Architecting oxidized alginate methacrylate hydrogels with tunable characteristics by altering the sequence of the cross-linking steps,methacrylation reaction time,and polymer concentration

Abstract

In this study, biodegradable oxidized methacrylated alginate (OMA) hydrogels with controllable mechanical properties were engineered. An ionic and photo cross-linking combination was employed to fabricate dual cross-linked hydrogels. By altering the degree of methacrylation and polymer concentration, hydrogels with an elastic modulus of 4.85 ± 0.13 to 21.02 ± 0.91 kPa, controllable swelling, and degradation kinetics, and cross-link density in the range of 1.0 × 10⁻⁵ to 6.5 × 10⁻⁵ mol/cm³ were obtained. Moreover, evaluating the effect of cross-linking sequence on the hydrogels’ mechanical properties demonstrated that in comparison to the hydrogels fabricated by ionic cross-linking followed by photo-polymerization, hydrogels produced by photo-polymerization followed by ionic cross-linking retain a stiffer gel network with more compact structure. Cytocompatibility examination was performed via MTT assay against L929 fibroblasts, and all the hydrogel samples demonstrated high cell viability (>80%). The findings demonstrate the significant effect of the sequence of cross-linking as a novel tool to tune the OMA hydrogel’s final properties which can serve as a useful platform for tissue engineering applications.

Keywords

Modified alginate photo cross-linking ionic cross-linking cross-linking sequence hydrogels

Introduction

Alginate is a natural polymer that possesses bio-compatibility, solubility, porosity, viscosity tunability with concentration, and shear thinning capability, making it suitable for biomedical applications.^1–6 Divalent cations can cross-link long chains of alginates, causing them to gel more quickly, promoting cell encapsulation, and increasing mechanical stability. Ca²⁺ is the most frequently utilized for cross-linking due to its non-toxicity.^7,8

Despite all these credentials, alginates are challenged by a lack of bio-activity and slow degradation.^9–12 As a result of the unpredictable loss of divalent cations into the environment, there is also little control over the mechanical and physiological properties of ionically cross-linked alginate hydrogels.¹³

Photo cross-linkable hydrogels have lately attracted more interest in regenerative medicine, as macromer solutions comprising cells and growth factors can be delivered minimally invasively and subsequently quickly cross-linked after short exposure to radiation sources. Most photo-initiators work in the ultraviolet (UV) region of the spectrum.^14–16 However, the UV or near-UV blue light irradiation can damage the cells, and DNA and result in the cancerization of the skin.^17,18 Utilizing visible-light responsive photo-initiators for polymerization is hence safer. As a result, photo-initiators having an absorbing peak greater than 405 nm, or higher than the wavelength of blue light, are favored. According to research, eosin Y (EY)-based photo-initiation is a highly biocompatible cross-linking method for tissue engineering that is responsive to green light (450–550 nm).^19,20 Proper concentrations (<0.5%) of EY have proven to be less cytotoxic for encapsulation of hMSCs.²¹

Employing a dual-crosslinking approach is a powerful tool to fabricate hydrogels with controllable mechanical properties. In this sense, Pereira et al.²² used a combination of UV and ionic cross-linking to produce pectin-methacrylate bioinks with elastic modulus in the range of 154.7–6667.9 Pa and tunable swelling and degradation kinetics . Kim et al. fabricated alginate-tyramine hydrochloride hydrogels by visible light cross-linking using riboflavin as the photo-initiator and CaCO₃ as the ionic cross-linking agent. They found out that photo cross-linked hydrogels demonstrated more elastic properties compared to the ionically cross-linked samples as confirmed by the cyclic compression test; However, they did not thoroughly discuss the possible theory behind this observation.²³ Additionally, none of these studies have concentrated on the effect of the sequence of cross-linking steps, as it can have a significant impact on the final properties of hydrogels.

This study aimed to architect biodegradable alginate hydrogels with tunable mechanical properties for tissue engineering applications. To achieve this goal, the alginate was oxidized to make it more prone to hydrolytic degradation by changing the chair conformation of the uronate residue to an open-chain adduct. Then, we performed the methacrylation of the oxidized alginate (OA) to produce hydrogels that can be chemically cross-linked under visible light. Theoretically, we hypothesized that adding methacrylate groups to alginate would enable the construction of a macromer with inherent ionic gelation properties, while also undergoing chemical cross-linking through photopolymerization. By analyzing the swelling trend, elastic modulus, and degradation kinetics of the fabricated hydrogels, the impact of increasing the degree of methacrylation (DM) and alginate concentration on the biopolymer cross-linking density was evaluated. Through various independent cross-linking chemistries, we showed the adaptability of oxidized methacrylated alginate (OMA) macromers for the fabrication of hydrogels. We also studied the chronological effect of ionic and photo cross-linking on the rheological properties, cross-linking density, and microstructure of the final hydrogel. According to our knowledge, this is the first report on studying the effect of the sequence of cross-linking on the final properties of the modified alginate hydrogels.

