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Keywords:

  • PLGA;
  • BMP-2;
  • scaffold;
  • controlled release;
  • calvarial defect;
  • bone formation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP-2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature-sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP-2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP-2 loaded into the scaffolds was released by the end of this time period. The released BMP-2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP-2-loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP-2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP-2 delivery for bone-regeneration applications. Copyright © 2012 John Wiley & Sons, Ltd.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Healing of bone fractures and reconstruction of critical-sized bone defects present a significant challenge. Although autologous bone grafts are routinely used, disadvantages such as limited graft accessibility have necessitated the development of alternative methods for bone repair (Haidar et al., 2009). One such method involves the use of three-dimensional (3D) polymer scaffolds, which have shown excellent potential in the field of tissue engineering for the purpose of bone regeneration (Salgado et al., 2004).

Growth factors can be used in bone tissue engineering to stimulate natural repair processes (Ryoo et al., 2006). However, the successful use of growth factor-loaded scaffolds for tissue repair is currently limited by issues of protein stability and high cost. These issues could potentially be overcome by using a sustained and localized delivery approach (Putney and Burke, 1998). Release of growth factors from engineered scaffolds in a sustained manner has potential for such a delivery system, as this would allow cells to migrate to the defect area, proliferate and differentiate, resulting in enhanced tissue repair (Issa et al., 2008).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGFβ) superfamily, which are involved in regulating differentiation processes of various cells during skeletal development and fracture repair (Duguy et al., 2000; Groeneveld and Burger, 2000). BMP-2 is considered one of the most important signalling molecules in bone regeneration and it has been observed to have a very strong osteoinductive activity (Fujimura et al., 1995; Boyne et al., 1997; Kusumoto et al., 1998; Okubo et al., 2000). The challenge in using BMP-2 for bone repair is the inherent short half-life the protein exhibits in vivo. Pharmacokinetic studies have demonstrated that the half-life of BMP-2 in non-human primates is approximately 7 min (Poynton and Lane, 2002). It is therefore necessary to use a carrier scaffold for controlled and sustained release of BMP-2.

Induction of new bone growth in vivo is influenced by the rate of BMP-2 release, with slow release yielding improved growth (La et al., 2010; Srouji et al., 2010). Biomaterials that can retain growth factors, such as BMP-2, at the site of interest for slow release have been shown to greatly enhance efficacy and reduce protein dose by localizing the bone morphogenetic stimulus (Chen and Mooney, 2003). For example, collagen sponges are currently used clinically to deliver BMP-2 for spinal fusion in the product INFUSE® (Medtronic). Whilst clinically effective, the dose of BMP-2 loaded into each collagen sponge is relatively high (12 mg), due to the fact that collagen itself does not support sustained delivery over a prolonged period of time (Geiger et al., 2003; Kato et al., 2006).

Various synthetic polymers have been investigated for use as scaffolds for bone tissue engineering, including poly(dl-lactic acid-co-glycolic acid) (PLGA). This polymer has a long history of clinical use as a degradable implant material (Coombes and Heckman, 1992; Howard et al., 2008). PLGA delivery systems for BMP-2 have shown promise for bone repair applications (Lee et al., 1994; Muschler et al., 1994).

As previously described by Dhillon et al. (2011), blending PLGA with a plasticizer, such as poly(ethylene glycol) (PEG), allows the production of temperature-sensitive particles with a reduced glass transition temperature (Tg) of 37°C. The particles become soft and cohesive when they reach their Tg, which causes them to adhere to each other and the hydrophilic PEG component to leach out. The decreased PEG concentration increases the Tg of the particles, which causes them to re-solidify to form strong porous scaffolds. The Dhillon et al. study showed that the scaffolds have mechanical properties which are suitable for use in bone tissue-engineering applications, with a maximum compressive strength of 2 MPa after 2 h at 37°C, which is within the range for human trabecular bone. The Young's modulus of the scaffolds after 2 h at 37°C is approximately 40 MPa.

