3D-printed PCL scaffolds functionalized with GelMA for sustained delivery of human dental follicle stem cell-derived conditioned medium in bone regeneration.

Repairing large segmental bone defects remains a significant clinical challenge due to the limited self-healing capacity of bone tissue. Although autologous bone grafting is still the clinical standard, it is limited by donor-site complications and availability. Three-dimensional (3D) printed scaffolds offer customizable architectures but often lack bioactivity. Human dental follicle stem cell-derived conditioned medium (hDFSC-CM) is enriched in osteogenic factors but suffers from rapid clearance at defect sites. Here, we engineered a bioactive composite scaffold by integrating a 3D-printed polycaprolactone (PCL) framework with a gelatin methacrylate (GelMA) hydrogel loaded with hDFSC-CM (PCL/GelMA@hDFSC-CM). This design leverages the mechanical strength of PCL and the bio-interfacial properties of GelMA to achieve sustained release of bioactive factors. In vitro, PCL/GelMA@hDFSC-CM significantly enhanced cell adhesion, proliferation, and osteogenic differentiation compared with PCL or PCL/GelMA controls. In vivo, it promoted defect bridging, mineral deposition, and bone regeneration in a rat tibial defect model. Mechanistically, RNA sequencing and functional validation revealed that these pro-osteogenic effects were mediated, at least in part, by activation of the JAK2/STAT3 signaling pathway. These findings demonstrate that functionalizing polymeric scaffolds with a hydrogel-based delivery system effectively harnesses the regenerative potential of stem cell secretomes, offering a promising cell-free strategy for bone tissue engineering.