脂肪干细胞/Ⅰ型胶原凝胶复合体的构建及其体内外成骨分化研究

Osteogenesis of adipose-derived stem cells(ADSCs)-collagenⅠgel composite in vitro and in vivo

目的探讨利用脂肪干细胞与Ⅰ型胶原凝胶复合构建新型骨组织工程复合体的可行性并对其体内外成骨分化情况进行分析。方法取3月龄日本大耳白兔肩胛部皮下脂肪,Ⅰ型胶原酶消化获得细胞,于体外依次进行骨、软骨、脂肪多向诱导分化鉴定;取第3代细胞与Ⅰ型胶原凝胶复合,于成骨诱导条件下体外培养,相差显微镜、扫描电镜观察复合情况并对其碱性磷酸酶、细胞外基质矿化程度进行检测,同时设立单层贴壁培养细胞作为对照(n=4);两周后移植于裸鼠皮下,12周后处死动物,依次行放射学、组织学检测观察成骨情况(n=3)。结果(1)所获细胞具备骨、软骨、脂肪多向分化潜能,为多能干细胞。(2)形态学观察显示脂肪干细胞呈三维立体生长状态,均匀、高密度悬浮于Ⅰ型胶原凝胶中。在体外成骨诱导条件下,其碱性磷酸酶活性以及外基质矿化程度均显著高于单层贴壁培养细胞(P<0.01,n=4)。(3)于裸鼠皮下移植12周后,放射学、组织学检测显示脂肪干细胞-Ⅰ型胶原凝胶复合体成骨明显(n=3)。结论(1)Ⅰ型胶原凝胶可显著促进脂肪干细胞的成骨分化。(2)作为一种新型的骨组织工程复合物,脂肪干细胞-Ⅰ型胶原凝胶复合体可应用于骨组织工程,特别是腔隙性骨缺损的修复过程。

Objective To construct a new bone tissue-engineering composite by combining adipose-derived stem cells （ADSCs） with collagen Ⅰ gel and further evaluate its osteogenesis in vitro and in vivo. Methods ADSCs were prepared by collagenase Ⅰ digestion of subcutaneous fat from the suprascapular site of Japanese white rabbits. Multipotent differentiation of harvested cells was determined through osteogenic,chondrogenic and adipogenic differentiation in vitro. The passage 3 cells were used to resuspend in collagen Ⅰ gel solution at 2 × 10^5 cells/ml. After gelation ,the ADSCs- collagen Ⅰ gel composite （ADSCs- COL） was cultured under osteogenic medium in vitro for 2 weeks with the ADSCs under monolayer culture as control （n = 4）. The osteogenesis of ADSCs-COL in vitro was evaluated through alkaline phosphatase （ALPase） activity and cell mineralization detection. The composite was implanted into the back of nude mice for 12 weeks （n = 3 ）. The ectopic bone formation was assayed by radiographic and histological analyses. Results The multipotency of ADSCs was confirmed in vitro under osteogenic,chondrogenic, and adipogenic medium respectively. The expression of ALPase activity and cell mineralization for the ADSCs-COL composite was statistically higher than ADSCs under monolayer culture （P 〈 0.01, n = 4 ）. Radiographic and histological analyses indicated that new bone tissue formed 12 weeks after implantation. Conclusion Collagen Ⅰ gel can facilitate the osteogenesis of ADSCs and the ADSCs-COL composite can be used in bone tissue engineering,especiaUy in lacunar bone defect site.