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

Articular cartilage has a complex, inhomogeneous, anisotropic structure with distinct superficial, middle, and deep zones, and limited intrinsic healing capacity. Surgical repair with autografts can succeed but is limited by donor-site morbidity, tissue availability, and mechanical mismatch. Allografts avoid donor-site issues but often provoke immunogenic rejection and show poor integration and mechanical performance. Besides cell-based therapies, biomaterial scaffolds that mimic cartilage composition and properties aim to promote in situ tissue regeneration. Gelatin (as collagen derivative) and hyaluronic acid (a native cartilage GAG) are promising bases, especially as photocurable derivatives (GelMA and HAMA) that can be crosslinked in situ to conform to irregular defects. This study hypothesized that incorporating an allogenic decellularized cartilage matrix (DCM) from α-1,3-galactose gene-knockout pigs would (i) reduce immunogenicity and inflammation and (ii) provide retained bioactive factors and GAGs to promote chondrocyte population and matrix regeneration. The objective was to synthesize, characterize, and evaluate GelMA/HAMA/DCM hybrid photocrosslinked hydrogels across formulations for physicochemical properties, in vitro cytocompatibility and chondrogenic differentiation of dental pulp stem cells (DPSCs), and in vivo cartilage repair in a rat knee defect model.

The paper situates the work within challenges of cartilage repair: complex cartilage zonal structure and limited self-healing impede outcomes with grafts. Previous biomaterials include chitosan-based scaffolds that are non-immunogenic, mimic GAGs, and support collagen II production, yet with limitations. Photocurable GelMA/HAMA systems have shown promise; Levett et al. combined GelMA and HAMA with chondroitin sulfate to support cell growth and integration. Other studies have used ECM-derived materials, but decellularization can reduce GAG/collagen and pure ECM often lacks tunable mechanics and shape fidelity, necessitating composites with synthetics (e.g., PCL), which may degrade too slowly in vivo. The paper builds on these by using an α-1,3-galactose-deficient porcine DCM to mitigate hyperacute rejection associated with α-Gal epitopes and hypothesizes retained growth factors/GAGs to drive chondrogenesis when combined with photocurable GelMA/HAMA.

