Baysal Eylem, Al-Sharabi Niyaz, Mustafa Kamal, Costea Daniela E, Brennan Meadhbh, Suliman Salwa
Center of Translational Oral Research (TOR)-Tissue Engineering Group, Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway.
The Gade Laboratory for Pathology and Center for Cancer Biomarkers (CCBIO), Department of Clinical Medicine, University of Bergen, Bergen, Norway.
Stem Cell Rev Rep. 2025 Nov 10. doi: 10.1007/s12015-025-11015-2.
Bone regeneration is a dynamic process regulated by the interplay between the immune and skeletal systems. Regulatory T cells (Treg), a specialized subset of CD4 T cells, play a crucial role in immunomodulation and bone regeneration by regulating the immune response and interacting with progenitor cells. However, the specific mechanisms through which Treg influence the osteogenic differentiation of bone marrow stromal cells (BMSC) remain unexplored. Treg were isolated from six healthy donors, expanded for 13 days, and starved for 24 h to collect Treg-conditioned media (Treg-CM). BMSC obtained from three different healthy donors were treated with Treg-CM at an optimized concentration (50 µg/mL) to assess its impact on BMSC metabolic activity, migration, and osteogenic differentiation. Label free proteomics and cytokine profiling were conducted to identify unique proteins and immunomodulatory factors in Treg-CM. The secretory cytokines of BMSC treated with Treg-CM were also analyzed. Treg-CM enhances BMSC osteogenic differentiation by upregulating the expression of key osteoblast-specific genes, increasing ALP activity, and facilitating calcium deposition. Proteomics identified unique proteins in Treg-CM that regulate cytoskeletal dynamics, metabolic processes and mRNA regulation, highlighting a complex mechanism underlying Treg-CM effects. Cytokine profiling provided key immune modulators in Treg-CM that regulate osteogenesis. Furthermore, elevated levels of MIP-1α and G-CSF were secreted by BMSC treated with Treg-CM further supporting its role in immune-mediated osteogenesis. Our findings reveal that Treg-CM enhances not only osteogenesis in vitro but also fosters a pro-regenerative microenvironment. This highlights its potential as a cell-free strategy for enhancing stem-cell based osteogenesis.
骨再生是一个由免疫系统和骨骼系统相互作用调节的动态过程。调节性T细胞（Treg）是CD4 T细胞的一个特殊亚群，通过调节免疫反应以及与祖细胞相互作用，在免疫调节和骨再生中发挥关键作用。然而，Treg影响骨髓间充质干细胞（BMSC）成骨分化的具体机制仍未得到探索。从六名健康供体中分离出Treg，扩增13天，饥饿24小时以收集Treg条件培养基（Treg-CM）。用优化浓度（50µg/mL）的Treg-CM处理从三名不同健康供体获得的BMSC，以评估其对BMSC代谢活性、迁移和成骨分化的影响。进行无标记蛋白质组学和细胞因子分析，以鉴定Treg-CM中的独特蛋白质和免疫调节因子。还分析了用Treg-CM处理的BMSC的分泌细胞因子。Treg-CM通过上调关键成骨细胞特异性基因的表达、增加碱性磷酸酶（ALP）活性和促进钙沉积来增强BMSC的成骨分化。蛋白质组学鉴定了Treg-CM中调节细胞骨架动力学、代谢过程和mRNA调节的独特蛋白质，突出了Treg-CM作用的复杂机制。细胞因子分析提供了Treg-CM中调节骨生成的关键免疫调节因子。此外，用Treg-CM处理的BMSC分泌的MIP-1α和G-CSF水平升高，进一步支持了其在免疫介导的骨生成中的作用。我们的研究结果表明，Treg-CM不仅增强体外成骨，还促进促再生微环境。这突出了其作为增强基于干细胞的骨生成的无细胞策略的潜力。
