Masquelet技术联合人工真皮对家兔骨与软组织缺损的治疗研究

Masquelet technique combined with artificial dermis for the treatment of bone and soft tissue defects in rabbits

目的探讨应用Masquelet技术联合人工真皮对家兔骨与软组织缺损的修复效果,观察诱导膜的微观结构及血管化情况,以期指导临床中对Gustilo-Anderson Ⅲ型开放性骨折伴大段骨缺损及软组织缺损的治疗。方法雄性家兔80只,体质量2.03～2.27 kg,平均2.11 kg。取64只兔双侧大腿随机分为实验组及对照组,余16只兔双侧为假手术组。所有兔均制备股骨骨质缺损及软组织缺损模型。实验组以Masquelet技术第一阶段方法[将聚甲基丙烯酸甲酯骨水泥填充于骨缺损区]联合人工真皮治疗;对照组单纯以Masquelet技术第一阶段方法治疗;假手术组不作处理,直接缝合伤口。术后2、4、6、8周各处死假手术组4只家兔,以及实验组/对照组16只家兔,于双侧股骨原手术部位取材,大体观察诱导膜及结合膜情况;对膜结构取材行常规HE染色观察其微观结构;行CD34免疫组织化学染色观察,并计数微血管密度(microvessel density,MVD)。结果大体观察示:术后4周,实验组人工真皮胶原蛋白海绵层完全消失,实验组及对照组均产生完整诱导膜结构;对照组膜结构呈半透明状,实验组膜结构较厚,呈淡红色不透明,伴小血管增生。术后6～8周,实验组和对照组膜结构均逐渐增厚。假手术组随时间变化仅观察到瘢痕组织增生。HE染色示:术后2周,实验组及对照组均可见大量肌纤维,少量胶原纤维增生伴炎性细胞浸润;假手术组大多为肌纤维,伴少量肌纤维间血管。术后4周,实验组较对照组胶原纤维增多,部分血管形成,对照组胶原纤维细胞核呈类圆形,实验组呈扁圆形。术后6、8周实验组和对照组胶原纤维均较前增多,对照组胶原纤维细胞核仍呈类圆形,实验组呈梭形且细胞核较同期对照组深染;两组均可观察到增生血管,实验组增生血管数目较对照组增多。假手术组仍可见大量成纤维细胞出现,未表现随时间变化的血管显著增生。CD34免疫组织化学染色示,术后随时间延长,各组MVD均呈逐渐增加趋势。术后2周假手术组MVD显著大于实验组和对照组,4、6、8周显著小于实验组和对照组,差异均有统计学意义(P<0.05)。实验组术后4、6周MVD显著大于对照组(P<0.05),2、8周两组MVD比较差异无统计学意义(P>0.05)。结论 Masquelet技术结合人工真皮治疗兔股骨骨质缺损及软组织缺损,在术后4～6周明显促进了膜结构的血管化过程。这两种方法结合对临床Gustilo-AndersonⅢ型开放性骨折伴骨及软组织缺损的治疗有指导意义。

Objective To investigate the effect of Masquelet technique combined with artificial dermis on repairing bone and soft tissue defects in rabbits, and to observe the microstructure and vascularization of induced membrane, so as to guide the clinical treatment of Gustilo-Anderson type Ⅲ open fracture with large bone defect and soft tissue defect. Methods Eighty male rabbits, weighing 2.03-2.27 kg(mean, 2.11 kg), were selected. The bilateral thighs of64 rabbits were randomly divided into experimental group and control group, the remaining 16 rabbits were sham operation group. Bone and soft tissue defect models of femur were made in all rabbits. In the experimental group, the first stage of Masquelet technique was used [polymethyl methacrylate bone cement was filled in bone defect area] combined with artificial dermis treatment;in the control group, the first stage of Masquelet technique was used only;in the sham operation group, the wound was sutured directly without any treatment. Four rabbits in sham operation group and 16 rabbits in the experimental group and control group were sacrificed at 2, 4, 6, and 8 weeks after operation, respectively.The induced membranes and conjunctive membranes were observed on both sides of the femur. The membrane structure was observed by HE staining, and the microvessel density(MVD) was counted by CD34 immunohistochemical staining.Results Gross observation showed that the spongy layer of collagen in the artificial dermis of the experimental group disappeared completely at 4 weeks after operation, and the induced membrane structure of the experimental group and the control group was complete;the membrane structure of the control group was translucent, and the membrane structure of the experimental group was thicker, light red opaque, accompanied by small vessel proliferation. The membrane structure of the experimental group and the control group increased gradually from 6 to 8 weeks after operation. In the sham operation group, only scar tissue proliferation was observed over time. HE staining showed that a large number of muscle fibers and a small amount of collagen fibers proliferation with inflammatory cell infiltration could be seen in the experimental group and the control group at 2 weeks after operation;most of the sham operation group were muscle fibers with a small amount of interfibrous vessels. At 4 weeks after operation, collagen fibers increased and some blood vessels formed in the experimental group. The nuclei of collagen fibers in the control group were round-like,while those in the experimental group were flat-round. At 6 and 8 weeks after operation, the collagen fibers in the experimental group and the control group increased. The nuclei of the collagen fibers in the control group were still round-like. The nuclei of the collagen fibers in the experimental group were fusiformis and deeply stained compared with those in the control group. The proliferation of blood vessels was observed in both groups, and the number of proliferation vessels in the experimental group was increased compared with that in the control group. In the sham operation group, a large number of fibroblasts still appeared, but no significant proliferation of blood vessels with time was observed. CD34 immunohistochemical staining showed that MVD in each group increased gradually with the prolongation of time after operation. MVD in the sham operation group was significantly higher than that in the experimental group and the control group at 2 weeks after operation, and significantly smaller than that in the experimental group and the control group at 4, 6, and 8 weeks after operation(P<0.05). MVD in the experimental group was significantly higher than that in the control group at 4 and 6 weeks after operation(P<0.05), but there was no significant difference in MVD between the two groups at 2 and 8 weeks(P>0.05). Conclusion Masquelet technique combined with artificial dermis in the treatment of femoral bone defect and soft tissue defect in rabbits can significantly promote the vascularization of membrane structure at 4-6 weeks after operation. The combination of these two methods has guiding significance for the treatment of Gustilo-Anderson type Ⅲ open fracture with bone and soft tissue defects.