Experimental section

Materials

The alginic acid sodium salt, sodium periodate (NaIO₄), calcium chloride (CaCl₂), methacrylic anhydride (MA), Eosin Y (EY), Triethanolamine (TEA), and 1-vinyl-2-pyrrolidone (NVP) were purchased from Sigma-Aldrich (St Louis, MO, USA). Sodium chloride (NaCl), ethanol, and Ethylene glycol were acquired from Merck Millipore (Darmstadt, Germany). Dulbecco’s modified eagle medium (DMEM F12), Fetal bovine serum (FBS), phosphate-buffered saline (PBS), and antibiotics were acquired from GIBCO (Grand Island, NY, USA).

Preparation of oxidized alginate (OA)

A modified version of a previously described procedure was used to react sodium alginate (viscosity 21.4 cP, 1 w/v % in H₂O at 25°C) with sodium periodate to prepare OA.^4,9,12 To achieve OA with a 5 percent theoretical oxidation degree (OD), a molar ratio of 0.05 of sodium periodate to a repeating unit of alginate was chosen. In a nutshell, 30 mL of deionized water (diH₂O) was used to dissolve 1.5 g of sodium alginate overnight. 0.081 g sodium periodate was dissolved in 30 mL diH₂O and poured into alginate solution while stirring in the dark at 25°C. To end the reaction after 24 h, 21 μL of ethylene glycol was added. The OA was precipitated and washed three times using ethanol, diH₂O, and NaCl. Subsequently, it was filtered (0.22 μm filter) and lyophilized.

Methacrylation of oxidized alginate

Photo-curable alginates were synthesized via a functionalization process using methacrylate anhydride (MA).^24,25 Briefly, 1 wt. % OA powder was dissolved in diH₂O and vigorously stirred before being combined with MA at a ratio of 15 mL MA/g of OA. The pH of the solution was maintained at 8.0 during the reaction period to speed up the reaction.²⁶ The impact of the reaction duration on the level of modification was evaluated using three different reaction times (6, 12, and 24 h). Then, it was completely mixed in 100 mL of 100% ethanol before being dried for an entire night at 50°C. The precipitated polymer was diluted in diH₂O and dialyzed (SnakeSkin Dialysis Tubing from Thermo Fisher Scientific, UK) against 0.3 wt. % NaCl solution in diH₂O for 7 days and with water changing each day. Lyophilization was used to recover the polymer.

Characterization of OA and OMAs

FT-IR Characterization

To confirm the successful synthesis of OA and OMAs, the Fourier transform infrared spectroscopy (FI-IR) test was carried out via a PerkinElmer model instrument from 4000 to 400 cm⁻¹ and with a resolution of 1 cm⁻¹ by creating a pallet of the sodium alginate, OA, and OMAs with potassium bromide (KBr).

¹H-NMR Characterization

Deuterium oxide (D₂O, 2 w/v %) was used to dissolve the OA and OMAs before they were put in an NMR tube. A Varian - INOVA 500 MHz NMR spectrometer (Varian Inc., Palo Alto, CA, USA) was utilized to record ¹H-NMR spectra in order to examine the OD and DM of the OMAs.

According to equations (1) and (2), the proportion of guluronic (G) in the alginate chain and the DM of the OMA macromers were computed from each corresponding spectrum ^25,27

G (%) = \frac{H_{G - 1}}{H_{M - 1} + H_{G - 1}}

(1)

D M (%) = \frac{\frac{H_{a} + H_{b}}{2}}{H_{G - 1}} \times G

(2)

where H_G‐1, H_M‐1, H_a, and H_b are the integrals of the proton in the guluronic blocks (4.90–5.40 ppm), a proton in the mannuronic blocks (4.30 ppm), and the two vinyl protons in the methacrylate branch (5.50–6.30 ppm), respectively.^25,27

UV-visible spectroscopy

Utilizing UV-visible absorption spectroscopy, the expenditure of NaIO₄ during the oxidation process was measured to determine the oxidation degree (OD) of OA.¹² By combining the same volumes of aqueous solutions of KI (20 w/v %) and starch (1 w/v %) PBS (pH = 7.0) as the solvent, the indicator solution was created. 1 mL of the solution was diluted to 250 mL with diH₂O before ending the oxidation process using ethylene glycol. After that, 3 mL of the diluted solution was combined with 1.5 mL of the indicator solution and 5 mL of diH₂O was used to make the final volume. A spectrophotometer (Thermo Fisher Scientific, USA) was used to test the triiodide-starch complex’s absorbance right away at 486 nm. (Equation (3))

Y O (%) = \frac{n_{c}}{n_{0}} \times 100

(3)

Where n_c is the number of consumed NaIO₄ (mol) and n₀ is the total amount of NaIO₄. This variable shows the number of glycol moieties that were converted into aldehyde groups. By measuring the amount of NaIO₄ consumed in the oxidation process using equation (4), the actual alginate OD was determined

O D (%) = \frac{m_{w} \times n_{c}}{m} \times 100

(4)

Where m is the mass of the initial sodium alginate (g), and m_w is the molecular weight of the alginate repeating unit (198.0198 g/mol).

