids could be reproducibly created with high uniformity. The endothelial cells were able to remain viable and maintain the 3-D structure in vitro. We have proved the concept to use SPIONs as a patterning agent to direct the attachment and self assembly of SPION-loaded endothelial cell spheroids on a pre-fabricated magnetic template for the formation of 3-D cell based structures. A magnetic-directed technique allows quick patterning of cell spheroids in accordance with desirable magnetic patterns, therefore, holding promise for scalable fabrication of complicated 3-D multi-cellular tissue structures. By varying the cell types and the pre-fabricated magnetic patterns, this magnetic-directed patterning strategy may find use in bioprinting and multi-cellular tissue graft fabrication.

The metallic materials used for implantable medical devices are predominantly stainless steels, Ti and its alloys, and Co-Cr alloys. The corrosion resistance of each of these materials is associated with a passive oxide film on its surface. Since corrosion resistance is crucial to implant performance, considerable effort has been focused on understanding the nature of the passive film present under physiological conditions. Surface analytical techniques and electrochemical impedance spectroscopy (EIS) have been used in a number of studies to investigate the passive film formed on metallic biomaterials in simulated physiological solutions. This review focuses on the surface characteristics of these materials with regard to composition, thickness, and impedance of the passive films. Of particular interest are changes in the films with surface treatment and the nature of the films developed over time in the simulated solutions.

The demand for scaffolds comprised of natural materials such as collagen has increased in recent years. However, many scaffolds rely on chemical or physical modifications in order to comply with the necessary requirements for biomedical engineering. We evaluated the in vivo biocompatibility and biodegradation of a novel, thin, mechanically stable and chemically non-crosslinked collagen cell carrier (CCC). CCC was implanted subcutaneously into 25 adult Lewis rats and biopsies were taken on days 7, 14, 21, 42, and 84 after surgery. For histological analysis, paraffin sections of implanted skin were immunolabelled for CD68 and stained by haematoxylin-eosin and Masson-Goldner's trichrome method. Macroscopic analysis of skin surface during wound healing process showed a normal physiological reaction. Biodegradation of CCC was completed 42 days after subcutaneous implantation. Histological evaluation revealed no evidence of encapsulation, scar formation, or long term vascularisation and inflammation. The collagen type I based biomaterial demonstrated a high in vivo biocompatibility, low irritability, complete resorption and replacement by autologous tissue. The in vivo biocompatibility and degradation behaviour encourage for further evaluation of CCC in surgical applications and regenerative medicine.

Bacterial cellulose (BC) is a natural biomaterial with unique properties suitable for tissue engineering applications, but it has not yet been used for preparing nerve conduits to repair peripheral nerve injuries. The objectives of this study were to prepare and characterize the Kampuchea-synthesized bacterial cellulose (KBC) and further evaluate the biocompatibility of KBC with peripheral nerve cells and tissues in vitro and in vivo. KBC membranes were composed of interwoven ribbons of about 20–100 nm in width, and had a high purity and the same crystallinity as that of cellulose Iα. The results from light and scanning electron microscopy (SEM), MTT assay, flow cytometry, and RT-PCR indicated that no significant differences in the morphology and cell function were observed between Schwann cells cultured on KBC membranes and glass slips. We also fabricated a nerve conduit using KBC, which was implanted into the spatium intermusculare of rats. At 1, 3 and 6 weeks post-implantation, clinical chemistry and histochemistry showed that there were no significant differences in blood counts, serum biochemical parameters, and tissue reactions between implanted rats and sham-operated rats. Collectively, our data indicated that KBC possessed good biocompatibility with primary cultured Schwann cells (SCs) and KBC did not exert hematological and histological toxic effects on nerve tissues in vivo.

The performance of neural electrodes implanted in the brain is often limited by host response in the surrounding brain tissue, including astrocytic scar formation, neuronal cell death, and inflammation around the implant. We applied conformal microgel coatings to silicon neural electrodes and examined host responses to microgel-coated and uncoated electrodes following implantation in the rat brain. In vitro analyses demonstrated significantly reduced astrocyte and microglia adhesion to microgel-coated electrodes compared to uncoated controls. Microgel-coated and uncoated electrodes were implanted in the rat brain cortex and the extent of activated microglia and astrocytes as well as neuron density around the implant were evaluated at 1, 4, and 24 weeks post-implantation. Microgel coatings reduced astrocytic recruitment around the implant at later time points. However, microglial response indicated persistence of inflammation in the area around the electrode. Neuronal density around the implanted electrodes was also lower for both implant groups compared to the uninjured control. These results demonstrate that microgel coatings do not significantly improve host responses to implanted neural electrodes and underscore the need for further improvements in implantable materials.

The aim of this study was to investigate whether ZK60, an extruded magnesium alloy, reacts in vivo with an appropriate host response, and to investigate how microarc oxidation (MAO) treatment influences this in vivo corrosion behavior. Twelve cylinders were machined from as-extruded ZK60, with six cylinders treated with MAO and six left untreated; poly-L-lactic acid (PLLA) pins were used as a control to compare biocompatibility. These cylinders were implanted into the right distal femur of mice along the transepicondylar axis from the medial condyle. Micro-computerized tomography (micro-CT) was used to quantitatively analyze corrosion in a non-destructive manner in vivo and the corrosion rate was calculated based on the volume measurements of the residual implants. The physiological response of the rats post-implantation was obtained by clinical observation and blood biochemical analysis. Histological analyses of the soft tissue around the implants were used to assess bone response in relation to the implants. The results obtained clearly indicate that the untreated ZK60 alloy showed high degradation rates in vivo, and that MAO treatment had a significant but unsatisfying effect on protecting the implant from further corrosion. Compared with PLLA, the ZK60 alloy showed good osteoconductivity and osteoinductivity, and, according to biochemical indicators, had good biocompatibility in vivo.

Collagen films have been widely used in the field of biomedical engineering. However, the poor mechanical properties of collagen have limited its application. Here, rod-like cellulose nanocrystals (CNCs) were fabricated and used to reinforce collagen films. A series of collagen/CNCs films were prepared by collagen solution with CNCs suspensions homogeneously dispersed at CNCs: collagen weight ratios of 1, 3, 5, 7 and 10. The morphology of the resulting films was analyzed by scanning electron microscopy (SEM), the enhancement of the thermomechanical properties of the collagen/CNCs composites were demonstrated by thermal gravimetric analysis (TGA) and mechanical testing. Among the CNCs contents used, a loading of 7 wt% led to the maximum mechanical properties for the collagen/CNCs composite films. In addition, in vitro cell culture studies revealed that the CNCs have no negative effect on the cell morphology, viability, and proliferation and possess good biocompatibility. We conclude that the incorporation of CNCs is a simple and promising way to reinforce collagen films without impairing biocompatibility. This study demonstrates that the composite films show good potential for use in the field of skin tissue engineering.

At approximately 50 μm thin, the human amniotic membrane (hAM) has been shown to be a versatile biomaterial with applications ranging from ocular transplants to skin and nerve regeneration. These investigations describe laminating layers of the hAM into a multilayered, conformation creating a thicker, more robust biomaterial for applications requiring more supportive structures. Amniotic membranes were decellularized using 4M NaCl and prepared as either flat single-layered sheets or rolled into concentric five-layered configurations. Constructs were seeded with human vascular smooth muscle cells and cultured over 40 days to quantify biological and mechanical changes that occurred during early remodeling events. By day 40 single layered constructs displayed a decreasing trend in cellular densities and glycosaminoglycan (GAG) concentration, comparative to multilayered constructs with increasing cell densities (from 9.1 to 32 x 10⁶ cells/g) and GAG concentrations (from 6.07 to 17.4 mg/g). Oxygen diffusion was calculated and found to be sufficient to maintain cell populations through the constructs full thickness. While an overall decrease in the modulus of elasticity was noted, the modulus in the failure range of rolled constructs stabilized at values 25 times higher than single-layered constructs. Rolled constructs typically displayed an up-regulation of contractile and matrix remodeling markers (α-actin, SM22 and type 1 collagen, MMP-2 respectively) indicating biological adaptation. Considerable design flexibility can be achieved by varying the number of scaffold layers, allowing the possibility of tuning the constructs physical dimensions, shape and tensile properties to suit specific targeted vascular locations.

Calcium-phosphate ceramics, which have a composition similar to bone mineral, represent a potentially interesting synthetic bone graft substitute. In the present study, three porous HA/β-TCP/Collagen ceramic scaffolds were developed, characterized and tested for their bone repairing capacity and osteoinductive potential in a New Zealand Rabbit model. The ratio of the ceramic's components HA/B-TCP/Collagen varied from 40/30/30 to 50/20/30 and 60/20/20 (in wt%), respectively. None of the ceramic scaffolds succeeded in completely bridging the 6 mm calvarian defect with new bone after 60 days implantation. 60/20/20 ceramic scaffolds showed significantly more bone formation in the pores and in the periphery of the graft than the other two materials. Histomorphometric analysis revealed that the 40/30/30 scaffold produced best bone to implant contact percentages (BIC 67.23 % ± 0.34 with higher quality, closer contact) in comparison with 50/20/30 (54.87 % ± 0.32), and 60/20/20 (48.53 % ± 0.31). Both physicochemical and structural properties of the ceramic composites affected their in vivo behavior, either dependently or independently, emphasizing the importance of assessing bone repair parameters individually. The scaffolds may offer clinical applications in reconstructive surgery for treating bone pathologies.

Improvements to clinically used biomaterials such as hydroxyapatite (HA) are of potential benefit to the patient. One modification, the addition of surface charges, has been shown to have an important role influencing cell response. In this study, porous HA scaffolds with both positive and negative surface charges were manufactured. The samples were sintered in air to produce porous HA ceramic scaffolds in the form of cylinders 12mm in height x 7mm in diameter. These were polarised with a d.c. voltage of 3kV/cm. MC3T3E1 cells were placed on either negative or positive ends of the charged (or unpoled control) HA scaffolds. At 7 days, picogreen analysis was performed to analyse the cell number at the negative (4mm), central (4mm) and positive (4mm) portions of the 12mm cylindrical scaffold. At 4 weeks, microCT analysis was performed to quantify the regional volume of mineralised matrix deposition on the 3D scaffold. At 7 days, there were significantly more cells present at the negative end of the scaffold when seeded from the negative end in comparison to the other samples tested. MicroCT data at 4 weeks correlated with this finding, demonstrating an increase in mineralised matrix at the negatively charged end of the scaffold seeded from the negative end in comparison to the positively charged and unpoled control scaffolds. The results indicate that the charge on HA influences cell activity and that this phenomenon can be translated to a clinically relevant porous scaffold structure.

Purposes: Both decalcified bone matrix (DBM) and fibrin gel possess good biocompatibility, so they are used as scaffolds to culture bone marrow mesenchymal stem cells(BMSCs). The feasibility and efficacy of using compound material being made of decalcified bone matrix and fibrin gel as a three-dimensional scaffold for bone growth were investigated.

Methods: BMSCs were isolated from the femur of rabbit, then seeded in prepared scaffolds after incubation for 28 days in vitro. In vivo: 30 New Zealand White Rabbits received bone defect in left radius and divided 3 treatment groups randomly: (1)BMSCs/decalcified bone matrix/fibrin glue as experimental group;(2) decalcified bone matrix /fibrin glue without cells as control group;(3) nothing was implanted into the bone defects as blank group. The observation period of specimens was 12^th weeks, and were analyzed bone formation in terms of serum proteomics (2D-PAGE and MALDI-TOF-TOF-MS), hematoxylin-eosin (HE) staining, ALP staining, Osteopontin immunofluorescence detection

Results: The experimental group present in three peculiar kinds of proteins, whose Geninfo identifier(GI) number were 136466, 126722803, and 126723746, respectively correspond to TTR protein, ALB protein, RBP4 protein; and the histological inspections were superior to the other group. The content of osteopontin in experimental group was significantly higher than control group(p<0.05).

Conclusion: The overall results indicated that a combined material being made of BMSCs/decalcified bone matrix /fibrin glue can result in successful bone formation and decalcified bone matrix /fibrin glue admixtures can be used as a scaffold for bone tissue engineering.

Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor β1 (TGF-β1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-β1, 5-fold higher retention was found in peptide. Excess unlabeled TGF-β1 minimally displaced retained radiolabeled TGF-β1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly 3-fold lower than agarose hydrogels, at 18% of loaded TGF-β1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-β1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-β1 degradation and release of small labeled species. TGF-β1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.

Commonly used aluminum hydroxide (Alum) adjuvant provokes a strong type 2 helper T cell (Th2) response for mediating antibody production, but is rather ineffective for disease prevention that requires type 1 helper T cell (Th1) response for mediating cellular immunity in human vaccination. Here, for the purpose of inducing Th1 anti-tumor immunity, a mesoporous silica (MS)-based adjuvant is prepared. Three kinds of MS particles with nearly identical particle size and surface area but different pore sizes of 4, 7 and 10 nm were prepared. No serious in vitro cytotoxicity was observed for the MS particles at 5, 20, 50 and 100 μg/mL. Pathogen-associated molecular patterns (PAMPs) were immobilized with apatite (Ap) on MS to prepare the MS-based and PAMP-loaded adjuvants (MS-Ap-PAMP adjuvants). Macrophage-like cells cultured in the presence of MS-Ap-PAMP adjuvant with a MS pore size of 10 nm showed the maximum in vitro immunogenic activity. Injection of the MS-Ap-PAMP adjuvant with a MS pore size of 10 nm in combination with liquid-nitrogen-treated tumor tissue (derived from Lewis lung carcinoma cells) to C57BL/6 mice markedly inhibited the development of re-challenged tumor in vivo, while no such antitumor immunity was induced in injection of Alum mixed with PAMP in combination with liquid-nitrogen-treated tumor tissue. The MS-Ap-PAMP adjuvant contributed to the elicitation of a potent systemic Th1 antitumor immunity in vivo.

Self-assembled, biodegradable materials that embed fragile, soluble or insoluble compounds of therapeutic interest have potential use as drug delivery systems. The bead-forming peptide Ac-X₃-gT can embed hydrophobic and hydrophilic payloads. Loaded peptide beads were internalized by THP-1 macrophages, THP-1 monocytes, and hepatocellular carcinoma cells (Huh7). Furthermore, paclitaxel and doxorubicin co-encapsulated in the peptide beads were delivered to THP-1 monocytes, causing a decrease in cell viability due to the activity of the anti-cancer drugs. In addition to the bead-forming peptide Ac-X₃-gT, the use of a positively charged peptide analogue increased the RNA/DNA embedding efficiency to 99% by charge compensation and micellar complexation. Internalization of the resulting gene delivery systems by Huh7 cells led to specific gene silencing either by embedded siRNA or plasmid encoding shRNA delivered in cells.

The new class of purely peptidic material caused no measurable toxicity during in vitro experiments, thereby indicating potential use as a drug delivery system for multi-drug delivery and gene therapy.

Biodegradable radiopaque iodinated poly(ester-urethane) (PU), consisting of poly(ε-caprolactone)-diol (PCL) and 4,4′-isopropylidinedi-(2,6-diiodophenol) (IBPA), has been successfully synthesized via a coupling reaction of PCL-diisocyanate and IBPA with varying compositions. The IBPA with four iodine atoms per molecule was applied as a chain extender to endow the iodine-containing poly(ester-urethane)s (I-PUs) with intrinsic X-ray visibility. The chemical structure and molecular weights of I-PUs were characterized by FT-IR, ¹H-NMR and GPC. The effects of IBPA on the physical properties of I-PUs were systematically studied by means of DSC, TGA and WAXD. The DSC results showed that the crystallization of PCL segments in I-PUs was restrained with increasing amount of IBPA, which was also confirmed by WAXD. In the X-radiography analysis, all the synthesized I-PUs exhibited high radiopacity compared with an Aluminum wedge of equivalent thickness. Enzymatic degradation tests showed that the incorporation of IBPA prolonged the degradation of I-PUs and distinct mass loss and degradation happened in the third month. Basic cytocompatibility conducted using rat adipose-derived cells proved that all the I-PUs and their biodegradation products were nontoxic. The radiopaque I-PUs is expected to possess a significant advantage over the traditional polymer counterparts in some related biomedical fields.

Biodegradable polymer scaffolds can be used to deliver soluble factors to enhance osseous remodeling in bone defects. To this end, we designed a poly(lactic-co-glycolic acid) (PLAGA) microsphere scaffold to sustain the release of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors. The microsphere scaffolds were created from fast degrading 50:50 PLAGA and/or from slow-degrading 85:15 PLAGA. Temporal and spatial regulation of bone remodeling depended on the use of appropriate scaffolds for drug delivery. The release profiles from the scaffolds were used to design an optimal delivery system to treat critical size cranial defects in a rodent model. The ability of local FTY720 delivery to maximize bone regeneration was evaluated with micro-computed tomography (microCT) and histology. Following 4 weeks of defect healing, FTY720 delivery from 85:15 PLAGA scaffolds resulted in a significant increase in bone volumes in the defect region compared to the controls. 85:15 microsphere scaffolds maintain their structural integrity over a longer period of time, and cause an initial burst release of FTY720 due to surface localization of the drug. This encourages cellular in-growth and an increase in new bone formation.

Immobilization of biomolecules onto implant surfaces is one of the most straightforward strategies to control the interaction between an implant and its biological environment. Recently, it was shown that the enzyme alkaline phosphatase (ALP) could be efficiently immobilized onto titanium implants in a single step using polydopamine. We hypothesized that such polydopamine-ALP coatings can enhance the early attachment of cells and increase mineralization. Therefore, the current study aimed at immobilization of ALP onto titanium by means of either one- or two-step polydopamine-assisted immobilization or electrospray deposition, the comparative characterization of these experimental substrates and subsequent cell behavioural analysis using primary osteoblast-like cells. Uncoated titanium and ALP-free polydopamine coatings served as controls. Despite significant ALP surface activity and lower water contact for angles ALP-containing surface modifications, only marginal effects on early cell behaviour (i.e. cell spreading) and osteogenic differentiation (i.e. proliferation, differentiation and mineralization) were observed in comparison to uncoated titanium.

The aim of this study was to compare the osteogenic capacity between human adipose tissue-derived mesenchymal stem cells (AT-MSCs) and their cocultures with human umbilical vein endothelial cells (HUVECs) in vitro and their biological performance in vivo. Firstly, the optimal cell ratio in cocultures for osteogenic differentiation was determined by seeding AT-MSCs and HUVECs in ratios varying from 100:0 to 0:100 on tissue culture plates. Afterwards, AT-MSCs and AT-MSCs/HUVECs (50:50) were seeded on porous titanium fiber mesh scaffolds (Ti) for both in vitro and in vivo osteogenic evaluation. For in vitro evaluation, cell osteogenic differentiation was assessed by ALP-activity and calcium assay. For in vivo evaluation, the scaffolds were implanted bilaterally into rat cranial defects (5 mm diameter) and bone formation was assessed histologically and histomorphometrically after 8 weeks. The ratio of 50:50 was chosen in the cocultures since this coculture condition retained similar amount of calcium deposition while using the least amount of AT-MSCs. Moreover, AT-MSCs showed higher osteogenic differentiation in comparison to AT-MSCs/HUVECs on Ti in vitro. Further, superior bone formation was observed in AT-MSCs compared to AT-MSCs/HUVECs in rat cranial defects. In conclusion, AT-MSCs showed significantly higher osteogenic potential compared to AT-MSCs/HUVECs both in vitro and in vivo.

A biomimetic hydrogel was integrated into microfluidic chips to monitor glioma cell alignment and migration. The extracellular matrix-based biomimetic hydrogel was remodeled by matrix metalloprotease (MMP) secreted by glioma cells and the hydrogel could thus be used to assess cellular behavior. Both static and dynamic cell growth conditions (flow rate of 0.1 mL/hr) were used. Cell culture medium with and without vascular endothelial growth factor (VEGF), insensitive VEGF and tissue inhibitor of metalloproteinases (TIMP) were employed to monitor cell behavior. A concentration gradient formed in the hydrogel resulted in differences in cell behavior. Glioma cell viability in the microchannel was 75~85%. Cells in the VEGF-loaded microchannels spread extensively, degrading the MMP-sensitive hydrogel, and achieved cell sizes almost 5-fold larger than seen in the control medium. Our integrated system can be used as a model for the study of cellular behavior in a controlled microenvironment generated by fluidic conditions in a biomimetic matrix.

Regenerative medicine treatments that combine the use of cells and materials may open new options for tissue/organ repair and regeneration. The microenvironment of mesenchymal stem cells (MSCs) strictly regulates their self-renewal and functions. In this study, when rat bone marrow derived MSCs (rBMSCs) and rat adipose tissue derived MSCs (rAMSCs) in passages 2-4 were cultured on different substrates, they presented the cellular functions to be dependent of substrate stiffness. The cells attached better on the softer substrate than on the stiffer one. The substrate stiffness had no significant influence on the proliferation of those cells. However, the substrate stiffness significantly promoted the osteogenic differentiation of the two kinds of stem cells. Furthermore, rBMSCs cultured on the same stiffness expressed more osteoblast-related markers than rAMSCs. In addition, combined biomaterials and biochemical reagents treatment yielded a stronger effect on osteogenic differentiation of MSCs than either treatment alone. These results have significant implications for further extending our capabilities in engineering functional tissue substitutes.

Processed nerve allografts are increasingly used as “off the shelf” nerve replacements for surgically bridging nerve gaps. Benchmarking the regenerative capacity of a commercially available human-derived nerve or xenograft in a rat nerve injury model would provide a convenient platform for future studies seeking to modify the processed nerve graft. Human and rat processed nerve grafts were used to bridge a 14 mm defect in a Sprague-Dawley rat sciatic nerve. Reversed autografts served as a positive control group. Twelve weeks following surgery, the distal nerve stumps were retrograde labeled and harvested for histology and histomorphometry. The cross-sectional areas of the human- and rat-derived processed nerve grafts were similar. Neuron counts and myelinated axon counts following use of the human-derived processed xenografts were decreased compared to those obtained from both the rat-derived processed nerve allografts and the autografts; the rat-derived processed nerve allografts were statistically equivalent to autografts. Measures of nerve fiber diameter and myelination revealed inferior axon regeneration maturity in both processed nerve grafts compared to autografts. Processed xenografts showed significantly reduced regeneration compared to autografts or processed allografts indicating that cross-species immunological reactions are important considerations in this rat model.

The clinical need for improved treatment options for patients with cartilage injuries has motivated tissue-engineering studies aimed at the in vitro generation of cell-based implants with functional properties. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to the scaffold. In the present study, chondrocyte-scaffold constructs were engineered by planting porcine chondrocytes into non-porous chitosan membranes and 3D porous chitosan scaffolds that were treated with or without biotin-conjugated anti-CD44 antibody-avidin binding system and avidin-biotin binding system. The spreading area, cell exfoliation rates, cell proliferation rates, histological analysis, DNA and glycosaminoglycan (GAG) content, and mRNA expression were investigated to evaluate the efficiency of biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of cell adhesion to scaffolds in the cartilage tissue. The results showed that the biotin-conjugated anti-CD44 antibody-avidin binding system improved cell adhesion to scaffolds effectively. These studies suggest that this binding system has the potential to provide improved tissue-engineered cartilage for clinical applications.

This study aimed to analyze the in vitro and in vivo release kinetics and evaluate the grades of repair induced by either the release of 50 ng of transforming growth factor-β1 (TGF-β1) or 2.5 or 5 µg of bone morphogenetic protein-2 (BMP-2) from a bilayer scaffold of segmented polyurethane/polylactic-co-glycolic (SPU/PLGA) in osteochondral defects, in a rabbit model. The scaffold consisted of a porous, bone-directed PLGA layer, overlaid with a cartilage-directed layer of growth factor (GF) loaded PLGA microspheres, dispersed in a matrix of SPU. The PLGA porous layer was fabricated by gas foaming. Microspheres were prepared by a double emulsion method. SPU was synthesized following the two-step method. GF release kinetics were assessed using iodinated (¹²⁵I) GFs. The in vivo release profiles of both GFs fitted to zero order kinetics, demonstrating a consistently good control of their release rates by SPU. Cartilage-like tissue, characterized by histological analysis, scoring, and immunolabeling of chondrogenic differentiation markers, was only observed after 12 weeks, maintaining integrity up to at least 24 weeks, independently of the GF and the dose of BMP-2. The biocompatibility and the resulting good quality, hyaline repair cartilage convert this system into a promising candidate for future applications in osteochondral lesions.

The conformation change of bovine serum albumin (BSA) induced by bioactive titanium surfaces, including alkali-acid treated titanium (AA-Ti) and alkali-heat treated titanium (AH-Ti), was studied, and its effects on the activity of MC3T3-E1 cell were evaluated. Pure titanium metal (P-Ti) was used as control. The AA-Ti could adsorb more BSA on its surface than AH-Ti and P-Ti. The α-helix part of the protein adsorbed on P-T has weakly decreased compared with native BSA, and it dramatically decreased on AA-Ti and AH-Ti. The β-sheet segment of proteins adsorbed on P-Ti and AH-Ti had obviously increased. Much more tryptophan residues were exposed after the protein conformation changed when it interacted with AH-Ti, and some tryptophan residues were enveloped after it interacted with AA-Ti and P-Ti. AA-Ti has more tryptophan residues enveloped than P-Ti. All titanium surfaces induced tyrosine residues exposed, especially for the P-Ti. The higher ratio of COO^-/NH₃⁺ for the proteins on P-Ti and AA-Ti indicated an orientation of proteins on P-Ti and AA-Ti, which makes more COO^- exposed. The lower ratio of COO^-/NH₃⁺ on AH-Ti indicates that more NH₃⁺ is exposed on its surface. The cell proliferation ability on different treated titanium surfaces coated with BSA followed by the order: P-Ti > AA-Ti > AH-Ti, which indicated that the protein conformation change on different bioactive titanium surfaces has great effect on the cell activity. Our results showed that the different biological response of bioactive titanium metals might depend on the protein conformation change induced by the surface structure.

