# Synthesis of Biomaterial-Based Hydrogels Reinforced with Cellulose Nanocrystals for Biomedical Applications

Authors: Pavan Kumar Dara; Mahadevan Raghavankutty; Karthik Deekonda; Anil Kumar Vemu; Visnuvinayagam Sivam; Suseela Mathew; Anandan Rangasamy; Ravishankar Chandragiri Nagarajarao; Senthilkumar Subramanian
Journal: International Journal of Polymer Science
 (2021)

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## Abstract

Cellulose nanocrystals (CNC) were prepared by formic acid hydrolysis and TEMPO- (2,2,6,6-tetramethyl-piperidine-1-oxyl-) mediated oxidation. The prepared CNCs were reinforced into biopolymers chitosan (CHI), alginate (ALG), and gelatin (GEL) to obtain “CNC-ALG-GEL” and “CNC-CHI-GEL” hydrogels. The synthesized hydrogels were characterized for physicochemical, thermal, and structural characterization using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), thermal gravity analysis (TGA), and X-ray diffraction (XRD) analyses. Notably, the reinforcement of CNC has not altered the molecular structure of a biopolymer as revealed by FT-IR analysis. The hydrogels reinforced with CNC have shown better thermal stability and miscibility as revealed by thermal gravity analysis. The physicochemical, thermal, and structural characterization revealed the chemical interaction and electrostatic attraction between the CNC and biopolymers. The biocompatibility was investigated by evaluating the viability of theL929 fibroblast cell, which represents good biocompatibility and nontoxic nature. These hydrogels could be implemented in therapeutic biomedical research and regenerative medicinal applications.


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## Body

## 1. Introduction

Cellulose is one of the naturally occurring renewable biopolymers. It plays a key position as an abundant raw organic material which is capable of meeting the demands for green and biobased products [1]. Nanocellulose materials such as cellulose nanocrystals (CNC) and cellulose nanofibrils (CNF) prepared from cellulose have gained tremendous attention in tissue engineering, biomedical applications, and regenerative medicine due to their superior biocompatibility and favourable rheological and biological active properties [2].CNCs are rod-shaped-like nanoparticles that can be obtained from different types of diversified rich cellulose bioresources such as rice husk, wheat straw, cotton, and wood pulp [3]. It possesses a diameter of 2-20 nm with a length of 100-500 nm. Generally, CNC can be extracted from cellulose biomaterial through acid hydrolysis using acids such as sulfuric acid, formic acid, hydrochloric acid, nitric acid, and phosphoric acid. Nevertheless, among all the acids, formic acid is mostly preferable due to its certain desirable abilities like being less corrosive, reusable, and easily recoverable and having a low boiling point [4]. Due to minimal cytotoxicity and immunogenicity properties, CNCs have the ability to be utilised as an extracellular matrix in tissue culture medium which enhances cell growth and cell proliferation [5].The immense capacity of CNC for surface charge modification allows researchers to improve their hydrophilicity and stability and thereby enhance the bioavailability of CNC [6]. The nanocrystals with high carboxyl content have been found to be suitable for drug delivery applications [7].Over the past years, many researchers have attempted to develop extraction methods for the preparation of CNC. Many researchers have developed biopolymer-based materials such as hydrogels and scaffolds for wound dressing, biomedical applications, and therapeutic research. Several research studies have been extensively made for the incorporation/fabrication/injection of CNC into biopolymers such as alginate, chitosan, gelatin, polyvinyl alcohol, and hydroxyapatite [2, 5, 8–11]. Jeddi and Mahkam synthesized nanocarboxymethyl cellulose from CNC to incorporate in bilayer alginate-chitosan hydrogel beads and successfully use as a carrier for the controlled delivery of dexamethasone [12]. In another study, Ntoutoume et al. demonstrated that the curcumin and cyclodextrin complexes with CNC have an antiproliferative effect on colorectal and prostatic cancer cell lines [13]. Further, according to Bertsch et al., the presence of CNC in the polymer complex enhanced the physiological and rheological characteristics of the wound dressing material [14]. Due to the outstanding mechanical properties of CNC, innovative research could evolve for the development of artificial organs such as blood vessels, nuclear pulposus, and soft tissues by the application of CNC [15]. With this rationale, the aim of the present research investigation was to synthesize the CNC from cellulose by formic acid hydrolysis and reinforcement of synthesized CNC into the biopolymers chitosan, alginate, and gelatin for the preparation of hydrogels. The physicochemical, structural rheological, and cytotoxicity properties were assessed that play an important role in biomedical and tissue engineering applications.


## 2. Materials and Methods

Low molecular weight chitosan (degree of deacetylation with 75–85%, 50–190 kDa), sodium alginate, ferric (III) chloride (FeCl3), formic acid, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and sodium bromide (NaBr) were purchased from Sigma, St. Louis, USA. All other chemicals and reagents used in the present study were of either analytical grade (AR) or guaranteed grade (GR).


