Delivery Of Bone Morphogenetic Protein-2 Research Articles

Effects of the combination of fibroblast growth factor-2 and bone morphogenetic protein-2 on the proliferation and differentiation of osteoprecursor cells.

Fibroblast growth factor (FGF) plays a critical role in bone growth; FGF-2 is known to be an important regulator of osteoblast activity because it stimulates osteoblast replication and decreases differentiation markers. Bone morphogenetic protein-2 (BMP-2) has been shown to be an active inducer of osteoblast differentiation and stimulates expression of mineralization-associated genes. The dose-dependent impact of FGF-2 and BMP-2 on the cellular proliferation and differentiation of osteoprecursor cells was evaluated. The alkaline phosphatase activity (ALP) test was performed to assess differentiation, and protein expressions related to bone formation were measured using the Western blot analysis. Cultures grown in the presence of FGF at 20 ng/mL showed significantly increased value when compared with control group and cultures loaded with FGF-2 at 20 ng/mL, and BMP-2 at 100 ng/mL showed significant decrease in cellular proliferation when compared with cultures loaded with FGF-2 at 20 ng/mL. The ALP activity increased when cells were treated with 10 and 100 ng/mL BMP-2, with relative ALP activity of 213.1% and 312.5%, respectively, when ALP activity of the uncontrolled control was considered 100%. However, when 100 ng/mL BMP-2 was combined with 20 ng/mL FGF-2, the relative increase reached up to 392.2%, but this did not reach a statistically significant increase when compared with 100 ng/mL BMP-2 alone. Within the limits of this study, BMP-2 significantly enhanced osteoblast differentiation but combined delivery of FGF-2 and BMP-2 did not produce synergistic effects on osteoblast differentiation under the current experimental condition.

Silk Fibroin-Based Complex Particles with Bioactive Encrustation for Bone Morphogenetic Protein 2 Delivery

Application of bone morphogenetic protein 2 (BMP-2) currently faces its challenges, and its efficacy of delivery has to be improved. The proper dosage of the powerful bioactive molecule is still under discussion and needs to be investigated further. In this work, pure silk fibroin particles and particles with calcium carbonate encrustation (complex particles) are designed, developed, and functionalized by BMP-2. These are used to deliver the bioactive molecule to mesenchymal stem cells (MSCs) to induce osteogenic differentiation. Results are compared with those of control groups of BMP-2 carriers under the same condition. Silk fibroin-based particles with size and component variations are prepared by self-assembly, desolvation, and soft template formation to improve BMP-2 loading efficiency. Results show that the particles significantly enhance osteogenic differentiation of MSCs, which is evident in the high ALP enzyme activity as well as the increased level of expression of osteogenic genes. Specifically, the combination of calcium compound and BMP-2 in the silk fibroin-calcium carbonate complex particles synergistically enhances osteogenesis. Release tests and mathematical modeling are applied to describe BMP-2 dissolution profiles, and the release mechanism is based on diffusion and polymer chain relaxation. In summary, the particles show high efficacies of BMP-2 delivery, and introduction of the complex particle can progressively enhance osteogenesis.

The effect of carrier for BMP-2 delivery on histological aspects of tissue-engineered bone

Bone morphogenetic protein-2 (BMP-2) that can regenerate bone by recruiting osteo-progenitor cells and inducing osteogenic differentiation has been highlighted as an alternative therapy to treat bone defects. The therapeutic effect of BMP-2 depends considerably on the delivery carriers. Previous studies demonstrated that collagen and fibrin have the potential to serve as a carrier for delivery of BMP-2. The successful bone regeneration by collagen and fibrin with BMP-2 implantation has been shown in animal models. However, those studies were not focused on the effect of the carriers on the quality of the regenerated bone. In this study, we directly compared the quality of regenerated bone when BMP-2 was delivered with collagen or fibrin, both of which are shown to be excellent carrier for BMP-2. The microstructure, surface morphology, BMP-2 release profile was examined in vitro. To evaluate the quality of regenerated bone, both types of BMP-2 carriers were implanted into the dorsal subcutaneous spaces of the mice. Four and six weeks after implantation, the regenerated bone was evaluated by computed tomography, histology, and histomorphometric analysis. The bone regeneration by the collagen with BMP-2 was higher bone density than bone regenerated by the fibrin with BMP-2. The results of this study show that carrier of BMP-2 is an important factor affecting the quality of engineered bone.