Cellular adhesiveness to biomaterial is one of the important properties to the success of tissue engineering. The cell-biomaterial interactions involve close cooperation of adhesion proteins, the plasma membrane, and cytoskeletons in order to form focal adhesions during the process of anchoring. Dynamic development of the plasma membrane in the process reflects the cellular biocompatibility and motility. The process of cell attachment beginning from seeding, contact, attachment and spreading has not been investigated. In this study, we monitored the whole process of cells attaching to the substrate surface by time-lapse confocal microscopy. We observed that the surface configuration of the substratum effects plasma membrane expansion and genomic materials distribution. In contrast to the cells grown on the plate, the cells attached on pillars are with rounded nuclei and with prominent lamellipodia spreading out. Membrane expansion is involved in dynamic development of the plasma membrane and lamellipodia formation for attachment, migration or proliferation and reflects the cellular physiology status of the cells. This study provides a platform for investigation of cell behavior and dynamic development of subcellular structures regarding cell-biomaterial interactions.

Objective: To explore the feasibility of constructing a functional biomaterial complex with regenerated silk fibroin membrane and immortalized chondrocytes in vivo.

Methods: RACs (rat auricular chondrocytes) were transfected with the lentivirus vector pGC-FU-hTERT-3FLAG or pGC-FU-GFP-3FLAG, encoding the human telomerase reverse transcriptase (hTERT) or GFP gene. The effects of regenerated silk fibroin film on the adhesion, growth of immortalized chondrocytes and expression of collagen II in vitro were analyzd with immunofluorescent histochemistry.

Results: Immortalized RACs were transformed. Induction by nutrient medium promoted higher expression levels of collagen II in transformed chondrocytes. The regenerated silk fibroin film was not cytotoxic to immortalized chondrocytes and had no adverse influence on their adhesion. Collagen II expression was good in the immortalized chondrocytes in vivo.

Conclusion: The construction of a silk-based biomaterial complex with immortalized chondrocytes may provide a feasible kind of functional biomaterial for the repair of cartilage defects in clinical applications.

Purpose: Use of textile structures for reinforcement of pelvic floor structures has to consider mechanical forces to the implant, which are quite different to the tension free conditions of the abdominal wall. Thus, biomechanical analysis of textile devices, have to include the impact of strain on stretchability and effective porosity.

Material and methods: Prolift® and Prolift + M®, developed for tension free conditions, were tested by measuring stretchability and effective porosity applying mechanical strain. For comparison we used Dynamesh – PR4®, which was designed for pelvic floor repair to withstand mechanical strain.

Results: Prolift® at rest showed moderate porosity with little stretchability but complete loss of effective porosity at strain of 4.9 N/cm. Prolift + M® revealed an increased porosity at rest, but at strain showed high stretchability, with subsequent loss of effective porosity at strain of 2.5 N/cm. Dynamesh PR4® preserved its high porosity even under strain, but as consequence of limited stretchability.

Conclusions: Though in tension free conditions Prolift® and Prolift + M® can be considered as large pore class I meshes, application of mechanical strain rapidly lead to collapse of pores. The loss of porosity at mechanical stress can be prevented by constructions with high structural stability. Assessment of porosity under strain was found helpful to define requirements for pelvic floor devices. Clinical studies have to prove whether devices with high porosity as well as high structural stability can improve the patients' outcome.

Hydroxyapatite (HA) biomaterials and allograft bone are common alternatives to autogenous grafts, however these materials lack the strong osteoinductive potential of autologous bone. Previous studies have established that polyglutamate domains, which bind selectively to HA, can be engineered onto bioactive peptides as a mechanism for coupling osteoinductive signals onto HA and allograft. In the current investigation, we adapted the polyglutamate approach to tailor delivery of a model collagen-derived peptide, DGEA, by manipulating the number of glutamates in the HA binding domain. Specifically, DGEA was modified with diglutamate (E2-DGEA), tetraglutamate (E4-DGEA) or heptaglutamate (E7-DGEA), and it was found that initial peptide binding to HA and allograft was significantly enhanced as the number of glutamates increased. We also determined that the rate of release of polyglutamate-DGEA from substrates over a 5-day interval increased proportionally as the number of glutamate residues was decreased. Additionally, we tuned the peptide release rate by creating mixtures of E2-DGEA, E4-DGEA and E7-DGEA, and observed that release kinetics of the mixtures were distinct from pure solutions of each respective peptide. These collective results suggest that variable length polyglutamate domains provide an effective mechanism for controlled delivery of osteoregenerative peptides on HA-containing bone graft materials.

Decellularized whole organs represent ideal scaffolds for engineering new organs and/or cell transplantation. Here, we investigate whether decellularized liver scaffolds provide cell-friendly biocompatible three-dimensional environment to support the proliferation and differentiation of hepatic progenitor cells. Mouse liver tissues are efficiently decellularized through portal vein perfusion. Using the reversibly immortalized mouse fetal hepatic progenitor cells (iHPCs), we are able to effectively recellularize the decellularized liver scaffolds. The perfused iHPCs survive and proliferate in the three-dimensional scaffolds in vitro for 2 weeks. When the recellularized scaffolds are implanted into the kidney capsule of athymic nude mice, cell survival and proliferation of the implanted scaffolds are readily detected by whole body imaging for 10 days. Furthermore, EGF is shown to significantly promote the proliferation and differentiation of the implanted iHPCs. Histologic and immunochemical analyses indicate that iHPCs are able to proliferate and differentiate to mature hepatocytes upon EGF stimulation in the scaffolds. The recellularization of the biomaterial scaffolds is accompanied with vascularization. Taken together, these results indicate that decullarized liver scaffolds effectively support the proliferation and differentiation of iHPCs, suggesting that decellularized liver matrix may be used as ideal biocompatible scaffolds for hepatocyte transplantation.

In this study we evaluated the effect of new plasma-nitrided Ti surfaces on the progression of osteoblast cultures, including cell adhesion, proliferation and differentiation. Ti surfaces were treated using two plasma nitriding protocols, hollow cathode for 3 h (HC 3h) and 1 h (HC 1h) and planar (Planar) for 1 h. Untreated Ti surfaces were used as Control. Cells derived from human alveolar and rat calvarial bones were cultured on Ti surfaces for periods of up to 14 days and the following parameters were evaluated: cell morphology, adhesion, spreading and proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, and gene expression of key osteoblast markers. Plasma nitriding treatments resulted in Ti surfaces with distinct physicochemical characteristics. The cell adhesion and ALP activity were higher on plasma-nitrided Ti surfaces compared with untreated one, while cell proliferation and extracellular matrix mineralization were not affected by the treatments. In addition, the plasma-nitrided Ti surfaces increased the ALP, reduced the osteocalcin and did not affect the Runx2 gene expression. We have shown that HC 3h and Planar Ti surfaces slightly favored the osteoblast differentiation process and then these surfaces should be considered for further investigation using preclinical models.

The purpose of this study was to investigate osteogenesis using an artificial fusion protein (AFP) composed of modified bone morphogenetic protein-2 (BMP-2) with a titanium (Ti) binding peptide (TBP) motif on a Ti surface in vivo. In the in vivo study, 5 μm thick Ti was coated with electron cyclotron resonance sputtering on a porous carbon scaffold which was then dipped in 1 of 3 different mixtures of collagen gel: 1, collagen gel only, 2, collagen gel with TBP, and 3, collagen gel with the AFP between BMP-2 and the TBP motif (AFP-TBP-BMP-2). These scaffolds were then implanted into rat abdominal muscles and were studied histologically at various times and the expression of several bone-related protein mRNAs was also analyzed. The Ti-coated scaffold of the collagen gel with AFP-TBP-BMP-2 produced cartilage in the muscle and the expression of ALP, BSP and Runx2 mRNAs was significantly increased. These results suggest that the scaffold of the collagen gel with AFP-TBP-BMP-2 accelerates osteogenesis in vivo.

Tissue engineering is a multidisciplinary science which combines a structural scaffold and cells to form a construct able to promote regeneration of injured tissue. Bioactive glass foam produced by sol-gel is an osteoinductive material with a network of interconnected macropores necessary for cell colonization. The use of human adipose-derived stem cell (hASC) presents advantages as the potential for a large number of cells, rapid expansion in vitro and the capability of differentiating into osteoblasts. The use of a bioreactor in three-dimensional cell culture enables greater efficiency for cell nutrition and application of mechanical forces, important modulators of bone physiology. The hASC seeded in a bioactive glass scaffold and cultured in osteogenic Leibovitz L-15 medium in a bioreactor with a flow rate of 0.1 mL/min demonstrated a significant increase in cell proliferation and viability and ALP activity peak after 14 days. The immunofluorescence assay revealed an expression of osteopontin, osteocalcin and type I collagen from 7 to 21 days after culture. The cells changed from a spindle shape to a cuboidal morphology characteristic of osteoblasts. The PCR assay confirmed that osteopontin, osteocalcin and ALP genes were expressed. These results indicate that hASCs differentiated into an osteogenic phenotype when cultured in bioactive glass scaffold, osteogenic Leibovitz L-15 medium and a perfusion bioreactor. Therefore, these results highlight the synergism between a bioactive glass scaffold and the effect of perfusion on cells and indicate the differentiation into an osteogenic phenotype.

Biodegradable stents can alleviate intestinal obstruction and stenosis in patients. The objective of this study was to develop a biodegradable polydioxanone stent using weft-knitting technology and then investigate its biodegradation behaviours in vitro. Polydioxanone monofilament with linear density of 100 ± 10 Tex was knitted into a tubular stent using a tubular weft-knitting machine. The physical and mechanical properties were evaluated according to British standard BS EN 13895:2003 and ISO 7198:1998. The biodegradation behaviours of polydioxanone weft-knitted stent in a phosphate buffer solution (pH = 6.8 ± 0.2, 37 ± 0.5 °C) were then investigated. The results showed that the stent maintained more than 60% of its original radial force above 12 weeks. During the 16 weeks of degradation, weight, crystallization and pH change indicated the degradation medium was diffused into the chain segments of low molecular weight due to the rupture of ester bonds in the monofilament. Fourier transform infrared spectroscopy results demonstrated that the chemical structure of polydioxanone polymer is stable during the in vitro degradation. In conclusion, this biodegradable stent can find valuable applications in treatment of intestinal obstruction and stenosis clinically.

Purpose: “TiGlass” was designed and was known to promote initial adhesion and increase migration of rat calvarial osteoblats. In this paper, migration study and a series of epifluorescence microscopic studies were conducted to find out the composition of focal adhesion on titanium surface.

Material and methods: The translucent titanium surface was applied in random migration analysis and immunofluorescence cell staining. In the immunofluorescent double staining, pFAK was tested with vinculin. Various integrin subunits were then tested with vinculin to study the composition of activated focal adhesions. Integrin subunit α5, αV were tested against β3; Integrin subunit α5, αV, β3, αVβ3 were tested with F-actin, respectively.

Results: The MG-63 cells began migration earlier and migrated faster on “TiGlass". Immunofluorescent double staining revealed all FAK in the focal adhesions were activated on both surfaces. The osteoblast was inferred to made adhesion to titanium and glass through integrins. The focal adhesions on glass were found to be composed of integrin subunits αV and β3. However, on “TiGlass", integrin subunits α5 might have supplemented the adhesion to titanium. Results from double staining of integrin subunits α5, αV, β3, and αVβ3 with F-actin also supported integrin subunits α5 might have involved in adhesion of titanium.

Generation of supporting structures which guide cell growth is a challenging task in the field of tissue engineering. Cell guidance properties of a scaffold are important in the field of neuronal regeneration. Those guiding structures can provide guidance just by mechanical stimulus or by chemical stimuli like cell signaling molecules. For an enhanced guidance chemical gradients are under investigation. With this study we show that UV-laser irradiation is a useful tool to activate polymer surfaces with a high temporal and spatial resolution. We demonstrated that poly(methyl metacrylate) (PMMA) and poly-ε-caprolactone (PCL) can be locally activated and functionalized with amine groups which can be used for immobilization of RGD peptide. The immobilized RGD was detected by neuronal B35 cells. By defined pulse accumulation functionalization density on the surface can be varied for the generation of gradients. We demonstrated that PMMA and PCL have different process windows for functionalization. While PMMA has a very small process window for activation, PCL allows the generation of stepwise functionalization.

The presented technology can help to develop assays for the analysis of cell migration and neuronal regeneration due to flexible patterning easily realized by changing the irradiation parameters.

Biofilm formation on medical devices is a common cause of implant failure, especially regarding implants that breach the epithelial tissue, so called transcutaneous implants. Nanotechnology and the development of new nanomaterials have given the opportunity to design nanotextured implant surfaces. Such surfaces have been studied using various in vitro methods showing that nanosized features strongly benefit bone cell growth. However, little is known on how nanostructured features affect biofilm formation. The aim of this study was therefore to examine the shape- and chemical-dependent effect of a nanostructured hydroxyapatite (HA) coating on the degree of Staphylococcus epidermidis biofilm formation. Three different types of nanosized HA particles having different shapes and calcium to phosphate ratios were compared to uncoated turned titanium using safranin stain in a biofilm assay and confocal laser scanning microscopy (CLSM) for assessment of biofilm biomass and bacterial volume, respectively. No difference in biofilm biomass was detected for the various surfaces after 6 h incubation with S. epidermidis. Additionally, image analysis of CLSM Z-stacks confirmed the biofilm assay and showed similar results. In conclusion, the difference in nanomorphology and chemical composition of the surface coatings did not influence the adhesion and biofilm formation of S. epidermidis.

Chitosan (Ch) is one of the most commonly used natural biomaterials. Osteo-differentiation of mesenchymal stem cells (MSCs) on Ch has drawn extensive interest. Hydroxyapatite (HA) is a component of skeleton and teeth with good biocompatibility. Combination with HA may be a good method to modify Ch to facilitate cellular behaviors and functions on it. In this study, chitosan/hydroxyapatite (Ch/HA) film was prepared and characterized. Its potential to benefit cellular behaviors and osteo-differentiation of MSCs was evaluated. Resultantly, physical properties of composite Ch/HA, including water-in-air contact angle, tensile strength, elastic modulus and breaking elongation were favorably modified. In cellular culture medium, Ch/HA films absorbed more Ca²⁺ than Ch films, and more HA crystalline growths on Ch/HA films. MTT and morphological features showed better proliferation and adhesion of MSCs on Ch/HA films. Osteo-differentiation of MSCs on Ch/HA was promoted, indicated by modified transcription level of osteocalcin, osteopontin, collagen I, alkaline phosphatase, and induced alkaline phosphatase activity. These data suggest biocompatibility of Ch is modified after being blended with HA, which promotes osteo-differentiation of MSCs. This can be a promising approach to modify Ch for its applications in bone tissue engineering.

Hydroxyapatite (HA) coatings are widely applied to enhance the level of osteointegration onto orthopaedic implants. Atmospheric plasma spray (APS) is typically used for the deposition of these coatings, however HA crystalline changes regularly occur during this high thermal process. This paper reports on the evaluation of a novel low temperature (<47 °C) HA deposition technique, called CoBlast, for the application of crystalline HA coatings. To-date, reports on the CoBlast technique have been limited to titanium alloy substrates. This study addresses the suitability of the CoBlast technique for the deposition of HA coatings on a number of alternative metal alloys utilised in the fabrication of orthopaedic devices. In addition to titanium grade 5, both cobalt chromium and stainless steel 316 were investigated. In this study HA coatings were deposited using both the CoBlast and plasma sprayed techniques, and the resultant HA coating and substrate properties were evaluated and compared. The CoBlast deposited HA coatings were found to present similar surface morphologies, interfacial properties and composition irrespective of the substrate alloy type. Coating thickness however displayed some variation with the substrate alloy, ranging from 2.0 to 3.0 μm. This perhaps is associated with the electro-negativity of the metal alloys. The APS treated samples exhibited evidence of both coating, and significantly, substrate phase alterations for two metal alloys; titanium grade 5 and cobalt chrome. Conversely, the CoBlast processed samples exhibited no phase changes to the substrates after depositions. The APS alterations were attributed to the brief, but high intensity temperatures experienced during processing.

An emerging approach towards development of injectable, self setting and fully biodegradable bone substitutes involves the combination of injectable hydrogel matrices with a dispersed phase consisting of nanosized calcium phosphate particles. Here, novel injectable composites for bone regeneration have been developed based on the combination of ultra pure alginate as the matrix phase, crystalline CaP (monetite and poorly crystalline hydroxyapatite) powders as both a dispersed mineral phase and a source of calcium for crosslinking alginate, GDL as acidifier and glycerol as both plasticizer and temporary sequestrant. The composites were maximized with respect to CaP content to obtain the highest amount of osteoconductive filler. The viscoelastic and physicochemical properties of the precursor compounds and composites were analyzed using rheometry, elemental analysis (for calcium release and uptake), acidity (by measuring pH in SBF), general biocompatibility (subcutaneous implantation in rabbits) and osteocompatibility (implantation in femoral condyle bone defect of rabbits). The gelation of the resulting composites could be controlled from seconds to tens of minutes by varying the solubility of the CaP phase (hydroxyapatite vs. monetite) or amount of GDL. All composites mineralized extensively in SBF for up to 11 days. In vivo, the composites also disintegrated upon implantation in subcutaneous or bone tissue, leaving behind less degradable but osteoconductive CaP particles. Although the composites need to be optimized with respect to the available amount of calcium for crosslinking of alginate, the beneficial bone response as observed in the in vivo studies render these gels promising for minimally invasive applications as bone filling material.

Human amniotic membrane (AM) has been widely used as graft biomaterial for a variety of clinical applications. But, there have some persistent problems related to the preparation, storage, and sterilization. To resolve these problems, we developed Hyper dry AM (HD-AM) using far-infrared rays, depression of air and microwaves and then sterilized by γ-ray irradiation.

To elucidate the benefit of HD-AM as biological materials, it was to compare to the histological and physical properties of HD-AM with a freeze-dried AM (FD-AM) as typical cryopreserved methods, evaluate the safety of HD-AM in vivo experiment used nude mice, and demonstrate the feasibility of HD-AM transplant in pterygium.

HD-AM has been kept the morphological structure of epithelium and connective tissues. The water permeability and the sieving coefficient of HD-AM were significantly lower than that of FD-AM. At 18 months after transplanted, single and multi layer of HD-AM in the intraperitoneal cavity was degraded without any infiltrated cells. For clinical treatment, recurrence of pterygium and regrowth of the subconjunctival fibrosis were not observed during the follow-up periods of 6 months after the surgery.

It was proposed that HD-AM was a safe and effective new biological material for clinical use including treatment for recurrent pterygium.

In view of the fact that bone healing can be enhanced due to external electric field application, it is important to assess the influence of the implant conductivity on the bone regeneration in vivo. In order to address this issue, the present study reports the in vivo biocompatibility property of multistage spark plasma sintered Hydroxyapatite (HA)-80 wt. % calcium titanate (CaTiO₃) composites and monolithic HA, which have widely different conductivity property (14 orders of magnitude difference). The ability of bone regeneration was assessed by implantation in cylindrical femoral bone defects of rabbit animal model for varying time period of 1, 4, and 12 weeks. The overall assessment of the histology results suggests that the progressive healing of bone defects around HA-80 wt.% CaTiO₃ is associated with a better efficacy w.r.t early stage neobone formation, which is histomorphometrically around 140% higher than monolithic HA. Overall, the present study demonstrates that the in vivo biocompatibility property of HA-80 wt.% CaTiO₃ with respect to local effects after 12 weeks of implantation is not compromised both qualitatively and quantitatively and a comparison with control implant (HA) points towards the critical role of electrical conductivity on better early stage bone regeneration.

The biologic/cytotoxic effects of dispersed nano graphene platelets (NGPs) to human osteosarcoma cells (MG63 cell line) were firstly studied by examining cell viability, cycle, apoptosis, the change of morphology, lactate dehydrogenase (LDH) release, alkaline phosphatase (ALP) activity and inflammation. The results shown that the cell cytotoxicity of the dispersed NGPs exhibited dose-dependent characters, which had no obvious cytotoxic effects to MG63 cells at the concentration less than 10 μg/mL, whereas could postpone cell cycle, promote cell apoptosis, damage cell microstructure, induce serious tumor necrosis factor-α (TNF-α) expression and greatly reduce ALP activity of MG63 cells at higher concentration of NGPs (>10 µg/ml ). Besides, NGPs had little influence on the LDH leakage. The cytotoxic mechanism of NGPs to MG63 cells was speculated to be intracellular activity with no physical damage of plasma membrane.

Failure rates of spinal fusion are high in smokers and diabetics. The authors are investigating the development of a piezoelectric composite biomaterial and interbody device design that could generate clinically relevant levels of electrical stimulation to help improve the rate of fusion for these patients. A lumped parameter model of the piezoelectric composite implant was developed based on a model that has been utilized to successfully predict power generation for piezoceramics. Seven variables (fiber material, matrix material, fiber volume fraction, fiber aspect ratio, implant cross-sectional area, implant thickness, and electrical load resistance) were parametrically analyzed to determine their effects on power generation within reasonable implant constraints. Influences of implant geometry and fiber aspect ratio were independent of material parameters. For a cyclic force of constant magnitude, implant thickness was directly and cross-sectional area inversely proportional to power generation potential. Fiber aspect ratios above 30 yielded maximum power generation potential while volume fractions above 15 percent showed superior performance. This investigation demonstrates the feasibility of using composite piezoelectric biomaterials in medical implants to generate therapeutic levels of direct current electrical stimulation. The piezoelectric spinal fusion interbody implant shows promise for helping increase success rates of spinal fusion.

Chitosan is a biodegradable and biocompatible natural scaffold material, which has numerous applications in biomedical sciences. In this study, the in vitro antioxidant activity of chitosan scaffold material was investigated by the chemiluminescence signal generated from the hydroxyl radical (•OH) scavenging assay. The scavenging mechanism was also discussed. The results indicated that the free radical scavenging ability of chitosan scaffold material significantly depends on the chitosan concentration and shows interesting kinetic change. Within the experimental concentration range, the optimal concentration of chitosan was 0.2 mg/mL. The molecular weight of chitosan also attributed to the free radical scavenging ability. Comparison between chitosan and its derivative found that carboxymethyl chitosan possessed higher scavenging ability.

This study evaluated the utility of the creation of a nanoporous TiO₂ surface to enhance the in vitro biocompatibility and in vivo osseoconductivity of polyetheretherketone (PEEK) implants by providing favorable sites for the effective immobilization of bone morphogenetic protein-2 (BMP-2). A uniform nanoporous TiO₂ layer with a pore diameter of ~ 70 nm was successfully created by anodizing a Ti film, which had been deposited onto a PEEK substrate via electron beam (e-beam) evaporation technique. This nanoporous, hydrophilic TiO₂ surface enabled the efficient immobilization of BMP-2, resulting in a remarkable enhancement in in vitro biocompatibility that was assessed in terms of cell attachment, proliferation and differentiation. The in vivo animal tests also confirmed that the nanoporous TiO₂ surface immobilized with BMP-2 could significantly enhance the osseoconductivity of PEEK implants. The BMP-immobilized PEEK implant with the nanoporous TiO₂ surface showed much higher bone-to-implant contact (BIC) ratio (60 %) than the bare PEEK (30 %), PEEK with the nanoporous TiO₂ surface (50 %) and even BMP- immobilized PEEK without the nanoporous TiO₂ surface (32 %).

We aimed to evaluate the feasibility of the application of the nano-hydroxyapatite/ Chitosan/poly(lactide-co-glycolide)(nHA/CS/PLGA)scaffold seeded with human umbilical cord mesenchymal stem cells (hUCMSCs) in bone tissue engineering. We prepared the nHA/CS/PLGA, nHA/PLGA, CS/PLGA and PLGA scaffolds, and tested their mechanical strength. We analyzed the surface antigen markers of hUCMSCs to determine their capability to differentiate into osteoblasts, chondrocytes, and adipocytes. The growth of hUCMSCs on the four types of scaffold was assayed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT assay)and observed using scanning electron microscopy (SEM). Quantitative analysis of alkaline phosphatase (ALP)activity and osteocalcin (OCN)content, as well as the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed. After 21 days, the subcutaneous implantations of the scaffolds samples seeded with hUCMSCs into nude mice were analyzed using immunohistochemical staining. The results showed that the mechanical strength of the nHA/CS/PLGA scaffold was enhanced. Furthermore, the nHA/CS/PLGA scaffolds were the most suitable for the adhesion, proliferation and osteogenic differentiation of hUCMSCs in vitro and nude mouse subcutaneous implantation. The enhanced osteogenic inductivity of the nHA/CS/PLGA scaffolds for hUCMSCs might result from the addition of nHA and CS.

