{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T23:54:32Z","timestamp":1787961272030,"version":"build-2784847793"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,4,23]],"date-time":"2021-04-23T00:00:00Z","timestamp":1619136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"<jats:p>Patients with a skull defect are at risk of developing cerebrospinal fluid leakage and ascending bacterial meningitis at &amp;gt;10% per year. However, treatment with stem cells has brought great hope to large-area cranial defects. Having found that transforming growth factor (TGF)-\u03b23 can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), we designed a hybrid TGF-\u03b23\/recombinant human-like collagen recombinant human collagen\/chitosan (CS) freeze-dried sponge (TRFS) loading hPDLSCs (TRFS-h) to repair skull defects in rats. CFS with 2% CS was selected based on the swelling degree, water absorption, and moisture retention. The CS freeze-dried sponge (CFS) formed a porous three-dimensional structure, as observed by scanning electron microscopy. In addition, cytotoxicity experiments and calcein-AM\/PI staining showed that TRFS had a good cellular compatibility and could be degraded completely at 90\u00a0days in the implantation site. Furthermore, bone healing was evaluated using micro-computed tomography in rat skull defect models. The bone volume and bone volume fraction were higher in TRFS loaded with hPDLSCs (TRFS-h) group than in the controls (<jats:italic>p<\/jats:italic> &amp;lt; 0.01, <jats:italic>vs.<\/jats:italic> CFS or TRFS alone). The immunohistochemical results indicated that the expression of Runx2, BMP-2, and collagen-1 (COL \u2160) in cells surrounding bone defects in the experimental group was higher than those in the other groups (<jats:italic>p<\/jats:italic> &amp;lt; 0.01, <jats:italic>vs.<\/jats:italic> CFS or TRFS alone). Taken together, hPDLSCs could proliferate and undergo osteogenic differentiation in TRFS (<jats:italic>p<\/jats:italic> &amp;lt; 0.05), and TRFS-h accelerated bone repair in calvarial defect rats. Our research revealed that hPDLSCs could function as seeded cells for skull injury, and their osteogenic differentiation could be accelerated by TGF-\u03b23. This represents an effective therapeutic strategy for restoring traumatic defects of the skull.<\/jats:p>","DOI":"10.3389\/fphar.2021.678322","type":"journal-article","created":{"date-parts":[[2021,4,23]],"date-time":"2021-04-23T07:29:58Z","timestamp":1619162998000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":17,"title":["Transforming Growth Factor-\u03b23\/Recombinant Human-like Collagen\/Chitosan Freeze-Dried Sponge Primed With Human Periodontal Ligament Stem Cells Promotes Bone Regeneration in Calvarial Defect Rats"],"prefix":"10.3389","volume":"12","author":[{"given":"Shiyi","family":"Huang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fenglin","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yating","family":"Cheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yangfan","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yini","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianzhong","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Bei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingxia","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yueping","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yadong","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Xiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,4,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"116159","DOI":"10.1016\/j.carbpol.2020.116159","article-title":"Structure-properties Relationship of Chitosan\/collagen Films with Potential for Biomedical