Experimental study on the mechanical properties of biological hydrogels of different concentrations

Abstract

BACKGROUND:

Biological hydrogels provide a conducive three-dimensional extracellular matrix environment for encapsulating and cultivating living cells. Microenvironmental modulus of hydrogels dictates several characteristics of cell functions such as proliferation, adhesion, self-renewal, differentiation, migration, cell morphology and fate. Precise measurement of the mechanical properties of gels is necessary for investigating cellular mechanobiology in a variety of applications in tissue engineering. Elastic properties of gels are strongly influenced by the amount of crosslinking density.

OBJECTIVE:

The main purpose of the present study was to determine the elastic modulus of two types of well-known biological hydrogels: Agarose and Gelatin Methacryloyl.

METHODS:

Mechanical properties such as Young’s modulus, fracture stress and failure strain of the prescribed gels with a wide range of concentrations were determined using tension and compression tests.

RESULTS:

The elastic modulus, failure stress and strain were found to be strongly influenced when the amount of concentration in the hydrogels was changed. The elastic modulus for a lower level of concentration, not considered in this study, was also predicted using statistical analysis.

CONCLUSIONS:

Closed matching of the mechanical properties of the gels revealed that the bulk tension and compression tests could be confidently used for assessing mechanical properties of delicate biological hydrogels.

Keywords

Agarose Gelatin Methacryloyl elastic modulus cell culture tension test compression test ANOVA analysis

1. Introduction

Hydrogels are delicate materials composed of water and a cross-linked hydrophilic polymer network. Hydrogels are unique biomaterials offering a combination of desirable characteristics such as high water content, biodegradability and porosity required for several biomedical applications [1]. Advanced understanding of developing hydrogel with mechanical properties closely matching with the surrounding environment has gained increased attention in the biomaterials field. Hydrogels with excellent biocompatible properties have attracted strong attention of researchers for applications in tissue engineering and regenerative medicine. Unique physical properties and the ability to mimic the tissue environment of hydrogels have made them a suitable candidate material in the biomedical field [2]. Hydrogel offers a conducive two or three-dimensional environment for human and animal cells [1, 3, 4].

Among other types of hydrogels, Agarose and Gelatin Methacryloyl (GelMA) hydrogels have been widely used for various biomedical applications due to their suitable biological properties and tunable physical characteristics [5, 6, 7, 8, 9, 10]. Agarose is commercially found in the form of white powder and is routines used for the separation of large molecules such as DNA by electrophoresis. GelMA hydrogels possess essential properties of native extracellular matrix (ECM) which facilitate cells to proliferate in GelMA-based scaffolds. Determination of mechanical properties of soft materials such as agarose gel and Gelatin based hydrogels are important for a variety of applications in biomedical engineering fields, food, and ballistic industry. Detailed information on the classification, preparation and properties of hydrogels and their role in tissue engineering, drug delivery systems, optics, diagnostics, and imaging can be found in recent studies [11, 12, 13, 14].

Physical properties of hydrogels, particularly elastic properties matching living tissues, have made them attractive in the field of tissue engineering. Biological tissues exhibit a wide range of values for modulus from hundreds of Pascals (Pa) for soft tissues such as brain and fat to tens of kilopascals (kPa) for stiffer tissues such as muscles [15]. The Young’s modulus of soft tissues was estimated in the range from 1 kPa to more than 1 MPa [16]. Preliminary results reported in the published literature have shown mixtures of agar and gelatin with to lie in this range [17]. The selection of environment for cell growth in clinical research with elastic properties closely matching living tissue is vital for developing tissues. Tuning the mechanical properties of hydrogels by varying the crosslinking density of the polymeric network has enabled them for broad applications in biomedical engineering. Selection of a hydrogel for optimum function in a specific application depends on mechanical and rheological properties, especially elastic modulus, Poisson’s ratio, density, strength and damping. Discrepancies in the mechanical properties of living cells and their surrounding environment may seriously affect cell proliferation. Stiffness of the hydrogel network has shown significant effect on proliferation of cells and other biological characteristics associated with gels [18, 19, 20, 21, 22]. The elastic properties of GelMA hydrogels and agarose gel can be modified by varying the hydrogel concentration [6, 23, 24].

