Self-multilayered cell sheet generated by dental pulp stem cells provides a mechanically quantifiable regenerative graft for bone tissue engineering

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.