Complexation and Sequestration of BMP-2 from an ECM Mimetic Hyaluronan Gel for Improved Bone Formation

Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.

Cartilage Tissue Formation from Dedifferentiated Chondrocytes by Codelivery of BMP-2 and SOX-9 Genes Encoding Bicistronic Vector

Articular cartilage, when damaged by degenerative disease or trauma, has limited ability for self-repair. Recently, many trials have demonstrated that gene therapy combined with tissue engineering techniques would be a promising approach for cartilage regeneration. Bone morphogenetic protein 2 (BMP-2) is an important signal for upregulation of osteogenesis and chondrogenesis of stem cells. Sex-determining region Y box gene 9 (SOX-9) has also been reported as one of the key transcription factors for chondrogenesis. We hypothesized that codelivery of BMP-2 and SOX-9 genes would result in improved efficiency of recovery of normal chondrogenic properties in dedifferentiated chondrocytes. To this aim, we constructed a bicistronic vector encoding the BMP-2 and SOX-9 genes linked to the "self-cleaving" 2A peptide sequence. After gene delivery to dedifferentiated chondrocytes using a microporator transfection system, we confirmed over 65% delivery efficiency of the BMP-2 and SOX-9 genes. According to RT-PCR analysis and Alcian blue staining, simultaneous delivery of BMP-2/SOX-9 resulted in significantly increased expression of chondrogenesis-related markers (type II collagen and aggrecan) and GAG matrix formation compared with individual delivery of the BMP-2 or SOX-9 gene. Six weeks after in vivo transplantation, BMP-2/SOX-9 genes also showed a significant increase in cartilage formation compared with the BMP-2 or SOX-9 gene. These results demonstrate that codelivery of two chondrogenic lineage-determining genes can enhance normal chondrogenic properties of dedifferentiated chondrocytes followed by improved cartilage formation.

Bisphosphonate-Linked Hyaluronic Acid Hydrogel Sequesters and Enzymatically Releases Active Bone Morphogenetic Protein-2 for Induction of Osteogenic Differentiation

Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analogue lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity, which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration.

The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil™) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.

Gene delivery of osteoinductive signals to a human fetal osteoblast cell line induces cell death in a dose-dependent manner.

Gene delivery provides a powerful tool for regulating tissue regeneration by activating or inhibiting specific genes associated with targeted signaling pathways. Up-regulating bone morphogenetic protein-2 (BMP-2) or silencing GNAS and Noggin gene expression in stem cells has been shown to enhance osteogenic differentiation and bone tissue formation. However, few studies have examined how such gene delivery would influence other differentiated cell types residing in the bone. In this study, we examined the effects of DNA delivery of BMP-2 and siRNA delivery of GNAS or Noggin on a widely used human fetal osteoblast cell line (hFOB1.19) using biomaterials-mediated gene delivery. Our results showed that both GNAS and Noggin siRNA delivery increased cell death in hFOB1.19 in a dose-dependent manner. In particular, groups treated with the highest doses of BMP-2, siGNAS or siNoggin showed a more than 50% decline in cell proliferation and a 90% decline in cell viability compared to untransfected and sham DNA/siRNA-transfected controls. TUNEL staining showed that BMP-2, siGNAS or siNoggin induced cell apoptosis in hFOBs. In contrast, cells transfected using sham DNA or siRNA showed no noticeable cell death or apoptosis. These results elucidate the nuanced responses of progenitor and immortalized cell populations to the delivery of exogenous osteoinductive genes. In particular, they highlight the differences between immortalized and primary cell lines and underscore the importance of targeted gene delivery mechanisms in the regeneration of injured bone tissue.

Ex Vivo Bone Morphogenetic Protein‐2 Gene Delivery Using Bone Marrow Stem Cells in Rabbit Maxillary Sinus Augmentation in Conjunction With Implant Placement

