Porous nanocomposites based on poly(ε-caprolactone fumarate) (PCLF) resin matrix; N-vinyl pyrrolidone (NVP) as a reactive diluents and nanohydroxyapatite (nHA) filler were developed for bone tissue engineering applications. Nanocomposite scaffolds with three different contents of nHA [5, 10, and 20 (w/w %)] were prepared by thermal crosslinking of PCLF followed by particulate leaching and characterized in terms of mechanical properties (cyclic loading) and in vitro cell-material interaction by MTT assay and alkaline phosphatase activity measurements. Five osteoblastic cell lines were used to investigate the ability of the nanocomposites to support cell attachment, spreading, and proliferation after 3, 7, and 14 days. By adding the nHA filler phase, elastic modulus of the nanocomposites increased significantly. Scaffolds showed comparable biocompatibility to neat nHA particles, commercial bone graft (Bio-Oss) and tissue culture polystyrene as control groups. According to the results it can be concluded that these scaffolds are potential candidates for bone substitution because of their mechanical strength and bioactivity. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Studies of the fracture behavior of cortical bone have determined multiple toughening mechanisms that are active during propagation of a crack. Common methods for measuring bone fracture toughness use single-notched specimens often in four-point (SN4PB) or three-point bending (SN3PB). A double-notch four-point bending (DN4PB) specimen is useful to study prefailure damage at the crack tip. Total failure occurs at one notch and only partial failure at the other allowing study of prefailure damage in the unbroken notch. There is no widely known method for calculating the fracture toughness of bone using a DN4PB specimen. A method for calculating the fracture toughness of cortical bone using a DN4PB is developed here and compared with results for a common SN3PB specimen. The new double-notch method permits using a single specimen to measure apparent fracture toughness and to study both pre- and postfailure microdamage in the bone matrix. When and how to use the new and the established test specimens for understanding bone mechanics is discussed. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Collagen, a major component of native extracellular matrix, has diverse biomedical applications. However, its application is limited due to lack of cost-effective production and risk of disease transmission from bovine sources currently utilized. This study describes fabrication and characterization of nano/micro fibrous scaffolds utilizing collagen extracted from fresh water fish origin. This is the first time collagen extracted from fresh water fish origin was studied for their biocompatibility and immunogenicity. The nano/micro fibrous collagen scaffolds were fabricated through self-assembly owing to its amphiphilic nature and were subsequently cross-linked. In vitro degradation study revealed higher stability of the cross-linked scaffolds with only ∼50% reduction of mass in 30 days, while the uncross-linked one degraded completely in 4 days. Further, minimal inflammatory response was observed when collagen solution was injected in mice with or without adjuvant, without significant dilution of sera. The fish collagen scaffolds exhibited considerable cell viability and were comparable with that of bovine collagen. SEM and fluorescence microscopic analysis revealed significant proliferation rate of cells on the scaffolds and within 5 days the cells were fully confluent. These findings indicated that fish collagen scaffolds derived from fresh water origin were highly biocompatible in nature. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl₂. After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Hydroxyapatite (HA), has been used commonly as a bone substitute and as a scaffold in bone tissue engineering. However it has certain drawbacks such as limited biodegradability and osteointegration properties. Other forms of HA, for example, carbonated hydroxyapatite (CHA) could prove to have enhanced bioactivity as they more closely mimic the chemical composition of the apatite found in human bone. The aim of this study was to test the efficacy of CHA in comparison to HA used as a control. The CHA (4.9 wt %) and the HA discs were seeded with MC3T3-E1 osteoblastic cells. Results revealed a trend of increased cell attachment on the HA discs at day 0, however, the cell proliferation on the CHA discs at 7 and 28 days showed no significant difference in comparison to the HA control. SEM of the CHA discs showed surface irregularities at 7 days indicating dissolution. Also at 7 days, SEM demonstrated cell attachment and extracellular matrix production on both the CHA and HA samples. There was no significant difference in the total amount of collagen produced in the CHA samples relative to the HA control samples at 28 days as evaluated by the hydroxyproline assay. Real time PCR revealed mRNA increase by 2.08, 7.62, and 9.86 fold for collagen I a1, collagen III a1, and osteocalcin respectively on the CHA as compared to the HA discs. This study demonstrates the use of CHA as a biocompatible material that has potentially increased biodegradation properties and osteogenic capability in comparison to HA. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

This study reports the use of a targeted cationic peptide with the ability to disrupt Staphylococcus epidermidis biofilm formation. Complications due to nosocomial infections of implanted medical devices pose a significant health risk to patients, with Staphylococcus epidermidis often implicated in the case of blood-contacting biomaterials. S. epidermidis virulence relies mainly on its ability to form a biofilm, the main component of which is polysaccharide intercellular adhesin (PIA). We utilized the synthetic β6-20 peptide, known to specifically bind S. epidermidis, in order to deliver a cationic polylysine peptide (G₃K₆) to the bacterial surface and disrupt the charge–charge interactions needed for PIA retention and biofilm stability. The effects of the β6-20-G₃K₆ peptide on biofilm formation were assessed using optical density, fluorescently labeled wheat germ agglutinin, nucleic acid stain (SYTO 9), and a metabolic assay (XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt). Biofilms formed in the presence of β6-20-G₃K₆ peptide (100 μM) resulted in a 37.9% reduction in PIA content and a 17.5% reduction of adherent bacteria relative to biofilms grown in the absence of peptide. These studies demonstrate the targeting ability of the β6-20 peptide towards biomaterial-adherent S.epidermidis, and highlight the potential for disrupting the early stages of biofilm formation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Microencapsulation has been a promising approach for drug delivery, cell implantation, cell-based gene therapy and large-scale cell culture. To make use of microcapsules more effectively, it is important to accurately construct the microcapsule membranes with desired properties including a certain thickness, strength, and so forth. To date single factor experiments have been widely used, however, they are time-consuming to obtain the desired membrane preparation conditions. Response surface methodology (RSM) is a mathematical and statistical technique for building empirical models that gained importance for optimizing reacting conditions. In this study, three signifficant effect factors that affect alginate-based microcapsule membrane properties, including membrane thickness, swelling degree, and mechanical stability, were determined with Plackett–Burman method, and then three empirical models were built to optimize the preparation conditions of the microcapsule membranes according to the responses of these three signifficant effect factors respectively with RSM. These models can be used to predict the characteristics of microcapsules under different membrane preparation conditions, which provide a guide for optimizing the microencapsulation technology. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