The determination of the mechanical properties of hydrogels has been the main topic of several research studies over the past three decades. Mechanical properties of interest, including elastic modulus of hydrogels, are determined using atomic force microscopy (AFM) indentation, dynamic mechanical analyzer, nanoindentation, rheometer, inflation test, spherical ball inclusion, and micropipette aspiration [6, 12, 25, 26, 27, 28, 29, 30, 31, 32, 33]. AFM with its continuous evolution has widely used to assess the mechanical properties of soft materials such as gel. In a recent study, elastic properties of several hydrogels based soft materials measured using AFM are reported [27]. The Young’s modulus of GelMA hydrogels was found to significantly increased with high higher crosslinking density [22]. Tensile testing, compression testing, and shear testing are the technique those can be used to measure the mechanical behavior of hydrogels [4, 34, 35, 36, 37, 38]; however, such studies are few and have several limitations including preparation and holding samples for testing. The elastic properties such as Young’s modulus, yield strength, ultimate tensile strength and failure strain were determined from the stress-strain plots. The preparation of standardized specimens for such testing and gripping of specimens during tests requires enormous effort and time.

Mechanical signals play important roles in several activities of cells involving growth, morphology, adhesion, migration and mutual communications between cells [24, 39]. The determination of mechanical properties of hydrogels may be challenging due to its soft nature. Technological advancement in developing state-of-the-art equipment has lead characterization of soft biological materials at micro and nano scale with minimum effort. Despite the availability of sophisticated equipment for the determination of elastic properties of hydrogels at micro and nano levels, inconsistent data has been reported in the published literature [6, 12, 25, 26, 27, 28, 29, 30, 31, 32, 33]. Obvious reasons for obtaining different results were the use of different techniques for measurements, type and concentration of gel and method of gel preparation. This study is a step forward to measure bulk elastic modulus of two types of gels using tensile and compression testing equipment and compare the results. The study provides engineering-based information on the relationship between stiffness and concentration of hydrogels for a specific application in cell culture and tissue engineering.

2. Materials and methods

2.1 Equipment and test procedure

Tensile tester (Tinius Olsen, H5KT) with a maximum loading capacity of 5 kN was used to test hydrogels samples in tension and compression. The load cell was based on strain gauge measurements with accuracy $\pm$ 0.2% of applied force. The internal sampling rate of the load cell was 1000 Hz. The experimental set up for the tension and compression tests is shown in Fig. 1. Gripping biological sample has been reported as one of the main challenges in performing tension tests due to crushing of the material at the grip positions as well as slippery nature of the specimen. A set of smooth holders were attached to the ends of tensile test specimen to avoid potential damage to the specimen during loading. The Tinius Olsen model 100R extensometer was used to measure stretch in the specimen. Extension was measured by attaching two counterbalanced extensometer clamps to the specimen at a pre-selected gauge length. Flat-faced cylindrical discs were used for compressing cylindrical specimens. Applied load on samples and contraction of specimen in compression tests were measured throughout the test. Specimens were stretched and compressed at control strain of 2 mm/min and were strained in steps of 2% until failure. All samples were tested at room temperature.

Figure 1.

Experimental set up for tensile and compression testing, (a) tension test, (b) compression test.

2.2 Gel preparation

To study the effect of different concentrations of agarose in the gel on the mechanical properties, gels were prepared from agarose powder (Analytical grade, Promega Corporation). Gels with 2%, 3%, 4%, 5% and 6% concentrations were prepared by dissolving appropriate amounts of agarose (w/v) into distilled deionized water and boiled the solution in microwave for around two minutes. Specimens with different stiffness were fabricated by varying the concentration of GelMA. To prepare GelMA hydrogels with different concentrations (10, 15, 20, 25 and 30%), GelMA precursor solutions were prepared by dissolving freeze-dried GelMA in deionized water at 40 ${}^{\circ}$ C and kept for 2 hours to form homogeneous solutions. The 5% GelMA hydrogels were very soft and were not used in the tests.