The repair of craniofacial bony defects is surgically challenging, due to the delicate and complex anatomy of the craniofacial skeleton. A calvarial wound model has many similarities to the craniofacial region (Terella et al., 2010). The mouse calvarial critical-size defect model is a useful in vivo model for evaluating the potential of biomaterials to deliver growth factors and regenerate bone (Aalami et al., 2004; Gupta et al., 2008). Biomaterials such as PLGA (Cowan et al., 2004), PEG-based hydrogels (Lutolf et al., 2003), demineralized bone matrix (Jones et al., 2007), hyaluronic acid hydrogels (Kim et al., 2007), chitosan gel (Stephan et al., 2010) and bovine gelatin (Ben-David et al., 2010) have previously been assessed using this model.

The objective of the current study was to investigate the ability of novel PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner, both in vitro and in vivo. The BMP-2 solution was mixed with the polymer particles prior to scaffold sintering, creating a unique method for the production of BMP-2-loaded scaffolds. The mouse calvarial critical-size defect model was used to assess the potential of these scaffolds for BMP-2 delivery and subsequent bone regeneration in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

PLGA/PEG particle production

Thermosensitive particles were fabricated from blends of 53 kDa PDLLGA (85:15 DLG 4CA; Lakeshore Biomaterials, USA) and PEG 400 (Sigma-Aldrich, UK). A mixture of 93.5%:6.5% PLGA:PEG w/v was blended at 80–90°C on a hotplate. The melted PLGA and PEG were mixed together by hand, using a PTFE-coated spatula, and allowed to cool. Polymer blend sheets were then ground into particles in a bench-top mill (Krups Mill F203) and the particles were sieved to obtain the 100–200 µm particle size fraction.

Scaffold preparation

Composite scaffolds were prepared in PTFE moulds. For in vitro release experiments, cylindrical scaffolds 12 mm in length and 6 mm in diameter were produced. The PLGA/PEG particles were mixed manually with BMP-2 solution (Medtronic, USA) or water (negative controls). A particles:solution ratio of 1:0.6 was used. The BMP-2-loaded scaffolds contained 750 µg BMP-2/scaffold. The particle paste was then packed into the mould, which was placed at 37°C for 2 h to allow scaffold formation.

For in vivo bone formation experiments, cylindrical scaffolds 1 mm thick and 4 mm in diameter were produced. The PLGA/PEG particles were mixed manually with BMP-2 solution (R&D Systems, USA), using a particles:solution ratio of 1:0.6. The BMP-2-loaded scaffolds contained 1 µg BMP-2/scaffold. The particle paste was then packed into the mould, which was placed at 37°C for 2 h to allow scaffold formation.

Scanning electron microscopy

Samples were mounted on aluminium stubs and were sputter-coated with gold at an argon current rate of 30 mA for 3 min. The structural morphology of the scaffolds was visualized using a scanning electron microscope (SEM; JEOL JSM-6060LV) at 10 kV.

Fourier transform infrared (FTIR) spectroscopy

PLGA and PLGA/PEG melt-blend (1 mg each) were combined with 10 mg KBr (Sigma-Aldrich), using a pestle and mortar. These samples were then pressed into disks, using a 6 mm die at a pressure of 10 tonnes for 2 min. PEG 400 was brought to room temperature and 5 µl of the material placed between two unpolished NaCl disks (Apollo Scientific). All samples were analysed using a Nicolet Avatar 360 FTIR instrument within the 400–4000 cm–1 region at room temperature.

In vitro BMP-2 release assay

For the BMP-2 release assay, PLGA/PEG scaffolds loaded with 750 µg BMP-2 and negative control scaffolds prepared without BMP-2 were placed into 20 ml Falcon tubes containing 10 ml phosphate-buffered saline (PBS; Invitrogen, UK). The PBS was removed daily for sampling and 10 ml fresh PBS was placed on each scaffold. Samples of the removed PBS were analysed, using a BMP-2 Quantikine ELISA kit (R&D Systems, UK). ELISA was performed according to the manufacturer's instructions and the optical density of each sample was measured at an optical wavelength of 450 nm (Tecan Infinite 200 plate reader).

