In vitro characterization and in vivo technical feasibility of biphasic GelMA/HAMA/hydroxyapatite cell-laden hydrogels for osteochondral regeneration: a pilot study in an ovine model

Background Durable osteochondral repair remains an unmet clinical need. Microfracture (MF) and autologous chondrocyte implantation are established options for selected lesions, although they do not reliably reproduce the native zonal architecture and biomechanics of osteochondral tissue. Injectable photopolymerizable hydrogels may offer a single-step intraoperative alternative, but clinical translation requires phase-specific formulations and protocols compatible with surgical workflow. Objectives To develop and characterize, in vitro , GelMA/HAMA hydrogels, with or without hydroxyapatite (HA) microparticles, loaded with ovine Wharton’s jelly mesenchymal stromal cells (WJ-MSCs), and to evaluate, in vivo , the technical feasibility of their sequential in situ delivery and visible-light photopolymerization in an ovine model of critical-size osteochondral defects. Methods Four cell-laden hydrogels (H1–H4) were characterized over 21 days for viability, stability, and chondrogenic/osteogenic differentiation. H1 (chondral) and H3 (subchondral) were then applied in situ as a biphasic implant (IMB) in bilateral critical-size defects (7 mm Ø × 10 mm depth) in two sheep, with layer-by-layer photopolymerization; MF served as the comparator. Exploratory assessment at 1.5 and 3 months used International Cartilage Repair Society (ICRS), Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) 2.0, and modified O'Driscoll scores. Results All formulations supported cell viability without significant differences. Regarding differentiation potential, Collagen II staining was qualitatively stronger in H1 than in H2, and H3, but not H4, showed progressive Alizarin Red positivity from day 7. In situ delivery of the IMB was feasible without perioperative complications. Exploratory assessment did not show a consistent advantage of the IMB over MF. Conclusion Available in vitro evidence supported the selection of H1 and H3 as candidate chondral and subchondral formulations on the basis of qualitative differentiation profiles. This pilot study supports the technical feasibility of sequential in situ delivery and photopolymerization of these GelMA/HAMA biphasic hydrogels loaded with WJ-MSCs in a large-animal model and provides preliminary descriptive data useful for designing larger, adequately powered confirmatory studies.