Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part B: Delivery systems for BMPs in orthopaedic and craniofacial tissue engineering

Localized and release-controlled delivery systems for the sustained expression of the biologic potency of rhBMPs are essential. A substantial number of biomaterials have been investigated thus far. Most fail after implantation or administration mainly due to either being too soft, difficult to control and/or stabilize mechanically. In the second part of this review, we review a representative selection of rhBMP-2 and rhBMP-7 carrier materials and delivery systems ranging from simple nano/microparticles to complex 3-D scaffolds in sites of orthopaedic and craniofacial bone regeneration and repair.     

Biological activity of rhBMP-2 released from PLGA microspheres

Human recombinant bone morphogenetic protein-2 (rhBMP-2) has been proven effective in stimulating the regeneration of bone in both skeletal and extraskeletal locations. Through encapsulation within, and release from, biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) microspheres, a proven vehicle for sustained delivery of various proteins, the local concentrations of rhBMP-2 could be maintained at optimal levels to stimulate bone regeneration and remodeling at the site of healing in diverse clinical settings. Thus the purpose of this work was to investigate the encapsulation of rhBMP-2 in PLGA microspheres and its biologic activity upon release. Using in vitro tests in simulated body fluids, the effect of rhBMP-2 released from PLGA microspheres upon osteoblast cell cultures was found to be statistically similar to the effect produced by positive controls consisting of nonencapsulated aqueous rhBMP-2 in simulated body fluids. This clarifies an important step in skeletal tissue engineering strategies aimed at the use of encapsulated rhBMP-2 to stimulate bone regeneration and remodeling.     

Ectopic bone formation in titanium mesh loaded with bone morphogenetic protein and coated with calcium phosphate

The osteoinductive properties of porous titanium fiber mesh, with or without a calcium phosphate coating and loaded with recombinant human bone morphogenic protein-2 (rhBMP-2) or rhBMP-2 and native bovine BMP (S-300) were investigated in a rat ectopic assay model. A total of 112 calcium phosphate-coated and 112 noncoated porous titanium implants, either loaded with rhBMP-2 and S-300 or loaded with rhBMP-2 alone, were subcutaneously placed in 56 Wistar-King rats. The rats were killed 5, 10, 20, and 40 days postoperatively, and the implants were retrieved.Histologic analysis demonstrated that all growth factor and carrier combinations induced ectopic cartilage and bone formation at 5 and 10 days, respectively. At 20 days, bone formation increased and was characterized by trabecular bone and bone marrow-like tissue. At 40 days, more lamellar bone and hemopoietic bone marrow-like tissue were present. At both times, more bone had been formed in calcium phosphate-coated implants than in noncoated samples. Further, in rhBMP-2 and S-300-loaded specimens, bone formation was higher than in rhBMP-2 only-loaded specimens. In rhBMP-2 only-loaded specimens, bone formation was mainly localized inside the mesh material, whereas in specimens loaded with both rhBMP-2 and S-300, the bone was localized inside and surrounding the titanium mesh. The histological findings were confirmed by calcium content and alkaline phosphatase activity measurements. In addition, all specimens showed osteocalcin expression as early as 5 days postoperatively.Our results show that the combination of titanium mesh with BMPs can induce ectopic bone formation and that this bone formation seems to be similar to "enchondral" ossification. In addition, a thin calcium phosphate coating can have a beneficial effect on the bone-inducing properties of a scaffold material. Finally, rhBMP-2 and native BMP act synergistically in ectopic bone induction.     

A biodegradable polymer as a cytokine delivery system for inducing bone formation

Bone morphogenetic proteins (BMPs) that have the potential to elicit new bone in vivo have been used in a tissue-engineering approach for the repair of bone injuries and bone defects. Although it is now possible to generate large amounts of recombinant human (rh) BMPs for medical use, the major challenge remains in the development of optimal local delivery systems for these proteins. Here we describe the development of a synthetic biodegradable polymer, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG). This polymer exhibits promising degradation characteristics for BMP delivery systems and good biocompatibility under test conditions. PLA-DX-PEG/rhBMP-2 composite implants induced ectopic new bone formation effectively when tested in vivo, and can repair large bone defects orthotopically. This polymeric delivery system represents an advance in the technology for the enhancement of bone repair.     

