The potential utility of hybrid photo-crosslinked hydrogels with non-immunogenic component for cartilage repair

Articular cartilage repair is a significant challenge in biomaterials science due to its complex structure and limited self-healing capabilities. Current strategies, such as autologous cartilage transplantation, face limitations like donor site morbidity and tissue availability. Allografts, while circumventing donor site issues, risk immunogenic rejection. Cell-based therapies and biomaterial-based tissue engineering approaches offer alternatives. Chitosan, with its biocompatibility and structural resemblance to glycosaminoglycans (GAGs), shows promise. Another promising approach involves photocurable gelatin (Gel) and hyaluronic acid (HA) hydrogels, which can be crosslinked in situ. This study builds upon previous research using photocurable Gel and HA, adding decellularized cartilage matrix (DCM) from α-1,3-galactose gene knockout pigs to enhance bioactivity and reduce immunogenicity. The hypothesis is that the growth factors and GAGs present in the DCM will promote regeneration and integration with surrounding tissue. The aim is to develop a biomimetic hydrogel suitable for cartilage tissue engineering.

Extensive research focuses on cartilage repair biomaterials, yielding numerous studies in the past 5 years. Synthetic polymers and hydrogels alone have proven inadequate. Bioderived hydrogels based on gelatin (Gel) and hyaluronic acid (HA), components of healthy cartilage, are promising. Photocurable derivatives of Gel and HA allow in situ crosslinking, filling irregularly shaped defects. The incorporation of chondroitin sulfate has been explored to support cell growth and integration. Decellularized cartilage matrices (DCMs) offer a potential source of growth factors and GAGs, but immunogenicity remains a concern. The use of α-1,3-galactose gene knockout pigs addresses this issue, offering non-immunogenic DCM. Existing studies have explored the use of decellularized extracellular matrices (ECM) for cartilage regeneration, but challenges remain in terms of mechanical properties and complete degradation.

The study involved the synthesis of gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) using methacrylic anhydride. Decellularized cartilage matrix (DCM) was obtained from α-1,3-galactose gene knockout pigs through a multi-step process involving enzymatic treatment and grinding. Eight different hydrogel formulations were created, varying the concentrations of GelMA (10% or 15%), HAMA (1%), and DCM (0%, 3%, 6%, or 12%). Photoinitiator was added, and the formulations were photo-polymerized in a Teflon mold. The hydrogels were characterized using NMR and FTIR spectroscopy to verify the composition. Decellularization efficiency was assessed through DNA quantification, histological staining (H&E, Masson, Safranin O, Alcian blue), and SEM. Physicochemical properties, including swelling behavior and compressive modulus, were determined. Cytotoxicity was evaluated using a Live/Dead assay and DNA quantification with DPSCs. Chondrogenic differentiation was assessed by RT-PCR analysis of marker genes (Sox9, Col2a1, ACAN, Coll2, ALP, Col10A1). In vivo studies involved creating cartilage defects in rat knee joints, implanting hydrogels, and assessing repair after 9 weeks using H&E and Safranin O staining, along with ICRS macroscopic scoring.

SEM revealed similar morphologies between pristine cartilage and DCM. DNA analysis confirmed effective decellularization. Hydrogels showed varying water absorption (40-110%), with higher DCM content correlating with increased hydrophilicity. Compressive modulus increased with GelMA and DCM content. All hydrogels degraded completely within 8 hours in vitro. SEM showed porous microstructure. In vitro biocompatibility studies with DPSCs showed no cytotoxicity and enhanced proliferation with increasing DCM concentration. RT-PCR indicated chondrogenic differentiation, particularly in hydrogels with higher GelMA and DCM content. The in vivo study using a rat model showed improved cartilage defect filling with higher DCM content; materials with 12% DCM completely filled the defects with cartilage-like tissue, whereas the control group showed incomplete filling. ICRS macroscopic scores supported these findings, with the highest scores observed for the 12% DCM group.

The study's findings address the research question by demonstrating the successful creation and characterization of biomimetic hydrogels for cartilage repair. The incorporation of non-immunogenic DCM significantly improves hydrogel properties and promotes cartilage regeneration, both in vitro and in vivo. The results suggest that these hydrogels offer an advantage over existing synthetic approaches by closely mimicking the natural composition and structure of cartilage, enhancing cell viability, proliferation, and differentiation. The observed complete degradation in vitro is favorable for tissue integration. The positive in vivo results in a rat model, however, need to be validated in larger animal models with longer follow-up periods. The observed layer-specific chondrogenic differentiation supports the development of layered hydrogels with tailored properties.

This study presents a novel approach to cartilage repair using hybrid photo-crosslinked hydrogels incorporating non-immunogenic porcine DCM. The findings demonstrate enhanced biocompatibility, improved mechanical properties, and significant promotion of chondrogenic differentiation. Future research should focus on larger animal models to validate the clinical potential of these hydrogels, especially exploring the use of layered hydrogels with varying compositions to precisely mimic the natural cartilage structure. The unique properties of these hydrogels indicate their potential as effective biomaterials for cartilage tissue engineering.

The in vivo study was conducted in a rat model, which may not fully replicate the complexities of human cartilage repair. The relatively short in vivo study duration (9 weeks) may not capture long-term effects. The study primarily focused on the effect of DCM concentration and did not explore other factors such as growth factor supplementation or different decellularization techniques. Larger animal models are needed for a comprehensive assessment of long-term integration and functional restoration.