A novel biodegradable Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy was successfully produced using a series of metallurgical processes; including melting, casting, rolling, and heat treatment. The hardness and ultimate tensile strength of the alloy sheets increased to 71.2HV and 320 MPa after rolling and then aging for 12 h at 175°C. These mechanical properties were sufficient for load-bearing orthopedic implants. A hydroxyapatite (HA) coating was deposited on the Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy using a novel coating process combining alkali heat pretreatment, electrodeposition, and alkali heat posttreatment. The microstructure, composition, and phases of the Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy and HA coating were characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD). The degradation, hemolysis, and cytocompatibility of the HA-coated and uncoated Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy were studied in vitro. The corrosion potential (E_corr) of Mg-4.0Zn-1.0Ca-0.6Zr alloy (−1.72 V) was higher than Mg (−1.95 V), Mg-0.6Ca alloy (−1.91 V) and Mg-1.0Ca alloy (−1.97 V), indicating the Mg-Zn-Ca-Zr alloy would be more corrosion resistant. The initial corrosion potential of the HA-coated Mg alloy sample (−1.51 V) was higher than the uncoated sample (−1.72 V). The hemolysis rates of the HA-coated and uncoated Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy samples were both <5%, which met the requirements for implant materials. The HA-coated and uncoated Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy samples demonstrated the same cytotoxicity score as the negative control. The HA-coated samples showed a slightly greater relative growth rate (RGR%) of fibroblasts than the uncoated samples. Both the HA-coated and uncoated Mg-4.0Zn-1.0Ca-0.6Zr (wt %) alloy provided evidence of acceptable cytocompatibility for medical applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Cell behavior depends strongly on the physical and chemical properties of the material surface, for example, its chemistry and topography. The authors have therefore assessed the influence of materials of different chemical composition (i.e., glass substrates with and without TiO₂ films in anatase form) and different surface roughness (R_a = 0, 40, 100, or 170 nm) on the adhesion, proliferation, and osteogenic differentiation of human osteoblast-like MG63 cells. On day 1 after seeding, the largest cell spreading area was found on flat TiO₂ films (R_a = 0 nm). On TiO₂ films with R_a = 170 nm, the cell spreading area was larger and the number of initially adhering cells was higher than the values on the corresponding uncoated glass. On day 3 after seeding, the cell number was higher on the TiO₂ films (R_a = 0 and 40 nm) than on the corresponding glass substrates and the standard polystyrene dishes. On day 7, all TiO₂ films contained higher cell numbers than the corresponding glass substrates, and the cells on the TiO₂ films with R_a = 40 and 100 nm also contained a higher concentration of β-actin. These results indicate that TiO₂ coating had a positive influence on the adhesion and subsequent proliferation of MG63 cells. In addition, on all investigated materials, the cell population density achieved on day 7 decreased with increasing surface roughness. The concentration of osteocalcin, measured per mg of protein, was significantly lower in the cells on rougher TiO₂ films (R_a = 100 and 170 nm) than in the cells on the polystyrene dishes. Thus, it can be concluded that the adhesion, growth, and phenotypic maturation of MG63 cells were controlled by the interplay between the material chemistry and surface topography, and were usually better on smoother and TiO₂-coated surfaces than on rougher and uncoated glass substrates. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Silver nanoparticles (AgNPs) have found a variety of uses including biomedical materials; however, studies of the cytotoxicity of AgNPs by size effects are only in the beginning stage. In this study, we examined the size-dependent cellular toxicity of AgNPs using three different characteristic sizes (∼ 10, 50, and 100 nm) against several cell lines including MC3T3-E1 and PC12. The cytotoxic effect determined based on the cell viability, intracellular reactive oxygen species generation, lactate dehydrogenase release, ultrastructural changes in cell morphology, and upregulation of stress-related genes (ho-1 and MMP-3) was fairly size- and dose-dependent. In particular, AgNPs stimulated apoptosis in the MC3T3-E1 cells, but induced necrotic cell death in the PC12 cells. Furthermore, the smallest sized AgNPs (10 nm size) had a greater ability to induce apoptosis in the MC3T3-E1 cells than the other sized AgNPs (50 and 100 nm). These data suggest that the AgNPs-induced cytotoxic effects against tissue cells are particle size-dependent, and thus, the particle size needs careful consideration in the design of the nanoparticles for biomedical uses. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

The conformational changes of fibronectin (FN) deposited on various block copolymers where one block is composed of poly(methyl methacrylate) (PMMA) and the other block is either poly(acrylic acid) (PAA) or poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated using a functionalized atomic force microscope (AFM) tip. The tip was modified with an antibody sensitive to the exposure of the arginine–glycine–aspartic acid (RGD) groups in FN. By studying the adhesive interactions between the antibody and the proteins adsorbed on the block copolymer surface and phase imaging, it was found that the triblock copolymers PAA-b-PMMA-b-PAA and PMMA-b-PHEMA-b-PMMA, which both have large domain sizes, are conducive to the exposure of the FN RGD groups on the surface. On the basis of these results, it is concluded that the surface chemistry as well as the nanomorphology dictated by the block copolymer arrangement could both tune protein conformation and orientation and optimize cell adhesion to the biomaterial surface. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

The purpose of this study was to evaluate the effects of composite wound dressing films made of silk fibroin (SF) containing hydroxyapatite (HA) or polarized HA (pHA) powders on endothelial cell (EC) behaviors that have important roles in the wound-healing process. XRD revealed the SF films to be semicrystalline, with a broad peak centered at about 20.7° which is characteristic of β-sheets embedded within an amorphous matrix. The SF composite films with 0.6 (w/v)% in concentration of HA powder (HA/SF) or pHA powder (pHA/SF) contained HA crystals of amorphous and silk II crystalline structures. SEM observation showed that there were differences in SF morphology between HA/SF and pHA/SF. The pHA/SF exhibited a furry texture around the pHA crystals, most likely due to the stored charged and zeta potentials. The HA/SF and pHA/SF films enhanced EC migration compared with that on the SF film. The number of migrated cells on the HA/SF and pHA/SF was ∼1.5 times larger than that on the SF. The quantitative analysis of the endothelial morphogenesis indicated that the pHA/SF film enhanced the formation of capillary-like structures compared with SF and HA/SF. Thus, pHA/SF may potentially stimulate and contribute to the enhancement of angiogenesis in the wound-healing process. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

