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

 Corresponding author: Adekunle Job Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Assessing the changes in mechanical properties of different Gellan gum hydrogels under tissue engineering conditions Adekunle Job * Department Technical Physics, University of Eastern Finland, Yliopistonranta 8, 70210, Kuopio Finland. World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 Publication history: Received on 12 March 2025; revised on 26 April 2025; accepted on 29 April 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.1.1349 Abstract 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 flatended 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. Keywords: Gallen Gum(GG); Hydrogel; Spermidine(SPD); Calcium(Ca); Crosslinker World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4034 1. Introduction For several years now, one of the most significant areas of medicine in the recent decades has been lauded as Tissue Engineering (TE) in the area of Regenerative Medicine (RM). While the creation of bio-engineered skin replacements was the primary focus of the initial therapeutically relevant TE studies, Tissue Engineering applications have now been progressively broadened to a wide spectrum of tissues and organs. The emergence of either mesenchymal adult stemcell technology or embryonic stem-cell technology has inspired various attempts to incorporate this potential tool with TE techniques and has united the two areas under the umbrella known as "regenerative medicine." The identification of telocytes, a novel cell type that has been characterized in several organs and identified by electron microscopy, opens another doorway to RM and serves as a classic illustration of translational medicine. In addition to cell-therapy techniques, the use of gene therapy in conjunction with TE has been researched to produce tissues and organs. A vital function is also played by the vascularization of constructions in addition to the matrix and cell replacements. As a result, new in vivo models of vascularization have emerged that enable axial vascularization followed by construct transplantation. Tissue engineering has elements such as cells, scaffold biomolecules, and bioreactors [1–5]. The idea of using cell-culture methods and biomaterials to create living tissue equivalents and/or organs in the lab, later dubbed "Tissue Engineering," was first proposed as a multidisciplinary approach to regenerate lost tissue or organ functions. It was envisioned as the solution to both the organ shortage in transplantation medicine and the donor site morbidity issues that had previously gone unresolved. Clinicians and experts from many different fundamental fields in biology, engineering, and applied sciences have collaborated extensively only on the concept of growing tissue in the lab. Therefore, in order to create biological replacements that will preserve and restore normal function in sick and wounded tissues, researchers working in the fields of tissue TE and RM are currently using the concepts of cell culture and transplantation, material science, and bioengineering.[6–10]. Though there have been great efforts and improvements in standardizing cell culture processes and developing customized biomaterials to replace lost organ functions, the translation of laboratory triumphs into clinical situations has not proven to be as successful as it may be. This is partly due to the three-dimensional orientation of the organs and tissue, which necessitates the existence of a microvascular network in order to sustain the survival of the cells even at the center of any given construct by providing adequate blood flow and oxygenation. Before extrinsic vascularization may occur, an internal vascular network must form. This is an inherent limitation on the ability of cells to survive in three-dimensional scaffolds in the first stages after the implantation of tissue-engineered substitutes into patient structures. [13]. 1.1. Instantaneous Modulus (Einst) The instantaneous modulus, often represented as E, serves as an indicator of a material's stiffness at a specific point along its stress-strain curve, reflecting the slope of the curve at that particular location. Hooke's law describes the stressstrain relationship for an elastic material as follows: σ=E⋅ε……………… (1.6) Here: -σ (sigma) signifies the applied stress to the material. - (E) represents the instantaneous modulus. - ε (epsilon) denotes the strain (deformation) experienced by the material. The calculation of the instantaneous modulus involves taking the derivative of the stress-strain curve at the designated point, expressed mathematically as: [116]. E=∆σ/ ∆ε ……………….