Effect of guided bone regeneration on bone quality surrounding dental implants

Bone quality as well as its quantity at the implant interface is responsible for determining stability of the implant system. The objective of this study is to examine the nanoindentation based elastic modulus (E) at different bone regions adjacent to titanium dental implants with guided bone regeneration (GBR) treated with DBM and BMP-2 during different post-implantation periods. Six adult male beagle dogs were used to create circumferential defects with buccal bone removal at each implantation site of mandibles. The implant systems were randomly assigned to only GBR (control), GBR with demineralized bone matrix (DBM), and GBR with DBM + recombinant human bone morphogenetic protein-2 (rhBMP-2) (BMP) groups. Three animals were sacrificed at each 4 and 8 weeks of post-implantation healing periods. Following buccolingual dissection, the E values were assessed at the defects (Defect), interfacial bone tissue adjacent to the implant (Interface), and pre-existing bone tissue away from the implant (Pre-existing). The E values of BMP group had significantly higher than control and DBM groups for interface and defect regions at 4 weeks of post-implantation period and for the defect region at 8 weeks (p < 0.043). DBM group had higher E values than control group only for the defect region at 4 weeks (p < 0.001). The current results indicate that treatment of rhBMP-2 with GBR accelerates bone tissue mineralization for longer healing period because the GBR likely facilitates a microenvironment to provide more metabolites with open space of the defect region surrounding the implant.     

Bone neogenesis in domes made of expanded polytetrafluoroethylene: efficacy of rhBMP-2 to enhance the amount of achievable bone in rats

For bone reconstructive purposes, it would be a great advantage to be able to gain bone without grafting. In experimental studies, barrier membranes have been used to accomplish this, however, with limited efficacy. In this study, the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the early onset of bone formation, as well as on the final amount of achievable bone, was investigated in an experimental osteo-neogenesis model. In 60 adult rats, dome-shaped barrier membranes made of expanded polytetrafluoroethylene (Gore-Tex membrane), with an inside volume of approximately 60 mm3, were placed on the left parietal bone. The domes were pretreated according to four different alternatives: (1) filled with autogenous blood only (n = 15); (2) filled with 5 microg of rhBMP-2 in an absorbable collagen sponge carrier (n = 15); (3) filled with 15 microg of rhBMP-2 in absorbable collagen sponge carrier (n = 15); or (4) filled with the absorbable collagen sponge carrier only (n = 15). The animals treated according to each alternative were then divided into three equal groups with five rats in each, and subsequently killed after 3, 6, or 12 weeks. The amount of bone formed within the domes was evaluated by light microscopy and computer-assisted image analysis. It was found that the amount of newly formed bone could be enhanced by approximately 100 percent by simultaneous implantation of rhBMP-2, irrespective of dose. The early onset of bone formation was, however, not affected by the rhBMP-2 supplementation. This finding was interpreted as being due to the delivery system used, because as long as the carrier was still present, no significant difference between the treatment groups was observed. The bone formed in domes with carrier implantation, with or without rhBMP-2, displayed more marrow spaces in comparison to controls. The combined treatment with barrier membranes and local delivery of rhBMP-2 may be a useful tool in reconstructive surgery, for instance replacing onlay grafting, especially when a more delicate anatomy is necessary, because membranes can be shaped in multiple ways.     

Bone morphogenetic protein-2 binds as multilayers to a collagen delivery matrix: an equilibrium thermodynamic analysis

Recombinant human bone morphogenetic protein-2 (rhBMP-2) promotes bone growth but must be retained at the delivery site for optimal efficacy in vivo. rhBMP-2 release from a collagen-based matrix has shown favorable pharmacokinetics. The present study assessed binding affinity and binding saturation of rhBMP-2 to a collagen matrix as a function of solution and rhBMP-2 isoform variables. Results indicate that rhBMP-2 binds to the collagen matrix with affinities on the order of 10(3) to 10(4) M(-1). Maximum binding, nu, was primarily a function of pH for heterogeneous rhBMP-2 and the extended (T(266)/T(266)) isoform. However, binding saturation of the 相似文献   

Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects

AimsBone defects induced by different causes are difficult to replace and repair. We sought to repair bone defects by transplantation of genetically modified adipose-derived stem cells (ADSC) and acellular bone matrix (ACBM).MethodsWe constructed the biologic material of ACBM and evaluated its mechanical properties, general biocompatibility and biosafety. ADSC isolated from minipigs were cultured in vitro and then transfected by recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) plasmids, respectively. Subsequently, the compounds of ACBM/ADSC/rhBMP-2/rhVEGF were used to repair bone defects of the ulna in minipigs. X-ray examination, radionuclide bone imaging and single photon emission computerized tomography (SPECT) were employed to monitor the therapeutic effects 2, 4, 8 and 12 weeks after operation. Histologic experiments were carried out 12 weeks after operation.ResultsACBM had no or weak antigenicity and the natural mechanical properties of ACBM were preserved. In vitro, ADSC transfected by rhBMP-2 and rhVEGF, respectively, could release rhBMP-2 or rhVEGF for at least 4 weeks. The X-ray, radionuclide bone imaging and SPECT examinations indicated that the compound of ACBM/ADSC/rhBMP-2/rhVEGF had better treatment effects on bone defects compared with the controls.ConclusionsScaffolds, seed cells and bioactive factors are key points in tissue engineering. This research indicates that ACBM is a good biologic material for tissue repair, and ACBM/ADSC/rhBMP-2/rhVEGF can accelerate bone formation significantly.     