This study evaluates the potential of bone morphogenetic protein 2 (BMP-2) gene-transduced bone marrow stem cells (BMSCs) to facilitate osseous healing after rabbit maxillary sinus augmentation in conjunction with implant placement. Autologous BMSCs derived from New Zealand white rabbits were cultured and transduced with BMP-2 using an adenovirus vector. Transduced BMSCs (BMP-2/BMSCs) were then combined with a deproteinized bovine bone mineral (DBBM) scaffold. Twenty-seven animals were randomly allocated into three groups: 1) control, sinus grafted with DBBM alone; 2) BMSC, sinus grafted with non-transduced BMSCs and DBBM; and 3) BMP-2/BMSC, sinus grafted with BMP-2/BMSCs and DBBM. During these procedures, a mini-implant was placed in the floor of the sinus. Animals were sacrificed at 2, 4, and 8 weeks after surgery. New bone area and bone-to-implant contact (BIC) were evaluated histomorphometrically. At 2 and 4 weeks, the BMP-2/BMSC group showed more new bone area and higher BIC than the other two groups. BMP-2/BMSCs were detected with confocal microscopy for up to 4 weeks, which indicates that transduced cells contributed to new bone formation. However, at 8 weeks, there was no difference in new bone area or BIC among the three groups. These results suggest that BMP-2 delivery using BMSCs may result in earlier and increased bone formation in the maxillary sinus. This finding may offer more stable bone support to implants and reduce healing times. However, this study also revealed limitations in the stimulatory effect of BMP-2/BMSCs, such as diminished activity over time in later healing stages.

Dual Delivery of BMP-2 and bFGF from a New Nano-Composite Scaffold, Loaded with Vascular Stents for Large-Size Mandibular Defect Regeneration

The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) from nano-composite scaffolds (PLGA/PCL/nHA) loaded with vascular stents (PLCL/Col/nHA) for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs), bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA). AlamarBlue assay and alkaline phosphatase (ALP) activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01). In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large bone defects in bone tissue engineering.

Creation of nanoporous TiO2surface onto polyetheretherketone for effective immobilization and delivery of bone morphogenetic protein

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

Deproteinized bovine bone functionalized with the slow delivery of BMP-2 for the repair of critical-sized bone defects in sheep

As an alternative to an autologous bone graft, deproteinized bovine bone (DBB) is widely used in the clinical dentistry. Although DBB provides an osteoconductive scaffold, it is not capable of enhancing bone regeneration because it is not osteoinductive. In order to render DBB osteoinductive, bone morphogenetic protein 2 (BMP-2) has previously been incorporated into a three dimensional reservoir (a biomimetic calcium phosphate coating) on DBB, which effectively promoted the osteogenic response by the slow delivery of BMP-2. The aim of this study was to investigate the therapeutic effectiveness of such coating on the DBB granules in repairing a large cylindrical bone defect (8mm diameter, 13mm depth) in sheep. Eight groups were randomly assigned to the bone defects: (i) no graft material; (ii) autologous bone; (iii) DBB only; (iv) DBB mixed with autologous bone; (v) DBB bearing adsorbed BMP-2; (vi) DBB bearing a coating but no BMP-2; (vii) DBB bearing a coating with adsorbed BMP-2; and (viii) DBB bearing a coating-incorporated depot of BMP-2. 4 and 8weeks after implantation, samples were withdrawn for a histological and a histomorphometric analysis. Histological results confirmed the excellent biocompatibility and osteoconductivity of all the grafts tested. At 4weeks, DBB mixed with autologous bone or functionalized with coating-incorporated BMP-2 showed more newly-formed bone than the other groups with DBB. At 8weeks, the volume of newly-formed bone around DBB that bore a coating-incorporated depot of BMP-2 was greatest among the groups with DBB, and was comparable to the autologous bone group. The use of autologous bone and BMP-2 resulted in more bone marrow formation. Multinucleated giant cells were observed in the resorption process around DBB, whereas histomorphometric analysis revealed no significant degradation of DBB. In conclusion, it was shown that incorporating BMP-2 into the calcium phosphate coating of DBB induced strong bone formation around DBB for repairing a critical-sized bone defect.

Adsorbed BMP-2 in polyelectrolyte multilayer films for enhanced early osteogenic differentiation of mesenchymal stem cells

The present work was focused on the development of multilayer films to adsorb and stabilize growth factors initially for delivery to mesenchymal stem cells (MSCs). The multilayer was constructed by poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) via layer-by-layer assembly. The adsorption and bioactivity of bone morphogenetic protein 2 (BMP-2) in the films were explored. Then, the response of MSCs to the adsorbed BMP-2 was evaluated. It was demonstrated that the adsorbed BMP-2 could retain its activity toward enhanced early osteogenic differentiation of MSCs. Alkaline phosphatase (ALP) activity of MSCs seeded on the BMP-2 loaded film was even higher than that on the non-loaded film with BMP-2 added to the culture media. The results confirmed that a localized and sustained delivery of BMP-2 was more effective in vivo. It is noteworthy that these films can be deposited on a wide range of substrates with different shapes, sizes and composition, and can load other growth factors for facilitating stem cell differentiation toward specific lineages. Thus, such a system may find diverse applications in local delivery of immobilized growth factors for medical implants and for tissue-engineered constructs.

