Abstract: BACKGROUND: Tendon injury repair is often compromised by inflammatory cascades and disordered collagen metabolism, leading to scar formation and mechanical deterioration. Curcumin exhibits anti-inflammatory, antioxidant, and pro-repair potential, but its rapid metabolism and low bioavailability limit clinical application.
OBJECTIVE: To construct a thermosensitive injectable curcumin-loaded chitosan/sodium β-glycerophosphate hydrogel and evaluate its efficacy in tendon repair.
METHODS: (1) Rat tendon stem cells were cultured with different concentrations of curcumin for 24 hours. Cell viability was assessed using the CCK-8 assay, and the 20 µmol/L concentration was selected for subsequent experiments. Rat tendon stem cells were cultured with 0 (control) and 20 µmol/L curcumin, and cell migration was assessed using a wound healing assay. Rat tendon stem cells were cultured in three groups: a control group received no treatment; a model group received tert-butyl hydroperoxide to induce oxidative stress, and a curcumin group received tert-butyl hydroperoxide plus 20 µmol/L curcumin. qRT-PCR was used to analyze the mRNA expression of matrix metalloproteinases 3, 13, type I collagen α1 chain, and type III collagen α1 chain. Western blot assay was used to analyze the protein expression of matrix metalloproteinases 3, 13, Bcl-2, Bax, type I collagen α1 chain, and type III collagen α1 chain. (2) Thermosensitive injectable chitosan/sodium β-glycerophosphate hydrogels and curcumin-loaded chitosan/sodium β-glycerophosphate hydrogels (final concentration 20 µmol/L) were prepared and characterized for their micromorphology and drug release. Rat tendon stem cells were co-cultured with either hydrogel, with cells cultured alone serving as the control group. Live/dead staining and cytoskeleton staining were used to assess the cytocompatibility of the hydrogels. (3) Sixty SD rats were randomly divided into a sham operation group (n=12, Achilles tendon rupture model established after incision and suture), a model group (n=12, Achilles tendon rupture model established), a simple hydrogel group (n=12, Achilles tendon stump injected with chitosan/sodium β-glycerophosphate thermosensitive injectable hydrogel, followed by a second injection 4 days later), a drug group (n=12, Achilles tendon stump injected with 20 μmol/L curcumin solution, followed by a second injection 4 days later), and a drug-loaded hydrogel group (n=12, Achilles tendon stump injected with curcumin-loaded chitosan/sodium β-glycerophosphate thermosensitive injectable hydrogel, followed by a second injection 4 days later). Eight weeks after surgery, tissue samples were collected for peritendinous tissue adhesion assessment, hematoxylin-eosin staining, Masson staining, immunohistochemical staining for cyclooxygenase-2 and type I collagen α1 chain, and biomechanical analysis.
RESULTS AND CONCLUSION: (1) Wound healing assay showed that curcumin promoted the migration of rat tendon stem cells. Compared with the model group, the curcumin group showed decreased mRNA and protein expressions of matrix metalloproteinase-3, matrix metalloproteinase-13, type III collagen α1 chain, and Bax protein (P < 0.05), while increased mRNA and protein expressions of type I collagen α1 chain and Bcl-2 protein (P < 0.05). This suggests that curcumin regulates tendon stem cell function through multiple targets. (2) Scanning electron microscopy revealed that the chitosan/sodium β-glycerophosphate thermosensitive injectable hydrogel exhibited a typical three-dimensional porous network morphology with uniform pore size distribution and interconnected pores. The curcumin-loaded chitosan/sodium β-glycerophosphate thermosensitive injectable hydrogel exhibited excellent drug sustained-release capacity. Live-dead and skeleton staining revealed that both hydrogels had good cytocompatibility. (3) The degree of peritendinous adhesion in the drug-loaded hydrogel group was lower than that in the model group, the simple hydrogel group, and the drug group. Hematoxylin-eosin and Masson staining revealed reduced inflammatory cell infiltration and orderly collagen deposition in the Achilles tendon tissue of the drug-loaded hydrogel group, indicating improved tissue repair quality. Immunohistochemical staining revealed lower cyclooxygenase-2 protein expression in the drug-loaded hydrogel group compared with the model group, the simple hydrogel group, and the drug group (P < 0.05), while higher expression of type I collagen α1 chain protein was observed in the drug-loaded hydrogel group compared with the model group, the simple hydrogel group, and the drug group (P < 0.05). The maximum tensile stress and elastic modulus in the drug-loaded hydrogel group were higher than those in the model group, the simple hydrogel group, and the drug group (P < 0.05). These results indicate that the curcumin-loaded chitosan/sodium β-glycerophosphate thermosensitive hydrogel synergistically reduces inflammation and promotes orderly collagen deposition, significantly improving the quality of tendon repair.

Key words: curcumin, thermosensitive hydrogel, tendon injury, oxidative stress, matrix metalloproteinases, collagen metabolism, tissue engineering, sustained drug release, chitosan