### 2.1. Preparation of Cellulose Nanocrystals

CNC was prepared by formic acid hydrolysis according to the following method as described by Du et al. [4]. Briefly, 3 gm of cellulose was added in 100 ml of 0.015 M FeCl3 containing 90 ml formic acid in a spherical flask. The flask was kept at 95°C for 4 h under stirring using a magnetic stirrer (Heidolph Instruments, GmbH and Co., Germany). The flask was cooled down to room temperature followed by centrifugation at 8000 rpm for 10 min using centrifuge (Sorvall Legend XTR, Thermo Fisher Scientific, New Hampshire, USA). The obtained white residue was washed repeatedly with distilled water until pH 6 was achieved. After separation of the supernatant, the white residue nanocrystals were freeze dried at −40°C for 72 h using a freeze dryer (Lark, Tamil Nadu, India) and stored at 4°C until further analysis.The obtained white residue nanocrystals were subjected to cationic modification using TEMPO and sodium hypochlorite (NaOCl) according to the following method as described by Akhlaghi et al. [6]. 1.5 gm of nanocrystals was added to 100 ml of deionised water and sonicated for 15 min. 20 mg of TEMPO and 400 mg of NaBr were added to the CNC suspension. The pH of the CNC suspension was adjusted to 10 using 1 M NaOH. The oxidation reaction was initiated by adding 10 ml of 13% NaOCl to the CNC suspension under gentle stirring continuously for 3 h, simultaneously maintaining constant pH 10. An aliquot of 10 ml of methanol was added to quench the reaction, and pH was adjusted to 7 using 1 M HCl. Finally, the oxidised nanocrystals were dialysed against deionised water using 14 kDa dialysis membranes (Sigma, St. Louis, USA). The resultant freeze-dried cellulose nanocrystals were designated as “CNC.”


### 2.2. Preparation of Hydrogels Reinforced with CNC

The biomaterial-based hydrogels reinforced with CNC were prepared according to the following method described by Wang et al. [16], with slight modifications. A 1% CNC (20 ml) was dispersed in 40 ml of 2% alginate and 2% chitosan homogenous solutions separately via sonication for 15 min and designated as “CNC-ALG” and “CNC-CHI,” respectively. Further, a 2% gelatin was dissolved in 40 ml of deionised water with 20 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and stirred continuously for 24 h at room temperature. Fish gelatin was used in the present study, which was prepared according to the following method as described by Dara et al. [17]. This gelatin solution with EDC was added to CNC-ALG and CNC-CHI solutions followed by the addition of 0.5 ml of 50 mM ZnSO4. The suspension was stirred continuously for 6 h at room temperature for ionic cross-linking. Finally, the obtained hydrogels were rinsed with 1x phosphate buffer saline (PBS) and designated as “CNC-ALG-GEL” and “CNC-CHI-GEL,” respectively. The hydrogels were stored at 4°C until further analysis. The procedure for the preparation of hydrogels is shown in Figure 1.Figure 1
Procedure for the preparation of hydrogels reinforced with CNC.


### 2.3. Particle Size Distribution and Zeta Potential Analysis

The particle size distribution (PSD) and zeta potential of CNC and hydrogel samples were determined by dynamic light scattering analyses (DLS) using a Zetasizer Nano Series (Malvern, Worcestershire, UK). For PSD analysis, the CNC and hydrogel samples were loaded into a quartz cuvette with 1 cm path-length and were subjected to DLS measurement with a detection angle of 90° at25±0.1°C. The Z-average hydrodynamic diameters and polydispersity index (PDI) of hydrogels were determined and recorded based on the scattering intensity. For analysing the zeta potential, the CNC and hydrogel samples were filtered using a 0.45 μm syringe filter membrane to remove large particles and measured with an angle of 15° at 25°C.


### 2.4. FT-IR Spectra and SEM Morphology

The FT-IR spectra of CNC and hydrogels were analysed using a FT-IR spectrometer (Bruker Optik GmBH, Germany), in the region of 4000 cm-1–500 cm-1 wave numbers at 4 cm-1 resolution. The surface morphology was examined using scanning electron microscopy (SEM JSM, Japan), and samples were observed at an accelerated 15 kV voltage.


### 2.5. X-Ray Diffraction (XRD) Pattern

X-ray powder diffraction patterns of CNC and hydrogels were recorded using an Analytical X’Pert Pro X-ray diffractometer. The X-ray source was Cu-Kα radiation (40 kV, 80 mA). The XRD patterns of samples were taken in the 2θ range of 10°–80° in a fixed time mode at room temperature at a scanning rate of 4° min-1.


### 2.6. Thermal Characteristics

Prior to the analyses, the CNC and hydrogel samples were conditioned at 25°C and 53% relative humidity. DTG (derivative thermogravimetry) and DTA (differential thermal analysis) of hydrogels were analysed using a thermogravimetric analyser (Seiko Exstar 6300, Italy). Samples were heated from 600°C, at 20°C/min heating rate, using nitrogen flow (250 ml/min). TheTo (onset temperature), Tm (melting temperature), and ∆H (enthalpy) of hydrogel samples were obtained from DTG/DTA curves.


### 2.7. Rheological Study

The rheological flow properties (shear stress and viscosity vs. shear rate) of CNC and hydrogels were measured using a Brookfield DV-III Ultra™ Programmable Rheometer (Brookfield Engineering Laboratories Inc., Middleboro, USA). The cone and plate spindle (CP-41 model) with measuring geometry of 5 cm was used with a gap of 0.05 mm and a shear rate ranging from 10 to 100 s-1. A flow curve was obtained by plotting shear stress and viscosity vs. shear rate values using a steady state flow program. The Herschel–Bulkley model was selected as the best-fit model based on standard error of shear stress-shear rate and shear stress-viscosity data.The Herschel–Bulkley model equation is as follows:(1)Ʈ=Ʈo+kDƞ,where Ʈ is the shear stress (Pa), Ʈo is the yield stress (Pa), D is the shear rate (s-1), k is the consistency coefficient, and ƞ is the flow behaviour index (dimensionless). The consistency coefficient (k) and flow behaviour index (ƞ) of CNC and hydrogels were determined by the steady state flow program software.


