Abstract
Tendon healing is limited by hypocellularity and low metabolic activity, resulting in poor regeneration. Mesenchymal stem cells (MSCs) offer potential for tendon repair, but reliable tenogenic differentiation protocols remain undefined. Photobiomodulation (PBM) has been proposed as an adjunct to assist differentiation, yet standardized parameters are lacking, particularly in 3D systems. This study evaluated the effects of PBM at 525 nm, 825 nm, and combined wavelengths, delivered at 5 J/cm2 and 10 J/cm2, on immortalized adipose-derived MSCs (iADMSCs) encapsulated in TrueGel3D hydrogels, with the goal of optimizing parameters to support tenogenic differentiation. Immortalized ADMSCs were characterized by immunofluorescence (CD44, CD90, and CD166) and encapsulated in hydrogels. Following a single PBM exposure, differentiation was induced with transforming growth factor-beta 1 and ascorbic acid for 3 days, followed by the addition of connective tissue growth factor for an additional 7 days. Morphology, membrane permeability, proliferation, and gene expression were assessed at days 1, 4, and 10. The cells adopted a spindle-shaped fibroblastic morphology, forming dense cellular networks throughout the hydrogel, although without alignment due to random RGD distribution. LDH release remained low across groups, confirming biocompatibility. Proliferation rates were not significantly different on day 1. By day 4, green and consecutive PBM at 10 J/cm2 and day 10 green PBM at 5 J/cm2 showed increased proliferation rates, respectively. PCR analysis showed co-expression of Scleraxis and Tenomodulin in all groups by day 10, confirming tenogenic differentiation. NIR and consecutive (10 J/cm2) PBM maintained Scleraxis expression over time, with NIR PBM enhancing Collagen I, III, Biglycan and Tenascin-C on day 1 and 4. However, consecutive PBM (10 J/cm2) maintained higher expression patterns more consistently compared to NIR on day 10. Thus, consecutive (525/825 nm) wavelengths at 10 J/cm2 proved effective in enhancing tenogenic marker expression for a single-dose PBM protocol.