Applications","volume":"237","author":"Andonegi","year":"2020","journal-title":"Carbohydr. Polym"},{"key":"B2","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1016\/j.biomaterials.2019.04.001","article-title":"Injectable Osteogenic Microtissues Containing Mesenchymal Stromal Cells Conformally Fill and Repair Critical-Size Defects","volume":"208","author":"Annamalai","year":"2019","journal-title":"Biomaterials"},{"key":"B3","doi-asserted-by":"publisher","first-page":"2041731417702531","DOI":"10.1177\/2041731417702531","article-title":"Therapeutic Potential of Dental Stem Cells","volume":"8","author":"Chalisserry","year":"2017","journal-title":"J. Tissue Eng."},{"key":"B4","doi-asserted-by":"publisher","first-page":"340","DOI":"10.1002\/jcb.24668","article-title":"Effect and Possible Mechanism of Network between microRNAs and RUNX2 Gene on Human Dental Follicle Cells","volume":"115","author":"Chen","year":"2014","journal-title":"J. Cel. Biochem."},{"key":"B5","doi-asserted-by":"publisher","first-page":"742","DOI":"10.3389\/fbioe.2020.00742","article-title":"Hybrid Freeze-Dried Dressings Composed of Epidermal Growth Factor and Recombinant Human-like Collagen Enhance Cutaneous Wound Healing in Rats","volume":"8","author":"Cheng","year":"2020","journal-title":"Front. Bioeng. Biotechnol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"2197","DOI":"10.1039\/c8bm00492g","article-title":"Electrospinning of In Situ Crosslinked Recombinant Human Collagen Peptide\/chitosan Nanofibers for Wound Healing","volume":"6","author":"Deng","year":"2018","journal-title":"Biomater. Sci"},{"key":"B7","doi-asserted-by":"publisher","first-page":"258","DOI":"10.1186\/s13287-017-0693-0","article-title":"TGF\u03b23 Recruits Endogenous Mesenchymal Stem Cells to Initiate Bone Regeneration","volume":"8","author":"Deng","year":"2017","journal-title":"Stem Cel Res. Ther."},{"key":"B8","doi-asserted-by":"publisher","first-page":"207","DOI":"10.1016\/B978-0-12-802973-2.00012-4","article-title":"Neuroepidemiology of Traumatic Brain Injury","volume":"138","author":"Gardner","year":"2016","journal-title":"Handb. Clin. Neurol."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1275","DOI":"10.1016\/j.ijbiomac.2020.02.023","article-title":"Assessment of Biological Properties of Recombinant Collagen-Hyaluronic Acid Composite Scaffolds","volume":"149","author":"He","year":"2020","journal-title":"Int. J. Biol. Macromol"},{"key":"B10","doi-asserted-by":"publisher","first-page":"5474","DOI":"10.1016\/j.biomaterials.2005.02.002","article-title":"Porosity of 3D Biomaterial Scaffolds and Osteogenesis","volume":"26","author":"Karageorgiou","year":"2005","journal-title":"Biomaterials"},{"key":"B11","doi-asserted-by":"publisher","first-page":"1444","DOI":"10.1111\/jcmm.12125","article-title":"Calcium Ions and Osteoclastogenesis Initiate the Induction of Bone Formation by Coral-Derived Macroporous Constructs","volume":"17","author":"Klar","year":"2013","journal-title":"J. Cel Mol Med"},{"key":"B12","doi-asserted-by":"publisher","first-page":"2773","DOI":"10.1016\/j.biomaterials.2013.12.062","article-title":"The Induction of Bone Formation by the Recombinant Human Transforming Growth Factor-\u03923","volume":"35","author":"Klar","year":"2014","journal-title":"Biomaterials"},{"key":"B13","doi-asserted-by":"publisher","first-page":"631","DOI":"10.1016\/j.jcyt.2019.03.004","article-title":"Sub-confluent Culture of Human Mesenchymal Stromal Cells on Biodegradable Polycaprolactone Microcarriers Enhances Bone Healing of Rat Calvarial Defect","volume":"21","author":"Lam","year":"2019","journal-title":"Cytotherapy"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.3390\/ijms20204982","article-title":"Transforming Growth Factor-\u03b23\/Chitosan Sponge (TGF-\u03b23\/CS) Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells","volume":"20","author":"Li","year":"2019","journal-title":"Int. J. Mol. Sci."