浓缩生长因子促进兔骨膜来源细胞的体外增殖、成骨分化和血管生成潜力。
Concentrated growth factor promotes proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells in vitro.
机构信息
Department of Prosthodontics, School of Stomatology, China Medical University, No. 117, Nanjing North Street, Heping District, Shenyang, Liaoning, 110002, People's Republic of China.
Department of Stomatology, First Affiliated Hospital of Jinzhou Medical University, Jinzhou, 121000, Liaoning, China.
出版信息
J Orthop Surg Res. 2019 May 22;14(1):146. doi: 10.1186/s13018-019-1164-3.
OBJECTIVE
The aim of this research is to investigate the effects of concentrated growth factor (CGF) on the proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells (PDCs) in vitro.
METHODS
PDCs were isolated from the femoral and tibial periosteum of rabbits and cultured with or without CGF membranes or CGF conditioned media. Scanning electron microscopy (SEM) was used for the structural characterization. Cell Counting Kit-8 assay was used to measure cell proliferation. Alkaline phosphatase (ALP) activity of PDCs was also measured. Immunohistochemistry was used to detect the expression of CD34. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), and Western blot were used to evaluate the secretion and expression levels of osteogenic differentiation markers (bone morphogenetic protein-2, type I collagen, osteocalcin) and angiogenesis markers (vascular endothelial growth factor, basic fibroblast growth factor) in supernatants and PDCs at days 3, 7, 14, and 21.
RESULTS
The SEM analysis showed a dense three-dimensional fibrin network in CGF, and CGF membranes were covered by PDCs with elongated or polygonal morphological features. Compared with the control group, CGF significantly promoted the proliferation of PDCs during the experimental period (p < 0.05). Immunohistochemistry revealed that PDCs were dispersedly distributed among the CGF substrates, and CD34-positive cells were also present. Moreover, CGF significantly increased the ALP activity and upregulated the expression and secretion of osteogenic differentiation and angiogenesis markers in PDCs at days 3, 7, 14, and 21 (p < 0.05).
CONCLUSION
CGF can increase the proliferation and promote the osteogenic differentiation and angiogenic potential of PDCs in vitro. These results indicate that CGF can be used as a new therapeutic means for biotechnological and clinical applications.
目的
本研究旨在探讨浓缩生长因子（CGF）对体外培养兔骨膜源性细胞（PDC）增殖、成骨分化和血管生成潜能的影响。
方法
从兔股骨和胫骨骨膜中分离出 PDC，分别在有无 CGF 膜或 CGF 条件培养基的情况下进行培养。扫描电子显微镜（SEM）用于结构特征分析。使用细胞计数试剂盒-8 （CCK-8） 法测量细胞增殖。还测量碱性磷酸酶（ALP）活性。免疫组织化学法检测 CD34 的表达。酶联免疫吸附试验（ELISA）、实时定量 PCR（qPCR）和 Western blot 用于评估培养第 3、7、14 和 21 天上清液和 PDCs 中骨形成分化标志物（骨形态发生蛋白-2、I 型胶原、骨钙素）和血管生成标志物（血管内皮生长因子、碱性成纤维细胞生长因子）的分泌和表达水平。
结果
SEM 分析显示 CGF 中有致密的三维纤维蛋白网络，CGF 膜被具有长形或多边形形态特征的 PDC 覆盖。与对照组相比，CGF 在实验期间显著促进了 PDC 的增殖（p<0.05）。免疫组织化学显示 PDC 分散分布在 CGF 基质中，也存在 CD34 阳性细胞。此外，CGF 显著增加了 PDC 在第 3、7、14 和 21 天的 ALP 活性，并上调了骨形成分化和血管生成标志物的表达和分泌（p<0.05）。
结论
CGF 可增加 PDC 的增殖，并促进其体外成骨分化和血管生成潜能。这些结果表明，CGF 可作为生物技术和临床应用的一种新的治疗手段。
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