{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T23:27:02Z","timestamp":1792106822735,"version":"4.4.0"},"reference-count":98,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2022,8,30]],"date-time":"2022-08-30T00:00:00Z","timestamp":1661817600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University","award":["IZKF OC-1"],"award-info":[{"award-number":["IZKF OC-1"]}]},{"name":"Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University","award":["OPSF597"],"award-info":[{"award-number":["OPSF597"]}]},{"name":"Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University","award":["IA 692092"],"award-info":[{"award-number":["IA 692092"]}]},{"name":"Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University","award":["IA 691513"],"award-info":[{"award-number":["IA 691513"]}]},{"name":"Federal Ministry of Education and Research (BMBF)","award":["IZKF OC-1"],"award-info":[{"award-number":["IZKF OC-1"]}]},{"name":"Federal Ministry of Education and Research (BMBF)","award":["OPSF597"],"award-info":[{"award-number":["OPSF597"]}]},{"name":"Federal Ministry of Education and Research (BMBF)","award":["IA 692092"],"award-info":[{"award-number":["IA 692092"]}]},{"name":"Federal Ministry of Education and Research (BMBF)","award":["IA 691513"],"award-info":[{"award-number":["IA 691513"]}]},{"name":"Medical Faculty of the RWTH Aachen University","award":["IZKF OC-1"],"award-info":[{"award-number":["IZKF OC-1"]}]},{"name":"Medical Faculty of the RWTH Aachen University","award":["OPSF597"],"award-info":[{"award-number":["OPSF597"]}]},{"name":"Medical Faculty of the RWTH Aachen University","award":["IA 692092"],"award-info":[{"award-number":["IA 692092"]}]},{"name":"Medical Faculty of the RWTH Aachen University","award":["IA 691513"],"award-info":[{"award-number":["IA 691513"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in \u03b1-MEM with 10% FCS, at 37 \u00b0C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM \u03b2-glycerophosphate and 50 \u00b5g\/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM\u2013Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.<\/jats:p>","DOI":"10.3390\/cells11172702","type":"journal-article","created":{"date-parts":[[2022,8,30]],"date-time":"2022-08-30T21:25:18Z","timestamp":1661894718000},"page":"2702","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":61,"title":["Physiological Mineralization during In Vitro Osteogenesis in a Biomimetic Spheroid Culture Model"],"prefix":"10.3390","volume":"11","author":[{"given":"Maximilian","family":"Koblenzer","sequence":"first","affiliation":[{"name":"Department of Anatomy and Cell Biology, Uniklinik RWTH Aachen, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marek","family":"Weiler","sequence":"additional","affiliation":[{"name":"Institute for Experimental Molecular Imaging, RWTH Aachen University, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6973-3080","authenticated-orcid":false,"given":"Athanassios","family":"Fragoulis","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Uniklinik RWTH Aachen, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephan","family":"R\u00fctten","sequence":"additional","affiliation":[{"name":"Electron Microscope Facility, Uniklinik RWTH Aachen, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7886-9211","authenticated-orcid":false,"given":"Thomas","family":"Pufe","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Uniklinik RWTH Aachen, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7308-5584","authenticated-orcid":false,"given":"Holger","family":"Jahr","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Uniklinik RWTH Aachen, 52074 Aachen, Germany"},{"name":"Department of Orthopaedic Surgery, Laboratory for Experimental Orthopedics, Maastricht University Medical Centre, 6229 ER Maastricht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,8,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nrrheum.2014.164","article-title":"Fracture healing: Mechanisms and interventions","volume":"11","author":"Einhorn","year":"2015","journal-title":"Nat. Rev. Rheumatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"170","DOI":"10.3389\/fcell.2018.00170","article-title":"Wnt Pathway in Bone Repair and Regeneration\u2014What Do We Know So Far","volume":"6","author":"Houschyar","year":"2018","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1038\/nrrheum.2016.48","article-title":"The genetics of bone mass and susceptibility to bone diseases","volume":"12","author":"Karasik","year":"2016","journal-title":"Nat. Rev. Rheumatol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.1056\/NEJMoa1708322","article-title":"Romosozumab or Alendronate for Fracture Prevention in Women with Osteoporosis","volume":"377","author":"Saag","year":"2017","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1016\/j.jbspin.2018.01.007","article-title":"High bone mass in adults","volume":"85","author":"Paccou","year":"2018","journal-title":"Jt. Bone Spine"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.ajpath.2013.03.006","article-title":"Understanding coupling between bone resorption and formation: Are reversal cells the missing link?","volume":"183","author":"Andersen","year":"2013","journal-title":"Am. J. Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1038\/s41574-020-0339-7","article-title":"A broader strategy for osteoporosis interventions","volume":"16","author":"Reid","year":"2020","journal-title":"Nat. Rev. Endocrinol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2023","DOI":"10.1007\/s00198-017-4009-0","article-title":"Identification and management of patients at increased risk of osteoporotic fracture: Outcomes of an ESCEO expert consensus meeting","volume":"28","author":"Kanis","year":"2017","journal-title":"Osteoporos. Int."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1016\/S2213-8587(17)30188-2","article-title":"Osteoporosis treatment: Recent developments and ongoing challenges","volume":"5","author":"Khosla","year":"2017","journal-title":"Lancet Diabetes Endocrinol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1177\/2045125312471963","article-title":"Lithium: The pharmacodynamic actions of the amazing ion","volume":"3","author":"Brown","year":"2013","journal-title":"Adv. Psychopharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"17406","DOI":"10.1073\/pnas.0505259102","article-title":"Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation and bone mass in mice","volume":"102","author":"Ai","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.22203\/eCM.v024a01","article-title":"In search of an osteoblast cell model for in vitro research","volume":"24","author":"Czekanska","year":"2012","journal-title":"Eur. Cells Mater."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1038\/nrm2236","article-title":"The third dimension bridges the gap between cell culture and live tissue","volume":"8","author":"Pampaloni","year":"2007","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.drudis.2012.10.003","article-title":"Three-dimensional cell culture: The missing link in drug discovery","volume":"18","author":"Breslin","year":"2013","journal-title":"Drug Discov. Today"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1089\/adt.2014.573","article-title":"Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors","volume":"12","author":"Edmondson","year":"2014","journal-title":"Assay Drug Dev. Technol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1083\/jcb.137.1.231","article-title":"Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies","volume":"137","author":"Weaver","year":"1997","journal-title":"J. Cell Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1136\/gutjnl-2013-306651","article-title":"Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays","volume":"64","author":"VanDussen","year":"2015","journal-title":"Gut"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2062","DOI":"10.1038\/s41596-018-0032-7","article-title":"Generation and assembly of human brain region-specific three-dimensional cultures","volume":"13","author":"Sloan","year":"2018","journal-title":"Nat. Protoc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1607","DOI":"10.1016\/j.cell.2018.11.012","article-title":"Inflammatory Cytokine TNF\u03b1 Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture","volume":"175","author":"Peng","year":"2018","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1038\/79439","article-title":"Three-dimensional cellular development is essential for ex vivo formation of human bone","volume":"18","author":"Kale","year":"2000","journal-title":"Nat. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"288","DOI":"10.3389\/fbioe.2019.00288","article-title":"Cell Condensation Triggers the Differentiation of Osteoblast Precursor Cells to Osteocyte-Like Cells","volume":"7","author":"Kim","year":"2019","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1016\/j.jbiosc.2021.09.004","article-title":"Comparative gene expression analysis for pre-osteoblast MC3T3-E1 cells under non-adhesive culture toward osteocyte differentiation","volume":"132","author":"Kim","year":"2021","journal-title":"J. Biosci. Bioeng."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"149","DOI":"10.22203\/eCM.v020a13","article-title":"Pellet culture model for human primary osteoblasts","volume":"20","author":"Richards","year":"2010","journal-title":"Eur. Cell Mater."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"268930","DOI":"10.1155\/2015\/268930","article-title":"A 3D Osteoblast In Vitro Model for the Evaluation of Biomedical Materials","volume":"2015","author":"Restle","year":"2015","journal-title":"Adv. Mater. Sci. Eng."