目的:评价富血小板血浆(platelet rich plasma,PRP)和骨髓基质细胞(marrow stromal cells,MSCs)构建的细胞-膜片包裹珊瑚支架后修复兔颅骨极限缺损的能力.方法:将体外培养扩增的兔MSCs悬液与同一供体来源的PRP混合,滴加到六孔培养板上,再滴加牛凝血酶溶液,形成凝胶样MSCs/PRP混合物,置于孵箱内培养.第1周用DMEM完全培养液,第2周用DMEM诱导培养液培养,形成细胞-膜片.将细胞-膜片取下后包裹珊瑚支架,植人供体兔颅骨直径15 mm的圆形缺损内,用自体颅骨和单纯珊瑚植入作对照.术后8周和16周取材,通过大体观察、组织学观察和组织形态测量分析等,评价其骨修复效果.结果:培养的细胞-膜片厚约2 mm,呈半透明胶冻样,有较好的韧性和可操作性.细胞-膜片/珊瑚组修复兔颅骨缺损效果明显优于单纯珊瑚修复组,在16周时得到完全的骨修复,与自体骨类似.结论:用MSCs和PRP在体外培养构建的细胞-膜片具有较强的成骨活性,包裹珊瑚支架后具有良好的骨修复能力.%Objective: The purpose of this study was to evaluate the efficacy of a hybrid scaffold/cell construction repairing bone defects in rabbits.Methods: Mesenchymal stromal cells (MSCs) suspended in 500μl medium was dissolved in platelet rich plasma (PRP).The mixture of MSCs and PRP was dripped into one 6-well plate and further mixed with prothrombin solution and cultured in a humidified incubator for 3 weeks.The culture medium was standard DMEM for the first week.Osteogenic DMEM was used as the culture medium for next two weeks.The medium was changed every other day.MSCs sheet formed and were detached intactly from the substratum using a cell scraper.The cell-sheet was then wrapped around the coral scaffold and the hybrid sheet-scaffold construct was implanted into a 15mm-diameter rabbit calvarial defect.Autograft and coral implants acted as controls.The new bone formation was investigated by gross examination, histological observation and histomorphometrie analyses in 8- and 16-week after operation.Results: Eight weeks after grafting, new bone in the test group was distributed throughout the coral scaffolds and its surface.New bone in the coral implant group was appeared only in the periphery region.Histomorphometrie data was revealed a significantly higher bone area in test group than in the coral group (P＜0.01).The bone defects of the test group were repaired completely with new bone at the end of sixteen weeks after grafting, while those of the coral group were repaired partly with bone, and fibrous tissue was evident in the central region of defects.The test group had obvious advantage over the coral group in term of bone regeneration (P＜0.01).There was no significant difference in bone formation between the test group and autograft group at 16 weeks after grafting (P＞0.05).Conclusion: The method of growing cell-sheet by combined MSCs and PRP is convenient and simple.The bone graft could bo engineered through combination of the sheet and coral scaffold.
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