脂肪干细胞／Ⅰ型胶原凝胶／聚乳酸聚乙醇酸-β-磷酸三钙骨组织工程复合体的构建及其异位成骨研究

Fabrication and ectopic bone formation of a novel bone tissue engineering construct by using collagen I gel to suspend adipose-derived stem cells into a porous PLGA-β-TCP scaffold

目的构建基于脂肪于细胞、Ⅰ型胶原凝胶以及聚乳酸聚乙醇酸-β-磷酸三钙支架（PLGA-β-TCP）的骨组织工程复合体并对其异位成骨进行研究。方法设计构建脂肪干细胞-Ⅰ型胶原凝胶／PLGA—β—TCP复合体（A组），同时设立单纯细胞／PLGA—β—TCP材料复合体（B组）、单纯Ⅰ型胶原凝胶／PLGA-β-TCP复合体（C组）以及单纯PLGA-β-TCP支架材料（D组）作为对照。扫描电镜、相差显微镜观察细胞材料复合情况并对脂肪干细胞增殖以及成骨分化进行分析。体外成骨诱导培养2周后移植于自体股部肌袋，8周后取出，依次行放射学、组织学定性及半定量分析。结果（1）体外成骨诱导2周，A组细胞增殖率慢于B组（P〈0．05，n=4），但其细胞总数远高于后者（P〈0．01，n=4）。A组碱性磷酸酶（ALPase）活性、细胞外基质矿化程度均显著高于B组（P〈0．01，n=4）。A组细胞悬浮于Ⅰ型胶原凝胶并均匀分布于材料孔隙中而B组细胞仅见黏附于材料表面。（2）移植8周，X线片示A组高密度钙化影形成，B、C、D组未见有阳性结果。A组材料孔隙中均匀充满新生骨组织，可观察到骨小梁样结构。B组仅在少数孔隙中有类骨组织形成，同时伴有结缔组织长入。A组新生骨面积百分比显著高于B组（P〈0．01，n=4）。结论通过应用Ⅰ型胶原凝胶来实现脂肪干细胞与PLGA-β-TCP多孔支架材料的均匀复合，能够有效促进脂肪干细胞在材料孔隙中的成骨分化及均质骨组织形成。

Objective To explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricatedby using collagen I gel to suspend rabbit adipose-derived stem cells （ASCs） into a porous PLGA-β-TCP scaffold （ASCs-COL/PLGA-β-TCP）. Methods Following groups were set up： ASCs- COL/PLGA-β-TCP composite group （group A）, ASCs and PLGA-β-TCP group （group B）, acellular collagen I gel and PLGA-β-TCP group （group C）, PLGA-β-TCP scaffold （group D）. Composites were cultured in vitro for 2 weeks under osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. Results During in vitro culture, ALPase activity and extracellular matrix mineralization in group A was significantly greater than in group B （P 〈0.01 ,n =4）. After in vivo implantation,the calcification level was radiographieally evident in group A, but whereas no apparent calcification was observed in groups B, C and D （ n = 4）. In group A, woven bone with a trabecular structure was formed, but only osteiod tissue was observed in group B. Bone forming area in group A was significantly larger than in group B （ P 〈 0.01, n = 4 ）. NO bone formation was observed in groups C or D （ n = 4 ）. Condusion By using collagen I gel to suspend ASCs into porous PLGA-β-TCP scaffold, osteogenic differentiation of ASCs can be improved and homogeneous bone tissue can be successfully formed in vivo.