2.3 Specimen preparation

Specimens for tension and compression were prepared by pouring gels into custom-made moulds fabricated in the workshop (see Fig. 2a). Specimens were kept hydrated in plastic bags to avoid dryness before using them in tests. Samples prepared for both types of tests are shown in Fig. 2b. The dog-bone shape specimen as shown in Fig. 2 was used in tension tests. The gage length of the specimen for tension tests was 25 mm long and 12 mm wide. Samples having diameter of 30 mm and height of 20 mm were used for compression tests.

Figure 2.

Preparation of specimens for tension and compression tests, (a) moulds filled with gel, (b) final shape of specimens.

3. Results

Mechanical properties such as Young’s modulus, fracture stress and failure strain for both types of gels were determined from stress-strain graphs obtained from tension and compression tests. The elastic moduli were calculated from the slope of linear portion of the stress and strain graphs. Typical engineering stress-strain graphs for both types of gels with minimum and maximum concentrations are shown in Figs 3 and 4. Some droplets of water were seen to squeeze from the pores of the agarose gel as soon as the compression plate touched the cylindrical specimen during compression test.

Figure 3.

Engineering stress strain diagrams of agarose gel, left – (6% agarose), right – (2% agarose).

Figure 4.

Engineering stress strain diagrams of GelMA, left – (30% GelMA), right – 10% (GelMA).

Each data point in the subsequent plots represents the average value of three consecutive tests.

The elastic moduli of the gels were measured as a function of percent agarose and GelMA as shown in Fig. 5. The elastic modulus was significantly increased with increase in concentration. The elastic modulus was found to increase from an average value of 90 kPa to 1030 kPa (more than 1000% increase) when concentration was changed from 2% to 6% in tension test. Similarly, the elastic modulus was noted to increase from an average value of 65 kPa to 980 kPa (around 1400% increases) for the same increase in concentration in compression test. The elastic moduli measured in both types of tests were found to be in good agreement. It was noticed that GelMA with 30% concentration had the largest Young’s modulus of 290 KPa in tension test and 245 kPa in compression test. As shown in Fig. 5, the Young’s modulus was significantly increased when the GelMA concentration was increased from 10% to 30%. The elastic modulus of both types of gels was found more in tension tests compared to compression tests.

Figure 5.

Variation of elastic modulus with concentration, (a) agarose gel, (b) GelMA.

Figure 6.

Effect of gel concentration on failure stress, (a) agarose gel, (b) GelMA.

Unlike elastic modulus, the failure stress which indicates the strength of a material was found more in compression tests compared to tension tests (Fig. 6). A prominent enhancement in failure stress was found with increase in concentration. The average value of stress (average value obtained from both types of tests) at fracture obtained from both types of tests was observed to increase from 49 kPa to 235 kPa when agarose concertation was increased from 2% to 6%. Similarly, the average stress at fracture obtained from both types of tests was increased from 32.5 kPa to 144 kPa when the GelMA concertation was increased from 10% to 30%. A significant decrease in failure strain with the increase in concentration was also observed for both types of gels (see Fig. 7). The failure strain, indicating ductility of a material, was found more in tension tests compared to compression tests for all levels of concentrations of agarose gel. Conversely, larger failure strain was noted in compression tests compared to tension tests for all levels of concentrations of GelMA.

Figure 7.

Effect of gel concentration on failure strain, (a) agarose gel, (b) GelMA.