For use in the alkaline phosphatase activity assay, PLGA/PEG scaffolds loaded with 750 µg BMP-2 and negative control scaffolds prepared without BMP-2 were placed into 20 ml Falcon tubes containing 10 ml Dulbecco's modified Eagle's medium (DMEM; Invitrogen, UK). The DMEM was removed daily for sampling and 10 ml fresh DMEM was placed on each scaffolds. These samples were referred to as ‘release media’. Prior to use in cell culture, the release media were supplemented with 10% v/v fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, UK).

C2C12 cell culture

C2C12 (American Type Culture Collection, VA, USA; cat. no. CRL-1722) cells are mouse myoblast cells that undergo BMP-2 induced osteogenic differentiation (Vinals et al., 2002). C2C12 cells were cultured in DMEM supplemented with 10% v/v FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were seeded into six-well tissue culture plates at 1 × 104 cells/well and cultured at 37°C in a humidified 5% CO2 atmosphere.

Detection of alkaline phosphatase (ALP)

The C2C12 cells were cultured until approximately 70% confluent. The medium on the cells was then replaced with the release medium from days 0, 1, 2, 4, 7, 10, 14, 20 and 22 from the BMP-2 release assay. Following a period of 96 h in culture, the cells were washed with PBS and 2 ml working pNPP solution was added to each well (SigmaFAST p-Nitrophenyl phosphate tablets, Sigma-Aldrich) (Jones et al., 1989). The plates were incubated for 30 min at room temperature and the absorbance was measured at an optical wavelength of 405 nm (Tecan Infinite 200 plate reader).

Culture of bone marrow-derived mesenchymal stem cells on PLGA/PEG scaffolds

PLGA/PEG particles were sterilized by treatment with ultraviolet light for 20 min prior to preparing scaffolds as described in Section 2.3. Triplicate scaffolds for cell-seeding and triplicate negative control scaffolds (without cells) were prepared for each time point (days 1, 3 and 7) and transferred to a 24-well plate. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs; purchased from TCS Cellworks, UK) were cultured in mesenchymal stem cell medium (TCS Cellworks). An hBM-MSC cell suspension was placed drop-wise onto the surface of each scaffold at a density of 2.5 × 105 cells in a volume of 50 µl/scaffold. The cell-seeded scaffolds were incubated for 2 h at 37°C with 5% CO2 to allow the cells to adhere to the scaffolds. Following incubation, 2 ml fresh medium was added to each well to cover both the cell-seeded and negative control scaffolds. The culture plates were then incubated at 37°C with 5% CO2.

Alamar blue cell viability assay

PLGA/PEG scaffolds were seeded with hBM-MSCs as described in Section 2.8. On days 1, 3 and 7 post-seeding, the medium was removed from the scaffolds and replaced with 2 ml 10% Alamar blue solution, prepared by adding 1 ml Alamar blue indicator dye (Invitrogen) to 9 ml PBS. The culture plate was then incubated at 37°C with 5% CO2 for 90 min. Following incubation, triplicate samples of 100 µl were transferred to a 96-well plate for analysis. Fluorescence was measured at 585 nm.

Cranial defect model

Athymic Nude-Foxn1nu mice, 8 weeks old and weighing 25 g (Harlan Laboratories, Jerusalem, Israel) were used for the critical-size calvarial defect model. The mice were anaesthetized with a 0.5 ml intraperitoneal (i.p.) injection of a 1:1 mixture of xylazine and ketamine. Two bilateral full-thickness circular defects (5 mm diameter and 1.5 mm thick) were created with a hand drill and trephine bit in the parietal bones of the skull on both sides of the sagittal suture line. Care was taken not to damage the sagittal suture or to interrupt the dura mater beneath the bone. A disc shaped PLGA/PEG scaffold specifically designed to fit the defects was transplanted in the defects. The transplanted discs were either loaded with 1 µg BMP-2 or were without BMP-2, as described earlier. Completely empty calvarial defects were used as a negative control for the experiment. Each group contained five replicates (empty negative controls, scaffolds alone and scaffolds containing BMP-2). The surgical procedure was approved according to institutional guidelines of the Animal Ethics Committee of the Technion–Israel Institute of Technology, Haifa, Israel.

