脂肪来源的干细胞复合壳聚糖支架促进大鼠颅骨缺损修复的实验研究

Adipose-derived stem cells loaded chitosan scaffold to promote the healing of calvarial defect：an experimental study

摘要:

目的：探讨以脂肪来源的干细胞（adipose-derived stem cells，ADSCs）为基础构建组织工程生物材料修复大鼠颅骨缺损的效果。方法：分离、培养ADSCs，并进行成骨、成脂、成软骨三系分化。制备壳聚糖多孔支架，扫描电镜观察接种ADSCs前后的微结构变化，以细胞活力试剂盒测试ADSCs接种于壳聚糖多孔支架后的细胞活力。取15只雄性Sprague Dawley（SD）大鼠予手术制备颅骨缺损模型，将缺损留空或植入壳聚糖支架，壳聚糖支架表面预先种植低密度（2.5×105）或高密度（1×106）ADSCs。术后8周收集样品用于微型计算机断层扫描分析，随后脱钙，石蜡包埋切片后行苏木素-伊红染色和免疫组化染色观察成骨指标骨涎蛋白。结果：ADSCs可以成功诱导成骨、成软骨和成脂肪分化；扫描电镜和细胞活力测试结果提示壳聚糖支架呈现多孔结构，ADSCs可黏附于支架并增殖，增殖效果与普通培养板相比无统计学差异。在大鼠颅骨缺损模型中，高密度ADSCs/壳聚糖支架组骨组织体积百分数分别比空白对照组和低密度ADSCs/壳聚糖支架组升高140.8%（P=0.014）和37.4%（P=0.036）。与空白缺损对照组相比，低密度ADSCs/壳聚糖支架组和高密度ADSCs/壳聚糖支架组骨密度分别升高29.4%（P=0.123）和64.1%（P=0.022）。组织学结果表明，骨缺损区新生骨组织的分布和数量与微型计算机断层扫描分析结果一致。成骨分化指标骨涎蛋白在高密度组表达有增高趋势。结论：高密度ADSCs复合壳聚糖支架可有效修复大鼠的颅骨缺损。

Abstract:

Objective：To investigate the effect of tissue engineering biomaterials based on adipose-derived stem cells（ADSCs） for healing calvarial defects. Methods：ADSCs were isolated，cultured and conducted the three-lineage differentiation of osteogenesis，adipogenesis and chondrogesis. Porous chitosan-based scaffolds were prepared and ADSCs were seeded onto the surface of scaffold. Microstructural changes before and after the implantation of ADSCs were detected by scanning electron microscope（SEM）. Cell viability after ADSCs loaded on scaffolds was determined by cell viability assays. A total of 15 male Sprague Dawley（SD） rats were selected to create cranial defect models，and the defects were left in empty or implanted with chitosan scaffolds which were seeded with low（2.5×105） or high densities（1×106） of ADSCs. After operation of 8 weeks，samples were collected for Micro-CT analysis. And then，those samples were decalcified，paraffin embedded and sectioned；hematoxylin-feosin staining and immunohistochemistry stain－ing were performed to observe the bone formation biomarker of bone sialoprotein. Results：ADSCs were able to induce osteoge－nesis，chondrogenesis and adipogenesis. Data from SEM and cell viability assays showed that chitosan-based scaffold had porous structure，and ADSCs were able to attach to the surface of scaf－folds and proliferate，and proliferation effects on scaffolds sur－face were not significantly different when compared with those on the culture plate. In the rat calvarial defect model，the bone volume ratio（BV/TV） in the high-density ADSCs/chitosan scaf－fold group was increased by 140.8%（P=0.014） and 37.4%（P=0.036） when compared with that in the blank control group and the low-density ADSCs/chitosan group，respectively. Compared with that in the control group，bone density in the low-density ADSCs/chitosan group and high-density ADSCs/chitosan group were in－creased by 29.4%（P=0.123） and 64.1%（P=0.022），respectively. Histological results showed that distribution and quantity of the new bone tissue in the bone defect area were consistent with results of Micro-CT analysis. Expression of bone formation markers of bone sialoprotein had an increasing tendency in the high-density group. Conclusion：High density of ADSCs-loaded chitosan scaffolds can effectively promote the healing of calvarial defect in rats.