Osteoarthritis (OA) is a prevalent and debilitating disease imposing a significant socioeconomic burden. Current treatment strategies, including microfracture, mosaicplasty, and transplantation, have limited success, often necessitating arthroplasty in advanced cases. Tissue engineering using stem cell-laden scaffolds offers a promising alternative but faces challenges such as low mechanical strength, poor cell survival, and ethical concerns. Cell-free approaches, which recruit endogenous mesenchymal stem cells (MSCs) using cell-homing agents, offer advantages. However, current strategies using cytokines or peptides can be costly and may disrupt subchondral bone homeostasis. This study aims to address these limitations by developing a cell-free hydrogel scaffold that features high durability, fast shape memory for minimally invasive surgery, effective cell recruitment, and chondrocyte differentiation.

The literature highlights the limitations of current cartilage regeneration techniques. Cell-laden hydrogels, while biocompatible, often lack sufficient mechanical strength. Stem cell-based therapies present challenges in terms of cell survival, immune rejection, and ethical considerations. Previous research demonstrated the cell-migration promoting properties of tannic acid (TA) and the chondrogenic differentiation properties of Kartogenin (KGN). Combining TA and KGN in a suitable hydrogel offers a potential solution for cell-free cartilage regeneration by sequentially recruiting MSCs and inducing their differentiation into chondrocytes.

A multiple hydrogen-bond crosslinked hydrogel (PTK) was synthesized through polyaddition of methylene diphenyl 4,4-diisocyanate (MDI), imidazolidinyl urea (IU), and poly(ethylene glycol) (PEG), incorporating TA and KGN. The hydrogel's properties were characterized through FT-IR spectroscopy, SEM, tensile testing, fatigue testing, lap shear testing, and UV-Vis spectroscopy and HPLC for drug release. In vitro studies assessed biocompatibility (MTT, Live/Dead assays, hemolysis), anti-oxidation (DPPH assay), anti-inflammation (IL-6 ELISA), and antibacterial activity (CFU counting, Live/Dead staining, SEM). Cell migration (scratch and Transwell assays) and chondrogenic differentiation (pellet culture) were evaluated in vitro using bone marrow mesenchymal stem cells (BMSCs). In vivo studies used a rat model with surgically induced cartilage defects to assess cartilage regeneration. The regenerated cartilage was evaluated macroscopically using ICRS scores, histologically (H&E, toluidine blue, Safranin O/Fast Green), immunohistochemically (COL II), and molecularly (RT-PCR, Western blot) for markers of chondrogenesis (ACAN, COL II, SOX9).

The PTK hydrogel exhibited a fracture strength of 1.1 MPa and withstood 28,000 loading-unloading cycles, exceeding the durability of previously reported hydrogels. It demonstrated fast shape memory recovery at body temperature (30 s), suitable for minimally invasive delivery. The hydrogel showed strong adhesion to cartilage tissue (19.2 kPa). TA exhibited a burst release followed by sustained release, while KGN showed sustained release for 60 days. The hydrogel demonstrated good biocompatibility, significant antioxidant and anti-inflammatory properties, and strong antibacterial effects against both gram-positive and gram-negative bacteria. In vitro studies showed that PTK hydrogel significantly promoted BMSC migration and chondrogenic differentiation. In vivo, PTK hydrogel significantly improved cartilage regeneration compared to controls, as evidenced by macroscopic observation, histological analysis (ICRS and OARSI scores), and molecular analysis (RT-PCR, Western blot) showing significantly increased expression of ACAN, COL II, and SOX9 in the PTK hydrogel group. The PTK group also showed the highest weight-bearing capacity.

The PTK hydrogel successfully addresses several limitations of current cartilage regeneration strategies. Its exceptional mechanical properties and shape memory make it suitable for minimally invasive surgery. The sequential release of TA and KGN effectively recruits MSCs and induces chondrogenesis, leading to significant cartilage regeneration in vivo. The superior performance of PTK hydrogel compared to controls and hydrogels with only TA or KGN highlights the synergistic effect of the two drugs and the importance of the hydrogel's properties. This study demonstrates a promising cell-free strategy for cartilage repair, which avoids the challenges associated with cell-based therapies.

This study successfully developed a novel, ultra-durable, cell-free bioactive hydrogel with fast shape memory and on-demand drug release for cartilage regeneration. The hydrogel's superior mechanical properties, biocompatibility, and ability to promote chondrogenesis make it a promising candidate for minimally invasive cartilage repair. Future studies should focus on optimizing the TA release profile to minimize the burst release, validating the minimally invasive delivery method in larger animal models, and exploring 3D-printing techniques to fabricate hydrogels with complex shapes to better mimic native cartilage structures.

The study used a rat model, which may not perfectly replicate the complexities of human cartilage regeneration. The burst release of TA, although effective in reducing inflammation, could be optimized for improved control over cell homing. The minimally invasive delivery was not fully demonstrated in vivo due to challenges in performing arthroscopic procedures in rats. Further studies in larger animal models are needed to fully assess the potential of minimally invasive delivery.