Design and properties of alginate/gelatin/cellulose nanocrystals interpenetrating polymer network composite hydrogels based on in situ cross-linking

doi:10.21203/rs.3.rs-2215053/v1

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Design and properties of alginate/gelatin/cellulose nanocrystals interpenetrating polymer network composite hydrogels based on in situ cross-linking

https://doi.org/10.21203/rs.3.rs-2215053/v1

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Alginate (Alg) hydrogels have attracted extensive attention in the biomedical field due to their biocompatibility. However, single Alg hydrogels exhibit weak mechanical strength, poor stability and cell adhesion, which severely restricts their biomedical application. For this reason, we designed alginate/gelatin/cellulose nanocrystals (Alg/G/CNCs) composite hydrogels by combining interpenetrating network (IPN) technology, cellulose nanocrystals (CNCs) reinforcement and in situ cross-linking method to improve the functional defects of Alg hydrogels. The structure and properties of the resultant Alg/G/CNCs composite hydrogels were comprehensively evaluated by FT-IR, TGA, XRD, swelling and degradability measurements, and cytocompatibility experiments. Alg/G/CNCs composite hydrogels with regular three-dimensional porous network (3D) structures was successfully fabricated through the ionic cross-linking of alginate and the covalent cross-linking of gelatin, followed by the reinforcement of colloidal cellulose nanocrystals (CNCs) that were prepared by sulfuric acid hydrolysis of microcrystalline cellulose (MCC). The addition of CNCs could generate interaction force with the polymer in the IPN matrix, which was able to regulate the physicochemical properties of the composite hydrogel to a certain extent. Moreover, with the increase of gelatin (G) content, the compressive strength of Alg/G/CNCs composite hydrogels gradually increased, while the swelling property decreased gradually. Meanwhile, Alg/G/CNCs composite hydrogels exhibited good cell adhesion, proliferation and differentiation properties. In particular, Alg/0.5G/CNCs composite hydrogels displayed the best cell proliferation effect, while Alg/2G/CNCs composite hydrogels revealed the most significant cell differentiation effect. Therefore, Alg/G/CNCs composite hydrogels could exhibit good mechanical properties and biocompatibility, which possessed great application potential in the field of tissue engineering.

alginate

gelatin

cellulose nanocrystals

interpenetrating network

in situ cross-linking

composite hydrogels

Hydrogel is a cross-linked polymer three-dimensional (3D) network system, which can absorb a large amount of water or tissue fluid in its network structure while maintaining a certain morphology [1]. And its outstanding feature is that its soft texture is able to fully simulate the extracellular matrix, so it can be widely used in water treatment, drug carriers, wound dressings, cell encapsulation, and tissue engineering [2, 3]. In recent years, many natural polymers have been reported as raw materials for the preparation of hydrogels, including hyaluronic acid, collagen and alginate [4]. Among these natural polymers, alginate, composed of 1,4-linked α-L-guluronic acid (G) and β-D mannuronic acid (M) units with the random sequence distributions, was regarded as an ideal biomaterial for the fabrication of biomedical hydrogels due to its attractive merits including source abundance, good biocompatibility, biodegradability, mild gelation conditions and encapsulation properties [5–9]. In spite of the desirable advantages of alginate for the preparation of hydrogels, it still undergoes the shortage of long-term stability in physiological medium, the limited mechanical properties and the lack of mammalian cell receptors, which severely restricts its biomedical application [10–12].

As another natural polymer, gelatin (GT), a linear polymer derived from collagen hydrolysis, has been widely used in biomedical field due to its low antigenicity, biocompatibility, and biodegradability [13, 14]. As it contains arginine-glycine-aspartic (RGD) polypeptide sequences that aid in cell adhesion, proliferation and differentiation, it is commonly applied to make composites with other materials to improve their biocompatibility [15]. Although gelatin can effectively improve the cell adhesion, proliferation and differentiation of composite materials in tissue engineering applications, gelatin as a tissue engineering material still suffer from fast biodegradation and low mechanical stiffness [16, 17]. For this reason, to overcome the functional defects of a single hydrogel material, people usually choose two or more materials for compounding to prepare a hydrogel with stronger mechanical properties. Those composite hydrogels often achieve desired mechanical properties by adding some reinforcing agents, and the use of large amounts of reinforcing agents usually endows the composites with some unique and excellent properties [8, 18].

Among the various methods for constructing composite hydrogels, the interpenetrating network (IPN) technology is regarded as one of the best approaches to prepare composite hydrogels with high mechanical properties [19]. IPN composite hydrogels are generally composed of at least two polymers through physical or chemical cross-linking to form an interpenetrating polymer network, and this interpenetrating polymer network structure significantly increases the mechanical strength and imparts elasticity to the hydrogel, which exhibits biomechanical properties similar to natural soft tissues [20–22]. Therefore, IPN hydrogels have potential applications in biomedical fields such as tissue engineering. Sun et al. reported a highly stretched SA/PAM composite hydrogel constructed by the IPN approach, which can be stretched to more than 20 times their length and has the fracture energy of approximate 9000 J/m², higher than the single alginate hydrogel or polyacrylamide hydrogel [23]. Fitzgerald et al. prepared the alginate-polyacrylamide composite hydrogel via IPN technology for the muscle tissue, which revealed remarkable mechanical properties and superior biological properties relative to themselves, because of the synergistic effect of IPN composites [24, 25].

