凹凸棒石/Ⅰ型胶原/聚己内酯复合修复兔骨缺损的实验研究

EXPERIMENTAL STUDY ON BONE DEFECT REPAIR WITH COMPOSITE OF ATTAPULGITE/COLLAGEN TYPE I/POLY(CAPROLACTONE) IN RABBITS

目的探讨凹凸棒石/Ⅰ型胶原/聚己内酯[attapulgite/collagen typeⅠ/poly(caprolactone),ATP/ColⅠ/PCL]支架材料修复兔桡骨缺损效果,及其作为骨替代材料的可行性。方法取ColⅠ、PCL按3∶2比例溶于六氟异丙醇后,添加ATP,制备ATP/ColⅠ/PCL支架材料;同法制备ColⅠ/PCL支架材料作为对照。扫描电镜观察两种支架材料结构。取24只2月龄雄性日本大耳白兔,于双侧前肢制备长15 mm的桡骨缺损模型。随机分为3组,A组6只(12侧)缺损不作任何处理作为对照;B、C组各9只(18侧),于缺损处分别植入ColⅠ/PCL、ATP/ColⅠ/PCL支架材料。术后观察动物一般情况,4、8、12周X线片观察骨缺损修复情况;12周取材行大体、扫描电镜、Micro-CT观察及组织学、免疫组织化学染色,观察骨缺损修复以及支架材料降解情况。结果扫描电镜示,两种支架材料均为多孔结构,ATP/ColⅠ/PCL支架材料结构较ColⅠ/PCL支架材料更致密。术后各组动物均存活至实验完成,无切口感染等现象。X线片检查示,随时间延长,C组缺损区骨髓腔连通,修复效果优于A、B组。术后12周,大体观察示B、C组支架材料与周围组织良好融合,A组缺损部位被结缔组织填满。扫描电镜示B、C组支架材料表面和孔隙间被大量细胞和组织覆盖。Micro-CT扫描示,C组骨缺损部位新生骨体积、骨矿物质含量、组织矿含量、骨小梁连接密度显著高于A、B组(P<0.05)。组织学和免疫组织化学染色示,A组缺损区域填充大量结缔组织,ALP、ColⅠ和OPN仅微弱表达;B组支架材料降解区域内有胶原纤维形成,与A组相比,ALP、ColⅠ和OPN表达增强;C组支架材料降解较B组慢,材料植入部位有新生骨组织形成,与A、B组相比,ALP表达减弱,而ColⅠ、OPN表达增强。结论 ATP/ColⅠ/PCL支架材料体内可降解,具有三维多孔致密结构,生物相容性良好,修复兔桡骨缺损效果较好,可以作为骨替代材料。

Objective To investigate the effect of repairing radial bone defect with scaffold material of attapulgite/collagen type I/poly （caprolactone） （ATP/Col I/PCL） in rabbits and the possibility as bone graft substitutes. Methods ATP/Col I/PCL materials were prepared via adding ATP to hexafluoroisopropanol after dissolved Col I/ PCL （3 ： 2）, and Col I/PCL materials via dissolving Col I/PCL （3 ： 2） in hexafluoroisopropanol served as control. The structure of scaffolds was observed under scanning electron microscope （SEM）. Twenty-four Japanese white rabbits （male, 2 months old） were used to establish the bilateral radius defect model of 15 mm in length, and randomly divided into group A （6 rabbits, 12 defects）, group B （9 rabbits, 18 defects）, and group C （9 rabbits, 18 defects）; then the Col I/ PCL scaffold was implanted in the bone defect area in group B, the ATP/Col I/PCL scaffold in group C, no treatment was done in group A as control. The general condition of rabbits was observed after operation, and bone defect repair was evaluated by X-ray at 4, 8, and 12 weeks. At 12 weeks, the tissue of defect area was harvested for the general, SEM, Micro- CT, histological, and immunohistochemical staining to observe defect repair and material degradation. Results SEM observation showed that two kinds of materials were porous structure, ATP/Col I/PCL structure was more dense than Col I/PCL. All animals survived to the end of experiment, and no incision infection occurred during repair process.X-ray films showed that the bone marrow cavity was re-opened in defect area of group C with time, the repair effect was superior to that of groups A and B. At 12 weeks after operation, general observation showed that scaffold material had good fusion with the surrounding tissue in groups B and C, defect was filled with connective tissue in group A. SEM indicated that the surface and pore of the scaffold were covered with a large number of cells and tissues in groups B and C. Micro-CT demonstrated that the new bone volume, bone mineral content, tissue mineral content, and connectivity density of group C were significantly higher than those of groups A and B （P〈0.05）. The observation of histology and immunohistochemical staining indicated that there were lots of connective tissues in defect area of group A, and ALP, Col I, and OPN were weakly expressed; there were many collagen fibers in scaffold degradation area in group B, and the expression levels of ALP, Col I, and OPN were higher than those of group A; there degrada was few new bone in group C, the tion rate of the scaffold was slower than that of group B, and the expression of Col I and OPN were enhanced, while ALP was weakened when compared with groups A and B. Conclusion ATP/Col I/PCL composite scaffold material can degrade in vivo, and has dense three-dimensional porous structure, good biocompatibility, and high potentiality of bone repair, so it can be used as bone substitute material.