Macrophages play a critical role in mediating not only normal tissue healing, but also the host reaction against biomaterial scaffolds. There is increasing interest in regenerative medicine to combine mesenchymal stromal/stem cells (MSCs) with biomaterial scaffolds to modulate inflammatory response while restoring tissue architecture. The objective of the current study was to investigate the interaction between MSCs, encapsulated in hyaluronan–based hydrogel, and differentiating macrophages as measured by extracellular matrix (ECM) gene expression and cytokine, chemokine and growth factors concentrations. Gene expression was analyzed using real-time PCR from MSCs embedded in Carbylan-GSX after 7 days of co-culture with or without CD14+ cells. Protein concentrations were measured using a Bio-plex assay from cell culture supernatants on days 3 and 7 of all conditions. Following seven days, we identified upregulation of collagen-I, collagen-III, pro-collagen, and matrix metalloproteinase-9 genes compared to control conditions. We demonstrate increased concentrations of immunoregulatory cytokines (IL-1β, TNF-α, MIP-1α, IFN-γ, IL-12, IL-10) and remodeling growth factors (VEGF, HGF) in MSC-3D constructs co-cultured with macrophages compared to control conditions, with some temporal variations. Our results indicate an alteration of expressions of ECM proteins important to tissue regeneration and cytokines critical to the inflammatory cascade when 3D constructs were cultured with differentiating macrophages.

Vascular endothelial growth factor (VEGF) is a powerful growth factor that promotes vascularization as well as osteoblastic differentiation and bone regeneration, all of which are key processes in the osseointegration of dental implants. Strategies to increase vascularization through delivery of VEGF may improve osseointegration, especially in patients with reduced bone healing potential. The aim of this study was to determine the potential of chitosan coatings on titanium to deliver VEGF and to support growth and matrix production of osteoblastic cells in vitro. Chitosan was chemically bonded to titanium coupons via silane-glutaraldehyde linker molecules and loaded with 0, 20, 50, or 100 ng of VEGF. Protein was released during a three day period with around 75% of VEGF (4.44, 11.37, and 22.10 ng/mL/cm² from the 20, 50 and 100 ng loaded levels respectively) released during the first 12 hours, and 90-95% of the VEGF released from the coatings by day 3. Saos-2 bone cells continued to proliferate over the 28 day period on the VEGF loaded chitosan coatings in contrast to cells seeded on uncoated titanium which plateaued after 14 days. Cells on uncoated titanium exhibited a peak in alkaline phosphatase expression at approximately 14 days, concomitant with the plateau in growth. While osteoblast-like cells on all chitosan coatings exhibited up to a 2-fold enhancement of the alkaline phosphatase activity and 10-fold increase in calcium deposition compared to uncoated controls, the incorporation of VEGF into the coatings did not enhance osteoblast matrix production over plain chitosan coatings throughout the study.

Poly(allylamine hydrochloride) (PAH) has found many applications both in biotechnology and biomedical fields. However, its high toxicity towards various mammalian cells significantly limits its effective usage. This study focuses on improving the biological properties of PAH by its modification to strong polyelectrolytes. The strong polycations were prepared by the direct quaternization of PAH amino groups or by the attachment of glycidyltrimethylammonium chloride to these groups. The biological properties, such as cytotoxicity towards human skin fibroblasts (HSFs), proliferation and migration of the cells on a polymeric surface, and antibacterial activities against two pathogenic bacteria, Staphylococcus aureus and Escherichia coli, were determined. All the modified polyelectrolytes are considerably less toxic to HSFs as compared to PAH. Moreover, the directly quaternized polycations are stronger biocides against S. aureus than the parent polymer. Contrary to PAH, thin films of the modified polyelectrolytes improve or do not affect HSFs proliferation and can stimulate cell migration into the wound, as was demonstrated using an in vitro model. The relationship between the structure of the modified polymers (amount and localization of the quaternary ammonium groups) and the biological activity is discussed. Due to the improved biological properties, the obtained polycations may be potentially useful for a variety of biotechnological and biomedical applications.

Though 2-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the 3-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide chemically conjugated to positively charged polyethyleneimine. We have achieved a conjugation ratio of 30 mol%, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol% and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool.

The postoperative outcome of flexor tendon healing remains limited by flexor tendon adhesion that reduces joint range of motion. Despite improvement in different methods, peritendinous adhesion formation continues to present a formidable challenge. Recent studies showed that transforming growth factor-β3 (TGF-β3) may be the key factor to reducing adhesion formation in skin or tendon. In this study, we designed a novel type of tissue engineering synovial sheath containing TGF-β3, to prevent flexor tendon adhesion. First, to achieve a stable release of TGF-β3, chitosan microspheres, prepared by crosslinking-emulsion, were used for the delivery of TGF-β3. Second, a three-dimensional chitosan scaffold was prepared by lyophilization, and TGF-β3 microspheres were carefully introduced into the scaffold. Then, synovial cells were cultured and then seeded into the TGF-β3 loaded scaffold to produce TGF-β3 controlled-released tissue engineering synovial sheath. Tests clearly demonstrated that the scaffold has good structure and compatibility with cells. These results expand the feasibility of combinative strategies of controlled protein release and tissue-engineered synovial sheath formation. Application of this scaffold to tendon repair sites may help to prevent adhesion of tendon healing.

This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli (E. coli) viability and reduced the colony forming units over a 3-day incubation period in an artificial urine solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the artificial urine solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.

Surface modification, as a means of enhancing soft tissue integration in titanium would have significant advantages including less marginal bone resorption, predictable esthetic outcome, improved soft tissue stability and seal against bacterial leakage.

The aim of this study was to evaluate the effects of laser-roughened titanium surfaces on human gingival fibroblast viability, proliferation and adhesion.

Titanium discs were ablated with impulse laser in four different patterns. Polished and sand-blasted titanium discs were used as control groups. Specimen surface properties were determined using optical profilometry and scanning electron microscopy. Human gingival fibroblast behaviour on modified surfaces was analyzed using cell adhesion, viability, proliferation and ELISA assays. Results suggested that modified Ti surfaces did not affect the viability of human gingival fibroblasts and improved adhesion was measured in laser treatment groups after 24 hours. However, proliferation study showed that the adsorbance of fibroblast cells after 72h cultured on polished titanium was higher and comparable with that of control cells. As for FAK, cells grown on laser modified surfaces had higher expression of FAK compared with polished titanium.

In conclusion, tested laser-treated surfaces seem to favor human gingival fibroblast adhesion. There were no significant differences between different laser treatment groups.

The biological response of osteoblast cells to implant materials depends on the topography and physico-chemistry of the implant surface and this determines the cell behavior such as shaping, adhesion and proliferation and finally the cell fate. In this study, Titanium (Ti) was anodized to create different topographies of titania nanotubes (TNTs) in order to investigate the cell behavior to them. TNTs with and without a highly ordered nanoporous layer on their top surface were fabricated using two-step and one-step anodizing processes, respectively. The TNTs without a highly ordered nanoporous layer on the top surface exhibited a rougher surface, higher surface energy and better hydrophilicity than the TNTs with such a layer. Osteoblast-like cells (SaOS2) were used to assess the biocompatibility of the TNTs with different topographies in comparison to bare cp-Ti. Results indicated that TNTs can enhance the proliferation and adhesion of osteoblast cells. TNTs without a highly ordered nanoporous layer exhibited better biocompatibility than the TNTs covered by such a nanoporous layer. Cell morphology observation using confocal microscopy and SEM indicated that SaOS2 cells that were adhered to the TNTs without the highly ordered nanoporous layer showed the longest filopodia compared to TNTs with a highly ordered nanoporous layer and bare cp-Ti.

The purpose of this study was to investigate the responses of the human osteosarcoma cell line MG63 to calcium silicate cements with different Si/Ca molar ratios and different surface roughness. In particular, the study evaluated integrin subunit levels, phosphor-focal adhesion kinase (pFAK) levels and protein production at the cell attachment stage. The results indicated that the surface roughness (variations within a factor of 10) of the cements did not play a prominent role in cell attachment and proliferation, but the effect of composition was highlighted. Increased pFAK and total integrin levels and promoted cell attachment and cell cycle progression were observed upon an increase in cement Si content. Cement with a higher Si content was beneficial for collagen type I (COL I) adsorption, COL I secretion and αlibβ3 subintegrin expression, whereas cement with a higher Ca content increased fibronectin (FN) adsorption, FN secretion and enhanced αvβ1 subintegrin levels. These results establish composition-dependent differences in integrin binding as a mechanism regulating cellular responses to biomaterial surfaces.

In this paper the pore structure and porosity parameters of Polycaprolactone (PCL) nano-microfibrous scaffolds are investigated using a predicting theoretical model and a non-destructive evaluation approach based on Confocal laser scanning microscopy (CLSM) and three-dimensional image analysis. Different fibrous scaffolds with different fiber diameters produced by Electrospinning process and their 3D–pore structure were evaluated theoretically and also compared to results of CLSM and Capillary Flow Porometery methods. The effect of polymer concentration on the pore structure of scaffolds was also investigated. The results showed that, the introduced approach not only can measure the pore size distribution of nanofibrous scaffolds, but also it can measure pore interconnectivity of fibrous scaffolds. Furthermore, the results showed that increasing the fiber diameter resulted from increasing the polymer concentration in solvent can effectively increase the pore dimensions within the scaffold structure.

Polyurethanes with regular and controlled block arrangement, i.e. alternating block polyurethanes (abbreviated as PUCL-alt-PEG) based on poly(ε-caprolactone) (PCL-diol) and poly(ethylene glycol) (PEG) was prepared via selectively coupling reaction between PCL-diol and diisocyanate end-capped PEG. Chemical structure, molecular weight, distribution and thermal properties were systematically characterized by FTIR, ¹H NMR, GPC, DSC and TGA. Hydrophilicity was studied by static contact angle of H₂O and CH₂I₂. Film surface was observed by scanning electron microscope (SEM) and atomic force microscopy (AFM), and mechanical properties were assessed by universal test machine. Results show that alternating block polyurethanes give higher crystal degree, higher mechanical properties and more hydrophilic and rougher (deep ravine) surface than their random counterpart, due to regular and controlled structure. Platelet adhesion illustrated that PUCL-alt-PEG has better hemocompatibility and the hemacompatibility was affected significantly by PEG content. Excellent hemocompatibility was obtained with high PEG content. CCK-8 assay and SEM observation revealed much better cell compatibility of fibroblast L929 and rat glial cells on the alternating block polyurethanes than that on random counterpart. Alternating block polyurethane PUC20-a-E4 with optimized composition, mechanical, surface properties, hemacompatibility and highest cell growth and proliferation was achieved for potential use in nerve regeneration.

Emulsion electrospinning has been sought as a method to prepare fibrous materials/scaffolds for growth factor delivery. Emulsion conditions, specifically sonication and the addition of a surfactant, were evaluated to determine their effect on the release and bioactivity of proteins from electrospun scaffolds. Polycaprolactone (PCL) and poly(ethylene oxide) (PEO/PCL) blends were evaluated where PEO, a hydrophilic polymer, was shown to enhance the incorporation of proteins. Electrospun scaffolds prepared with the addition of the non-ionic surfactant Span® 80 at a concentration greater than the critical micelle concentration (CMC) followed by mild sonication (10% amplitude) released lysozyme, the model protein, with a higher level of bioactivity as compared to other surfactant and sonication conditions. These conditions were then used to prepare emulsions of platelet-derived growth factor-BB (PDGF-BB) in PEO/PCL blends. Electrospun mats prepared by emulsions consisting of PDGF-BB incorporated with Span® 80 and sonicated at 10% amplitude exhibited a controlled release of PDGF-BB over 96 hours as compared to a more rapid release from solutions that were not emulsified (Direct Addition) or emulsions that did not receive Span® 80 or sonication. Bioactive PDGF-BB incorporated in electrospun scaffolds enhanced the osteogenic differentiation of human mesenchymal stem cells as evidenced by increased alkaline phosphatase activity, as well as improved cell attachment and reorganized cytoskeletal filaments. The findings in this study provide improved methods for achieving controlled release of bioactive proteins from electrospun materials.

A nerve conduit is designed to improve peripheral nerve regeneration by providing guidance to the nerve cells. Conductivity of such guides is reported to enhance this process. In the present study a nerve guide was constructed from poly(2-hydroxyethyl methacrylate) (pHEMA) which was loaded multiwalled carbon nanotubes (mwCNT) to introduce conductivity. PHEMA hydrogels were designed to have a porous structure to facilitate the transportation of the compounds needed for cell nutrition and growth and also for waste removal. We showed that when loaded with relatively high concentrations of mwCNTs (6%, w/w in hydrogels), the pHEMA guide was more conductive and more hydrophobic than pristine pHEMA hydrogel. The mechanical properties of the composites were better when they carried mwCNT. Elastic Modulus of 6% mwCNT loaded pHEMA was 2 fold higher (0.32±0.06 MPa) and similar to that of the soft tissues. Electrical conductivity was significantly improved (11.4 fold), from 7 x 10^-3 Ω^-.cm^- (pHEMA) to 8.0 x 10^-2 Ω^-.cm^- (6% mwCNT loaded pHEMA). Upon application of electrical potential, the SHSY5Y neuroblastoma cells seeded on mwCNTs carrying pHEMA maintained their viability, whereas those on pure pHEMA could not, indicating that mwCNT helped conduct electricity and make them more suitable as nerve conduits if electrical induction is to be tested.

A combined experimental and computational approach was employed to investigate the feasibility and effectiveness of characterizing carbonated apatite (CAp) by infrared (IR) spectroscopy. First, an experimental comparative study was conducted to identify characteristic IR vibrational bands of carbonate substitution in the apatite lattice. The IR spectra of pure hydroxyapatite (HA), carbonate adsorbed on the HA surface, a physical mixture of HA and sodium carbonate monohydrate, a physical mixture of HA and calcite, synthetic CAps prepared using three methods (precipitation method, hydrothermal route and solid-gas reaction at high temperature) and biological apatites (human enamel, human cortical bone, and two animal bones) were compared. Then, the IR vibrational bands of carbonate in CAp were calculated with density functional theory (DFT). The experimental study identified characteristic IR bands of carbonate that cannot be generated from surface adsorption or physical mixtures and the results show that the bands at ~880 cm^-1, 1413 cm^-1, and 1450 cm^-1 should not be used as characteristic bands of CAp since they could result from carbonate adsorbed on the apatite crystals surface or present as a separate phase. The combined experimental and computational study reveals that the carbonate v₃ bands at ~1546 cm^-1 and 1465 cm^-1 are respectively the IR signature bands for type-A CAp and type-B CAp.

Scaffolds for bone tissue engineering applications should have suitable degradability in favor of new bone ingrowth after implantation into bone defects. In this study, degradation behavior of polyurethane composites composed of tri-block copolymer Poly(caprolactone)-Poluronic-Poly(caprolactone) (PCL-Pluronic-PCL, PCFC) and nano-hydroxyapatite (n-HA) was investigated. The water contact angle and water absorption were measured to reveal the effect of n-HA content on the surface wettability and swelling behavior of the n-HA/PCFC composites, respectively. The weight loss in three degradation media with pH value of 4.0, 7.4 and 9.18, was also studied accordingly. Fourier transform infrared (FT-IR) analysis, differential scanning calorimeter (DSC), X-ray diffraction (XRD), thermal-gravimetric analysis (TGA) and scanning electron microscopy (SEM) were used to investigate the change of chemical structure and micro-morphology after the n-HA/PCFC composite with 30% HA was degraded for different time intervals. Meanwhile, in vivo degradation was conducted by subcutaneous implantation. The weight loss and morphology change during observation periods were also studied.

This study investigated the adhesive behaviors of normal and abnormal hematopoietic cells on nanotopographical materials. Previously, electrospun nanofiber scaffolds (NFS) were used to capture and expand hematopoietic stem cells in vitro, here we demonstrate that NFS could also serve as a useful bioadhesive platform for capturing functionally adherent leukemia cells. Collagen-blended PLGA NFS enabled more rapid and efficient capture of K562 leukemia cells than tissue culture polystyrene (TCP) surfaces with up to 70% improved adhesion and shorter time. Cellular extensions, stronger adhesion and enhanced cell-cell interactions were observed in K562 cells captured on NFS. While NFS promoted hematopoietic progenitor cell proliferation, it inhibited leukemia cell proliferation and affected cell cycle status by shifting more cells toward the G0/G1 phase. The expression of α-integrins was equally high in both captured and un-captured leukemia cell populations demonstrating no relation to its adhesive nature. Hematopoietic morphological signatures of NFS captured cells presented no impact on cell differentiation. We conclude that electrospun NFS not only serves as an excellent platform for capturing functionally adherent leukemia cells but for studying the impact of niche-like structure in the nanoscale.

Composite porous scaffolds have attracted extensive attention in the biomedical materials field. The aim of this research was to prepare a novel tri-component composite porous scaffold and to evaluate its relevant properties. The porous scaffold was composed of chitosan (CS), silk fibroin (SF) and nanohydroxyapatite particles (nHA), which we named CS/SF/nHA scaffold, and prepared via salt fractionation method combined with lyophilization. The porous structure was achieved using a porogen (salt) and the pore size was controlled by the size of porogen. To evaluate the characteristics of the tri-component scaffold, three bi-component scaffolds, CS/SF, CS/nHA and SF/nHA, were simultaneously prepared for comparison. The scaffolds were subjected to morphological, micro-structural and biodegradation analysis. Results demonstrated that all of the scaffolds had pore sizes of 100-300 µm and a porosity of 90.5-96.1%. The biodegradation characteristics of all scaffolds meet the requirements of good biomedical materials. The investigation of the mechanical properties showed that the tri-components scaffold has better properties than the bi-component scaffolds. The in vitro biocompatibility with osteoblast-like MG-63 cells showed that all the scaffolds are suitable for cell attachment and proliferation, however, the CS/SF/nHA composite porous scaffold is much more effective than the others.

Titanium dioxide nanoparticules (TiO₂ NPs) are widely used in toothpastes, sunscreens and products for cosmetic purpose that the human employ daily. While the neurotoxicity induced by TiO₂ NPs has been demonstrated, very little is known about the molecular mechanisms underlying the brain cognition and behavioral injury. In this study, mice were exposed to 2.5, 5, and 10 mg/kg BW TiO₂ NPs by nasal administration for 90 consecutive days, respectively, and their brains' injuries and brain gene-expressed profile were investigated. Our findings showed that TiO₂ NPs could be translocated and accumulated in brain, led to oxidative stress, overproliferation of all glial cells, tissue necrosis as well as hippocampal cell apoptosis. Furthermore, microarray data showed significant alterations in the expression of 249 known function genes, including 113 genes up-regulation and 136 genes down-regulation following exposure to 10 mg/kg BW TiO₂ NPs, which were associated with oxidative stress, immune response, apoptosis, memory and learning, brain development, signal transduction, metabolic process, DNA repair, response to stimulus and cellular process. Especially, significant increases in Col1a1, Sgk1, Ctnnb1, Csrnp1, Ddit4, Cyp2e1, and Krit1 expressions and great decreases in Drd2, Neu1, Fcrls, and Dhcr7 expressions following long-term exposure to TiO₂ NPs resulted in neurogenic disease states in mice. Therefore, these genes may be potential biomarkers of brain toxicity caused by TiO₂ NPs exposure, and the application of TiO₂ NPs should be carried out cautiously.

Since spinal cord injury is a complicated problem, neural tissue repair and regeneration strategies have received a great deal of attention. In this study, a 3D nanofibrous core-sheath scaffold with nanorough sheath and aligned core were fabricated by a combined electrospinning method with water vortex and two-nozzle system. In-vitro and in-vivo biological tests were carried out on the poly (lactic-co-glycolic acid) (PLGA) scaffolds. The cell morphology and proliferation evaluation of nerve cells on 3D PLGA scaffolds were studied. Cells were properly orientated along the aligned fiber direction of the scaffold. In animal studies, adult rats received a complete lateral hemisection at the T9-T10 level. Scaffolds were engrafted to bridge 3 mm defects of 10 adult rat spinal cords; 10 rats were used as controls. For 8 weeks, motor and sensory recovery by open field locomotor scale, narrow beam and tail flick tests were assessed. Locomotor and sensory scores of grafted animals were significantly better than the control group. Histological findings demonstrated that the scaffold supports the axonal regeneration of injured spinal cords and regenerating axons were seen to enter the graft and extend along its length.

The fundamental building blocks of hierarchically structured bone tissue are mineralized collagen fibrils with calcium phosphate nanocrystals that are biologically “engineered” through biomineralization. In this study, we demonstrate an original invention of dicalcium phosphate anhydrate (DCPA)/poly(lactic acid) (PLA) composite nanofibers, which mimics the mineralized collagen fibrils via biomimetic in-situ synthesis and electrospinning for hard tissue regenerative medicines. The interaction of the Ca²⁺ ions and the carbonyl groups in the PLA provides nucleation sites for DCPA during the in-situ synthesis process. This resulted in the improved dispersion of DCPA nanocrystallites in the intra-nanoporous PLA nanofibers through electrospinning, compared to the severely agglomerated clusters of DCPA nanoparticles fabricated by conventional mechanical blending/electrospinning methods. The addition of poly(ethylene glycol) (PEG), as a co-polymer source, generated more stable and efficient electrospun jets and aided in the electrospinability of the PLA nanofibers incorporating the nanocrystallites. It is expected that the uniformly distributed DCPA nanocrystallites and its unique nanocomposite fibrous topography will enhance the biological performance and the structural stability of the scaffolds used for hard tissue reconstruction and regeneration.

Driven by the complications occurring with bare metal stents (BMSs) and drug-eluting stents (DESs), concerns have been raised over strategies for long-term safety, with respect to preventing or inhibiting stent thrombosis (ST), restenosis and in-stent restenosis (ISR) in particularly. Surface modification is very important in constructing a buffer layer at the interface of the organic and inorganic materials, and in ultimately obtaining long-term biocompatibility. In this review, we summarize the developments in surface modification of implanted cardiovascular metal stents. This review focuses on the modification of metal stents via coating drugs or biomolecules to enhance anti-thrombosis, anti-restenosis and/or endothelialization. In addition, we indicate the probable future work involving the modification of the metallic blood-contacting surfaces of stents and other cardiovascular devices that are under development.

Transplantation of cells within poly(ethylene glycol) (PEG) hydrogel scaffolds as effective immunoisolation barriers is becoming increasingly important strategy for tissue engineering and regenerative medicine. In these applications, crosslink density of these membranes has significant effect on the control of diffusion of many biomolecules such as nutrients, cellular wastes and hormones. When these networks are designed with crosslink density gradients, alterations in network structure may have an effect on biomolecule diffusivity. The goal of this work was to synthesize PEG hydrogels via surface initiated photopolymerization for use in applications involving physiological protein delivery and cell encapsulation. For this purpose, PEG hydrogels of differing crosslink density gradients were formed via surface initiated photopolymerization, and the diffusion of model proteins with various molecular weights were observed through these PEG hydrogel scaffolds with defined properties. Diffusion coefficients were on the order of 10^-7-10^-8 cm²/s and protein diffusion time scales varied from 5 min to 30 h. The results confirm that synthetic PEG hydrogels with crosslink density gradients are promising for controlled release of bioactive molecules and for covalent incorporation of ligands to support cell viability.

In this work, we expand on our understanding of the thrombogenicity of coatings prepared with three different recombinant elastin-like polypeptides (ELPs). The bulk platelet response of the ELP coatings was characterized following whole blood contact under physiological shear flow (300 s^-1) using flow cytometry. Prolonged exposure to shear flow (one-hour) indicated that materials coated with the longer ELP coatings (ELP2 and ELP4) had less bulk platelet activation and microparticle formation than materials coated with the shorter ELP1. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the binding of the platelet membrane receptor GPIIb/IIIa to ELP-adsorbed fibrinogen (Fg) surfaces. Compared to the shorter ELPs, a lower amount of Fg adsorbed to the ELP4 coated material and ELP4 appeared to form a softer, more structurally flexible coating layer. When Fg was adsorbed to the ELP coated surface it demonstrated an altered binding for GPIIb/IIIa that was inhibited in the presence of an AGDV-containing peptide but not an RGD-containing peptide. Conversely, on the shorter ELP coatings, binding of GPIIb/IIIa to an adsorbed Fg layer was partially inhibited in the presence of an RGD-containing peptide. These results indicate that both the quantity and conformational state of Fg varies when adsorbed to surfaces coated with ELPs of varying sequence length, which may be mediating their platelet response. Collectively, the findings reinforce the applicability of the ELPs as potential thromboresistant coatings, especially with the use of the longer polypeptide – ELP4. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A.

The advantages of endothelialization of a stent surface in comparison with the bare metal and drug eluting stents used today include reduced late-stent restenosis and in-stent thrombosis. In this paper, we study the effect of surface topology and functionalization of tantalum (Ta) with cyclic-(arginine-glycine-aspartic acid-D-phenylalanine-lysine (cRGDfK)) on the attachment, spreading, and growth of vascular endothelial cells. Self-assembled nano-dimpling on Ta surfaces was performed using a one-step electropolishing technique. Next, cRGDfK was covalently bonded onto the surface using silane chemistry. Our results suggest that nano-texturing alone was sufficient to enhance cell spreading, but the combination of a nano-dimpled surfaces along with the cRGDfK peptide may produce a better endothelialization coating on the surface in terms of higher cell density, better cell spreading, and more cell-cell interactions, when compared to using cRGDfK peptide functionalization alone or nano-texturing alone. We believe that future research should look into how to implement both modifications (topographic and chemical modifications) to optimize the stent surface for endothelialization. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A.

