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Traditional treatments often fail to fully restore bone health and may cause side effects, prompting the exploration of regenerative therapies. Adipose-derived stem cells (ADSCs) offer potential for osteoporosis treatment, but their natural inclination toward adipogenic rather than osteogenic differentiation poses a challenge. This study investigates a novel approach combining differentiation inducers (DIs), three-dimensional (3D) hydrogel scaffolds, and photobiomodulation (PBM) to promote osteogenic differentiation of immortalised ADSCs. A dextran-based 3D hydrogel matrix, supplemented with a DI cocktail of dexamethasone, \u03b2-glycerophosphate disodium, and ascorbic acid, was used to foster osteogenesis. PBM was applied using near-infrared (825 nm), green (525 nm), and combined wavelengths at fluences of 3 J\/cm2, 5 J\/cm2, and 7 J\/cm2 to enhance osteogenic potential. Flow cytometry identified osteoblast-specific markers, while inverted light microscopy evaluated cellular morphology. Reactive oxygen species assays measured oxidative stress, and quantitative polymerase chain reaction (qPCR) revealed upregulated gene expression linked to osteogenesis. The findings demonstrate that integrating DIs, 3D hydrogels, and PBM effectively drives osteogenic differentiation in immortalised ADSCs. The PBM enhanced osteogenic marker expression, induced morphological changes, and upregulated gene activity, presenting a promising framework for bone regeneration. Future research should assess the stability and functionality of these differentiated cells and explore their applicability in preclinical models of bone injury or degeneration. This integrative approach demonstrated specific efficacy in promoting the osteogenic differentiation of ADSCs, highlighting its potential application in developing targeted treatments for osteoporosis.<\/jats:p>","DOI":"10.3390\/ijms252413350","type":"journal-article","created":{"date-parts":[[2024,12,12]],"date-time":"2024-12-12T08:36:32Z","timestamp":1733992592000},"page":"13350","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Synergistic Effects of Photobiomodulation and Differentiation Inducers on Osteogenic Differentiation of Adipose-Derived Stem Cells in Three-Dimensional Culture"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1588-2697","authenticated-orcid":false,"given":"Daniella","family":"Da Silva","sequence":"first","affiliation":[{"name":"Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, P.O. Box 17011, Johannesburg 2028, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anine","family":"Crous","sequence":"additional","affiliation":[{"name":"Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, P.O. Box 17011, Johannesburg 2028, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5002-827X","authenticated-orcid":false,"given":"Heidi","family":"Abrahamse","sequence":"additional","affiliation":[{"name":"Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, P.O. 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