Mertz Edward L, Makareeva Elena, Mirigian Lynn S, Leikin Sergey
Eunice Kennedy Shriver National Institute of Health and Human Development National Institutes of Health Bethesda MD USA.
JBMR Plus. 2022 Dec 13;7(1):e10701. doi: 10.1002/jbm4.10701. eCollection 2023 Jan.
Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features in vivo, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and in situ messenger RNA (mRNA) hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form bona fide bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like structure with chondrocyte-to-osteoblast transdifferentiation. The two nonmineralizing cell-ECM structures may mimic periosteal cambium and pathological, nonmineralizing osteoid. Importantly, the most commonly used culture conditions (10mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of bona fide bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
二维（2D）成骨细胞/骨细胞培养中矿化结节与骨生物学、病理学和工程学的相关性是一个存在了数十年的问题，但似乎仍缺乏全面的答案。已报道了类骨细胞、细胞外基质（ECM）和矿物质，但也有非类骨细胞的报道。许多研究基于与体内少数选定骨特征的相似性描述了看似类骨的细胞-ECM结构，但没有研究同时检查多个骨特征，也没有系统地研究同一培养物中共存的所有类型的结构。在这里，我们报告基于光学和电子显微镜、拉曼显微光谱、基因表达和原位信使核糖核酸（mRNA）杂交对2D培养物进行的这种全面分析。我们证明，来自小鼠颅骨的原代细胞的2D培养确实形成了真正的骨。其中的细胞、ECM和矿物质表现出与膜内成骨一致的形态、结构、超微结构、组成、时空基因表达模式和生长。然而，这种骨只是同一2D培养物中共存的至少五种不同类型的细胞-ECM结构之一，它们在与骨的相似性和矿化能力方面差异很大。我们表明，其他两种矿化结构可能代表具有软骨细胞向成骨细胞转分化的异常（破坏的）骨和软骨样结构。两种非矿化细胞-ECM结构可能模拟骨膜形成层和病理性、非矿化类骨质。重要的是，最常用的培养条件（10mMβ-甘油磷酸）会诱导所有细胞-ECM结构的人工矿化，然后它们变得几乎无法区分。因此，我们讨论了促进真正骨形成的条件和方法，以及将其与其他细胞-ECM结构区分开来的简单方法。我们的发现可能会改善基于2D培养的成骨细胞分化和功能分析，并将这些培养物的应用扩展到一般骨生物学和组织工程研究。发表于2022年。本文是美国政府的作品，在美国属于公共领域。由Wiley Periodicals LLC代表美国骨与矿物质研究学会发表。
