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Abstract：
Disordered and hypomineralized woven bone formation by dysfunctional mesenchymal stromal cells (MSCs) characterize delayed fracture healing and endocrine -metabolic bone disorders like fibrous dysplasia and Paget disease of bone. To shed light on molecular players in osteoblast differentiation, woven bone formation, and mineralization by MSCs we looked at the intermediate filament desmin (DES) during the skeletogenic commitment of rat bone marrow MSCs (rBMSCs), where its bone-related action remains elusive.
Monolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs.
Immunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine - metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up.
Monolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs.
Immunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine - metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up.
摘要：
■由功能失调的间充质基质细胞（MSC）形成的无序和低矿化编织骨表征骨折愈合延迟和内分泌代谢骨紊乱，如纤维发育不良和骨Paget病。为了阐明成骨细胞分化中的分子参与者，编织骨形成，和MSCs的矿化我们观察了大鼠骨髓MSCs（rBMSCs）骨骼形成过程中的中间丝结蛋白（DES），它与骨骼相关的作用仍然难以捉摸。
■免疫表型和形态学特征的单层培养物，成年雄性rBMSCs显示结蛋白（DES）与波形蛋白的共定位，F-肌动蛋白,和runx2在所有细胞形态中，每个都有助于稀疏和密集的殖民地。这些细胞的蛋白质组学分析揭示了一个拓扑相关的相互作用组，专注于细胞骨架和相关酶//伴侣/信号分子连接DESrunx2和碱性磷酸酶（ALP）。成骨分化导致矿化的编织骨结节局限于致密的菌落，在控制方面明显更小，更循环。它还显着增加了集落形成效率和DES免疫反应性密集集落的数量，以及共定位DES/runx-2和DES/ALP的免疫染色。这些数据证实了前成骨细胞和成骨细胞分化，编织骨形成，和矿化，支持DES作为导致rBMSCs成骨命运的分子途径的参与者。
■免疫细胞化学和形态计量学研究结合蛋白质组学和生物信息学分析支持DES可能充当rBMSCs成骨定型上游信号的概念。因此，我们认为成骨细胞的代谢改变，编织的骨头，功能失调的BMSCs的矿化可能早期通过DES表达//水平的变化来揭示。不愈合骨折和内分泌-代谢性骨疾病，如纤维异常增生和骨Paget病，可能会利用这些分子证据进行早期诊断和随访。
■免疫表型和形态学特征的单层培养物，成年雄性rBMSCs显示结蛋白（DES）与波形蛋白的共定位，F-肌动蛋白,和runx2在所有细胞形态中，每个都有助于稀疏和密集的殖民地。这些细胞的蛋白质组学分析揭示了一个拓扑相关的相互作用组，专注于细胞骨架和相关酶//伴侣/信号分子连接DESrunx2和碱性磷酸酶（ALP）。成骨分化导致矿化的编织骨结节局限于致密的菌落，在控制方面明显更小，更循环。它还显着增加了集落形成效率和DES免疫反应性密集集落的数量，以及共定位DES/runx-2和DES/ALP的免疫染色。这些数据证实了前成骨细胞和成骨细胞分化，编织骨形成，和矿化，支持DES作为导致rBMSCs成骨命运的分子途径的参与者。
■免疫细胞化学和形态计量学研究结合蛋白质组学和生物信息学分析支持DES可能充当rBMSCs成骨定型上游信号的概念。因此，我们认为成骨细胞的代谢改变，编织的骨头，功能失调的BMSCs的矿化可能早期通过DES表达//水平的变化来揭示。不愈合骨折和内分泌-代谢性骨疾病，如纤维异常增生和骨Paget病，可能会利用这些分子证据进行早期诊断和随访。