(1.6.1) The initial instantaneous modulus (Eoinst) is determined at the intersection of the fitted linear line, while the straindependent instantaneous modulus (Eԑinst) is obtained by measuring the slope of this line at each stress relaxation phase [116]. The overall instantaneous modulus (Einst) is then given by the sum of Eoinst and Eԑinst : Einst= Eԑinst + Eoinst ………………….(1.6.2) World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4035 Figure 1 The instantaneous modulus derived from loading in several steps. Each "point" in the right picture is formed by the red rising portion in the left figure [116] 1.2. Equilibrium Modulus (Eeq) The equilibrium modulus, indicated as Eeq, describes the material's stiffness once it has attained a state of mechanical equilibrium, typically following some deformation. Its computation involves utilizing stress and strain values at a specific point and the origin (unstressed state) [116]. The formula for calculating the equilibrium modulus is as follows: Eeq=σ/ε ……………….(1.6.3) Where: - σ represents the stress at the point where equilibrium is achieved. - ε stands for the strain at the point where equilibrium is reached. The equilibrium modulus offers an average stiffness measurement spanning from the origin to the equilibrium point, providing insight into the overall stiffness of the material under specific loading conditions [116]. To characterize the equilibrium modulus, a common approach involves employing multi-step stress relaxation or creep tests. Following the application of displacements or stress to the material, a relaxation period is allowed. [116] The equilibrium modulus is then calculated using the stress-strain ratio, determined by the slope of a linear line fitted to equilibrium points, expressed as: Eeq=∆σeq/∆εeq ……………(1.6.4) Figure 2 Calculating the elastic equilibrium modulus using stress relaxation [116] It's important to note that the terms "instantaneous modulus" and "equilibrium modulus" are frequently employed when dealing with linear elastic materials. However, real materials can display more intricate behaviors, including nonlinearity, viscoelasticity, or plasticity, necessitating the use of more advanced modeling and testing approaches. World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4036 Additionally, it is crucial to maintain consistency in the units of stress and strain in these formulas, typically using Pascals (Pa) for stress and dimensionless units for strain since it represents a ratio of lengths [116]. By calculating the ratio of the difference between the peak stress in the second step and the equilibrium stress in the first step to the strain amplitude, the instantaneous modulus was found. By evaluating the slope of the linear fit at the equilibrium locations, the equilibrium modulus was computed. The mean values of stress for the last five seconds were used to approximate the equilibrium stresses for each step. Elastic and isotropic behavior of the hydrogel was considered for both the immediate and equilibrium loading phases. Thus, the Hayes model was used to modify the predicted instantaneous and equilibrium moduli. It was assumed that the instantaneous and equilibrium moduli had a Poisson's ratio of 0.5 and 0.1 respectively [118]. In this project work we made use of the same step method to calculate our instantaneous and equilibrium moduli however we determined our instantaneous and equilibrium moduli with Poisson ratio of 0.5 2. Materials and Methods The materials used for hydrogel preparation were provided by Prof. Kellomäki at the BioMediTech Institute, Faculty of Biomedical Sciences and Engineering, Tampere University of Technology. These materials included sterile-filtered 5 mg/mL Gellan Gum (Gelzan™ CM Gelrite, Sigma-Aldrich) in a HEPES/sucrose solution (HEPES 25 mM, sucrose 100 mg/mL), spermidine trihydrochloride in HEPES/sucrose buffer (2 mM), and calcium chloride (10 mM). Teflon molds (10 mm diameter, 2 mm thickness) were also utilized. Hydrogels were incubated in chondrogenic media, composed of α-Minimum Essential Medium, 1x Insulin-TransferrinSelenium +1, 0.3% Penicillin/Streptomycin, 50 μg/mL ascorbic acid-2-phosphate, 55 μg/mL sodium pyruvate, and 23 μg/mL L-proline. 2.1. Hydrogel Preparation Two types of hydrogels were prepared: (1) Gellan Gum crosslinked with calcium (GG/Ca) and (2) Gellan Gum crosslinked with spermidine (GG/SPD). Both hydrogels were prepared following an identical protocol, maintaining a polymer-to-crosslinker volume ratio of 5:1 (e.g., 1000 μL GG and 200 μL CL). Samples were prepared in a 24-well plate, with 10 technical replicates per plate. The hydrogels were incubated at 37°C and monitored over a 28-day period, with mechanical testing conducted on days 1, 3, 7, 14, and 28. 