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In this study, we developed a microfluidic culture device for the spatial regulation of mesenchymal stem cell (MSC) differentiation within a single-phase hydrogel by controlling concentration gradients of differentiation-inducing factors. The system comprised a PDMS culture chamber with dual-flow of culture media to generate concentration gradients of chondrogenic and osteogenic inductive factors. Numerical simulations were performed to characterize the flow velocity and distribution of factors aligned with the dual-flow direction in the hydrogel. The numerical results confirmed that the dual-flow successfully induced concentration differences between the opposite sides of the hydrogel. Bovine bone marrow-derived MSCs encapsulated in 3% agarose gel were cultured for 20 days under continuous dual-flow containing chondrogenic and osteogenic differentiation media. Histological analysis showed that the localized calcium deposition was aligned with the flow direction on the osteogenic differentiation side. In contrast, sulfated glycosaminoglycan (sGAG) synthesis was observed on the chondrogenic differentiation side. These results demonstrate that the developed culture system can successfully reconstruct an osteochondral-like interface from MSCs within a single-phase hydrogel.<\/jats:p>","DOI":"10.3390\/mi17091073","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T08:07:41Z","timestamp":1789027661000},"page":"1073","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of Microfluidic Culture System for Spatially Regulated Differentiation of Mesenchymal Stem Cells Toward Osteochondral Interface Regeneration"],"prefix":"10.3390","volume":"17","author":[{"given":"Toshifumi","family":"Ohi","sequence":"first","affiliation":[{"name":"Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"conceptualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0749-2699","authenticated-orcid":false,"given":"Shogo","family":"Miyata","sequence":"additional","affiliation":[{"name":"Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1039\/C6BM00068A","article-title":"Articular cartilage: From formation to tissue engineering","volume":"4","author":"Foster","year":"2016","journal-title":"Biomater. 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