In this article, we firstly synthesized oil-soluble PbS quantum dots (QDs) emitting in the near-infrared (NIR) spectral range through a two-phase method, which exhibit a conveniently tunable photoluminescence (PL) emission (from ∼750 to 872 nm) with a narrow PL bandwidth, as well as a high (up to 40%) PL quantum yield (QY). Next, the as-prepared oil-soluble NIR PbS QDs were applied to the in vivo imaging of tumors by entrapping in biodegradable micelles (N-succinyl-N′-octyl nanomicelles, SOC) which had hydrophobic inner cores. Transmission electron microscope results show well dispersed spherical shaped QDs-loaded SOC micelles with 100 nm diameter. The QY of PbS QDs entrapped into SOC has no significant change compared to free QDs. Besides, both in vitro and in vivo acute toxicity results demonstrated that the QDs-loaded micelles have low cytotoxicity. Furthermore, in vivo imaging of PbS QDs-loaded SOC injected intravenously into tumor-bearing nude mice showed the NIR QDs-loaded micelles can accumulate in the tumor tissue due to the enhanced permeability and retention effect of SOC micelles. These results confirm that the as-prepared PbS QDs could be used as fluorescence probes to study the biodistribution of nanocarriers and their intracellular pathways, as well as their passive targeted behavior to tumors in preclinical research. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Cellulose perforated by micro-channels (∅ ∼500 μm) has been investigated as a potential future scaffold material for meniscus implants. Scaffolds seeded with 3T6 fibroblasts were cultivated with mechanical stimulation in a compression bioreactor for enhanced collagen production. Constructs under dynamic compression at a frequency of 0.1 Hz and compression strain of 5% were compared to static cultures used as controls. The three-dimensional distributions of collagen fibers and fibroblasts in the cellulose scaffolds were studied under native, soft-matter conditions by combined second harmonic generation and coherent antiStokes Raman scattering microscopy, requiring no artificial sample preparation. Results showed that the micro-channels facilitated the alignment of cells and collagen fibers and that collagen production was enhanced by mechanical stimulation. Thus, cell-seeded, micro-channeled cellulose scaffolds provided guided tissue growth required to obtain an ultrastructure mimicking that of the meniscus. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Chitosan/tripolyphosphate/chondroitin sulfate (Chi/TPP/CS) nanoparticles were prepared by an ionic gelation method to obtain a controlled release of proteins. Using Nel-like molecule-1 (Nell-1), a novel osteogenic protein, as a model protein, it was demonstrated that adjusting the composition of the particles modulated the protein association and release kinetics of incorporated proteins. Increasing the amounts of Chi crosslinking agents, TPP and CS, in the particles achieved sustained protein release. An increase in crosslinking density decreased degradation rates of the particles. Furthermore, the bioactivity of the protein was preserved during the encapsulating procedure into the particles. To demonstrate the feasibility of Chi/TPP/CS nanoparticles as sustained release carriers for tissue engineering scaffold applications, protein-loaded nanoparticles were successfully incorporated into collagen hydrogels or prefabricated porous poly(lactide-co-glycolide) (PLGA) scaffolds without obstructing the integrity of the hydrogels or porous structure of the scaffolds. Thus, we expect that these particles have a potential for efficient protein carriers in tissue engineering applications, and will be further evaluated in vivo. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Mussel inspired proteins have been demonstrated to serve as a versatile biologic adhesive with numerous applications. The present study illustrates the use of such Mussel inspired proteins (polydopamine) in the fabrication of functionalized bio-inspired nanomaterials capable of both improving cell response and sustained delivery of model probes. X-ray photoelectron spectroscopy analysis confirmed the ability of dopamine to polymerize on the surface of plasma-treated, electrospun poly(ε-caprolactone) (PCL) fiber mats to form polydopamine coating. Transmission electron microscopy images demonstrated that self-polymerization of dopamine was induced by pH shift and that the thickness of polydopamine coating was readily modulated by adjusting the concentration of dopamine and reaction time. Polydopamine coatings were noted to affect the mechanical properties of underlying fiber mats, as mechanical testing demonstrated a decrease in elasticity and increase in stiffness of polydopamine-coated fiber mats. Polydopamine coatings were also utilized to effectively immobilize extracellular matrix proteins (i.e., fibronectin) on the surface of polydopamine-coated, electrospun fibers, resulting in enhancement of NIH3T3 cell attachment, spreading, and cytoskeletal development. Comparison of release rates of rhodamine 6G encapsulated in coated and uncoated PCL fibers also confirmed that polydopamine coatings modulate the release rate of loaded payloads. The authors further demonstrate the significant difference of rhodamine 6G adsorption kinetics in water between PCL fibers and polydopamine-coated PCL fibers. Taken together, polydopamine-mediated surface modification to electrospun fibers may be an effective means of fabricating a wide range of bio-inspired nanomaterials with unique properties for use in tissue engineering, drug delivery, and advanced biomedical applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Revision surgery for particle-induced implant loosening in total joint replacement is expected to increase dramatically over the next few decades. This study was designed to investigate if local tissue and serum markers of bone remodeling reflect implant fixation following administration of lipopolysaccharide (LPS)-doped polyethylene (PE) particles in a rat model. Twenty-four rats received bilateral implantation of intramedullary titanium rods in the distal femur, followed by weekly bilateral intra-articular injection of either LPS-doped PE particles (n = 12) or vehicle that contained no particles (n = 12) for 12 weeks. The group in which the particles were injected had increased serum C-terminal telopeptide of type I collagen (CTX-I), decreased serum osteocalcin (OC), increased peri-implant eroded surface, decreased peri-implant bone volume, and decreased mechanical pull-out strength compared to the controls. Implant fixation strength was positively correlated with peri-implant bone volume and serum OC and inversely correlated with serum CTX-I, while energy to yield was positively correlated with serum OC and inversely correlated with the number of tartrate-resistant acid phosphatase positive cells at the interface and the amount of peri-implant eroded surface. There was no effect on trabecular bone volume at a remote site. Thus, the particle-induced impaired fixation in this rat model was directly associated with local and serum markers of elevated bone resorption and depressed bone formation, supporting the rationale of exploring both anticatabolic and anabolic strategies to treat and prevent particle-related implant osteolysis and loosening, and indicating that serum markers may prove useful in tracking implant fixation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Osseointegrated implants (OI)s for transfemoral prosthetic attachment offer amputees an alternative to the traditional socket attachment. Potential benefits include a natural transfer of loads directly to the skeleton via the percutaneous abutment, relief of pain and discomfort of residual limb soft tissues by eliminating sockets, increased sensory feedback, and improved function. Despite the benefits, the skin-implant interface remains a critical limitation, as it is highly prone to bacterial infection. One approach to improve clinical outcomes is to minimize stress concentrations at the skin-implant interface due to shear loading, reducing soft tissue breakdown and subsequent risk of infection. We hypothesized that broadening the bone base at the distal end of the femur would provide added surface area for skin adhesion and reduce stresses at the skin-implant interface. We tested this hypothesis using finite element models of an OI in a residual limb. Results showed a dramatic decrease in stress reduction, with up to ∼90% decrease in stresses at the skin-implant interface as cortical bone thickness increased from 2 to 8 mm. The findings in this study suggests that surgical techniques could stabilize the skin-implant interface, thus enhancing a skin-to-bone seal around the percutaneous device and minimizing infection. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Poly(ε-caprolactone) (PCL) is a promising material for tissue engineering applications; however, it can be difficult to create scaffolds with the morphology, hydrophilicity, and mechanical properties necessary to support tissue growth. Typically, pure PCL scaffolds have good cellular adhesion, but somewhat low mechanical properties (elastic modulus and tensile strength). This study addresses these issues by incorporating Al₂O₃ whiskers as reinforcements within PCL membranes generated by electrospinning. Membranes were prepared with Al₂O₃ content ranging from 1 to 20 wt % and characterized using XRD, TEM, and SEM to determine composition and morphology. The Al₂O₃ whiskers were well dispersed within the PCL fibers, and the membranes had a highly porous morphology. The elastic modulus was significantly improved by the well aligned whisker reinforcements as verified by tensile testing. The cell morphology and proliferation studies demonstrate Al₂O₃ whisker reinforced PCL scaffolds maintained the good biocompatibility. These improvements demonstrate that Al₂O₃ whisker reinforced PCL scaffolds can be considered as a biocompatible material for tissue engineering and dental applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

To understand the chronic spleen injury induced by intragastric administrations with 2.5, 5, and 10 mg kg⁻¹ body weight titanium dioxide nanoparticles (TiO₂ NPs) for 90 consecutive days, histopathological and ultrastructure changes, hematological parameters, lymphocyte subsets, the inflammatory, and apoptotic cytokines in the mouse spleen were investigated. Our findings indicate that TiO₂ NPs exposure results in the significant increase in the spleen indices, histopathological changes, and splenocyte apoptosis in spleen. Especially, in these TiO₂ NPs-treated mice, immunoglobulin, blood cells, platelets, hemoglobin, lymphocyte subsets (such as CD3, CD4, CD8, B cell, natural killer cell) of mice were significantly decreased. Furthermore, TiO₂ NPs exposure can significantly increase the levels of nucleic factor-κB, tumor necrosis factor-α, macrophage migration inhibitory factor, interleukin-2, interleukin-4, interleukin-6, interleukin-8, interleukin-10, interleukin-18, interleukin-1β, cross-reaction protein, transforming growth factor-β, interferon-γ, Bax, and CYP1A1 expression, whereas decrease the levels of Bcl-2 and heat shock protein 70 expression. These findings suggest that long-term exposure to low dose TiO₂ NPs may result in spleen injury and reduction of immune capacity, TiO₂ NP-induced injury in spleen may result from alteration of inflammatory and apoptotic cytokines expression, and workers and consumers should take great caution when handling nanomaterials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