Materials and hydrogel preparation: Photocurable precursors were synthesized. Gelatin methacrylate (GelMA) was prepared by reacting gelatin (20 g) in DPBS (200 mL) at 50 °C with methacrylic anhydride (16 mL) for 3 h, followed by dilution, dialysis, freezing, and lyophilization (yield 16.27 g). Hyaluronic acid methacrylate (HAMA) was synthesized from HA (2 g) in PBS (200 mL) with methacrylic anhydride (2 mL) for 24 h at 4 °C while maintaining pH 8–10, followed by dialysis and lyophilization (yield 1.84 g). DCM: Cartilage (~200 g) from ribs of α-1,3-Gal gene-knockout pigs (Nanjing Medical University) was processed: PBS washes, 10 mM Tris-HCl (45 °C, 24 h), 0.25% trypsin (37 °C, 24 h), protease inhibitor treatment, freeze-thaw, grinding, sieving to particles <40 μm, storage at −80 °C. Ethical approval WIUCAS 20033115 and 20200331. Hydrogel formulations: Eight formulations were prepared with constant HAMA 1% (m/v) and variable GelMA (10% or 15%) and DCM (0, 3, 6, 12% m/v). Series A: 10% GelMA/1% HAMA with 0, 3, 6, 12% DCM. Series B: 15% GelMA/1% HAMA with 0, 3, 6, 12% DCM. Water content by mass ranged ~78–90%. Photoinitiator Irgacure 2959 at 0.5% (m/v) was added. Samples were cast in Teflon molds and photocrosslinked under 365 nm UV at 18 mW/cm² for 2 min. Characterization: Spectroscopy: 1H NMR (500 MHz, D2O) confirmed methacrylate vinyl peaks (5.2–5.7 ppm) for GelMA and HAMA. FTIR (500–4000 cm−1) analyzed compositions. Decellularization verification: SEM imaging of pristine versus decellularized cartilage; DNA content quantified by Quant-iT PicoGreen dsDNA assay (10 mg samples, triplicates) with absorbance read at 520 nm; histology (H&E, Masson trichrome, Safranin O, Alcian blue) assessed cellular remnants and matrix retention. Physical properties: Swelling tests: Disks (8 mm diameter, 1 mm height) weighed (W0), incubated in PBS at 37 °C, reweighed at 6–60 h to compute swelling ratio P = (Wt − W0)/W0 × 100%. Compression: Cylinders (5 mm diameter × 5 mm height) tested on Instron 5944 at 2 mm/min; apparent Young’s modulus taken from 5–10% strain region; n=5 per composition. Enzymatic degradation: Samples incubated in 0.5 mg/mL collagenase type II at 37 °C; mass remaining measured at 2, 4, 6, 8 h (R = Wt/W0 × 100%); n=3. SEM microstructure: Freeze-dried fractured samples sputter-coated with Pt and imaged (Hitachi SU 8010); pore size distributions estimated. Cell studies (in vitro): DPSCs (Chinese Academy of Sciences Stem Cell Bank) cultured in α-MEM with 10% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2. Hydrogel coatings were formed in 24-well plates by in-well photocrosslinking. After ethanol sterilization and PBS washing, DPSCs were seeded (5 × 10^3 cells/well). Viability and morphology: Live–Dead staining (AO/EB) and cytoskeleton staining (rhodamine for F-actin, DAPI for nuclei) at 1, 3, 7, 14 days with fluorescence microscopy. Proliferation: Quant-iT PicoGreen dsDNA assay at days 7 and 14. Chondrogenic differentiation: DPSCs cultured on materials for 7, 14, 21 days. RNA extracted (TRIzol), cDNA synthesized, and RT-PCR performed with SYBR Green for Sox9, ACAN, Col2a1, Col2, ALP, Col10A1 (primers listed), GAPDH as reference; cycling: 95 °C 15 min, 40 cycles of 95 °C 10 s, 60 °C 15 s, 72 °C 15 s; melting curve 75–95 °C. Relative expression calculated by ΔΔCt. Qualitative GAG/cartilage matrix deposition assessed by Safranin O and Alcian blue staining at days 1, 7, 14 (supplementary). In vivo rat knee defect model: Adult male Sprague–Dawley rats (n=20; ~260 g) received full-thickness cylindrical cartilage defects (2 mm diameter × 2 mm depth) in the right knee femoral condyle. Disks from Series B materials (formulations 5–8) were prepared and cored to 2 mm diameter; implanted into defects (four rats per material); four sham controls. Anesthesia with 10% chloral hydrate (4 μL/g); penicillin prophylaxis. Animals recovered without complications; sacrificed at 9 weeks (chloral hydrate, 8 μL/g). Gross inspection and histology: H&E and Safranin O/Fast Green staining; ICRS macroscopic repair scoring performed by two blinded observers. Statistical analysis with GraphPad Prism; data mean ± SD; significance thresholds p < 0.05, 0.01, 0.001.

- Decellularization efficacy: DNA content decreased from 813.9 ± 26.3 ng/mg (native cartilage) to 85.8 ± 5.7 ng/mg (DCM), p < 0.001; H&E showed near absence of nuclei with preserved ECM; Masson staining indicated collagen retention; Safranin O and Alcian blue confirmed cartilage matrix and proteoglycans present post-decellularization. - Swelling behavior: Water uptake in PBS (37 °C) ranged ~45–110% by mass. DCM increased hydrophilicity; in Series A, swelling rose from 48% (no DCM) to 110% ± 3% (material 4: 10% GelMA/1% HAMA/12% DCM). Series B showed a smaller increase (60% to ~75% from material 5 to 8). - Compressive properties: Apparent Young’s modulus increased with GelMA and DCM content. Series A (10% GelMA): from 66 ± 10 kPa (no DCM) to 135 ± 20 kPa (12% DCM). Series B (15% GelMA): from 179 ± 22 kPa to 305 ± 30 kPa (12% DCM). GelMA increase (10%→15%) raised stiffness by ~2–3×; adding DCM increased stiffness by factors ~2.8 (Series A) and ~1.7 (Series B). - Enzymatic degradability: In collagenase II (0.5 mg/mL), Series A samples fully degraded within 6 h; Series B within 8 h. Higher GelMA content prolonged degradation; DCM content had minor impact on degradation kinetics. - Microstructure: All hydrogels were highly porous (initial water 78–90% by mass). SEM indicated bimodal pore sizes ~140 ± 20 μm and ~175 ± 25 μm; higher DCM content (materials 4, 8) correlated with smaller pores. - In vitro cytocompatibility and proliferation: Live–Dead showed viable cells across all materials/timepoints. Rhodamine/DAPI staining revealed robust cytoskeletal organization and increasing confluence by day 7–14, most pronounced on higher DCM content hydrogels (3, 4, 7, 8). PicoGreen assays showed increased DNA content with increasing DCM at days 7 and 14; higher GelMA (15%) further supported proliferation (e.g., material 8 > 4 at day 14). - Chondrogenic differentiation: Sox9 (early marker) significantly upregulated at day 7, up to 4.5-fold for 12% DCM (materials 4, 8). Col2a1 and Col2 (middle/late markers) showed clear upregulation at days 14 and 21, with significant increases (up to ~7-fold) in 15% GelMA materials (5–8), enhanced by higher DCM. ACAN (late marker) upregulated at days 14 and 21, particularly in Series B, with limited sensitivity to DCM level within series. Hypertrophy markers (ALP, Col10A1) showed only slight increases by day 21, indicating minimal hypertrophic differentiation under these conditions. Safranin O and Alcian blue staining showed increased matrix deposition with higher DCM and longer culture. - In vivo repair (rat knee): All implants (materials 6–8) filled defects macroscopically at 9 weeks; controls (material 5 and sham) were only partially filled. Histology showed greater tissue filling and cell invasion with higher DCM; Safranin O/Fast Green indicated bone-like tissue (blue/green) predominance at 0–6% DCM but cartilage (red) predominance only at 12% DCM (material 8). No signs of inflammation were observed. ICRS macroscopic repair scores were highest for material 8 (15% GelMA/1% HAMA/12% DCM), exceeding other groups (p values indicated).

