Modified silicon carbide NPs reinforced nanocomposite hydrogels based on alginate-gelatin by with high mechanical properties for tissue engineering

2.1 Materials

Sodium alginate (low viscosity derived from brown algae), Gelatin (type B from bovine skin), acetone, potassium periodate (KIO₄), silver nitrate (AgNO₃), ethyl alcohol, n-propanol, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), ethylene glycol, sodium chloride, N-hydroxysuccinimide (NHS) were acquired from Merck and Sigma-Aldrich Company and employed without additional refinement. Silicon Carbide Nanopowder/Nanoparticles (SiC, beta, 99+%, <80 nm, cubic) were purchased from US Research Nanomaterials, Inc.

2.2 Alginate oxidation

2.01 g of sodium alginate and 11.2 ml of n-propanol were blended with DI-water in a 250-mL beaker to obtain 225 ml in total. The mix was kept at 30 °C in the dark under stirring (5 h) to dissolve alginate completely. 1.16 g of Potassium periodate (KIO₄) dispersed in 30 ml DI-water was combined with alginate solution. The mixture was kept in the dark for 24 h. The reaction was quenched by adding 1 ml of Ethylene glycol (EG), and the mix was agitated for another 30 min. 6.5 g of Sodium chloride (NaCl) was dissolved in the above suspension to purify the polymer, which was next gently added to 400 ml agitated ethyl alcohol. The white precipitate was dissolved in DI-water with 3.3 g of NaCl and reprecipitated in 250 ml ethyl alcohol. The precipitate was dissolved in DI-water again and precipitated in 200 ml acetone. Eventually, the precipitate was rinsed in agitated ethyl alcohol for 15 min, refined, and dried at 25 °C (Rogalsky et al., 2011). The lack of periodate was controlled by combining 500 µL fractional of the dialyzate to 500 µL of a 1% silver nitrate solution, and assuring the nonexistence of any precipitate (Balakrishnan & Jayakrishnan, 2005).

2.3 OA/GEL/SiC scaffolds formation

4 ml of 6 wt% of OA solution was agitated with 4 ml of 15 wt% of GEL at 37 °C. The cross-linker, including a mixture of 0.1 g EDC and 0.05 g NHS, was added to the above solution. The first gelation was observed in 4–5 s and kept at 37° C, resulting in the creation of a perfect gel after 2 min. Different weight percentages of SiC (0.25%, 1.0%, and 1.5%) was added to the 4 ml of 6 wt% of OA solution and agitated for 5 min. Next, 4 ml of 15 wt% of GEL was added to the suspension and stirred for another 5 min. The final solutions were mixed for 2 min by adding EDC and NHS as cross-linker agents. The samples were freeze-dried for 24 h. Three-dimensional scaffolds with a diameter of about 2 cm and a thickness of 1 cm were produced.

2.4 Swelling behavior and in vitro biodegradation

The water absorption of hydrogels was evaluated by the gravimetric technique. About 0.2 g (M₀) of the hydrogels were incubated in 10 ml PBS for 24 h to attain equilibrium swelling. The buoyant was removed and the swollen hydrogel were weighed (M_s). The swelling ratio (SR) was expressed by the following equation (Nguyen & Lee, 2011):

Mass degradation/erosion degrees were additionally evaluated likewise at various periods up to 21 days. All tests were accomplished five times.

2.5 Materials characterizations

Fourier transform infrared spectroscopy (Shimadzu Varian 4300 spectrophotometer) was utilized to investigate the chemical composition of oxidized alginate and the fabricated hydrogels applying KBr pellets in the wavenumber between 4000 and 400 cm⁻¹. A field emission scanning electron microscopy (TESCAN MIRA 3 FE-SEM) was used to study the morphological and structural of lyophilized hydrogels. The lyophilized hydrogels were cross-sectioned, coated by gold (Au), and detected by FE-SEM at an accelerating voltage of 15 kV. High-resolution transmission electron microscopy (EM 208, Philips HR-TEM with an accelerating voltage of 100 kV) was utilized to observe carbon nitride quantum dots. A Physica MCR 300 Rheometer (Anton Paar Ltd., Austria) was utilized to measure the oscillatory rheological properties of the hydrogels.