The integration and long-term functional retention of tissue implants are both strongly linked to the implant material characteristics. As a first approach, the cytocompatibility and bioactivity of such materials are evaluated using in vitro-based cell culture models. Typically, in vitro bioactivity is assessed by seeding single cells onto the test material to evaluate certain parameters such as cell adhesion, survival, proliferation, and functional differentiation. Probably, due to the reduction from three dimensional (3D) toward the two dimensional (2D) situation the data obtained from 2D culture models falls short of predicting the in vivo behavior of the biomaterial in question. In this study, a three dimensional (3D) in vitro cell culture model was applied to evaluate the bioactivity of well characterized fiber-based scaffolds using scaffold colonization as a bioactivity indicator. Cell behavior in this culture model was evaluated against a classical comparable, 2D cell culture system using polyethylene terephthalat and polyamide 6.6 fabrics. By using the 3D culture model, however, differences in cell population performance as a function of fiber diameter and mesh angle were evident. The use of 3D cell culture model clearly outperformed typical cell culture setup as means to evaluate cell population–scaffold interaction. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

The application of porous hollow fibers has recently been extended to the controlled release of biologics such as protein growth factors and lipid angiogenesis promoters. Release of these materials tends to occur more rapidly than would be predicted by conventional diffusion-based models of controlled release. Analysis of other modalities of transport as well as structural analysis of the controlled release system itself was performed to provide insight into the observed controlled release behavior from such systems. Specifically, it was discovered that osmotic-driven processes play a significant role in controlled release of proteins including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). It was also found that the fiber pore microstructure and (more importantly) macrostructure influences release behavior. Model-guided design was implemented to adjust the physical properties of the fiber wall, leading to a release system that is better able to sustain the delivery of VEGF. This model may be used to more easily achieve a desired complex release behavior when used in combination with external regulation of the reservoir. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

We synthesized poly(amidoamine)s with pendant primary amines and flexible backbone (polymers 1–3) by Michael polyaddition of N-tert-butyloxycarbonyl (N-Boc) protected diamine to 1,6-Bis(acrylamido)hexane, followed by the deprotection of N-Boc under acidic conditions. The physicochemical properties of polymers 1–3, including buffer capacity, DNA-binding capacity, cytotoxicity, particle sizes, and zeta potentials of polycation/DNA complexes, were explored. All the three polymers possess high buffer capacity and excellent DNA-binding capacity. In vitro MTT assay revealed that these synthesized poly(amidoamine)s were less cytotoxic than commercial branched PEI (25 kDa). These poly(amidoamine)s with pendant primary amines and flexible backbone were evaluated as in vitro nonviral gene delivery vectors for 293T and COS-7 cells. All the three polymers exhibited high transfection efficiencies, which were even higher than branched PEI (25 kDa) at optimized conditions. Further evidences from confocal laser scanning microscope (CLSM) demonstrated that the high transfection efficiencies of polymers 1–3 were due to the efficient uptake and intracellular trafficking of plasmid DNA in the cells during the transfection. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Tunable mesoporosity and nanoporosity of stimuli pH-responsive (N-vinyl imidazole-ran-acryloylmorpholine) hydrogels studied in terms of %swelling at various ionic strength, pH, temperature, and crosslinker concentration values were investigated. Hydrogel properties including diffusional exponent, number of links between two crosslinks, rms end-to-end distance and mesh size of gels were evaluated. The structural sequence of the scaffolds was tested and verified using Kelen–Tudos technique, and Alfrey–Price relationship. Hydrogels were characterized using FTIR, thermogravimetric analysis, differential scanning calorimetry, and freeze-dried Scanning electron micrographs techniques. The reversible pH responsiveness and possible mesoporous and nanoporous (i.e., 0.88–4.03 nm) structures suggest their suitable candidate in membrane technology and/or is an adequate drug delivery vehicle in drug delivery systems. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Reconstruction of composite defects involving bone and soft tissue presents a significant clinical challenge. In the craniofacial complex, reconstruction of the soft and hard tissues is critical for both functional and aesthetic outcomes. Constructs for space maintenance provide a template for soft tissue regeneration, priming the wound bed for a definitive repair of the bone tissue with greater success. However, materials used clinically for space maintenance are subject to poor soft tissue integration, which can result in wound dehiscence. Porous materials in space maintenance applications have been previously shown to support soft tissue integration and to allow for drug release from the implant to further prepare the wound bed for definitive repair. This study evaluated solid and low porosity (16.9% ± 4.1%) polymethylmethacrylate space maintainers fabricated intraoperatively and implanted in a composite rabbit mandibular defect model for 12 weeks. The data analyses showed no difference in the solid and porous groups both histologically, evaluating the inflammatory response at the interface and within the pores of the implants, and grossly, observing the healing of the soft tissue defect over the implant. These results demonstrate the potential of porous polymethylmethacrylate implants formed in situ for space maintenance in the craniofacial complex, which may have implications in the potential delivery of therapeutic drugs to prime the wound site for a definitive bone repair. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

The objective of this study was to investigate the bioactivity and protein-resistant properties of dual functioning surfaces modified with PEG for protein resistance and corn trypsin inhibitor (CTI) for anticoagulant effect. Surfaces on gold substrate were prepared with varying ratios of free PEG to CTI-conjugated PEG. Two methods designated, respectively, “sequential” and “direct” were used. For sequential surfaces, PEG was first immobilized on gold and the surfaces were incubated with CTI at varying concentration. For direct surfaces, a PEG–CTI conjugate was synthesized and gold surfaces were modified using solutions of the conjugate of varying concentration. The CTI density on these surfaces was measured using radiolabeled CTI. Water contact angles were measured and the thickness of PEG–CTI layers was determined by ellipsometry. Fibrinogen adsorption from buffer and human plasma, and adsorption from binary solutions of fibrinogen and α-lactalbumin were investigated using radiolabeling methods. Bioactivity of the surfaces was evaluated via their effects on FXIIa inhibition and plasma clotting time. It was found that as the ratio of CTI-conjugated PEG to free PEG increased, bioactivity increased but protein resistance was relatively constant. It is concluded that on these surfaces conjugation of PEG to CTI does not greatly compromise the protein resistance of the PEG but results in improved interactions between the CTI and the “target” protein FXIIa. At the same CTI density, sequential surfaces were more effective in terms of inhibiting FXIIa and prolonging clotting time. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

The particle size and surface properties of gold nanoparticles are critical factors for the interactions between nanoparticles and cells. To produce noncytotoxic gold nanoparticles, a straightforward method for the synthesis of gold nanoparticles designed involving the reduction and stabilization by a protein such as a lysozyme in conjunction with microwave irradiation. The cooperative combination of a lysozyme with a high affinity for metal ions and the microwave irradiation allowed to form biocompatible gold nanoparticles in an aqueous system. In addition, the cell toxicity and the cellular uptake pathways of the gold nanoparticles synthesized against mouse embryonic fibroblast NIH-3T3 cells were studied and found to be taken up by receptor-mediated endocytosis. In addition, the lysozyme-stabilized gold nanoparticles are accumulated in the cytoplasm as well as the nucleus without any significant cytotoxicity. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

