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Many studies describe the use of the supplements ascorbic acid, dexamethasone, and \u03b2-glycerophosphate in varying combinations and concentrations. To clarify their individual and combined effects on MC3T3-E1 cells, we conducted a detailed study focusing on cell morphology, mineralization, and gene expression of key osteogenic markers at four time points. Cell morphology was analyzed using light and scanning electron microscopy. Mineralization was assessed via Alizarin Red staining for calcium and von Kossa staining for phosphate. Additionally, energy-dispersive X-ray analysis was used to detect calcium and phosphorus in the cell layer. Sirius Red staining was applied to characterize the fiber formation observed under electron microscopy. To evaluate the influence of the supplements on gene expression, real-time quantitative PCR was performed for the osteogenic markers alkaline phosphatase, bone sialoprotein, bone morphogenetic protein 2, collagen 1a2, osteocalcin, osterix, and runt-related transcription factor 2. Osteogenic differentiation of MC3T3-E1 cells could be induced by application of ascorbic acid and \u03b2-glycerophosphate leading to upregulation of osteogenic markers, changes in cellular morphology, and mineralizing activity. Nevertheless, this effect could be further increased by additional use of dexamethasone. While most osteogenic marker genes were already upregulated on day 7, we did not see the first signs of mineralization until day 14. We recommend using ascorbic acid, \u03b2-glycerophosphate, and dexamethasone in order to get an osteogenic phenotype of MC3T3-E1 on day 14 at the earliest.<\/jats:p>","DOI":"10.1007\/s11626-026-01194-8","type":"journal-article","created":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T14:51:22Z","timestamp":1784559082000},"page":"1112-1130","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The influence of specific groups of osteogenic supplements on the differentiation of MC3T3-E1 cells over time"],"prefix":"10.1007","volume":"62","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-3029-1529","authenticated-orcid":false,"given":"Erik","family":"Betzold","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Bubel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norbert","family":"P\u00fctz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0669-6881","authenticated-orcid":false,"given":"Matthias","family":"Hannig","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8870-6216","authenticated-orcid":false,"given":"Emmanouil","family":"Liodakis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1288-0696","authenticated-orcid":false,"given":"Tim","family":"Pohlemann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9668-4694","authenticated-orcid":false,"given":"Wolfgang","family":"Metzger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,20]]},"reference":[{"key":"1194_CR1","doi-asserted-by":"publisher","first-page":"244","DOI":"10.1016\/j.bone.2014.11.003","volume":"71","author":"WN Addison","year":"2015","unstructured":"Addison WN, Nelea V, Chicatun F, Chien YC, Tran-Khanh N, Buschmann MD, Nazhat SN, Kaartinen MT, Vali H, Tecklenburg MM, Franceschi RT, McKee MD (2015) Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone. 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After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.","order":4,"name":"Ethics","label":"Declaration of generative AI and AI-assisted technologies in the writing process","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Clinical trial number: not applicable.","order":5,"name":"Ethics","label":"Statement on clinical trial","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests","order":6,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}]}}