A cardiac patch is a construct devised in regenerative medicine to replace necrotic heart tissue after myocardial infarctions. The cardiac patch consists of a scaffold seeded with stem cells. To identify the best scaffold for cardiac patch construction we compared polyurethane, Collagen Cell Carriers, ePTFE, and ePTFE SSP1-RGD regarding their receptiveness to seeding with mesenchymal stem cells isolated from umbilical cord tissue. Seeding was tested at an array of cell seeding densities. The bioartificial patches were cultured for up to 35 days and evaluated by scanning electron microscopy, microscopy of histological stains, fluorescence microscopy, and mitochondrial assays. Polyurethane was the only biomaterial which resulted in an organized multilayer (seeding density: 0.750 × 10⁶ cells/cm²). Cultured over 35 days at this seeding density the mitochondrial activity of the cells on polyurethane patches continually increased. There was no decrease in the E Modulus of polyurethane once seeded with cells. Seeding of CCC could only be realized at a low seeding density and both ePTFE and ePTFE SSP1-RGD were found to be unreceptive to seeding. Of the tested scaffolds polyurethane thus crystallized as the most appropriate for seeding with mesenchymal stem cells in the framework of myocardial tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

A constitutive framework is provided for the characterization of the mechanical behavior of colonic tissues, as a fundamental tool for the development of numerical models of the colonic structures. The constitutive analysis is performed by a multidisciplinary approach that requires the cooperation between experimental and computational competences. The preliminary investigation pertains to the review of the tissues histology. The complex structural configuration of the tissues and the specific distributions of fibrous elements entail the nonlinear mechanical behavior and the anisotropic response. The identification of the mechanical properties requires to perform mechanical tests according to different loading situations, as different loading directions. Because of the typical functionality of colon structures, the tissues mechanics is investigated by tensile tests, which are performed on taenia coli and haustra specimens from fresh pig colons. Accounting for the histological investigation and the results from the mechanical tests, a specific hyperelastic framework is provided within the theory of fiber-reinforced composite materials. Preliminary analytical formulations are defined to identify the constitutive parameters by the inverse analysis of the experimental tests. Finite element models of the specimens are developed accounting for the actual configuration of the colon structures to verify the quality of the results. The good agreement between experimental and numerical model results suggests the reliability of the constitutive formulations and parameters. Finally, the developed constitutive analysis makes it possible to identify the mechanical behavior and properties of the different colonic tissues. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

This article reports the surface modification of electrospun polycarbonate urethane membrane with polydimethyl siloxane (PDMS) using plasma-induced grafting technique for biomedical applications. The nonwoven membranes were characterized for their structure, performance, and compatibility with cells. The surface modification was confirmed by means of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and energy dispersive X-ray analysis (EDXA). ATR-FTIR and EDXA analyses displayed characteristic absorption peaks of PDMS for the membrane. The structure and morphology of the developed membranes were studied using scanning electron microscope and microcomputed tomography (µCT). Scanning electron microscopy and µCT revealed the fibrous morphology and percentage porosity of the membranes before and after plasma modification. Static mechanical tests showed that the tensile strength was greater than 8 MPa. Physical characterization of the membranes after immersion in hydrolytic and oxidative media supports their biostability. Cytotoxicity of the membrane was evaluated using L929 fibroblast cells, and the results indicated that the membrane is cytocompatible. Accordingly, these results highlight the potential of this fibrous membrane for biomedical applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Cerebral aneurysms treated by traditional endovascular methods using platinum coils have a tendency to be unstable, either due to chronic inflammation, compaction of coils, or growth of the aneurysm. We propose to use alternate filling methods for the treatment of intracranial aneurysms using polyurethane-based shape memory polymer (SMP) foams. SMP polyurethane foams were surgically implanted in a porcine aneurysm model to determine biocompatibility, localized thrombogenicity, and their ability to serve as a stable filler material within an aneurysm. The degree of healing was evaluated via gross observation, histopathology, and low vacuum scanning electron microscopy imaging after 0, 30, and 90 days. Clotting was initiated within the SMP foam at time 0 (<1 h exposure to blood before euthanization), partial healing was observed at 30 days, and almost complete healing had occurred at 90 days in vivo, with minimal inflammatory response. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bone regenerative medicine, based on the combined use of cells and scaffolds, represents a promising strategy in bone regeneration. Hydrogels have attracted huge interests for application as a scaffold for minimally invasive surgery. Collagen and oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels are the representatives of two main categories of hydrogels, that is, natural- and synthetic-based hydrogels. With these the optimal cell-loading (i.e., cell distribution inside the hydrogels) method was assessed. The cell behavior of both bone marrow- and adipose tissue-derived mesenchymal stem cells (BM- and AT-MSCs) in three loading methods, which are dispersed (i.e., homogeneous cell encapsulation, D), sandwich (i.e., cells located in between two hydrogel layers, S), and spheroid (i.e., cell pellets encapsulation, Sp) loading in two hydrogel systems (i.e., collagen and OPF), was compared. The results suggested that the cell behavior was influenced by the hydrogel type, meaning cells cultured in collagen hydrogels had higher proliferation and osteogenic differentiation capacity than in OPF hydrogels. In addition, AT-MSCs exhibited higher proliferation and osteogenic properties compared to BM-MSCs. However, no difference was observed for mineralization among the three loading methods, which did not approve the hypothesis that S and Sp loading would increase osteogenic capacity compared to D loading. In conclusion, D and Sp loading represents two promising cell loading methods for injectable bone substitute materials that allow application of minimally invasive surgery for cell-based regenerative treatment. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In this investigation, an ecofriendly method for the synthesis of silver nanoparticles (AgNPs) using biodegradable gelatin as a stabilizing agent is reported. Here, we prepared thermosensitive silver nanocomposite hydrogels composed of gelatin and N-isopropylacrylamide. In this green process AgNPs were formed from Ag⁺ ions and reduced with leaf [Azadirachta indica (neem leaf)] extracts, resulting in a hydrogel network. The Ag⁰ nanoparticles affect the hydrogel strength and improved the biological activity (inactivation effect of bacteria) of the biodegradable hydrogels. The resulted hydrogel structure, morphology, thermal, swelling behavior, degradation, and antibacterial properties were systematically investigated. The biodegradable thermosensitive silver nanocomposite hydrogels developed were tested for antibacterial activities. The results indicate that these biodegradable silver nanocomposite hydrogels are suitable potential candidates for antibacterial applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

How do we quantify cellular alignment? Cellular alignment is an important technique used to study and promote tissue regeneration in vitro and in vivo. Indeed, regenerative outcomes are often strongly correlated with the efficacy of alignment, making quantitative, automated assessment an important goal for the field of tissue engineering. There currently exist various classes of algorithms, which effectively address the problem of quantifying individual cellular alignments using Fourier methods, kernel methods, and elliptical approximation; however, these algorithms often yield population distributions and are limited by their inability to yield a scalar metric quantifying the efficacy of alignment. The current work builds on these classes of algorithms by adapting the signal processing methods previously used by our group to study the alignment of cellular processes. We use an automated, ellipse-fitting algorithm to approximate cell body alignment with respect to a silk biomaterial scaffold, followed by the application of the normalized cumulative periodogram criterion to produce a scalar value quantifying alignment. The proposed work offers a generalized method for assessing cellular alignment in complex, two-dimensional environments. This method may also offer a novel alternative for assessing the alignment of cell types with polarity, such as fibroblasts, endothelial cells, and mesenchymal stem cells, as well as nuclei. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Magnesium and its alloys are candidate materials for biodegradable implants; however, excessively rapid corrosion behavior restricts their practical uses in biological systems. For such applications, surface modification is essential, and the use of anticorrosion coatings is considered as a promising avenue. In this study, we coated Mg with hydroxyapatite (HA) in an aqueous solution containing calcium and phosphate sources to improve its in vitro and in vivo biocorrosion resistance, biocompatibility and bone response. A layer of needle-shaped HA crystals was created uniformly on the Mg substrate even when the Mg sample had a complex shape of a screw. In addition, a dense HA-stratum between this layer and the Mg substrate was formed. This HA-coating layer remarkably reduced the corrosion rate of the Mg tested in a simulated body fluid. Moreover, the biological response, including cell attachment, proliferation and differentiation, of the HA-coated samples was enhanced considerably compared to samples without a coating layer. The preliminary in vivo experiments also showed that the biocorrosion of the Mg implant was significantly retarded by HA coating, which resulted in good mechanical stability. In addition, in the case of the HA-coated implants, biodegradation was mitigated, particularly over the first 6 weeks of implantation. This considerably promoted bone growth at the interface between the implant and bone. These results confirmed that HA-coated Mg is a promising material for biomedical implant applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The effect of chloride-substitution on bioactivity and osteoconductivity of hydroxyapatite (OHAp) was newly investigated. Chloride-substituted hydroxyapatites (ClAp) with low and high chloride concentrations were synthesized by reacting Ca(OH)₂ and H₃PO₄ with NH₄Cl of low and high concentrations, with subsequent sintering. As a control, pure OHAp was prepared under the same conditions but without addition of NH₄Cl. The ClAp showed markedly enhanced bioactivity in simulated body fluid (SBF) as the chloride substitution was increased. In contrast, OHAp did not show any bioactivity at all within the testing period. The solubility tests in deionized water also showed that the higher the chloride-substituting amount, the higher the dissolution amounts of the constituent elements of apatite, which directly affect bioactivity by increasing the degree of supersaturation of apatite in SBF. In addition, ClAp also showed noticeably higher osteoconductivity within the 4 weeks of implantation in calvarial defects of New Zealand white rabbits, compared with that of OHAp. The total system energy of the apatite calculated by the ab initio method showed that the higher the chloride-substituting amount, the higher the total system energy, which suggests that the ClAp was energetically less stable compared with OHAp. This result demonstrates the higher solubility of ClAp over that of OHAp in SBF and deionized water. The improved solubility of the OHAp enhances its bioactivity and consequent osteoconductivity. Taken together, it can be concluded that ClAp has encouraging potential for use as a bone grafting material due to its highly enhanced bioactivity and osteoconductivity compared with pure OHAp. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bioactive glasses are used clinically for bone regeneration, and their bioactivity and cell compatibility are often characterized in vitro, using physiologically relevant test solutions. The aim of this study was to show the influence of varying medium characteristics (pH, composition, presence of proteins) on glass dissolution and apatite formation. The dissolution behavior of a fluoride-containing bioactive glass (BG) was investigated over a period of one week in Eagle's Minimal Essential Medium with Earle's Salts (MEM), supplemented with either, (a) acetate buffer, (b) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, (c) HEPES + carbonate, or (d) HEPES + carbonate + fetal bovine serum. Results show pronounced differences in pH, ion release, and apatite formation over 1 week: Despite its acidic pH (pH 5.8 after BG immersion, as compared to pH 7.4–8.3 for HEPES-containing media), apatite formation was fastest in acetate buffered (HEPES-free) MEM. Presence of carbonate resulted in formation of calcite (calcium carbonate). Presence of serum proteins, on the other hand, delayed apatite formation significantly. These results confirm that the composition and properties of a tissue culture medium are important factors during in vitro experiments and need to be taken into consideration when interpreting results from dissolution or cell culture studies. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Successful repair of craniofacial and periodontal tissue defects ideally involves a combined therapy that includes inflammation modulation, control of soft tissue infiltration, and bone regeneration. In this study, an anti-inflammatory polymer, salicylic acid-based poly(anhydride-ester) (SAPAE) and a three-dimensional osteoconductive ceramic scaffold were evaluated as a combined guided bone regeneration (GBR) system for concurrent control of inflammation, soft tissue ingrowth, and bone repair in a rabbit cranial defect model. At time periods of 1, 3, and 8 weeks, five groups were compared: (1) scaffolds with a solid ceramic cap (as a GBR structure); (2) scaffolds with no cap; (3) scaffolds with a poly(lactide-glycolide) cap; (4) scaffolds with a slow release SAPAE polymer cap; and (5) scaffolds with a fast release SAPAE polymer cap. Cellular infiltration and bone formation in these scaffolds were evaluated to assess inflammation and bone repair capacity of the test groups. The SAPAE polymers suppressed inflammation and displayed no deleterious effect on bone formation. Additional work is warranted to optimize the anti-inflammatory action of the SAPAE, GBR suppression of soft tissue infiltration, and stimulation of bone formation in the scaffolds and create a composite device for successful repair of craniofacial and periodontal tissue defects. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Poly(β-amino ester) (PBAE) biodegradable hydrogel systems have garnered much attention in recent years due to their appealing properties for biomedical applications. These hydrogel systems exhibit properties similar to natural soft tissue, degrade in aqueous environments, and have easily tunable properties that have been well studied and understood. In most cases, tissue engineering scaffolds must possess a three-dimensional interconnected porous network for tissue ingrowth and construct vascularization. Here, PBAE properties were explored and systems were selected to serve as both the pore-forming agent and the outer matrix of a scaffold that exhibits controlled pore opening upon degradation. To our knowledge, this is the first demonstration of a biodegradable hydrogel porogen system entrapped in a degradable hydrogel outer matrix. Scaffolds were prepared, and the degradation, compressive moduli, and porosity were analyzed. An added advantage of a degradable porogen is the potential for controlled drug release, and a model protein was released from the porogen particles to demonstrate this application. Finally, pluripotent cells seeded onto predegraded scaffolds were viable during the first 24 h of exposure, and furthermore, cell tracking confirmed the presence of cells within the pores of the scaffold. Overall, these present studies demonstrate the possibility of using these biodegradable hydrogel porogen-matrix systems as tissue engineering scaffolding materials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Lung bioengineering based on decellularized organ scaffolds is a potential alternative for transplantation. Freezing/thawing, a usual procedure in organ decellularization and storage could modify the mechanical properties of the lung scaffold and reduce the performance of the bioengineered lung when subjected to the physiological inflation-deflation breathing cycles. The aim of this study was to determine the effects of repeated freezing/thawing on the mechanical properties of decellularized lungs in the physiological pressure–volume regime associated with normal ventilation. Fifteen mice lungs (C57BL/6) were decellularized using a conventional protocol not involving organ freezing and based on sodium dodecyl sulfate detergent. Subsequently, the mechanical properties of the acellular lungs were measured before and after subjecting them to three consecutive cycles of freezing/thawing. The resistance (R_L) and elastance (E_L) of the decellularized lungs were computed by linear regression fitting of the recorded signals (tracheal pressure, flow, and volume) during mechanical ventilation. R_L was not significantly modified by freezing-thawing: from 0.88 ± 0.37 to 0.90 ± 0.38 cmH₂O·s·mL⁻¹ (mean ± SE). E_L slightly increased from 64.4 ± 11.1 to 73.0 ± 16.3 cmH₂O·mL⁻¹ after the three freeze-thaw cycles (p = 0.0013). In conclusion, the freezing/thawing process that is commonly used for both organ decellularization and storage induces only minor changes in the ventilation mechanical properties of the organ scaffold. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Modification of carbon nanotubes (CNTs) with carboxyl group is one of the widely used strategies to increase their water dispersibility. Various molecules can be further coupled to the surface of carboxylated CNTs for the desired applications. However, the effect of carboxylation of CNTs on their cytotoxicity is far from being completely understood. In this study, the impact of carboxylated multiwalled CNT (MWCNT-COOH) on human normal liver cell line L02 was studied and compared with pristine multiwalled CNT (p-MWCNT). The data accumulated in this study revealed that modification with carboxyl group reduced the toxicity of MWCNT on L02 cells, probably due to the decreased activation of mitochondria mediated apoptotic pathway. Both p-MWCNT and MWCNT-COOH, when reaching to certain concentration, induced significant decrease in the mitochondrial membrane potential, enhanced release of cytochrome c from the mitochondria to cytoplasm as well as activation of caspase-9, and -3. However, the changes induced by MWCNT-COOH were significantly milder than that by p-MWCNT. Our observation suggests that carboxylated MWCNTs might be safer for in vivo application as compared with p-MWCNT. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The insertion of cochlear implants into the inner ear often causes inflammation and fibrosis inside the scala tympani and thus growth of fibrous tissue on the implant surface. This deposition leads to the loss of function in both electrical and laser-based implants. The design of this study was to realize fibroblast growth inhibition by dexamethasone (Dex) released from the base material of the implant [polydimethylsiloxane (PDMS)]. To prevent cell and protein adhesion, the PDMS was coated with a hydrogel layer [star-shaped polyethylene glycol prepolymer (sPEG)]. Drug release rates were studied over 3 months, and surface characterization was performed. It was observed that the hydrogel slightly smoothened the surface roughened by the Dex crystals. The hydrogel coating reduced and prolonged the release of the drug over several months. Unmodified, sPEG-coated, Dex-loaded, and Dex/sPEG-equipped PDMS filaments were cocultivated in vitro with fluorescent fibroblasts, analyzed by fluorescent microscopy, and quantified by cell counting. Compared to the unmodified PDMS, cell growth on all modified filaments was averagely 95% ±standard deviation (SD) less, while cell growth on the bottom of the culture dishes containing Dex-loaded filaments was reduced by 70% ±SD. Both, Dex and sPEG prevented direct cell growth on the filament surfaces, while drug delivery was maintained for the duration of several months. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Biodegradable polymer scaffolds are being extensively investigated for uses in tissue engineering because of their versatility in fabrication methods and range of achievable chemical and mechanical properties. In this study, poly(lactic-co-glycolic acid) (PLGA) was used to make various types of microspheres that were processed into porous scaffolds that possessed a wide range of properties. A heat sintering step was used to fuse microspheres together around porogen particles that were subsequently leached out, allowing for a 10-fold increase in mechanical properties over other PLGA scaffolds. The sintering temperature was based on the glass transition temperature that ranged from 43 to 49°C, which was low enough to enable drug loading. Degradation times were observed to be between 30 and 120 days, with an initial compressive modulus ranging from 10 to 100 MPa, and after 5 days of degradation up to 10 MPa was retained. These scaffolds were designed to allow for cell ingrowth, enable drug loading, and have an adjustable compressive modulus to be applicable for soft or hard tissue implants. This study combined well-established methods, such as double emulsion microspheres, polymer sintering, and salt leaching, to fabricate polymer scaffolds useful for different tissue engineering applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Techniques to pattern cells on biocompatible hydrogels allow for the creation of highly controlled cell microenvironments within materials that mimic the physicochemical properties of native tissues. Such technology has the potential to further enhance our knowledge of cell biology and to play a role in the development of novel tissue engineering devices. Light is an ideal stimulus to catalyze pattern formation since it can be controlled spatially as well as temporally. Herein, we have developed and enhanced a hydrogel cell patterning strategy. It is based on photoactive caged RGDS peptides incorporated into a hyaluronic acid (HA) hydrogel, which can be subsequently activated with near-UV light to create cell-adhesive regions within an otherwise non-adhesive hydrogel. With this strategy, we have been able to pattern multiple cell populations—either in contact with one another or held apart—on an underlying chemically patterned HA hydrogel. Furthermore, the hydrogel cell pattern could be altered with time, even 2 weeks after initial seeding, to create additional adhesive regions to regulate the direction of cell growth and migration. These dynamic hydrogel cell patterns, created with a standard fluorescence microscope, were shown to be robust and lasted at least 3 weeks in vitro. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Composite porous scaffolds have attracted extensive attention in the biomedical material field. The aim of this research was to prepare a novel tri-component composite porous scaffold and to evaluate its relevant properties. The porous scaffold was composed of chitosan (CS), silk fibroin (SF), and nanohydroxyapatite particles (nHA), which we named CS/SF/nHA scaffold and prepared via salt fractionation method combined with lyophilization. The porous structure was achieved using a porogen (salt), and the pore size was controlled by the size of porogen. To evaluate the characteristics of the tri-component scaffold, three bi-component scaffolds, CS/SF, CS/nHA, and SF/nHA, were simultaneously prepared for comparison. The scaffolds were subjected to morphological, micro-structural, and biodegradation analyses. Results demonstrated that all of the scaffolds had pore sizes of 100–300 μm and a porosity of 90.5–96.1%. The biodegradation characteristics of all scaffolds meet the requirements of good biomedical materials. The investigation of the mechanical properties showed that the tri-component scaffold has better properties than the bi-component scaffolds. The in vitro biocompatibility with osteoblast-like MG-63 cells showed that all the scaffolds are suitable for cell attachment and proliferation; however, the CS/SF/nHA composite porous scaffold is much more effective than the others. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

An injectable hydrogel via hydrazone cross-linking was prepared under mild conditions without addition of cross-linker or catalyst. Hydrazine and aldehyde modified poly(aspartic acid)s were used as two gel precursors. Both of them are water-soluble and biodegradable polymers with a protein-like structure, and obtained by aminolysis reaction of polysuccinimide. The latter can be prepared by thermal polycondensation of aspartic acid. Hydrogels were prepared in PBS solution and characterized by different methods including gel content and swelling, Fourier transformed-infrared spectroscopy, and in vitro degradation experiment. A scanning electron microscope viewed the interior morphology of the obtained hydrogels, which showed porous three-dimensional structures. Different porous sizes were present, which could be well controlled by the degree of aldehyde substitution in precursor poly(aspartic acid) derivatives. The doxorubicin-loaded hydrogels were prepared and showed a pH-sensitive release profile. The release rate can be accelerated by decreasing the environmental pH from a physiological to a weak acidic condition. Moreover, the cell adhesion and growth behaviors on the hydrogel were studied and the polymeric hydrogel showed good biocompatibility. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of this study was to investigate the use of bioactive RGD-containing elastin-like recombinamers (ELR-RGDs) as a substrate that can maintain human retinal pigment epithelial cell (hRPE) phenotype and growth pattern. Results obtained are compared with previously published behavior of ARPE19 cells. The extension of these results to hRPE is required because ARPE19 cells cannot be used clinically to treat age-related macular degeneration. hRPE cells were isolated, cultured, seeded, and grown on surface of glass, treated polystyrene (TCP), and solvent-cast ELR-RGD and ELR-IK film with no specific sequence. Cells were analyzed to study cell adhesion, proliferation, morphology, and RPE65 protein expression by staining with diamidino-2-phenylindole, Rhodamine-Phalloidin, and anti-RPE65 antibody at 12, 24, 72, 120, 168, and 360 h. hRPE cells always grew better on ELR-RGD than on glass and ELR-IK but not on TCP. The kinetic hRPE growth curves confirmed that growth differences started to appear at 24 h for these surfaces in ascending order of cell growths, namely glass, ELR-IK, ELR-RGD, and TCP. There was a clear difference at 360 h. ELR-RGD maintained hRPE cells stable morphology and RPE65 protein expression. ELR-RGD seems to be a good substrate for growing hRPE cells with stable morphology and RPE65 protein expression. As such, this work confirms our hypothesis regarding ELR-RGD substrates viability, which can be used as a Bruch's membrane prosthesis for further studies in animals. However, these results must subsequently be extrapolated to use of hRPE cells in animals to evaluate them as a transplantation vehicle in human. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