In recent years, the addition of nanoparticles to develop nanocomposite hydrogels can effectively improve the mechanical strength of hydrogels [26, 27]. As one of the most abundant biopolymers in nature, cellulose nanocrystals (CNCs) have various characteristics different from traditional materials, such as abundant sources, renewable, good biocompatibility, biodegradability, and barrier properties., high specific surface area, etc. It has been found that CNCs have a positive effect on the gelation mechanism of hydrogels, which can improve the stability of hydrogels and increase the mechanical strength of materials [28–30]. Therefore, CNCs are capable of being used as a reinforcing agent in the preparation of tissue engineering composite hydrogels. Gao et al. [31] enhanced the mechanical properties of gelatin/bioactive glass nanocomposite hydrogels by the addition of CNCs. The experimental results showed that even the incorporation of a small amount of CNCs into the nanocomposite hydrogel could greatly improve the mechanical strength of the nanocomposite hydrogel. Kumar et al. [32] used freeze-drying technology to prepare polyacrylamide/alginate/silica/cellulose nanocrystals composite hydrogels for the first time, which displayed high mechanical strength, thermal stability and good biocompatibility. Shaheen et al. [33] obtained the chitosan/alginate/hydroxyapatite/cellulose nanocrystal composite hydrogel with good porosity and high mechanical strength by freeze-drying technology. This composite scaffold could realize cell adhesion and proliferation, and possessed potential application value in the field of bone tissue engineering.

Additionally, it has to be noted that the ionic crosslinking mode of alginate is one of the main factors affecting its hydrogel structure, morphology and mechanical properties [24]. The commonly used exogenous calcium ion cross-linking mainly relies on the penetration of calcium ions for cross-linking, which is susceptive to cause defects such as uneven cross-linking and hydrogel deformation. However, some studies have reported that in situ cross-linking by the endogenous mode of alginate can effectively improve the mechanical strength and mechanical integrity of hydrogels [25, 34]. Especially, hydroxyapatite (HAP) as the main inorganic component of bone tissue is also an insoluble calcium salt itself which can be used to achieve in situ cross-linking of alginate with D-glucono-δ-lactone (GDL) [35].

In this study, we intended to design alginate/gelatin/cellulose nanocrystals (Alg/G/CNCs) composite hydrogels by combining interpenetrating network technology, CNCs reinforcement and in situ cross-linking. The CNCs with certain carboxyl groups were prepared by the methods of sulfuric acid hydrolysis according to our previous work [36]. The objective of present work is to improve the mechanical properties and biocompatibility of alginate hydrogels, thereby improving the functional defects of alginate hydrogels in biomedical applications. In view of the superior properties of interpenetrating network technology in the construction of composite materials, the Alg/G composite hydrogel constructed by the interpenetrating network method was used as the matrix in this study. The structure and properties of the prepared composite hydrogels were comprehensively evaluated by FT-IR, TGA and XRD analyses, swelling and degradability measurements and cytocompatibility experiments. This comprehensive analysis and resolution of the functional defects of alginate hydrogels in biomedical applications from the perspectives of structure and performance is rarely reported so far.

2.1 Materials and reagents

Sodium alginate (M_W=632500, biochemical grade), gelatin (GT, type B, biochemical grade), hydroxyapatite (HAP, ≥ 97%), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 98%), D-glucono-δ-lactone (GDL, ≥ 99%), N-hydroxysuccinimide (NHS, 98%) and glutaraldehyde (analytical grade, 50% in H₂O) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Microcrystalline cellulose (particles size: 20 µm) was purchased from Sigma-Aldrich company (New York, USA). Sulfuric acid (analytical grade) and anhydrous calcium chloride (analytical grade) were purchased from Xilong Chemical Co., Ltd. (Wuhan, China). Mouse osteoblastic MC3T3-E1 cells were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). DMEM medium, trypsin (0.25% trypsin-EDTA), fetal bovine serum, streptomycin and penicillin were purchased from Amresco Co., Ltd. (USA). Cell Counting Kit-8 (CCK-8) and alkaline phosphatase (ALP) assay kit were bought from Gibco (Thermo Fisher Scientific, USA).

2.2 Preparation and Characterization of CNCs

The synthesis of CNCs was achieved by sulfuric acid hydrolysis of microcrystalline cellulose (MCC) according to our previous methods [36, 37]. The preparation process is shown in Scheme 1. Firstly, 10 g of MCC powder was added to 100 mL of 64 wt % sulfuric acid solution, and then placed in a constant temperature water bath at 45°C. After mechanical stirring for 90 min, the mixture was poured into 800 mL of ultrapure water to quench the hydrolysis reaction. Afterwards, the precipitate was centrifuged at 8000 r/min, then washed and centrifuged with ultrapure water for many times until the upper layer liquid turned light blue. The upper layer light blue liquid was collected and placed in a dialysis bag with the molecular weight cut-off of 8000. Finally, it was dialyzed against ultrapure water for 7 days, sonicated in an ice-water bath for half an hour, and then placed in a refrigerator for later use.

