{
  "abstract": "Background: Mesenchymal stromal cell (MSC) sheets are promising regenerative grafts, but fragile scaffold-free sheets remain difficult to harvest, manipulate, and prepare as mechanically reliable grafts for transplantation. We found that human dental pulp stem cells (DPSCs) cultured on type I collagen-coated cultureware in xenogeneic serum-free medium (COL-XFM) autonomously generated a thick, handleable multilayered tissue without manual stacking or exogenous scaffold support. Here, we define this tissue as a DPSC-derived self-multilayered cell sheet (SMCS) and examine its formation, mechanical properties, and graft-organizing function.\n\nMethods: SMCS formation was optimized by seeding-density analysis under COL-XFM culture. The DPSC-optimized condition was then used to compare DPSCs with bone marrow-derived MSCs (BMSCs) and umbilical cord-derived MSCs (UCSCs). We developed a simple surface-contact puncture-force assay to quantify localized rupture resistance during sheet puncture. The day 8 (D8)–day 10 (D10) transition from a two-dimensional (2D) monolayer to three-dimensional (3D) SMCS was examined by analyzing type I collagen (COL1) production, TGF-β signaling, integrin α2 expression, and integrin α2-negative cell sorting. Graft-organizing function was tested by wrapping biphasic calcium phosphate (BCP) granules with SMCS and evaluating ectopic hard tissue formation in mice.\n\nResults: At a defined seeding density, DPSCs reached confluence at the D8 critical point and generated harvestable SMCS by D10, whereas BMSCs and UCSCs did not form SMCS. The puncture-force assay quantified a significantly higher peak puncture force in SMCS than in BMSC monolayers, converting subjective handleability into a quantitative mechanical measure. The D8–D10 2D-to-3D transition was marked by the COL1 Matrix Burst, dependence on TGF-β signaling, and integrin α2-dependent structural integrity. SMCS wrapped BCP granules into cohesive, cell-rich grafts before transplantation. In vivo, SMCS grafts generated significantly more new hard tissue than both DPSC and BMSC suspension grafts, demonstrating an effect beyond the DPSC source alone.\n\nConclusions: This study defines DPSC self-multilayering as a distinct, density-gated, matrix-anchored process of self-organization that generates SMCS. A novel standardized puncture-force assay established SMCS as a mechanically quantifiable, surgically handleable cell sheet. By organizing BCP granules and enhancing in vivo hard tissue formation, SMCS provides a practical regenerative graft for bone tissue engineering.\n\nGraphical Abstract:\n\nSupplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13287-026-05304-x.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Life Science Dentistry, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo, 102-8159 Japan",
    "Department of Developmental and Regenerative Dentistry, The Nippon Dental University School of Life Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo, 102-8159 Japan"
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 2,
  "authors": [
    {
      "affs": [
        0,
        1
      ],
      "name": "Mai Mochizuki",
      "orcid": "0000-0003-2071-8733"
    },
    {
      "affs": [
        1
      ],
      "name": "Taka Nakahara",
      "orcid": "0000-0002-4637-1072"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/2brhdbcnzvg9c.bib",
  "cited_by": 0,
  "doi": "10.1186/s13287-026-05304-x",
  "fulltext_url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC13733114/",
  "id": "2brhdbcnzvg9c",
  "issns": [
    "1757-6512"
  ],
  "landing_url": "https://doi.org/10.1186/s13287-026-05304-x",
  "language": "en",
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "heading",
      "Research"
    ],
    [
      "openalex",
      "1307|Mesenchymal stem cell research"
    ]
  ],
  "openalex": "W7213968435",
  "pdf_url": null,
  "pmcid": "PMC13733114",
  "pmid": "42983583",
  "published": "2026-09-22",
  "publisher": "BMC",
  "ris": "https://research.draconitic.com/paper/2brhdbcnzvg9c.ris",
  "sources": [
    {
      "first_seen": "2026-11-08T12:22:58Z",
      "native_id": "PMC13733114",
      "source": "pmc",
      "url": "https://doi.org/10.1186/s13287-026-05304-x"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "openalex:1307|Mesenchymal stem cell research",
      "subfield": "cell-biology"
    }
  ],
  "title": "Self-multilayered cell sheet generated by dental pulp stem cells provides a mechanically quantifiable regenerative graft for bone tissue engineering",
  "title_text": "Self-multilayered cell sheet generated by dental pulp stem cells provides a mechanically quantifiable regenerative graft for bone tissue engineering",
  "type": "peer-reviewed",
  "updated_at": "2026-11-08T12:22:58Z",
  "url": "https://research.draconitic.com/paper/2brhdbcnzvg9c",
  "venue": "Stem Cell Research & Therapy",
  "versions": []
}