{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T08:22:06Z","timestamp":1791879726945,"version":"4.3.4"},"reference-count":51,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T00:00:00Z","timestamp":1737676800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005980","name":"Kementerian Pendidikan dan Kebudayaan","doi-asserted-by":"publisher","award":["753\/UN.3\/PT\/2022"],"award-info":[{"award-number":["753\/UN.3\/PT\/2022"]}],"id":[{"id":"10.13039\/501100005980","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003710","name":"Korea Health Industry Development Institute","doi-asserted-by":"publisher","award":["HI22C1394"],"award-info":[{"award-number":["HI22C1394"]}],"id":[{"id":"10.13039\/501100003710","id-type":"DOI","asserted-by":"publisher"}]},{"name":"the PUA Scheme of Airlangga Research Fund 2023"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Hydroxyapatite (HA) is widely used as a bone graft. However, information on the head-to-head osteoinductivity and in vivo performance of micro- and nanosized natural and synthetic HA is still lacking. Here, we fabricated nanosized bovine HA (nanoBHA) by using a wet ball milling method and compared its in vitro and in vivo performance with microsized BHA, nanosized synthetic HA (nanoHA), and microsized synthetic HA (HA). The results showed that the wet ball milling method successfully reduced the particle size of BHA to 40 nm without changing its natural characteristics. NanoBHA was able to maintain cell viability and induce cell proliferation and calcium deposits. NanoBHA promoted osteogenic differentiation via OPN as a specific regulator, with a 13-fold greater expression level. NanoBHA and HA also activated ERK1\/2 indicated corresponding to the proliferation-differentiation and death of cells, respectively. The calvarial bone defect model showed that nanoBHA induced bone growth based on CT images, which is in line with the histological results showing the presence of bone cells and connective tissue at the nanoBHA implantation site. In conclusion, natural HA outperformed synthetic HA. Our findings will attract interest in further research into nanomaterials and their mechanism of action in bone remodeling.<\/jats:p>","DOI":"10.1371\/journal.pone.0311652","type":"journal-article","created":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T19:04:54Z","timestamp":1737745494000},"page":"e0311652","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":13,"title":["Comparative study of bovine and synthetic hydroxyapatite in micro- and nanosized on osteoblasts action and bone growth"],"prefix":"10.1371","volume":"20","author":[{"given":"Maria Apriliani","family":"Gani","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6634-7523","authenticated-orcid":true,"given":"Gyubok","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chrismawan","family":"Ardianto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fedik Abdul","family":"Rantam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Lucia Ardhani Dwi","family":"Lestari","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafi","family":"Addimaysqi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"I Ketut","family":"Adnyana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kangwon","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8468-8441","authenticated-orcid":true,"given":"Junaidi","family":"Khotib","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,1,24]]},"reference":[{"key":"pone.0311652.ref001","doi-asserted-by":"crossref","first-page":"580","DOI":"10.1016\/j.actbio.2023.08.041","article-title":"Hydroxyapatite 3D-printed scaffolds with Gyroid-Triply periodic minimal surface (TPMS) porous structure: Fabrication and an in vivo pilot study in sheep","volume":"170","author":"I Bouakaz","year":"2023","journal-title":"Acta Biomater"},{"key":"pone.0311652.ref002","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1016\/j.jcms.2019.01.040","article-title":"The impact of cranioplasty on the patients\u2019 quality of life","volume":"47","author":"PV Worm","year":"2019","journal-title":"Journal of Cranio-Maxillofacial Surgery"},{"key":"pone.0311652.ref003","doi-asserted-by":"crossref","first-page":"S56","DOI":"10.1016\/j.injury.2011.06.011","article-title":"The use of bone-graft substitutes in large bone defects: Any specific needs?","volume":"42","author":"GM Calori","year":"2011","journal-title":"Injury"},{"key":"pone.0311652.ref004","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.bone.2015.07.007","article-title":"Osteoinduction of bone grafting materials for bone repair and regeneration","volume":"81","author":"E Garc\u00eda-Gareta","year":"2015","journal-title":"Bone"},{"key":"pone.0311652.ref005","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nrrheum.2014.164","article-title":"Fracture healing: Mechanisms and interventions","volume":"11","author":"TA Einhorn","year":"2015","journal-title":"Nat Rev Rheumatol"},{"issue":"Suppl 3","key":"pone.0311652.ref006","doi-asserted-by":"crossref","first-page":"131","DOI":"10.2215\/CJN.04151206","article-title":"Normal bone anatomy and physiology","volume":"3","author":"B. 