2.6 Mechanical features

A Physica MCR 300 Rheometer (Anton Paar Ltd., Austria) was used to measure the rheological attributes of the hydrogels utilizing a circular disk parallel plate with a diameter of 25 mm and a gap of 0.5 mm. An amplitude sweep was conducted at a consistent angular frequency of 1 Hz to define the limit of linear viscoelasticity. The strain amplitude was kept at 0.1% during the test. The contribution of the liquid-like form (viscous modulus (G“)) and solid-like form (elastic modulus (G')) were noted through temperature sweep from 20 to 50 °C at a speed of 1 °C min⁻¹ to assess thermogelling attributes (angular frequency = 1 Hz). Each following rheological test was conducted below simulated physiological states (in PBS pH = 7.4 at 37 °C), considering the possible utilization of hydrogels. The oscillatory rheological determination as a function of time was conducted at a consistent frequency of 1 Hz to evaluate the time of gelation. The gel point or gelation time was specified as the time that the loss modulus and shear storage modulus were identical (Macaya et al., 2011). The hydrogels were swollen for 1 h in 1 ml PBS and moved to the rheometer stage for performing crosslinked hydrogels. Next, frequency sweep analyses in the linear viscoelastic area were performed to determine the dynamic viscoelasticity at 37 °C on a broad range of frequencies (0.1–100 Hz).

2.7 In vitro biological assays

The cytotoxicity of scaffolds with and without SiC NPs was evaluated utilizing the methyl thiazolyl tetrazolium (MTT) assay. In brief, L929 cells with a density of 5 × 10³ cells/mL were seeded in 96-well microplates in complete Roswell Park Memorial Institute media (RPMI) comprising 1% penicillin–streptomycin and 10% heat-inactivated fetal bovine serum (FBS). The plates were incubated with 5% CO₂ in a 95% humidified atmosphere at 37 °C for 48 h. Scaffolds were immersed in 70% ethanol for 1 h, rinsed five times with cold sterile phosphate-buffered saline (PBS), and added into the medium with successive concentrations (20, 50, and 100 μg/mL), and cultured for 24 and 72 h. After incubation time the supernatants were trashed, 100 μL in complete RPMI media comprising 10% MTT stock solution were added per wells. Cells were incubated at 37 °C for 4 h again. The medium was discharged and the reaction was terminated by adding 100 μL dimethyl sulfoxide (DMSO) per wells to solubilize formed formazan crystals. Finally, the optical densities were measured by a microplate reader (Microplate Reader Gmi. Ltd., USA, Awareness Technology Stat Fax 2100) at 570 nm with background subtraction at 630 nm after 1 h. The cell viability percentage was assessed for the control.

To study the morphology of the cells attached to the scaffolds, the scaffolds were soaked in 70% ethanol for 1 h and rinsed five times with sterilized PBS. Sterilized scaffolds were placed in 6-well cell culture plates (Corning-Costar, Corning, NY). Cells were seeded with 5 ml cell suspension comprising 5 × 10³ cells/ml on scaffolds for 24 h. Scaffolds were monitored by Phase-contrast microscope (Olympus America, Inc). Cell-seeded scaffolds were picked up from the media and fixed with glutaraldehyde as fixator. In summary, scaffolds were gently rinsed with PBS (pH = 7.4) three times then soaked in 2.5% glutaraldehyde solution at 4 °C for 12 h. The fixative was discharged and was dehydrated carefully with ethanol at a series of concentrations and placed for drying overnight in a desiccator. The morphology of the scaffold surface was studied by scanning electron microscopy (SEM, MIRA3-TESCAN).

