In Vitro Release and In Vivo Study of Recombinant TGF-β and EGCG from Dual Self-Cross-Linked Alginate-Di-Aldehyde In Situ Injectable Hydrogel for the Repair of a Degenerated Intervertebral Disc in a Rat Tail.

Background and Objective: Intervertebral disc degeneration (IVDD) is a leading cause of lower back pain with limited regenerative treatments. Among emerging regenerative approaches, growth factor-based therapies, such as recombinant human transforming growth factor-beta (Rh-TGF-β), have shown potential for disc regeneration but are hindered by rapid degradation and uncontrolled release by direct administration. Additionally, mechanical stress elevates heat shock protein 90 (HSP-90), impairing cell function and extracellular matrix (ECM) production. This study aimed to investigate a dual self-cross-linked alginate di-aldehyde (ADA) hydrogel system for the sustained delivery of Rh-TGF-β and epigallocatechin gallate (EGCG) to enhance protein stability, regulate release, and promote disc regeneration by targeting both regenerative and stress-response pathways. Methods: ELISA and UV-Vis spectrophotometry assessed Rh-TGF-β and EGCG release profiles. A rat tail IVDD model was established with an Ilizarov-type external fixator for loading, followed by hydrogel treatment with or without bioactive agents. Disc height, tissue structure, and protein expression were evaluated via radiography, histological staining, immunohistochemistry, and Western blotting. Results: The hydrogel demonstrated a biphasic release profile with 100% Rh-TGF-β released over 60 days and complete EGCG release achieved within 15 days. Treated groups showed improved disc height, structural integrity, and proteoglycan retention revealed by histological analysis and elevated HSP-90 expression by immunohistochemistry. In contrast, Western blot analysis confirmed that EGCG effectively downregulated HSP-90 expression, suggesting a reduction in mechanical stress-induced degeneration. Conclusions: ADA hydrogel effectively delivers therapeutic agents, offering a promising strategy for IVDD treatment.