Biglycan Reconstitutes a Neonatal ECM Signaling Microenvironment to Drive Stem Cell-Mediated Tendon Regeneration via a Scaffold-Free Cell Sheet Platform.

Unlike newborns, tendon injuries in adults usually lead to fibrotic scarring rather than functional regeneration. This difference is primarily due to the loss of neonatal extracellular matrix (nECM) signaling in adulthood. In this study, we investigated the molecular mechanisms by which a key neonatal ECM proteoglycan, biglycan (Bgn), orchestrates the behavior of tendon stem/progenitor cells (TSPCs) within a scaffold-free 3D cell sheet microenvironment that recapitulates native tendon architecture. Through immunofluorescence screening, we confirmed that Bgn is the predominant proteoglycan in neonatal rat Achilles tendons. Functional validation showed that adding Bgn to cell sheet cultures promoted TSPCs proliferation, maintained stem cell properties, induced tendon differentiation, and encouraged anisotropic alignment-effects similar to those of intact neonatal ECM. Immunodepletion experiments confirmed the causal role of Bgn. Notably, transplanting Bgn-conditioned TSPCs sheets into a rat full-thickness Achilles tendon defect model significantly restored final tensile load, collagen maturation, and gait function. These outcomes were statistically indistinguishable from those of the uninjured contralateral limb. These findings confirm that Bgn-functionalized cell sheet therapy is a viable translational strategy that can effectively recreate a natural 3D regenerative microenvironment. This work sheds light on the mechanisms involved in the determination of stem cell fate by the ECM and establishes Bgn-functionalized cell sheet therapy as a translatable, scaffold-free strategy for overcoming fibrotic repair and restoring functional tendon architecture.