Micro-computed tomography (μCT)

At 6 weeks post-surgery, the skulls (n = 5) were fixed in 10% neutral buffered formalin and analysed by a micro-computed tomography (μCT) imaging system (μCT 40, Scanco Medical AG, Brüttisellen, Switzerland). The scanner was operated in the high-resolution mode at energy of 55 kVp and an intensity of 145 μA, using a 200 ms acquisition time and no frame averaging, providing a nominal isotropic resolution of 30 µm. A constrained Gaussian filter (Sigma 1.2, support 1) was used to partly suppress the noise in the images. The mineralized bone tissue was segmented from the non-mineralized tissues by using a global threshold (21% of maximal grey value). The defect region was then identified by a cylindrical contour and the bone volume (BV) was calculated within this fixed volume of interest (VOI). Bone coverage was illustrated from a projection of the cranium in the superior–inferior direction to create a high-resolution pseudoradiograph (Lutolf et al., 2003). The obtained values for the PLGA/PEG scaffolds alone and the PLGA/PEG scaffolds loaded with BMP-2 were then expressed as a percentage more than the negative control (empty defects) group.

Histology

After μCT imaging, the entire skull was decalcified with 10% ethylene diaminetetra-acetic acid (EDTA). The orientation and alignment of the femoral bones were carefully taken into consideration during paraffin embedding, in order to be able to view the defect. Latitudinal serial sections (6 µm thick) were stained with haematoxylin and eosin (H&E) for general histology.

Statistics

Results were reported as mean ± SEM. Multiple comparison analysis was conducted using analysis of variance (ANOVA) with Dunnett's correction, where significant values were set at p < 0.05. The statistics were performed using GraphPad prism analysis software (v 5.01, GraphPad Software Inc., CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

PLGA/PEG FTIR analysis

FTIR results analysis shows that all the characteristic PLGA absorbance bands are conserved following the melt-blend procedure. There is little influence of PEG on the PLGA/PEG absorption spectrum, which is most likely due to the low amount of PEG polymer present in the melt-blend (6.5%) (Figure 1C). Detailed results of the FTIR analysis are available in the Supporting information (Figure S1).

image

Figure 1. PLGA/PEG scaffolds. Scaffolds were fabricated by sintering 100–200 µm PLGA/PEG particles mixed with saline (particles:saline ratio = 1:0.6) in Teflon moulds at 37°C for 2 h (A). PLGA/PEG scaffold visualized by scanning electron microscopy (SEM) at a magnification of ×20 and ×100 (insert, B). All the characteristic PLGA absorbance bands are conserved following the melt-blend procedure. There is little influence of PEG on the PLGA/PEG absorption spectrum, which is most likely due to the low amount of PEG polymer present in the melt-blend (6.5%) (C)

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BMP-2 release from PLGA/PEG scaffolds

PLGA/PEG scaffolds were fabricated by sintering PLGA/PEG particles mixed with BMP-2 solution or water (negative controls) in Teflon moulds at 37°C (Figure 1A). The porosity of the scaffolds was caused by the manner in which the sintered particles pack together, leaving voids (Figure 1B). Triplicate scaffolds were loaded with BMP-2 by mixing BMP-2 solution with PLGA/PEG particles prior to sintering at 37°C. To determine the ability of the scaffolds to release BMP-2, the PBS surrounding the scaffolds was sampled for BMP-2 content compared to triplicate negative control scaffolds over a 22-day time period. Following an initial burst release of 13% after 12 h, the scaffolds continued to release BMP-2 in a sustained manner at ca. 0.01–8%/day over a 23-day period (Figure 2). By the end of the release study, almost 70% of the BMP-2 had been released from the scaffolds.

