Abstract
<title>Abstract</title> <p>Background Anterior cruciate ligament (ACL) regeneration remains challenging due to poor tendon–bone healing. Adipose-derived mesenchymal stem cells (ADSCs) combined with smart hydrogels offer a promising strategy, but their efficacy and mechanisms in ACL repair require further investigation. Objective To construct a PLGA-PEG-PLGA thermosensitive hydrogel loaded with ADSCs and evaluate its ability to promote ACL regeneration in a rabbit model, focusing on cell proliferation, migration, JNK signaling, and anti-apoptotic effects. Methods PLGA-PEG-PLGA copolymer was synthesized and characterized for gelation behavior, porosity, swelling, and degradation. Rabbit ADSCs were isolated, cultured, and seeded into the hydrogel. In vitro, cell adhesion, proliferation (CCK-8), migration (scratch assay), viability (live/dead staining), and JNK1 protein expression (Western blot) were assessed. In vivo, an ACL transection model was established in rabbits. The ADSCs/hydrogel composite was injected into the injury site (experimental group), while the control group received no intervention. Tendon–bone healing was evaluated at 6 and 12 weeks post-surgery using histology (HE staining), immunohistochemistry (JNK), TUNEL apoptosis assay, and biomechanical testing. Results The hydrogel exhibited thermosensitive gelation at 28.5°C, porous structure (pore diameter ~ 158 µm, porosity ~ 84.6%), and favorable swelling/degradation properties. In vitro, the hydrogel significantly promoted ADSCs proliferation (OD value at 72 h: 0.541 vs. 0.433 in control, p < 0.05), migration (48 h migration rate: 78.5% vs. 53.2%), and upregulated JNK1 protein expression (0.560 vs. 0.460 in control, p < 0.05). Cell viability remained > 90% after 72 h. In vivo, the experimental group showed narrower tendon–bone interface gaps, more organized collagen fibers, higher JNK expression (68.4% vs. 42.2% at 6 weeks,p < 0.05), lower apoptosis index (12.4% vs. 25.7% at 6 weeks), and significantly enhanced biomechanical properties (maximum load at 12 weeks: 268.75 N vs. 182.36 N,p < 0.05) compared to controls. Conclusion The injectable PLGA-PEG-PLGA/ADSCs hydrogel effectively promotes ACL regeneration in a rabbit model by enhancing ADSCs proliferation/migration, activating JNK signaling, and reducing local apoptosis, thereby improving tendon–bone healing and biomechanical strength.This study provides experimental evidence for a minimally invasive cell-based therapy for ACL injury.</p>