The 5% GelMA hydrogels samples were soft and it was difficult to make specimen with enough strength to be used in the tests. Since GelMA with concentration closed to 5% are widely used in cell culture experiments, the elastic modulus of the gel with prescribed concentration was predicted. To predict the elastic modulus of 5% GelMA, a regression model was develop based on the collected data. The ANOVA analysis of non-linear regression models for GelMA is shown in Table 1. For both types of tests, $R^{2}$ and adjusted $R^{2}$ values were significantly high ( $>$ 95%) indicating that the data was closed to the fitted non-linear regression line. Further, the $p$ -value for both types of tests was below 0.05, indicating the results of ANOVA analysis statistically significant. The non-linear plots generated based on the collected data are shown in Fig. 8. To test the accuracy of the regression model, elastic modulus obtained in tension and compression tests were predicted based on the non-linear equations. Absolute percentage error was calculated using Eq. (1). The analysis found that the average absolute percentage errors in the predicted data were 9.18% and 8.65% for tensile modulus and compressive modulus respectively.

$\displaystyle\%\textit{Error}=\left|\frac{\textit{Observed value}-\textit{% Predicted value}}{\textit{Observed value}}\right|\times 100$ (1)

Table 1

ANOVA analysis for elastic modulus GelMA

Tension test
Analysis of variance
Compression test $=$ 21.00 $+$ 1.129 Concentration $+$ 0.2143 Concentration ${}^{\wedge}$ 2
R-Sq $=$ 98.3% R-Sq (adj) $=$ 96.5%
Source	DF	SS	MS	F	$P$
Regression	2	43547.1	21773.6	102.98	0.010
Error	2	422.9	211.4
Total	4	43970.0
R-Sq $=$ 99.0% R-Sq (adj) $=$ 98.1%
Tension test $=$ 51.00 $-$ 5.957 Concentration $+$ 0.4714 Concentration ${}^{\wedge}$ 2
Compression test
Analysis of variance
Source	DF	SS	MS	F	$P$
Regression	2	23924.3	11962.1	56.20	0.017
Error	2	425.7	212.9
Total	4	24350.0

Figure 8.

Fitted line plot for elastic modulus GelMA.

Based on the generated regression models, the predicted elastic modulus for 5% GelMA was found 32.8 kPa in tension and 33.6 kPa in compression.

4. Discussion

The elastic modulus of hydrogels with high degree of concentration was significantly higher than those with low and medium concentration. Concertation of the agarose below 2% and GelMA below 10% resulted in soft gels and were found unsuitable for tension and compression tests. Both types of tests revealed increased stiffness of the hydrogels with increased level of concentration. Increase in the fracture stress was due to the increased rigidity of the gel network for higher levels of concentration.

Results on elastic modulus of agarose gel obtained from both testing techniques were closed to those reported in the published literature [6, 40, 41]. The average elastic moduli of 2% and 4% concentrations of agarose gel were found 340 kPa and 950 kPa, respectively [41]. In the same study, the average peak stress for 2% and 4% concentrations were found 50 kPa and 125 kPa, respectively in tension tests. The values obtained for gels with lower concentration were consistent with the data obtained in the current study. The Young’s moduli of agarose gel with concentration ranging from 2% to 8% was found 50 kPa and 450 kPa using quasi-static uniaxial compression test [42]. The Young’s moduli measured using concentration below 6% were closed to the values obtained in the current study. However, the strain at failure in the current study was slightly higher. In a recent study on the measurements of mechanical properties of agarose gel in tension tests, the average elastic moduli with concentrations 1.5%, 2%, 2.5%, 3% were found 184.8 kPa, 288.5 kPa, 451 kPa and 629 kPa, respectively [6]. Similarly, the average elastic moduli with concentrations 2% and 3% were found 235 kPa and 516 kPa, respectively in compression tests. In the same study, the average failure stress with concentrations 2% and 3% were found 126 kPa and 227 kPa, respectively in tension tests. All these values were comparatively higher than those obtained in the current study. In another study [43], while measuring the storage modulus of agarose gel using ultrasound wave propagation, the modulus was found to linearly increase when concentration was changed from 0.5% to 5%.

