PDF(Pubmed)
Abstract：
UNASSIGNED: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year).
UNASSIGNED: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail.
UNASSIGNED: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture.
UNASSIGNED: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.
UNASSIGNED: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail.
UNASSIGNED: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture.
UNASSIGNED: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.
摘要：
■没有足够的体外骨模型来适应成骨细胞的长期培养并支持它们向骨细胞的分化。对骨骼疾病有效疗法的需求增加，以及在研究中取代动物的伦理要求，保证了这种模式的发展。在这里，我们提出了一个深入的协议来准备，创造和保持三维，在体外，支持成骨细胞生成和成骨细胞长期存活（>1年）的自结构化骨模型。
■将成骨细胞接种在纤维蛋白水凝胶上，浇铸在两个β-磷酸三钙锚之间。针对这些自结构化骨模型(SSBM)构造优化的分析方法，包括RT-qPCR，免疫荧光染色和XRF，有详细描述。
■随着时间的推移，细胞重组并用富含胶原蛋白的基质代替初始基质，矿化一个；并在培养12周内证明向骨细胞分化。
■虽然使用次生人类细胞系（hFOB1.19）进行了优化，该协议很容易容纳来自其他物种（大鼠和小鼠）和起源（原发性和继发性）的成骨细胞。这个简单的，简单的方法创建了可重复的体外骨骼模型，这些模型对外部刺激有反应，为进行临床前可翻译研究提供了一个通用的平台。
■将成骨细胞接种在纤维蛋白水凝胶上，浇铸在两个β-磷酸三钙锚之间。针对这些自结构化骨模型(SSBM)构造优化的分析方法，包括RT-qPCR，免疫荧光染色和XRF，有详细描述。
■随着时间的推移，细胞重组并用富含胶原蛋白的基质代替初始基质，矿化一个；并在培养12周内证明向骨细胞分化。
■虽然使用次生人类细胞系（hFOB1.19）进行了优化，该协议很容易容纳来自其他物种（大鼠和小鼠）和起源（原发性和继发性）的成骨细胞。这个简单的，简单的方法创建了可重复的体外骨骼模型，这些模型对外部刺激有反应，为进行临床前可翻译研究提供了一个通用的平台。