image

Figure 2. BMP-2 release from PLGA/PEG scaffolds in vitro. Release was measured over a 22-day time period, using a BMP-2 Quantikine ELISA. The cumulative release is shown as a percentage of the total amount of BMP-2 added to the scaffolds. Data generated from triplicate scaffolds; error bars represent SD (where not visible, the error bars are very small)

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BMP-2-induced osteogenesis in C2C12 cells

During the BMP-2 release assay the medium was removed and replaced daily. The samples were referred to as ‘release media’. The release media from days 0 (4 h), 1, 2, 4, 7, 10, 14, 20 and 22 were used to culture C2C12 cells for 96 h. The p-nitrophenyl phosphate (pNPP) assay was then performed to detect the biochemical activity of alkaline phosphatase (ALP). Results obtained in this assay showed a nine-fold increase in ALP activity in cells which were incubated with the release medium gathered from the BMP-2 release assay, compared that shown by cells which were incubated with negative control release medium (Figure 3).

image

Figure 3. BMP-2-induced osteogenesis in C2C12 cells. Release medium from the BMP-2 release assay was used to culture C2C12 cells for 96 h. The pNPP assay was then performed to detect the presence of ALP. Higher ALP activity was detected in samples from cells cultured in release medium from BMP-2-loaded scaffolds. Data generated from triplicate scaffolds; error bars represent SD (where not visible, the error bars are very small)

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Proliferation of bone marrow-derived mesenchymal stem cells on PLGA/PEG scaffolds

To determine the ability of the PLGA/PEG scaffolds to support cell growth, triplicate PLGA/PEG scaffolds were seeded with hBM-MSCs, as described in Section 2.8. The Alamar blue assay was performed as described in section 2.9 to determine the number of viable cells on the scaffolds on days 1, 3 and 7 post-seeding (Figure 4). The cell numbers increased significantly (p < 0.001) from day 1 to day 3 and from day 3 to day 7, demonstrating proliferation of the hBM-MSCs on the PLGA/PEG scaffolds in culture.

image

Figure 4. Proliferation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on PLGA/PEG scaffolds; hBM-MSCs were cultured on PLGA/PEG scaffolds for 7 days. The Alamar blue assay was performed on days 1, 3 and 7 post-seeding to determine the number of viable cells on the scaffolds. Error bars represent SD. ***p < 0.001

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In vivo evaluation of PLGA/PEG containing BMP-2

PLGA/PEG scaffolds loaded with BMP-2 and negative control PLGA/PEG scaffolds without BMP-2 were implanted in a mouse calvarial bone defect model for 6 weeks. The defect repair was evaluated using μCT. BMP-2-containing scaffolds were found to induce 55% more new bone volume in the total defect volume (p < 0.01) compared to empty defects and 24% more bone compared to PLGA/PEG scaffolds without BMP-2 (Figure 5A–C). Control PLGA/PEG scaffolds that did not contain BMP-2 induced 31% more new bone volume compared to empty defects (p < 0.05). Following the μCT analysis, the skulls were taken for general histological analysis, which showed that adding BMP-2 to the implants resulted in better defect closure and yielded thicker and healthier new bone in the defect area, compared to PLGA/PEG scaffolds without BMP-2. Histology of the empty defects group showed no healing (Figure 5D).

image

Figure 5. PLGA/PEG-loaded scaffolds induce bone formation in vivo. μCT image of a mouse skull with two empty CSDs (A). μCT image of a mouse skull with CSDs implanted with or without BMP-2-loaded scaffolds (B). μCT analysis of cranial CSDs transplanted with or without BMP-2 loaded PLGA/PEG scaffolds compared to empty cranial CSDs controls (C). Histological analysis of the different experimental groups (D). *p < 0.05, **p < 0.01

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Since the time when Urist (1965), for the first time, successfully used demineralized bone matrix to induce ectopic bone formation in muscle tissue, experimental and clinical studies on BMP have been a focus of intensive investigation in orthopaedic surgery. BMP-2 has strong osteoinductive potential, although its clinical use is currently restrained by the lack of a suitable delivery system that maintains BMP-2 at the defect site for a sufficient time and with an appropriate dose (Woo et al., 2001). An ideal carrier matrix needs to provide localized, delayed and active BMP-2 delivery. Moreover, it should withstand the applicable load at the site of implantation and support cell requirements, attachment, proliferation and differentiation (Burg et al., 2000; Kirker-Head, 2000; Lu et al., 2003; Yokota et al., 2003). We hypothesized that BMP-2 loaded PLGA/PEG scaffolds, prepared by mixing BMP-2 solution with PLGA/PEG particles prior to scaffold sintering, could provide a sustained release system complying with these requirements.

