(H-316) Adaptable Sliding Hydrogels with Tunable Stress Relaxation for Enhancing Cell-Based Cartilage Regeneration

Introduction:: Hyaline cartilage is essential for proper joint function as it lubricates and protects the underlying bone from high mechanical loading. However, cartilage has a limited capacity for regeneration, and injury to the tissue significantly increases one’s risk for developing Osteoarthritis. As current treatment options are limited to pain and symptom management, there exists a critical need for therapeutic options that restore cartilage function and structure. When designing hydrogels to improve cell-based cartilage regeneration, inspiration can be drawn from cues within native cartilage extracellular matrix (ECM). Cartilage is viscoelastic and demonstrates stress relaxation behavior in response to applied stresses. Using ionically-crosslinked alginate hydrogels, it has recently been shown that faster stress relaxation enhances chondrocyte-based cartilage formation in vitro. This suggests that including stress relaxation in hydrogel design may benefit and accelerate cell-based cartilage regeneration [1]. However, the effect of tuning stress relaxation on Mesenchymal Stem Cell (MSC) chondrogenesis remains unknown. Our lab has previously reported PEG-based sliding hydrogels (SG) as a 3D cell niche with mobile crosslinks which enhanced MSC chondrogenesis compared to conventional 3D PEG hydrogels with non-mobile crosslinks [2]. Unlike alginate hydrogels, SG is crosslinked by irreversible covalent bonds and does not exhibit stress relaxation. We hypothesize that introducing viscoelasticity into SG via reversible, dynamic crosslinks will further accelerate cell-based cartilage regeneration in a dose-dependent manner. The goal of this study is to develop adaptable sliding hydrogels (ASG) with dynamic crosslinks to assess the effect of tuning stress relaxation on cell-based cartilage formation in vitro using chondrocytes and MSCs.

Materials and Methods:: To introduce stress relaxation in ASG, reversible hydrazone crosslinks were used. Polyrotaxane was functionalized with hydrazine, and 8-arm polyethylene glycol (PEG) aldehydes were used as crosslinkers. Tunable stress relaxation was achieved by varying the ratio of alkyl aldehyde (AA) and benzaldehyde (BA) crosslinkers while maintaining crosslinking density. The stress relaxation behavior of ASG was determined using shear-based rheology. To assess cartilage formation, both bovine juvenile chondrocytes and human adult MSCs were used. Outcomes were assessed using histology, immunostaining, biochemical assays, and mechanical testing. Changes in cell volume and cell proliferation were also measured via immunohistochemical staining. To probe the biophysical mechanism by which ASG modulates cell-based cartilage regeneration, immunohistochemical staining was performed for mechanosensing markers across multiple subcellular levels from cell-gel interface, cytoskeleton to the nucleus.

Results, Conclusions, and Discussions:: Shear rheology validates that varying the ratios of AA:BA (0:100, 40:60, and 80:0) results in ASG hydrogels with tunable stress relaxation (Fig. 1B). Increasing stress relaxation in ASG significantly accelerates chondrocyte-based cartilage production in a dose-dependent manner, as shown by staining for sulfated glycosaminoglycans (sGAG) and collagen production per cell (data not shown) (Fig. 2A). Impressively, the ASG fast group undergoes a 50-fold increase in Young’s modulus by day 21 (Fig. 2B), indicating robust ECM deposition. Tuning stress relaxation in ASG induces minimal changes in chondrocyte volume yet is sufficient to substantially accelerate neocartilage deposition. When human MSCs are encapsulated in ASG, we also observe an increase cartilage matrix deposition, though the speed and amount of newly produced cartilage are decreased compared to chondrocytes. We further demonstrate that stress relaxation increases MSC proliferation (Fig. 3A). Our results demonstrate for the first time that stress relaxation enhances MSC-based cartilage formation in a dose-dependent manner. Using immunostaining of LaminA/C (Fig. 3B), a marker for nuclear mechanosensing, we identify that cells in ASG experience greater nuclear mechanical loading, highlighting the role of canonical mechanosensing pathways in chondrogenesis. Together, these results demonstrate ASG as a promising new biomaterial for enhancing cell-based cartilage regeneration and identify nuclear mechanosensing as a biophysical mechanism for enhanced cartilage formation.

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