Self-multilayered cell sheet generated by dental pulp stem cells provides a mechanically quantifiable regenerative graft for bone tissue engineering
- 1Department of Life Science Dentistry, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo, 102-8159 Japan
- 2Department 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
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.
Methods: 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.
Results: 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.
Conclusions: 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.
Graphical Abstract:
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13287-026-05304-x.
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BibTeX
@article{mochizuki2026selfmultilayered,
title = {{Self-multilayered cell sheet generated by dental pulp stem cells provides a mechanically quantifiable regenerative graft for bone tissue engineering}},
author = {Mai Mochizuki and Taka Nakahara},
journal = {Stem Cell Research \& Therapy},
year = {2026},
month = sep,
doi = {10.1186/s13287-026-05304-x},
url = {https://doi.org/10.1186/s13287-026-05304-x},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Self-multilayered cell sheet generated by dental pulp stem cells provides a mechanically quantifiable regenerative graft for bone tissue engineering
AU - Mai Mochizuki
AU - Taka Nakahara
PY - 2026
DA - 2026/09/22
JO - Stem Cell Research & Therapy
DO - 10.1186/s13287-026-05304-x
UR - https://doi.org/10.1186/s13287-026-05304-x
AB - 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. Methods: 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. Results: 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. Conclusions: 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. Graphical Abstract: Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13287-026-05304-x.
SN - 1757-6512
ER -