3.1 FTIR spectra of hydrogels

The architectural variations in gelatin, alginate, OA, and fabricated hydrogels were studied by FTIR spectroscopy (Fig. 1). The FTIR spectrum of gelatin shows that the region of 3000–3600 cm⁻¹ and 1100–1700 cm⁻¹ contains the most potentially useful information bearing on the structure of gelatin. The band identified as N—H stretching vibrations are considered to be hydrogen bonded. The frequency N—H band was observed at 3431 cm⁻¹ that is a characteristic of N—H stretching of secondary amide II bands. The spectrum of gelatin also shows the peaks at 1631 and 1639 cm⁻¹ due to C⚌O stretching (amide I bands). The characteristic bands at 1550 cm⁻¹ and 1500 cm⁻¹ corresponded to N—H bending and amide II bands (Pawde & Deshmukh, 2008). N—H out of plane wagging is located at 670 cm⁻¹. The peaks at 2922 cm⁻¹ and 2850 cm⁻¹ are related to C—H stretching (Tengroth et al., 2005). The band at 1240 cm⁻¹ presented amide III. The amide I band predominantly arises from the protein amide C⚌O stretching vibration, and amide II absorption is caused by N—H bending and C—N stretching vibrations, and the amide III peak is made up of C—N stretching vibration (Baniasadi et al., 2016). Fig. 1 shows some changes in characteristic bands of alginate spectrum after the oxidation, equivalent to reported literature (Jejurikar et al., 2012; Sarker et al., 2014). As displayed in the OA spectrum, the specific absorption bands correlated to AL structure lightly change, and a unique band arrives at 1731 cm⁻¹ assigned to the aldehyde group. The absorption bands at 1384 cm⁻¹ and 1631 cm⁻¹ are also existing in OA, which are assigned to symmetric and asymmetric —COO— stretching vibrations on the AL structure, respectively (Emami et al., 2018). The peaks at around 3000 cm⁻¹ are attributed to asymmetric (2945 cm⁻¹) and symmetric (2850 cm⁻¹) stretching modes of —CH₂ groups (Kuzmanović et al., 2017). Hence, the oxidation reaction via potassium periodate did not influence the carboxyl groups in AL. The new peak at 1731 cm⁻¹ in the OA shows the appearance of aldehyde groups (—CHO). In some circumstances, this band is not recognized because of the hemiacetal configuration of hydroxyl groups with free aldehydes groups on the neighboring D-glucuronic acid subunits (Jejurikar et al., 2012; Vieira et al., 2008). The broad characteristic band at 3420 cm⁻¹ is associated with the —OH stretching vibration. The stretching vibration of the H-C aldehyde can be seen around 2830 cm⁻¹ (Bock & Gierlinger, 2019). The characteristic peaks at 1631 and 1384 cm⁻¹ indicate the vibration of C⚌N of gelatin, showing the production of Schiff’s base. As displayed in Fig. 1, a small shift is visible in the characteristic peak of OA in the spectrum of OA-GEL cross-linked hydrogel. Furthermore, the aldehyde group band of OA at 1731 cm⁻¹ has vanished, and a new band emerged at 1585 cm⁻¹ assigned to CN double bond (Baniasadi et al., 2016). The new band is because of the Schiff-Base reaction within the amine group of gelatin and the aldehyde group of OA (Sarker et al., 2014), which proved that the cross-linking of OA and GEL occurred. Absorption peak at 566 cm⁻¹ corresponded to the stretching vibration of Si—C bond. The addition of SiC NPs did not change the FTIR spectra of the hydrogels due to the low amounts of SiC in the hydrogel network.

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Fig. 1

FTIR spectrum of gelatin, alginate, oxidized alginate, and the hydrogels.

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3.2 Mechanical properties

Viscoelastic behavior of the injectable hydrogels was assessed utilizing frequency sweep tests to evaluate the dynamic storage modulus (G') and the loss modulus (G“), which are presented in Fig. 2a. Likewise, G' is much higher than G” in all hydrogels, showing that the samples are very elastic. By raising the SiC contents, the magnitude of the moduli is significantly higher, increasing by around 5 orders of magnitude associated with the hydrogel without SiC (Table 1 and Fig. 3a). Increasing the SiC contents can drive to a higher physical crosslinking formation as it promotes mechanical behavior and gel formation within the presence of different reactive elements.

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Fig. 2

Rheological properties of the hydrogels by (a) frequency sweep, (b) time sweep, and (c) temperature sweep.

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Fig. 3

Cross-section morphology of freeze dried hydrogels (a) uncrosslinked, (b) crosslinked, (c) containing 0.25% SiC, (d) 1.0% SiC, (e) 1.5% SiC, and (f) size distribution diagram of the samples.