When PLGA/PEG particles are mixed with a carrier solution at room temperature, a particulate paste is formed which can be moulded into any size or shape. The paste then hardens into a scaffold at 37°C due to the temperature induced PEG-leaching. The incorporation of any dose of one or multiple growth factors into the scaffolds can be achieved. The protein adsorbs onto the surface of the particles and is physically entrapped within the scaffold as it forms. Release of the protein occurs via diffusion through the pores of the scaffold and then as the scaffold erodes due to polymer degradation. In terms of ease of preparation, this method is similar to incorporation of growth factors into hydrogels, and is in contrast to most particle-based delivery systems which rely on the more complex process of growth factor encapsulation (Kirby et al., 2011).

The protein-polymer interaction between BMP-2 and PLGA microparticles has been described as being a combination of hydrophobic, ionic and hydrogen-bonding interactions (Duggirala et al., 1996). By mixing the BMP-2 solution with the PLGA/PEG particles prior to scaffold formation, the BMP-2 interacts with the PLGA/PEG particles by adsorption and/or absorption. In the current study, BMP-2 was successfully loaded into PLGA/PEG scaffolds, and the results demonstrate the ability of the scaffolds to release BMP-2 in a sustained manner over a 22-day time period in vitro. The released BMP-2 yielded high ALP activity in cultures of C2C12 cells, as observed in the ALP activity assay. This demonstrates that the BMP-2 released from the PLGA/PEG scaffolds is active and capable of inducing osteogenic differentiation.

To date, most studies on BMP-2 delivery for bone repair have used a relatively high dose, which makes treatment costly and raises the issue of safety (Bodde et al., 2008). The loading doses previously used have ranged from several tens to several hundreds of micrograms (Miki and Imai, 1996; Hollinger et al., 1998; Schmoekel et al., 2004; Bodde et al., 2008). Recent studies have investigated the use of lower doses in the range 0.5–5 µg (Chung et al., 2007; Issa et al., 2008; Young et al., 2009; Patterson et al., 2010).

In this study, the PLGA/PEG scaffolds were loaded with 1 µg BMP-2. Using this low dose of BMP-2, and without cell delivery, we achieved 55% increase in bone volume compared to empty defects, 6 weeks after implantation. Interestingly, PLGA/PEG scaffolds alone resulted in 31% increase in bone volume compared to the empty defects group, highlighting the osteoconductive scaffold characteristics which are favourable for bone tissue-engineering applications.

The results obtained in this in vivo experiment describe substantial bone regeneration in comparison with other studies delivering similar amounts of BMP-2 in a calvarial defect model. For example, only 10–20% bone repair was observed in this model when BMP-2 was delivered using BMP-2-loaded gelatin microparticles incorporated in a poly(propylene fumarate) scaffold (Young et al., 2009). As with our study, 1 µg BMP-2 was loaded per scaffold.

In summary, the current study demonstrates the feasibility of utilizing a novel PLGA/PEG scaffold for BMP-2 delivery supporting bone formation in vivo. This carrier scaffold for BMP-2 can provide a promising surgical tool for bone tissue engineering directed at orthopaedic and cranio-maxillofacial clinical applications.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work has benefited from research funding from the European Community's Seventh Framework Programme in the project Angioscaff NMP-LA-2008-214402, as well as from BBSRC and ERC grants. The excellent technical assistance of Janette Zavin and Dr Srouji Samer is also acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

PLGA/PEG FTIR analysis

Figure S1. Characteristic PLGA absorbance bands are conserved following the melt-blend procedure

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