Molecular weight measurement

Using an Agilent series 1100 device equipped with PL Aquagel-OH Mixed-H column (Agilent Technologies, USA), gel permeation chromatography (GPC) was performed to assess the molecular weight and molecular weight distribution of alginate and OA. The eluent utilized was diH₂O, the flow rate was set at 1 mL/min, and polyethylene glycol standard samples were used for calibration. Weight average molecular weight (M_w), number average molecular weight (M_n), and the polydispersity index (PDI = M_w/M_n) were determined.

Preparation of the hydrogel

The single or dual cross-linked OMA hydrogels were developed by ionic and photo cross-linking mechanisms. OMA samples were dissolved in diH₂O at 4 and 6 wt. % polymer concentrations. 0.5 mM of EY, 1.5 wt. % of TEA, and 1.5 wt. % of 1-vinyl-2-pyrrolidinone were added to the macromer solution and agitated in order to trigger the photo-crosslinking reaction. The blue light (80 mW/cm², 485 nm) was irradiated for 6 min and hydrogels were stabilized for roughly 6 h to finish the reaction. 20 mM of CaCl₂ was added to the OMA solutions and vigorously swirled while stabilizing for around 6 h to induce ionic cross-linking. Two different strategies were utilized to prepare dual cross-linked OMA hydrogels. Hydrogels were fabricated by either ionic cross-linking followed by photo cross-linking (IP) or photo cross-linking followed by ionic cross-linking (PI). For the PI sequence, after photo cross-linking hydrogels were immersed in 20 mM CaCl₂ solution for 2 min. By varying the concentrations of OMA, methacrylation reaction time, and sequence of cross-linking, eight experimental groups were characterized (Table 1).

Table 1.

Experimental groups.

Abbreviations	Polymer conc. (% w/w)	Methacrylation time	Cross-linking sequence
OMA-4%-6hr-PI	4	6	“Photo cross-linking followed by ionic cross-linking”
OMA-4%-12hr-PI	4	12	“Photo cross-linking followed by ionic cross-linking”
OMA-4%-24hr-PI	4	24	“Photo cross-linking followed by ionic cross-linking”
OMA-6%-6hr-PI	6	6	“Photo cross-linking followed by ionic cross-linking”
OMA-6%-12hr-PI	6	12	“Photo cross-linking followed by ionic cross-linking”
OMA-6%-24hr-PI	6	24	“Photo cross-linking followed by ionic cross-linking”
OMA-4%-24hr-IP	4	24	“Ionic cross-linking followed by photo cross-linking”
OMA-6%-24hr-IP	6	24	“Ionic cross-linking followed by photo cross-linking”

Characterization of OMA hydrogels

Rheological properties

Utilizing a strain-controlled AR-2000ex rheometer (TA Instruments, NewCastle, DE, USA) with parallel plate geometry (4 mm plate radius, 0.7–0.8 mm gap), the rheological properties of the dual-crosslinked OMA hydrogels were assessed. The characterization was made using a dynamic frequency sweep test, where a sinusoidal shear strain was applied over a range of frequencies (0.6–10 rad/s), with a constant peak amplitude (0.1%). Dynamic strain sweep tests (up to 10% strain at 10 rad/s frequency) revealed that the strain applied was within the linear viscoelastic region (LVR).

Mechanical testing

Compressive strengths of OMA hydrogels with various polymer concentrations, methacrylation reaction times, and sequence of cross-linking were measured using Universal Test Machine STM-1 (Santam Co., Iran). Dual cross-linked OMA hydrogels with 20 mm diameter and 10 mm thickness were fabricated. Programming required the probe to drop at a constant rate of 0.5 mm/s. The experiment was conducted at 25°C. The stress-strain curves were utilized to determine the Young modulus (E), compressive strength (σ_F), and toughness moduli (U_T). The initial slope of the graph in the elastic region (up to ε = 15%) was used to determine E. The value of the applied stress at the fracture point is represented by the symbol σ_F. U_T was then computed as the total area under the curve (0 ≤ σ ≤ σ_F).

