Induction Of Vascularized Bone Grafts In Muscle Using RhBMP-7 And RhVEGF-165

BackgroundsrhBMP-7,a kind of osteoinductive factor,can induce heterotopic bone formation in muscles,when being carried on BioOss which is a kind of bovine bone minerals.In this way,a vascularized tissue engineered bone graft can be prefabricated in muscles for further transplantation.This technique was applied clinically to prefabricate a large-size tissue engineered bone graft in latissmus dorsi muscle,with which a mandibular defect was successfully repaired.However,the rhBMP-7-induced bone formation as well as vessel density is still low in the central area of the implanted scaffold,especially when the scaffold is large.Therefore,it is necessary and scientifically meaningful to improve bone formation and vascularization of the heterotopic-engineered bone graft.ObjectivesA main objective of this study was to investigate the possibility of improving bone quantity,vascularization and the compression resistance of the in-muscle engineered bone graft by combination of rhBMP-7 and rhVEGF-165,an intense angiogenic factor.The other objective was to investigate the potential advantages of combination of autogenous sponge bone granules(AB) and BioOss over BioOss alone as the scaffold in the heterotopic prefabrication of large engineered bone graft in muscles.MethodsEight house pigs were applied as the experimental model in the present study.Six various groups of scaffold were fabricated.These scaffolds were composed of the external scaffold that was a micro-mesh titanium box of 2.0×2.0×1.0cm~3 in size and the internal scaffold which was filled in the titanium box and varied between different groups of scaffold.The various internal scaffolds were:(1) 660μg rhBMP-7-carrying BioOss granules,(2) BioOss granules and AB granules,(3) 660μg rhBMP-7-carrying BioOss granules and AB granules,(4) 660μg rhBMP-7 and 4μg rhVEGF 165-carrying BioOss granules,(5) 4μg rhVEGF165-carrying BioOss granules and AB granules,and (6) 660μg rhBMP-7 and 4μg rhVEGF165-carrying BioOss granules and AB granules.The six groups of scaffold were implanted into the bilateral lattissmus dorsi muscles in all pigs to prefabricate tissue-engineered bone grafts.Polychrome fluorescence labeling was performed sequently.After 12 weeks the implanted constructs were retrieved and used to make undecalcified sections and standard cylinders. Microradiography,fluorescence microscopy and histological microscopy were performed based on the sections to qualitatively investigate the implanted constructs. Further,histomorphometry was done to examine bone distribution density(BD), residual BioOss density(RBOB),percentage of osteointegrated BioOss(POB) and the early-stage mineral apposition rate(MAR).The cylinders were used to test the compressive strength(CS).ResultsBone formation began at week 2,was active at week 4 and 5 but rare at week 7,as was indicated by polychrome fluorescence labeling.Application of rhVEGF-165,along with rhBMP-7,led to rich and evenly distributed multiple color bands,indicating that rhVEGF-165 propelled early-stage bone formation which was induced by rhBMP-7. However,the application of AB failed to benefit early bone formation.Histologically,the combination of osteogenic and angiogenic factors,as well as the application of AB,led to evenly distributed and mature bone and vessels within the scaffolds.Moreover,the application of AB led to more adipocytes and increased the maturity of the bone structure.The combination of osteogenic and angiogenic factors enhanced osteointegration of the BioOss granules,whatever AB/BioOss or BioOss alone as the internal scaffold.The nonintegrated BioOss granules were degraded by osteoclasts in a very slow rate.When loaded on BioOss granules,the combination of osteogenic and angiogenic factors led to higher BD(31.93%±4.31%) than the application of rhBMP-7 alone (t=3.567,P=0.023).Among the four groups with AB/BioOss as the internal scaffold,the combined application of osteogenic and angiogenic factors reached the highest BD (32.66%±4.55%)(F=3.341,P=0.041).However,the combination of AB and BioOss failed to show any advantage over BioOss alone in BD.When the BioOss granules were loaded with both rhBMP-7 and rhVEGF-165, POB was 67.12%±15.29%and higher than those loaded with rhBMP-7 alone(t=2.866, P=0.045).The application of AB granules failed to enhance POB.The combination of rhBMP-7 and rhVEGF-165 failed to show any advantage over rhBMP-7 alone of improving early-stage mineral apposition rate.However,the rate became higher when AB granules were applied along with rhBMP-7 and rhVEGF-165 (t=2.630,P=0.046).CS was 4.71±1.42MPa when BioOss granules were loaded with both rhBMP-7 and rhVEGF-165,and was higher than when with rhBMP-7 alone(t=2.991,P=0.040). The further application of AB,with CS of 5.84±1.60MPa,seemed to enhance compressive strength,although still not enough to reach statistical significance (P=0.057). Conclusions The combination of rhBMP-7 and rhVEGF-165 benefits the early-stage bone formation in muscles.The combination of rhBMP-7 and rhVEGF-165 enhances mineralization, vascularization and BioOss osteointegration of the heterotopic induced bone graft in muscles.The combination of rhBMP-7 and rhVEGF-165 improves compressive strength of the heterotopic tissue engineered bone graft in muscles.The application of autogenous spongy bone(AB) granules fails to improve bone quantity of the growth factor(s)-induced bone graft in muscles.The application of AB granules improves the maturity of the growth factor(s)-induced bone graft in muscles.The application of AB granules might enhance the compressive strength of the growth factor(s)-induced bone graft in muscles.The osteogenic factor rhBMP-7 and the angiogenic factor rhVEGF-165 are synergic in inducing large-size bone graft in muscles.The present study proves the potentiality of prefabricating a highly mineralized and vascularized tissue-engineered bone graft in heterotopic sites(muscles) by the combination of osteogenic and angiogenic factors,which can be transplanted to repair bone defect.