Fibroblast Growth Factor-2 and Bone Morphogenetic Protein-2 Have a Synergistic Stimulatory Effect on Bone Formation in Cell Cultures From Elderly Mouse and Human Bone

Combined regimens of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) were investigated to stimulate osteogenic differentiation. In young mouse calvaria-derived cells, FGF-2 (0.16ng/mL) in combination with BMP-2 (50ng/mL) did not enhance mineralization, but in old mouse cells it resulted in more mineralization than BMP-2 alone. In young long bone mouse cultures, FGF-2 enhanced mineralization relative to BMP-2 alone, but in old cultures, lower dose of FGF-2 (0.016ng/mL) was necessary. In neonatal mouse calvarial cells, sequential delivery of low-dose FGF-2 and low-dose BMP-2 (5ng/mL) was more stimulatory than co-delivery. In young human cultures, 0.016ng/mL of FGF-2 did not enhance mineralization, in combination with 5ng/mL of BMP-2, but in older cultures, codelivery of FGF-2 and BMP-2 was superior to BMP-2 alone. In conclusion, BMP-2 treatment alone was sufficient for maximal mineralization in young osteoblast cultures. However, coadministration of FGF-2 and BMP-2 increases mineralization more than BMP-2 alone in cultures from old and young mouse long bones and old humans but not in young mouse calvarial cultures.

Study of Bone Morphogenetic Protein-2 Delivery with Different TiO&lt;SUB&gt;2&lt;/SUB&gt; Nanotube Structures

Study of Bone Morphogenetic Protein-2 Delivery with Different TiO&lt;SUB&gt;2&lt;/SUB&gt; Nanotube Structures

Evaluation of a Thiolated Chitosan Scaffold for Local Delivery of BMP-2 for Osteogenic Differentiation and Ectopic Bone Formation

Thiolated chitosan (Thio-CS) is a well-established pharmaceutical excipient for drug delivery. However, its use as a scaffold for bone formation has not been investigated. The aim of this study was to evaluate the potential of Thio-CS in bone morphogenetic protein-2 (BMP-2) delivery and bone formation. In vitro study showed that BMP-2 interacted with the Thio-CS and did not affect the swelling behavior. The release kinetics of BMP-2 from the Thio-CS was slightly delayed (70%) within 7 days compared with that from collagen gel (Col-gel, 85%), which is widely used in BMP-2 delivery. The BMP-2 released from Thio-CS increased osteoblastic cell differentiation but did not show any cytotoxicity until 21 days. Analysis of the in vivo ectopic bone formation at 4 weeks of posttransplantation showed that use of Thio-CS for BMP-2 delivery induced more bone formation to a greater extent (1.8 fold) than that of Col-gel. However, bone mineral density in both bones was equivalent, regardless of Thio-CS or Col-gel carrier. Taken together, Thio-CS system might be useful for delivering osteogenic protein BMP-2 and present a promising bone regeneration strategy.

Beyond osteogenesis: an in vitro comparison of the potentials of six bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) other than the clinically available BMP-2 and BMP-7 may be useful for improving fracture healing through both increasing osteogenesis and creating a favorable healing environment by altering cytokine release by endogenous cells. Given the spectrum of potential applications for BMPs, the objective of this study was to evaluate various BMPs under a variety of conditions to provide further insight into their therapeutic capabilities. The alkaline phosphatase (ALP) activity of both C2C12 and human adipose-derived stem cells (hASCs) was measured after exposure of increasing doses of recombinant human BMP-2, -4, -5, -6, -7, or -9 for 3 and 7 days. BMPs-2, -4, -5, -6, -7, and -9 were compared in terms of their ability to affect the release of stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (b-FGF) from human bone marrow stromal cells (hBMSCs). Gene expression of ALP, osteocalcin, SDF-1, VEGF, and b-FGF following shRNA-mediated knockdown of BMP-2 and BMP-6 in hBMSCs or human osteoblasts under osteogenic differentiation conditions was also evaluated. Collectively, BMPs-6 and -9 produced the greatest osteogenic differentiation of C2C12 and hASCs as determined by ALP. The hBMSC secretion of SDF-1 was most affected by BMP-5, VEGF by BMP-4, and b-FGF by BMP-2. The knockdown of BMP-2 in BMSCs had no effect on any of the genes measured whereas BMP-6 knockdown in hBMSCs caused a significant increase in VEGF gene expression. BMP-2 and BMP-6 knockdown in human osteoblasts caused significant increases in VEGF gene expression and trends toward decreases in osteocalcin expression. These findings support efforts to study other BMPs as potential bone graft supplements, and to consider combined BMP delivery for promotion of multiple aspects of fracture healing.