Gel fraction, swelling ratio, and degradation profiles of hydrogels

The photo cross-linked hydrogels were freeze-dried and weighed (W_i) before being submerged in diH₂O at 37°C for 24 h. After being dried once more to eliminate the soluble components, the submerged hydrogels were reweighed (W_e). The gel fraction percentage of photo-cross-linked OMA hydrogels was determined as follows (equation (5))

G e l f r a c t i o n (%) = \frac{W_{e}}{W_{i}} \times 100

(5)

To measure the swelling ratio of the dual cross-linked OMA samples, the hydrogels were submerged in diH₂O at 37°C. The swelling ratio of samples was obtained by equation (6)

Q = \frac{W_{s} - W_{d}}{W_{d}} \times 100

(6)

Where W_d and W_s stand for the hydrogels’ weight at dried and swollen state at time t, respectively.

For the in vitro degradation tests, the samples were freeze-dried and weighed (W_i), then incubated in diH₂O at 37°C. Every 2 days, the diH₂O was changed. The samples were taken out, washed with diH₂O, freeze-dried, and weighed at specified intervals (W_d). The weight loss percentage (W_L) was computed using equation (7)

W L = (\frac{W_{i} - W_{d}}{W_{i}}) \times 100

(7)

All experiments were repeated three times to confirm reproducibility.

Morphology

Using field emission scanning electron microscopy (FE-SEM, Tescan Mira3 LMU), the OMA hydrogels' porous and interconnected architectures were explored. After being cross-linked by visible light and calcium ions, dual cross-linked OMA hydrogels were lyophilized, and the cross-sections of the hydrogels were examined.

Mesh size and cross-linking density

The OMA hydrogels mesh size was calculated from the swelling and rheometry data due to the anionic contributions of the alginate polymers according to the previous literature. ^28–31 The number average molecular weight between cross-links (M_c) for dual cross-linked OMA hydrogels was computed according to the equation below ¹³ equation (8)

M_{c} = \frac{C_{p} R T}{G^{'}}

(8)

T stands for the measurement temperature (298 K), C_p for the OMA concentration (g/m³), R for the gas constant (8.314 J mol⁻¹. K⁻¹), and G´ for the storage moduli measured at 1 Hz in the linear viscoelastic region (LVR). Mesh size (ζ) is then determined via Flory’s theory as follows³² (equation (9))

ζ = {Q_{v}}^{1 / 3} L (\frac{2 M_{c}}{M_{r}}) {C_{n}}^{1 / 2}

(9)

Where L is the repeating unit’s Kuhn length (5.15 Å), M_r is its molecular weight (194 g/mol), and C_n is Flory’s characteristic ratio, which measures how far the polymer chain extends in a disordered state (22.5),³³ and Q_v is the volumetric swelling ratio (after equilibrium (72 h)) calculated via the following equation³² (equation (10))

Q_{v} = \frac{\frac{W_{s} - W_{d}}{ρ_{w}} + \frac{W_{d}}{ρ_{p}}}{\frac{W_{d}}{ρ_{p}}}

(10)

Where ρ_w is the water’s density (1 g/cm³) and ρ_P is the alginates’ density (1.6 g/cm³).

The cross-link density can then be calculated as follows (equation (11))

ν = \frac{ρ_{p}}{M_{c}}

(11)

Cytocompatibility study

The specimens were placed in a full culture medium and incubated for 7 days to test the potential cytotoxicity of the emitted degradation byproducts under ISO10993-5.⁴ After that, samples were taken out, and the extract was used to assess indirect cytocompatibility. Briefly, in 96-well plates, 2 × 10⁴ cells/well were incubated for 24 h. The 7-days extract was then added to the medium, and the cells were then cultured for a further 24 h. The tissue culture plate (TCP) control group and different experimental groups were subjected to MTT evaluation.

Statistical analysis

Statistical analysis was completed in the IBM SPSS Statistics 26 software. The findings were presented as the mean ± standard deviation (SD) (n = 3). With a 95% confidence interval, the independent sample t test and one-way analysis of variance (ANOVA) were employed. Statistically significant differences were designated by the symbols p < .05 (*), p < .01 (**), and p < .001 (***).

Results and discussion

Alginate functionalization

To prepare bio-degradable photo cross-linkable OMA macromers, alginate was oxidized, and then methacrylation was performed, as demonstrated in Figure 1(a).

The successful functionalization was confirmed by comparing the FT-IR plots of sodium alginate, OA, and OMA (Figure 1(b)).³⁴ The symmetric C-O-C mode region at 887 cm⁻¹ also showed spectral alteration. A newly formed distinctive peak associated with the aldehyde unit was also not seen in the 1725-1751 cm⁻¹ range. In contrast to the dried specimens that are often utilized for FT-IR spectra, this peak was only discovered under specimens that had been equilibrated in ambient settings. Aldehyde and hemiacetal group equilibrium in OA was suggested by Jejurikar et al. as a potential explanation for this phenomenon. For OMA-24 h, the rise at 1741 cm⁻¹ was assigned to the stretching of the carbonyl of the ester unit, confirming the methacrylate functionalization of OA. For OMA samples with lower methacrylation reaction time (OMA-6 h and OMA-12 h), new characteristic peaks were not evident which is probably due to the lower DM of these samples (data not shown).