2.2. Mixing Technique Hydrogel components were combined and mixed in a glass vial before being transferred into the molds. Additional components were incorporated into the polymer solutions before the addition of crosslinkers to ensure homogeneity. Once crosslinkers were added, the solutions were briefly stirred and transferred to the molds using a pipette. The gelation onset occurred within 30 to 60 seconds. Following confirmation of gel formation, 1 mL of chondrogenic media was added under aseptic conditions. Media was refreshed every three days until mechanical testing. Figure 3 Schematic representation of hydrogel-plated plating gel, ensuring homogenous gelation supported by Teflon at 37 °C. [117] World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4037 2.3. Mechanical Studies A Mach-1 (Micromechanical System, Biomomentum) biomechanical testing device was employed for stress relaxation testing. Data obtained from these tests were analyzed to determine the equilibrium modulus and instantaneous modulus over time. Each hydrogel type (GG/Ca and GG/SPD) was tested using five replicates per well plate. 2.3.1. Mechanical Testing Hydrogel mechanical properties were assessed using a Mach-1 device comprising a PM500-C Precision Motion Controller (Newport Corporation, USA), a Model V500Css Actuator (Newport), and a 0.15 kg single-axis load cell (Biomomentum, Quebec, Canada). A cylindrical flat-ended indenter (3 mm diameter) was used for indentation testing. Figure 4 (A ) Actuator, (B) Motion Controller, (C) Indenter Razer, dia. 3mm Stress relaxation tests were conducted at room temperature under wet conditions. Samples were retrieved at designated time points (days 1, 3, 7, 14, and 28) and subjected to indentation testing. The well plates were placed on the Mach-1 device stage, and the indenter was aligned with each hydrogel sample. Each sample underwent a three-step indentation process, consisting of 5% compression per step, resulting in a total indentation of 15%, followed by a 240second relaxation period per step, yielding a total test duration of 270 seconds per sample. Force and indentation depth data were continuously recorded and analyzed to determine instantaneous and equilibrium modulus values. Figure 5 Basic template for the biomechanics setup application 2.4. Statistical Analysis Data were expressed as mean ± standard deviation (SD). Each experiment included five replicates per sample group. Group differences were analyzed using an Excel spreadsheet (R package 'stats' version 4.2.1), followed by t-tests (Tukey's test) for multiple comparisons. The Pearson correlation coefficient was employed to assess correlations between modulus values and time. ANOVA was used to evaluate fluctuations in modulus values over time, with statistical significance set at p < 0.05. World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4038 Equilibrium modulus values were averaged and compared between GG/SPD and GG/Ca samples over time to assess stability, stiffness, and indentation resistance. For example, equilibrium modulus values (± SD) for GG/Ca were 0.0014 ± 0.00009 MPa on day 1 and 0.00137 ± 0.0002 MPa on day 7, which were lower than those observed for GG/SPD (0.0015 ± 0.0003 MPa on day 1 and 0.0014 ± 0.0002 MPa on day 7). 2.5. Expected Outcome This study aims to provide fundamental insights into the evolution of hydrogel mechanical properties over the incubation period. The findings will contribute to a deeper understanding of hydrogel behavior in long-term culture, informing future studies conducted by the Biophysics of Bone and Cartilage (BBC) research group. 3. Results 3.1. Hydrogel Appearance and Dimensions Hydrogels were prepared using molds with dimensions of 2 mm in thickness and 10 mm in diameter (Figure 6). Figure 6A presents a schematic representation of hydrogel formation in well plates, while Figure 6B illustrates the mold and its dimensions. Figures 6C and 6D depict the hydrogels within the mold and after removal, respectively, following overnight incubation in culture media. The pink coloration of the gel resulted from the presence of phenol red in the culture medium. Figure 6 (A) Schematic of gel formation, (B) mold dimensions, (C) hydrogel within mold, (D) hydrogel post-removal retaining mold shape 3.2. Mechanical Properties of the Hydrogels Indentation testing was conducted using the Mach-1 device to assess the mechanical properties of the hydrogels. A multi-step stress relaxation protocol was applied, in which the indenter compressed the hydrogel samples within a confined space. The applied force at each step is illustrated in Figure 7A. Typically, the equilibrium modulus of hydrogels is characterized using multi-step stress relaxation. However, Figure 