The CNCs suspension concentration was diluted to 0.005% (w/v), and the hydrodynamic diameter and zeta potential of CNCs were measured by a Zetasizer nano ZS90 laser particle size analyzer (Malvern Instrument, UK) at 25°C. The morphology and structure of CNCs were examined by a JSM-7100F scanning electron microscope (SEM, JEOL, Japan), a Dimension Icon atomic force microscope (AFM, Bruker, Germany) and a JEM-2100 transmission electron microscope (TEM, JEOL, Japan).

2.3 Fabrication of Alg/G/CNCs composite hydrogels

As illustrated in Scheme 2, Alg/G/CNCs interpenetrating network composite hydrogels were fabricated by a two-step method using sodium alginate, gelatin (G), and CNCs as raw materials. Firstly, 100 ml of CNCs solution with a concentration of 0.3% (w/v) was prepared, and 137.0 mg of HAP, 1.5 g of gelatin and 1.5 g of sodium alginate were sequentially added under the action of sonication at 45°C and intensely stirred to form Alg/G/CNCs/HAP homogeneous solution. Then, 15 mL of the mixture was injected into a 50 mL beaker with a 30 mL sterile syringe, and 73.0 mg of GDL was added to induce in-situ ion crosslinking of alginate under the action of magnetic stirring. After magnetic stirring for 4 min, a certain volume of viscous solution was sucked out with a 5 mL sterile syringe and quickly transferred to a 12-well tissue culture plate, sealed with plastic wrap to prevent the liquid from volatilizing. The alginate composite hydrogels were obtained after cross-linking at 4°C for 24 h. Furthermore, a mixed cross-linking solution containing 10 mmol/L EDC and 10 mmol/L NHS was prepared in the presence of 0.1% (w/v) CaCl₂ to prevent the alginate composite hydrogels from swelling during the covalent cross-linking process. The gelatin was covalently cross-linked by dipping the alginate composite hydrogels into the EDC/NHS solution. After soaking for 12 h, they were taken out and washed three times with ultrapure water to remove unreacted reagents and by-products. Finally, the product was freeze-dried to obtain the alginate/gelatin/cellulose nanocrystals interpenetrating network composite hydrogels (Alg/G/CNCs (1:1)). For comparison, Alg/0.5G/CNCs (1:0.5) and Alg/2G/CNCs (1:2) composite hydrogels as well as Alg and Alg/G (1:1) in the absence of CNCs were also prepared by the same method. Table 1 listed the compositions of these five different hydrogels.

Table 1

Composition of five different types of hydrogels
Sample	Alginate (w/v)	Gelatin (w/v)	CNCs (w/v)	Final volume
Alg	1.5% (1.5 g)	0% (0 g)	0%	100 mL
Alg/G	1.5% (1.5 g)	1.5% (1.5 g)	0%	100 mL
Alg/0.5G/CNCs	1.5% (1.5 g)	0.75% (0.75 g)	0.3%	100 mL
Alg/G/CNCs	1.5% (1.5 g)	1.5% (1.5 g)	0.3%	100 mL
Alg/2G/CNCs	1.5% (1.5 g)	3% (3 g)	0.3%	100 mL

2.3 Characterization of the composite hydrogels

The porous morphology and pore structure of various composite hydrogels after freeze-drying were observed by a JSM-7100F scanning electron microscope (JEOL, Japan). The functional groups and inter-component interactions of the composite hydrogels were characterized by a Nicolet 6700 FT-IR spectroscopy (Thermo Fisher Scientific, USA). The test samples were prepared by the KBr tablet method. The test conditions were carried out under the conditions of a resolution of 4 cm^− 1, 32 scan times and a scanning wavenumber range of 4000 ~ 400 cm^− 1. XRD analysis of various hydrogel powders was performed using a Rigaku UItima-IV X-ray diffraction (Japan). Cu-Kα radiation was used as the X-ray source, the test voltage and current were 40 kV and 40 mA, respectively, and the XRD patterns of the samples were collected at room temperature at a scanning speed of 1.8°/min over a 2θ range of 5 ~ 65°. Furthermore, a TA Q600 thermogravimetric analyzer (USA) was used to analyze the thermal stability of various composite hydrogels under N₂ atmosphere at a heating rate of 10°C/min with the test range of 25 ~ 500°C. Subsequently, the compression tests of various composite hydrogels were performed using a WDW-1 computer-controlled electronic universal tensile testing machine (Jinan Yinuo Century Testing Instrument Co., Ltd., China). The freeze-dried composite hydrogel was placed under the compression disc, and it was longitudinally compressed perpendicular to the surface of the sample. When the sample was compressed to a strain greater than 60%, it was stopped, and the compression speed was 5 mm/min. The highest linear point on the recorded stress-strain curve was the compressive strength of the sample. Each sample was measured five times in parallel and then averaged.

2.4 Swelling behaviors of the composite hydrogels

The swelling behavior of the composite hydrogels was studied by gravimetric method. After the composite hydrogels were freeze-dried, their weight was recorded as W_{Dry weight}. Then, these composite hydrogels were immersed in PBS solution (pH = 7.4) at 37°C. At the set time interval (0.5, 1, 2, 4, 7, 25 h), these samples were taken out. The excess PBS solution on the surface of the sample was wiped off with filter paper, and then the wet samples were weighed as W_{Wet weight}. The swelling rate of each sample at each time interval was calculated according to the following Eq. (1). Three parallel determinations were performed for each sample, and the average value was taken.