The ¹H-NMR figure of Alg, OA, and designed OMA samples are displayed in Figure 1(c). The saccharide units of the alginate backbone are represented by distinctive peaks in all spectra between 3.50 and 5.20 ppm. Additionally, the methacrylate grafted groups' vinyl protons (5.50–6.30 ppm) and methyl protons (1.60–2.00 ppm) can be seen in the OMA’s spectra as unique signals.²⁶

Using equations (2) and (3), the G percentage of the alginate backbone and the DM of OMA samples were computed from Figure 2(c), respectively (Section 2.4.2). G was determined as 43.58%, while the DM of each OMA macromer was calculated as 15.86%, 21.58%, and 28.13% for OMA-6h, OMA-12h, and OMA-24h, respectively.

The ¹H-NMR spectra of OA did not show any significant peak which is due to the low oxidation degree (OD) of Alginate (<5%). UV-vis spectroscopy was employed to evaluate the actual OD of alginate.

The amount of unreacted sodium periodate was attained by UV-vis spectroscopy. Afterward, the corresponding YO and actual OD were determined using equations (3) and (4), respectively. The results are shown in Table 2. As is shown, the YO was 95.2 ± 0.6%, and the actual OD was 4.76.

Table 2.

Oxidation condition of alginate.

Theoretical OD (%)	Total periodate (mmol)	Unconsumed periodate (mmol)	Consumed periodate (mmol)	YO (%)	Actual OD (%)
5	0.379	0.018	0.361	95.2	4.76

The M_w and PDI of alginate, OA, and OMA determined via GPC are summarized in Table 3. Native alginate had an M_w of 198.03 kDa (PDI = 1.51). In order to introduce hydrolyzable acetal-like connections in polymer chains, the oxidation reaction opens a sugar ring in the alginate molecule. The oxidation of alginate resulted in the formation of a polymer with an M_w of 115.87 kDa (PDI = 1.85). Alginate’s molecular weight is reduced during the oxidation process as a result of sodium periodate’s activation of alginate breakdown .³⁵ Methacrylation reaction slightly increased the M_w of OA probably due to the introduction of methacrylate groups in the polymer chain. The calculated DM of OMA hydrogels is also presented in Table 3.

Table 3.

Weight average molecular weight (M_w) and PDI of alginate (Alg), oxidized alginate (OA), and oxidized methacrylated alginate (OMA). Calculated DM of OMA hydrogels are also presented.

Code	M_w (kDa)	PDI	DM (%)
Alg	198.03	1.51	—
OA	115.87	1.85	—
OMA-6h	121.22	2.04	15.86
OMA-12h	123.34	3.12	21.58
OMA-24h	124.73	2.76	28.13

Single and dual-cross-linked hydrogels preparation

OMA hydrogels were prepared via a dual cross-linking method involving a photo cross-linking and an ionic cross-linking step. Photo-polymerization was performed via a photo-initiation system based on EY, TEA, and 1-vinyl-2-pyrrolidinone by irradiation under a visible light source. Visible light excites the photo-initiator (EY) from the ground state into a triplet state (color change from red to yellow). Excited EY then extracts hydrogen atoms from the co-initiator (TEA). Afterward, the deprotonated TEA radical creates a radical center on the methacrylate groups of OMA. 1-vinyl-2-pyrrolidinone acts as a co-monomer, generating radicals to boost the cross-linking process under visible light. The wavelength of peak absorbance (λ_max) of EY is located around 475 nm, and the maximum radiation of the radiation source is detected at 481 nm, indicating that the maximum radiation wavelength of the LED source overlaps the maximum absorption wavelength of EY (data not shown). The color change of the hydrogel due to the excitation of EY after photo-crosslinking is shown in Figure 2(a).

An indirect MTT method was performed to evaluate the optimum EY concentration without diminishing the viability of L-929 fibroblast cells. According to the cell viability data (Figure 2(c)), a 0.05 mM concentration of EY was chosen for the photo cross-linking of hydrogels. A higher concentration of EY (0.75 mM) resulted in cell viability values under 80%.

To improve the mechanical strength of OMA hydrogels, an ionic cross-linking step using CaCl₂ was performed. 20 mM CaCl₂ concentrations were used for the ionic cross-linking step as higher concentrations (40 mM) led to the creation of inhomogeneous gel as a result of rapid cross-linking (Figure 2(c)).

