| It is still a great challenge to repair defects of the craniomaxillofacial skeleton. Autogenous bone grafts and allografts, in spite of their acceptable reparation effect, are limited by their intrinsic weakness. Self-setting calcium phosphate cement (CPC) is a kind of bone substitute with excellent osteoconductivity, biocompatibility and malleability. It also has capacity to carry bioactive factors. Yet its micropores are too small, its porosity is low and its degradation is too slow. The bioactive factor loaded on CPC is released so slowly, resulting in poor bioactive effect. Therefore, the present study was:(1) to explore the feasibility of improving the physical and chemical properties of CPC, as well as the release of its carrying bioactive factor, through incorporating rhBMP-2(and rhVEGF165)-loaded gelatin microparticles (GM) into CPC,(2) to investigate the effect of rhBMP-2(and rhVEGF165)-loaded CPC/GM composite biomaterial in the reparation of the critical-size cranial bone defect and in the heterotopic bone regeneration in muscles. Part1:Fabrication and feature study of the rhBMP-2-loaded CPC/GM composite biomaterialThe rhBMP-2/CPC/GM and rhBMP-2/GM cylinders were examined by SEM scanning, compression test and porosity measuring. Their parameters like setting time, surface feature, compression strength, porosity were compared. The results showed that the rhBMP-2/CPC/GM composite biomaterial possessed an extended setting time (30.3±1.7min vs.37.8±2.1min) and decreased compression strength (13.18±2.60MPa vs.30.05±5.84MPa). However, its porosity was significantly improved (44.83%±7.99%vs.18.63%±4.26%). Moreover, macropores of100-350μm in size formed after the gelatin microparticles melted. These are the features beneficial to new bone in-growth.Part2:Biocompatibility of the composite biomaterial and release dynamics and bioactivity of the loading bioactive factorsAt first, various composite biomaterials were immersed in PBS and the bioactive factor release of the biomaterials was examined by ELISA of the soaking solution. Then, the cell culture media that were soaked with various biomaterials (rhBMP-2/CPC, rhBMP-2/CPC/GM and CPC/GM) were used to culture rabbit bone marrow stromal cells (BMSCs). Their effects on cell proliferation and differentiation were study with MTS and ALP activity measuring. The results demonstrated that, from the third day on, rhBMP-2release were higher from the rhBMP-2/CPC/GM cylinders than from the rhBMP-2/CPC ones. An almost linear relation was observed between the cumulative growth factor release and time from d7to d28. The soaking media showed neither negative nor positive effect on cell proliferation. The soaking media of rhBMP-2/CPC and rhBMP-2/CPC/GM both induced osteogenic differentiation of rBMSCs, indicated by their improved ALP activities. The results also showed that the bioactivity was dose-dependent. Part3:Bioactive factor-carrying CPC/GM composite biomaterials in repairing critical-size cranial bone defects in the rabbit modelThe rabbit critical-size cranial bone defects (Φ8mm) were filled with various kinds of paste (GM/CPC, rhBMP-2/GM/CPC, rhVEGF165/GM/CPC and rhBMP-2/rhVEGF165/GM/CPC). The specimens were harvested after12weeks and investigated with histology and histomorphometry. The results showed that bone formation and material degradation were improved by the bioactive factor loading. There was a synergic effect between rhBMP-2and rhVEGF165in this bone repairing model.Part4:Bioactive factor-loading CPC/GM composite biomaterials in heterotopical bone regenerationFour groups of scaffolds (GM/CPC, rhBMP-2/GM/CPC, rhVEGF165/GM/CPC and rhBMP-2/rhVEGF165/GM/CPC) were fabricated in the human mandibular condyle shape. The scaffolds were implanted into the latissimus dorsi muscles in7rabbits. The implants were retrieved after12weeks and examined with histology of the decalcified sections. The results showed that there was a little, yet limited, new bone in the groups of scaffolds loaded with rhBMP-2. No bone was found in the other two groups.In summary, the present study proved that the incorporation of bioactive factor-carrying GM into CPC improves porosity and pore size, and consequently the speed of biomaterial degradation, all beneficial to new bone in-growth. The CPC/GM composite biomaterial also has advantage over pure CPC in carrying rhBMP-2and rhVEGF165, with an improved factor release and higher bioactivity. The bioactive factor-carrying CPC/GM composite biomaterials are superior to CPC/GM in repairing cranial bone defect. They also have the potential for bone tissue engineering. |
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