### 2.8.In Vitro Degradation

In vitro degradation of CNC and hydrogels was studied using the enzymatic digestion method. A known weight of the hydrogel sample was taken and placed in an aliquot (10 ml) of PBS with 10 U/ml of trypsin at 37°C for the duration of 7 days. The weight of hydrogel samples was taken periodically.


### 2.9. Cytotoxicity

Cytotoxicity/biocompatibility of CNC and hydrogels was investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the method followed Dara et al. [18]. The viability of cells was calculated and was expressed in percentage (%).
(2)Cellviablity%=ODofsampleODofcontrol×100.


### 2.10. Statistical Analysis

One-way ANOVA was used to analyse the data. Experiments were carried out in triplicates independently. Significant differences between the means of triplicates were determined by Duncan’s multiple comparison test using statistical software IBM SPSS 20 (SPSS Inc., Illinois, USA).


## 2.1. Preparation of Cellulose Nanocrystals

CNC was prepared by formic acid hydrolysis according to the following method as described by Du et al. [4]. Briefly, 3 gm of cellulose was added in 100 ml of 0.015 M FeCl3 containing 90 ml formic acid in a spherical flask. The flask was kept at 95°C for 4 h under stirring using a magnetic stirrer (Heidolph Instruments, GmbH and Co., Germany). The flask was cooled down to room temperature followed by centrifugation at 8000 rpm for 10 min using centrifuge (Sorvall Legend XTR, Thermo Fisher Scientific, New Hampshire, USA). The obtained white residue was washed repeatedly with distilled water until pH 6 was achieved. After separation of the supernatant, the white residue nanocrystals were freeze dried at −40°C for 72 h using a freeze dryer (Lark, Tamil Nadu, India) and stored at 4°C until further analysis.The obtained white residue nanocrystals were subjected to cationic modification using TEMPO and sodium hypochlorite (NaOCl) according to the following method as described by Akhlaghi et al. [6]. 1.5 gm of nanocrystals was added to 100 ml of deionised water and sonicated for 15 min. 20 mg of TEMPO and 400 mg of NaBr were added to the CNC suspension. The pH of the CNC suspension was adjusted to 10 using 1 M NaOH. The oxidation reaction was initiated by adding 10 ml of 13% NaOCl to the CNC suspension under gentle stirring continuously for 3 h, simultaneously maintaining constant pH 10. An aliquot of 10 ml of methanol was added to quench the reaction, and pH was adjusted to 7 using 1 M HCl. Finally, the oxidised nanocrystals were dialysed against deionised water using 14 kDa dialysis membranes (Sigma, St. Louis, USA). The resultant freeze-dried cellulose nanocrystals were designated as “CNC.”


## 2.2. Preparation of Hydrogels Reinforced with CNC

The biomaterial-based hydrogels reinforced with CNC were prepared according to the following method described by Wang et al. [16], with slight modifications. A 1% CNC (20 ml) was dispersed in 40 ml of 2% alginate and 2% chitosan homogenous solutions separately via sonication for 15 min and designated as “CNC-ALG” and “CNC-CHI,” respectively. Further, a 2% gelatin was dissolved in 40 ml of deionised water with 20 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and stirred continuously for 24 h at room temperature. Fish gelatin was used in the present study, which was prepared according to the following method as described by Dara et al. [17]. This gelatin solution with EDC was added to CNC-ALG and CNC-CHI solutions followed by the addition of 0.5 ml of 50 mM ZnSO4. The suspension was stirred continuously for 6 h at room temperature for ionic cross-linking. Finally, the obtained hydrogels were rinsed with 1x phosphate buffer saline (PBS) and designated as “CNC-ALG-GEL” and “CNC-CHI-GEL,” respectively. The hydrogels were stored at 4°C until further analysis. The procedure for the preparation of hydrogels is shown in Figure 1.Figure 1
Procedure for the preparation of hydrogels reinforced with CNC.


## 2.3. Particle Size Distribution and Zeta Potential Analysis

The particle size distribution (PSD) and zeta potential of CNC and hydrogel samples were determined by dynamic light scattering analyses (DLS) using a Zetasizer Nano Series (Malvern, Worcestershire, UK). For PSD analysis, the CNC and hydrogel samples were loaded into a quartz cuvette with 1 cm path-length and were subjected to DLS measurement with a detection angle of 90° at25±0.1°C. The Z-average hydrodynamic diameters and polydispersity index (PDI) of hydrogels were determined and recorded based on the scattering intensity. For analysing the zeta potential, the CNC and hydrogel samples were filtered using a 0.45 μm syringe filter membrane to remove large particles and measured with an angle of 15° at 25°C.


## 2.4. FT-IR Spectra and SEM Morphology

The FT-IR spectra of CNC and hydrogels were analysed using a FT-IR spectrometer (Bruker Optik GmBH, Germany), in the region of 4000 cm-1–500 cm-1 wave numbers at 4 cm-1 resolution. The surface morphology was examined using scanning electron microscopy (SEM JSM, Japan), and samples were observed at an accelerated 15 kV voltage.


## 2.5. X-Ray Diffraction (XRD) Pattern

X-ray powder diffraction patterns of CNC and hydrogels were recorded using an Analytical X’Pert Pro X-ray diffractometer. The X-ray source was Cu-Kα radiation (40 kV, 80 mA). The XRD patterns of samples were taken in the 2θ range of 10°–80° in a fixed time mode at room temperature at a scanning rate of 4° min-1.