The schematic illustration (Figure 2(d)) depicts OMA macromers' versatility in preparing hydrogels through independent cross-linking chemistries. Two different sequences of cross-linking were employed for the preparation of OMA hydrogels.

Gel fraction, swelling ratio, and degradation profiles

To examine the impacts of DM and cross-linking time on the creation of the hydrogel, the sol-gel fraction was established as an indication of the double bond conversion and cross-linking efficiency. Gel fraction plots of sole photo cross-linked OMA samples revealed that DM, irradiation time, and polymer concentration affect sol-gel conversion (Figure 3(a) and (b)). Because of the development of a densely cross-linked hydrogel network, correlated with reduced swelling and improved resistance to enzymatic breakdown, the gel fraction raised with the increase of the DM (Figure 3(c) and (d)). Similar to this, increased DM resulted in a shorter minimum irradiation time necessary to create stable gel networks. For hydrogels prepared from OMA-6%-6 h, a stable gel was only obtained for cross-linking times higher than 240 s (gel fraction = 19.2 ± 1.3). The minimum cross-linking time needed to create stable hydrogels was decreased to 150 s (OMA-6%-12 h, gel fraction = 21.9% ± 1.0) and 60 s (OMA-6%-24 h, gel fraction = 28.1% ± 2.1) by merely increasing the DM. After 330 s of irradiation, the gel fractions calculated for OMA-6%-24 h reached a plateau value, which was set as the photo-polymerization time. With a 150 s cross-linking time, OMA-4%-6 h and OMA-6%-6 h hydrogels produced gel networks that were loosely cross-linked and had low shape fidelity after immersion in diH₂O water, preventing the evaluation of gel content.

To enhance the mechanical stability of the OMA hydrogels, 20 mM Ca²⁺ ions were introduced into the OMA hydrogels after photo cross-linking. The swelling content variation of the hydrogels examined in time echoes alterations in their chemical and physical nature. The swelling ratios of OMA samples in diH₂O are depicted in Figure 3(c). All hydrogels demonstrated fast swelling and reached the equilibrium stage within 72 h. Compared to other hydrogels, OMA-4%-6 h exhibited much faster swelling kinetics and reached a maximum swelling ratio of 641% ± 27 after 72 h. It was not possible to measure the swelling ratio of OMA-4%-6h after 2 weeks due to the complete degradation of the sample. The swelling ratio of OMA-6%-24h increased during the course of 2 weeks and reached 421% ± 16 at the end of week two. The swelling ratio of all the other samples increased up to 1 week and then decreased probably due to the degradation of these samples.

As an indicator of degradation, the weight loss (%) of OMA hydrogels over time was evaluated (Figure 3(d)). As can be observed, OMA-4%-6h demonstrated the sharpest trend with a mass loss of 100% after 1 week, whereas OMA-6%-24h demonstrated the slowest degradation kinetics (63% ± 5 at three weeks). The cross-linked hydrogels' rate of degradation reduced as DM and macromer concentrations increased (Figure 3(d)).

Partially OA was employed to accelerate and make the biodegradation of hydrogel more palatable for in vivo applications. The weight loss of unmodified alginate (MA-4%-24h) was 22% lower than OMA-6%-24h and 42% lower than the OMA-4%-24h sample indicating the significant role of oxidation in improving the in-vitro degradability of alginate hydrogels. Within 3 weeks, every hydrogel lost more than half of its weight, equal to the period required for different tissue regeneration and the release of the new matrix.

Mechanical characteristics of OMA hydrogels

The elastic modulus (G´) and loss modulus (G´´) versus frequency plots of 6% OMA hydrogels with different methacrylation reaction times are demonstrated in Figure 4(a). The increase in the methacrylation reaction time, i.e. the increase in the DM, enhanced the storage modulus of OMA hydrogels. An increase in the DM results in the presence of more methacrylate groups in the polymer backbone. These methacrylate functionalities can form cross-links after irradiation under the light source; hence increase in their numbers results in the creation of a denser network, leading to a higher elastic modulus. The G´ of OMA-6%-12h and OMA-6%-24h were almost three times and seven times higher than that of OMA-6%-6h (@ 1 Hz), respectively. The storage modulus of all samples was considerably greater than the loss modulus. This indicates that the OMA hydrogels present a dominant elastic behavior compared to the viscous behavior demonstrating the applicability of these hydrogels for tissue engineering applications.

