纳米"羟基磷灰石-胶原蛋白-壳聚糖"材料复合骨髓间充质干细胞修复小鼠胫骨缺损

目的 应用纳米级材料“羟基磷灰石-胶原蛋白-壳聚糖”复合骨髓间充质干细胞（BMSCs）修复小鼠胫骨5mm的缺损，探讨其对长骨缺
损的修复效果。方法 原代分离培养小鼠BMSCs，进行干细胞相关生物学特性检测。制备纳米级材料羟基磷灰石-胶原蛋白-壳聚糖的新型复合材
料，扫描电子显微镜下观察材料结构，检测支架材料的孔隙率、降解率。制备小鼠胫骨5mm离断性缺损模型30只，分3组进行骨修复,每组10只：
A组（纳米羟基磷灰石-胶原蛋白-壳聚糖材料复合BMSCs）；B组（单纯纳米羟基磷灰石-胶原蛋白-壳聚糖材料）；C组（单一纳米级羟基磷灰石
复合BMSCs）。于8周时各组动物麻醉并取材，HE染色观察缺损区成骨情况，X线检测骨修复情况。结果 原代分离的BMSCs具有成纤维细胞形态，
表达间充质干细胞的表面标记，符合干细胞的生物学特性。制备的纳米级羟基磷灰石-胶原蛋白-壳聚糖支架具有丰富的孔隙，支架内部孔隙间
有广泛的交通，孔隙大小均匀，直径为100~200μm，孔隙率72.3%～92.7%之间，平均为82.5%左右，支架降解率为(60.3±5.4)%。修复8周后，
A组可见支架材料大部分降解，无炎性反应，并见大量成熟板层骨样结构。 X线评分显示A组在缺损区可见新生骨痂且新骨密度增高，形成骨桥
并与两端桥接，而B、C组效果不理想，与A组相比差异明显 (P＜0.01)。 结论 纳米级材料“羟基磷灰石-胶原蛋白-壳聚糖”复合BMSCs对于小
鼠胫骨骨缺损修复效果良好。

Nano-hydroxyapatite-collegen-chitosan substance compound with bone marrow mesenchymal stem cells to repair tibia defect in mice

Objective To use nano-hydroxyapatite-collegen-chitosan compounded with bone marrow mesenchymal stem cells (BMSCs)
to repair tibia bulk defect in mice, and to investigate the application effectiveness of the composite to the long bone defect.
Methods BMSCs of mice were isolated and cultured. Biological characteristics of the stem cells were detected. The
nano-hydroxyapatite-collegen-chitosan was prepared. The structure, poriness and degradation rate of the composite were observed
under a scanning electronic microscope（SEM）. Male mice with a 5 mm tibia defect were randomly divided into three groups:
group A treated with the nano-hydroxyapatite-collegen-chitosan compound with BMSCs; group B with the nano-hydroxyapatite-collegen-chitosan
and group C with the nanoscale hydroxyapatite compound with BMSCs. At 8 weeks after restoration, the mice were anesthetize
and samples were collected. HE staining and X-ray were used to observe the osteogenesis and healing process in the defect
area. Results The BMSCs presented fibroblast morphology and expressed the surface markers of mesenchymal stem cells which were
of the biological characteristics of stem cells. The nano-hydroxyapatite-collegen-chitosan had abundant pores and poriness
with a relatively even diameter of 100-200μm and an appearance ratio of 72.3%-92.7% (an average of about 82.5%). The degradation
rate was（60.3±5.4）% . There were extensive communications inside the scaffold. At the 8th week after transplantation, there
was no obvious inflammatory response but mass mature osteoid structure, and the scaffold degraded mostly in group A. X-ray
showed that the defect area had new callus，increased new bone density and the bridge grafting in group A. The above featrues
were less obvious in groups B and C than that in group A(P＜0.01). Conclusion Nano-hydroxyapatite-collegen-chitosan compounded
with BMSCs works well for repairing the tibia bulk defect in mice.