Layered Double Hydroxide-Engineered Oxidized Hyaluronic Acid/Gelatin/Carboxymethyl Chitosan Injectable Hydrogel Incorporating Salvianolic Acid B and Dexamethasone for Cartilage Regeneration

Introduction
Articular cartilage defects remain a major clinical challenge due to the limited intrinsic regenerative capacity, frequently leading to osteoarthritis and joint dysfunction. Current treatments provide only temporary symptomatic relief without restoring cartilage structure and function. This study aims to develop an injectable hydrogel system capable of localized drug delivery and structural support to promote cartilage regeneration.
Methods
An injectable Schiff-base crosslinked hydrogel composed of oxidized hyaluronic acid (OHA), gelatin (GEL), and carboxymethyl chitosan (CMCs) was prepared, with layered double hydroxide nanoparticles (LDHs) incorporated to enhance stability of the hydrogel and enable controlled release of salvianolic acid B (SalB) and dexamethasone (DEX). The chemical change of OHA were determined using titration method and FT-IR analysis. The morphology of LDHs and hydrogel was characterized by SEM and TEM. Gelation behavior, in vitro injectability, self-healing ability, and tissue adhesion were comprehensively evaluated. Biocompatibility was assessed via the CAM assay and in vivo subcutaneous injection in mice. The angiogenic activity of the SalB-loaded hydrogel was examined using scratch wound-healing assay.
Results
FT-IR analysis confirmed oxidation of OHA ≈70% and formation of imine bonds within the hydrogel network. SEM and TEM revealed the nanoscale size and hexagonal morphology of LDHs, as well as homogeneous porous microstructure of the hydrogel. The hydrogel exhibited controlled in situ gelation, excellent injectability, self-healing capability, and strong tissue adhesion. Sustained release of SalB and DEX was achieved over 14 days. The incorporation of LDHs significantly improved structural stability and drug-release behavior compared to LDH-free systems. In vivo studies demonstrated stable gel formation without signs of adverse tissue reactions. Additionally, the hydrogel did not affect chick embryo development and significantly promoted angiogenesis.
Conclusions
The synthesized hydrogel integrates in situ gelation, sustained dual-drug delivery, and excellent biocompatibility, offering a promising minimally invasive platform for cartilage regeneration.