Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Rheumatology and Clinical Immunology, Berlin, Germany.
German Rheumatism Research Centre (DRFZ) Berlin, a Leibniz Institute, Berlin, Germany.
ALTEX. 2020;37(4):561-578. doi: 10.14573/altex.1910211. Epub 2020 Jun 9.
The aim of the study was to establish an in vitro fracture hematoma (FH) model that mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, which were incubated in osteogenic medium under normoxia/hypoxia and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocyte populations decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia, while cytokine/chemokine secretion remained unchanged. Dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression, and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for modeling the initial phase of fracture healing in vitro and that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and could reduce the number of animal experiments in early preclinical studies.
本研究旨在建立一种体外骨折血肿（FH）模型，以模拟人体骨折间隙的体内情况，从而评估治疗骨折愈合障碍的药物疗效和效果。将人外周血和间充质基质细胞（MSCs）凝结以产生体外 FH 模型，将其在常氧/低氧条件下孵育于成骨培养基中，并分析细胞组成、基因表达和细胞因子/趋化因子分泌情况。为了评估该模型，我们研究了地塞米松（损害骨折愈合）和去铁胺（促进骨折愈合）的影响。在低氧条件下，MSC 成为主要的细胞群，而白细胞群的频率降低。成骨、血管生成、炎症、迁移和低氧适应的标记基因表达随时间推移和与常氧相比显著增加，而细胞因子/趋化因子分泌保持不变。与 MSC 相比，地塞米松更有利于免疫细胞的频率，抑制成骨和成血管基因表达，并增强炎症细胞因子的分泌。相反，去铁胺更有利于 MSC 的频率，增强成骨标记物 RUNX2 和低氧适应标记物的表达。总之，我们证明了缺氧是体外模拟骨折愈合初始阶段的重要因素，并且骨折愈合破坏和促进物质都可以像体内情况一样影响体外模型。因此，我们得出结论，我们的模型能够部分模拟人类 FH，并可以减少早期临床前研究中动物实验的数量。