S$\text{welling ratio=}\frac{{\text{W}}_{\text{Wet weight}}\text{-}{\text{W}}_{\text{Dry weight}}}{{\text{W}}_{\text{Dry weight}}}\text{×100}\text{\%}$ (1)

2.5 In vitro degradation study of the composite hydrogels

A 14-day degradability study was performed by measuring the weight change of the composite hydrogels after incubation in simulated body fluids (SBF) that was prepared based on previous method [38]. The freeze-dried composite hydrogel was weighed and recorded as W_{Initial weight}. Then, it was immersed in pH 7.4 SBF solution at 37°C for a period of 2 ~ 14 d. At the set time interval, it was taken out, washed with ultrapure water for several times and freeze-dried to record its weight as W_{Dry weight}. Similarly, each sample was subjected to three parallel experiments to obtain the average value, and the biodegradation ratio of the sample was calculated by the following Eq. (2).

$$\text{Degradation ratio=}\frac{{\text{W}}_{\text{Initial weight}}\text{-}{\text{W}}_{\text{Dry weight}}}{{\text{W}}_{\text{Initial weight}}}\text{×100\%}$$

2.6 Cytocompatibility measurement of the composite hydrogels

Mouse osteoblast MC3T3-E1 cells, which can simulate the growth process of osteoblasts, were selected as the model cells and cultured in the medium composed of 90% DMEM, 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin to test the cytocompatibility of the composite hydrogel. The composite hydrogels with a diameter of 14 mm and a height of 3 mm were prepared and irradiated with cobalt 60 at an intensity of 8 kGy, and then placed in the 24-well tissue culture plates. Subsequently, MC3T3-E1 cells were seeded on the composite hydrogel at a density of 5×10⁴ per well, while the same cells were seeded on the material-free tissue culture plate as the blank control. Meanwhile, the culture medium was then supplemented to maintain the total amount of medium up to 500 µL per well. And the cells were cultured in an incubator containing 5% CO₂, 95% air, and 100% relative humidity at 37°C, and the medium was changed every 2 days.

After 2 days of culture, the cells were fixed by glutaraldehyde chemical crosslinking and freeze-dried, then the adhesion and spreading of the cells on the composite hydrogel were observed by scanning electron microscopy (SEM). Afterwards, the Cell counting Kit-8 (CCK-8) assay was used to detect the proliferation activity of the cells on the composite hydrogel according to our previous work [39]. After 2 and 7 days of incubation, 50 µL of CCK-8 reagent was added to 500 µL of medium in each well of 24-well culture plates, and the culture was incubated for 4 h at 37°C in an incubator. Finally, 100 µL of solution per well was transferred to a 96-well plate. The OD value was measured by a Bio-rad X-mark microplate reader (USA) at a wavelength of 450 nm.

The osteogenic differentiation activity of the cells on the composite hydrogel was examined by the alkaline phosphatase (ALP) kit. The culture medium was supplemented with 0.2 µmol/L dexamethasone and 8.0 mmol/L β-glycerophosphate to promote cell differentiation. After incubation for 7 d, the cells were washed with PBS and disrupted in a 0.2% (w/w) Triton X-100 ice bath for 30 min, followed by centrifugation at 12000 r/min for 5 min at 4 ° C. Furthermore, 50 µL of centrifugation supernatant was placed on a 96-well plate, then 50 µL of ALP staining reagent was added and fully mixed. After incubation at 37 ° C for 30 min, the OD value was determined by X-mark microplate reader at 405 nm wavelength, which could be used as an indicator of the relative activity of ALP.

3.1 Colloidal properties of CNCs

The particle size distribution of CNCs aqueous dispersions was investigated by dynamic light scattering (DLS), showing a bimodal distribution, as shown in Fig. 1a, in which the peak with larger peak area corresponded to an average hydrodynamic diameter of 89.2 nm (PDI = 0.23). The appearance of this peak indicated the presence of aggregates, which was attributed to the large specific surface area characteristic of CNCs, and they may be caused by the presence of a large number of hydroxyl groups in CNCs through hydrogen bonding association [40]. At the same time, it can be observed from Fig. 2b that the zeta potential of the CNCs aqueous dispersion was − 37.3 mV, indicating that during the sulfuric acid hydrolysis of MCC, the hydroxyl groups on the cellulose polymer molecular chain underwent an esterification reaction with sulfuric acid groups (Fig. 1c), making the CNCs negatively charged. In addition, the stability of CNCs suspension was higher than that of MCC solution observed from Figs. 2a and 2b, which may be due to the negative charge on the surface of CNCs. This made the double-layer electrostatic repulsion between CNCs and realized the stable existence of CNCs in aqueous solution, which also played an important role in preventing the aggregation of nanoparticles [41].

From Figs. 2a and 2b, it can be also observed that the CNCs dispersion was light blue, while the MCC dispersion was white. As the negative charge of CNCs in the suspension produced the repulsive forces, no precipitate was formed after long-term storage, so the CNCs suspension revealed good stability. SEM images of MCC showed irregular shapes with the size ranging from 8 to 53 µm (Fig. 2c). Nonetheless, the CNCs prepared by sulfuric acid hydrolysis of the amorphous region of MCC exhibited "needle-like" or "rod-like" morphology as observed by SEM, TEM and AFM (Figs. 2d ~ 2f). However, it can be also observed that the CNCs exhibited a layered structure in both SEM images or TEM images and AFM images, which may be ascribed to the existence of hydrogen bonding between the nanofibers.