A collagen–silica xerogel hybrid membrane was fabricated by a sol–gel process for guided bone regeneration (GBR). The silica xerogel synthesized by the sol–gel method was distributed uniformly within the collagen matrix in the form of nanoparticles. The hybridization of the silica xerogel with collagen improved the biological properties of the membrane significantly. Preosteoblast cells were observed to adhere well and grow much more actively on the hybrid membrane than on the pure collagen membrane. In particular, the hybrid membrane containing 30% of the silica xerogel showed the highest level of osteoblast differentiation. Moreover, the GBR ability, as assessed by the in vivo animal test, was superior to that of the pure collagen membrane. These findings suggest that the collagen–silica xerogel hybrid can be used as a GBR membrane. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

This study investigated the effects of magnesium ion (Mg) incorporation into the surface of deproteinized porcine cancellous bone in the bone healing of rabbit calvarial defects with the expectation of utilizing the integrin–ligand binding enhancement effect of Mg, and compared its bone healing capacity with that of untreated porcine cancellous bone and deproteinized bovine bone (Bio-Oss). Hydrothermal treatment was performed to produce Mg-incorporated porcine bone using an alkaline Mg-containing solution. The surface morphology and chemical composition of the samples were investigated using scanning electron microscopy, energy-dispersive X-ray spectrometry, and X-ray photoelectron spectroscopy. Defects 7 mm in diameter were created in the calvaria of 14 adult male New Zealand White rabbits and were filled with (1) untreated porcine bone (PB), (2) Bio-Oss, and (3) Mg-containing porcine bone (MG). The percentage of newly formed bone (NB%) was evaluated histomorphometrically at 2 and 4 weeks after implantation. Hydrothermal treatment resulted in a Mg-containing surface in porcine bone covered with nanostructures ∼100 nm in size. The MG group supported better new bone formation compared with the other groups. Osteoconductive new bone formation was observed in the central defect area in the MG group at an early healing time-point. Histomorphometric analysis revealed significantly greater NB% in the MG group when compared with the untreated PB and Bio-Oss groups at 4 weeks (p < 0.05). The Mg-incorporated porcine bone with surface nanostructures achieved rapid new bone formation in the osseous defects of rabbit calvaria compared with untreated xenografts of porcine and bovine origin. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

This article has been retracted because the authors and editors of the Journal of Biomedical Materials Research, Part A have agreed there is overlap with another article by the same group in the Journal of Controlled Release, 2006; 114(2):209–222.

Adipose tissue is a readily available source of multipotent adult stem cells for use in tissue engineering/regenerative medicine. Various growth factors have been used to stimulate acquisition of endothelial characteristics by adipose-derived stem cells (ADSC). Herein, we study the growth and endothelial differentiation potential of ADSC seeded onto a porous polycaprolactone (PCL) scaffold. The objective of this study is to demonstrate that PCL is a good material to be used as a scaffold to support reconstruction of new endothelial tissue using adipose stem cells. We found that undifferentiated ADSC adhere and grow on PCL. We show that, after culture in endothelial differentiation medium, ADSC were positive to LDL uptake and expressed molecular markers characteristic of endothelial cells (CD31; eNOS and vWF). In addition, our study defines the time required for the differentiation of ADSC directly onto PCL. This study suggests that PCL can be used as a scaffold to generate endothelial tissue in vitro. PLC has excellent mechanical properties and a slow degradation rate. Moreover, based on our results, we propose that PCL could be used to graft scaffolds coated with endothelial cells derived from ADSC stem cells. Endothelial cells-coated PCL could find several applications to replace damaged area of the body; for example, a possible use could be the generation of vascular grafts. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Tympanic membrane (TM) perforation is still one of the most common otology complications. New designs of biomaterials, and lately tissue-engineered composites and grafts, have thoroughly revolutionized the management of TM perforation. In this study, we examined a biologically modified collagen-immobilized polydimethyl siloxane patch to repair TM perforation. In vitro potential of the aforementioned patch as a scaffold to support fibroblast cell growth and adhesion was assessed. An in vivo assay of the patch for initiating repair of TM perforations also was investigated. In vitro assay showed that the patch has significantly increased cell adhesion and growth in comparison with unmodified ones (p < 0.05). In vivo study also showed an overall closure rate of TM perforation of 70% and an average gain of 15.75 ± 4.29 dB in air-bone gap. This study shows that the preliminary in vivo evaluation of a modified siloxane patch in humans had promising results and is comparable to existing biomaterial patches. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Crosslinked vitamin-E-stabilized polyethylene acetabular cups were compared with both commercially available conventional and custom-crosslinked polyethylene acetabular cups in terms of wear behavior, in a hip joint simulator for five millions cycles, using bovine calf serum as lubricant. We correlated the wear experiments results with the chemical characterization of the investigated materials: Fourier transformed infrared (FTIR) spectroscopic analyses, differential scanning calorimetry, and crosslink density measurements were used to assess the chemical characteristics of the pristine materials. In addition, further FTIR analyses and cyclohexane extraction were carried out after the simulator experiments. Lipids absorption was observed in all tested specimens and it has been shown to strongly affect the results of the wear test. Corrected gravimetric wear measurements showed that vitamin-E blended, crosslinked polyethylene wore more than the traditional crosslinked polyethylene but exhibited a much lower wear than conventional ultrahigh-molecular weight polyethylene. The chemical analyses showed that the addition of vitamin E reduced the crosslinking efficiency. Given the correlation between crosslink density and wear resistance, this gave an explanation for the observed wear performances. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

The ultimate success of in vivo organ formation utilizing ex vivo expanded “starter” tissues relies heavily upon the level of vascularization provided by either endogenous or artificial induction of angiogenic or vasculogenic events. To facilitate proangiogenic outcomes and promote tissue growth, an elastomeric scaffold previously shown to be instrumental in the urinary bladder regenerative process was modified to release proangiogenic growth factors. Carboxylic acid groups on poly(1,8-octanediol-co-citrate) films (POCfs) were modified with heparan sulfate creating a heparan binding POCf (HBPOCf). Release of proangiogenic growth factors vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF-1) from HBPOCfs demonstrated an approximate threefold increase over controls during a 30-day time course in vitro. Atomic force microscopy demonstrated significant topological differences between films. Subcutaneous implantation of POCf alone, HBPOCf, POCf-VEGF, and HBPOCf-VEGF within the dorsa of nude rats yielded increased vascular growth in HBPOCf-VEGF constructs. Vessel quantification studies revealed that POCfs alone contained 41.1 ± 4.1 vessels/mm², while HBPOCf, POCf-VEGF, and HBPOCF-VEGF contained 41.7 ± 2.6, 76.3 ± 9.4, and 167.72 ± 15.3 vessels/mm², respectively. Presence of increased vessel growth was demonstrated by CD31 and vWF immunostaining in HBPOCf-VEGF implanted areas. Data demonstrate that elastomeric POCfs can be chemically modified and possess the ability to promote angiogenesis in vivo. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