7B depicts an invalid result due to an abnormal stress relaxation pattern, inconsistent with the expected three-step protocol validated in Figures 8, 9, and 7A. Consequently, only samples exhibiting expected mechanical responses were included in further analysis. World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4039 Figure 7 (A) Typical stress relaxation response, (B) Abnormal stress relaxation response (excluded from analysis) 3.2.1. Equilibrium Elastic Modulus of the Hydrogels Over the Incubation Period The equilibrium modulus values (Table 1) indicate that on Day 1, the modulus of GG crosslinked with spermidine (GG/SPD) was slightly higher than that of GG crosslinked with calcium (GG/Ca). By Day 3, the modulus of GG/Ca exceeded that of GG/SPD, but this trend was not sustained, as GG/SPD exhibited a higher modulus on Day 7. From Day 14 onward, GG/Ca demonstrated a consistent increase, surpassing GG/SPD through Day 28. Despite these variations, statistical analysis revealed no significant difference between the two crosslinkers, as indicated by p-values exceeding the 0.05 significance threshold. This suggests that the observed differences in equilibrium modulus between GG/SPD and GG/Ca are not statistically significant and should not be considered conclusive. Table 1 Equilibrium Modulus (MPa) for GG/Ca and GG/SPD (Averaged) Equilibrium Modulus(MPa) Ca SPD Time (Days) Mean (µ) ±Std Mean (µ) ± Std Difference Pvalue 1 0,00142534 9,07142E-05 0,001493667 0,000261774 -0,000068327 0,63508484 3 0,001440113 0,000189421 0,00143656 0,000193493 3,5528E-06 0,979707445 7 0,001378935 0,000170837 0,001412426 0,00028787 -3,3491E-05 0,846391846 14 0,001481232 0,000133703 0,001402372 0,000257827 7,88602E-05 0,601893198 28 0,001452707 0,000241861 0,001300737 0,000121923 0,00015197 0,294345953 The plot below, Fig.8, practically revealed how the various crosslinked hydrogel changes through the time of incubation and the application of indentation compression that is from day 1,3,7,14 and 28 respectively. In order to further buttress or prove if there is significant difference between the values recorded with both crosslinkers on GG, we took a step further to elaborate this by looking at the Pearson correlation coefficient (r), where we noticed a decrease in equilibrium of GG/SPD with time and no change for those crosslinked with GG/Ca with r of -0.25 and 0.0837, respectively, which indicate that there is low positive negative correlation between them, we know that Pearson coefficient helps to quantify a correlation, and to show the significance, we now looked at that the P values which are p 0.6907 for GG/Ca and p 0.228 for GG/SPD which are both higher than 0.05, shows that the correlation is not statistically significant as confirmed by their P values. However we derived the Pearson correlation and its P value from the raw data which was plotted (scatter plot) against the independent variable which are the days. Remember that P value helps to assess whether a correlation is real, meaning "r" and the P value should be analyzed together not individually Fig.9 [120]. World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4040 Figure 8 Equilibrium modulus (MPa) of crosslinked GG/Ca and GG/SPD Vs time Figure 9 Evaluating Gel's Equilibrium Modulus Changes Over Time Using Pearson Correlation In Table 1, we previously described the behavior of hydrogels GG/Ca and GG/SPD at a 5:1 ratio over the course of the incubation period under an indentation mechanical compression protocol. The data revealed fluctuations in the modulus values across the days, with notable exceptions on days 14 and 28, where the GG/Ca hydrogel consistently exhibited higher values. Figure 12 further illustrates the relationship between the various hydrogels (GG/Ca and GG/SPD) across the incubation days. To provide a clearer understanding of these trends, we applied Pearson's correlation coefficient and its corresponding p-value to assess the relationship and statistical significance. However, our analysis showed that the observed correlation was not statistically significant. Furthermore, box plots were generated (Fig. 10 and Fig. 11) for each of the hydrogels, GG/Ca and GG/SPD, to further illustrate their relationship over time using the raw data. These box plots indicate that the data did not follow a clear trend relative to the duration of the incubation period. To provide a more rigorous analysis, we performed an analysis of variance (ANOVA) on the data for each hydrogel, crosslinked with the same crosslinker, as shown in Fig. 10 (GG/Ca) and Fig. 11 (GG/SPD). This was done to assess whether the fluctuations observed in the data held statistical significance, as discussed in paragraph 1 of section 3.2.1. The