人体脂肪基质细胞复合藻酸盐异位软骨生成的研究

Heterotopic chondrogenesis of human adipose tissue-derived stromal cells loading on alginate gel

目的 分离培养人体脂肪基质细胞,经成软骨定向诱导后复合藻酸盐凝胶,研究其异位软骨生成能力。方法 将脂肪抽吸术获取的人体脂肪机械分割,通过Ⅰ型胶原酶消化后得到脂肪基质细胞,在BGJb培养基中原代培养10 d,消化传代后在含体积分数10％FBS及6.25 mg/L胰岛素,10μg/LTGF-β1,50 mg/L维生素C的DMEM/F12培养基中诱导培养14 d,获取的细胞按1×1010/L的细胞密度与质量分数为1.2％的藻酸钠复合,加入102mmol/L CaCl2充分混匀,使之交联,将细胞-藻酸盐凝胶1 ml注入每只体重为25 g的BALB/C裸小鼠背部皮下(共4只),同时在裸小鼠臀部两侧皮下各注入相同量的无细胞藻酸盐及单纯细胞作为自身对照。术后4、8周各处死2只动物,取材固定、脱钙、包埋后切片染色,镜下观察。结果 经连续诱导培养14 d的脂肪基质细胞已具明显成骨表型(细胞基质中富含硫酸软骨素及Ⅱ型胶原)。4、8周时,注射细胞-藻酸盐凝胶标本均显示明显的软骨形成,肥大的软骨细胞位于富含硫酸蛋白多糖的基质中,而自身对照注射的无细胞藻酸盐及单纯细胞在术后3-4周已完全吸收。结论 脂肪基质细胞经过诱导培养后具有向软骨细胞表型分化的潜能。

Objective To isolate and chondro-inductive culture of human adipose tissue-derived stromal cells and to study their heterotopic chondrogenesis by loading them on alginate gel. Methods Liposuction human adipose tissues were minced and digested with collagenase type I . The obtained stromal cells were primarily cultured in BGJb medium for ten days. Secondary harvested cells were cultured in DMEM-F12 medium supplemented with 10% FBS, 6.25 mg/L insulin, 10 mg/L TGF-pl, 50 mg/L of freshly prepared L-ascorbate for 14 days. After in vitro assay of chondrogenic phenotypes, the cells at density of 1010/L were mixed with 1. 2% alginate soldium and 102 mmol/L CaCl2. The cross-linking cell-alginate gel were injected into four BALB/C athymic mice subcutaneously (1ml for each mouse). Meanwhile, the auto-controls were set by injecting equal dose of simple alginate gel and pure cells in two opposite buttocks of the same mouse subcutaneously. Two mice were sacrificed at fourth and eighth week postoperatively and all samples were removed, fixed, embedded in paraffin and cut into sections of 5μm thick. HE staining, Alcian blue and modified Masson' s trichrome staining were employed to observe chondrogenesis histologically. Results Alcian blue and immunocytochemical staining revealed chondroitin sulfate and collagen Ⅱ in cell matrix after having been chondro-inductive cultured for 14 days. At intervals of fourth and eighth week, heterotopic chondrogenese is (cartilage formed) within cell-alginate injected sites were found in all mice but negatively in auto-controls. Histologically the hypertrophic chondrocytes were among cartilage matrix in different staining. All alginate gel and solitary cells absorbed within two to three weeks postoperatively in auto-controls. Conclusion It seems that stromal cells derived from human adipose tissue presents a potential for chondrogenic differentiation.