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Another essential aspect of injectable hydrogel design is the gelation time. The hydrogel formation should be gradually enough so that biomolecules and cells can be incorporated uniformly inside them, and they must remain liquid during surgery and injections. Nevertheless, after injection, they should immediately transform into the gel, therefore, that they cannot abandon the injection part (Epstein-Barash et al., 2012). The time sweep of the crosslinked hydrogel and hydrogel containing 1.5% SiC was performed to check the time of gelation at human body temperature (37 °C). Before gelation, the G“ is higher than the G', which means a liquid form, and the predominant viscoelasticity during the gelation is begun. G' grows quicker than G” by progressing the time, suggesting transition into a solid-like phase with great elasticity (Fig. 2b). The gelation time of crosslinked hydrogel is 135 s, which is sufficient for acting as an injectable hydrogel. The gelation time of hydrogel has decreased to 100 s by adding SiC NPs due to the formation of physical crosslinking of SiC with the hydrogel network. In clinical treatments, quick gelation is aspired to restrict the diffusion of bioactive molecules/cells or hydrogel precursors to the surrounding tissue (Chiu et al., 2009; Choi et al., 2003; Hiemstra et al., 2007).

The temperature sweep analysis was utilized to study the thermoresponsive behavior of the crosslinked hydrogel and hydrogel containing 1.5% SiC. The temperature was boosted from 20 to 50 °C at a rate of 1 °C min⁻¹ (Fig. 2c). G' was lower than G“ at lower temperatures, signifying a liquid-like form. G' grown prominently faster than G” throughout the heating procedure. At greater temperatures, G' enhanced greatly larger than G“, indicating a gel-like form. The G' and G” are nearly parallel straight lines under the gelation temperature, and the amount of G' is more than G“ after the sol/gel transformation. When the temperature is above the gel point, the elastic modulus is greater than the viscous modulus recommending transformation into the solid state. The crossing point of G' and G” is the temperature of gelation (35 °C) and is usually indicated as the sol/gel conversion temperature (Li et al., 2015). The addition of silicon carbide nanoparticles has brought the gelation temperature closer to body temperature (37 °C). The gelation temperature of the hydrogel containing 1.5% SiC was obtained at 37 °C.

3.3 Structure of hydrogels

As one of the main features for cartilage tissue engineering, hydrogels must possess extremely porous and interconnected pore composition to assure a suitable biological environment for cell proliferation, cell attachment, and supplying the nutrient flow passage (Zhu et al., 2009). The pore size and morphological structure depend on the content and molecular mass of the polymer–ceramic hydrogels and the freeze-drying pace (Pekor et al., 2008). The scaffolds were fabricated by the freeze-drying process to achieve extremely porous structures. Fig. 3 reveals the cross-sectional structures of hydrogels without and with crosslinkers (Fig. 3a and 3b) and the hydrogels containing 0.25, 1.0, and 1.5% SiC NPs (Fig. 3(c-e)). The uncrosslinked hydrogel has a porous structure with average pore sizes of about 290 µm (Fig. 3a). The addition of crosslinking agents reduced the pore sizes to be 235 µm (Fig. 3b). As indicated in Fig. 3(c-e), a highly interconnected and an almost uniform porous composition was observed by increasing the amount of SiC. The SEM images reveal that the hydrogels are greatly porous, and the average pores are in the range of 145–200 µm in size, which are essential for cartilage tissue engineering (Zhu et al., 2009). Besides, the average of pores was reduced by increasing the SiC content.

Fig. 4 reveals the EDS spectra of as-prepared hydrogels. As can be seen, the spectrum of crosslinked hydrogel without SiC contains C, O, N, and Na elements. In addition to C, O, N elements, Si element exists in the hydrogels containing SiC, and by increasing the amount of SiC, the intensity of Si peaks are improved. Besides, a small amount of Au is observed in all hydrogels, which is related to the coating of the hydrogels with Au to conduct the samples

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Fig. 4

EDS spectra of freeze dried hydrogels (a) crosslinked, (b) containing 0.25% SiC, (c) 1.0% SiC, and (d) 1.5% SiC.