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"672518","DOI":"10.3389\/fmolb.2021.672518","article-title":"Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering","volume":"8","year":"2021","journal-title":"Front. Mol. Biosci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"9309","DOI":"10.1038\/s41598-019-45797-6","article-title":"The two faces of titanium dioxide nanoparticles bio-camouflage in 3D bone spheroids","volume":"9","author":"Souza","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Luttrell, L.M., Dar, M.S., Gesty-Palmer, D., El-Shewy, H.M., Robinson, K.M., Haycraft, C.J., and Barth, J.L. (2019). Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells. PLoS ONE, 14.","DOI":"10.1101\/410597"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1089\/ten.tea.2011.0545","article-title":"MC3T3-E1 osteoprogenitor cells systemically migrate to a bone defect and enhance bone healing","volume":"18","author":"Gibon","year":"2012","journal-title":"Tissue Eng. Part A"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1083\/jcb.96.1.191","article-title":"In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria","volume":"96","author":"Sudo","year":"1983","journal-title":"J. Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"13","DOI":"10.22203\/eCM.v023a02","article-title":"Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche","volume":"23","author":"Birmingham","year":"2012","journal-title":"Eur. Cell Mater."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1002\/jbmr.5650070613","article-title":"Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development","volume":"7","author":"Quarles","year":"1992","journal-title":"J. Bone Miner. Res."},{"key":"ref_32","first-page":"881","article-title":"Divide, accumulate, differentiate: Cell condensation in skeletal development revisited","volume":"39","author":"Hall","year":"1995","journal-title":"Int. J. Dev. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1016\/S0092-8674(00)80257-3","article-title":"Osf2\/Cbfa1: A transcriptional activator of osteoblast differentiation","volume":"89","author":"Ducy","year":"1997","journal-title":"Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"18","DOI":"10.5897\/JDBTE2013.0065","article-title":"Markers Are Shared Between Adipogenic and Osteogenic Differentiated Mesenchymal Stem Cells","volume":"5","author":"Buhrman","year":"2013","journal-title":"J. Dev. Biol. Tissue Eng."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1177\/0022034511435260","article-title":"Osteoinduction: A review of old concepts with new standards","volume":"91","author":"Miron","year":"2012","journal-title":"J. Dent. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1002\/jbmr.5650050806","article-title":"Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization","volume":"5","author":"Weinreb","year":"1990","journal-title":"J. Bone Miner. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1858","DOI":"10.1128\/MCB.15.4.1858","article-title":"Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene","volume":"15","author":"Ducy","year":"1995","journal-title":"Mol. Cell. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1007\/s00223-002-1057-y","article-title":"Von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation","volume":"72","author":"Bonewald","year":"2003","journal-title":"Calcif. Tissue Int."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.ddtec.2006.06.006","article-title":"The use of micro-CT to study bone architecture dynamics noninvasively","volume":"3","author":"Waarsing","year":"2006","journal-title":"Drug Discov. Today Technol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1093\/toxsci\/kfz055","article-title":"Nrf2 Ameliorates DDC-Induced Sclerosing Cholangitis and Biliary Fibrosis and Improves the Regenerative Capacity of the Liver","volume":"169","author":"Fragoulis","year":"2019","journal-title":"Toxicol. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Fragoulis, A., Biller, K., Fragoulis, S., Lex, D., Uhlig, S., and Reiss, L.K. (2021). Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22157853"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"Research0034","DOI":"10.1186\/gb-2002-3-7-research0034","article-title":"Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes","volume":"3","author":"Vandesompele","year":"2002","journal-title":"Genome Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.ab.2004.02.002","article-title":"An Alizarin red-based assay of mineralization by adherent cells in culture: Comparison with cetylpyridinium chloride extraction","volume":"329","author":"Gregory","year":"2004","journal-title":"Anal. Biochem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1007\/s004180050447","article-title":"In situ measurement of collagen synthesis by human bone cells with a Sirius Red-based colorimetric microassay: Effects of transforming growth factor \u03b22 and ascorbic acid 2-phosphate","volume":"112","author":"Jundt","year":"1999","journal-title":"Histochem. Cell Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"8299","DOI":"10.1038\/s41598-019-44575-8","article-title":"Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology","volume":"9","author":"Hwang","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Kubo, Y., Beckmann, R., Fragoulis, A., Conrads, C., Pavanram, P., Nebelung, S., Wolf, M., Wruck, C.J., Jahr, H., and Pufe, T. (2022). Nrf2\/ARE Signaling Directly Regulates SOX9 to Potentially Alter Age-Dependent Cartilage Degeneration. Antioxidants, 11.","DOI":"10.3390\/antiox11020263"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"925","DOI":"10.3727\/096368910X539119","article-title":"Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles","volume":"20","author":"Hellingman","year":"2011","journal-title":"Cell Transpl."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"328","DOI":"10.7150\/thno.13624","article-title":"Imalytics Preclinical: Interactive Analysis of Biomedical Volume Data","volume":"6","author":"Gremse","year":"2016","journal-title":"Theranostics"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"45204","DOI":"10.1038\/srep45204","article-title":"Acceleration of bone regeneration by activating Wnt\/\u03b2-catenin signalling pathway via lithium released from lithium chloride\/calcium phosphate cement in osteoporosis","volume":"7","author":"Li","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1186\/s13287-020-02094-8","article-title":"Impact of 3D cell culture on bone regeneration potential of mesenchymal stromal cells","volume":"12","author":"Bicer","year":"2021","journal-title":"Stem Cell Res. Ther."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"6","DOI":"10.3389\/fphar.2018.00006","article-title":"Three-Dimensional","volume":"9","author":"Langhans","year":"2018","journal-title":"Front. Pharm."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/8756-3282(88)90005-1","article-title":"Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells","volume":"9","author":"Bhargava","year":"1988","journal-title":"Bone"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"558","DOI":"10.22203\/eCM.v021a42","article-title":"Developmental plasticity of human foetal femur-derived cells in pellet culture: Self assembly of an osteoid shell around a cartilaginous core","volume":"21","author":"Wilson","year":"2011","journal-title":"Eur. Cells Mater."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1177\/1087057117696795","article-title":"Three-Dimensional Cell Cultures in Drug Discovery and Development","volume":"22","author":"Fang","year":"2017","journal-title":"SlAS Discov."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1002\/biot.200700228","article-title":"Recent advances in three-dimensional multicellular spheroid culture for biomedical research","volume":"3","author":"Lin","year":"2008","journal-title":"Biotechnol. J."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1016\/j.devcel.2009.11.008","article-title":"The role of hypoxia in development of the Mammalian embryo","volume":"17","author":"Dunwoodie","year":"2009","journal-title":"Dev. Cell."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"3917","DOI":"10.1242\/dev.008441","article-title":"HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis","volume":"134","author":"Amarilio","year":"2007","journal-title":"Development"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1083\/jcb.200612023","article-title":"Hif-1alpha regulates differentiation of limb bud mesenchyme and joint development","volume":"177","author":"Provot","year":"2007","journal-title":"J. Cell Biol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1002\/(SICI)1521-1878(200002)22:2<138::AID-BIES5>3.0.CO;2-4","article-title":"All for one and one for all: Condensations and the initiation of skeletal development","volume":"22","author":"Hall","year":"2000","journal-title":"Bioessays"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1002\/jcp.1041430304","article-title":"Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix","volume":"143","author":"Owen","year":"1990","journal-title":"J. Cell. Physiol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"3111","DOI":"10.1096\/fasebj.4.13.2210157","article-title":"Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation","volume":"4","author":"Stein","year":"1990","journal-title":"Faseb J."