The aim of this study is to estimate the increase of bone-inductive potency by human demineralized dentin matrix (DDM) with recombinant human bone morphogenetic protein-2 (BMP-2). Human teeth were crushed, completely demineralized in 0.6M HCl, and freeze-dried. The tooth-derived material is called DDM. The shape of DDM was a particle type and its size varied from 0.4 to 0.8 mm. The BMP-2 dose-dependent study in the rat subcutaneous tissues demonstrated that the volume of induced bone and marrow increased at a dose-dependent manner. The time-course study of bone induction by the BMP-2 (5.0 μg)/DDM (70 mg) was estimated histologically and biochemically. Histological findings showed that the BMP-2/DDM increased bone and marrow sequentially between the DDM particles. Calcium content in the BMP-2/DDM-induced tissue was compatible to the histological findings. ALP activity in the BMP-2/DDM showed a maximal value at 1 week and gradually decreased. The morphometric analysis demonstrated that the BMP-2/DDM showed 66.9%, 79.0% in the volume of bone and marrow, and 32.4%, 21.0% in that of DDM at 8, 32 weeks, respectively. We confirmed that BMP-2 significantly accelerated bone formation in the acid-insoluble human-dentin carriers. These results indicate that human DDM should be an effective carrier for delivering BMP-2 and superior scaffold for bone-forming cells. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Extracellular matrix is the gold standard for tissue regeneration. In this study, we directly made the extracellular matrix of the tissue or organ into scaffold for spinal cord injuries, a strategy that is seldomly tried in spinal cord engineering. The aim of this study was to determine if the chemically extracted acellular muscle could be a potential scaffold for spinal cord injury. The chemically extracted acellular muscle was implanted in the lateral hemisected adult rat thoracic spinal cord. Control rats were similarly injured. After 1 and 4 weeks, scaffold integration and biocompatibility, axon sprouting, and myelination were evaluated. The chemically extracted acellular muscle scaffolds were found to be well integrated with the host tissue. Sprouting axons grew into the full length of the scaffold in a strikingly parallel and linear fashion. A few remyelinated axons were also detected in the scaffolds. The tracing results in another six rats showed that labeled fibers entered the chemically treated muscle grafts. Furthermore, there were no apparent quantitative differences in the ED-1 and glial fibrillary acidic protein positive cells between groups. Neuron counting showed more surviving neurons in the acellular muscle treated group than those of the injured only group. Vascularization of the grafts was also confirmed. These findings clearly demonstrated that chemically extracted acellular muscle grafts provided useful biomatrices to enhance axon sprouting in the injured spinal cord. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Most implanted electrical devices use encapsulant as insulation. The encapsulant may remain functional for many years, bonded to the metallic surfaces, but eventually become partly detached allowing corrosion to occur. To understand whether the corrosion products will cause toxic effects, we need to know how quickly they will permeate through the encapsulant. In these experiments, silicone capsules (the encapsulant) containing metal compounds were left in jars of initially pure water for 6 months, and the concentration of the metal in the water was measured. The amount of metal depended on the type of compound; for the organometallic compounds tested, permeation was very rapid. However, for most of the other compounds, whether oxides or salts, the amount of metal was below the control level and therefore could have been the result of contamination. These compounds were tin sulfate and oxide (<10²), lead nitrate and oxide (<10²), copper sulfate (<10³), and nitrates of bismuth (<10¹), chrome (<10²), nickel (<10³) and zinc (<10²). The numbers in brackets are the maximum mass (ng) of permeated metal after 6 months. Three silver compounds were tested but without proper controls; however, the amount of permeated silver appeared to be low: silver oxide (1.3 × 10²), silver nitrate (6.3 × 10¹), and silver chloride (6 × 10⁰). The resolution of this method is limited by contamination that is detected by control capsules. The conclusion is that compounds that are likely corrosion products permeate through silicone encapsulant at a low rate and seem unlikely to cause toxic effects. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

This study used a rat subcutaneous implantation model to investigate gradual in situ pore formation in a self-regulating degradable chitosan-based material, which comprises lysozyme incorporated into biomimetic calcium phosphate (CaP) coatings at the surface to control the scaffold degradation and subsequent pore formation. Specifically, the in vivo degradation of the scaffolds, the in situ pore formation, and the tissue response were investigated. Chitosan or chitosan/starch scaffolds were studied with and without a CaP coating in the presence or absence of lysozyme for a total of six experimental groups. Twenty-four scaffolds per group were implanted, and eight scaffolds were retrieved at each of three time points (3, 6, and 12 weeks). Harvested samples were analyzed for weight loss, microcomputed tomography, and histological analysis. All scaffolds showed pronounced weight loss and pore formation as a function of time. The highest weight loss was 29.8% ± 1.5%, obtained at week 12 for CaP chitosan/starch scaffolds with lysozyme incorporated. Moreover, all experimental groups showed a significant increase in porosity after 12 weeks. At all time points no adverse tissue reaction was observed, and as degradation increased, histological analysis showed cellular ingrowth throughout the implants. Using this innovative methodology, the ability to gradually generate pores in situ was clearly demonstrated in vivo. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment—30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison with collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin—transketolase (TKT), the myofibroblast marker—α-smooth muscle actin (SMA), and four matrix proteins—collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN), and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared with the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared with the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype, and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDP peptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Although transplantation of human embryonic stem cells (hESCs)-derived neural precursors (NPs) has been demonstrated with some success for nervous repair in small animal model, control of the survival, and directional differentiation of these cells is still challenging. Meanwhile, the notion that using suitable scaffolding materials to control the growth and differentiation of grafted hESC-derived NPs raises the hope for better clinical nervous repair. In this study, we cultured hESC-derived NPs on Tussah silk fibroin (TSF)-scaffold of different diameter (i.e., 400 and 800 nm) and orientation (i.e., random and aligned) to analyze the effect of fiber diameter and alignment on the cell viability, neuronal differentiation, and neurite outgrowth of hESC-derived NPs. The results show that TSF-scaffold supports the survival, migration, and differentiation of hESC-derived NPs. Aligned TSF-scaffold significantly promotes the neuronal differentiation and neurite outgrowth of hESC-derived neurons compared with random TSF-scaffold. Moreover, on aligned 400 nm fibers cell viability, neuronal differentiation and neurite outgrowth are greater than that on aligned 800 nm fibers. Together, these results demonstrate that aligned 400 nm TSF-scaffold is more suitable for the development of hESC-derived NPs, which shed light on optimization of the therapeutic potential of hESCs to be employed for neural regeneration. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

In this study, a new functionalized peptide RLN was designed containing the bioactive motif link N, the amino terminal peptide of link protein. A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16. The characterization of LN-NS was tested using atomic force microscopy (AFM). The biocompatibility and bioactivity of this nanofiber scaffold for rabbit nucleus pulposus cells (NPCs) were also evaluated. This designer functionalized nanofiber scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously. In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS-guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs. Our findings demonstrate that the functionalized nanofiber scaffold containing link N had excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

The objective of this study was to investigate the effect of chemical structure, ion concentration, and ion type on the release rate of biologically available ions useful for remineralization from microcapsules with ion permeable membranes. A heterogeneous polymerization technique was utilized to prepare microcapsules containing either an aqueous solution of K₂HPO₄, Ca(NO₃)₂, or NaF. Six different polyurethane-based microcapsule shells were prepared and characterized based on ethylene glycol, butanediol, hexanediol, octanediol, triethylene glycol, and bisphenol A structural units. Ion release profiles were measured as a function of initial ion concentration within the microcapsule, ion type, and microcapsule chemical structure. The rate of ion release increased with initial concentration of ion stored in the microcapsule over a range of 0.5–3.0M. The monomer used in the synthesis of the membrane had a significant effect on ion release rates at 3.0M salt concentration. At 1.0M, the ethylene glycol released ions significantly faster than the hexanediol-, octanediol-, and butanediol-based microcapsules. Ion release was fastest for fluoride and slowest for phosphate for the salts used in this study. It was concluded that the microcapsules are capable of releasing calcium, phosphate, and fluoride ions in their biologically available form. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Development of transferable biocompatible membranes, which can be used for melanocyte culture and transplantation, is considered a feasible approach to increase the success rate for vitiliginous treatment. In this study, a crosslinked chitosan membrane (CCM) was produced via physical crosslinking of chitosan with sodium sulfate. The physical and mechanical properties as well as growth and phenotype expression of melanocytes on the CCM were investigated. The CCM supported growth and proliferation of melanocytes with the existence of melanin granules in the cytoplasm. The melanocytes remained active after transplantation. The CCM absorbed water approximately doubled from its original weight and permitted ∼2400 g/m² per day of water vapor transmission, suggesting that the CCM can function as an efficient wound dressing. Dynamic mechanical and tensile measurement results showed that the CCM possessed favorable wet strength for cell culture, separation, transfer, and transplantation application. This transferable CCM has the potential to be applied for vitiligo treatment. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

