Institute of Animal Physiology and Genetics, Academy of Sciences, Liběchov, Czech Republic.
Physiol Res. 2011;60(3):559-71. doi: 10.33549/physiolres.932028. Epub 2011 Mar 14.
Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.
间充质干细胞（MSCs）已被反复证明能够修复骨缺损。本研究旨在体外研究小型猪 MSCs 的成骨分化及其分化的标志物。经流式细胞术鉴定的 MSCs 接种于培养塑料（胶原蛋白 I 和纤连蛋白包被/未包被）或血浆凝块（PC）/血浆藻酸盐凝块（PAC）支架上，并在成骨培养基中分化。在分化的三周内，通过茜素红染色观察结节的形成和钙的沉积。此外，通过荧光定量检测碱性磷酸酶（ALP）活性。通过免疫组织化学和 Western Blot 分析检测骨桥蛋白、骨粘连蛋白和骨钙素的表达。我们发现，在分化过程中，2D 和 3D 环境中的骨桥蛋白表达减少。在 2D 系统中，初始较弱的骨粘连蛋白信号在分化的第 7 天或第 14 天达到高峰，依赖于胶原蛋白 I 和纤连蛋白的包被。ALP 的最高活性在成骨分化的第 21 天检测到。与 PAC 支架相比，PC 支架在体外为 MSCs 的成骨分化提供了更好的条件。我们还观察到胶原蛋白 I 和纤连蛋白对加速小型猪 MSC 成骨分化的预期作用。我们的结果表明，小型猪 MSCs 在 2D 和 3D 环境中具有相似的成骨分化能力，但在支架和 ECM 包被单层中的成骨标志物的表达比在没有 ECM 的单层中更早开始。