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"2734","DOI":"10.2741\/3882","article-title":"Induction and patterning of intramembranous bone","volume":"16","year":"2011","journal-title":"Front. Biosci. (Landmark Ed.)"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1186\/1475-925X-13-136","article-title":"A facile in vitro model to study rapid mineralization in bone tissues","volume":"13","author":"Deegan","year":"2014","journal-title":"Biomed. Eng. Online"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"5487","DOI":"10.1016\/j.biomaterials.2007.08.033","article-title":"Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system","volume":"28","author":"Curcio","year":"2007","journal-title":"Biomaterials"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1046\/j.1469-7580.2003.00247.x","article-title":"Apoptosis during intramembranous ossification","volume":"203","author":"Palumbo","year":"2003","journal-title":"J. Anat."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1016\/S0092-8674(00)80259-7","article-title":"Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development","volume":"89","author":"Otto","year":"1997","journal-title":"Cell"},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Komori, T. (2019). Regulation of Proliferation, Differentiation and Functions of Osteoblasts by Runx2. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20071694"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"3133","DOI":"10.1242\/dev.002709","article-title":"Regulation of skeletogenic differentiation in cranial dermal bone","volume":"134","author":"Abzhanov","year":"2007","journal-title":"Development"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1111\/joa.12095","article-title":"An investigation of cellular dynamics during the development of intramembranous bones: The scleral ossicles","volume":"223","author":"Jabalee","year":"2013","journal-title":"J. Anat."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.bone.2014.08.007","article-title":"Uncovering the periosteum for skeletal regeneration: The stem cell that lies beneath","volume":"70","author":"Roberts","year":"2015","journal-title":"Bone"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1016\/j.bone.2004.07.014","article-title":"Periosteum: Biology, regulation, and response to osteoporosis therapies","volume":"35","author":"Allen","year":"2004","journal-title":"Bone"},{"key":"ref_72","doi-asserted-by":"crossref","unstructured":"Alcorta-Sevillano, N., Mac\u00edas, I., Infante, A., and Rodr\u00edguez, C.I. (2020). Deciphering the Relevance of Bone ECM Signaling. Cells, 9.","DOI":"10.3390\/cells9122630"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1007\/s00223-013-9698-6","article-title":"Role of osteopontin in modulation of hydroxyapatite formation","volume":"93","author":"Hunter","year":"2013","journal-title":"Calcif. Tissue Int."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s00223-001-1121-z","article-title":"Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone","volume":"71","author":"Boskey","year":"2002","journal-title":"Calcif. Tissue Int."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"15872","DOI":"10.1074\/jbc.M701116200","article-title":"Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity","volume":"282","author":"Addison","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1006\/cbir.1994.1088","article-title":"Why does bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption","volume":"18","author":"Roach","year":"1994","journal-title":"Cell Biol. Int."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1042\/BJ20140702","article-title":"Osteopontin mediates mineralization and not osteogenic cell development in vitro","volume":"464","author":"Holm","year":"2014","journal-title":"Biochem. J."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1016\/j.bone.2003.11.027","article-title":"Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-E1 pre-osteoblastic cells","volume":"34","author":"Huang","year":"2004","journal-title":"Bone"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"757","DOI":"10.3389\/fphar.2020.00757","article-title":"The Bone Extracellular Matrix in Bone Formation and Regeneration","volume":"11","author":"Lin","year":"2020","journal-title":"Front. Pharm."