Repair Of Rat Calvarial Defects Using Recombinant Human Bone Morphogenetic Protein-2/Nacre/Poly(DL-lactic Acid-co-glycolic Acid) Scaffold

Objective: To prepare Nacre/Poly-lactide-co-glycolic acid (Nacre/PLGA) scaffoldcombined with recombinant human bone morphogeneticprotein-2(rhBMP-2), and tostudy the biocompatibility and osteogenic activity of the scaffold in vitro and vivo.Method:1. Different proportional nacre powder was mixed with PLGA by phaseseparation/salt leaching technology. The morphology of rhBMP-2/Nacre/PLGAscaffold was detected by SEM. In addition, porosity, hydrophilic, biomechanicalstrength were tested. The release characteristics of rhBMP-2in the Nacre/PLGAscaffold which has the best mechanical properties were tested by ELISA.2. MC3T3-E1cells were cultured in the extracts which were obtained from rhBMP-2/Nacre/PLGA, and the relative growth rate was measured by CCK-8assay. Adhesionof the cells in the material surface was detected by scanning electron microscope, aswell as the situation of cells growth in the material was observed by fluorescentmicroscope. Mineralized nodules induced by MC3T3-E1cells were detected withAlizarin red stained. In addition, the area of calcium nodules was measured by IPPsoftware. The material was implanted subcutaneously in the New Zealand rabbitmodels. Inflammatory response and ectopic bone forming capability were detected byHE slice in1and4weeks, as well as comparing with the control groups.3. Two critical bone defects were established on the Sprague-Dawley rat cranial. Thescaffolds were implanted in the defects, and the rats were sacrificed at6and12weeks.The interface between material and bone were observed by SEM. The penetratingstrength of new bone was measured by universal testing machines at6and12weeks.New bone was detected by CBCT and histology at6and12weeks. The results werecompared with Nacre/PLGA groups.Results:1. Nacre/PLGA scaffolds were prepared successfully by phase separation/salting technology. The composite possessed three-dimensional porous structure withlarge pore size of150~250μm and small pore size of10to30μm, all of the porositieswere higher than86%. No statistically significant difference was found in the fivegroups. No significant difference was found among1:1,3:2,2:1of Nacre/PLGA groups in hydrophilic (P>0.05), however, significant difference were foundcompared with pure PLGA group. The3/2group of Nacre/PLGA showed goodmechanical strength, and the strength were3.8718±0.1981MPa,15.7334±1.0198MPa and4.0468±0.0894MPa for compression strength, flexural strength andtensile strength respectively. A burst release phenomenon of rhBMP-2in the3/2group was detected in the first seven days, and rhBMP-2was released stably afterthen.2All growth rates of MC3T3-E1cells cultured in extract of rhBMP-2/Nacre/PLGAon day1,3,5,7were more then92%, and the toxic levels were1grade. MC3T3-E1cells can growth on the surface and in the porosity of the rhBMP-2/Nacre/PLGAcomposite. MC3T3-E1cells differentiation were induced by the extracts ofrhBMP-2/Nacre/PLGA, and the difference in area of mineralized nodules betweenrhBMP-2/Nacre/PLGA and Nacre/PLGA was statistically significant (P <0.05). Theinflammatory response was not observed but Ectopic bone formation was detected atthe forth week in rhBMP-2/Nacre/PLGA groups.3. A critical bone defect with the diameter of5mm at both sizes was established incranial of SD rats. The interface between rhBMP-2/Nacre/PLGA and autologous bonewas superior to the Nacre/PLGA. There was statistically significant difference inpenetrating strength of the new bone between rhBMP-2/Nacre/PLGA and Nacre/PLGA at6weeks and12weeks (P <0.05). The HE slice display showed that the newbone grew into the material when it was implanted into the critical crania defects at6weeks. The new bone which was induced by rhBMP-2/Nacre/PLGA were integratedinto autologous bone at12weeks, the result indicated that the osteogenesis effect ofrhBMP-2/Nacre/PLGA was better than Nacre/PLGA.Conclusion: rhBMP-2/Nacre/PLGA scaffold prepared by the phase separation/saltleaching technology has good biological property, biocompatibility, and it can repaircritical crania defects in rats effectively. The rhBMP-2/Nacre/PLGA could be apromising scaffold material for bone tissue engineering.