},{"key":"B15","doi-asserted-by":"publisher","first-page":"975","DOI":"10.1002\/JPER.19-0273","article-title":"Sulfonated Chitosan Oligosaccharide Alleviates the Inhibitory Effect of Basic Fibroblast Growth Factor on Osteogenic Differentiation of Human Periodontal Ligament Stem Cells","volume":"91","author":"Li","year":"2020","journal-title":"J. Periodontol."},{"key":"B16","doi-asserted-by":"publisher","first-page":"627","DOI":"10.1002\/stem.1909","article-title":"Concise Reviews: Characteristics and Potential Applications of Human Dental Tissue-Derived Mesenchymal Stem Cells","volume":"33","author":"Liu","year":"2015","journal-title":"Stem Cells"},{"key":"B17","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41421-019-0132-8","article-title":"Bi-directional Differentiation of Single Bronchioalveolar Stem Cells during Lung Repair","volume":"6","author":"Liu","year":"2020","journal-title":"Cell Discov"},{"key":"B18","doi-asserted-by":"publisher","first-page":"638","DOI":"10.1016\/j.bioactmat.2020.08.028","article-title":"Application of Bioactive Hydrogels Combined with Dental Pulp Stem Cells for the Repair of Large Gap Peripheral Nerve Injuries","volume":"6","author":"Luo","year":"2021","journal-title":"Bioact. Mater"},{"key":"B19","doi-asserted-by":"publisher","first-page":"1731289","DOI":"10.1155\/2018\/1731289","article-title":"Potential Roles of Dental Pulp Stem Cells in Neural Regeneration and Repair","volume":"2018","author":"Luo","year":"2018","journal-title":"Stem Cell Int"},{"key":"B20","doi-asserted-by":"publisher","first-page":"4660","DOI":"10.2174\/0929867327666200420091312","article-title":"Functional Chitosan-Based Materials for Biological Applications","volume":"27","author":"Ma","year":"2020","journal-title":"Curr. Med. Chem"},{"key":"B21","doi-asserted-by":"publisher","first-page":"1023","DOI":"10.4252\/wjsc.v12.i9.1023","article-title":"Mass Acquisition of Human Periodontal Ligament Stem Cells","volume":"12","author":"Maeda","year":"2020","journal-title":"World J. Stem Cell"},{"key":"B22","doi-asserted-by":"publisher","first-page":"461","DOI":"10.1016\/j.biomaterials.2009.09.063","article-title":"The Effect of Mean Pore Size on Cell Attachment, Proliferation and Migration in Collagen-Glycosaminoglycan Scaffolds for Bone Tissue Engineering","volume":"31","author":"Murphy","year":"2010","journal-title":"Biomaterials"},{"key":"B23","doi-asserted-by":"publisher","first-page":"e12396","DOI":"10.1111\/cpr.12396","article-title":"Oestrogen Retains Human Periodontal Ligament Stem Cells Stemness in Long-Term Culture","volume":"51","author":"Ou","year":"2018","journal-title":"Cell Prolif"},{"key":"B24","doi-asserted-by":"publisher","first-page":"40","DOI":"10.3390\/cells9010040","article-title":"Profile of Matrix-Remodeling Proteinases in Osteoarthritis: Impact of Fibronectin","volume":"9","author":"P\u00e9rez-Garc\u00eda","year":"2019","journal-title":"Cells."},{"key":"B25","doi-asserted-by":"publisher","first-page":"5415","DOI":"10.1039\/c5tb00637f","article-title":"3D Printing Technology to Control BMP-2 and VEGF Delivery Spatially and Temporally to Promote Large-Volume Bone Regeneration","volume":"3","author":"Park","year":"2015","journal-title":"J. Mater. Chem"},{"key":"B26","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1016\/j.biomaterials.2016.01.071","article-title":"Tissue Segregation Restores the Induction of Bone Formation by the Mammalian Transforming Growth Factor-\u0392(3) in Calvarial Defects of the Non-human Primate Papio ursinus","volume":"86","author":"Ripamonti","year":"","journal-title":"Biomaterials"},{"key":"B27","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1111\/jcpe.12642","article-title":"Cementogenesis and Osteogenesis in Periodontal Tissue Regeneration by