While investigating the influence of GelMA substrate on morpohology of PC12 cells, the Young’s modulus of GelMA with concentrations ranging from 5% to 30% was measured in tension tests [23]. The Young’s modulus of 10% GelMA was 34.9 KPa which was closed to the values obtained in this study (approximately 45 kPa). There was a difference of more than 50 kPa between the values obtained in [23] and the current study when GelMA of 30% concentrations was tested. Other studies [44, 45] have also found similar results as obtained in the current study. The difference between failure strain of agarose gel in both types of tests conducted in the current study was due to the early cracking of the specimens in compression tests. The differences between elastic modulus using two techniques are attributed to the slip of the specimen in tension tests and local swelling of the material in compression tests. Although not studied here, the swelling characteristic of hydrogels is also an important parameter influencing mechanical properties of gels [46]. The swelling behavior of hydrogels depends on their structural properties such as interaction with the solvent, cross-linking density and hydrophilicity [47].

Precise measurement of elasticity and reproducibility of results for the substrate are important for the applicability of hydrogels for a specific cell response. All techniques previously employed to assess mechanical properties of hydrogels have several limitations. Significant variation in the results on mechanical properties of gel measured at macro, micro and nano levels are reported in the published literature. Serval standardized procedures have been suggested in the published literature with the aim to overcome inconsistency and reproducibility of the results on mechanical properties of hydrogel measured by AFM and nanoindentation [48]. The force constants used in the analysis and cantilever’s spring constant, which can affect the force and indentation values, were the prime factors for obtaining unmatched results. In addition, variability in elasticity measurements may also be attributed to the inherent errors in the equipment and experimental protocol followed. Another possible reason for obtaining inconsistent data may be the use of different types and sizes of indenters, sample position during measurement, maximum loading force, and the use of different contact models for analyzing the data.

One of the prime objectives of determination of elastic properties of hydrogel substrate is to investigate the effect of gel stiffness on migration, proliferation, and morphology of cells. The use of appropriate gel stiffness is crucial for the design and optimization of tissue engineering scaffolds for variety of applications. The cell adhesion rate was lowered by increasing stiffness of the substrate while the cell spreading area and neurite length showed complex behavior [23]. Majority of research have demonstrated stiffness in the range of 1 kPa to 200 kPa suitable for unrestricted cell grow. Greater attachment of PC12 cells was observed on 5% GelMA [23]. Substrate stiffness from 0.041 MPa to 2.7 MPa was found to promote spreading, proliferation, mesendodermal gene expression, and terminal osteogenic differentiation of Embryonic stem cells [49]. Cell attachment was found to be unaffected by the stiffness of the substrate [49]. Structural stability of hydrogel, pore size and density may seriously affect spreading, migration and proliferation of cells [44, 46].

Almost all studies pertinent to the investigation of substrate stiffness on cell viability, adhesion and spreading have demonstrated similar results. Since significant change in the elastic modulus was observed by alteration of concentration in both types of gels with large intervals (1% for agarose and 5% for GelMA), it is necessary to conduct further study by narrowing down this interval. This will provide more optimized value of elastic modulus for a specific application where cell growth under control mechanical properties of the hydrogels is to be used. Results obtained in this study can be used as a benchmark for specific applications in tissue engineering as well as for constitutive modelling of hydrogels material in future studies.

5. Conclusions

The elastic response of agarose and GelMA hydrogels were found using tensile and compression tests. Mechanical properties such as Young’s modulus, fracture stress and failure strain were found sensitive to the amount of concentration in the gels. The elastic modulus of GelMA for a 5% concentration level was predicted using statistical analysis. Increased level of concentration resulted in stiffer microenvironment of the gels. This study has determined mechanical properties of the prescribed gels for a wide range of concentration and may serve as a benchmark for future investigations pertinent to the effect of substrate stiffness on the growth characteristics, proliferation, gene expression and morphology of cells. The relationship between viscoelastic properties of hydrogels of different concentrations on cell growth has to be investigated in future studies.

Footnotes

Acknowledgments

The authors would like to thank Engr. Mohammed Al-Hadhrami, College of Engineering, Sultan Qaboos University for his support in the experiments. The authors would like to acknowledge financial support from Sultan Qaboos University (Grant No: CL/SQU-GCC/17/03) and Qatar University (Grant No: GCC-2017-005).

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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