## 2.6. Thermal Characteristics

Prior to the analyses, the CNC and hydrogel samples were conditioned at 25°C and 53% relative humidity. DTG (derivative thermogravimetry) and DTA (differential thermal analysis) of hydrogels were analysed using a thermogravimetric analyser (Seiko Exstar 6300, Italy). Samples were heated from 600°C, at 20°C/min heating rate, using nitrogen flow (250 ml/min). TheTo (onset temperature), Tm (melting temperature), and ∆H (enthalpy) of hydrogel samples were obtained from DTG/DTA curves.


## 2.7. Rheological Study

The rheological flow properties (shear stress and viscosity vs. shear rate) of CNC and hydrogels were measured using a Brookfield DV-III Ultra™ Programmable Rheometer (Brookfield Engineering Laboratories Inc., Middleboro, USA). The cone and plate spindle (CP-41 model) with measuring geometry of 5 cm was used with a gap of 0.05 mm and a shear rate ranging from 10 to 100 s-1. A flow curve was obtained by plotting shear stress and viscosity vs. shear rate values using a steady state flow program. The Herschel–Bulkley model was selected as the best-fit model based on standard error of shear stress-shear rate and shear stress-viscosity data.The Herschel–Bulkley model equation is as follows:(1)Ʈ=Ʈo+kDƞ,where Ʈ is the shear stress (Pa), Ʈo is the yield stress (Pa), D is the shear rate (s-1), k is the consistency coefficient, and ƞ is the flow behaviour index (dimensionless). The consistency coefficient (k) and flow behaviour index (ƞ) of CNC and hydrogels were determined by the steady state flow program software.


## 2.8.In Vitro Degradation

In vitro degradation of CNC and hydrogels was studied using the enzymatic digestion method. A known weight of the hydrogel sample was taken and placed in an aliquot (10 ml) of PBS with 10 U/ml of trypsin at 37°C for the duration of 7 days. The weight of hydrogel samples was taken periodically.


## 2.9. Cytotoxicity

Cytotoxicity/biocompatibility of CNC and hydrogels was investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the method followed Dara et al. [18]. The viability of cells was calculated and was expressed in percentage (%).
(2)Cellviablity%=ODofsampleODofcontrol×100.


## 2.10. Statistical Analysis

One-way ANOVA was used to analyse the data. Experiments were carried out in triplicates independently. Significant differences between the means of triplicates were determined by Duncan’s multiple comparison test using statistical software IBM SPSS 20 (SPSS Inc., Illinois, USA).


## 3. Results and Discussion

### 3.1. Particle Size Distribution and Zeta Potential Analysis

Particle size distribution is one of the most important characteristic properties that determine its functionality, whereas the zeta potential defines the potential difference between the dispersion medium and the layer of fluid attached to the dispersed nanocrystals in a colloidal system of charged particles [10]. The Z-average hydrodynamic diameter, polydispersity index (PDI), and zeta potential data of hydrogels are displayed in Table 1. The Z-average hydrodynamic diameter and zeta potential of synthesized CNC were found to be 192 nm and -48.40 mV, respectively. The particle size of the CNC depends on several factors such as type of acid used for the hydrolysis, reaction temperature, and reaction time [1]. In the present study, formic acid was used for the hydrolysis, and the reaction time and temperature were 4 h and 95°C, respectively. The smaller particle size and higher zeta potential of synthesized CNC were most likely due to the improved dispersibility and conversion of hydroxyl groups to carboxyl groups on the biopolymer cellulose surface during the TEMPO-mediated oxidation process [19]. The Z-average hydrodynamic diameter and zeta potential of hydrogels CNC-ALG-GEL and CNC-CHI-GEL were found to be 415 nm and 572 nm and -61.6 mV and +16.9 mV, respectively. It can be noted that the changes in the particle size and zeta potential of CNC-ALG-GEL and CNC-CHI-GEL represent the colloidal electrostatic interactions between the incorporated CNC, chitosan, and alginate biopolymers. The negative zeta potential value of CNC-ALG-GEL (-61.6 mV) indicates the presence of carboxyl groups, and the positive value of CNC-CHI-GEL confers the cationic characteristics of chitosan chains [18, 20]. Further, the lowest zeta potential (+16.9 mV) of CNC-CHI-GEL might be due to the neutralization of amino groups on biopolymer chitosan that results from the separation from CNC and aggregation at the surface of nanocrystals [21]. Nevertheless, the nature of the biopolymers used for the surface modification defines the surface charge. In the present study, the positive zeta potential of CNC-CHI-GEL and CHI-GEL represents the cationic characteristic of chitosan, whereas the negative zeta potential of CNC-ALG-GEL and ALG-GEL could be originated from the carboxylic groups of alginate. The smaller particle size of CNC-ALG-GEL might be due to more affinity and adsorption of the cellulose nanocrystal to the surface of the biopolymer. Moreover, the swelling of cellulose nanocrystals tends to create repulsion between polymeric chains which results in increasing the particle size [15]. It can be noticed that the synthesized CNC and hydrogels were monodispersed which was evident by the low PDI (<0.5). Furthermore, the detailed morphological studies of CNC and hydrogels were confirmed by SEM. From this data, it can be deduced that the biopolymer hydrogels were highly stable due to reinforcement of CNC.Table 1
Particle size distribution and zeta potential of hydrogels.
SamplesParticle size (d·nm)PDIZeta potential (mV)CNC192±6.820.275±0.0013−48.4±0.62ALG-GEL251±7.800.376±0.0110−24.4±2.06CHI-GEL308±9.870.405±0.023661.5±5.87CNC-ALG-GEL415±14.300.464±0.0215−61.6±7.89CNC-CHI-GEL572±13.740.488±0.025516.9±1.96


