de Beer Jamie, Pepper Michael S
Institute for Cellular and Molecular Medicine, Department of Medical Immunology, and SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
Methods Mol Biol. 2025;2938:145-154. doi: 10.1007/978-1-0716-4607-6_17.
Osteogenesis, which refers to the formation of bone tissue, can be studied in vitro to elucidate the mechanisms of bone development, identify key factors and cytokines, and enhance potential regenerative medicine applications in tissue engineering and transplantation. Adipose-derived stromal/stem cells (ASCs) are particularly promising for these studies due to their capacity to differentiate into various mesodermal lineage cells, including osteoblasts. To induce osteogenic differentiation, ASCs are cultured in a complete growth medium (CGM) supplemented with β-glycerophosphate, ascorbate-2-phosphate and dexamethasone over a period of at least 3 weeks. Osteogenic differentiation can be assessed through the deposition of hydroxyapatite, a key component of mineralized bone matrix, and the activity of alkaline phosphatase, an enzyme crucial for matrix calcification. This study provides a protocol for differentiating ASCs into osteoblasts using osteogenic differentiation medium (ODM) and evaluating the differentiation using Alizarin Red S (ARS) staining and alkaline phosphatase (ALP) assays. The methodology includes the preparation of CGM and ODM, as well as the specific procedures for ARS staining and ALP activity assays. The ARS staining involves fixation of cells, staining, and quantification of hydroxyapatite deposition, while the ALP assay measures enzyme activity. Both assays are normalized to cell counts, determined through DAPI staining and microscopic analysis. The results of these assays are intended to provide an accurate evaluation of osteogenic differentiation efficiency in ASCs.
骨生成是指骨组织的形成，可在体外进行研究，以阐明骨发育的机制，确定关键因子和细胞因子，并加强组织工程和移植中潜在的再生医学应用。脂肪来源的基质/干细胞（ASC）因其能够分化为包括成骨细胞在内的各种中胚层谱系细胞，在这些研究中特别有前景。为诱导成骨分化，将ASC在补充有β-甘油磷酸、抗坏血酸-2-磷酸和地塞米松的完全生长培养基（CGM）中培养至少3周。成骨分化可通过羟基磷灰石（矿化骨基质的关键成分）的沉积以及碱性磷酸酶（对基质钙化至关重要的一种酶）的活性来评估。本研究提供了一种使用成骨分化培养基（ODM）将ASC分化为成骨细胞并使用茜素红S（ARS）染色和碱性磷酸酶（ALP）测定法评估分化的方案。该方法包括CGM和ODM的制备，以及ARS染色和ALP活性测定的具体步骤。ARS染色包括细胞固定、染色以及羟基磷灰石沉积的定量，而ALP测定则测量酶活性。两种测定均根据通过DAPI染色和显微镜分析确定的细胞计数进行标准化。这些测定的结果旨在准确评估ASC中的成骨分化效率。
