Introduction: Hydrogels are a suitable class of materials for cell-based cartilage repair, as they provide a 3D environment that can stimulate encapsulated cells to proliferate, differentiate, and form new tissue . Their mechanical properties however are generally poor, and most measures taken to improve the mechanical properties make it a less permissive environment for cellular processes. There is a need for strategies that reinforce hydrogels without compromising chondrocyte performance. Here, we studied the reinforcement of cell-laden gelatine-methacrylamide (gelMA) hydrogels by thermal (physical) gelation prior to UV-initiated (chemical) crosslinking. Experimental Methods: Gelatine was mefhacrylamide-functionalised as reported previously. Solutions of 10% or 20% gelMA + 0.1% Irgacure 2959 photo-initiator were crosslinked by exposure to 15 min UV-A, either in the sol state (purely chemical gel = C) or after thermal ageing at 5 °C (physical-chemical duakrosslinked gel = PC). Compressive moduli of C and PC gels were measured after 24 h free swelling at 37 °C, whilst stiffness development of purely physical gels (P) was measured by oscillatory shear rheometry at 5 °C. All gels were subjected to proteolytic degradation in 0.05% trypsin + 0.006% EDTA. Chondrocytes were isolated from equine articular cartilage and passaged once before encapsulation in gels at 5.106 cells/mL under the same conditions as described above. After 24 h, live/dead and Alamar blue assays were performed. Cell-laden constructs (10% C, 10% PC and 20% C) were cultured in chondrogenic medium for 6 weeks, then assayed for DNA and glycosaminoglycan (GAG) content using Picogreen and DMMB, respectively. Results and Discussion: After photo-crosslinking, 10% gelMAgels crosslinked directly from the sol state (Figure 1, C) had a stiffness of 24 kPa. On the contrary, when allowed to gel thermally prior to photo-crosslinking, the stiffness increased up to 165 kPa for 10% dual-crosslinked gels (PC), reaching similar values as 20% gelMA gels crosslinked from the sol state (not shown). By comparison, the sum of stiffness of a purely physical gel and a purely chemical gel is only 51 kPa (P+C). Figure 1: Stiffness development of dual-crosslinked 10% gelMA gels, as a function of thermal gelation time prior to photo-initiated crosslinking. P = purely physical gel (thermal gelation) C = purely chemical gel (crosslinked from solution) P + C = sum of physical and chemical gel (for reference) PC = dual-crosslinked gel Whilst the stiffness of 10% PC gels was similar to that of 20% C gels, proteolytic degradation was much faster, and more akin to 10% C gels (Figure 2). Faster enzymatic degradation indicates the 10% dual-crosslinked gels being more permissive to matrix remodelling by encapsulated cells than 20% chemical gels. This was not yet corroborated in terms of cartilage tissue formation by encapsulated equine chondrocytes; after 6 weeks of culture, GAG/DNA was similar for all conditions evaluated. Nevertheless, the cold treatment did not reduce cell viability, nor chondrogenesis. Experiments are ongoing to reveal differential behaviour within the different gel types by other cell types. Figure 2: Proteolytic degradation of gelMAgels. Mass loss of dual-crosslinked (PC) 10 % gelMAgels compared to 10% and 20% only chemically crosslinked (C) gels. Discussion: Hydrogels that allow cells to develop new tissue are generally mechanically weak. Here, we present a straightforward method for the reinforcement of cell-laden gelMAgels, which showed seven-fold increase in stiffness whilst having no negative influence on cell viability, and only slightly decreased enzymatic degradation rates. Conclusion: Thermal ageing of gelatine-methacrylamide gels prior to UV-initiated crosslinking leads to a large Increase in compressive stiffness, whilst still providing a permissive environment for enzymatic degradation, and for survival and tissue formation by encapsulated cells.
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