真皮来源细胞亚群修复小鼠颅骨缺损

Dermis-derived cell subpopulation is used to repair mouse calvarial defects

背景:考虑到皮肤是全身最大的器官,拥有丰富的皮肤毛细血管网,可能存在足够的成体干细胞用于组织工程。目的:探讨真皮骨形成蛋白受体ⅠB亚型阳性细胞的成骨潜能和修复骨缺损的能力。方法:采用组织学方法分析骨形成蛋白受体ⅠB亚型细胞在皮肤中的定位和表达情况。新鲜皮肤组织制成单细胞悬液,以表面蛋白骨形成蛋白受体ⅠB亚型作为分选标志,采用免疫磁珠分选获得骨形成蛋白受体ⅠB亚型细胞。在体外进行成骨诱导分化,并分别通过碱性磷酸酶染色和茜素红染色检测其碱性磷酸酶和钙结节的表达。将骨形成蛋白受体ⅠB亚型细胞复合于珊瑚支架后修复小鼠颅骨缺损,术后6周通过组织学方法,术后24周通过影像学方法评估其修复骨缺损的能力。结果与结论:骨形成蛋白受体ⅠB亚型细胞在皮肤中呈单个分散存在,位于真皮网状层。通过免疫磁珠分选可获得表达骨形成蛋白受体ⅠB亚型的细胞亚群。体外成骨诱导后,碱性磷酸酶染色呈阳性表达,茜素红染色显示大量的钙结节形成,证实其在体外具有成骨分化能力。体内修复颅骨缺损6周后组织学结果显示在骨形成蛋白受体ⅠB亚型阳性细胞复合珊瑚组可见大量新生骨形成;24周影像结果显示骨形成蛋白受体ⅠB亚型阳性细胞复合珊瑚组的颅骨骨缺损组织基本完全修复。结果表明真皮来源的骨形成蛋白受体ⅠB亚型阳性细胞亚群拥有成骨的潜能,其可能是骨组织工程适合的种子细胞。

BACKGROUND： In consideration of skin as the largest organ all over the body and its abundant vessels and vessel plexuses, there would be sufficient adult stem cells for tissue engineering. OBJECTIVE： To investigate the osteogenic potential of dermis-derived bone morphogenetic protein receptor subtype IB （BMPR-IB） positive cells. METHODS： In current study, histochemical analysis was adopted to study the localization and expression of BMPR-IB＋ cells in skin. Fresh skin samples were digested into single cell suspension. Then, the surface marker BMPR-IB was used to isolate cell subpopulation by magnetic activated cell sorting from freshly prepared single cell suspension. After that, the osteogenic potential in vitro and in vivo was tested. Alkaline phosphatase staining and alizarin red staining were performed after osteogenic induction in vitro. The BMPR-IB＋ cells were seeded onto coral scaffolds, and the scaffolds were used to repair critical-sized calvarial defects of mice. Histochemicalanalysis was performed at 6 weeks postoperatively and micro-CT analysis was carried out at 24 weeks postoperatively to evaluate the ability of bone repairment. RESULTS AND CONCLUSION： We localized BMPR-IB cells in situ by immunohistochemistry that turned out to be expressed in the reticular layer of dermis and by single cells. Cell subpopulation which expressed BMPR-IB could be sorted by magnetic activated cell sorting. Alkaline phosphatase staining was obviously positive and lots of calcium modules were confirmed by alizarin red staining after osteogenic induction, indicating that BMPR-IB＋ cells had the osteogenic potential in vitro. Histochemical analysis demonstrated that plenty of new bone formation was found in BMPR-IB＋ cells group after 6 weeks in vivo. Micro-CT analysis revealed that BMPR-IB＋ cells-coral scaffold complex could repair calvarial defects successfully after 24 weeks in vivo. These results indicated that dermis-derived BMPR-IB＋ cells possessed adequate osteogenic potential. Moreover, they might be promising seed cells for bone tissue engineering.