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Janovszky","year":"2019","journal-title":"Materials"},{"key":"pone.0311652.ref021","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1016\/j.cap.2007.10.076","article-title":"Synthesis and characterization of nanocrystalline hydroxyapatite from natural bovine bone","volume":"8","author":"A Ruksudjarit","year":"2008","journal-title":"Current Applied Physics"},{"key":"pone.0311652.ref022","doi-asserted-by":"crossref","first-page":"2422","DOI":"10.1021\/am4000694","article-title":"Effect of calcium phosphate particle shape and size on their antibacterial and osteogenic activity in the delivery of antibiotics in vitro","volume":"5","author":"V Uskokovi\u0107","year":"2013","journal-title":"ACS Appl Mater Interfaces"},{"key":"pone.0311652.ref023","doi-asserted-by":"crossref","first-page":"840","DOI":"10.1016\/j.sjbs.2020.11.020","article-title":"Spectral characterization of hydroxyapatite extracted from Black Sumatra and Fighting cock bone samples: A comparative analysis","volume":"28","author":"KC Vinoth Kumar","year":"2021","journal-title":"Saudi J Biol Sci"},{"key":"pone.0311652.ref024","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1100\/tsw.2010.43","article-title":"Spectroscopic assessment of normal cortical bone: Differences in relation to bone site and sex","volume":"10","author":"N Kourkoumelis","year":"2010","journal-title":"ScientificWorldJournal"},{"key":"pone.0311652.ref025","doi-asserted-by":"crossref","first-page":"25","DOI":"10.3390\/ma10030272","article-title":"Structural evolution during milling, annealing, and rapid consolidation of nanocrystalline Fe-10Cr-3Al powder","volume":"10","author":"R Kumar","year":"2017","journal-title":"Materials"},{"key":"pone.0311652.ref026","unstructured":"ISO. Biological evaluation of medical devices\u2014Part 5: Tests for in vitro cytotoxicity. Biological evaluation of medical devices. Switzerland: International Organization for Standardization; 2009."},{"key":"pone.0311652.ref027","first-page":"23","article-title":"Antibacterial and Cytotoxicity Evaluation of New Hydroxyapatite-Based Granules Containing Silver or Gallium Ions with Potential Use as Bone Substitutes","author":"K Pajor","year":"2022","journal-title":"Int J Mol Sci"},{"key":"pone.0311652.ref028","doi-asserted-by":"crossref","first-page":"653","DOI":"10.2147\/IJN.S39299","article-title":"Highly biocompatible, nanocrystalline hydroxyapatite synthesized in a solvothermal process driven by high energy density microwave radiation","volume":"8","author":"D Smolen","year":"2013","journal-title":"Int J Nanomedicine"},{"key":"pone.0311652.ref029","doi-asserted-by":"crossref","first-page":"111668","DOI":"10.1016\/j.ecoenv.2020.111668","article-title":"Effects of cadmium on osteoblast cell line: Exportin 1 accumulation, p-JNK activation, DNA damage and cell apoptosis","volume":"208","author":"L Ou","year":"2021","journal-title":"Ecotoxicol Environ Saf"},{"key":"pone.0311652.ref030","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1016\/j.actbio.2008.07.023","article-title":"Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells","volume":"5","author":"Z Shi","year":"2009","journal-title":"Acta Biomater"},{"key":"pone.0311652.ref031","doi-asserted-by":"crossref","first-page":"2769","DOI":"10.1016\/j.actbio.2011.03.019","article-title":"Nanoscale hydroxyapatite particles for bone tissue engineering","volume":"7","author":"H Zhou","year":"2011","journal-title":"Acta Biomater"},{"key":"pone.0311652.ref032","first-page":"234","article-title":"Comparison of proliferation and osteoblast differentiation of marrow-derived mesenchymal stem cells on nano- and micro-hydroxyapatite contained composite scaffolds","volume":"8","author":"MB Eslaminejad","year":"2010","journal-title":"Iran J 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Nano"},{"key":"pone.0311652.ref036","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1166\/jbn.2019.2677","article-title":"Nano-hydroxyapatite modulates osteoblast differentiation through autophagy induction via mTOR signaling pathway","volume":"15","author":"R Wang","year":"2019","journal-title":"J Biomed Nanotechnol"},{"key":"pone.0311652.ref037","doi-asserted-by":"crossref","first-page":"8352","DOI":"10.1073\/pnas.140021997","article-title":"Phosphate is a specific signal for induction of osteopontin gene expression","volume":"97","author":"GR Beck","year":"2000","journal-title":"Proc Natl Acad Sci U S A"},{"key":"pone.0311652.ref038","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1016\/j.bone.2010.12.002","article-title":"Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1\/2 pathway is modulated by calcium","volume":"48","author":"S Khoshniat","year":"2011","journal-title":"Bone"},{"key":"pone.0311652.ref039","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.biomaterials.2015.06.039","article-title":"Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression","volume":"65","author":"SW Ha","year":"2015","journal-title":"Biomaterials"},{"key":"pone.0311652.ref040","doi-asserted-by":"crossref","first-page":"1461","DOI":"10.1007\/s10853-013-7864-x","article-title":"Extracting hydroxyapatite and its precursors from natural resources","volume":"49","author":"M Akram","year":"2014","journal-title":"J Mater Sci"},{"key":"pone.0311652.ref041","doi-asserted-by":"crossref","first-page":"e01588","DOI":"10.1016\/j.heliyon.2019.e01588","article-title":"Syntheses of hydroxyapatite from natural sources","volume":"5","author":"NAS Mohd 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