### 3.2. FT-IR Spectra

FT-IR spectral analysis was performed to study the alterations in the functional groups of the hydrogels that would have occurred due to cross-linking of CNC. The FT-IR spectra of CNC were found to have absorption bands at wave numbers at 3334 cm-1, 2898 cm-1, 1654 cm-1, 1427 cm-1, 1315 cm-1, 1150 cm-1,1030 cm-1, and 897 cm-1, which can be attributed to O-H, C-H, COO-, O-H bending, C-O stretching, CO-O-CO stretching, and C-H bending, respectively (Figure 2). The spectral data of CNC were in accordance with the spectral data of CNC extracted from rice husk [3]. The absorption band of CNC at a wave number at 1710 cm-1 can be attributed to stretching of carbonyl groups (C=O) resulting from the TEMPO reaction [22]. The characteristic absorption peaks of CNC-CHI-GEL and CHI-GEL at wavenumber≈1170cm−1 can be assigned to the free amine group (-NH2) of the chitosan molecule, whereas on the other hand, the absorption peak of CNC-ALG-GEL and ALG-GEL wavenumber≈1450cm−1 could be related to the carboxylic group (-COOH) of alginate [5]. In line with these reports, our findings correlated well with the results of zeta potential analyses as reported earlier. The pattern of absorption bands of all the hydrogels was in accordance with the FT-IR spectra of fish gelatin [17]. It can be noted that the appearance of a broad absorption band at wavenumber≈1625cm−1 of all hydrogel samples is an indicator of the successful formation of a complex between carboxylic groups of CNC and amino groups of biopolymers. Furthermore, it is stated that the intensity of the peak at region ~1625 cm-1 of all hydrogel samples represents the successful cross-linking of the gelatin chain with the biopolymer hydrogels by overlapping the amide group of the gelatin molecule [3]. In another study by Wang et al., it was mentioned that the broad absorption peaks in the region (3200 cm-1–2900 cm-1) of the spectrum of hydrogels illustrate the strong O-H bond interaction between the biopolymer-gelatin backbone and CNC molecules [16]. The absorption bands of CNC at regions ~1425 cm-1 and ~1225 cm-1 were shifted to different frequencies in hydrogel samples after reinforcement, and this emphasizes the formation complex inter- and intrahydrogen bonding between CNC and polymer matrix [23]. The FT-IR results of the present study clearly indicated that the reinforcement of CNC has not altered the network structure of biopolymer molecules.Figure 2
FT-IR spectra of CNC and hydrogels.


### 3.3. SEM Morphology

The morphology and microstructures of CNC and hydrogels were depicted in Figure3. The morphology of CNC was found to have roughness with fractured surface. It is well known that the morphology of nanocrystals depends upon the severity of hydrolysis used for the reaction [4]. In the present study, the formic acid hydrolysis method was used for the preparation of CNC. The SEM micrographs of ALG-GEL, CNC-ALG-GEL, CHI-GEL, and CNC-CHI-GEL appeared to be smooth and also showed porous structures compared to the micrograph of CNC, which indicated the blend miscibility, high homogeneity, and in situ dissolution of biopolymers to form blend mixtures [22]. The impact of smoothness and porosity was high in CNC-ALG-GEL and CNC-CHI-GEL hydrogels as compared to ALG-GEL and CHI-GEL. It is noticed that certain factors such as hydrogen bonding and complexity of CNC with biopolymers define the network structure formation of hydrogel membranes [24]. The uniform distribution of pores in the CNC-ALG-GEL and CNC-CHI-GEL hydrogels reflected the embedded nature of CNC which resulted from strong interfacial adhesion between the CNC and the biopolymer matrix. Technically, the roughness and smoothness of hydrogel membranes depend upon the concentration of CNC added to hydrogels [9]. In the present study, the interpenetrated networks in the hydrogel membranes due to the reinforcement of CNC were a good indication of superior mechanical properties and good stability against degradation, which correlated well with an earlier report [16]. Moreover, the hydrogels with these characteristics also support other features such as easy injectability and cost-effectiveness which are more suitable for tissue engineering and regenerative medicine applications [1].Figure 3
SEM of CNC and hydrogels.


### 3.4. X-Ray Diffraction (XRD) Pattern

As shown in Figure4, the CNC showed three cellulose characteristic peaks at 2θ=14.2°, 16.4°, and 22.5°, where the prominent characteristic sharp peak at 2θ=22.5° attributing to (200) plane reflection of crystal lattice of alpha-cellulose and hemicelluloses [25]. The peak at 2θ=20° of CHI-GEL indicates the semiamorphous nature of chitosan, whereas the peak at 2θ=19.9° of ALG-GEL indicates the amorphous and crystalline nature of alginate [5, 18]. The characteristic peak of CNC at 2θ=22° appeared in the CNC-ALG-GEL and CNC-CHI-GEL which indicated the presence of CNC in the hydrogel membrane matrix. However, this characteristic peak was slightly shifted, and the disparity in crystallinity can be due to intermolecular interactions. It was noticed that the intensity of the peak at 2θ=22° of CNC-ALG-GEL was lesser as compared to that of CNC-CHI-GEL, which could be due to the difference in the percolation phenomenon of CNC in contact with alginate [8]. The intensity of the peak at 2θ=22° of CNC-ALG-GEL was found to be increased as compared to that of CNC-CHI-GEL, due to the transcrystallization effect of nanocrystals. Furthermore, it can also be stated that the interaction between the CNC and the biopolymer matrix was one of the probable reasons for the increment of crystallinity [9]. However, XRD data in the present study revealed that the reinforcement of CNC does not change the structural uniformity of the hydrogel-based biopolymer matrix by improving the molecular ordering and mechanical properties as well as the amorphous nature of the polymer matrix.Figure 4
XRD of CNC and hydrogels.


