Parker H Petit Institute for Bioengineering and Bioscience, Georgia, Institute of Technology, 315 Ferst Drive NW, Atlanta, GA 30332, USA.
J Tissue Eng Regen Med. 2012 Dec;6 Suppl 3:s34-46. doi: 10.1002/term.513. Epub 2012 Mar 30.
The goal of this study was to develop a method for increasing the yield of multipotent adipose-derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271(+), CD73(+) and CD45(-)) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11(+) and OCN(+). Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µM) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two-dimensional (2D) cultures as a function of alkaline phosphatase-specific activity and osteocalcin production. In addition, cells were seeded onto three-dimensional (3D) poly-ε-caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro-CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol-treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy.
本研究的目的是开发一种从皮下脂肪中提高多能脂肪间充质干细胞（ASCs）和成骨前体细胞（OPCs）产量的方法。从大鼠腹股沟脂肪中去除成熟脂肪细胞和造血细胞后，通过其对塑料的附着、表面标志物特征（CD271(+)、CD73(+)和 CD45(-)）和分化为脂肪细胞、软骨细胞和成骨细胞的能力来验证剩余细胞群体中的 ASCs。OPCs 被定义为 E11(+)和 OCN(+)。将贴壁细胞在生长培养基 (GM) 或成骨培养基 (OM) 中培养，并在 7 天内用白藜芦醇（0、12.5 和 25 µM）处理；通过流式细胞术评估 ASCs 和 OPCs。在二维（2D）培养中，通过碱性磷酸酶特异性活性和骨钙素产生来确定成骨潜能。此外，将细胞接种到三维（3D）聚己内酯支架上，并在动态条件下培养；通过微 CT 在 4、8 和 12 周时量化矿化。白藜芦醇增加了群体中 ASCs 的百分比（群体%）和 GM 和 OM 中 ASCs 的数量，但仅增加了 GM 中 OPCs 的数量。在两种培养基类型中，白藜芦醇均增加了碱性磷酸酶活性和骨钙素水平。在 3D 培养中，白藜芦醇处理的细胞在早期时间点显著增加了矿化基质体积。白藜芦醇通过富集 ASC 和 OPC 群体并增强成骨分化对贴壁细胞产生双相作用。白藜芦醇预处理在早期时间点诱导更多的矿化，代表了一种可行的临床技术，可用于自体干细胞治疗的骨科和牙科应用。
