富血小板血浆构建组织工程化细胞-膜片的结构和组成  

Structure and composition of the cell-sheet constructed by combination of marrow stromal cells and platelet-rich plasma

作　　者：黄辉[1,2] 苏寒[3] 董震[3] 张森林[3] 

机构地区：[1]南京医科大学口腔医学院,南京210002 [2]解放军第八一医院口腔科 [3]南京军区南京总医院口腔科,南京210002

出　　处：《医学研究生学报》2010年第6期587-590,共4页Journal of Medical Postgraduates

基　　金：南京军区医学科学技术研究"十一五"计划课题(06MA128);南京军区医学科技创新资助课题(07M087);南京军区南京总医院重点科研课题(Z2008044)

摘　　要：目的应用细胞-膜片技术可以显著降低传统组织工程方法中的细胞流失问题。文中分析应用富血小板血浆(platelet rich plasma,PRP)和骨髓基质细胞(marrowstromal cell,MSC)构建的细胞-膜片的结构和组成。方法将体外培养扩增的兔MSC悬液与PRP混合,滴加到6孔培养板上,再滴加牛凝血酶溶液,形成凝胶样MSC/PRP混合物,置于孵箱内培养。第1周用DMEM完全培养液,后2周改用DMEM诱导培养液,形成细胞-膜片。通过大体观察、组织学观察和扫描电镜观察等方法,了解细胞-膜片的结构和组成。以乏血小板血浆(platelet poor plasma,PPP)与MSC在体外培养构建的膜、单纯MSC体外培养形成的膜和兔颅骨骨膜作对照。结果 MSC/PRP组膜片厚约2 mm,呈半透明胶冻样,有较好的韧性和可操作性;组织学观察显示纤维网络样支架中有大量梭形的成骨细胞;扫描电镜观察显示:纤维网状支架中有散在的梭形细胞附着,并可见絮状的骨基质形成。MSC/PPP组膜片的纤维网络样支架中仅有少量细胞分布。MSC组膜片中可见大量的成骨细胞多层密集排列,未见纤维网络样结构,膜片较薄。骨膜中有大量纤维组织紧密排列,其中有散在的成骨细胞。结论用PRP和MSC在体外培养构建的细胞-膜片,由网络状纤维组织及散布其中的梭形成骨细胞所构成,有良好的可操作性。Objective Cell-sheet technology can significantly reduce the loss of cells in conventional tissue engineering.The purpose of this study was to investigate the structure of the cell-sheet constructed by combined marrow stromal cells（MSC） and platelet-rich plasma（PRP）.Methods A suspension of 5×106 MSC in 500 μl medium was dissolved in 500 μl of PRP.The mixture of MSC and PRP was plated in one well of a 6-well plate and cultured in a humidified incubator for 3 weeks.Standard DMEM was used as culture media for first week and osteogenic DMEM for the next two.The cell-sheet was formed and its structure and composition were examined by macroscopic,histological and scanning electron microscopic observations.The sheet constructed by combination of MSC and platelet-poor plasma（PPP） or by simple MSC and periosteum were used as controls.Results The sheet of the MSC/PRP group was 2.0 mm thick,translucent and jelly-like,with good mechanical properties and a great number of spindle-like osteoblasts and some flocculent bone matrices on the network-like fiber frame,while that of the MSC/PPP group exhibited only a few spindle-like osteoblasts.The sheet of the MSC group was thinner than that of the MSC/PRP group,composed of multilayered spindle-like osteoblasts,and without the network-like fiber frame.The periosteum comprised a great deal of compact fibrous tissues and sporadic spindle-like osteoblasts.Conclusion The cell-sheet constructed by combination of MSC and PRP comprises large numbers of spindle-like osteoblasts and some flocculent bone matrices on the network-like fiber frame,and has a good manipulability.

关 键 词：骨组织工程 富血小板血浆 骨髓基质细胞 细胞膜片技术 

分 类 号：R318[医药卫生—生物医学工程]