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"77s","DOI":"10.1016\/8756-3282(95)00183-E","article-title":"Osteoblast and chondroblast differentiation","volume":"17","author":"Aubin","year":"1995","journal-title":"Bone"},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"448","DOI":"10.1038\/382448a0","article-title":"Increased bone formation in osteocalcin-deficient mice","volume":"382","author":"Ducy","year":"1996","journal-title":"Nature"},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.1038\/s41598-018-19253-w","article-title":"Biomolecular regulation, composition and nanoarchitecture of bone mineral","volume":"8","author":"Poundarik","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"2538","DOI":"10.1021\/bi00539a038","article-title":"Calcium-dependent alpha-helical structure in osteocalcin","volume":"21","author":"Hauschka","year":"1982","journal-title":"Biochemistry"},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"144855","DOI":"10.1016\/j.gene.2020.144855","article-title":"Alkaline phosphatase: Structure, expression and its function in bone mineralization","volume":"754","author":"Vimalraj","year":"2020","journal-title":"Gene"},{"key":"ref_85","doi-asserted-by":"crossref","unstructured":"Jansen, J.H., van der Jagt, O.P., Punt, B.J., Verhaar, J.A., van Leeuwen, J.P., Weinans, H., and Jahr, H. (2010). Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: An in vitro study. BMC Musculoskelet. Disord., 11.","DOI":"10.1186\/1471-2474-11-188"},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1002\/jor.20991","article-title":"Stretch-induced inhibition of Wnt\/beta-catenin signaling in mineralizing osteoblasts","volume":"28","author":"Jansen","year":"2010","journal-title":"J. Orthop. Res."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1046\/j.1432-0436.2003.700602.x","article-title":"In vitro differentiation of embryonic stem cells into mineralized osteoblasts","volume":"71","author":"Kempka","year":"2003","journal-title":"Differentiation"},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1016\/j.bone.2014.11.003","article-title":"Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone","volume":"71","author":"Addison","year":"2015","journal-title":"Bone"},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1089\/ten.2006.0141","article-title":"MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects","volume":"13","author":"Cowan","year":"2007","journal-title":"Tissue Eng."},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1001\/jamapsychiatry.2022.0337","article-title":"Association of Lithium Treatment With the Risk of Osteoporosis in Patients With Bipolar Disorder","volume":"79","author":"Rohde","year":"2022","journal-title":"JAMA Psychiatry"},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1016\/j.bone.2009.12.031","article-title":"Psychotropic drugs have contrasting skeletal effects that are independent of their effects on physical activity levels","volume":"46","author":"Warden","year":"2010","journal-title":"Bone"},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1007\/s00774-016-0783-6","article-title":"Lithium chloride enhances bone regeneration and implant osseointegration in osteoporotic conditions","volume":"35","author":"Jin","year":"2017","journal-title":"J. Bone Miner. Metab."},{"key":"ref_93","doi-asserted-by":"crossref","unstructured":"Hongisto, V., Jernstr\u00f6m, S., Fey, V., Mpindi, J.P., Kleivi Sahlberg, K., Kallioniemi, O., and Per\u00e4l\u00e4, M. (2013). High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0077232"},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1006\/bbrc.2000.4169","article-title":"Lithium inhibits glycogen synthase kinase-3 by competition for magnesium","volume":"280","author":"Ryves","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"1867","DOI":"10.1359\/JBMR.050614","article-title":"Downregulation of Wnt signaling by increased expression of Dickkopf-1 and -2 is a prerequisite for late-stage osteoblast differentiation of KS483 cells","volume":"20","author":"Karperien","year":"2005","journal-title":"J. Bone Miner. Res."},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1016\/j.bone.2005.02.001","article-title":"Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts","volume":"36","author":"Vaes","year":"2005","journal-title":"Bone"},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1016\/j.bbrc.2019.12.066","article-title":"Three-dimensional spheroids of mesenchymal stem\/stromal cells promote osteogenesis by activating stemness and Wnt\/\u03b2-catenin","volume":"523","author":"Imamura","year":"2020","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"7132529","DOI":"10.1155\/2016\/7132529","article-title":"Real-Time Analysis of Endogenous Wnt Signalling in 3D Mesenchymal Stromal Cells","volume":"2016","author":"Saleh","year":"2016","journal-title":"Stem. Cells Int."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/11\/17\/2702\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:20:12Z","timestamp":1760142012000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/11\/17\/2702"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,8,30]]},"references-count":98,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2022,9]]}},"alternative-id":["cells11172702"],"URL":"https:\/\/doi.org\/10.3390\/cells11172702","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,8,30]]}}}