血清浓度影响骨髓基质干细胞体外软骨分化的实验研究

An experimental study on influence of concentration of fetal bovine serum on chondrogenesis of BMSCs

目的 探讨含有不同浓度胎牛血清的成软骨分化诱导液对骨髓基质干细胞（BMSCs）体外分化的影响，明确体外培养用血清在细胞分化中的作用，为软骨的体外组织构建提供技术参数。取第2代猪BMSCs，以5×10^7个细胞/cm^3的密度接种到聚羟基乙酸（PGA）制成的圆盘状支架上（直径5mm，厚度2mm），7d后分别应用含0％、5％、10％血清浓度的诱导液（诱导因子包括TGF-1、IGF-Ⅰ及地塞米松）进行体外持续性诱导培养。8周取材行组织湿重测量、GAG含量定量分析、大体观察、组织学、组织化学及免疫组织化学检测。结果 10％血清组复合物外观瓷白色，坚硬细腻，体积和外形均无明显变化。组织学及免疫组织化学检测结果显示有典型的软骨陷窝，大量的软骨特异性细胞外基质成分，组织湿重和GAG定量亦明显高于其它两组；3％血清组复合物体积缩小．有少量陷窝样结构及软骨特异性胞外基质聚集；而无血清组复合物缩小，松软易碎，未见典型的软骨陷窝和软骨特异性基质成分。结论 体外诱导培养BMSCs构建组织工程软骨过程中，10％浓度的血清成分有利于BMSCs成软骨分化。

Objective To explore the chondrogenetic effect of induce media containing different concentrations of fetal bovine serum （FBS） on BMSCs differentiation in vitro and provide technical parameters for cartilage engineering in vitro. Methods Passage 2 BMSCs of swine were seeded at the density of 5 × 10^7 cells/ cm^3 to disc-shaped PGA scaffolds with a diameter of 5mm and a thickness of 2mm. After 7days, the scaffolds were induced in media with TGF-β1 , IGF-Ⅰ, dexomethasone, and different concentrations of FBS： 0% in A group, 5% in B group, and 10% in C group. Specimens were collected after 8 weeks for gross observation, size evaluation, wet weight, glycoaminoglycan （GAG） content, histology assessment, and immunohistology of type 0282 collagen. Results The compound of C group showed china-white color, hard and fine texture, no obvious change in size and shape, typical lacuna structures, cartilage specific ECM, and significantly higher wet weight and GAG content. The compound of B group showed reduced size, fewer lacuna structures and some cartilage specific ECM. And the compound of A group showed greatly reduced size, soft and loose texture, and no typical lacuna structure or cartilage specific ECM. Conclusions FBS was indispensable to cbondrogenetic media for invitro tissue engineering of cartilage with BMSCs.