Recombinant Human Transforming Growth Factor-\u03b2 (3)","volume":"44","author":"Ripamonti","year":"2017","journal-title":"J. Clin. Periodontol"},{"key":"B28","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1016\/j.biomaterials.2016.07.018","article-title":"The Synergistic Induction of Bone Formation by the Osteogenic Proteins of the TGF-\u03b2 Supergene Family","volume":"104","author":"Ripamonti","year":"","journal-title":"Biomaterials"},{"key":"B29","doi-asserted-by":"publisher","first-page":"1354","DOI":"10.1016\/j.ijbiomac.2016.01.112","article-title":"Chitosan Based Biocomposite Scaffolds for Bone Tissue Engineering","volume":"93","author":"Saravanan","year":"2016","journal-title":"Int. J. Biol. Macromol."},{"key":"B30","doi-asserted-by":"publisher","first-page":"875","DOI":"10.1016\/j.bjoms.2017.09.007","article-title":"Three-dimensional Macroporous Materials for Tissue Engineering of Craniofacial Bone","volume":"55","author":"Shakya","year":"2017","journal-title":"Br. J. Oral Maxillofac. Surg"},{"key":"B31","doi-asserted-by":"publisher","first-page":"e12917","DOI":"10.1111\/cpr.12917","article-title":"Nanotextured Silk Fibroin\/hydroxyapatite Biomimetic Bilayer Tough Structure Regulated Osteogenic\/chondrogenic Differentiation of Mesenchymal Stem Cells for Osteochondral Repair","volume":"53","author":"Shang","year":"2020","journal-title":"Cel Prolif"},{"key":"B32","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1016\/j.ijbiomac.2020.01.262","article-title":"Preparation and Characterization of Collagen\/hyaluronic Acid\/chitosan Film Crosslinked with Dialdehyde Starch","volume":"149","author":"Sionkowska","year":"2020","journal-title":"Int. J. Biol. Macromol"},{"key":"B33","doi-asserted-by":"publisher","first-page":"249","DOI":"10.4041\/kjod.2012.42.5.249","article-title":"Skeletal Myogenic Differentiation of Human Periodontal Ligament Stromal Cells Isolated from Orthodontically Extracted Premolars","volume":"42","author":"Song","year":"2012","journal-title":"Korean J. Orthod."},{"key":"B34","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1111\/j.1432-0436.2007.00233.x","article-title":"Development of a Multipotent Clonal Human Periodontal Ligament Cell Line","volume":"76","author":"Tomokiyo","year":"2008","journal-title":"Differentiation"},{"key":"B35","doi-asserted-by":"publisher","first-page":"4987","DOI":"10.3390\/ijms20204987","article-title":"Human Oral Stem Cells, Biomaterials and Extracellular Vesicles: A Promising Tool in Bone Tissue Repair","volume":"20","author":"Trubiani","year":"2019","journal-title":"Int. J. Mol. Sci."},{"key":"B36","doi-asserted-by":"publisher","first-page":"5819","DOI":"10.1016\/j.biomaterials.2011.04.071","article-title":"Comparison of Different Tissue-Derived Stem Cell Sheets for Periodontal Regeneration in a Canine 1-wall Defect Model","volume":"32","author":"Tsumanuma","year":"2011","journal-title":"Biomaterials"},{"key":"B37","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1016\/j.stem.2012.07.003","article-title":"Notch and TGF\u03b2 Form a Reciprocal Positive Regulatory Loop that Suppresses Murine Prostate Basal Stem\/progenitor Cell Activity","volume":"11","author":"Valdez","year":"2012","journal-title":"Cell Stem Cell"},{"key":"B38","doi-asserted-by":"publisher","first-page":"e1801455","DOI":"10.1002\/adhm.201801455","article-title":"PGS Scaffolds Promote the In Vivo Survival and Directional Differentiation of Bone Marrow Mesenchymal Stem Cells Restoring the Morphology and Function of Wounded Rat Uterus","volume":"8","author":"Xiao","year":"2019","journal-title":"Adv. Healthc. Mater"},{"key":"B39","doi-asserted-by":"publisher","first-page":"33","DOI":"10.1016\/j.clineuro.2015.05.031","article-title":"Early Cranioplasty vs. Late Cranioplasty for the Treatment of Cranial