Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
German Rheumatism Research Centre (DRFZ) Berlin, a Leibniz Institute, Berlin, Germany.
J Bone Miner Res. 2021 Jun;36(6):1189-1201. doi: 10.1002/jbmr.4267. Epub 2021 Feb 25.
After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (μCT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1β were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. © 2021 American Society for Bone and Mineral Research (ASBMR).
创伤后，骨折间隙内形成的骨折血肿包含所有启动骨再生的重要成分（免疫/干细胞、介质）。因此，它非常重要，但也最容易受到负面影响。为了研究骨折间隙内骨与免疫细胞的相互作用，最新的体外系统应能够再现细胞和体液相互作用以及理化微环境（例如缺氧）。在这里，我们首先开发并表征了无支架骨样构建体（SFBC），该构建体是使用宏观间充质凝聚方法从骨髓间充质基质细胞（MSCs）中产生的。SFBC 表现出弥漫性矿化，表现为骨体积增加（μCT、组织学）和成骨标志物（RUNX2、SPP1、RANKL）表达增加。由人外周血（免疫细胞）与 MSC 混合而成的骨折血肿（FH）模型在缺氧条件下与 SFBC 共培养。结果，FH 模型在共培养 12 和 48 小时后显示出成骨（RUNX2、SPP1）、血管生成（MMP2、VEGF）、HIF 相关（LDHA、PGK1）和炎症（IL6、IL8）标志物的表达增加。FH 的成骨和血管生成基因表达表明 SFBC 的骨诱导潜力和生物功能。在共培养 24 和 48 小时后，可在上清液中检测到 IL-6、IL-8、GM-CSF 和 MIP-1β。为了确认我们的模型对修饰物质（例如治疗药物）的反应性，我们使用了众所周知的诱导细胞缺氧适应反应的去铁胺（DFO）。事实上，DFO 特别增加了 FH 模型中的缺氧适应性、成骨和血管生成过程，但对 SFBC 几乎没有影响，这表明共培养系统中的反应动力学不同。因此，根据我们的数据，我们已经成功地在体外模拟了初始骨折愈合阶段的过程，并得出结论，初始骨折愈合阶段骨与免疫细胞之间的相互作用对于临床前研究尤为重要。