### 3.5. Thermal Characteristics

DTG/DTA curves of hydrogel samples at 20°C/min heating rate under inert atmosphere are depicted in Figures5(a) and 5(b). The thermal characteristic data illustrated that all the hydrogel samples have shown three major degradation steps. Thermal degradation of polymers includes dehydration followed by decomposition along with breakage of intra- and interlinkages in the structure of polymers [26]. The first degradation step of CNC was shown between 50°C and 300°C, which might be due to the loss of water bound to the polymer. The second degradation step was in the range of 300–360°C, and the maximum degradation temperature at this point was found to be 343°C. The final and third degradation step was found to be approximately at 450°C. The first degradation steps of hydrogels ALG-GEL, CHI-GEL, CNC-ALG-GEL, and CNC-CHI-GEL were found to be 82°C, 67°C, 75°C, and 68°C, respectively. The maximum degradation temperatures of hydrogels during the second step were observed to be 256°C, 274°C, 319°C and 336°C, respectively. The maximum degradation temperature of hydrogels hardly changes with the incorporation of CNC in the biopolymer matrix. It indicated that the CNC retained the thermal stability of hydrogels due to the strong interactions between CNC and biopolymers [27]. The significant weight loss of CHI-GEL and CNC-CHI-GEL hydrogels at the 200-400°C temperature range can be attributed to the depolymerization of chitosan through deacetylation and cleavage of glycosidic linkages via dehydration and deamination, whereas in the case of ALG-GEL and CNC-ALG-GEL, it could be due to chain scission and ring opening reactions of alginate [9]. Maximum weight loss was observed to be in the second degradation step which could be attributed to degradation and dislocation of backbone linkages as well as functional groups of biopolymers [26]. The final and third degradation steps of hydrogels ALG-GEL and CHI-GEL were found to be at 472°C and 619°C, respectively, whereas for CNC-ALG-GEL and CNC-CHI-GEL hydrogels, they were at 441°C and 512°C, respectively. It can be observed that the reinforcement of CNC to the hydrogels has reduced the degradation temperature. This might be due to the decomposition of CNC derivatives that indicated the carbonization of polymeric biomaterial [28]. This difference in the degradation temperature and weight loss between the hydrogels might be due to the difference in the formation of hydrogen bonding between the CNC and molecules of biopolymers, and cross-linkers [29].Figure 5
(a) DTA curves of hydrogels (A) CNC, (B) ALG-GEL, (C) CHI-GEL, (D) CNC-ALG-GEL, and (E) CNC-CHI-GEL at 20°C/min heating rate. (b) DTG curves of hydrogels (A) CNC, (B) ALG-GEL, (C) CHI-GEL, (D) CNC-ALG-GEL, and (E) CNC-CHI-GEL at 20°C/min heating rate.
(a)(b)Figures5(a) and 5(b) and Table 2 illustrate the To (onset temperature), Tm (melting temperature), and ∆H (enthalpy) of hydrogels. To, Tm, and ∆H (enthalpy) of polymeric blends usually represent the kinetic hindrances that take place during crystallization and melting. CNC showed a Tm value of 370°C that corresponds to the decomposition of glycosyl units and the formation of carbonaceous residues [30]. The CNC-CHI-GEL hydrogels reinforced with CNC were found to have a high Tm value compared to that of CHI-GEL, whereas CNC-ALG-GEL was marginally lesser than ALG-GEL. In a previous study by Rescignano et al., it was noted that the Tm values of chitosan and alginate biomaterials incorporated with CNC were found to be 289°C and 317°C, respectively [20]. In the present study, the Tm values of chitosan and alginate hydrogels reinforced with CNC were observed to be 359°C and 417°C, respectively. The disparity in Tm values might be due to the formation of intermolecular hydrogen interactions between chitosan, alginate, and CNC at elevated temperature. As the Tm value goes beyond the elevated temperature, the biopolymer gets the tendency to donate hydrogen bonds and results in the production of more -OH groups, and these groups facilitate more interaction and influence the melting temperature of biopolymeric materials [8]. Moreover, chemically modified CNC incorporated in biopolymers influence the Tm, due to strong interactions between chemically modified CNC and polymer matrices which could be originated from Tm difference [6]. Notably, the chemically modified CNC do not have any impact on the To of biopolymers [1]. The other resultant exothermic peaks of hydrogel membranes might be due to the decomposition of alginate as well as the breakdown of intermolecular structure of gelatin [31]. It is demonstrated from the present data that the biomaterial-based hydrogels reinforced with cellulose nanocrystals showed better thermal stability and miscibility as revealed by thermal gravity analysis.Table 2
Thermal characteristics of CNC and hydrogels.
SamplesTo (°C)Tm (°C)ΔH (mJ/mg)CNC313.2±14.86370.4±16.82−618±23ALG-GEL230.4±11.24486.7±17.80−5675±209CHI-GEL237.7±12.45340.9±29.87−889±236CNC-ALG-GEL237.8±13.56471.1±14.30−4992±215CNC-CHI-GEL246.9±10.41359.9±13.74−707±55