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3.4 Swelling behavior

Swelling performance is a significant factor in cartilage tissue engineering. Ensuring the passage of nutrients through the scaffold is essential. The uptake ability of PBS defines the scaffold's water absorption capacity, which is an essential parameter that emphasizes the biological activity of the hydrogels. Fig. 5a unveils the swelling ratio of hydrogels after 24 h immersion in PBS at 37 °C. The highest swelling ratio was observed for crosslinked hydrogel without NPs (1060%) due to the large pore sizes, and the hydrogel containing 1.5% SiC NPs showed the lowest swelling ratio (714%) because of smaller pores. Loading SiC into hydrogel decreased the water uptake in the hydrogels (Xiao et al., 2012).

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Fig. 5

(a) Swelling ratio of the hydrogels, (b) in vitro biodegradation after various incubation times in PBS at 37 °C.

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Fig. 5b reveals in vitro degradation of nanocomposite hydrogels over 21-day incubation in PBS buffer solution at 37 °C. In the first five days, all specimens showed an increase in mass, which may be due to swelling. The weight of all hydrogels incubated in PBS solution started to reduce continuously with time, and crosslinked hydrogel revealed the fastest and greatest mass loss between all hydrogels, which completely dissolved after 21 days. Therefore, the degradation rate of hydrogel with higher SiC content (1.5%) was slower than that of lower SiC content hydrogels. The lowest degradation was recognized for higher SiC content, which directly affected the total time needed to destroy the component (Kirchmajer et al., 2013).

3.5 Cell viability and adhesion

Nowadays it is clear that nanoparticles were shown to elicit cellular activities including cell differentiation, proliferation, and attachment in the transplanted tissue. In this investigation, SiC NPs embedded composite was applied to improve the cellular activities of the hydrogels. As indicated in both SEM and phase-contrast microscopy images, the potential ability of nanoparticles to increase cell adhesion is well observed (Fig. 6). Embedded SiC NPs supply a more efficient surface for cell adhesion than hydrogel without SiC. As presented in Fig. 7, the cell viability percentage of scaffolds was determined after 24 and 72 h of incubation. The cell viability was 87.90%, 86.26%, 83.92%, and which resultant in adding 100, 50, and 25 (μg/ml) of scaffold comprising 1.5% SiC NPs after 24 h, sequentially. Besides, the hydrogel without SiC exhibited 81.03%, 82.46%, and 83.59% cell viability after 24 h of incubation. The cell viability was 90.3%, 89.3%, and 87.9%, which is reached by adding 100, 50, and 25 (μg/ml) of the hydrogel comprising 1.5% SiC after 72 h incubation, sequentially. Moreover, the crosslinked scaffold (without SiC) unveiled 74.1%, 78.3%, and 81.0% cell viability after 72 h. Cell adhesion and proliferation significantly improved compared to crosslinked hydrogel without NPs (P-value < 0.05). Therefore, applying more NP concentration was effective to induce a significantly high level of live cells as compared with the blank (crosslinked hydrogel without NPs). OA/GEL composites embedded nanoparticles have showed excellent physical properties for tissue engineering in comparison with hydrogels without nanoparticles. Besides the improvement of physical properties, integrated SiC NPs into the hydrogel also improve cellular interaction, adhesion, viability, and differentiation which could impact the extracellular matrix deposition. Owning to the previous study, combining nanostructures in three-dimensional engineered tissues could decrease inflammation to transplanted tissues by the counterbalance of immune cell viability and cell adhesion (Divieto & Sassi, 2015). By comparing the results obtained for nanocomposites, it was confirmed that SiC NPs elicited the cell growth and viability (Hasan et al., 2018; Li et al., 2020; Smith et al., 2009).

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Fig. 6

Phase-contrast microscopy of hydrogel without SiC (a), and with 1.5% SiC (b), FESEM images of hydrogel without SiC (c), and with 1.5% SiC (d,e).

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Fig. 7

Cell viability of hydrogels (a) after 24 h, and (b) 72 h incubation.

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CRediT authorship contribution statement

Mojgan Ghanbari: Investigation, Formal analysis, Methodology, Writing – original draft, Software. Masoud Salavati-Niasari: Writing – review & editing, Conceptualization, Supervision, Project administration, Visualization, Data curation, Validation, Resources. Fatemeh Mohandes: Data curation, Validation, Resources. Zohreh Firouzi: Methodology.