Objective: To investigate the optimal mixing ratio of recombinant human bone morphogenetic protein 2 (rhBMP-2) with porous calcium phosphate cement (PCPC) and autologous bone as bone grafting material for the repair of large bone defects using Masquelet technique. The effect of platelet-rich plasma (PRP) on the healing of bone defects was evaluated under the optimal ratio of mixed bone.
Methods: Fifty-four New Zealand White rabbits were taken to establish a 2 cm long bone defect model of the ulna and treated using the Masquelet technique. Two parts of the experiment were performed in the second phase of the Masquelet technique. First, 36 modeled experimental animals were randomly divided into 4 groups ( n=9) according to the mass ratio of autologous bone and rhBMP-2/PCPC. Group A: autologous bone (100%); group B: 25% autologous bone+75% rhBMP-2/PCPC; group C: 50% autologous bone+50% rhBMP-2/PCPC; group D: 75% autologous bone+25% rhBMP-2/PCPC. The animals were executed at 4, 8, and 12 weeks postoperatively for general observation, imaging observation, histological observation (HE staining), alkaline phosphatase (ALP) activity assay, and biomechanical assay (three-point bending test) were performed to assess the osteogenic ability and to determine the optimal mixing ratio. Then, 18 modeled experimental animals were randomly divided into 2 groups ( n=9). The control group was implanted with the optimal mixture ratio of autologous bone+rhBMP-2/PCPC, and the experimental group was implanted with the optimal mixture ratio of autologous bone+rhBMP-2/PCPC+autologous PRP. The same method was used to observe the above indexes at 4, 8, and 12 weeks postoperatively.
Results: The bone healing process from callus formation to the cortical connection at the defected gap could be observed in each group after operation; new bone formation, bridging with the host bone, and bone remodeling to normal bone density were observed on imaging observation; new woven bone, new capillaries, bone marrow cavity, and other structures were observed on histological observation. The ALP activity of each group gradually increased with time ( P<0.05); the ALP activity of group A was significantly higher than that of the other 3 groups at each time point after operation, and of groups C and D than group B ( P<0.05); there was no significant difference between groups C and D ( P>0.05). Biomechanical assay showed that the maximum load in three-point bending test of each group increased gradually with time ( P<0.05), and the maximum loads of groups A and D were significantly higher than that of groups B and C at each time point after operation ( P<0.05), but there was no significant difference between groups A and D ( P>0.05). According to the above tests, the optimal mixing ratio was 75% autogenous bone+25% rhBMP-2/PCPC. The process of new bone formation in the experimental group and the control group was observed by gross observation, imaging examination, and histological observation, and the ability of bone formation in the experimental group was better than that in the control group. The ALP activity and maximum load increased gradually with time in both groups ( P<0.05); the ALP activity and maximum load in the experimental group were significantly higher than those in the control group at each time point after operation ( P<0.05), and the maximum load in the experimental group was also significantly higher than that in group A at 12 weeks after operation ( P<0.05).
Conclusion: In the second phase of Masquelet technique, rhBMP-2/PCPC mixed with autologous bone to fill the bone defect can treat large bone defect of rabbit ulna, and it has the best osteogenic ability when the mixing ratio is 75% autologous bone+25% rhBMP-2/PCPC. The combination of PRP can improve the osteogenic ability of rhBMP-2/PCPC and autologous bone mixture.
目的: 探讨使用Masquelet技术修复大段骨缺损时，使用重组人BMP-2（recombinant human BMP-2，rhBMP-2）复合多孔磷酸钙人工骨（porous calcium phosphate cement，PCPC）与自体骨作为植骨材料具有最佳成骨能力时的混合比例；并评估在最佳混合比例下联合应用富血小板血浆（platelet-rich plasma，PRP）对骨缺损愈合的影响。.
方法: 取54只新西兰大白兔建立尺骨2 cm长骨缺损模型，利用Masquelet技术进行治疗，在Masquelet技术第二阶段进行两部分实验。首先取36只已造模实验动物，根据植入自体骨及rhBMP-2/PCPC质量比不同随机分为4组（ n=9）。A组：自体骨（100%）；B组：25%自体骨+75%rhBMP-2/PCPC；C组：50%自体骨+50%rhBMP-2/PCPC；D组：75%自体骨+25%rhBMP-2/PCPC。术后4、8、12周处死动物行大体观察、影像学检查、组织学观察（HE染色）、ALP活性检测及生物力学检测（三点弯曲试验）评估成骨能力，确定自体骨和rhBMP-2/PCPC最佳混合比例。然后取18只已造模实验动物随机分为2组（ n=9），对照组植入最佳混合比例自体骨+rhBMP-2/PCPC，实验组植入最佳混合比例自体骨+rhBMP-2/PCPC+自体PRP。术后4、8、12周同上法进行各项指标观测。.
结果: 术后各组大体观察到缺损断端骨痂形成至骨皮质连接的骨愈合过程；影像学检查可见新骨生成、与宿主骨形成桥接以及骨质重塑至正常骨密度；组织学观察可见术后形成新的编织骨、新生毛细血管、骨髓腔等结构；各组ALP活性随时间延长均逐渐增加（ P<0.05），术后各时间点A组ALP活性显著高于其余3组，C、D组高于B组（ P<0.05），C、D组间差异无统计学意义（ P>0.05）；生物力学检测表明各组三点弯曲试验最大载荷随时间延长均逐渐增加（ P<0.05），术后各时间点A、D组最大载荷显著高于B、C组（ P<0.05），A、D组间差异无统计学意义（ P>0.05）。根据上述检测结果得出最佳混合比例为75%自体骨+25%rhBMP-2/PCPC。后续实验大体观察、影像学检查及组织学观察均观察到实验组和对照组新骨形成过程，且实验组成骨能力优于对照组；随时间延长，两组ALP活性和最大载荷均逐渐增加（ P<0.05），术后各时间点实验组ALP活性和最大载荷均显著高于对照组（ P<0.05），术后12周实验组最大载荷亦显著高于A组（ P<0.05）。.
结论: 在Masquelet技术第二阶段，采用75%自体骨+25%rhBMP-2/PCPC混合物填充骨缺损部位治疗兔尺骨大段骨缺损具有最佳成骨能力；结合PRP可以提高rhBMP-2/PCPC与自体骨混合物的成骨能力。.
Keywords: Bone defect; Masquelet technique; platelet-rich plasma; porous calcium phosphate cement; rabbit; recombinant human bone morphogenetic protein 2.