When surface-reactive (bioactive) coatings are applied to medical implants by means of the sol–gel dip-coating technique, the biological proprieties of the surface of the implant can be locally modified to match the properties of the surrounding tissues to provide a firm fixation of the implant. The aim of this study has been to synthesize, via sol–gel, organoinorganic nanoporous materials and to dip-coat a substrate to use in dental applications. Different systems have been prepared consisting of an inorganic zirconium-based matrix, in which a biodegradable polymer, the poly-ε-caprolactone was incorporated in different percentages. The materials synthesized by the sol–gel process, before gelation, when they were still in sol phase, have been used to coat a titanium grade 4 (Ti-4) substrate to change its surface biological properties. Thin films have been obtained by means of the dip-coating technique. A microstructural analysis of the obtained coatings was performed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. The biological proprieties have been investigated by means of tests in vitro. The bone-bonding capability of the nanocomposite films has been evaluated by examining the appearance of apatite on their surface when plunged in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. The examination of apatite formation on the nanocomposites, after immersion in SBF, has been carried out by SEM equipped with energy-dispersive X-ray spectroscopy. To evaluate cells–materials interaction, human osteosarcoma cell line (Saos-2) has been seeded on specimens and cell vitality evaluated by WST-8 assay. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Fluorhydroxyapatite/strontium-substituted hydroxyapatite (FHA/SrHA) biphasic coatings with F and Sr elements incorporated simultaneously into one coating layer were prepared on titanium substrate via colloidal sol–gel method. The bioactivity of the as-prepared FHA/SrHA biphasic coatings was evaluated in vitro by immersion in simulated body fluid (SBF). All the biphasic coatings exhibited great ability to induce apatite precipitation on their surfaces. In vitro cell responses were evaluated using osteoblast-like MG63 cells in terms of cell proliferation and differentiation (alkaline phosphatase activity and osteocalcin level). The biphasic coatings show significantly positive effects on the viability and functional activity of osteoblastic cells with clear evidence that an optimum SrHA amount dose exists, indicating that the coexistence of FHA and SrHA had a synergistic stimulatory effect. This finding suggests the potential use of this colloidal sol–gel derived FHA/SrHA biphasic coatings for hard tissue applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Collagen has been utilized as a scaffold for tissue engineering applications due to its many advantageous properties. However, collagen in its purified state is mechanically weak and prone to rapid degradation. To mitigate these effects, collagen can be crosslinked. Although enhanced mechanical properties and stability can be achieved by crosslinking, collagen can be rendered less biocompatible either due to changes in the overall microstructure or due to the cytotoxicity of the crosslinkers. We have investigated crosslinking collagen using gold nanoparticles (AuNPs) to enhance mechanical properties and resistance to degradation while also maintaining its natural microstructure and biocompatibility. Rat tail type I collagen was crosslinked with AuNPs using a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Several characterization studies were performed including electron microscopy, collagenase assays, ROS assays, and biocompatibility assays. The results demonstrated that AuNP-collagen scaffolds had increased resistance to degradation as compared to non-AuNP-collagen while still maintaining an open microstructure. Although the biocompatibility assays showed that the collagen and AuNP-collagen scaffolds are biocompatible, the AuNP-collagen demonstrated enhanced cellularity and glycoaminoglycans (GAG) production over the collagen scaffolds. Additionally, the Reactive Oxygen Species (ROS) assays indicated the ability of the AuNP-collagen to reduce oxidation. Overall, the AuNP-collagen scaffolds demonstrated enhanced biocompatibility and stability over non-AuNP scaffolds. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Locally applied simvastatin is known to promote bone regeneration; however, the lack of suitable delivery systems has restricted its clinical use. In this study we demonstrate for the first time the use of premixed acidic calcium phosphate cement (CPC) as a delivery system for water-solubilized simvastatin. Freeze-dried simvastatin β-hydroxy acid (SVA) was added to the premixed cement paste in four different doses (1, 0.5, 0.25, and 0 mg SVA/g cement). The addition of the drug did not alter the cement setting time (38 min), compression strength (5.54 MPa), or diametral tensile strength (2.62 MPa). In a release study conducted in phosphate buffered saline at 37°C, a diffusion-controlled release was observed for over a week. Furthermore, the osteogenic effect of the released SVA was demonstrated in vitro. Cell proliferation, alkaline phosphatase activity, and mineralization were assayed after incubation with cement extracts. The lower doses of SVA (0.5 and 0.25 mg SVA/g cement) showed an approximately fourfold increase in mineralization as compared to the control. In conclusion, our findings suggest that premixed acidic CPC is a good option for local delivery of SVA, due to its ability of slowly releasing the drug, leading to a prolonged stimulation of osteogenesis. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of this study was to evaluate the modulation of matrix metalloproteinase-2 (MMP-2) and −9 (MMP-9) expression in newly formed bone tissue at the interface between implants derived from castor oil (Ricinus communis) polymer and the tibia medullary canal. Forty-four rabbits were assigned to either Group 1 (n = 12; control) or Group 2 (n = 30), which had the tibial medullary canals reamed bilaterally and filled with polymer. CT scans showed no space between the material surface and the bone at the implant/bone marrow interface, and the density of the tissues at this interface was similar to the density measured of other regions of the bone. At 90 days postimplantation, the interface with the polymer presented a thick layer of newly formed bone tissue rich in osteocytes. This tissue exhibited ongoing maturation at 120 and 150 days postimplantation. Overall, bone remodeling process was accompanied by positive modulation of MMP-2 and low MMP-9 expression. Differently, in control group, the internal surface close to the medullary canal was lined by osteoblasts, followed by a bone tissue zone with few lacunae filled with osteocytes. Maturation of the tissue of the medullary internal surface occurred in the inner region, with the bone being nonlamellar. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan–plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan–pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan–pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stability, and transfection efficiency were compared in different groups. The results indicated that chitosan of 800 kDa at N/P ratio of 4 and pH 7.0 was optimal to prepare chitosan–pDNA nanoparticles. These nanoparticles showed high DNA loading efficiency (95.8 ± 1.5%) and high gene transfection efficiency (24.5 ± 1.6%). After the expanded chondrocytes were transfected by chitosan–pDNA nanoparticles, MMP-3-610 and MMP-13-2024 groups showed greater suppression in mRNA and protein levels. The results indicated that chitosan–pDNA nanoparticles encoding shRNA targeting MMP-3 and -13 had great potential in silencing the dedifferentiation-related genes for regenerating prolonged and endurable cartilage. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Layered adipose-derived stem cell (ADSC) sheet transplantation is attracting attention as a new stem cell therapeutic strategy for damaged hearts. To prolong the function of tissue-engineered constructs after transplantation, a rapid and sufficient vascularization of engrafted tissue is essential. The in vitro formation of network structures derived from endothelial cells (ECs) in grafts before transplantation contributes to the induction of functional anastomosis in vivo. This study compared the angiogenic potential of ADSC sheets containing dissociated ECs (non-prevascular cell-sheets) and networked ECs (prevascular cell-sheets) after transplantation. For preparing the two different types of ECs-containing layered cell-sheets, human umbilical vein endothelial cells (HUVECs) were sandwiched between two human ADSC sheets. Non-prevascular cell-sheets were obtained immediately after sandwiching without further cultivation. Prevascular cell-sheets were harvested form temperature-responsive culture dishes following re-cultivation for allowing them to form an EC network structure. In transplant experiments in the subcutaneous tissues of immune-deficient rat for 4 days, prevascular cell-sheets were observed to promote neovascularization with HUVEC-lined microvessels. In contrast, neovessels were hardly observed in non-prevascular cell-sheets. These results suggested that prefabricated EC network in layered cell-sheet was effective for making a rapid connection to the host vasculature in the early post-transplanted period. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In this systematic review, we aimed to compare conical versus nonconical implant–abutment connection systems in terms of their in vitro and in vivo performances. An electronic search was performed using PubMed, Embase, and Medline databases with the logical operators: “dental implant” AND “dental abutment” AND (“conical” OR “taper” OR “cone”). Names of the most common conical implant–abutment connection systems were used as additional key words to detect further data. The search was limited to articles published up to November 2012. Recent publications were also searched manually in order to find any relevant studies that might have been missed using the search criteria noted above. Fifty-two studies met the inclusion criteria and were included in this systematic review. As the data and methods, as well as types of implants used was so heterogeneous, this mitigated against the performance of meta-analysis. In vitro studies indicated that conical and nonconical abutments showed sufficient resistance to maximal bending forces and fatigue loading. However, conical abutments showed superiority in terms of seal performance, microgap formation, torque maintenance, and abutment stability. In vivo studies (human and animal) indicated that conical and nonconical systems are comparable in terms of implant success and survival rates with less marginal bone loss around conical connection implants in most cases. This review indicates that implant systems using a conical implant–abutment connection, provides better results in terms of abutment fit, stability, and seal performance. These design features could lead to improvements over time versus nonconical connection systems. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of the study is to estimate the effects of the water-holding capability of the polyvinyl formal (PVF) sponges on osteogenic response in vitro experiments. The rat bone marrow stem cells (BMCs) were seeded and cultured for up to 4 weeks under static conditions in osteogenic media to evaluate the adhesion, proliferation, differentiation, and mineralization on the Dextran-coated PVF sponges with or without water-holding capability. The BMCs seeded onto the PVF sponges with water-holding capability showed more significant increases in DNA content, alkaline phosphatase (ALP) activity, osteocalcin content, and calcium deposition than those without water-holding capability. These results suggest that the Dextran-coated PVF sponges with high water-holding capability would have potential uses as both a new scaffold to bone tissue engineering and as a new biomaterial. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

This study examined the effect of hyaluronan (HA) molecular weight on immune response. HA with molecular weights ranging from the unitary disaccharide unit (400 Da) up to 1.7 × 10⁶ Da and with very low endotoxin contamination level (less than 0.03 EU/mg) was used. Primary human monocyte/macrophage cultures were assayed for IL-1β production under a variety of inflammatory conditions with or without HA. Under the highest inflammatory states, production of interleukin 1β (IL-1β) was suppressed in the presence of high molecular weight hyaluronan (HMW-HA) and in the presence of low molecular weight hyaluronan (LMW-HA) at mg/mL concentrations. There was variability in the sensitivity of the response to HA fragments with MW below 5000 Da at micromolar concentrations. There was variability in IL-1β cytokine productions from donor to donor in unstimulated human cell cultures. This study supplements our previous published study that investigated the immunogenic effect of HA molecular weights using murine cell line RAW264.6, rat splenocytes, and rat adherent differentiated primary macrophages. These data support the hypothesis that if the amount of endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue macrophage-mediated inflammatory reactions. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bilayered porous scaffolds have recently attracted interest because of their considerable promise for repairing osteochondral defects. However, determination of optimal pore size in bilayered porous scaffolds remains an important issue. This study investigated the in vivo effects of pore size in bilayered scaffolds using a rabbit model of osteochondral defects. We fabricated five types of integrated bilayered poly(lactide-co-glycolide) (PLGA) scaffolds with different pore sizes in the chondral and osseous layers (50–100 µm, 100–200 µm, 200–300 µm, and 300–450 µm). A subset of bilayered scaffolds seeded with or without allogenic bone marrow mesenchymal stem cells (BMSCs) was implanted in rabbit osteochondral defects. All of the cell/scaffold composite constructs supported the simultaneous regeneration of articular cartilage and subchondral bone, but the best results were observed in cell-seeded PLGA scaffolds with 100–200 µm pores in the chondral layer and 300–450 µm pores in the osseous layer. Our study supports the concept that the effects of pore size on osteochondral repair should be taken into consideration during scaffold design for tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2013.

Tracheomalacia is a relatively rare problem, but can be challenging to treat, particularly in pediatric patients. Due to the presence of mechanically deficient cartilage, the trachea is unable to resist collapse under physiologic pressures of respiration, which can lead to acute death if left untreated. However, if treated, the outcome for patients with congenital tracheomalacia is quite good because the cartilage tends to spontaneously mature over a period of 12 to 18 months. The present study investigated the potential for the use of degradable magnesium-3% yttrium alloy (W3) to serve as an extraluminal tracheal stent in a canine model. The host response to the scaffold included the formation of a thin, vascularized capsule consisting of collagenous tissue and primarily mononuclear cells. The adjacent cartilage structure was not adversely affected as observed by bronchoscopic, gross, histologic, and mechanical analysis. The W3 stents showed reproducible spatial and temporal fracture patterns, but otherwise tended to corrode quite slowly, with a mix of Ca and P rich corrosion product formed on the surface and observed focal regions of pitting. The study showed that the approach to use degradable magnesium alloys as an extraluminal tracheal stent is promising, although further development of the alloys is required to improve the resistance to stress corrosion cracking and improve the ductility. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2013.

In recent years, major advances have been accomplished in abdominal wall reconstruction. Introduction of newer prostheses have improved outcomes, but elimination of mesh-related morbidity is still an elusive issue. It is believed that host foreign body reaction to prosthesis plays an important role in the biology of these complications, so understanding of the molecular mechanisms behind mesh–tissue interactions may be a key for upcoming therapies. It appears that increasing biocompatibility of both synthetic prosthesis and biologic scaffolds might be the main avenues to achieve better outcomes. This manuscript provides an overview of major effectors of wound healing with particular emphasis on how their modulation might improve outcomes in tissue remodeling and mesh integration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bone cell response to 3D bioinspired scaffolds was tested on osteoblast culture supernatants and by means of quantitative polymerase chain reaction (qPCR). Foaming and freeze-drying method was optimized in order to obtain three-dimensional interconnected porous scaffolds of gelatin at different contents of nanocrystalline hydroxyapatite (HA). Addition of a non toxic crosslinking agent during foaming stabilized the scaffolds, as confirmed by the slow and relatively low gelatin release in phosphate buffer up to 28 days. Micro-computed tomography reconstructed images showed porous interconnected structures, with interconnected pores displaying average diameter ranging from about 158 to about 71 μm as the inorganic phase content increases from 0 to 50 wt %. The high values of connectivity (>99%), porosity (> 60%), and percentage of pores with a size in the range 100–300 μm (>50%) were maintained up to 30 wt % HA, whereas higher content provoked a reduction of these parameters, as well as of the average pore size, and a significant increase of the compressive modulus and collapse strength up to 8 ± 1 and 0.9 ± 0.2 MPa, respectively. Osteoblast cultured on the scaffolds showed good adhesion, proliferation and differentiation. The presence of HA promoted ALP activity, TGF-β1, and osteocalcin production, in agreement with the observed upregulation of ALP, OC, Runx2, and TGF-β1 gene in qPCR analysis, indicating that the composite scaffolds enhanced osteoblast activation and extra-cellular matrix mineralization processes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In situ ring-opening polymerization of ε-caprolactone (ε-CL) was performed to coat β-tricalcium phosphate (β-TCP) scaffolds fabricated by robocasting in order to enhance their mechanical performance while preserving the predesigned macropore architecture. Concentrated colloidal inks prepared from β-TCP commercial powders were used to fabricate porous structures consisting of a three-dimensional mesh of interpenetrating rods. Then, ε-CL was in situ polymerized within the ceramic structure using a lipase as catalyst and toluene as solvent, to obtain a highly homogeneous coating and full impregnation of in-rod microporosity. The strength and toughness of scaffolds coated by ε-polycaprolactone (ε-PCL) were significantly increased (twofold and fivefold increase, respectively) over those of the bare structures. Enhancement of both properties is associated to the healing of preexisting microdefects in the bioceramic rods. These enhancements are compared to results from previous work on fully impregnated structures. The implications of the results for the optimization of the mechanical and biological performance of scaffolds for bone tissue engineering applications are discussed. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in three-dimensional scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and open-cell polylactic acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At 4 weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bone tissue engineering approaches using polymer/ceramic composites show promise as effective biocompatible, absorbable, and osteoinductive materials. A novel class of in situ polymerizing thiol-acrylate based copolymers synthesized via an amine-catalyzed Michael addition was studied for its potential to be used in bone defect repair. Both pentaerythritol triacrylate-co-trimethylolpropane tris(3-mercaptopropionate) (PETA-co-TMPTMP) and PETA-co-TMPTMP with hydroxyapatite (HA) composites were fabricated in solid cast and foamed forms. These materials were characterized chemically and mechanically followed by an in vitro evaluation of the biocompatibility and chemical stability in conjunction with human adipose-derived mesenchymal pluripotent stem cells (hASC). The solid PETA-co-TMPTMP with and without HA exhibited compressive strength in the range of 7–20 MPa, while the cytotoxicity and biocompatibility results demonstrate higher metabolic activity of hASC on PETA-co-TMPTMP than on a polycaprolactone control. Scanning electron microscope imaging of hASC show expected spindle shaped morphology when adhered to copolymer. Micro-CT analysis indicates open cell interconnected pores. Foamed PETA-co-TMPTMP HA composite shows promise as an alternative to FDA-approved biopolymers for bone tissue engineering applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

There is a great demand for dental implants with the ability to accelerate periimplant bone regeneration. Modification of surface micro- and nanotopographies has been revealed to affect bone cell metabolism. In this study, we utilized dielectric barrier discharge (DBD) technology to modify commercially pure titanium (Ti-tr) surfaces and then investigated the cytocompability of DBD-modified Ti surface when compared with machined (Ti-m) and polished (Ti-p) Ti surfaces. These three kinds of Ti plates exhibited different surface energies and topographies at the micro- and nanoscale levels. The DBD-treated pure Ti surface significantly enhances cell adhesion, spread, and proliferation of MC3T3-E1 preosteoblast cells compared with the Ti-p and Ti-m surfaces, suggesting that Ti-tr has better cytocompatibility compared with the other two surfaces. Preosteoblast cells on Ti-m surface exhibited higher alkaline phosphatase activity than cells on Ti-tr and Ti-p surfaces 14 days after seeding. No significant difference in alkaline phosphatase activity was observed between cells grown on Ti-tr and Ti-p surfaces. Our study demonstrated that DBD modification significantly enhanced cell adhesion, spread, and proliferation of preosteoblasts with no negative effects on cell differentiation. Microtopography and nanotopography of the surfaces of different materials and chemical/energetic properties have a synergistic effect on cell attachment, proliferation, and differentiation. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The goal of our study was to compare the biological responses of cells cultured on polyethylene glycol (PEG) hydrogels functionalized with varying concentrations of the widely used adhesion peptide, RGD, and the cell-binding domain of fibronectin, III_9-10. We used Michael addition chemistry to covalently link cysteines in GRGDSPC and glutathione S-transferase (GST) tagged III_9-10 (GST-III_9-10), to the acrylate groups in PEG diacrylate (PEGDA). Conjugation of GST-III_9-10 to PEGDA occurred through cysteine residues in GST. Ellman's reagent and immunoblotting studies demonstrated an efficiency of 90% or more for PEG conjugation of 1 μM GST-III_9-10 or GRDGSPC in 10% (wt/vol) PEGDA at 37°C for 1 h. Circular dichroism and limited proteolysis demonstrated that conjugating PEGDA to GST-III_9-10 did not significantly perturb its native secondary structure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis characterization of the wash solution of PEG hydrogels after photopolymerization demonstrated that >95% of the 1 μM GST-III_9-10 was incorporated into the PEG hydrogels after cross-linking. PEG hydrogels derivatized with 1 μM GST-III_9-10 had significantly higher cell adhesion and spreading than PEG hydrogels with 1 μM GRGDSPC. A comparable adhesion response between GRGDSPC and GST-III_9-10 was obtained when the former was at millimolar and the latter at micromolar concentration. The amount and type of conjugate in the PEG hydrogel derivative was statistically more significant than hydrogel rigidity in stimulating the biological responses observed. This report presents new evidence of the robustness of III_9-10 in mediating cell adhesion and spreading on PEG hydrogels. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In this article, we report the development of the fast incorporation of primary amine functional groups into a polylactide (PLA) surface using the post-discharge jet region of an atmospheric-pressure nitrogen-based dielectric barrier discharge (DBD). Plasma treatments were carried out in two sequential steps: (1) nitrogen with 0.1% oxygen addition, and (2) nitrogen with 5% ammonia addition. The analyses show that the concentration of N/C ratio, surface energy, contact angle, and surface roughness of the treated PLA surface can reach 19.1%, 70.5 mJ/m², 38° and 73.22 nm, respectively. In addition, the proposed two-step plasma treatment procedure can produce a PLA surface exhibiting almost the same C₂C₁₂ cell attachment and proliferation performance as that of the conventional gelatin coating method. Most importantly, the processing/preparation time is reduced from 13–15 h (gelatin coating method) to 5–15 min (two-step plasma treatment), which is very useful in practical applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

pH-sensitive pullulan-doxorubicin (DOX) conjugates were synthesized by attaching DOX onto pullulan derivate through hydrazone bond that was stable under neutral environment but readily cleaved under mildly acidic condition. By changing the feed ratio of DOX to the pullulan derivate, conjugates with drug-loading content up to 30 wt % were obtained. In aqueous solution, the conjugates spontaneously formed uniform core-shell structured nanoparticles with DOX as core and pullulan as shell. The diameters of the nanoparticles ranged from 50 to 110 nm according to the drug-loading content. In vitro releasing experiments showed that more than 75% DOX released within 2 h at pH 5.0, while less than 15% DOX released after 12 h at pH 7.4. This pH-responsive manner of DOX release might assist the quick diffusion of DOX from the acidic endosome/lysosome and the intracellular transfer into the nucleus. Pullulan on the nanoparticles surface provided the nanoparticles with active targeting property to hepatic cells through specific interaction with asialoglycoprotein receptors on the membrane of hepatic cells, without the necessity of introducing any extra ligand. These pullulan-DOX conjugate nanoparticles were expected to be promising drug delivery system for liver targeting antitumor chemotherapy. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Monitoring the degradation of calcium phosphate-based bone substitute materials in vivo by means of noninvasive techniques (e.g., radiography) is often a problem due to the chemical resemblance of those substitutes with the mineral phase of bone. In the view of that, the present study aimed at enhancing the radiopacity of calcium phosphate cement enriched with poly(lactic-co-glycolic acid) (CPC-PLGA) microspheres, by adding tantalum oxide (Ta₂O₅) or the more traditional radiopacifier barium sulfate (BaSO₄). The radiopacifying capacity of these radiopacifiers was first evaluated in vitro by microcomputed tomography (μCT). Thereafter, both radiopacifiers were tested in vivo using a distal femoral condyle model in rabbits, with subsequent ex vivo μCT analysis in parallel with histomorphometry. Addition of either one of the radiopacifiers proved to enhance radiopacity of CPC-PLGA in vitro. The in vivo experiment showed that both radiopacifiers did not induce alterations in biological performance compared to plain CPC-PLGA, hence both radiopacifiers can be considered safe and biocompatible. The histomorphometrical assessment of cement degradation and bone formation showed similar values for the three experimental groups. Interestingly, μCT analysis showed that monitoring cement degradation becomes feasible upon incorporation of either type of radiopacifier, albeit that BaSO₄ showed more accuracy compared to Ta₂O₅. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The interactions between bone tissue and orthopedic implants are strongly affected by mechanical forces at the bone-implant interface, but the interplay between surface topographies, mechanical stimuli, and cell behavior is complex and not well understood yet. This study reports on the influence of mechanical stretch on human mesenchymal stem cells (hMSCs) attached to metallic substrates with different roughness. Controlled forces were applied to plasma membrane of hMSCs cultured on smooth and rough stainless steel surfaces using magnetic collagen-coated particles and an electromagnet system. Degree of phosphorylation of focal adhesion kinase (p-FAK) on the active form (Tyr-397), prostaglandin E₂ (PGE₂) and vascular endothelial growth factor (VEGF) levels increased on rough samples under static conditions. Cell viability and fibronectin production decreased on rough substrates, while hMSCs maturated to the osteoblastic lineage to a similar extent on both surfaces. PGE₂ production and osteoprotegerin/receptor activator of nuclear factor kappa-B ligand ratio increased after force application on both surfaces, although to a greater extent on smooth substrates. p-FAK on Tyr-397 was induced fairly rapidly by mechanical stimulation on rough surfaces while cells cultured on smooth samples failed to activate this kinase in response to tensile forces. Mechanical forces enhanced VEGF secretion and reduced cell viability, fibronetin levels and osteoblastic maturation on smooth surfaces but not on rough samples. The magnetite beads model used in this study is well suited to characterize the response of hMSCs cultured on metallic surfaces to tensile forces and collected data suggest a mechanism whereby mechanotransduction driven by FAK is essential for stem cell growth and functioning on metallic substrates. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Nanoscale topography of artificial substrates can greatly influence the fate of stem cells including adhesion, proliferation, and differentiation. Thus the design and manipulation of nanoscale stem cell culture platforms or scaffolds are of great importance as a strategy in stem cell and tissue engineering applications. In this report, we propose that a graphene oxide (GO) film is an efficient platform for modulating structure and function of human adipose-derived stem cells (hASCs). Using a self-assembly method, we successfully coated GO on glass for fabricating GO films. The hASCs grown on the GO films showed increased adhesion, indicated by a large number of focal adhesions, and higher correlation between the orientations of actin filaments and vinculin bands compared to hASCs grown on the glass (uncoated GO substrate). It was also found that the GO films showed the stronger affinity for hASCs than the glass. In addition, the GO film proved to be a suitable environment for the time-dependent viability of hASCs. The enhanced differentiation of hASCs included osteogenesis, adipogenesis, and epithelial genesis, while chondrogenic differentiation of hASCs was decreased, compared to tissue culture polystyrene as a control substrate. The data obtained here collectively demonstrates that the GO film is an efficient substratum for the adhesion, proliferation, and differentiation of hASCs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Hollow fiber artificial lungs are increasingly being used for long-term applications. However, clot formation limits their use to 1–2 weeks. This study investigated the effect of nitric oxide generating (NOgen) hollow fibers on artificial lung thrombogenicity. Silicone hollow fibers were fabricated to incorporate 50 nm copper particles as a catalyst for NO generation from the blood. Fibers with and without (control) these particles were incorporated into artificial lungs with a 0.1 m² surface area and inserted in circuits coated tip-to-tip with the NOgen material. Circuits (N = 5/each) were attached to rabbits in a pumpless, arterio-venous configuration and run for 4 h at an activated clotting time of 350–400 s. Three control circuits clotted completely, while none of the NOgen circuits failed. Accordingly, blood flows were significantly higher in the NOgen group (95.9 ± 11.7, p < 0.01) compared to the controls (35.2 ± 19.7; mL/min), and resistance was significantly higher in the control group after 4 h (15.38 ± 9.65, p < 0.001) than in NOgen (0.09 ± 0.03; mmHg/mL/min). On the other hand, platelet counts and plasma fibrinogen concentration expressed as percent of baseline in control group (63.7 ± 5.7%, 77.2 ± 5.6%; p < 0.05) were greater than those in the NOgen group (60.4 ± 5.1%, 63.2 ± 3.7%). Plasma copper levels in the NOgen group were 2.8 times baseline at 4 h (132.8 ± 4.5 μg/dL) and unchanged in the controls. This study demonstrates that NO generating gas exchange fibers could be a potentially effective way to control coagulation inside artificial lungs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Polymer-based dental restorative materials are designed to polymerize in situ. However, the conversion of methacrylate monomer to polymer is never complete, and leakage of the monomer occurs. It has been shown that these monomers are toxic in vitro; hence concerns regarding exposure of patients and dental personnel have been raised. Different monomer methacrylates are thought to cause toxicity through similar mechanisms, and the sequestration of cellular glutathione (GSH) may be a key event. In this study we examined the commonly used monomer methacrylates, 2-hydroxyethylmethacrylate (HEMA), triethylenglycol-dimethacrylate (TEGDMA), bisphenol-A-glycidyl-dimethacrylate (BisGMA), glycerol-dimethacrylate (GDMA) and methyl-methacrylate (MMA). The study aimed to establish monomers' ability to complex with GSH, and relate this to cellular toxicity endpoints. Except for BisGMA, all the monomer methacrylates decreased the GSH levels both in cells and in a cell-free system. The spontaneous formation of methacrylate-GSH adducts were observed for all methacrylate monomers except BisGMA. However, we were not able to correlate GSH depletion and toxic response measured as SDH activity and changes in cell growth pattern. Together, the current study indicates mechanisms other than GSH-binding to be involved in the toxicity of methacrylate monomers. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Materials used for medical devices are usually tested for their biocompatibility, before use. However, it is known that long-term implantation in the body may lead to degradation of the material leading to an adverse tissue response. The failure of silicone breast implants due to excessive fibrosis and contracture has led to studies to delineate the cause of fibrosis around this material. To detect the biological moieties involved, conditioned media from RAW 264.7 macrophages seeded over commercially available silicone tissue expander material was added to L929 fibroblasts. Ultrahigh-molecular-weight polyethylene and tissue culture grade polystyrene served as the control materials. The gene expression of fibrogenic cytokines, interleukin-6 (IL-6), and transforming growth factor beta (TGFβ) in the RAW macrophages and myofibroblast marker alpha smooth muscle actin (αSMA) in L929 cells were quantitated by real time polymerase chain reaction. Protein expression analysis of αSMA was carried out by immunocytochemical staining and confocal microscopy. An in vitro degradation study of silicone expander material in pseudoextracellular fluid (PECF) and the αSMA expression in fibroblasts incubated with the silicone extract containing PECF has revealed the role of silicone leachants in induction of myofibroblasts. This in vitro expression study revealed the additional profibrotic role of IL-6 in fibroblast to myofibroblast transition and the synergy between material aspects and biomolecules in regulating fibrosis around Silicone implants. These findings may help in targeting newer biological moieties in the profibrotic pathway and in devising better manufacturing processes aiding the life of millions of patients. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The increasing interest in using chitosan nanoparticles for controlled drug delivery is hampered by its blood incompatibility, especially for intravenous applications. This study investigated the effects of processing solvents (acetic acid/lactic acid), dispersing media (acidic medium/saline), and surface modifiers (polyethylene glycol, polyvinyl alcohol, and ethylenediaminetetraacetatic acid) on the hemocompatibility of chitosan. Blood compatibility of chitosan nanoparticles prepared by ionotropic gelation with altered surface chemistry was evaluated by assessing their hemolytic activity, platelet aggregation, coagulation, and cytokine induction. It was observed that nanoparticles prepared in lactic acid and dispersed in saline did not show hemolysis, platelet aggregation, or coagulation, whereas nanoparticles prepared in acetic acid showed strong hemolysis. Surface modifiers were not observed to significantly affect blood compatibility, with the exception of EDTA, which delayed blood clotting times. Thus, chitosan nanoparticles prepared in lactic acid and dispersed in saline may be an ideal nanocarrier for parenteral applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