A conjugate of distearoylphosphoethanolamine-polyethylene glycol with 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was synthesized and incorporated into micelles and liposomes to create mineral-binding nanocarriers for therapeutic agents. The micelles and liposomes were used to encapsulate the anticancer drug doxorubicin (DOX) and a model protein lysozyme (LYZ) by using lipid film hydration (LFH) and reverse-phase evaporation vesicle (REV) methods. The results indicated that the micelles and LFH-derived liposomes were better at DOX loading than the REV-derived liposomes, while the REV method was preferable for encapsulating LYZ. The affinity of the micellar and liposomal formulations to hydroxyapatite (HA) was assessed in vitro, and the results indicated that all the thiolBP-incorporated nanocarriers had stronger HA affinity than their counterparts without thiolBP. The thiolBP-decorated liposomes also displayed a strong binding to a collagen/HA composite scaffold in vitro. More importantly, thiolBP-decorated liposomes gave increased retention in the collagen/HA scaffolds after subcutaneously implantation in rats. The designed liposomes were able to entrap the bone morphogenetic protein-2 in a bioactive form, indicating that the proposed nanocarriers could deliver bioactive factors locally in mineralized scaffolds for bone tissue engineering. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine–glycine–aspartic acid (RGD) and lysine–arginine–serine–arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48–780.28 N mm), bone-to-implant contact (BIC) (35.22%–41.49%), and new bone fill (28.58%–30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

The main disadvantage of apatitic calcium phosphate cements (CPCs) is their slow degradation rate, which limits complete bone regeneration. Carbonate (CO) is the common constituent of bone and it can be used to improve the degradability of the apatitic calcium phosphate ceramics. This study aimed to examine the effect of calcite (CaCO₃) incorporation into CPCs. To this end, the CaCO₃ amount (0–4–8–12 wt %) and its particle size (12.0-μm-coarse or 2.5-μm-fine) were systematically investigated. In comparison to calcite-free CPC, the setting time of the bone substitute was delayed with increasing CaCO₃ incorporation. Reduction of the CaCO₃ particle size in the initial powder increased the injectability time of the paste. During hardening of the cements, the increase in calcium release was inversely proportional to the extent of CO incorporation into apatites. The morphology of the carbonate-free product consisted of large needle-like crystals, whereas small plate-like crystals were observed for carbonated apatites. Compressive strength decreased with increasing CaCO₃ content. In vitro accelerated degradation tests demonstrated that calcium release and dissolution rate from the set cements increased with increasing the incorporation of CO, whereas differences in CaCO₃ particle size did not affect the in vitro degradation rate under accelerated conditions. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Establishing sufficient vascularization in scaffold remains a challenge for tissue-engineering. To improve angiogenesis, we incorporated vascular endothelial growth factor (VEGF) in collagen-coating over the porous polycaprolactone (PCL) scaffolds. The release kinetics of loaded VEGF from collagen-coated PCL (col-PCL) scaffolds was same as from scaffolds without the collagen. The bioactivity of VEGF delivered by the col-PCL scaffolds was confirmed by human umbilical vein endothelial cell (HUVEC) proliferation and chorioallantoic membrane (CAM) assay. The col-PCL scaffolds were implanted subcutaneously in mice for 7 and 14 days. At day 7, vascularization within scaffolds loaded with VEGF was superior to that in the scaffolds without VEGF. However, the vessel connectivity to host circulatory system was incomplete and restricted to the scaffold edges. At day 14, blood vessels in scaffolds reached density similar to the subcutaneous tissue and were perfusable throughout the implant thickness. Prewashing the scaffolds with saline to remove the unbound growth factor decreased the initial burst release and sustained the VEGF-mediated angiogenesis in vivo. In conclusion, our study demonstrates that physically adsorbed VEGF stimulated angiogenesis in collagen-coated PCL scaffolds. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Magnetic Fluid Hyperthermia (MFH) is a promising approach towards adjuvant cancer therapy that is based on the localized heating of tumors using the relaxation losses of iron oxide magnetic nanoparticles (MNPs) in alternating magnetic fields (AMF). In this study, we demonstrate optimization of MFH by tailoring MNP size to an applied AMF frequency. Unlike conventional aqueous synthesis routes, we use organic synthesis routes that offer precise control over MNP size (diameter ∼10 to 25 nm), size distribution, and phase purity. Furthermore, the particles are successfully transferred to the aqueous phase using a biocompatible amphiphilic polymer, and demonstrate long-term shelf life. A rigorous characterization protocol ensures that the water-stable MNPs meet all the critical requirements: (1) uniform shape and monodispersity, (2) phase purity, (3) stable magnetic properties approaching that of the bulk, (4) colloidal stability, (5) substantial shelf life, and (6) pose no significant in vitro toxicity. Using a dedicated hyperthermia system, we then identified that 16 nm monodisperse MNPs (σ–0.175) respond optimally to our chosen AMF conditions (f = 373 kHz, H_o = 14 kA/m); however, with a broader size distribution (σ–0.284) the Specific Loss Power (SLP) decreases by 30%. Finally, we show that these tailored MNPs demonstrate maximum hyperthermia efficiency by reducing viability of Jurkat cells in vitro, suggesting our optimization translates truthfully to cell populations. In summary, we present a way to intrinsically optimize MFH by tailoring the MNPs to any applied AMF, a required precursor to optimize dose and time of treatment. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Hydroxyapatite (HA) nanoparticles have been reported to exhibit anti-tumor effects on various human cancers, but the effects of HA on glioma cells remain unclear. The aim of this study was to explore whether HA can inhibit the proliferation and induce the apoptosis of C6 cells. Use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that HA induced C6 cell death in a concentration-dependent and time-dependent manner. Results from hoechst 33342 staining and flow cytometry assay showed that HA induced C6 cell apoptosis significantly. Meanwhile, the flow cytometric assay gave clear indication that HA induced intracellular accumulation of reactive oxygen species (ROS). The measurement of superoxide dismutase (SOD) generation showed that HA decreased the total SOD of cellular levels. Interestingly, pretreatment of N-(mercaptopropionyl)-glycine (N-MPG), known as a type of ROS scavenger formulations, could somehow inhibit C6 cell apoptosis induced by HA. These results may provide potential anti-glioma treatment in the future. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

