Assessing the changes in mechanical properties of different Gellan gum hydrogels under tissue engineering conditions

Background: Tissue engineering (TE) seeks to develop biological substitutes that restore, maintain, or improve tissue function. A significant challenge in TE is designing scaffolds that replicate the biomechanical environment of native tissues, especially in load-bearing applications. Hydrogels, particularly Gellan Gum (GG), are promising scaffold materials due to their high water content and tunable viscoelastic properties. Crosslinking agents such as calcium (Ca²⁺) and spermidine (SPD) play a crucial role in modulating these properties. Purpose: This study aims to compare the mechanical behavior of GG hydrogels crosslinked with calcium and spermidine over a 28-day period under chondrogenic conditions. The goal is to assess the impact of crosslinker type on mechanical stability and determine their suitability as scaffolds for cartilage tissue engineering. Method: GG was dissolved at a concentration of 5 mg/mL in HEPES/sucrose buffer and crosslinked using either 10 mM CaCl₂ or 2 mM SPD. Polymer and crosslinker solutions were mixed at a 5:1 volume ratio and cast into cylindrical molds (10 mm diameter, 2 mm height). Hydrogels were cultured in chondrogenic media at 37°C and sampled on Days 1, 3, 7, 14, and 28. Stress relaxation tests were conducted using a Mach-1 Micromechanical Testing System with a 3 mm flat-ended cylindrical indenter, applying 5%, 10%, and 15% compression steps followed by 240-second holds. Instantaneous modulus (Einst) and equilibrium modulus (Eeq) were calculated using Hayes’ analytical solution with Poisson's ratios of 0.1 and 0.5, respectively. Data were statistically analyzed using Pearson correlation and ANOVA (significance set at p < 0.05). Results: Both GG/Ca and GG/SPD hydrogels remained structurally stable over 28 days. GG/SPD initially exhibited a slightly higher Eeq (0.0015 ± 0.0003 MPa) than GG/Ca (0.0014 ± 0.00009 MPa) on Day 1. However, from Day 3 onwards, GG/Ca hydrogels consistently showed higher stiffness values. Over time, GG/Ca displayed a slight increase in Eeq, while GG/SPD remained unchanged or decreased marginally. Similar trends were observed for Einst. Pearson correlation analysis indicated a weak negative correlation between time and Eeq in GG/SPD (r = -0.25), and a weak positive correlation in GG/Ca (r = 0.08), neither of which were statistically significant. ANOVA revealed no significant changes in moduli over time for either group (p > 0.05). Conclusion: GG hydrogels crosslinked with calcium or spermidine exhibit stable mechanical properties under prolonged chondrogenic culture. Although GG/Ca hydrogels showed a slight increase in stiffness over time, both formulations maintained mechanical integrity and are viable candidates for cartilage tissue engineering. Further studies are required to explore their biological performance and regenerative potential.