3.2 Morphologies and pore structure of the composite hydrogels

Figure 3 presents the macroscopic morphologies of different types of the composite hydrogels. It can be observed that all of hydrogels exhibited good 3D morphologies. With the addition of CNCs, the transparency of the Alg/G/CNCs composite hydrogel decreased, showing milky white and light blue. When the content of CNCs was constant, the Alg/G/CNCs composite hydrogel appeared milky white with the increase of gelatin content. The macroscopic morphologies and SEM images of internal pores of various hydrogels after freeze drying were shown in Fig. 4. From Fig. 4a, it was obviously that the hydrogels still maintained good cylindrical morphologies after freeze-drying. This was mainly due to the ionic cross-linking of alginate and the covalent cross-linking of gelatin under the action of HAP/GDL endogenous ionic cross-linking and EDC/NHS covalent cross-linking, thereby constructing an interpenetrating network system.

Moreover, the various hydrogels after freeze-drying possessed the regular three-dimensional porous network (3D) structures, which was attributed to the sublimation of ice crystals from the hydrogel during the freeze-drying process. It is known in the biomedical field that a good 3D porous structure for hydrogels is beneficial for cell growth and delivery of nutrients and metabolic wastes [39]. Compared with Alg hydrogels, Alg/G composite hydrogels had smooth pore walls with different pore sizes. Meanwhile, with the addition of CNCs to the Alg/GT matrix to form Alg/G/CNCs, it can be observed that the addition of CNCs made the pore size of the composite hydrogel more uniform and the pore walls seemed to become rough. The reason may be that the addition of CNCs strengthened the pore structure of the hydrogels, forming ordered ice crystals and making the pore size more uniform, which was beneficial for cell adhesion and extracellular matrix production [42]. Meanwhile, the addition of CNCs also reduced the pore size of the composite hydrogels, which was probably due to the fact that CNCs themselves acted as a filler, so that the gaps in the interpenetrating network formed by Alg/G were filled by CNCs, thereby reducing the pore size. In addition, the comparison between Figs. 4d ~ 4f showed that with the increase of gelatin content, the pore wall thickened, the pore structure decreased, and the porosity decreased. Moreover, these nanocomposites did not show obvious agglomeration, which indicated that the CNCs were uniformly dispersed in these alginate/gelatin composite matrices. Compared with Alg/0.5G/CNCs composite hydrogels, Alg/G/CNCs composite hydrogels had a smaller pore size, which may be on account of the filling effect of gelatin and the intermolecular interaction that reduced the pore size. Meanwhile, Alg/2G/CNCs composite hydrogels possessed a larger pore size, because the composite hydrogel contained a large amount of gelatin, and the covalent cross-linking of gelatin under the action of EDC/NHS made them have strong resistance to deformability, resulting in a larger pore size.

3.3 FT-IR analysis

The FT-IR spectra of SA, G and CNCs were displayed in Fig. 5a. SA exhibited its characteristic absorption peaks at 3427 and 2926 cm^− 1 which were attributed to O-H and C-H stretching vibrations, respectively, while the peaks at 1621 and 1417 cm^− 1 were the asymmetric and symmetric stretching vibration absorption peaks of COO⁻ [43]. The peaks at 1095 and 1033 cm^− 1 may be due to the C-O and C-C stretching vibrations on the pyran ring [44, 45]. The characteristic peaks of CNCs at 3410 and 2903 cm^− 1 were assigned to the -OH and C-H stretching vibration absorption peaks, and the absorption peak at 1163 cm^− 1 was related to the C-O-C stretching vibration. Moreover, the absorption peaks at 1113 and 1059 cm^− 1 were ascribed to the C–O stretching vibrations of primary and secondary hydroxyl groups, respectively [46]. G had a broad absorption peak at 3325 cm^− 1, which was owing to the presence of -OH and -NH₂ in the molecular structure. The peak at 3079 cm^− 1 was owing to the N-H stretching vibration, while the characteristic peak at 2939 cm^− 1 was due to the C-H stretching vibration. The absorption peaks at 1653, 1545, and 1239 cm^− 1 corresponded to the amide I band (υ_C=O and υ_C−N), the amide II band (mainly δ_N−H) and the amide III band (υ_C−N), respectively [45, 47].