Repair of bone defects using synthetic mimetics of collagenous extracellular matrices

We have engineered synthetic poly(ethylene glycol) (PEG)-based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.     

Reconstruction of radial bone defect using gelatin sponge and a BMP-2 combination graft

Many bioactive molecules like recombinant human bone morphogenetic protein 2 (rhBMP-2) have been developed for mineralized bone grafts, for which proper scaffolds are necessary to successfully apply the bioactive molecules. In this study, we tested the osteogenic efficacy of rhBMP-2 produced in-house in combination with gelatin sponge as the scaffold carrier in a rabbit radial defect model. The efficacy of the rhBMP-2 was determined by alkaline phosphatase activity assay of C2C12 cells. Two groups of ten rabbits each were treated with rhBMP-2/gelatin sponge, or gelatin sponge only. At 4 weeks, rhBMP-2/gelatin sponge grafts showed more bone regeneration than gelatin sponge grafts, as determined by X-ray radiography, micro-computed tomography, and histological analyses. At 8 weeks, rhBMP-2/gelatin sponge grafts exerted much stronger osteogenic effects. The study demonstrates the improved osteogenic efficacy of the rhBMP-2/gelatin sponge grafts in a rabbit radial bone defect model acting as a bone-inductive material. [BMB Reports 2013; 46(6): 328-333]     

Protein-based tissue engineering in bone and cartilage repair

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.     

Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction

In this study, an attempt was made to transform a muscle vascularized pedicle raised on host vessels into a vascularized bone flap, using recombinant human bone morphogenetic protein 2 (rhBMP-2). The purpose of this study was to produce new bone vascularized in nature to increase the survival rate of the subsequently grafted bone and to fabricate the newly formed bone into the desired shape. Silicone molds in the shape of a rat mandible were used to deliver rat bone matrix impregnated with or without rhBMP-2. A muscle pedicle the same size as the mold was raised on the saphenous vessels in the rat thigh and then sandwiched in the center of the silicone molds. The molds were sliced in half and each section was filled with rat bone matrix that was impregnated either with 25 microg of rhBMP-2 for the experimental group or with diluting material alone for the control group. The sandwiched flaps were then secured by tying them to the adjacent muscles and were harvested at 2 and 4 weeks after surgery. Three and six rats were used in the control and experimental groups at each time point, respectively. Bone formation was assessed in the ex vivo specimens by macroscopic, radiologic, and histologic evaluation. Macroscopically, the continuation of the vascular pedicle was clearly visible for both the control and experimental muscle flaps. However, no evidence of muscle-tissue transformation was observed in the control flaps, whereas all the flaps treated with rhBMP-2 produced new bone that replicated the shape of the mold exactly and had saphenous vessels supplying the newly formed bone. This study demonstrates that this experimental model has the potential to be therapeutically applied for effective bone reconstruction.     

Surface-induced conformational and functional changes of bone morphogenetic protein-2 adsorbed onto single-walled carbon nanotubes

Efficient immobilization of bone morphogenetic protein-2 (BMP-2) onto matrix is of crucial importance in the development of BMP-2-based bone tissue scaffold/implant. This often ties with precise control of desirable protein conformation and retention of protein activity. Recently, great attentions were paid to the regulation of protein conformation by tailoring the nanoscale surface properties. In this contribution, with hydrophilic COOH- and hydrophobic CH₃-terminated single-walled carbon nanotubes (SWNTs-COOH and SWNTs-CH₃) as models, we investigated the nanoscale interface-induced changes of adsorption dynamics, conformation, and bioactivity of recombinant human BMP-2 (rhBMP-2). Our data showed that SWNTs-COOH and SWNTs-CH₃ bound rapidly to and induced unfolding of rhBMP-2 molecules, which promoted their interactions with corresponding receptors on cell surface and thus enhanced their bioactivities. In contrast, rhBMP-2 showed stronger affinity to the COOH-terminated surface than that terminated with CH₃ groups, while better enhanced bioactivity on the SWNTs-CH₃ surfaces. After released from SWNTs, the unfolded rhBMP-2 refolded and their activities from SWNTs-COOH and SWNTs-CH₃ were reduced to 90% and 70% of the native rhBMP-2, respectively. Based on these results obtained, a model of the binding characteristics of rhBMP-2 onto SWNTs with different chemistry is presented. This study demonstrates the possibility of simple tailor-made nanoscale chemical surfaces to modulate the binding, conformation and bioactivity of BMP-2, allowing fabrication of BMP-2-based bone tissue scaffolds with high osteoinductivity and low BMP-2 dosage.     

