Fibroblast stress responses to 3D-spheroid culture and thermal challenge, and stem-cell secretome-mediated repair: an exploratory in vitro investigation.

This study was designed as two complementary and exploratory components: (i) to evaluate transcriptional stress responses associated with different 3D fibroblast spheroid formation strategies and thermal challenge, and (ii) to independently assess the functional effects of stem-cell-derived conditioned media on fibroblast regenerative behavior. Oral fibroblasts are crucial for wound healing but face mechanical and thermal stress during dental procedures. Heat shock proteins (HSPs) mediate stress adaptation, but their response may be influenced by the 3D culture method used for in vitro modelling. Separately, stem-cell secretome, delivered via conditioned media (CM), shows promise for enhancing fibroblasts' regenerative capacity. This study aimed: (i)to investigate the effects of 3D spheroid formation methods and thermal stress on HSP70/90 expression in oral fibroblasts; and (ii)to independently assess the dose-dependent influence of dental pulp (DPSC) and Wharton's jelly (WJSC) stem cell CM on fibroblast proliferation and wound healing in a 2D monolayer system. For the first objective, oral fibroblasts were cultured as 3D spheroids via manual clumping or self-assembly and subjected to thermal stress, with HSP70/90 expression quantified by RT-qPCR. For the second objective, fibroblast monolayers were treated with graded concentrations (10-80%) of DPSC-CM or WJSC-CM, and regenerative characteristics were assessed via proliferation and wound-healing assays. Manual clumping of spheroids induced significantly higher baseline HSP70 expression compared to 2D monolayer controls (p = 0.011), while no significant difference in baseline HSP70 was observed between manual and self-assembled spheroids (p = 0.634). Manual spheroids also demonstrated a greater HSP response following thermal challenge. In the separate 2D experiments, both DPSC-CM and WJSC-CM significantly enhanced fibroblast proliferation in a dose-and time-dependent manner. Wound closure was also significantly enhanced, peaking at an optimal concentration of 40% for DPSCs and at 10% for WJSCs with evidence of a non-linear dose-response. Together, these exploratory findings suggest that spheroid formation methodology and stem cell-derived paracrine factors may independently influence fibroblast stress sensitivity and regenerative function, warranting further mechanistic and protein-level validation.