组织工程化骨构建及其修复羊跖骨标准性骨缺损的放射学评估

背景骨髓间充质干细胞具有多向分化潜能,与可降解的多孔β-磷酸三钙陶瓷复合后具有修复节段性骨缺损的能力,为临床上修复各种原因导致的骨缺损提供了新的思路.目的从放射学角度探讨用多孔β-磷酸三钙和自体骨髓间充质干细胞复合植入羊体内修复标准性骨缺损的效果.设计以骨折愈合不同时期的骨痂生长情况为观察对象的随机对照.单位武汉大学人民医院骨科,解放军军事医学科学院基础医学研究所组织工程中心.材料实验于2002-03/2003-09在解放军军事医学科学院基础医学研究所组织工程中心完成.选取健康成年中国绵羊20只,随机分为空白对照组4只,单纯植入组8只,复合植入组8只.方法全麻无菌条件下,抽取羊骨髓10～15 mL,分离并培养出间充质干细胞,与多孔β-磷酸三钙陶瓷复合,构建组织工程化骨.各组动物均在跖骨中段锯取21 mm长的跖骨干缺损,复合植入组植入β-磷酸三钙陶瓷与自体间充质干细胞复合体,单纯植入组植入β-磷酸三钙陶瓷,空白对照组缺损区空缺,然后逐层缝合.分别在术后即刻及1,3,6个月摄片观察,进行放射影像学评估(骨缺损区每面骨连接形成即得1分,在缺损区的任一面上均无骨连接为O分,缺损区的前、后、侧面及中央均形成骨连接为4分),图像分析X线阻射密度比较骨缺损的修复情况.主要观察指标各组绵羊术后一般情况及大体观察、骨缺损区放射学分析结果.结果实验纳入绵羊20只,全部进入结果分析.①各组绵羊术后一般情况术后2～6 h苏醒,切口无感染,内固定无松动.术后1周精神已逐渐恢复正常,伤肢能落地,但不能负重,2周后可以部分负重,3周有轻微跛行,4周后能自由活动,无跛行.②各组绵羊术后6个月大体观察结果单纯植入组植入物表面白色的透明软骨样组织逐渐钙化,两端有骨桥与宿主骨相连,但仍可见有陶瓷材料颗粒存在;复合植入组植入物已不见,材料与宿主骨的界面消失,骨缺损部与宿主骨基本一致.空白对照组在术后各时间点均无骨组织形成.③各组绵羊术后不同时间骨缺损区放射学评分比较复合植入组术后3,6个月均明显高于单纯植入组[(2.3±0.3),(1.8±0.5);(3.3±0.5),(2.6±0.6)分,P均＜0.05].④各组绵羊术后不同时间骨痂厚度与阻射密度相对值测量结果复合植入组术后6个月骨痂厚度明显低于单纯植入组(4.62,7.64,P＜0.05),阻射密度相对值明显高于单纯植入组(70.4±1.5,61.18±1.2,P＜0.05).结论放射学评估及骨缺损密度测量能够较好地反映骨缺损修复的动态发展过程,多孔β-磷酸三钙陶瓷和自体骨髓间充质干细胞复合植入羊体内,可使大节段骨缺损较快得到修复.

Construction of tissue engineering bone as well as radiological appraisal in the repair of standard sheep metatarsus bone defect

BACKGROUND:Bone marrow derived mesenchymal stem cells (BMMSCs) have multi-differentiation potentials, possessing a repairing capability for the sectional bone defects if combined with degradable porous β-tricalcium phosphate china. This provides a new idea in clinical repair of various bone defects.OBJECTIVE: To explore in radiology the curative effect of implanting porous β-tricalcium phosphate and autogenous BMMSCs compound to treat bone defects.DESIGN: A randomized controlled study with callus growth at various healing periods as subjects for observation.SETTING: Department of Orthopaedics, Renmin Hospital, Wuhan University; Tissue Engineering Center, Research Institute of Basic Medicine,Academy of Military Medical Sciences of Chinese PLAMATERIALS: This experiment was carried out at the Tissue Engineering Center, Research Institute of Basic Medicine, Academy of Military Medical Sciences of Chinese PLA between March and September 2002. Twenty China healthy adult sheep were randomized into the groups of blank control group (4 sheep), simple implantation group (8 sheep) and complex implantation group (8 sheep).METHODS: Under general anesthesia and aseptic condition, 10-15 mL of sheep marrow was extracted; MSCs were separated and cultured before combined with porous β-tricalcium phosphate china for tissue engineering bone construction. Rats in each group were cut off 21mm long metatarsus in the middle section of metatarsus bonestem. Β-tricalcium phosphate china and autogenous MSC compound was implanted into the sheep of the complex implantation group; β-tricalcium phosphate china was implanted into the simple implantation group; and the bone defects in the blank control group remained untouched. Then the incision was sutured.X-ray filming was carried out right after the operation, as well as 1, 3,and 6 months after the operation for radiological appraisal (scored for1 if bone union formed in one surface of bone defect, but scored 0 if no boneunion formed in any surface of bone defect, and scored 4 if bone union formed in front, back, lateral surfaces and the center of bone defect), Xray radiation-resisting density was analyzed to compare the results of bone defect repair.MAIN OUTCOME MEASURES: The postoperative general condition and general observation, as well as the results of the radiological analysis of the bone defects of all sheep.RESULTS: Totally 20 sheep were brought into this xperiment and all entered the stage of result analysis. ① The postoperative general condition:Sheep regained consciousness 2-6 hours after the operation without incision infection and loosing of internal fixation. Their spirit gradually was back to normal 1 week after the operation, at which time the injured legs could touch ground but were incapable of bearing load, and the affected legs could bear load 2 weeks after the operation, walked slightly lamely 3 weeks after the operation, and even moved freely without limp 4 weeks after the operation. ②The general condition 6 months after the operation: In pure implantation group, the surface white hyaline cartilage-like tissues were gradually calcified, with both ends connected with the host bone by bone bridge, but china granules could still be easily observed; while no implantation substance could be observed in compound implantation group,with the boundary between implantation substance and host bone vanished,and bone defect became basically the same as host bone. However there was no bone tissue formed in bone defect at various postoperative time points in the blank control group. ③ Radiological analysis of the bone defects at various postoperative time points: The radiological rating score was obviously higher in complex implantation group at the time poin ts of 3, 6 months after the operation compared with the pure implantation group [(2.3±0.3), (1.8±0.5); (3.3±0.5), (2.6±0.6), P ＜ 0.05]. ④ Radiological analysis of bone callus thickness and the relative value of radiation-resisting density at various postoperative time points: The bone callus thickness in the complex implantation group was obviously lower than that of the pure implantation group at the postoperative time point of 6 months (4.62 vs 7.64, P ＜ 0.05), with relative value of radiation-resisting density obviously higher than that of the pure implantation group (70.4±1.5 vs 61.18±1.2, P ＜ 0.05).CONCLUSION: Radiological appraisal and bone defect density measurement can well reflect the dynamical repairing process of bone defects; the implantation of porous β-tricalcium phosphate china and autogenous BMMSCs compound into sheep can enhance the repair of large sectional bone defect.