All biomedical materials are recognized as foreign entities by the host immune system despite the substantial range of different materials that have been developed by material scientists and engineers. Hydrophobic biomaterials, hydrogels, biomaterials with low protein binding surfaces, and those that readily adsorb a protein layer all seem to incite similar host responses in vivo that may differ in magnitude, but ultimately result in encapsulation by fibrotic tissue. The recognition of medical materials by the host is explained by the very intricate pattern recognition system made up of integrins, toll-like receptors, scavenger receptors, and other surface proteins that enable leukocytes to perceive almost any foreign body. In this review, we describe the various pattern recognition receptors and processes that occur on biomedical material surfaces that permit detection of a range of materials within the host. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In this study, a hierarchical nano/microfibrous chitosan/collagen scaffold that approximates structural and functional attributes of native extracellular matrix has been developed for applicability in skin tissue engineering. Scaffolds were produced by electrospinning of chitosan followed by imbibing of collagen solution, freeze-drying, and subsequent cross-linking of two polymers. Scanning electron microscopy showed formation of layered scaffolds with nano/microfibrous architechture. Physicochemical properties of scaffolds including tensile strength, swelling behavior, and biodegradability were found satisfactory for intended application. 3T3 fibroblasts and HaCaT keratinocytes showed good in vitro cellular response on scaffolds thereby indicating the matrices, cytocompatible nature. Scaffolds tested in an ex vivo human skin equivalent wound model, as a preliminary alternative to animal testing, showed keratinocyte migration and wound re-epithelization—a prerequisite for healing and regeneration. Taken together, the herein proposed chitosan/collagen scaffold, shows good potential for skin tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Electrospun scaffolds are widely used in tissue engineering; however, a common problem is the poor cell infiltration because of the small pore size and tightly packed structure of these fibrous scaffolds. To address this issue, a novel technique was developed to fabricate electrospun silk fibroin (SF) scaffolds with rather macropores and high porosity using electrospraying-generated PEO microparticles as porogen. The morphology and pore size of MPES scaffolds were evaluated by scanning electron microscopy. It was revealed that MPES scaffold had a relatively loose structure with an increase of mean pore size (i.e., approx. 30 μm of MPES vs. approx. 5 μm of traditional electrospun scaffolds (TES) and porosity (i.e., 95% vs. 84% of TES). Culture of mouse 3T3 fibroblast in TES and MPES scaffold revealed that both scaffolds could support cell attachment, spread and proliferation. Yet, cell inflitration in vitro under the static culture condition only occurred in the MPES scaffold. Subcutaneous implantation of scaffolds in rats further confirmed that the tissue ingrowth was more efficient in the MPES scaffold compared to TES scaffold. Thus, the use of PEO microparticles as porogen was a feasible and effective method for creating macroporous electrospun SF scaffold, which provided an alternative to address the limitation of cell infiltration associated with electrospun fibrous scaffold. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

A mineralized collagen (MC) coating on metallic implants has shown great potential as orthopedic material due to high biological responses. However, their drug delivery capacity remains unsatisfactory since a serious burst release may occur and long-term release is hard to be achieved. Aiming to improve the drug-eluting capability, we incorporated drug-loaded PLGA-PEG-PLGA (PPP) micelles into the thin coating. The in vitro release profiles showed that the burst release in the initial 8 h of the modified coating decreased from 81% to 58% compared to MC coating alone; meanwhile, the release duration was prolonged from 3 days to 1 week. Additionally, the release kinetics of vancomycin hydrochloride (VH, the model drug) could be adjusted by changing the size and concentration of PPP micelles. Interestingly, less initial release of VH caused by micelle immobilization did not affect the antibacterial activity in the early stage of implantation according to the antimicrobial test. The cytocompatibility assay demonstrated that the VH-loaded PPP micelles did not have negative effect on the bioactivity of coating which greatly enhanced cell activity compared to bare Ti substrates. Thus, the MC coatings with PPP micelles could be an effective implant route for bone repair. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Preparation of novel antibacterial and cytocompatible polyurethane membranes as occlusive dressing, which can provide moist and sterile environment over mild exudative wounds is considered in this work. In this regard, an epoxy-terminated polyurethane (EPU) prepolymer based on castor oil and glycidyltriethylammonium chloride (GTEAC) as a reactive bactericidal agent were synthesized. Polyurethane membranes were prepared through cocuring of EPU and different content of GTEAC with 1,4-butane diamine. The physical and mechanical properties, as well as cytocompatibility and antibacterial performance of prepared membranes were studied. Depending on their chemical formulations, the equilibrium water absorption and water vapor transmission rate values of the membranes were in ranges of 3–85% and 53–154g m⁻² day⁻¹, respectively. Therefore, these transparent membranes can maintain for a long period the moist environment over the wounds with low exudates. Detailed cytotoxicity analysis of samples against mouse L929 fibroblast and MCA-3D keratinocyte cells showed good level of cytocompatibility of membranes after purification via extraction of residual unreacted GTEAC moieties. The antibacterial activity of the membranes against Escherichia coli and Staphylococcus aureus bacteria was also studied. The membrane containing 50% GTEAC exhibited an effective antibacterial activity, while showed acceptable cytocompatibility and therefore, can be applied as an antibacterial occlusive wound dressing. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

To overcome release of silk sericin (SS) from semi-interpenetrating polymer network (semi-IPN) SS/poly(N-isopropylacrylamide) (PNIPAm) hydrogels, natural biocompatible genipin (GNP) was adopted as cross-linking agent of SS. The GNP/SS/PNIPAm hydrogels with various GNP contents were prepared by radical polymerization. Depending on GNP content, the resultant hydrogels present white, yellow, or dark blue. Required time of color change for GNP/SS mixture solution shortened with increasing GNP ratio. The GNP/SS/PNIPAm hydrogels present good oscillatory shrinking–swelling behavior between 20 and 37°C. The behaviors of L929 cell proliferation, desorption, and transshipment on the surface of hydrogels and tissue culture polystyrene were investigated by 3-(4,5-dimethy thioazol-2-yl)-2,5-di-phenytetrazoliumromide and scanning electron microscopy method. In comparison with pure SS/PNIPAm hydrogels, the introduction of certain GNP can accelerate cell adhesion and proliferation. Due to reversible change between hydrophobicity and hydrophilicity, by lowering temperature to 4°C from 37°C, L929 cells could spontaneously detach from the surface of hydrogels without the need for trypsin or ethylenediaminetetraacetic acid. The detached cells could subsequently be recultured. The prepared hydrogel and detached cells have potential applications in biomedical fields, such as organs or tissue regeneration and cancer or disease therapy. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Calcium phosphate cement (CPC) has been widely used in orthopedic and dental applications. A critical limitation of CPC is low strength and high susceptibility to severe fracture. Surgeons can use it only to reconstruct non-stress bearing bone, raising the need for a tougher new generation of CPC. Fibers have been used as a reinforcement of CPC to improve the strength of a pure CPC scaffold. The RGD peptides (Arg-Gly-Asp) have been used to improve the biocompatibility of the scaffold, via physical adsorption. The purpose of this study was to develop a novel CPC scaffold reinforced by RGD peptide-bearing chitosan fibers (RGD-fiber-CPC). Our data showed that the RGD-fiber-CPC scaffold had an increased flexural strength, and stimulated new bone formation in an animal model. The RGD-fiber-CPC is a novel bone graft substitute in orthopedic surgery. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Nanomaterials are attractive for use in biological systems due to their ability to control the microenvironment of cells. Additionally, nanofibers can mimic fibrous characteristics of natural tissues. This study was conducted to assess astrocyte activity and infiltration behavior on Spirulina extract-embedded polycaprolactone (SP-PCL) nanofiber. Astrocytes moved along with the nanofiber, and developed an elongated and stellate shape, which is similar to those in the natural neural tissue. In addition, the expression of GFAP, a biomarker representing the activation of astrocytes, was gradually up-regulated with the increase of the concentration of Spirulina extract, indicating that Spirulina extract can control astrocyte activation. Overall, the results presented herein indicate that SP-PCL nanofiber could be used in astrocyte tissue engineering for neuronal regeneration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Osteoarthritis is a type of arthritis that is caused by breakdown of cartilage, with eventual loss of the cartilage of the joints. The ability of self-repair in damaged cartilage tissue is limited; the aim of this work is to fabricate and characterize an oxidized hyaluronic acid/resveratrol (Oxi-HA/Res) hydrogel for future applications in cartilage tissue engineering. Under physiological conditions, the Oxi-HA/Res hydrogel was prepared by chemical crosslinking of Oxi-HA with resveratrol solution and characterized by Fourier transform infrared spectrometry assay; the biocompatibility and gene expression of chondrocytes within the Oxi-HA-Res hydrogel then analyzed. The cell viability and cytotoxicity assays showed that the Oxi-HA/Res hydrogel has good biocompatibility. Oxi-HA/Res hydrogel can upregulate expression of type II collagen, aggrecan, and Sox-9 genes; while down-regulating IL-1β, MMP-1, MMP-3, MMP13 gene expression. It can also reduce LPS-induced inflammation and chondrocyte damage. The results of this study showed that the Oxi-HA/Res hydrogel is biocompatible with chondrocytes, allows for extracellular matrix synthesis, and also reduce LPS-induced inflammation and damage. These results suggest that Oxi-HA/Res hydrogel may be a potential suitable cell carrier for chondrocyte cells in the treatment of cartilage defect. However, further in vivo study is mandatory for future possible clinical applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Surface wear of corresponding tribological pairings is still a major problem in the application of artificial joint surgery. This study aims at developing wear reduced surfaces to utilize them in total joint arthroplasty. Using a pico-second laser, samples of medical CoCrMo metal alloy and Al₂O₃ ceramic were patterned by laser material removal. The subsequent tribological investigations employed a ring-on-disc method. The results showed that those samples with modified surfaces show less mass or volume loss than those with a regular, smooth surface. Using calf serum as lubricating medium, the volume loss of the structured CoCrMo samples was eight times lower than that of regular samples. By structuring Al₂O₃ surfaces, the wear volume could be reduced by 4.5 times. The results demonstrate that defined surface channels or pits enable the local sedimentation of wear debris. Thus, the amount of free debris could be reduced. Fewer abrasives in the lubricated so-called three-body-wear between the contact surfaces should result in less surface damage. Apart from direct influences on the wear behavior, less amounts of free debris of artificial joints should also be beneficial for avoiding undesired reactions with the surrounding soft tissues. The results from this study are very promising. Future investigations should involve the use of simulators meeting the natural conditions in the joint and in vivo studies with living organisms. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Hydroxyapatite (HAp) is often used as a bone-implant material because it is biocompatible and osteoconductive. However, HAp possesses poor rheological properties and it is inactive against disease-causing microbes. To improve these properties, we developed a green method to synthesize multifunctional composites containing: (1) cellulose (CEL) to impart mechanical strength; (2) chitosan (CS) to induce antibacterial activity thereby maintaining a microbe-free wound site; and (3) HAp. In this method, CS and CEL were co-dissolved in an ionic liquid (IL) and then regenerated from water. HAp was subsequently formed in situ by alternately soaking [CEL+CS] composites in aqueous solutions of CaCl₂ and Na₂HPO₄. At least 88% of IL used was recovered for reuse by distilling the aqueous washings of [CEL+CS]. The composites were characterized using FTIR, XRD, and SEM. These composites retained the desirable properties of their constituents. For example, the tensile strength of the composites was enhanced 1.9 times by increasing CEL loading from 20% to 80%. Incorporating CS in the composites resulted in composites which inhibited the growth of both Gram positive (MRSA, S. aureus and VRE) and Gram negative (E. coli and P. aeruginosa) bacteria. These findings highlight the potential use of [CEL+CS+HAp] composites as scaffolds in bone tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Cochlea implants (CI) restore the hearing in patients with sensorineural hearing loss by electrical stimulation of the auditory nerve via an electrode array. The increase of the impedance at the electrode–tissue interface due to a postoperative connective tissue encapsulation leads to higher power consumption of the implants. Therefore, reduced adhesion and proliferation of connective tissue cells around the CI electrode array is of great clinical interest. The adhesion of cells to substrate surfaces is mediated by extracellular matrix (ECM) proteins. Protein repellent polymers (PRP) are able to inhibit unspecific protein adsorption. Thus, a reduction of cell adhesion might be achieved by coating the electrode carriers with PRPs. The aim of this study was to investigate the effects of two different PRPs, poly(dimethylacrylamide) (PDMAA) and poly(2-ethyloxazoline) (PEtOx), on the strength and the temporal dynamics of the initial adhesion of fibroblasts. Polymers were immobilized onto glass plates by a photochemical grafting onto method. Water contact angle measurements proved hydrophilic surface properties of both PDMAA and PEtOx (45 ± 1° and 44 ± 1°, respectively). The adhesion strength of NIH3T3 fibroblasts after 5, 30, and 180 s of interaction with surfaces was investigated by using single cell force spectroscopy. In comparison to glass surfaces, both polymers reduced the adhesion of fibroblasts significantly at all different interaction times and lower dynamic rates of adhesion were observed. Thus, both PDMAA and PEtOx represented antiadhesive properties and can be used as implant coatings to reduce the unspecific ECM-mediated adhesion of fibroblasts to surfaces. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The goal of periodontal tissue engineering is to regenerate alveolar bone, root cementum and periodontal ligament. To achieve this goal, bioactive scaffolds play an important role in inducing in vitro osteogenic/cementogenic gene expression of periodontal ligament cells (PDLCs) and in vivo bone/cementum formation. Diopside (DIOP: CaMgSi₂O₆) ceramics have shown excellent in vitro bioactivity for potential bone repair application. However, there is no study about DIOP porous scaffolds for periodontal tissue engineering. The aim of this study is to prepare DIOP scaffolds and investigate their in vitro and in vivo osteogenesis/cementogenesis for periodontal regeneration application. DIOP scaffolds with highly porous architecture were prepared and β-tricalcium phosphate (β-TCP) scaffolds were used for the control. The interaction of DIOP scaffolds with PDLCs was studied by investigating cell attachment, proliferation and ostegenic/cementogenic differentiation of PDLCs. DIOP scaffolds were implanted into the periodontal defects of beagle dogs to evaluate their in vivo osteogenesis/cementogenesis by hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase staining, and immunohistochemistry (type I collagen: Col I; cementum attachment protein) analyses. The results have shown that DIOP scaffolds supported the attachment and proliferation of PDLCs. DIOP scaffolds significantly enhanced osteogenesis/cementogenesis-related gene expression (Col 1, Runx2, transforming growth factor beta 1, and bone morphogenetic protein 2) of PDLCs, compared to β-TCP scaffolds. The in vivo study showed that DIOP scaffolds induced new bone and cementum regeneration of periodontal tissue defects. The rate of new bone and cementum in DIOP scaffolds is comparable to that in conventional β-TCP scaffolds. Our results indicated that silicate-based DIOP ceramics could not only be used for bone tissue engineering, but also for periodontal tissue engineering due to their excellent in vitro and in vivo osteogeneis/cementogenesis. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The Drosophila melanogaster Hox protein ultrabithorax (Ubx) has the interesting ability to hierarchically self-assemble in vitro into materials that have mechanical properties comparable to natural elastin. Ubx materials can be easily functionalized by gene fusion, generating potentially useful scaffolds for cell and tissue engineering. Here, we tested the cytocompatibility of fibers composed of Ubx or an mCherry-Ubx fusion protein. Fibers were cultured with three primary human cell lines derived from vasculature at low passage: umbilical vein endothelial cells, brain vascular pericytes, or aortic smooth muscle cells. No direct or indirect toxicity was observed for any cell line, in response to fibers composed of either plain Ubx or mCherry-Ubx. Cells readily adhered to Ubx fibers, and cells attached to fibers could be transferred between tissue cultures without loss of viability for at least 96 h. When attached to fibers, the morphology of the three cell lines differed somewhat, but all cells in contact with Ubx fibers exhibited a microtubular network aligned with the long axis of Ubx fibers. Thus, Ubx fibers are cytocompatible with cultured primary human vascular cells. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Several strategies have been investigated to enhance axonal regeneration after spinal cord injury, however, the resulting growth can be random and disorganized. Bioengineered scaffolds provide a physical substrate for guidance of regenerating axons towards their targets, and can be produced by freeze casting. This technique involves the controlled directional solidification of an aqueous solution or suspension, resulting in a linearly aligned porous structure caused by ice templating. In this study, freeze casting was used to fabricate porous chitosan-alginate (C/A) scaffolds with longitudinally oriented channels. Chick dorsal root ganglia explants adhered to and extended neurites through the scaffold in parallel alignment with the channel direction. Surface adsorption of a polycation and laminin promoted significantly longer neurite growth than the uncoated scaffold (poly-L-ornithine + Laminin = 793.2 ± 187.2 μm; poly-L-lysine + Laminin = 768.7 ± 241.2 μm; uncoated scaffold = 22.52 ± 50.14 μm) (P < 0.001). The elastic modulus of the hydrated scaffold was determined to be 5.08 ± 0.61 kPa, comparable to reported spinal cord values. The present data suggested that this C/A scaffold is a promising candidate for use as a nerve guidance scaffold, because of its ability to support neuronal attachment and the linearly aligned growth of DRG neurites. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Increased titanium surface hydrophilicity has been shown to accelerate dental implant osseointegration. Macrophages are important in the early inflammatory response to surgical implant placement and influence the subsequent healing response. This study investigated the modulatory effect of a hydrophilic titanium surface on the inflammatory cytokine expression profile in a human macrophage cell line (THP-1). Genes for 84 cytokines, chemokines, and their receptors were analyzed following exposure to (1) polished (SMO), (2) micro-rough sand blasted, acid etched (SLA), and (3) hydrophilic-modified SLA (modSLA) titanium surfaces for 1 and 3 days. By day 3, the SLA surface elicited a pro-inflammatory response compared to the SMO surface with statistically significant up-regulation of 16 genes [Tumor necrosis factor (TNF) Interleukin (IL)-1β, Chemokine (C-C motif) ligand (CCL)-1, 2, 3, 4, 18, 19, and 20, Chemokine (C-X-C motif) ligand (CXCL)-1, 5, 8 and 12, Chemokine (C-C motif) receptor (CCR)-7, Lymphotoxin-beta (LTB), and Leukotriene B4 receptor (LTB4R)]. This effect was countered by the modSLA surface, which down-regulated the expression of 10 genes (TNF, IL-1α and β, CCL-1, 3, 19 and 20, CXCL-1 and 8, and IL-1 receptor type 1), while two were up-regulated (osteopontin and CCR5) compared to the SLA surface. These cytokine gene expression changes were confirmed by decreased levels of corresponding protein secretion in response to modSLA compared to SLA. These results show that a hydrophilic titanium surface can modulate human macrophage pro-inflammatory cytokine gene expression and protein secretion. An attenuated pro-inflammatory response may be an important molecular mechanism for faster and/or improved wound healing. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