A new technique for seeding chondrocytes onto solvent-preserved human meniscus using the chemokinetic effect of recombinant human bone morphogenetic protein-2

Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients?? cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human bone morphogenetic protein-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto 10 ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering.     

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days. With the unembedded microspheres releasing faster than those embedded in CMC In vivo. the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2 PLGA microspheres in CMC was an effective implantable protein-delivery system for the use in bone repair. Published: October 7. 2001.     

Effects of rhBMP-2 and osteopromotive membranes on experimental bone grafting.

A problem with using autogeneic bone for onlay grafting is its degree of incorporation and volumetric persistence. This study explored whether improved graft volumetric maintenance can be achieved by recombinant human bone morphogenetic protein-2 (rhBMP-2) and whether a combination of rhBMP-2 and osteopromotive membranes is advantageous in this respect. Unicortico-cancellous bone grafts were positioned below the temporal muscle bilaterally in 48 adult Lewis rats. The recipient bone was ground, and the grafted area was randomly treated according to one of eight different alternatives. Two doses of rhBMP-2 (4 microg/80 microl or 32 microg/80 microl buffer) in a collagenous carrier were tested, with or without coverage with expanded polytetrafluoroethylene membranes. As controls, membrane and carrier, alone or in combination, and onlay grafts only were used. The results were evaluated after 4 and 20 weeks by routine histologic examination and immunohistochemical labeling for various bone and cartilage matrix proteins. After 4 weeks, rhBMP-2-treated grafts showed complete integration, whereas for controls, only the membrane-alone group attained incorporation after 20 weeks. The combined treatment with high-dose rhBMP-2 and membrane demonstrated, as compared with the remaining rhBMP-2 groups, pronounced bone formation and less graft resorption, resulting in maintained or increased graft size. This was observed after 4 weeks, and the result remained at 20 weeks. Treatment with high-dose rhBMP-2 without membrane placement, in contrast, resulted in extensive resorption and graft size reduction at 20 weeks. Control groups showed less success in graft size persistence. It was concluded that a combination of high-dose rhBMP-2 and osteopromotive membranes had a synergistic effect, leading to rapid, complete graft integration and size maintenance. In the absence of a membrane, the protein seemed to accelerate the remodeling of the graft.     

Engineering clinically relevant volumes of vascularized bone

Vascularization remains one of the most important challenges that must be overcome for tissue engineering to be consistently implemented for reconstruction of large volume bone defects. An extensive vascular network is needed for transport of nutrients, waste and progenitor cells required for remodelling and repair. A variety of tissue engineering strategies have been investigated in an attempt to vascularize tissues, including those applying cells, soluble factor delivery strategies, novel design and optimization of bio‐active materials, vascular assembly pre‐implantation and surgical techniques. However, many of these strategies face substantial barriers that must be overcome prior to their ultimate translation into clinical application. In this review recent progress in engineering vascularized bone will be presented with an emphasis on clinical feasibility.     

Prefabrication of bone by use of a vascularized periosteal flap and bone morphogenetic protein.

The purpose of this pilot study was to prefabricate a vascularized bone graft by using a vascularized periosteal flap containing osteoprogenitor cells, a structural matrix, and recombinant human bone morphogenetic protein-2 (rhBMP-2). In a rat model, a periosteal flap vascularized by the saphenous artery and vein was dissected off the medial surface of the tibia. This flap consisted of three layers-periosteum, muscle, and fascia-and was tubed on itself to form a watertight chamber that was then transferred on its vascular pedicle to the groin. A total of 78 vascularized periosteal chambers were constructed in 39 animals and divided into 10 groups. In group 1, the periosteal chamber was left empty. Groups 2, 3, and 4 consisted of the periosteal flap and rhBMP-2, but in group 3, the proximal vascular pedicle was ligated, and in group 4, the flap was harvested without the periosteal layer and turned inside out. Groups 5 through 10 consisted of the vascularized periosteal flap containing several different structural matrices (calcium alginate spheres, polylactic acid, or demineralized bone matrix) with or without rhBMP-2. Animals were killed at 2, 4, or 8 weeks in each group. The presence and density of any new bone formation was evaluated both radiologically and histologically. Significant bone formation was seen only in those periosteal flaps containing rhBMP-2 and either the calcium alginate or polylactic acid matrix. New bone formation increased both radiologically and histologically from 2 weeks to 8 weeks only in the periosteal flaps containing the polylactic acid matrix and rhBMP-2. This preliminary study therefore suggests that four factors-blood supply, osteoprogenitor cells in the periosteal layer, a biodegradable matrix, and rhBMP-2-are required for optimal prefabrication of a vascularized bone graft.