The FT-IR spectra of Alg, Alg/G, Alg/0.5G/CNCs, Alg/G/CNCs and Alg/2G/CNCs composite hydrogels were shown in Fig. 5b. The Alg/G composite hydrogels showed typical characteristic peaks of SA and G. However, compared with G, the intensity of the amide II band in the Alg/G composite hydrogel decreased, which may be due to the covalent cross-linking of G in the EDC/NHS system, and the formation of amide bonds after cross-linking, proving the interaction of this group in the cross-linking reaction. Meanwhile, compared with the Alg hydrogel, the Alg/G composite hydrogel appeared new absorption peaks at 1637 and 1560 cm^− 1 corresponding to the stretching vibration of C = O and N-H, respectively, indicating that G was successfully dispersed into the alginate matrix, and the IPN composite hydrogel was formed under the action of the cross-linking agent [45]. In addition, after HAP/GDL physical cross-linking and EDC/NHS covalent cross-linking to form IPN composite hydrogels, the absorption peaks of various composite hydrogels at 1545 and 1621 cm^− 1 shifted to the high wavenumber direction to 1560 and 1653 cm^− 1, indicating that the interaction between the components of the IPN hydrogel was enhanced, proving that the interaction force was generated between these groups [48]. Furthermore, compared with the Alg/G composite hydrogels, the addition of CNCs made the Alg/G/CNCs composite hydrogels display new absorption peaks at 1163 and 1059 cm^− 1, which were attributed to the characteristic peaks of CNCs, indicating that the CNCs as fillers had been uniformly dispersed in the Alg/G matrix. Meanwhile, similar results were observed in the FT-IR spectra of Alg/0.5G/CNCs and Alg/2G/CNCs composite hydrogels. Moreover, the addition of CNCs broadened the peak at 3600 − 2900 cm^− 1, implying that strong hydrogen bonds were generated between SA, G and CNCs molecular chains after CNCs were added to the Alg/G matrix [23, 48].

3.4 XRD analysis

The crystal structures of SA, MCC, and as-prepared CNCs were quantitatively analyzed by XRD, and the results were shown in Fig. 6a. It can be observed that the positions of the crystal diffraction peaks of MCC and CNCs were basically similar, and there were crystal diffraction peaks at 2θ = 14.9°, 16.7°, 22.6° and 34.6°, which were assigned to (1$\stackrel{-}{1}$0), (110), (020) and (004) crystal planes, respectively, indicating that the hydrolysis of MCC by sulfuric acid to prepare CNCs did not change the structure of cellulose crystals [37]. SA revealed two weak peaks at 2θ = 13.7° and 21.6°, indicating its amorphous crystalline property [10]. Meanwhile, the XRD patterns of each composite hydrogel are shown in Fig. 6b. The Alg/G composite hydrogel showed two broad peaks at 2θ = 14.8° and 22°, and these characteristic peaks belonged to the triple helix crystal structure of calcium alginate and G, respectively [49]. It was observed that the peak intensity at 14.8° was weakened, and this change may be due to the formation of the IPN composite matrix or the formation of intermolecular interactions between SA and G under the action of cross-linking, which destroyed the crystal structure of calcium alginate [43]. In addition, the Alg/0.5G/CNCs, Alg/G/CNCs and Alg/2G/CNCs composite hydrogels exhibited the characteristic peaks of CNCs at around 2θ = 22° which were caused by α-cellulose and semi-cellulose from the (200) plane reflection of the cellulose lattice [50], indicating that CNCs were present in these IPN composite hydrogel matrices. However, the position of this characteristic peak was slightly shifted, and the crystallinity difference may be resulted from the intermolecular interactions.

3.5 Thermal stability analysis

Figure 7 shows the thermogravimetric curves of different hydrogels measured at a heating rate of 10°C/min under N₂ atmosphere. It was observed from the TGA curve that the weight loss rate of each sample was in the range of 58%~65% at 500°C. From the DTG curve, it can be seen that, except for the Alg hydrogel indicating two main weight loss stages, all of other IPN composite hydrogels revealed three main degradation stages. The highest degradation temperatures in the first degradation stage of Alg, Alg/G and Alg/0.5G/CNCs, Alg/G/CNCs and Alg/2G/CNCs composite hydrogels were 55.8 ℃, 53.5 ℃, 51 ℃, 46.5 ℃ and 66.5 ℃, respectively, which was mainly ascribed to the removal of physical adsorption water [10]. While the second degradation stage was between 200 and 300°C and the significant degradation occurred during this stage, which may be attributed to the chain scission and ring-opening reactions of alginate [39]. Compared with Alg hydrogel, IPN composite hydrogels including Alg/G and Alg/0.5G/CNCs, Alg/G/CNCs and Alg/2G/CNCs possessed a third degradation stage between 300 ℃ and 400 ℃. This may be due to the scission of the polymer molecular chain and the scission of peptide bonds (gelatin degradation) [51]. At the same time, it is also shown that the formation of interpenetrating network structure between G and SA or CNCs under the action of cross-linking improved the thermal stability of the composite hydrogels. With the increase of G content, Alg/2G/CNCs showed the highest thermal stability performance, which may be owing to electrostatic interaction or intermolecular hydrogen bonding between G and SA or CNCs, or due to the formation of tough and tight network structures through the crosslinking effect, which helped to improve the thermal stability performance of the composite hydrogels [36, 38].