Defect: A Systematic Review","volume":"136","author":"Xu","year":"2015","journal-title":"Clin. Neurol. Neurosurg"},{"key":"B40","doi-asserted-by":"publisher","first-page":"103","DOI":"10.2165\/00063030-200418020-00004","article-title":"The Application of Recombinant Human Collagen in Tissue Engineering","volume":"18","author":"Yang","year":"2004","journal-title":"BioDrugs"},{"key":"B41","doi-asserted-by":"publisher","first-page":"1168","DOI":"10.3390\/ma10101168","article-title":"Electrospun Zein\/gelatin Scaffold-Enhanced Cell Attachment and Growth of Human Periodontal Ligament Stem Cells","volume":"10","author":"Yang","year":"2017","journal-title":"Materials (Basel)"},{"key":"B42","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1016\/j.carbpol.2014.07.018","article-title":"A Comparison of Physicochemical Properties of Sterilized Chitosan Hydrogel and its Applicability in a Canine Model of Periodontal Regeneration","volume":"113","author":"Zang","year":"2014","journal-title":"Carbohydr. Polym"},{"key":"B43","doi-asserted-by":"publisher","first-page":"1224","DOI":"10.1016\/j.ijbiomac.2019.10.169","article-title":"The Application of Hyaluronic Acid in Bone Regeneration","volume":"151","author":"Zhai","year":"2020","journal-title":"Int. J. Biol. Macromol"},{"key":"B44","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1016\/j.jcyt.2014.10.016","article-title":"Sweat Gland Regeneration after Burn Injury: Is Stem Cell Therapy a New Hope?","volume":"17","author":"Zhang","year":"","journal-title":"Cytotherapy"},{"key":"B45","doi-asserted-by":"publisher","first-page":"585","DOI":"10.1080\/09205063.2015.1047667","article-title":"Comparison of Two Proanthocyanidin Cross-Linked Recombinant Human Collagen-Peptide (RHC) - Chitosan Scaffolds","volume":"26","author":"Zhang","year":"","journal-title":"J. Biomater. Sci. Polym. Ed."},{"key":"B46","doi-asserted-by":"publisher","first-page":"387","DOI":"10.1097\/00002281-200307000-00004","article-title":"Molecular and Cellular Biology of New Bone Formation: Insights into the Ankylosis of Ankylosing Spondylitis","volume":"15","author":"Zhang","year":"2003","journal-title":"Curr. Opin. Rheumatol."},{"key":"B47","doi-asserted-by":"publisher","first-page":"065005","DOI":"10.1088\/1748-605X\/ab9422","article-title":"Efficient Regeneration of Rat Calvarial Defect with Gelatin-Hydroxyapatite Composite Cryogel","volume":"15","author":"Zhang","year":"2020","journal-title":"Biomed. Mater."},{"key":"B48","doi-asserted-by":"publisher","first-page":"1634","DOI":"10.1089\/scd.2018.0119","article-title":"An In Vitro Comparative Study of Multisource Derived Human Mesenchymal Stem Cells for Bone Tissue Engineering","volume":"27","author":"Zhang","year":"2018","journal-title":"Stem Cell Dev"},{"key":"B49","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1016\/j.colsurfb.2016.12.041","article-title":"Improving Osteogenesis of Three-Dimensional Porous Scaffold Based on Mineralized Recombinant Human-like Collagen via Mussel-Inspired Polydopamine and Effective Immobilization of BMP-2-Derived Peptide","volume":"152","author":"Zhou","year":"2017","journal-title":"Colloids Surf. B Biointerfaces"}],"container-title":["Frontiers in Pharmacology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2021.678322\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,4,23]],"date-time":"2021-04-23T07:30:00Z","timestamp":1619163000000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2021.678322\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,23]]},"references-count":49,"alternative-id":["10.3389\/fphar.2021.678322"],"URL":"https:\/\/doi.org\/10.3389\/fphar.2021.678322","relation":{},"ISSN":["1663-9812"],"issn-type":[{"value":"1663-9812","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,23]]},"article-number":"678322"}}