The findings support the hypothesis that incorporating decellularized cartilage matrix from α-1,3-galactose knockout pigs into photocurable GelMA/HAMA hydrogels augments their performance for cartilage repair. DCM increased hydrophilicity and stiffness in a controllable manner, while preserving enzymatic degradability. In vitro, DPSCs exhibited strong viability and proliferation trends that scaled with DCM content, and transcriptional profiles indicative of chondrogenesis (Sox9 early; Col2a1/Col2 and ACAN later) with minimal hypertrophy. These results are consistent with retained bioactive cues (GAGs, growth factors) in DCM facilitating chondrogenic differentiation. In vivo, DCM content modulated repair quality: only the highest DCM (12%) yielded cartilage-like tissue filling the defect, with improved macroscopic ICRS scores and no evident inflammatory response, aligning with the reduced immunogenicity anticipated from α-Gal deficiency. Collectively, the hybrid hydrogels mimic key compositional and water-rich features of cartilage, can be photocrosslinked in situ to conformal shapes, and provide a favorable microenvironment for cartilage-like tissue formation and integration. Despite promising results, translation requires caution: small-animal models and 9-week endpoints limit predictive value for human clinical scenarios, and mechanical testing used apparent moduli under unconfined compression rather than physiologic loading. Nevertheless, the data suggest design principles for next-generation scaffolds, including DCM content tuning and potential zonal layering to match cartilage gradients.

This study developed and comprehensively evaluated hybrid photocrosslinked hydrogels comprising GelMA, HAMA, and non-immunogenic porcine DCM. DCM enhanced hydrophilicity and mechanical stiffness while maintaining degradability, supported DPSC viability and chondrogenic differentiation in vitro, and improved cartilage repair in a rat knee defect model in a DCM dose-dependent manner, with 12% DCM showing the most cartilage-like regeneration and highest ICRS scores. These hydrogels mimic native cartilage constituents and can be cured in situ, offering practical advantages for irregular defects. Future work should validate efficacy and integration in larger, load-bearing animal models over longer durations, optimize graded, multi-layer constructs to mimic cartilage zonation (varying water content and stiffness), and further elucidate retained bioactive components within DCM and their mechanistic roles.

- The study used a small-animal (rat) model with a 9-week endpoint, limiting extrapolation to human clinical outcomes and long-term durability under load. - Mechanical testing employed unconfined compression yielding apparent moduli; physiological loading and confined behavior were not assessed. - Enzymatic degradation was tested in vitro with collagenase II; in vivo degradation kinetics may differ. - Histology could not directly verify the identity and functionality of specific cytokines/growth factors retained in DCM. - In vitro hypertrophy markers were only modestly assessed up to 21 days; longer-term phenotypic stability remains to be demonstrated. - Only one DCM source (α-1,3-Gal KO porcine) and particle size (<40 μm) were tested; batch variability and processing parameters may influence outcomes.