A two-photon excitation difluoroboron dye activated in the near infrared region for biological image analysis was synthesized in this study. Cell affinity, membrane interaction, and the endocytosis pathway of PAMAM dendrons were investigated using only covalent two-photon dyes (TPD) at the periphery of the PAMAM dendrons. Generation 3 TPD-labeled PAMAM dendrons (BG3) exhibited multivalency binding on the HeLa cell membranes from the cell affinity study in the fixation of HeLa cells. Photo-stimulation on the membrane of the living HeLa cell was observed by confocal optical imaging in situ, using the two-photon model, when incubated with BG3. Analyses of cell membrane integrity via lactate dehydrogenase (LDH) assay confirmed membrane damage at two photon excitation model. However, no variation in the cell was observed using the one-photon excitation model. These results indicated a high degree of dendrons uptake by cells through binding to the cell membrane following the endocytotic pathway. Furthermore, the wide excitation fluorescence spectrum of difluoroboron dye provides dual imaging with which to study the endocytosis of TPD-labeled PAMAM dendrons using a single near infrared laser. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Electrospinning is often used to create scaffolding as a biomimetic of the extracellular matrix of tissues. A frequent limitation of this technique for three-dimensional tissue modeling is poor cell infiltration throughout the void volume of scaffolds. Here, we generated low-temperature electrospun silk scaffolds and compared these with conventional electrospun silk scaffolds in terms of mechanical properties, void volume, cell infiltration, cell viability, and potential to support mucosal models under three-dimensional culture conditions. Low-temperature electrospun silk scaffolds supported fibroblast attachment and infiltration throughout the volume of the scaffolds, while conventional electrospun scaffolds exhibited limited cell infiltration with fibroblasts attaching exclusively to the seeding surface of the scaffolds. The porosity of low-temperature electrospun scaffolds was 93% compared with 88% of conventional electrospun silk scaffolds. Uniaxial tensile testing showed a 3.5-fold reduction in strength of low-temperature electrospun silk compared with the conventional in terms of peak stress and modulus but no significant change in strain at break. Mucosal modeling with fibroblast-keratinocyte or fibroblast-carcinoma cocultures showed similar results, with cell infiltration occurring only in low-temperature electrospun scaffolds. Cell viability was confirmed using live/dead staining after 21 days in culture. Furthermore, low-temperature electrospun silk scaffolds were able to support keratinocyte differentiation, as judged by involucrin immunoreactivity. The low-temperature electrospun silk scaffold that we have developed eliminates the limitation of electrospun silk scaffolds in terms of cell infiltration and, therefore, can potentially be used for a wide range of tissue engineering purposes ranging from in vitro tissue modeling to in vivo tissue regeneration purposes. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Titanium alloys are still on the top list of fundamental materials intended for dental, orthopedics, neurological, and cardiovascular implantations. Recently, a special attention has been paid to vanadium-free titanium alloy, Ti6Al7Nb, that seems to represent higher biocompatibility than traditional Ti6Al4V alloy. Surprisingly, these data are not thoroughly elaborated in the literature; particularly there is a lack of comparative experiments conducted simultaneously and at the same conditions. Our study fills these shortcomings in the field of blood contact and microbiological colonization. To observe platelets adhesion and biofilm formation on the surfaces of compared titanium alloys, fluorescence microscope Olympus GX71 and scanning electron microscope HITACHI S-3000N were used. Additionally, flow cytometry analysis of platelets aggregation and activation in the whole blood after contact with sample surface, as an essential tool for biomaterial thrombocompatibility assessment, was proposed. As a result of our study it was demonstrated that polished surfaces of Ti6Al7Nb and Ti6Al4V alloys after contact with whole citrated blood and E. coli bacterial cells exhibit a considerable difference. Overall, it was established that Ti6Al4V has distinct tendency to higher thrombogenicity, more excessive bacterial biofilm formation and notable cytotoxic properties in comparison to Ti6Al7Nb. However, we suggest these studies should be extended for other types of cells and biological objects. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Thermoresponsive hydrogels are attractive for their injectability and retention in tissue sites where they may serve as a mechanical support and as a scaffold to guide tissue remodeling. Our objective in this report was to develop a thermoresponsive, biodegradable hydrogel system that would be capable of protein release from two distinct reservoirs—one where protein was attached to the hydrogel backbone, and one where protein was loaded into biodegradable microparticles mixed into the network. Thermoresponsive hydrogels consisting of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and biodegradable methacrylate polylactide were synthesized along with modified copolymers incorporating 1 mol % protein-reactive methacryloxy N-hydroxysuccinimide (MANHS), hydrophilic acrylic acid (AAc), or both. In vitro bovine serum albumin (BSA) release was studied from hydrogels, poly(lactide-co-glycolide) microparticles, or microparticles mixed into the hydrogels. The synthesized copolymers were able to gel below 37°C and release protein in excess of 3 months. The presence of MANHS and AAc in the copolymers was associated with higher loaded protein retention during thermal transition (45% vs. 22%) and faster release (2 months), respectively. Microspheres entrapped in the hydrogel released protein in a delayed fashion relative to microspheres in saline. The combination of a protein-reactive hydrogel mixed with protein-loaded microspheres demonstrated a sequential release of specific BSA populations. Overall the described drug delivery system combines the advantages of injectability, degradability, extended release, and sequential release, which may be useful in tissue engineering applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Naturally occurring biomaterial scaffolds derived from extracellular matrix (ECM) have been the topic of recent investigation in the context of rotator cuff tendon repair. We previously reported a method to treat fascia ECM with high molecular weight tyramine substituted-hyaluronan (TS-HA) for use as a tendon augmentation scaffold. The presence of crosslinked TS-HA in fascia was associated with an increased macrophage and giant cell response compared to water-treated controls after implantation in a rat abdominal wall model. The objective of this study was to determine the extent to which TS-HA treatment was associated with mechanical property changes of fascia after implantation in the rat model. Fascia samples in all groups demonstrated time-dependent decreases in mechanical properties. TS-HA-treated fascia with crosslinking exhibited a lower toe modulus, a trend toward lower toe stiffness, and a higher transition strain than water-treated controls not only after implantation, but also at time zero. TS-HA treatment, with or without crosslinking, had no significant effect on time-zero or post-implantation load relaxation ratio, load relaxation rate, linear-region stiffness, or linear-region modulus. Our findings demonstrated that the particular TS-HA treatment employed in this study decreased the low-load elastic mechanical properties of fascia ECM, in keeping with the heightened macrophage and giant cell host response seen previously. This work provides a starting point and guidance for investigating alternative HA treatment strategies. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

A nonfouling peptide grafted polymer was synthesized that can promote endothelial cell (EC) binding. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate, and CGRGDS peptide. The peptide was incorporated into the polymer system either by a chain transfer reaction or by coupling to an acrylate-PEG-N-hydroxysuccinimide (NHS) comonomer. The introduction of PEG chains minimizes protein adsorption. Human umbilical vein ECs and endothelial colony forming cells were cultured on these surfaces in short term and long-term studies. A difference in number and morphology of ECs was observed depending on the method of peptide incorporation. Both cell types adhered better to polymer films containing NHS coupled RGD peptide after 2 h even in the presence of albumin but significant cell detachment occurred after 4 days. Polymer solutions were electrospun into fibrous scaffolds. Both nonfouling and peptide binding characteristics were retained after processing. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

This article provides the transcript for the Panel on Developing a Biomaterials Curriculum held at the 2011 annual meeting of the Society for Biomaterials in Orlando, FL. The panelists were Thomas R. Harris of Vanderbilt University, Jack Lemons of the University of Alabama, Birmingham, Antonios G. Mikos of Rice University, David A. Puleo on the University of Kentucky, Frederick J. Schoen of Harvard Medical School, and Johnna S. Temenoff of Georgia Tech/Emory. The panelists, each an expert in engineering education and textbook author, presented their perspectives on key issues of developing undergraduate and graduate curricula that contain a biomaterials focus. The presentations were followed by a lively and informative discussion with the audience. A redacted portion of this discussion is included. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.