3.6 Mechanical property analysis

As a biomedical material, the composite hydrogel should have suitable mechanical strength to support cell growth and support tissue load [35]. The compressive strength of the composite hydrogel was measured by a universal material testing machine with the results shown in Fig. 8a. Although the homogeneous Alg hydrogel was prepared by the endogenous ionic crosslinking method, its compressive strength was still only 0.109 MPa. However, the compressive strength of the Alg/G composite hydrogels prepared by the interpenetrating network technology was significantly improved to 0.701 MPa, which was mainly attributed to the covalent crosslinking of GT by EDC/NHS and the construction of the interpenetrating polymer network, which enhanced the mechanical properties of alginate ionically crosslinked networks. Compared with the Alg/G composite hydrogel, the compressive strength of the Alg/G/CNCs composite hydrogel increased to 0.982 MPa, indicating that the addition of CNCs enhanced the mechanical strength of the Alg/G/CNCs composite hydrogel. This may be attributed to the fact that the addition of CNCs to the IPN hydrogel matrix as a reinforcing agent led to its formation of the denser and highly entangled networks, thereby increasing the compressive strength [52]. Moreover, it further indicated that the mechanical strength of the composite hydrogels depended not only on the reinforcement of the nano-fillers, but also on the interaction with the polymer matrixes [53, 54]. For the Alg/G/CNCs series composite hydrogels, it was found that the compressive strength of the Alg/2G/CNCs composite hydrogels with the highest gelatin content reached a maximum of 1.750 MPa, which was attributed to the covalent cross-linking effect of a large amount of G in the EDC/NHS to form the network structures with higher crosslink density and more entanglement. Another notable explanation was that after G was added to the alginate matrixes, their mechanical properties were greatly improved due to the filling effect of G and the intermolecular interaction force, thereby indicating the better mechanical strength.

3.7 Swelling behavior analysis

Swellability is an important indicator for evaluating biomedical materials due to its important role in the transfer of nutrients, metabolic waste, tissue fluid absorption and cell penetration [55–57]. As shown in Fig. 8b, the Alg hydrogel exhibited uncontrollable swelling behavior due to the strong hydrophilicity of SA, which reached about 29 times its own weight after half an hour, and excessive swelling would destroy its mechanical integrity, which was not conducive to its biological application. However, under the action of EDC/NHS cross-linking agent, the free amine and carboxyl groups in the Alg/G IPN composite hydrogel matrix formed a network structure through covalent cross-linking, which reduced the number of free hydrophilic groups. As a result, the Alg/G composite hydrogel exhibited controllable swelling behavior. Therefore, the IPN structure formed between the polymer molecules in the composite hydrogel Alg/G matrix significantly improved the uncontrollable swelling behavior of the Alg hydrogel. At the same time, it can be observed that the Alg/G/CNCs composite hydrogel exhibited lower swelling than the Alg/G composite hydrogel, which may be due to the addition of CNCs that enhanced the interaction between the SA and G to form the denser network structures. In addition, CNCs as fillers partially occupy the network space of the hydrogel, resulting in the hydrogel with smaller pore size, thereby reducing the water absorption of the Alg/G/CNCs composite hydrogel [42, 52, 58], and further controlling the swelling of the IPN composite hydrogel. The above analysis results implied that the construction of IPN and the addition of CNCs could effectively regulate the swelling behavior of alginate hydrogels.

3.8 In vitro degradability analysis

In order to more truly reflect the degradation behavior of the composite hydrogels in the human body, these composite hydrogels were immersed in the SBF solution at 37°C to study their degradation rate within 14 days. As shown in Fig. 9, it was found that in SBF solution, with the increase of G content in the composite hydrogel component, the degradation rate increased significantly, which was attributed to the sensitivity of G to water. This result was due to the hydrolysis mechanism, that is, as G began to be removed from the IPN hydrogel, the space it occupied became void, which further promoted the decomposition of the rest of the sample [55, 59]. Furthermore, the composite hydrogels containing CNCs were found to have a lower degradation rate, which may be ascribed to their uniform dispersion and hydrophobicity [60], or it may be that CNCs enhanced the interaction between the polymer matrixes, thereby improving the stability of the composite hydrogel in SBF solution. Additionally, crystallinity and hydrophilicity were also important factors affecting the degradation rate. Since the crystalline regions of CNCs were more resistant to hydrolysis, their crystallinity and crystal size would reduce the degradation rate of the polymer matrix [61].

3.9 In vitro cytocompatibility of the composite hydrogels

In vitro cytocompatibility measurement were performed with mouse osteogenic MC3T3-E1 cells to examine the in vitro biocompatibility of the composite hydrogels to evaluate their potential applications in the biomedical field. As shown in Fig. 10, after 2 d of culture, MC3T3-E1 cells exhibited good attachment ability on these composite hydrogels. At the same time, cells were found to proliferate at the pores of the composite hydrogels, and these results suggested that the porous network structures of the composite hydrogels could provide a favorable 3D environment for cell attachment and proliferation. However, due to the lack of cell recognition sites in SA, there was less cell adhesion on the surface of Alg hydrogels, while G had the RGD polypeptide sequence, which could significantly enhance the adhesion of cells to the Alg/N composite hydrogel [62]. At the same time, it was observed that the cell adhesion on the Alg/0.5G/CNCs and Alg/G/CNCs composite hydrogels was significantly better than other hydrogels. These results may be due to their pore size suitable for cell growth, and the mutual bonding between CNCs and the polymer matrixes to form a highly entangled network on the pore wall, making the nanoscale roughness on the pore wall, which was conducive to cell adhesion and the production of extracellular matrix [42, 63].

The CCK-8 kit was used to detect the in vitro cytotoxicity of the composite hydrogels with the results shown in Fig. 11a. Taking the tissue culture plate as the control group, after culturing for 2 d, the absorbance of the composite hydrogels in each group was higher than that of the control group, indicating that these composite hydrogels had good biocompatibility. More interestingly, when cultured for 5 d, in contrast, the Alg/0.5G/CNCs composite hydrogel containing G and CNCs with relatively large pore size had the highest absorbance, indicating that its cell viability was higher than other experimental groups. This result showed that appropriate G content, pore size and CNCs content had positive effects on the cell proliferation [64, 65].