The storage and loss modulus versus frequency graphs of 4% and 6% OMA hydrogels with 24 h reaction times and different ionic and photo-cross-linking reaction sequences are depicted in Figure 4(b). Similar to the DM, increasing the OMA concentration also enhanced the storage modulus of OMA hydrogels. Increasing the polymer concentration enhances the fixed negative charges in hydrogels resulting in an increase in counterions and intermolecular interactions between OMA chains, which can improve the stiffness of the network and enhance the effect of Ca²⁺ ions. Additionally, increasing polymer concentration can increase the viscosity of the hydrogel solutions and also the number of C = C bonds for the photo-polymerization reaction, leading to the formation of a more dense network structure. The G´ of dual-cross-linked hydrogels prepared by the IP sequence displayed a relatively softer gel network in comparison to the hydrogels prepared by the PI sequence. Moreover, the G´ of PI samples was almost independent of frequency, while G´ of IP samples demonstrated a more frequency-dependent behavior. These findings imply that the dual-cross-linked gel network is less prone to further stiffening following ionic gelation for viscosity modification, most likely as a result of the spatial restrictions in the polymer backbone brought on by the cross-linking reactions. MA takes on an external orientation regarding the OMA backbone after being grafted to alginate. The carbon-carbon double bond is completely accessible for the intended photo cross-linking reaction because there are no visible intramolecular interactions that could hinder its free rotation.²⁵ The introduction of Ca²⁺ for ionic-cross linking forms an egg-box structure^36,37 which is anticipated to hamper the C = C availability for the later photo-cross-linking reaction. This intramolecular interaction could make the grafted MA group rigid, imposing an additional impediment to OMA chains to react with one another. Taking the aforementioned information into consideration, the results might provide an explanation for the observed trend in the mechanical properties: G´_IP < G´_PI. On the one hand, when photo-polymerization is the first cross-linking step, the C = C bonds of the OMA macromer are favorably oriented to establish a cross-linking reaction with their homologs, showing the additional advantage of being free to rotate. As a result, the PI hydrogels are expected to have a higher number of cross-linking joints (i.e., higher cross-linking density), giving support to the enhanced mechanical properties it displayed. On the other hand, when ionic cross-linking is the first cross-linking step, the poor orientation of the C = C reactive bonds coupled with a spatial hindrance in the polymer structure ensemble the possibility of photo-cross-linking to a great extent. This results in lower cross-linking density and poor mechanical properties of the final IP hydrogels. The inhomogeneity of the OMA hydrogels at the sub-molecular level due to the fast cross-linking kinetics of CaCl₂ could be another reason for the observed trend. However, as observed in Figure 2(c), no inhomogeneity was observed at the macro-molecular level.

The synthetic hydrogels must have adequate compressive strength and acceptable elasticity to withstand the authentic tissue’s contraction force. The attained stress-strain plots and the mechanical features thereby calculated are demonstrated in Figures 5(a)–(d). All samples exhibited the conventional stress-induced brittle material evolution, including elastic (ε ≤ 0.15), viscous (0.15 < ε ≤ εF), and fracture deformations (ε > ε_F), as documented for hydrogels of similar type.^25,38,39 The compressive modulus (Figure 5(b)) of hydrogels is improved by increasing OMA concentration and the DM of hydrogels, owing to the enhanced cross-linking density of the hydrogels. OMA hydrogels with an elastic modulus of 4.85 ± 0.13 to 21.02 ± 0.91 kPa were obtained by varying the DM. It was not possible to evaluate the compressive modulus of the OMA-4%-6h sample due to its poor mechanical stability. The compressive strength and toughness modulus (Figure 5(c), and (d)) were discovered to exhibit a similar upward trajectory as OMA concentration and DM increased (p < .001).

OMA-4%-24hr-IP and OMA-6%-24hr-IP demonstrated a lower Young’s modulus, compressive strength, and toughness modulus compared to OMA-4%-24hr-PI and OMA-6%-24hr-PI, respectively indicating the lower cross-linking density of samples fabricated by the IP sequence (Statistically significant (p < .05)). Furthermore, the IP samples were more inelastic compared to the PI samples which is in agreement with previous literature for ionic cross-linked alginate (more viscose behavior) hydrogels compared with photo cross-linked hydrogels (more elastic behavior).

The molecular weight between cross-links (M_c), cross-linking density (υ), and mesh size (ζ) were calculated using the obtained swelling ratios and moduli data. As expected, As expected, increasing methacrylation reaction time (i.e. increasing DM) decreased the M_c, ζ, and increased υ of the final hydrogel. M_c values determined (g/mol) for OMA-4% with different DM were 152,466.7 ± 6434.3, 80,571.5 ± 2439.4, 24,469.9 ± 1050.3 g/mol for 6 h, 12 h, and 24 h methacrylation reaction time, respectively. For OMA-6%, M_c values were 145,739.5 ± 6958.74, 71,813.7 ± 3284.8, and 24,652.5 ± 1234.7 g/mol for 6 h, 12 h, and 24 h methacrylation reaction time, respectively. Increasing OMA concentration did not significantly change the M_c and υ of hydrogels. However, ζ of samples were decreased.

