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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.
Baysal et al. (Mon,) studied this question.