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The aim of this study was to investigate different methods of mineralization in three-dimensional microporous bacterial nanocellulose with the intention of mimicking the composition, structure, and biomechanical properties of natural bone. To generate the 3D microporous biomaterial, porogen particles were incorporated during BNC fermentation with the Komagataeibacter medellinensis strain. Calcium phosphates (CPs) were deposited onto the BNC scaffolds in five immersion cycles, alternating between calcium and phosphate salts in their insoluble forms. Scanning electron microscopy (SEM) showed that the scaffolds had different pore sizes (between 70 and 350 \u00b5m), and their porous interconnectivity was affected by the biomineralization method and time. The crystals on the BNC surface were shown to be rod-shaped, with a calcium phosphate ratio similar to that of immature bone, increasing from 1.13 to 1.6 with increasing cycle numbers. These crystals also increased in size with an increasing number of cycles, going from 25.12 to 35.9 nm. The main mineral phase observed with X-ray diffraction was octacalcium dihydrogen hexakis phosphate (V) pentahydrate (OCP). In vitro studies showed good cellular adhesion and high cell viability (up to 95%) with all the scaffolds. The osteogenic differentiation of human bone marrow mesenchymal stem cells on the scaffolds was evaluated using bone expression markers, including alkaline phosphatase, osteocalcin, and osteopontin. In conclusion, it is possible to prepare 3D BNC scaffolds with controlled microporosity that allow osteoblast adhesion, proliferation, and differentiation.<\/jats:p>","DOI":"10.3390\/polym15092012","type":"journal-article","created":{"date-parts":[[2023,4,25]],"date-time":"2023-04-25T01:37:01Z","timestamp":1682386621000},"page":"2012","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Biomineralization in Three-Dimensional Scaffolds Based on Bacterial Nanocellulose for Bone Tissue Engineering: Feature Characterization and Stem Cell Differentiation"],"prefix":"10.3390","volume":"15","author":[{"given":"Ana","family":"Ca\u00f1as-Guti\u00e9rrez","sequence":"first","affiliation":[{"name":"Research Group on New Materials (GINUMA), Faculty of Engineering, Universidad Pontificia Bolivariana, Circular 1 No. 70-01, Medell\u00edn 050031, Colombia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lenka","family":"Toro","sequence":"additional","affiliation":[{"name":"Biomedical Engineering Research Group (GIBEC), EIA University, Km 2 + 200 on the Way to the Jos\u00e9 Mar\u00eda C\u00f3rdova Airport, Alto de Las Palmas, Envigado 055428, Colombia"},{"name":"Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, 84505 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7014-3213","authenticated-orcid":false,"given":"Cristina","family":"Fornaguera","sequence":"additional","affiliation":[{"name":"Grup d\u2019Enginyeria de Materials (Gemat), Institut Qu\u00edmic de Sarri\u00e0 (IQS), Universitat Ramon Llull (URL), Via Augusta 390, 08017 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4003-0381","authenticated-orcid":false,"given":"Salvador","family":"Borr\u00f3s","sequence":"additional","affiliation":[{"name":"Grup d\u2019Enginyeria de Materials (Gemat), Institut Qu\u00edmic de Sarri\u00e0 (IQS), Universitat Ramon Llull (URL), Via Augusta 390, 08017 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7755-5846","authenticated-orcid":false,"given":"Marlon","family":"Osorio","sequence":"additional","affiliation":[{"name":"Research Group on New Materials (GINUMA), Faculty of Engineering, Universidad Pontificia Bolivariana, Circular 1 No. 70-01, Medell\u00edn 050031, Colombia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8994-597X","authenticated-orcid":false,"given":"Cristina","family":"Castro-Herazo","sequence":"additional","affiliation":[{"name":"Research Group on New Materials (GINUMA), Faculty of Engineering, Universidad Pontificia Bolivariana, Circular 1 No. 70-01, Medell\u00edn 050031, Colombia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Arboleda-Toro","sequence":"additional","affiliation":[{"name":"Group of Biosocial Studies of the Body-EBSC-, Faculty of Dentistry, Universidad de Antioquia Calle 64 No. 52-59, Medell\u00edn 050010, Colombia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.mser.2014.04.001","article-title":"Biomimetic porous scaffolds for bone tissue engineering","volume":"80","author":"Wu","year":"2014","journal-title":"Mater. 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