摘要
目的 探讨软骨细胞与脂肪基质细胞（adipose-derived stromal cells,ADSCs）共培养体外构建软骨的可行性,并阐明软骨细胞提供的软骨微环境能否诱导ADSCs向软骨细胞分化并形成软骨组织.方法 分别培养扩增人ADSCs与猪耳软骨细胞,将2种细胞按7：3（ADSCs：软骨细胞）比例混匀,以5.0×107/ml的细胞终浓度接种于聚羟基乙酸/聚乳酸（PGA/PLA,直径8 mm,高2 mm）支架作为共培养组,以相同终浓度的单纯软骨细胞和单纯ADSCs分别接种相同支架作为阳性对照组及阴性对照组,以30％上述浓度（1.5×107/ml）的单纯软骨细胞接种作为低浓度软骨细胞对照组.每组各接种6例标本,每例接种细胞悬液200μl.全部标本均于体外培养8周时取材,通过大体观察、组织学、免疫组化及湿重、蛋白多糖定量检测等方法对新生软骨进行初步评价.多样本t检验统计分析各组湿重及蛋白多糖含量差异.结果 各组细胞均与材料粘附良好.共培养组及阳性对照组体外培养8周时基本保持了复合物初始大小和形状,大体观察2组均形成了较成熟软骨组织,组织学显示大量软骨基质和软骨陷窝形成,免疫组化显示软骨特异性细胞外基质Ⅱ型胶原分泌.定量测定结果表明,共培养组的平均湿重为（174±12） mg,平均蛋白多糖含量为（7.6±0.4） mg,两者分别达到阳性对照组的75％ （P＜ 0.01）和79％ （P＜ 0.01）.阴性对照组（单纯ADSCs组）明显皱缩变形,组织学未见成熟软骨陷窝.低浓度软骨细胞组明显变薄,新生软骨平均湿重为（85 ±5） mg,是阳性对照组的37％ （P＜ 0.01）,只在局部形成了不连续的软骨组织.结论 软骨细胞与ADSCs共培养能够在体外构建较成熟的软骨组织,软骨细胞能够诱导ADSCs成软骨分化及体外形成软骨组织.
Objective To explore the feasibility of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells （ADSCs） so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs.Methods Human ADSCs and porcine auricular chondrocytes were in vitro expanded respectively and then were mixed at the ratio of 7：3 （ADSCs： chondrocytes）.200 μl mixed cells （5.0 × 107/ml） were seeded onto a polyglycolic acid/polylactic acid （PGA/PLA）scaffold,8 mm in diameter and 2 mm in thickness,as coculture group. Chondrocytes and ADSCs with the same cell number were seeded respectively onto the scaffold as positive control group and negative control group.200 μl chondrocytes （1.5 × 107/ml） were seeded as low concentration chondrocyte group.There were 6 specimens in each group.A11 specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS.Gross observation, histology,immunohistochemistry,wet weight measurement and glycosaminoglycan（GAG） quantification were used to evaluate the results.Multiple-sample t-test statistics analysis was done to compare the difference of wet weight and glycosaminoglycan（GAG） content between the groups.Results Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In co-culture group and positive control group,cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 8weeks,cartilage-like tissue formed in gross appearance and histological features,and abundant type Ⅱcollagen could be detected by immunohistochemistry.Wet weight and glycosaminoglycan（GAG） content of co-culture group were respectively（ 174 ± 12） mg and（7.6 ±0.4） mg.There were respectively 75% （P 〈0.01） and 79% （P 〈 0.01 ）of those of positive control group.In negative control group,however,constructs shrunk gradually without mature cartilage lacuna in histology. In low concentration chondrocyte group,constructs also shrunk obviously with small amount of cartilage formation at the edge area of the construct,and wet weight was （ 85 ± 5 ） mg,which was 37% （ P 〈 0.01 ） of that of positive control group.Conclusions Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the in vitro chondrogenesis of ADSCs.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2012年第1期49-54,共6页
Chinese Journal of Plastic Surgery
关键词
组织工程
软骨细胞
脂肪基质细胞
共同培养技术
Tissue engineering
Chondrocytes
Adipose-derived stromal cells
Coculture techniques