Combined delivery of BMP-2 and bFGF from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo

During the process of bone regeneration, a multitude of morphogenetic signaling factors regulate cellular behavior and ultimately tissue response. These factors are presented to cells under strong spatial and temporal control, which stresses the relevance of controlled delivery of multiple growth factors for bone tissue regeneration. This demand for biomimetic delivery has prompted the development of a novel generation of biomaterials that is capable of delivering multiple growth factors in a controlled manner. Therefore, the current study has exploited the strong capacity of colloidal gels solely made of oppositely charged gelatin nanospheres to obtain controlled release of angiogenic and osteogenic growth factors. The release kinetics of dual delivery of osteogenic bone morphogenetic protein-2 (BMP-2) and angiogenic basic fibroblast growth factor (bFGF) were investigated in vitro by radiolabeling the respective growth factors and monitoring their release in vitro. Furthermore, the effect of single or dual delivery of BMP-2 and bFGF on bone regeneration was evaluated in vivo using a rat femoral condyle defect model. The in vitro results confirmed that the delivery kinetics of BMP-2 and/or bFGF are more dependent on the degree of crosslinking than on the type of gelatin. Sequential release characterized by rapid release of angiogenic bFGF and more sustained release of BMP-2 was obtained by loading bFGF onto cationic nanospheres of low crosslinking density and BMP-2 onto anionic nanospheres of high crosslinking density. The in vivo study demonstrated the biocompatibility and biodegradability of bare colloidal gelatin gels, and did not show any adverse effects on the process of bone healing after 4week of implantation since the volumes of new bone formation were comparable to empty control defects. An obvious stimulatory effect on bone regeneration was observed for the colloidal gels loaded with BMP-2, whereas bFGF-loaded colloidal gelatin gels did not influence the rate of bone regeneration. In contrast, the combined delivery of BMP-2 and bFGF resulted into an inhibitory effect on osteogenesis under the current experimental conditions. Summarizing, the current study proved that nanostructured colloidal gelatin gels are suitable carriers for programmed and sustained release of multiple therapeutic proteins for tissue regeneration.

Transient Brown Adipocyte-Like Cells Derive from Peripheral Nerve Progenitors in Response to Bone Morphogenetic Protein 2

Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling.

Evaluation of collagen/heparin coated TCP/HA granules for long-term delivery of BMP-2

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. The purpose of this study was to develop a sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2). We covalently attached heparin to a cross-linked collagen type I coated tricalciumphosphate/hydroxyapatite (TCP/HA) bone substitute and subsequently loaded it with BMP-2. To systematically evaluate the contribution of each component with respect to the binding and release of BMP-2, six constructs were prepared and characterized: TCP/HA, TCP/HA with collagen (TCP/HACol), and TCP/HA with collagen and heparin (TCP/HAColHep) with and without BMP-2 (B). More BMP-2 bound to the TCP/HAColHep + B (92.9 ± 4.8 ng BMP-2/mg granule) granules as compared to the TCP/HACol + B (69.0 ± 9.6 ng BMP-2/mg granule) and TCP/HA + B granules (62.9 ± 5.4 ng BMP-2/mg granule). No difference in release pattern was found between the TCP/HA + B and TCP/HACol + B granules. Up to day 14, BMP-2 was still bound to the TCP/HAColHep + B granules, whereas most BMP had been released from TCP/HACol + B and TCP/HA + B granules at that time. After 21 days most BMP-2 also had been released from the TCP/HAColHep + B granules. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and could possibly enhance bone regeneration efficacy for the treatment of large bone defects.