An understanding of neovascularization and/or angiogenesis in cancer is acutely required for effective cancer therapy due to concerns about tumor growth and metastasis. In particular, integrin αvβ3 is closely associated with cell migration and invasion during angiogenesis. Hence, we developed aptamer_αvβ3-conjugated magnetic nanoparticles (Apt_αvβ3-MNPs) to enable precise detection of integrin-expressing cancer cells using magnetic resonance imaging. Apt_αvβ3-MNPs exhibited not only cytocompatibility, but also an efficient targeting ability with high magnetic sensitivity through in vitro/in vivo studies. The results of this study demonstrate that Apt_αvβ3-MNPs have the potential to be used for accurate tumor diagnosis and therapy. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Nanostructured porous silica coatings were synthesized on titanium by the combined sol–gel and evaporation-induced self-assembly process. The silica-coating structures were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and nitrogen sorptometry. The effect of the nanoporous surface on apatite formation in simulated body fluid, protein adsorption, osteoblast cell adhesion behavior, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) is reported. Silica coatings with highly ordered sub-10 nm porosity accelerate early osteoblast adhesive response, a favorable cell response that is attributed to an indirect effect due to the high protein adsorption observed on the large-specific surface area of the nanoporous coating but is also probably due to direct mechanical stimulus from the nanostructured topography. The nanoporous silica coatings, particularly those doped with calcium and phosphate, also promote the osteogenic differentiation of hBMSCs with spontaneous mineral nodule formation in basal conditions. The bioactive surface properties exhibited by the nanostructured porous silica coatings make these materials a promising alternative to improve the osseointegration properties of titanium dental implants and could have future impact on the nanoscale design of implant surfaces. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of this study was to evaluate in vivo the influence of the native oxide layer on osseointegration and new bone formation on the surface of atmospheric plasma-sprayed porous titanium coatings. Porous titanium coatings were deposited on all implant surfaces, and half of the samples were subsequently submitted to oxide layer removal treatment. Samples were implanted onto the cortical bone of sheep (tibia) and evaluated at 30 and 60 days. Implants were removed en bloc and the attachment of bone to implants was examined by tensile pull-out test (osseointegration assessment), light microscopy, scanning electron microscopy (histological analysis), and instrumented hardness tests (mechanical properties of mature and newly formed bone tissue). Coatings submitted to oxide layer treatment presented higher osseointegration values at both healing periods and showed more mature and mineralized bone tissue when compared with nontreated coatings. Our findings showed that the use of acid-etching in association with atmospheric plasma spraying techniques improves osseointegration of titanium implants. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Tissue engineered hydrogels hold great potential as nucleus pulposus substitutes (NP), as they promote intervertebral disc (IVD) regeneration and re-establish its original function. But, the key to their success in future clinical applications greatly depends on its ability to replicate the native 3D micro-environment and circumvent their limitation in terms of mechanical performance. In the present study, we investigated the rheological/mechanical properties of both ionic- (iGG-MA) and photo-crosslinked methacrylated gellan gum (phGG-MA) hydrogels. Steady shear analysis, injectability and confined compression stress-relaxation tests were carried out. The injectability of the reactive solutions employed for the preparation of iGG-MA and phGG-MA hydrogels was first studied, then the zero-strain compressive modulus and permeability of the acellular hydrogels were evaluated. In addition, human intervertebral disc (hIVD) cells encapsulated in both iGG-MA and phGG-MA hydrogels were cultured in vitro, and its mechanical properties also investigated under dynamic mechanical analysis at 37°C and pH 7.4. After 21 days of culturing, hIVD cells were alive (Calcein AM) and the E′ of ionic-crosslinked hydrogels and photo-crosslinked was higher than that observed for acellular hydrogels. Our study suggests that methacrylated gellan gum hydrogels present promising mechanical and biological performance as hIVD cells were producing extracellular matrix. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Skeletal stem cell (SSC) growth on a novel porous HA/TCP scaffold has been investigated in vivo. The effect of porosity on osteogenic differentiation was assessed by comparing two groups of scaffolds with differing porosity but controlled pore size. Histology, microCT, scanning electron microscopy, and biochemical analysis were used to assess SSC proliferation and differentiation. The 45 pores per inch (ppi) scaffold demonstrated a greater increase in density than the 30 ppi scaffold following in vivo culture, and a reduction in dimensions of the pores and channels of the higher porosity scaffold was observed, indicating generation of new tissue within the pores. All scaffolds supported SSC proliferation but the higher scaffold porosity augmented osteogenic differentiation. ALP specific activity was enhanced on the 45 ppi scaffold compared to the 30 ppi scaffold. These studies demonstrate the importance of porosity in scaffold design and impact therein for tissue engineering application. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening, and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of proinflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Peripheral nerves are often subjected to mechanical stretching, which in excess results in various degrees of impairment of their function. An understanding of the biomechanical behavior of peripheral nerves is important to the prevention of nerve injury during surgical manipulation. Here, in vitro mechanical properties and viscoelastic behavior of human ulnar/median nerves were measured with a tensile tester. In vivo stress and deformation of an ulnar nerve was also examined in continuity during a surgical procedure. Finite element models were developed to determine in vitro and in vivo viscoelastic parameters of the nerves. The results show that in vitro mechanical properties of fresh ulnar nerve are different from those measured in vivo. Several factors that are possibly attributed to the difference were analyzed. The in situ strain of the nerves is one of the major factors that must be considered to obtain accurate strain–stress relationship in the in vivo measurement. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Both physiological and pathological tissue remodeling (e.g., during wound healing and cancer, respectively) require new blood vessel formation via angiogenesis, but the underlying microenvironmental mechanisms remain poorly defined due in part to the lack of biologically relevant in vitro models. Here, we present a biomaterials-based microfluidic 3D platform for analysis of endothelial sprouting in response to morphogen gradients. This system consists of three lithographically defined channels embedded in type I collagen hydrogels. A central channel is coated with endothelial cells, and two parallel side channels serve as a source and a sink for the steady-state generation of biochemical gradients. Gradients of vascular endothelial growth factor (VEGF) promoted sprouting, whereby endothelial cell responsiveness was markedly dependent on cell density and vessel geometry regardless of treatment conditions. These results point toward mechanical and/or autocrine mechanisms that may overwhelm pro-angiogenic paracrine signaling under certain conditions. To date, neither geometrical effects nor cell density have been considered critical determinants of angiogenesis in health and disease. This biomimetic vessel platform demonstrated utility for delineating hitherto underappreciated contributors of angiogenesis, and future studies may enable important new mechanistic insights that will inform anti-angiogenic cancer therapy. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The current nano-biotechnologies interfacing synthetic materials and cell biology requires a better understanding of cell–surface interactions on the micro-to-nanometer scale. Cell–substrate interactions are mediated by the presence of proteins adsorbed from biological fluids to the substrate. The effect of nanotopography and surface chemistry on protein adsorption as well as the mediation effect on subsequent cellular communication with substratum is not well documented. This review discusses the role of physicochemical properties of cell–surface interactions and state-of-the-art methods currently available for micro-nanoscale surface fabrication and patterning. We also briefly discuss the current surface patterning techniques that allow the combination of a fine and independent control on nanotopography and chemistry to understand the effect of surface nanoscale substrate morphology on cell–surface interactions which has never been realized in previous reports. In addition, we discuss the influence of various chemical patterning and modulation of the nano-topography of surfaces on cell functionality and phenotype. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Anodization of titanium and its alloys, under controlled conditions, generates a nanotubular architecture on the material surface. The biological consequences of such changes are poorly understood, and therefore, we have analyzed the cellular and molecular responses of osteoblasts that were plated on nanotubular anodized surface of a titanium-zirconium (TiZr) alloy. Upon comparing these results with those obtained on acid etched and polished surfaces of the same alloy, we observed a significant increase in adhesion and proliferation of cells on anodized surfaces as compared to acid etched or polished surface. The expression of genes related to cell adhesion was high only on anodized TiZr, but that of genes related to osteoblast differentiation and osteocalcin protein and extracellular matrix secretion were higher on both anodized and acid etched surfaces. Examination of surface morphology, topography, roughness, surface area and wettability using scanning electron microscopy, atomic force microscopy, and contact angle goniometry, showed that higher surface area, hydrophilicity, and nanoscale roughness of nanotubular TiZr surfaces, which were generated specifically by the anodization process, could strongly enhance the adhesion and proliferation of osteoblasts. We propose that biological properties of known bioactive titanium alloys can be further enhanced by generating nanotubular surfaces using anodization. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Three-dimensional tissue engineered constructs provide a platform to examine how the local extracellular matrix (ECM) contributes to the malignancy of cancers such as human glioblastoma multiforme. Improved resolution of how local matrix biophysical features impact glioma proliferation, genomic and signal transduction paths, as well as phenotypic malignancy markers would complement recent improvements in our understanding of molecular mechanisms associated with enhanced malignancy. Here, we report the use of a gelatin methacrylate (GelMA) platform to create libraries of three-dimensional biomaterials to identify combinations of biophysical features that promote malignant phenotypes of human U87MG glioma cells. We noted key biophysical properties, namely matrix density, crosslinking density, and biodegradability, that significantly impact glioma cell morphology, proliferation, and motility. Gene expression profiles and secreted markers of increased malignancy, notably VEGF, MMP-2, MMP-9, HIF-1, and the ECM protein fibronectin, were also significantly impacted by the local biophysical environment as well as matrix-induced deficits in diffusion-mediated oxygen and nutrient biotransport. Overall, this biomaterial system provides a flexible platform to explore the role biophysical factors play in the etiology, growth, and subsequent invasive spreading of gliomas. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of this work is to investigate the use of microtopographies in providing physical cues to modulate the cellular response of human mesenchymal stem cells on ceramics. Two microgrooved patterns (100 μm/50 μm, 10 μm/10 μm groove/pitch) were transcribed reversely onto alumina green ceramic tapes via an embossing technique followed by sintering. Characterization of the micropatterned alumina surfaces and their cellular response was carried out. Spread and polygonal cell morphologies were observed on the wider groove (50 μm/100 μm) surface. Cells seeded onto the narrow groove (10 μm/10 μm) surface aligned themselves alongside the grooves, resulting in more elongated cell morphology. More osteoid matrix nodules shown by osteopontin and osteocalcin biomarkers were detected on the larger grooved surfaces after cell culture of 21 days, indicating a greater level of osteogenicity. This study has shown that micropatterned wider groove (50 μm) topographies are more suitable surfaces for improving osseointegration of ceramic implants. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

This study investigated the influence of the surface microporosity of beta-tri-calcium phosphate (β-TCP) ceramics on the resorption capacity of osteoclasts. This was achieved by first compacting commercially available β-TCP powder into disks that were sintered at various temperatures, thereby yielding different surface microporosities. Scanning electron microscopy (SEM) and subsequent image processing verified different degrees of surface microporosity on the disks. Rabbit osteoclasts in a bone marrow derived cell suspension were then seeded onto these disks and incubated for 48 h. Tartrate resistant acid phosphatase (TRAP) staining confirmed the presence of osteoclasts on all disks. Actin ring staining that detected actively resorbing OCs showed an inverse linear correlation between the number of actively resorbing osteoclasts (percentage of total OCs on the surfaces) with surface microporosity. These findings should be taken into consideration for the design and/or production of new β-TCP bone graft substitutes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

A microspheres-aggregated scaffold with ultra big pores (over 300 μm) and fuzzy microspheres is fabricated by incubating polycaprolactone (PCL)/tetrahydrofuran (THF) solution in a −20°C refrigerator, following by freeze-drying. Formation of the scaffold is mainly governed by the crystallization of the PCL polymer at appropriate conditions. All the 10–20% PCL/THF solutions yield the microspheres-aggregated scaffolds when the initial solution temperature is higher than 37°C, whereas the 10–15% solutions form dense membranes when the initial solution temperature is below 25°C. The size of the microspheres and pores is as large as 70–150 μm and 170–816 μm, respectively. The PCL microspheres-aggregated scaffold can better support the adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs) compared to the traditional porous scaffold obtained by a porogen leaching method. The tendencies of chondrogenesis and osteogenesis differentiation of BMSCs are observed on the microspheres-aggregated scaffold and the ordinary porous scaffold, respectively. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Cell–cell interaction plays an important role in the control of cell functions. The precise control of cell–cell interaction will provide a useful tool to elucidate its influence on stem cell differentiation. In this study, four types of micropatterned surfaces were prepared by ultraviolet photolithography to investigate the effect of cell–cell interaction on the osteogenic differentiation of human mesenchymal stem cells (MSCs). Single MSCs adhered on the micropatterned surfaces following the micropatterns. Single cells on the isolated, barbell, linear, and honeycomb dot micropatterns had zero, one, two, and three cell–cell interaction partners, respectively. The number of cell–cell interaction of single MSCs was controlled by the different micropatterns, which showed evident effects on actin filament structure assembly and osteogenic differentiation of MSCs. MSCs with two and three interaction partners showed a significantly higher rate of osteogenic differentiation than did isolated single cells and cells with only one interaction partner. Thus, the osteogenic differentiation of MSCs was enhanced with increased cell–cell interaction. These results highlight the importance of cell–cell interaction in stem cell differentiation. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Insufficient implant stability is an important determinant in the failure of cementless prostheses. To improve osseointegration, we aim at generating a bioactive implant combining a macroporous titanium (TT) with a biocompatible hydrogel to encapsulate osteo-inductive factors and osteoprogenitor cells. Amidation and cross-linking degree of an amidated carboxymethylcellulose hydrogel (CMCA) were characterized by FT-IR spectrometry and mechanical testing. Bone marrow mesenchymal stem cells (BMSCs) from osteoarthritic patients were cultured on CMCA hydrogels, TT, and TT loaded with CMCA (TT + CMCA) with an optimized concentration of SrCl₂ to evaluate cell viability and osteo-differentiation. Amidation and cross-linking degree were homogeneous among independent CMCA batches. SrCl₂ at 5 μg/mL significantly improved BMSCs osteo-differentiation increasing calcified matrix (P < 0.01), type I collagen expression (P < 0.05) and alkaline phosphatase activity. TT + CMCA samples better retained cells into the TT mesh, significantly improving cell seeding efficiency with respect to TT (P < 0.05). BMSCs on TT + CMCA underwent a more efficient osteo-differentiation with higher alkaline phosphatase (P < 0.05) and calcium levels compared to cells on TT. Based on these in vitro results, we envision the association of TT with strontium-enriched CMCA and BMSCs as a promising strategy to generate bioactive implants promoting bone neoformation at the implant site. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In direct skeletal attachment (DSA) of limb prostheses, a construct is implanted into an amputee's residuum bone and protrudes out of the residuum's skin. This technology represents an alternative to traditional suspension of prostheses via various socket systems, with clear indications when the sockets cannot be properly fitted. Contemporary DSA was invented in the 1990s, and several implant systems have been introduced since then. The current review is intended to compare the design features of implants for DSA whose use in humans or in animal studies has been reported in the literature. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Two lipid-solid dispersion loading Norcantharidin sustained-released microspheres of alginate-chitosan (NCTD/LSD-ACMs) were prepared via the emulsification-gelation method. The effects of microspheres for transarterial hepatic chemoembolization were evaluated in VX2 rabbit liver cancer model. The VX2 animal model was established by biopsy needle, divided randomly into four groups, and disposed with three preparations including NCTD/LSD-ACMs (60–120 μm), NCTD/LSD-ACMs(120–200 μm), and NCTD solution through the hepatic arteries compared with the untreated group (control group). The serum of all rabbits before and at 3, 7, and 14 days after embolization was collected to determine the level of aspartate aminotransferase (AST). The AST level increased in the three treated groups on the first day compared with the control group (p < 0.05), and was higher in the two embolization groups (with no significant difference, p >0.05) than that in the NCTD group (p < 0.05). The tumor growth rates, which were significantly decreased in the two embolization groups compared with that in the control group, and the degree of liver cell necrosis assessed by the histopathological specimens, were used to evaluate the embolization effect. Liquefactive necrosis and coagulative necrosis were observed in the two embolization groups. The results showed that NCTD/LSD-ACMs are a potential candidate for embolization of liver cancer. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Despite the therapeutic benefits of both mechanical circulatory assist devices and nitinol stents with titanium (Ti) outer surfaces, problems remain with thrombosis at the blood-contacting surface. Covering these surfaces with a layer of endothelium would mimic the native lining of the cardiovascular system, potentially decreasing thrombotic complications. Since surface topography is known to affect the phenotype of a seeded cell layer and since stents and ventricular assist devices exhibit surface protrusions, we tested the hypothesis that endothelial cells (ECs) have altered function on Ti surfaces with protrusions of 1.25, 3, and 5 μm height, compared with smooth Ti surfaces. ECs and nuclei were more aligned and ECs were more elongated on all patterned surfaces. Cell area was reduced on the 3 and 5 μm features. Expression of eNOS and COX2 was not altered by patterned surfaces, but expression of KLF-2 was higher on 1.25 and 5 μm features. Nitric oxide production following exposure to flow was higher on the 5 μm features. These results show that some antithrombogenic functions of ECs are significantly enhanced for ECs cultured on surface protrusions, and no functions are diminished, informing the future design of implant surfaces for endothelialization. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of this study is to investigate the influence of macrophages on osteoblast (OB) performance and differentiation. In this regard, we studied the secretion of growth factors including bone morphogenetic proteins (BMPs) from before and after activation of macrophages. We also evaluated osteogenic markers in the co-culture of macrophages and OBs. The macrophages were seeded on microparticles (MPs) based on chitosan (CS). Two types of MPs were fabricated including CS MPs and 10% calcium phosphate (CaHPO₄)-incorporated CS MPs. Macrophage seeded on MPs was activated using lipopolysaccharide (LPS). The expression of BMP-2, BMP-6, BMP-7, and transforming growth factor beta (TGF-β) from macrophages seeded and cultured on hybrid MPs before and after activation of LPS at predetermined times was quantified using a quantitative reverse transcription-polymerase chain reaction (RT-PCR). All of the above growth factors were expressed from MP–macrophage cultures before LPS activation. Osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and collagen I (COL-I) in the cultures of MP–OB–macrophage were quantified using a quantitative RT-PCR at days 2, 4, and 7. We found an elevation of gene expression of ALP and COL-1 in the co-cultures of OB–macrophage on MPs compared to OB on MP cultures. These data suggest that macrophages enhance expression of osteogenic markers in OBs, and demonstrate the importance of the role of macrophages in bone regeneration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Acellular dermal matrices (ADM) are commonly used in reconstructive procedures and rely on host cell invasion to become incorporated into host tissues. We investigated different approaches to adipose-derived stem cells (ASCs) engraftment into ADM to enhance this process. Lewis rat adipose-derived stem cells were isolated and grafted (3.0 × 10⁵ cells) to porcine ADM disks (1.5 mm thick × 6 mm diameter) using either passive onlay or interstitial injection seeding techniques. Following incubation, seeding efficiency and seeded cell viability were measured in vitro. In addition, Eighteen Lewis rats underwent subcutaneous placement of ADM disk either as control or seeded with PKH67 labeled ASCs. ADM disks were seeded with ASCs using either onlay or injection techniques. On day 7 and or 14, ADM disks were harvested and analyzed for host cell infiltration. Onlay and injection techniques resulted in unique seeding patterns; however cell seeding efficiency and cell viability were similar. In-vivo studies showed significantly increased host cell infiltration towards the ASCs foci following injection seeding in comparison to control group (p < 0.05). Moreover, regional endothelial cell invasion was significantly greater in ASCs injected grafts in comparison to onlay seeding (p < 0.05). ADM can successfully be engrafted with ASCs. Interstitial engraftment of ASCs into ADM via injection enhances regional infiltration of host cells and angiogenesis, whereas onlay seeding showed relatively broad and superficial cell infiltration. These findings may be applied to improve the incorporation of avascular engineered constructs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Despite the inherent ability of bone to regenerate itself, there are a number of clinical situations in which complete bone regeneration fails to occur. In view of shortcomings of conventional treatment, gene therapy may have a place in cases of critical-size bone loss that cannot be properly treated with current medical or surgical treatment. The purpose of this review is to provide an overview of gene therapy in general, nonviral techniques of gene transfer including physical and chemical methods, RNA-based therapy, therapeutic genes to be transferred for bone regeneration, route of application including ex vivo application, and direct gene therapy approaches to regenerate bone. ©2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Poly(ester-urethane-urea) (PEUU) is one of many synthetic biodegradable elastomers under scrutiny for biomedical and soft tissue applications. The goal of this study was to investigate the effect of the experimental parameters on mechanical properties of PEUUs following exposure to different degrading environments, similar to that of the human body, using linear regression, producing one predictive model. The model utilizes two independent variables of poly(caprolactone) (PCL) type and copolymer crystallinity to predict the dependent variable of maximum tangential modulus (MTM). Results indicate that comparisons between PCLs at different degradation states are statistically different (p < 0.0003), while the difference between experimental and predicted average MTM is statistically negligible (p < 0.02). The linear correlation between experimental and predicted MTM values is R² = 0.75. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Acid functional hydrogels are a type of materials with many advantages. Over the last years, increasing attention for the synthesis of dendronized polymers has been drawn due to their unique properties of high multivalence in the same surface as compared with conventional polymers. In this study, we report the preparation of novel acid dendronized hydrogels using a dendritic monomer obtained from Behera's amine. The swelling and rheological performance, the non-toxicity over fibroblast cells and the drug encapsulation capacity of the novel hydrogels suggests that the new materials can achieve great potential as carrier for drug delivery and other potential biomedical applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Nanoparticulate titanium dioxide (nano-TiO₂) is a widely used powerful nanoparticulate material with high stability, anticorrosion, and photocatalytic property. However, it is possible that during nano-TiO₂ exposure, there may be negative effects on cardiovascular system in intoxicated mice. The present study was therefore undertaken to determine nano-TiO₂-induced oxidative stress and to determine whether nano-TiO₂ intoxication alters the antioxidant system in the mouse heart exposed to 2.5, 5, and 10 mg/kg body weight nano-TiO₂ for 90 consecutive days. The findings showed that long-term exposure to nano-TiO₂ resulted in obvious titanium accumulation in heart, in turn led to sparse cardiac muscle fibers, inflammatory response, cell necrosis, and cardiac biochemical dysfunction. Nano-TiO₂ exposure promoted remarkably reactive oxygen species production such as superoxide radicals, hydrogen peroxide, and increased malondialdehyde, carbonyl and 8-OHdG levels as degradation products of lipid, protein, and DNA peroxidation in heart. Furthermore, nano-TiO₂ exposure attenuated the activities of antioxidative enzymes, such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, glutathione-S-transferase, and levels of antioxidants including ascorbic acid, glutathione, and thiol in heart. Therefore, TiO₂ NPs exposure may impair cardiovascular system in mice, and attention should be aroused on the application of nano-TiO₂ and their potential long-term exposure effects especially on human beings. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

We present a concept for a new regenerative and resorbable prosthesis for tendon and ligament and characterize the physicomechanical and biological behavior of one of its components, a hollow braid made of poly-lactide acid (PLA) which is the load-bearing part of the prosthesis concept. The prosthesis consists of a braid, microparticles in its interior serving as cell carriers, and a surface non-adherent coating, all these parts being made of biodegradable materials. The PLA braid has a nonlinear convex stress–strain behavior with a Young modulus of 1370 ± 90 MPa in the linear, stretched state, and after 12 months of hydrolytic degradation the modulus shows a reduction by a factor of four. Different disinfection methods were tested as to their efficiency in cleansing the braid and preparing it for cell culture. Fibroblasts of L929 line were grown on the PLA braid for 14 days, showing good adherence and proliferation. These studies validate the PLA braid for the intended purpose in the regenerative prosthesis concept. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

One mechanism of the failure of blood-contacting devices is clotting. Nitric oxide (NO) releasing materials are seen as a viable solution to the mediation of surface clotting by preventing platelet activation; however, NO's involvement in preventing clot formation extends beyond controlling platelet function. In this study, we evaluate NO's effect on factor XII (fibrinogen) adsorption and activation, which causes the initiation of the intrinsic arm of the coagulation cascade. This is done by utilizing a model plasticized poly(vinyl) chloride (PVC), N-diazeniumdiolate system and looking at the adsorption of fibrinogen, an important clotting protein, to these surfaces. The materials have been prepared in such a way to eliminate changes in surface properties between the control (plasticized PVC) and composite (NO-releasing) materials. This allows us to isolate NO release and determine the effect on the adsorption of fibrinogen, to the material surface. Surprisingly, it was found that an NO releasing material with a surface flux of 17.4 ± 0.5 × 10⁻¹⁰ mol NO cm⁻² min⁻¹ showed a significant increase in the amount of fibrinogen adsorbed to the material surface compared to one with a flux of 13.0 ± 1.6 × 10⁻¹⁰ mol NO cm⁻² min⁻¹ and the control (2334 ± 496, 226 ± 99, and 103 ±31% fibrinogen adsorbed of control, respectively). This study suggests that NO's role in controlling clotting is extended beyond platelet activation. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Hair follicle transplantation is often used in the treatment of androgenetic alopecia (AGA). However, the only source of hair follicles is from human donors themselves, which limits the application of this approach. One possible solution is to reconstitute hair follicle from dissociated cells. Currently, a number of microscale technologies have been developed to create size and shape controlled microenvironments in tissue engineering. Photopolymerizable PEGDA hydrogels are often selected as promising scaffolds in engineered microtissues due to their biocompatibility and adjustable mechanical properties. Here, we fabricated an array of PEGDA microwells with center islets that mimic the architecture of human hair follicles using soft lithography. Dermal and epithelial cells were seeded in different compartments of the microstructured mould to mimic mesenchymal and epithelial compartmentalization in native hair follicles. We demonstrated that these compartmentalized microstructures support cell proliferation and cell survival over 14 days, and spreading of dermal fibroblasts was observed. This hydrogel micromould provides a potentially useful tool for engineering 3D hair follicle-mimicking complex cultures in vitro. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Decellularized tissues have been successfully used in tissue engineering and regenerative medicine for the purpose of removing antigens present in the cellular components. However, this decellularization technique uses ionic solutions or chemical treatments such as enzyme treatments that might damage the biophysical properties or reduce the physical strength of tissue. This study aimed to improve the strength of decellularized tissues. We designed a tissue bioreactor that can repeatedly deliver physical stimulation, such as tensile and torsional deformation, to the upper and lower parts of a tissue. To decellularized porcine Tibialis tendons, we used an enzymatic solution to remove the primary cells, and then applied ultrasonic cleansing using a combination of ionic solution and distilled water to destroy residual cells by differing from the osmotic pressure between the inside and outside of the cell membrane. The total DNA content of decellularized tissue was decreased by 77% compared with that of the original tissue and the ultimate tensile strength of the decellularized tissue was 20% lower than that of the normal tissue. Decellularized tissues were then cultivated in the tissue bioreactor with repeated physical stimulation of 110% tension, 90° torsion, and frequency of once per a second, and the ultimate tensile strength was found to be greater than that of the normal ligament at 7 day culture. This study showed that decellularization using enzyme and mechanical treatment is safe and use of a tissue bioreactor can increase the physical strength of tendons, making this a potential mechanism to reconstruct human ligaments. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bioresorption is a biological mechanism by which biomaterials are resorbed and thereby disappear from implantation sites partially or completely over a period of time. Osteoclast-medicated bioresorption is a possible new advantage to incorporate material degradation into remodeling in bone metabolism process. The purpose of this study was to investigate the osteoclastogenesis and bioresorption of synthesized calcium phosphate materials. Differentiation into mature human osteoclasts on carbonated hydroxyapatite (CA) was significantly enhanced compared to hydroxyapatite (HA) and β-tricalcium phosphate, based on the quantitative gene expressions of molecular markers for osteoclast differentiation. Osteoclasts adhered and differentiated into giant multinuclear TRAP-positive cells on every type of synthesized sample based on the histological analysis. Morphological observations using fluorescence and quantitative analysis revealed that the actin rings of osteoclasts on CA were thick, small in diameter and co-localized with vinculin, similar to the rings found on bone slices. In contrast, the actin rings of osteoclasts on HA and culture dishes were thin and large in diameter. Scanning electron microscopic images and quantitative analysis indicated that the resorption pits on CA were significantly deeper than those on HA due to the enhanced tight sealing ability between osteoclasts and their substrate. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Hydroxyapatite (HA)-based biocomposites have been widely investigated for a multitude of applications and these studies have been largely driven to improve mechanical properties (toughness and strength) without compromising cytocompatibility properties. Apart from routine cell viability/proliferation analysis, limited efforts have been made to quantify the fate processes (cell proliferation, cell cycle, and cell apoptosis) of human fetal osteoblast (hFOB) cells on HA-based composites, in vitro. In this work, the osteoblast cell fate process has been studied on a model hydroxyapatite–titanium (HA–Ti) system using the flow cytometry. In order to retain both HA and Ti, the novel processing technique, that is, spark plasma sintering, was suitably adopted. The cell fate processes of hFOBs, as evaluated using a flow cytometry, revealed statistically insignificant differences among HA–10 wt % Ti and HA and control (tissue culture polystyrene surface) in terms of osteoblast apoptosis, proliferation index as well as division index. For the first time, we provide quantified flow cytometry results to demonstrate that 10 wt % Ti additions to HA do not have any significant influence on the fate processes of human osteoblast-like cells, in vitro. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Because of their suitable bio-mechanical properties, polymeric materials, such as Poly(L-lactic acid) (PLLA), and poly (lactic-co-glycolic acid) (PLGA), are often used in the biomedical field, in particular for cardiovascular applications. Implanted materials induce several events related to the inflammatory reaction, such as macrophage adhesion and activation with following cytokine release. This work considered the effect of macrophage adhesion and related cytokine release on endothelial cells (PAOEC) proliferation and migration. Slight differences have been shown by the macrophages reaction when in contact with PLLA, PLGA, or PLLA/PLGA blend. However, these differences showed to differently enhance endothelial cells behavior in terms of wound healing. These data suggest the inflammatory reaction as a useful way to consider concerning materials biocompatibility, in order to optimize the endothelial regeneration following vascular prosthetic implants. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Although hydrogel formulations that may be applied to many mucosal surfaces are now readily accessible, little research effort has been concentrated on the development of systems that may be usefully employed for the prolonged hydration of the oral cavity. To this end, and set within the context of oral care in general, this review considers the requirements for the design of hydrogel formulations with an affinity for buccal cells and details methods for evaluating the performance of these formulations as treatments for the management of xerostomia. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Ideally, bone substitute materials would undergo cell-mediated degradation during the remodeling process of the host bone tissue while being replaced by newly formed bone. In an attempt to exploit the capacity of Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) to stimulate osteoclast-like cells formation, this study explored different loading methods for RANKL in injectable calcium phosphate cement (CPC) and the effect on release and biological activity. RANKL was loaded via the liquid phase of CPC by adsorption onto or incorporation into poly(lactic-co-glycolic acid) (PLGA) microspheres with two different morphologies (i.e., hollow and dense), which were subsequently embedded in CPC. As controls nonembedded PLGA-microspheres were used as well as plain CPC scaffolds with RANKL adsorbed onto the surface. RANKL release and activity were evaluated by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) and osteoclast-like cells formation in cell culture experiments. Results indicated that sustained release of active RANKL can be achieved upon RANKL adsorption to PLGA microspheres, whereas inactive RANKL was released from CPC-PLGA formulations with RANKL incorporated within the microspheres or within the liquid phase of the CPC. These results demonstrate that effective loading of RANKL in injectable CPC is only possible via adsorption to PLGA microspheres, which are subsequently embedded within the CPC-matrix. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Large-diameter metal-on-metal (MoM) bearings evolved from the success of hip resurfacing. These implants were used in revision surgery in cases with well-fixed acetabular cups but loose or failed femoral stems, to avoid cup revision. Early data showed low rates of dislocation and potentially low wear profiles due to better fluid film lubrication. The risk of impingement was also thought to be low due to the increased head-neck ratio. Subsequently large-diameter MoM heads gained popularity in primary hip replacement. Recent data has emerged on the unacceptably high revision rates among patients with large-diameter MoM total hip arthroplasties (THAs), high blood levels of metal ions, and adverse tissue reactions. The head–neck (cone–taper) modular interface probably represents the weak link in large metal heads that have been used on conventional tapers. Increased torque of the large head, micromotion, and instability at the cone–taper interface, synergistic interactions between corrosion and wear, edge loading, low clearance, and psoas impingement are the likely causes for early failure of these prostheses. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

