1.1 本文件提供了如何进行 体外 用包括间充质基质细胞在内的祖干细胞进行成骨细胞分化试验。 1.2 本文描述了添加到成骨细胞分化试验的细胞培养基中的各种成骨补充剂的作用，以鼓励和支持祖细胞分化为产生基质的成骨细胞。 1.3 本文件提供了成骨补充剂浓度的建议，这些补充剂可以防止人工矿藏的沉淀，这些矿藏不是由成骨细胞直接产生的，也与细胞的成骨基因表达无关。 1.4 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.5 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认标准化原则制定的。 ====意义和用途====== 4.1 本指导文件描述了用于 体外 成骨细胞分化试验，可用于筛选来自各种人类或动物来源的祖细胞干细胞的成骨能力，包括混合组织源性结缔组织祖细胞群，或可通过培养扩增、处理、转染或基因修饰选择性分离或操纵的细胞群。 4.2 成骨细胞分化试验可称为成骨试验或矿化试验。 4.3 重要的是仔细选择用于 体外 成骨细胞分化试验，因为大量成骨培养基成分可能导致营养不良、病理性或人工钙基沉淀物，这些沉淀物并不表明培养中的细胞分化为功能性成骨细胞 ( 1. ) . 4. 例如，当在培养基中使用高浓度的β-甘油磷酸作为细胞分泌的碱性磷酸酶的底物时，游离磷酸盐显著增加，然后随Ca沉淀 ++ 培养基中形成磷酸钙晶体的离子与祖细胞的分化状态无关 ( 2. , 3. ) . 4.4 碱性磷酸酶的产生是与成骨细胞分化相关的早期事件，但也可以通过向培养基中添加成骨补充剂地塞米松来刺激其他细胞类型。碱性磷酸酶在钙化沉积物自然出现在骨中之前促进其形成，这通常与矿化基质产生的成骨细胞的骨唾液蛋白和骨钙素表达一致。因此，这些类型的钙化/矿藏被认为是营养不良、病理性或人为的，因为它们不是由成熟的成骨细胞引起的。钙测量，如实践中所述 F2997 因此，使用荧光图像分析对祖细胞成骨培养中的钙沉积进行量化，如果在没有基因或蛋白质表达数据的情况下单独使用，可能会导致对骨祖细胞分化状态的潜在错误解释。 4.5 除了筛选未分化干细胞的多潜能外，成骨细胞分化分析有助于评估细胞培养基质或生物材料支架或药物或生物分子（如细胞因子或生长因子）的骨诱导性。 4.6 体外 成骨细胞分化试验不能预测 体内 骨形成，但用于比较目的，以标准化不同类型、来源或传代的祖细胞、生物材料或生物分子的类型和浓度之间的性能。

1.1 This document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells including mesenchymal stromal cells. 1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts. 1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the cells. 1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can be used to screen for the osteogenic capability of progenitor stem cells from various human or animal sources, including mixed tissue-derived connective tissue progenitor populations, or cell populations that may be selectively isolated or manipulated through culture expansion, processing, transfection, or genetic modification. 4.2 The osteoblast differentiation assay may be referred to as an osteogenesis assay or a mineralization assay. 4.3 It is important to carefully select the components and conditions used for in vitro osteoblast differentiation assays since high amounts of osteogenic medium components can lead to dystrophic, pathologic, or artifactual calcium-based precipitates that do not indicate differentiation of the cells in culture to functional osteoblasts ( 1 ) . 4 For example, when high concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca ++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell ( 2 , 3 ) . 4.4 Alkaline phosphatase production is an early event associated with osteoblast differentiation, but it can also be stimulated in other cell types by the addition of the osteogenic supplement dexamethasone to the medium. Alkaline phosphatase enhances the formation of calcified deposits prior to their natural occurrence in bone that typically coincides with bone sialoprotein and osteocalcin expression by mineralized matrix-producing osteoblasts. These kinds of calcified/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. A calcium measurement, such as that described in Practice F2997 for the Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis, may thus result in a potentially false interpretation of the differentiation status of osteoprogenitor cells if used in isolation without gene or protein expression data. 4.5 In addition to screening for multipotentiality of undifferentiated stem cells, osteoblast differentiation assays are useful for assessing the osteoinductivity of cell culture substrates or biomaterial scaffolds or drugs or biomolecules, such as cytokines or growth factors. 4.6 In vitro osteoblast differentiation assays are not predictive of in vivo bone formation, but are useful for comparison purposes to standardize performance between different types, sources or passages of progenitor cells, biomaterials, or types and concentrations of biomolecules.