Alkaline phosphatase (ALP) is an important biomarker of early osteoblast differentiation. After 7 d of culture, the ALP activity of MC3T3-E1 cells on the composite hydrogel was improved compared with the control group, as shown in Fig. 11b. Meanwhile, it was observed that the ALP activity of Alg/G, Alg/G/CNCs and Alg/2G/CNCs composite hydrogels was significantly higher than that of Alg hydrogel and Alg/0.5G/CNCs composite hydrogels. Among them, Alg hydrogel showed low ALP activity due to its poor cell adhesion, and Alg/0.5G/CNCs composite hydrogels with lower G content exhibited similar ALP activity to Alg hydrogel. On the contrary, Alg/2G/CNCs composite hydrogels exhibited the highest ALP activity during cell differentiation. This result may be related to the G content in the composite hydrogel, and the addition of an appropriate amount of G could promote cell differentiation. Furthermore, it is found that the ALP activity on the Alg/G/CNCs composite hydrogel was significantly higher than that of the Alg/G composite hydrogel, which may be ascribed to the CNCs contained in the composite hydrogel, indicating that an appropriate amount of CNCs was beneficial to promote cell differentiation [38, 39]. Therefore, the above results indicated that various physical and biochemical factors of biomaterials played a crucial role in the survival, proliferation and differentiation of cells.

In this paper, aiming at the functional defects of alginate hydrogels in the field of biomedical applications, Alg/G/CNCs composite hydrogels were designed and fabricated by IPN technology, CNCs reinforcement and in situ cross-linking approach. Meanwhile, the structure and properties of the prepared Alg/G/CNCs composite hydrogels were comprehensively characterized by FT-IR, TGA, XRD, swelling and degradability measurements, and cytocompatibility experiments. The characterization results showed the reinforcement of colloidal CNCs was successfully prepared by sulfuric acid hydrolysis of MCC. The addition of CNCs could generate interaction force with the polymer in the IPN matrix, which was able to regulate the physicochemical properties of the composite hydrogel to a certain extent. Moreover, with the increase of gelatin (G) content, the compressive strength of Alg/G/CNCs composite hydrogels gradually increased, while the swelling property decreased gradually. Meanwhile, Alg/G/CNCs composite hydrogels exhibited good cell adhesion, proliferation and differentiation properties. In particular, Alg/0.5G/CNCs composite hydrogels displayed the best cell proliferation effect, while Alg/2G/CNCs composite hydrogels revealedthe most significant cell differentiation effect. Therefore, Alg/G/CNCs composite hydrogels based on the interpenetrating network technology could improve the physicochemical properties of Alg hydrogels and enhance their applications in the biomedical field.

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Consent for publication

All authors have read and approve the publication of the article.

Availability of data and materials

The raw/processed data and materials required to reproduce these findings cannot be shared at this time due to technical or time limitations.

Competing interests

There are no conflicts to declare.

Funding

This work receives the financial support from the National Natural Science Foundation of China (51963009) and the Natural Science Foundation of Hainan Province (220MS035).

Authors’ contributions

Z.L., H.W. and X.C. wrote the original manuscript text, Y.L., D.L., and X.S. prepared figures 1-11, and H.Y. and Q.L. supervised the manuscript. All authors reviewed the manuscript.

Acknowledgments

We gratefully thank the financial support from the National Natural Science Foundation of China (51963009), the Natural Science Foundation of Hainan Province (220MS035), the Innovation and Scientific Research Projects for Undergraduates of Hainan Province, the Open Fund for Innovation and Entrepreneurship of College Students of Hainan Normal University (hscy2022-14) and the Innovation and Scientific Research Projects for Undergraduates of Hainan Normal University (hscx2022-30).

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Scheme 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

scheme1.png
Scheme 1. Preparation process of CNCs from MCC by sulfuric acid hydrolysis.
scheme2.png
Scheme 2. Schematic diagram of the formation of Alg/G/CNCs interpenetrating network composite hydrogels through the in situ cross-linking method.

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Design and properties of alginate/gelatin/cellulose nanocrystals interpenetrating polymer network composite hydrogels based on in situ cross-linking

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Abstract

Figures

1. Introduction

2. Experimental Procedure

2.1 Materials and reagents

2.2 Preparation and Characterization of CNCs

2.3 Fabrication of Alg/G/CNCs composite hydrogels

2.3 Characterization of the composite hydrogels

2.4 Swelling behaviors of the composite hydrogels

2.5 In vitro degradation study of the composite hydrogels

2.6 Cytocompatibility measurement of the composite hydrogels

3. Results And Discussion

3.1 Colloidal properties of CNCs

3.2 Morphologies and pore structure of the composite hydrogels

3.3 FT-IR analysis

3.4 XRD analysis

3.5 Thermal stability analysis

3.6 Mechanical property analysis

3.7 Swelling behavior analysis

3.8 In vitro degradability analysis

3.9 In vitro cytocompatibility of the composite hydrogels

4. Conclusions

Declarations

References

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Supplementary Files

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