ANOVA results revealed that the fluctuations were not statistically significant, suggesting that the modulus does not show consistent increases or decreases over time. Specifically, the P-values for GG/Ca (0.940) and GG/SPD (0.831) were both greater than 0.05, indicating no significant fluctuations in the data for either hydrogel. World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4041 Figure 10 Comparison of equilibrium modulus(MPa) of GG/Ca changes across the days Figure 11 Comparison of equilibrium modulus(MPa) of GG/SPD changes across the days 4. Discussion Certain inorganic and biological amine cations are known to crosslink negatively charged polymers, forming physical hydrogels via ionotropic gelation [115]. In this study, we employed spermidine, an endogenous polyamine, and calcium, an inorganic cation, as crosslinkers for Gellan Gum (GG) hydrogels. While hydrogels can be formed by simply adding a crosslinker to GG, we also incubated the hydrogel in chondrogenic media. Although not part of our experiment, other studies have included various biological molecules in hydrogels, and GG has shown promise in tissue engineering (TE) applications. The method used here for preparing GG crosslinked with Ca and SPD is flexible, practical, and costeffective. GG is FDAand EMA-approved as a gelling agent in food, cosmetics, and pharmaceuticals, and its use in TE has yielded encouraging results. Hydrogels are categorized into weak gels, which flow under stress, and true gels, which fracture and become self-supporting under severe stress. Our hydrogels are considered true gels, maintaining their structure even when handled with tweezers or cast [109]. We evaluated the mechanical properties of the hydrogels under cultured conditions. Although all materials were based on GG, we assessed the elastic behavior of the crosslinkers (Ca and SPD) using stress relaxation tests, determining the World Journal of Advanced Research and Reviews, 2025, 26(01), 4033-4048 4048 [105] Neves, N. M. & Reis, R. L. (2016). Biomaterials from Nature for Advanced Devices and Therapies. [Online]. [106] Prajapati, V. D. et al., (2013). An insight into the emerging exopolysaccharide Gellan Gum as a novel polymer, Carbohydrate Polymers ,. 93 (2): p. 670. [107] López-Cebral R. et al. (2013). Spermidine-crosslinked hydrogels as novel potential platforms for pharmaceutical applications. J Pharm Sci. 2013;102:2632–2643. doi:10.1002/jps.23631 [108] Vigani, B. et al., (2022). Spermidine Crosslinked Gellan Gum-Based ‘Hydrogel Nanofibers’ as Potential Tool for the Treatment of Nervous Tissue Injuries: A Formulation Study. International journal of nanomedicine. [Online] 173421–3439. [109] Koivisto, J. T. et al., (2017). Bioamine-crosslinked Gellan Gum hydrogel for neural tissue engineering. Biomedical materials (Bristol). [Online] 12 (2), 025014–. [110] Cao, N. et al., (2012). Influence of Calcium Ions on Cell Survival and Proliferation in the Context of an Alginate Hydrogel. ISRN chemical engineering. [Online] 20121–9. [111] Cencetti, C. et al., (2011). Preparation and characterization of a new Gellan Gum and sulphated hyaluronic acid hydrogel designed for epidural scar prevention. Journal of materials science. Materials in medicine. [Online] 22 (2), 263–271. [112] Gering, C. (2023). Design Strategies for Polysaccharide Hydrogels Used in Soft Tissue Engineering: Modification, Testing and Applications of Gellan Gum. [113] Koivisto, J. (2019). Development and Characterization of Gellan Gum Based Hydrogels for Soft Tissue Engineering Applications. [114] Kirchmajer, D.M. et al., (2014). Enhanced gelation properties of purified Gellan Gum. Carbohydrate research, 388, pp.125-129 [115] El-Sherbiny IM. et al., (2011). Ionotropically crosslinked pH-sensitive IPN hydrogel matrices as potential carriers for intestine-specific oral delivery of protein drugs. Drug Dev Ind Pharm 37(2):121–130. [116] Ebrahimi, M. (2022). Structure, composition and function of human tibiofemoral joint cartilage : experimental and computational analysis of healthy and osteoarthritic tissue. Kuopio: University of Eastern Finland. [117] Gering, C. et al., (2017). ‘Reproducible preparation method of hydrogels for cell culture applications – Case study with spermidine crosslinked Gellan Gum’, in IFMBE Proceedings. [Online]. 2017 pp. 811–814. [118] Ahearne, M.et al., (2013). A comparison of fibrin, agarose and Gellan Gum hydrogels as carriers of stem cells and growth factor delivery microspheres for cartilage regeneration. Biomedical materials (Bristol). [Online] 8 (3), 035004-. [119] Abdelsalam, A. (2021) Modelling light interaction in healthy and degenerated articular cartilage. [120] https://opexresources.com/interpreting-pearson-coefficient/.