### 3.6. Rheological Study

Different sorts of physicodynamic or computer rheological models such as Herschel–Bulkley, Newtonian, Bingham, Casson, and Power law were usually used to present the flow profile of blends and hydrogels to describe shear stress and shear behaviour. The Herschel–Bulkley rheological model is mostly preferred due to its accuracy. In the present study, the rheological Herschel–Bulkley model was used to analyse the experimental result data (shear stress and viscosity-shear rate) of hydrogel solutions (Table3).The yield stress (Ʈo), flow behaviour index (ƞ), and consistency coefficient (k) were also calculated using the software provided with the rheometer. The yield stress (Ʈo) represents the interaction potential of colloidal particles in the biopolymer and also plays an important role in demonstrating the macrolattice structure of biopolymers [32]. The rheological Herschel–Bulkley model exhibited yield stress values for hydrogel samples which were studied (Table 3). The Ʈo and consistency coefficient (k) values of ALG-GEL and CHI-GEL were found to have higher values as compared to those of CNC-ALG-GEL and CNC-CHI-GEL hydrogels, respectively. It has been stated that the method of analysis and experimental condition influences the yield stress [33]. Several research studies have conflated views on the yield stress which could be linked with the transition between liquid and solid states and/or two same fluids with different viscosities [34].Table 3
Herschel–Bulkley parameters for hydrogels.
HydrogelsƮokƞ’R2CNC0.030.741.380.99ALG-GEL0.141.401.040.99CNC-ALG-GEL0.040.961.260.99CHI-GEL0.0820.50.800.99CNC-CHI-GEL0.0611.71.060.99Ʈo: yield stress; k: consistency coefficient; ƞ: flow behavior index; R2: regression coefficient.The ALG-GEL exhibited shear thickening and Newtonian behaviour, whereas the CHI-GEL exhibited shear thinning and pseudoplastic behaviour, which represents an irreversible structural breakdown (Figure6). The Newtonian behaviour of ALG-GEL at low shear rates was characterized by shear rate-independent viscosity. The shear thinning behaviour of CHI-GEL indicates the disrupted intermolecular junctions with a slower rate of reformations, which tends to drop in viscosity at a lower shear rate [35]. As the shear rate increases, the viscosity of CNC-ALG-GEL and CNC-CHI-GEL hydrogels tends to decrease. This might be due to the distributed and weak nature of the hydrogel network, and this leads to release of the entrapped liquid which resists the flow behaviour, thus inducing the lower viscosity [36]. This nonlinear relationship represents the higher viscosity of hydrogels which is a strong indication on the impact of cross-linking [22]. The viscosity of a shear thinning liquid has an inversely proportional relationship with the shear rate, whereas a shear thickening liquid has a directly proportional relationship between viscosity and the shear rate [37]. Pseudoplastic nature is important for topical formulations that have viscous behaviour under static conditions; nevertheless, this viscous behaviour tends to decrease at a higher shear rate which results in better spreadability and improves the drug permeation of specific substances upon topical application [11]. By the reinforcement of CNC to hydrogels, the viscosity vs. shear rate curve of CNC-ALG-GEL and CNC-CHI-GEL hydrogels has transformed to a plateau shape, due to the breaking down of CNC domains by shearing force [38]. With regard to the flow behaviour index, ƞ=1 for a Newtonian fluid and ƞ<1 for a shear thinning fluid or rheofluid, whereas ƞ>1 for shear thickening or rheothickening fluid. In the present study, ƞ values of CNC-ALG-GEL and CNC-CHI-GEL hydrogels indicated a Newtonian and shear thickening nature. This phenomenon of transition represents the network structure formed by CNC within alginate, chitosan, and gelatin which was constructed by strong hydrogen interactions and eventually maintains the viscosity [39, 40]. However, the increase in ƞ values of CNC-ALG-GEL and CNC-CHI-GEL hydrogels was also an indication of the shear thickening nature [2]. The biomaterial-based hydrogels reinforced with CNC showed a dependent behaviour which results from rheological characterization.Figure 6
Flow properties (shear stress vs. shear rate and viscosity vs. shear rate) of hydrogels. (a) CNC, (b) ALG-GEL, (c) CHI-GEL, (d) CNC-ALG-GEL, and (e)CNC-CHI-GEL.


### 3.7.In Vitro Degradation

The ALG-GEL and CHI-GEL hydrogels were found to have 50% of weight loss from day 1 to day 7 (Figure7). This illustrates that the majority of gelatin content in the respective hydrogel samples were susceptible to enzymatic degradation. In the hydrogel samples (CNC-ALG-GEL and CNC-CHI-GEL), 15% of weight loss were significantly observed from day 1 to day 7. In a study by Wang et al. [16], the hydrogels were prepared using alginate and gelatin biopolymers; moreover, the hydrogels without CNC were found to have 50% weight loss, and the hydrogels with CNC have significantly improved resistance to biodegradation. This improved resistance to enzymatic degradation might be due to homogenous dispersion and hydrophobicity [16]. It was believed that the CNC might have helped in creating the interpenetrated matrix between biomaterials alginate, chitosan, and gelatin due to cross-linking and can be expected to distribute uniformly by hydrogen bonding and hydrophobic interactions. Wen et al. [41] developed interpenetrated network (IPN) hydrogels with 25% of weight loss observed within 2 h of enzymatic degradation. They stated that the biomaterials blended with gelatin tend to have superior mechanical characteristics which resulted from interpenetrated matrix, and this provides good stability against degradation [41]. Furthermore, the crystallinity and hydrophilicity are important factors that influence the degradation rate. In addition, the surface hydrophilicity tends to cause the invasion of the degradation medium and water into the amorphous regions of CNC [42]. In this viewpoint of degradation, the biomaterial-based hydrogels reinforced with CNC are mostly recommended for bone and tissue engineering applications.Figure 7
In vitro degradation of CNCs and hydrogels.


