Mechanically Tunable and Cellularly Adhesive Highly Entangled Hydrogels with Degradable Crosslinks

Introduction: Highly entangled (HE) hydrogels are promising biomaterials for orthopedics, as they retain high stiffness and toughness when fully hydrated, minimizing the reduction in mechanical strength often exhibited by hydrogels in a swollen state. Moreover, their low-friction characteristics make them suitable for cartilage repair in joints. Existing research highlights their potential in the medical field but focuses on non-clinical uses such as improved plastics and ionic conductance. As such, they have not yet been tested in biomedical applications. Therefore, the objective of this study was to advance HE hydrogels for orthopedic applications by developing degradable and cell-adherent variants. We hypothesized that increasing crosslink density (crosslink-to-hydrogel volume ratio) would allow the hydrogel to maintain its mechanical properties longer as it degrades in biological fluids.

Materials and Methods: Hydrogel Formulation: Non-degradable HE hydrogels were synthesized using polyacrylamide (PAAM, Sigma-Aldrich, A8887) with N,N'-Methylenebisacrylamide (MBAA, Sigma-Aldrich, M7279) crosslinks and 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, Sigma-Aldrich, 410896) photoinitiator. Degradable variants incorporated Poly(ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, 475629) crosslinks. Tensile-Testing: The effect of PEGDA-HE crosslink density was assessed by analyzing hydrogels with crosslink-to-hydrogel volume ratios of 2.5x10^-4, 1.50x10^-4, 1.25x10^-5, and 2.50x10^-6. Samples were evaluated at 1, 2, and 7 days after swelling in Phosphate-Buffered-Saline (PBS) to study degradation effects on elastic modulus and toughness using a CellScale™ UniVert system (n=3 per group). Biofunctionalization: HE hydrogels were coated with sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate (Sulfo-SANPAH, Sigma-Aldrich, 803332) and UV-exposed (254nm). Fibronectin (FN, Sigma-Aldrich, F0635) rat plasma was applied, followed by incubation in cell media to allow for cell adhesion. Cell Imaging: 3T3 fibroblasts were seeded onto 8mm hydrogels and incubated in a cell medium. Variants included MBAA-HE, PEGDA-HE (1.50x10^-4), and PEGDA-HE (1.25x10^-5), divided into +FN and -FN groups (n=3 per group). On Day 7, fibroblasts were imaged using a Leica Mica system.

Results and Discussion: During degradation, high-crosslink-density variants, PEGDA-HE (2.5x10^-4) and PEGDA-HE (1.50x10^-4), maintained stable, high elastic modulus from Day 1 to Day 7 and low toughness. This stability is likely due to the formation of rigid polymer networks, which require more hydrolysis reactions to degrade. In contrast, the low crosslink-density variants, PEGDA-HE (1.25x10^-5) and PEGDA-HE (2.50x10^-6) exhibited decreasing elastic modulus and increasing toughness, indicative of the loosening of the HE network. Swollen weight ratios of high crosslink-density variants remained consistent, while those of low crosslink-density variants increased rapidly, further suggesting the formation of a looser polymer network as hydrophilic polyacrylamide likely became more exposed to the buffer solution.

The fibroblast population on PEGDA-HE (+FN, 1.50x10-4) hydrogels was not statistically different from PEGDA-HE (+FN, 1.25x10^-5) hydrogels, contrasting the hypothesis that higher-density hydrogels support more cells during degradation. Interestingly, fibroblasts on PEGDA-HE (+FN, 1.50x10^-4) were significantly more circular than those on PEGDA-HE (+FN, 1.25x10^-5). This suggests that fibroblast behavior is influenced by stiffness and crosslink density: stiffer, higher-density PEGDA-HE hydrogels may enhance cell adhesion but result in rounded cells, whereas softer, lower-density hydrogels support more elongated, and active fibroblasts.

Conclusions: This study demonstrates that hydrolytically degradable PEGDA-HE hydrogels can be formulated and adjusted in crosslink density to maintain high mechanical strength during degradation while promoting cell adhesion.