骨形态发生蛋白2诱导慢性腱病大鼠肌腱干细胞体外成骨、成软骨分化

背景：目前临床对于慢性腱病缺乏有效的治疗手段，原因在于其发病机制至今尚未阐明。目的：研究体外骨形态发生蛋白2对胶原酶诱导的大鼠慢性腱病模型髌腱来源肌腱干细胞的成骨、成软骨分化的作用。方法：从大鼠慢性腱病模型的髌腱中分离培养出原代肌腱干细胞，传代培养至第3代细胞，行成骨、成脂、成软骨诱导分化鉴定其干细胞的特性。将肌腱干细胞(P3)单层培养至细胞融合，用重组人骨形态发生蛋白2干预。7 d后分别行茜素红染色，并行茜素红染色定量分析。将肌腱干细胞体外三维微球培养后分为2组，诱导组用重组人骨形态发生蛋白2干预，对照组不进行干预。21 d后三维微球行苏木精-伊红染色，阿利辛蓝染色以及Sox9和Ⅱ型胶原免疫组织化学染色。结果与结论：慢性腱病大鼠来源原代肌腱干细胞体外培养呈克隆样集落生长，传代后细胞主要表现为多突的纺锤形和星形的扁平细胞，具有成纤维细胞样的特征。肌腱干细胞(P3)成脂诱导10 d，油红O染色阳性；成骨诱导7 d，茜素红染色阳性；成软骨诱导14 d，苏木精-伊红染色阳性可见软骨样细胞，Ⅱ型胶原免疫组化染色阳性。单层培养的肌腱干细胞用重组人骨形态发生蛋白2诱导7 d茜素红染色阳性，对照组为阴性，茜素红染色定量检测显示差异有显著性意义。重组人骨形态发生蛋白2诱导肌腱干细胞21 d，苏木精-伊红染色可见软骨样细胞形成、阿利辛蓝染色可见细胞内糖胺多糖沉积、Sox9和Ⅱ型胶原免疫组织化学染色均呈阳性。可见体外重组人骨形态发生蛋白2可以诱导慢性腱病来源的肌腱干细胞成骨、成软骨分化。这为进一步研究慢性腱病的发病机制提供了细胞生物学依据。中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：

Bone morphogenetic protein 2 stimulated osteo-chondrogenic differentiation of patellar tendon-derived stem cells isolated from a failed tendon-healing animal model of tendinopathy

BACKGROUND:The pathogenesis of tendinopathy remains unclear and hence treatment of tendinopathy is usually palliative.OBJECTIVE:To investigate the effects of bone morphogenetic protein 2 on the osteogenic and chondrogenic differentiation of patellar tendon-derived stem cells isolated from collagenase-induced tendinopathy rats in vitro.  METHODS:Patellar tendon-derived stem cells were isolated from patellar tendons of collagenase-induced tendinopathy rats. The multi-differentiation potential of patellar tendon-derived stem cells at passage 3 was identified by osteogenic, adipogenic and chondrogenic differentiation assays. The patellar tendon-derived stem cells were cultured to the 3rd passage in complete culture medium, and then the cells were divided into two groups with (experimental group) or without recombinant human bone morphogenetic protein 2 (control group) until the cells reached confluence for 7 days. Their osteogenic response to bone morphogenetic protein 2 in vitro  was examined by alizarin red S staining of calciumnodule formation and quantification assay. The patellar tendon-derived stem cell pellets were cultured in complete culture medium with (experimental group) or without bone morphogenetic protein 2 (control grup) for 21 days. Chondrogenic differentiation of the cell pellets was evaluated by hematoxylin-eosin staining, alcian blue staining, immunohistochemical staining for Sox9 and collagen type II. RESULTS AND CONCLUSION: Primary patellar tendon-derived stem cells from the tendinopathy rats cultured in vitro showed clonal growth; after passage, spindle fibroblast-like and flat-like cells were detectable. The cells were positive for oil red O staining at 10 days after adipogenic induction, positive for alizarin red staining at 7 days after osteogenic induction, and positive for hematoxylin-eosin staining and immunohistochemical staining of collagen type II at 14 days after chondrogenic induction. After patellar tendon-derived stem cells were induced with recombinant human bone morphogenetic protein 2 for 7 days, the result of alizarin red staining was positive in the experimental group, but negative in the control group without recombinant human bone morphogenetic protein 2. The difference in the result of alizarin red staining between the two groups was statistically significant. After patellar tendon-derived stem cells were induced with recombinant human bone morphogenetic protein 2 for 21 days, the results of hematoxylin-eosin staining, alcian blue staining, immunohistochemical staining for Sox9 and collagen type II were all positive. In conclusion, bone morphogenetic protein 2 could stimulate the osteogenic and chondrogenic differentiation of patellar tendon-derived stem cells isolated from collagenase-induced tendinopathy rats in vitro, which can help to better understand the pathogenesis of tendinopathy.