Assistant Professor Beth Israel Deaconess Medical Center Boston, Massachusetts, United States
Introduction: Highly entangled (HE) hydrogels are promising biomaterials for the field of 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 particularly suitable for cartilage repair and replacement 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 evaluating their biocompatibility and developing degradable and cell-adherent variants. We hypothesized that increasing crosslink density (crosslink-to-hydrogel volume ratio) would allow the hydrogel to maintain its structure and 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 2.5x10E-4, 1.50x10E-4, 1.25x10E-5, and 2.50x10E-6 (Fig.1A). 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 medium at 37°C to allow for cell adhesion (Fig.1A).
Cell Imaging: 3T3 fibroblasts were seeded onto 8mm hydrogels and incubated in cell medium at 37 °C. Variants included MBAA-HE, PEGDA-HE (1.50x10E-4), and PEGDA-HE (1.25x10E-5), divided into +FN and -FN groups (n=3 per group). On Day 7, fibroblasts were imaged using a Leica Mica system.
WST-1 Assay: 3T3 fibroblasts were exposed to conditioned media from MBAA-HE, low entanglement (PA-W20), and tough (TG) hydrogels. This setup allowed a comparison of HE hydrogel biocompatibility (Fig.1A).
Statistical Analysis: One-way analysis of variance (ANOVA) tests were used in mechanical testing and cell adhesion to compare groups, with post hoc t-tests and Bonferroni correction. Two-way ANOVA was used for WST-1 analysis (Prism 8, GraphPad).
Results, Conclusions, and Discussions: Results and
Discussion:
During degradation, high-crosslink-density variants, PEGDA-HE (2.5x10E-4) and PEGDA-HE (1.50x10E-4), maintained stable, high elastic modulus from Day 1 to Day 7 (Fig.2A) and low toughness (Fig.2B). 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 (1.25x10E-5) and PEGDA-HE (2.50x10E-6), exhibited decreasing elastic modulus and increasing toughness (Fig.2A, Fig.2B), 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 (Fig.2E).
The fibroblast population on PEGDA-HE (+FN, 1.50x10E-4) hydrogels was not statistically different from PEGDA-HE (+FN, 1.25x10E-5) hydrogels, contrasting the hypothesis that higher-density hydrogels support more cells during degradation (Fig.3A). Interestingly, fibroblasts on PEGDA-HE (+FN, 1.50x10E-4) were significantly more circular than those on PEGDA-HE (+FN, 1.25x10E-5) (Fig.3B). 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 more rounded cells, whereas softer, lower-density hydrogels support fewer, more elongated, and active fibroblasts.
WST-1 assay results demonstrate that HE hydrogels, as well as PA-W20, and TG hydrogels, exhibit biocompatibility at dilution ratios of 1:10, 1:100, 1:500, and 1:1000 of conditioned media to unconditioned media. The absorbance values for these hydrogels were comparable to or exceeded those of the negative control (2.831 ± 0.01768), indicating biocompatibility at lower concentrations (Fig.4A). However, 1:1 dilutions of all tested hydrogels yielded absorbance values lower than the negative control but significantly higher than the positive control, suggesting that biocompatibility at higher concentrations requires further characterization.
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.
Acknowledgements (Optional): We thank Dr. David Mooney, Elise Ansart, Nicolas Hesse, and the Office of Harvard Undergraduate Research and Fellowships (URAF).