The chronology of cross-linking showed an important impact on the M_c, υ, and ζ of the final hydrogel. The M_C, υ, and ζ data for OMA-6%-24hr-PI were 24,652.5 ± 1234.7 g/mol, 6.5 × 10⁻⁵ ± 3.1 × 10⁻⁶ mol/cm³, and 20.5 ± 0.8 nm, respectively, and for OMA-6%-24hr-IP were 560,95.9 ± 2543.4 g/mol, 2.8 × 10⁻⁵ ± 1.2 × 10⁻⁶ mol/cm³, and 33.1 ± 1.5 nm, respectively. As it can be inferred from these data, the sequence of photo cross-linking followed by ionic cross-linking resulted in the formation of a much stiffer hydrogel network.

Using the dual cross-linking method, it was possible to architect OMA hydrogels with controllable cross-link density and mesh size in the range of 1.0 × 10⁻⁵ to 6.5 × 10⁻⁵ and 77.0 to 20.5, respectively.

Internal morphology of OMA hydrogels

SEM graphs revealed that alterations in the DM and the chronology of cross-linking also had an impact on the microstructure and pore size of OMA hydrogels. The SEM images of the lyophilized dual cross-linked 6% OMA hydrogels are demonstrated in Figures 6(a)–(d). The average pore sizes of OMA samples are shown in Figure 6(e). As can be inferred from the graphs, increasing the DM decreased pore size and enhanced the average number of pores, indicating the formation of a more compact structure. The OMA hydrogel produced with photo-polymerization followed by ionic gelation had a smaller pore size in comparison to the samples produced with ionic gelation followed by photo-polymerization. This result is in accordance with the elastic modulus, swelling, and weight loss data denoting the formation of a denser network when the photo cross-linking is the first gelation step. The smallest pore size belonged to the OMA-6%-24hr-PI sample with 20.1 ± 1.6 μm.

Cytocompatibility of OMA hydrogels

The cytotoxicity of OMA hydrogels in vitro was evaluated by an indirect MTT method using L-929 fibroblast cells (Figure 6(f)). The cell viabilities of all hydrogels were above 80% indicating that the hydrogels had good biocompatibility. The cell viability value of OMA-4%-6hr-PI compared to the tissue culture plate (TCP) was not statistically meaningful (p > .05). However, the cytocompatibility of hydrogels fell with the rise of hydrogel concentration (p < .05). This is probably due to the increment in the hydrophobic nature of hydrogels caused by increasing the polymer concentration and the introduction of methacrylate moieties which is in accordance with previous findings .^10,22

Conclusion

In this study, modifications of alginate were performed to fabricate hydrogels with tunable mechanical and biological characteristics. A dual-crosslinking method using CaCl₂ and visible light was employed to prepare OMA hydrogels. The impact of varying the degree of methacrylation (DM) and OMA concentration on the mechanical and biological traits of the final hydrogel was evaluated by examining hydrogel mechanical properties, swelling behavior, degradation profiles, and microstructure. The increase in the DM and macromer concentration led to a reduction in swelling ratio, an increment in cross-linking density, and elastic modulus of the final hydrogel. Utilizing this method, the elastic modulus of hydrogels can be modulated between 4.85 ± 0.13 to 21.02 ± 0.91 by changing polymer concentration and DM. The effect of the ionic and photo cross-linking sequence on the cross-link density of the final hydrogel was also investigated. The elastic modulus of hydrogels prepared by ionic cross-linking followed by photopolymerization was higher than hydrogels prepared by the inverse sequence. SEM images obtained from the samples demonstrated the formation of a more compact structure with a smaller pore size for samples with higher DM and macromer concentrations. These results indicated that the sequence of cross-linking has a significant role in the properties of the final hydrogel probably due to spatial hindrance imposed by ionic cross-linking. Furthermore, L-929 fibroblasts demonstrated high cell viability (>80% for all samples) when incorporated into the OMA hydrogels. In the next step, this dual-cross-linked OMA hydrogel with improved mechanical properties and physiological constancy will be applied as a bioink for 3D extrusion bioprinting.

Footnotes

Acknowledgements

The authors would like to express their gratitude to the Cell and Tissue Engineering Laboratory of Sharif University of Technology (SUT) for providing the resources and support.

CRediT authorship contribution statement

Amirhossein Jalali Kandeloos: Investigation, Methodology, Formal analysis, data curation, Writing - original draft. Saeed Bastani: Project administration, Conceptualization, Supervision, Validation. Shohreh Mashayekhan: Conceptualization, Supervision, Writing - review & editing. Conflicts of interest

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

S Bastani

S Mashayekhan

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