A poly (lactide-co-glycolide) (PLGA) scaffold filled with fibrin gel, mesenchymal stem cells (MSCs) and poly(ethylene oxide)-b-poly (L-lysine) (PEO-b-PLL)/pDNA-TGF-β1 complexes was fabricated and applied in vivo for synchronized regeneration of cartilage and subchondral bone. The PEO-b-PLL/pDNA-TGF-β1 complexes could transfect MSCs in vitro to produce TGF-β1 in situ and up regulate the expression of chondrogenesis-related genes in the construct. The expression of heterogeneous TGF-β1 in vivo declined along with the prolongation of implantation time, and lasted for 3 and 6 weeks in the mRNA and protein levels, respectively. The constructs (Experimental group) of PLGA/fibrin gel/MSCs/(PEO-b-PLL/pDNA-TGF-β1 complexes) were implanted into the osteochondral defects of rabbits to restore the functional cartilages, with gene-absent constructs as the Control. After 12 weeks, the Experimental group regenerated the neo-cartilage and subchondral bone with abundant deposition of glycosaminoglycans (GAGs) and type II collagen. The regenerated tissues had good integration with the host tissues too. By contrast, the defects were only partially repaired by the Control constructs. qRT-PCR results demonstrated that expression of the chondrogenesis-marker genes in the Experimental group was significantly higher than that of the Control group, and was very close to that of the normal cartilage tissue. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In this study, PEGylated nanoparticles quercetin drug delivery vehicles were investigated as carriers for anticancer drugs induced programed cell death (PCD). PEG2000-DPSE-coated quercetin nanoparticles were prepared and tumor cell killing efficacy was studied on glioma C6 cells and assayed for cell survival, apoptosis, or necrosis. The levels of ROS production and mitochondrial membrane potential (ΔΨm) were determined. Western blot assayed p53, p-p53, cytochrome C, and caspase proteins expression were also studied. Results indicate that PEG2000-DPSE-QUE-NPS showed dose-dependent cytotoxicity to C6 glioma cells and enhanced ROS accumulation induced upregulation of p53 protein, which was accompanied with an increase in cytochrome c and caspase-3 protein levels. These results support the hypothesis that quercetin nanoparticles-coated PEG2000-DPSE remarkably enhanced anticancer effect of induced programed cell death on C6 glioma cells. Overall, PEG2000-DPSE-coated quercetin nanoparticles showed promising potential as a drug carrier for cancer therapy. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The fixation of cementless endoprostheses requires excellent fixation at the bone implant interface. Although the surface structures of these implants are designed to promote osteoblastic differentiation, poor bone quality may prevent or delay osseointegration. There is evidence that RGD peptides known as recognition motifs for various integrins, promote cellular adhesion, influence cellular proliferation, and differentiation of local cells. In this study, five different metal surfaces were analyzed: Sandblasted (TiSa) and polished (TiPol) Ti6Al4V, porocoated (CCPor) and polished (CCPol) cobalt chrome and polished stainless steel (SS) were coated by ethanol amine and poly(ethylene glycol) to attach covalently RGD peptides. Human mesenchymal stromal cells of healthy donors were cultivated onto prior functionalized metal surfaces for 14 days without osteogenic stimulation. Cell proliferation and differentiation were quantitatively evaluated for native (I), NaOH pre-activated (II), NaOH pre-activated, and PEG-coated (III) as well as for RGD (IV) coated surfaces. The RGD immobilization efficiency was analyzed by epi-fluorescence spectroscopy, cell morphology was documented by light and scanning electron microscopy. The RGD-binding efficiency was TiSa > TiPol > SS > CCPor > CCPol. RGD coated surfaces showed the highest average cell proliferation on CCPol > SS > CCPor > TiSa ≥ TiPol, whereas cellular differentiation mostly correlated with the observed proliferation results, such as CCPol > TiSa > SS > CCPor > TiPol. Considering statistical analyses (significance level of α = 0.05), the RGD-coating of all biometals in comparison and in respect of their particular controls showed no significant improvement in cellular proliferation and osteoblastic differentiation. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Technological innovations in biomaterial sciences harness nanoparticle (NP) production, manipulation, and deposition with supreme precision, enabling the development of industrial processes. This review first discusses the basic components of this approach, introducing cluster sources, experimental apparatus, and growth mechanisms for NP formation. The second part of this review provides an overview of how the nanoscale bottom-up engineering can control protein adsorption, which in turn determines the fate of nanostructured coating for prokaryotic and mammalian (primary and stem) cell interactions. In addition, we briefly address the implications of the cluster beam deposition technique for nanostructuration of biocompatible microdevices and its potential as a facile coating method to promote protein–surface interactions for microarray applications in biotechnology. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

A new approach to pattern cells using photochemistry and self-assembled monolayer (SAM) was described in this study. Photocleavable 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC) protected amine on an alkanethiol-gold SAM was developed for cell patterning. The cleavage of NVOC and the deprotection of amines on the SAM were controlled spatially by two sequential UV exposures with a photomask. Biomolecule patterning was achieved by introducing cell nonadhesive poly(ethylene glycol) after the first exposure and subsequently cell adhesive protein laminin after the second exposure to create surface cell adhesiveness differential for cell patterning. UV–Vis spectrophotometry was used to determine the photolysis of caged self-assembled molecules; in addition, water contact angle, atomic force microscopy, cyclic voltammetry, and X-ray photoelectron spectroscopy were used to characterize properties of different surfaces. To test the efficacy of resulting surfaces in patterning cells, a neuron-like cell line, PC12 cell line, was used. The in vitro cell studies showed successful PC12 cell patterns on the photoactive SAM surfaces. This patterning technique is unique in that it does not rely on cell adhesive or nonadhesive properties of the starting base material as both cell adhesive and cell nonadhesive molecules were individually introduced onto the base material surface through photo-uncaging at preselected regions for the ultimate cell patterning. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Osteoblast-like cells together with a suitable scaffold can aid to the regeneration of bone defects. A suitable scaffold could be starch poly(ε-caprolactone) (SPCL) fiber meshes, which have shown a high potential to support bone formation in previous in vitro and in noncritical sized in vivo studies. The aim of this study was to assess the effect of these scaffolds alone or combined with osteoblast-like cells in the regeneration of a critical-sized cranial defect in male Fisher rats. Empty defects and defects filled with cell-free scaffolds were used as controls groups. Samples were analyzed by microcomputed tomography (micro-CT) and histological analyses. Histological analyses revealed that all study groups showed new bone formation from the defect edges toward the interior of the defects. In addition, bone was formed in the center of the scaffolds, especially in the groups containing preloaded osteoblast-like cells. Micro-CT reconstructions showed that bone formation increased over time and was enhanced with the inclusion of preloaded osteoblast-like cells compared with SPCL scaffolds alone. According to these results, the preloaded osteoblast-like cells contributed to the bone regeneration process in a critical-sized bone defect. Furthermore, SPCL fiber meshes proved to be an osteoconductive material to use for bone regeneration purposes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Improvement of oral epithelial adhesion to titanium (Ti) may significantly enhance the efficacy of dental implants. Here, we investigated whether insulin-like growth factor-1 (IGF-1) improved the sealing of the peri-implant epithelium (PIE) around the implant. Right maxillary first molars were extracted from rats and replaced with experimental implants. After 4 weeks of IGF-1 treatment, the implant-PIE interface exhibited a band of immunoreactive laminin-332 (Ln-5), similar to the tooth-junctional epithelium interface, that was partially absent in the untreated group. Immunoelectron microscopy showed a characteristic Ln-5-positive band including hemidesmosomes at both the apical and upper portions of the implant-PIE interface in the IGF-1-treated group. We also investigated the effects of IGF-1/PI3K inhibitors on the dynamics of rat oral epithelial cells (OECs) grown on Ti plates. In OECs cultured with IGF-1, adhesion protein expression increased, cell adherence to Ti plates was higher, and proliferation was faster, whereas migration and apoptosis were induced in the absence of IGF-1 or in the presence of both IGF-1 and a PI3K inhibitor. These data suggest that PI3K mediates the promotive effects of IGF-1, and that IGF-1 is effective at enhancing epithelial integration around Ti implants. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Natural hydrogels have been investigated for three-dimensional tissue reconstruction and regeneration given their ability to emulate the structural complexity of multi-component extracellular matrices (ECM). Hydrogels rich in ECM can be extracted and assembled from soft tissues, retain a composition specific to the tissue source, and stimulate vascularized tissue formation. However, poor mechanical properties and rapid degradation hinder their performance in regenerative applications. This study investigates the effect of glutaraldehyde (GA) crosslinking on the mechanical properties, biological activity, and degradation of dermis-isolated ECM-rich hydrogels. Compression tests indicated that hydrogel elastic moduli and yield stress values increased significantly with GA exposure time. Lyophilization was shown to decrease yield stress values with respect to non-lyophilized gels. Crosslinked ECM, unlike non-crosslinked gels, was resistant to pepsin degradation in vitro. In a rodent subcutaneous implant model, crosslinking for 0.5 hours or longer drastically slowed degradation relative to controls. Inflammation was low and mature vascularized granulation tissue was observed in all gels, with an increase in vessel density at 1 week in crosslinked gels relative to controls. These results support the potential use of dermis-derived hydrogels as materials for tissue engineering applications and suggest that crosslinking can enhance mechanical properties and prolong hydrogel lifetime while promoting vascularized tissue formation. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Mechanical mismatch, along with inadequate hemocompatibility and endothelialization, contribute to the high failure rate of many synthetic vascular grafts. However, due to the dueling nature of these requirements (i.e., inhibiting platelet adhesion frequently means inhibiting endothelial cell (EC) adhesion), the creation of materials that simultaneously satisfy the mechanical and biological design criteria needed for small diameter vascular grafts has been an elusive goal. In this work, we demonstrate the ability of polyurethane (PU) containing hyaluronic acid (HA) in its backbone structure to reduce protein adsorption, platelet and bacterial adhesion, and fibroblast and macrophage proliferation while allowing the retention of both ECs and vascular-appropriate mechanics. Irrespective of HA molecular weight (MW), PU-HA materials selectively supported the growth of ECs relative to fibroblasts, reduced platelet adhesion, and performed comparably to negative controls with respect to bactericidal activity. The extent of EC growth on the PU-HA materials did differ with HA MW, with a lower HA MW yielding improved EC growth in both two-dimensional (2-D) films and 3-D electrospun fibrous scaffolds. These findings illustrate that HA incorporated into the backbone of a synthetic polymer structure can retain bioactivity, with subtle differences in HA MW significantly impacting the physical and biological properties of the biomaterial; in particular, PU modified with low-MW HA appears promising for vascular graft applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

In this article, porous poly(D,L-lactide-co-glycolide) (PLGA) microsphere scaffolds with a size of ∼ 400 μm and pores of ∼ 20 μm were prepared for constructing injectable three-dimensional hepatocyte spheroids. The porous sites of PLGA microspheres provided a spatial space for hepatocyte distribution. Hepatocytes spheroids were cocultured with human umbilical vein endothelial cell, bone marrow mesenchymal stem cell, or NIH/3T3 cells by combining the porous PLGA microspheres with the relatively hydrophobic culture strategy. The combination of open porous microspheres, hepatocytes, and nonparenchymal cells was demonstrated for application in functional hepatic tissue reconstruction. Hepatocellular-specific functions can sustained up to 2 weeks in the support of coculturing with nonparenchymal cells. The spheroidal hepatocyte coculture system had the advantages of an injectable delivery, higher cell seeding density, protection from exerted shear stress, better exchange of nutrients, oxygen and metabolites, and heterotypic cell–cell contact within and between microspheres. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bioceramic processing using rapid prototyping technique (RPT) results in a fragile device that requires thermal treatment to improve the mechanical properties. This investigation evaluates the effect of thermal treatment on the mechanical, porosity, and bioactivity properties as well as the cytotoxicity of a porous silica-calcium phosphate nanocomposite (SCPC) implant prepared by RPT. Porous SCPC implant was subject to 3-h treatment at 800°C, 850°C, or 900°C. The compressive strength (s) and modulus of elasticity (E) were doubled when the sintering temperature is raised from 850 to 900°C measuring (s = 15.326 ± 2.95 MPa and E = 1095 ± 164 MPa) after the later treatment. The significant increase in mechanical properties takes place with minimal changes in the surface area and the percentage of pores in the range 1–356 μm. The SCPC implant prepared at 900°C was loaded with rh-BMP-2 and grafted into a segmental defect in the rabbit ulna. Histology analyses showed highly vascularized bone formation inside the defect. Histopathological analyses of the liver, spleen, kidney, heart, and the lung of rabbits grafted with and without SCPC demonstrated healthy tissues with no signs of toxicity or morphology alterations. Results of the study suggest that it is possible to engineering the mechanical properties of the SCPC implant without compromising its bioactivity. The enhanced bone formation inside the porous SCPC facilitated cell-mediated graft resorption and prohibited any accumulation of the material in the body organs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Poly-L-lysine-modified iron oxide nanoparticle (IONP-PLL), which is formed by modifying poly-L-lysine to the surface of iron oxide nanoparticles, can deliver exogenous genes to cells in vitro and in vivo. However, there is relatively little information available about how is IONP-PLL uptaken by cells. In this study, we are focusing on the transferrin receptor (TFR) mediated and TFR-independent cellular internalization of IONP-PLL. The cells were incubated with 1 µM of IONP-PLL with or without transferrin bound. Transferrin–TFR pathway blockers, such as NH₄Cl, CH₃NH₂, or trypsin, were added to the media and their effects were observed. Atomic absorption spectrophotometer was used to quantify the cellular concentration of iron. The cellular concentrations of iron were evaluated at 37°C or 4°C. (1) Transferrin–IONP-PLL uptake into cells was reliant on time and temperature. (2) The addition of blockers, either NH₄CL, CH₃NH₂, or trypsin, decreased the cellular transferrin-dependent IONP-PLL uptake, but not completely blocked the entry of IONP-PLL. (3) When the cells were culture at pH 6.5, under conditions which the binding of iron and transferrin were inhibited, IONP-PLL still had the capacity to enter into cells with time and temperature-dependent manner. These results suggest that the cellular internalization of IONP-PLL, much like iron ion, were mediated by TFR-dependent endocytosis and TFR-free uptake. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

We evaluated the advantages of three-dimensional (3D) culture in a collagen hydrogel for stem cell differentiation, including the morphology of differentiated cells, differentiation efficiency of stem cells from aged rat and cells after passaging and freeze/thawing. Rat mesenchymal stem cells (MSCs) from young and aged rats, and MSCs after passaging and freeze/thawing were induced to differentiate into osteoblasts in 3D and 2D cultures, and histological studies were performed. Differentiation efficiency was evaluated by markers of osteoblastic differentiation including Runx2 and osterix gene expressions, osteocalcin secretion and calcium deposition. MSCs were stained positive for alkaline phosphatase in 3D and 2D cultures. However, the morphology of differentiated cells in 3D culture, which was different from that in 2D culture, was similar to that of osteoblasts in vivo. Markers of osteoblastic differentiation in MSCs from aged rats in 3D culture were higher than those in MSCs from young rats in 2D culture. Markers of osteoblastic differentiation in MSCs after passaging and freeze/thawing in 3D culture were higher than those in nonpassaged MSCs in 2D culture. These results indicate that 3D culture in a collagen hydrogel has advantages for the differentiation of MSCs into osteoblasts with a similar phenotype to that of in vivo, when using even MSCs from aged donors or after passaging and freeze/thawing. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

This study investigates the roles of orthopedic biomaterial particles [Ti-alloy, poly(methyl methacrylate) (PMMA), ultrahigh-molecular-weight polyethylene (UHMWPE), Co–Cr alloy] on the differentiation and functions of bone marrow stromal cells (BMSCs). Cells were isolated from femurs of BALB/c mice and cultured in complete osteoblast-induction medium in presence of micron-sized biomaterial particles at various doses. 3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase assay were performed for cell proliferation and cytotoxicity. Differentiation and function of osteoblasts were evaluated by alkaline phosphatase (ALP), osteocalcin, RANKL, OSX, and Runx2 expressions. Murine interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α in culture media were determined by enzyme-linked immunosorbent assay. Challenge with low doses of Ti, UHMWPE, or Co–Cr particles markedly promoted the bone marrow cell proliferation while high dose of Co–Cr significantly inhibited cell growth (p < 0.05). Cells challenged with low dose of PMMA or UHMWPE particles (0.63 mg/mL) exhibited strong ALP activity, whereas Ti and Co–Cr groups showed minimal effects (p < 0.05). UHMWPE and Ti particles also promoted higher expression of proinflammatory cytokines. Real-time polymerase chain reaction data suggested that cells treated with low dose (0.5 mg/mL) particles resulted in distinctly diminished RANKL expression compared to those exposed to high concentrated (3 mg/mL) particles. In conclusion, various types of wear debris particles behaved differently in the differentiation, maturation, and functions of osteogenic cells; and the particulate debris-interacted BMSCs may play an important role in the pathogenesis and process of the debris-associated aseptic prosthetic loosening. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The aim of this study was to investigate the ability of nano-sized collagen I molecules (nanoparticles or nanofibrils) and a 5-azacytidine (5-aza) treatment to enhance the differentiation of rat mesenchymal stem cells (MSCs) toward a cardiomyogenic phenotype in vitro. Second passaged MSCs were cocultured with nano-sized collagen I molecules for 24 h and then treated with 10 μM 5-aza for 24 h. The results demonstrated that the size of the cells increased significantly and acquired a flattened, triangular-shaped morphology after treatment with nano-sized collagen I molecules and 5-aza. The cells are connecting with adjoining cells by forming myotube-like structures. Additional treatment of the MSCs with nano-sized collagen I fibrils significantly increased two transcription factors GATA-4 (12.6-fold increase) and Nkx2.5 (4.8-fold increase) expressions compared with MSC groups treated only with 5-aza at 3-day culturing. Furthermore, MSCs pretreated with nano-sized collagen fibrils significantly increased the expressions of cardiac genes of troponin I, β-myosin heavy chain, and cardiac α-actin compared with MSC groups treated only with 5-aza (all, p < 0.01 or better). These results indicate that culturing MSCs with nano-sized collagen I molecules, which may act as scaffolds or soluble protein ingredients, leads to alterations in gene expression and affects the differentiation fate induced with 5-aza. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Controlling topographic features at all length scales is of great importance for the interaction of cells with tissue regenerative materials. We utilized an indirect three-dimensional printing method to fabricate polymeric scaffolds with pre-defined and controlled external and internal architecture that had an interconnected structure with macro- (400–500 μm) and micro- (∼25 μm) porosity. Polycaprolactone (PCL) was used as model system to study the kinetics of tissue growth within porous scaffolds. The surface of the scaffolds was decorated with TiO₂ and bioactive glass (BG) nanoparticles to the better match to nanoarchitecture of extracellular matrix (ECM). Micrometric BG particles were also used to reveal the effect of particle size on the cell behavior. Observation of tissue growth and enzyme activity on two-dimensional (2D) films and three-dimensional (3D) scaffolds showed effects of nanoparticle inclusion and of surface curvature on the cellular adhesion, proliferation, and kinetics of preosteoblastic cells (MC3T3-E1) tissue growth into the pore channels. It was found that the presence of nanoparticles in the substrate impaired cellular adhesion and proliferation in 3D structures. Evaluation of alkaline phosphate activity showed that the presence of the hard particles affects differentiation of the cells on 2D films. Notwithstanding, the effect of particles on cell differentiation was not as strong as that seen by the curvature of the substrate. We observed different effects of nanofeatures on 2D structures with those of 3D scaffolds, which influence the cell proliferation and differentiation for non-load-bearing applications in bone regenerative medicine. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 α3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 α3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 α3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 α3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The inflammatory response to titanium and hydroxyapatite (HA)-coated titanium in living tissue is controlled by a number of humoral factors, of which monocyte chemoattractant protein-1 (MCP-1) has been specifically linked to the recruitment of monocytes. These cells subsequently mature into tissue-bound macrophages. Macrophages adhering to the proteins adsorbed at the implant surface play a pivotal role in initiating the rejection or integration of the foreign material. Despite this, little is known about the initial inflammatory events that occur in soft tissues following the implantation of titanium and HA-coated titanium implants. In this study, circular discs of commercially pure titanium (c.p. Ti) with either a thin crystalline HA coating or amorphous HA coating or uncoated were implanted subcutaneously into rats. The implants were retrieved after 24 and 72 h. The lactate dehydrogenase (LD) activity, DNA content, expression of MCP-1, interleukin-10 (IL-10), tumor necrosis factor α (TNF-α), as well as monocyte and polymorphonuclear granulocyte counts in the exudate surrounding the implants were analyzed. There were significantly higher DNA and LD levels around the titanium implants at 24 h compared with HA-coated titanium. A rapid decrease in MCP-1 levels was observed for all the implants over the period of observation. No statistically significant differences were found between the two HA-coated implants. Our results suggest a difference in the early soft-tissue response to HA-coated implants when compared with titanium implants, expressed as a downregulation of inflammatory cell recruitment. This suggests that thin HA coatings are promising surfaces for soft tissue applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Titanium and titanium alloy implants that have been demonstrated to be more biocompatible than other metallic implant materials, such as Co–Cr alloys and stainless steels, must also be accepted by bone cells, bonding with and growing on them to prevent loosening. Highly ordered nanoporous arrays of titanium dioxide that form on titanium surface by anodic oxidation are receiving increasing research interest due to their effectiveness in promoting osseointegration. The response of bone cells to implant materials depends on the topography, physicochemistry, mechanics, and electronics of the implant surface and this influences cell behavior, such as adhesion, proliferation, shape, migration, survival, and differentiation; for example the existing anions on the surface of a titanium implant make it negative and this affects the interaction with negative fibronectin (FN). Although optimal nanosize of reproducible titania nanotubes has not been reported due to different protocols used in studies, cell response was more sensitive to titania nanotubes with nanometer diameter and interspace. By annealing, amorphous TiO₂ nanotubes change to a crystalline form and become more hydrophilic, resulting in an encouraging effect on cell behavior. The crystalline size and thickness of the bone-like apatite that forms on the titania nanotubes after implantation are also affected by the diameter and shape. This review describes how changes in nanotube morphologies, such as the tube diameter, the thickness of the nanotube layer, and the crystalline structure, influence the response of cells. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.