### 3.8. Cytotoxicity

Thein vitro cytotoxicity of CNC and hydrogel membranes was investigated using the L929 cell line by MTT assay, and absorbance was read at 570 nm to evaluate cell viability [43]. In this method, the viability of the L929 fibroblast cell line was observed after 24 h of incubation. According to GB/T 16886.5-2003 (ISO 10993-5:1999), the biomaterial-based hydrogel membranes with cell viability more than 75% can be considered as noncytotoxic and are recommended for biomedical applications. The cell viabilities of ALG-GEL and CHI-GEL hydrogels were significantly higher than those of CNC (p<0.05). The CNC-ALG-GEL and CNC-CHI-GEL hydrogel membranes reinforced with CNC had 96% and 97% cell viabilities, respectively, which were significantly higher than CNC, ALG-GEL, and CHI-GEL hydrogels (p<0.05) (Figures 8(a) and 8(b)).Figure 8
(a) Cell viability assay (different letters indicate significant difference;p<0.05). (b) Fluorescence microscope images of CNC and hydrogel membranes at ×10 magnification.
(a)(b)The alginate and chitosan bionanocomposite membranes reinforced with CNC enhanced the viability ofNIH-3T3 fibroblasts [16, 24]. In an another study by Shaheen et al., the chitosan-alginate-hydroxyapatite scaffolds fabricated with CNC were found to have 100% cell viability and promising cell growth and cell adherence [5]. In a previous study of our lab, promising biocompatibility characteristics of polyvinyl alcohol/starch polymer were proven [44]. It was noticed that the mechanical properties and zwitterion polymer network provide the suitable environment for cell growth and proliferation [23]. Interaction of viable cells with the substratum matrix of hydrogel membranes reinforced with CNC via physicobiochemical and molecular interactions and surface topological parameters has an essential role in the cytocompatibility of biomaterial membranes such as cell adhesion, differentiation, proliferation, and migration [5]. Furthermore, the pore size of hydrogel membranes is mostly controlled by the zeta potential which enables the transportation of necessary bioactive molecules and migration of cells within the matrix of hydrogel membranes. In the present study, the zeta potential values of CNC-ALG-GEL, and CNC-CHI-GEL were quite different from those of ALG-GEL and CHI-GEL which might be due to the reinforcement of CNC. Surface charge modification by TEMPO was also most likely a probable reason for changes in zeta potential values. It was noted from a previous study that the CNC and gelatin promote the cell growth by forming mimetics of native bone tissue [45].


## 3.1. Particle Size Distribution and Zeta Potential Analysis

Particle size distribution is one of the most important characteristic properties that determine its functionality, whereas the zeta potential defines the potential difference between the dispersion medium and the layer of fluid attached to the dispersed nanocrystals in a colloidal system of charged particles [10]. The Z-average hydrodynamic diameter, polydispersity index (PDI), and zeta potential data of hydrogels are displayed in Table 1. The Z-average hydrodynamic diameter and zeta potential of synthesized CNC were found to be 192 nm and -48.40 mV, respectively. The particle size of the CNC depends on several factors such as type of acid used for the hydrolysis, reaction temperature, and reaction time [1]. In the present study, formic acid was used for the hydrolysis, and the reaction time and temperature were 4 h and 95°C, respectively. The smaller particle size and higher zeta potential of synthesized CNC were most likely due to the improved dispersibility and conversion of hydroxyl groups to carboxyl groups on the biopolymer cellulose surface during the TEMPO-mediated oxidation process [19]. The Z-average hydrodynamic diameter and zeta potential of hydrogels CNC-ALG-GEL and CNC-CHI-GEL were found to be 415 nm and 572 nm and -61.6 mV and +16.9 mV, respectively. It can be noted that the changes in the particle size and zeta potential of CNC-ALG-GEL and CNC-CHI-GEL represent the colloidal electrostatic interactions between the incorporated CNC, chitosan, and alginate biopolymers. The negative zeta potential value of CNC-ALG-GEL (-61.6 mV) indicates the presence of carboxyl groups, and the positive value of CNC-CHI-GEL confers the cationic characteristics of chitosan chains [18, 20]. Further, the lowest zeta potential (+16.9 mV) of CNC-CHI-GEL might be due to the neutralization of amino groups on biopolymer chitosan that results from the separation from CNC and aggregation at the surface of nanocrystals [21]. Nevertheless, the nature of the biopolymers used for the surface modification defines the surface charge. In the present study, the positive zeta potential of CNC-CHI-GEL and CHI-GEL represents the cationic characteristic of chitosan, whereas the negative zeta potential of CNC-ALG-GEL and ALG-GEL could be originated from the carboxylic groups of alginate. The smaller particle size of CNC-ALG-GEL might be due to more affinity and adsorption of the cellulose nanocrystal to the surface of the biopolymer. Moreover, the swelling of cellulose nanocrystals tends to create repulsion between polymeric chains which results in increasing the particle size [15]. It can be noticed that the synthesized CNC and hydrogels were monodispersed which was evident by the low PDI (<0.5). Furthermore, the detailed morphological studies of CNC and hydrogels were confirmed by SEM. From this data, it can be deduced that the biopolymer hydrogels were highly stable due to reinforcement of CNC.Table 1
Particle size distribution and zeta potential of hydrogels.
SamplesParticle size (d·nm)PDIZeta potential (mV)CNC192±6.820.275±0.0013−48.4±0.62ALG-GEL251±7.800.376±0.0110−24.4±2.06CHI-GEL308±9.870.405±0.023661.5±5.87CNC-ALG-GEL415±14.300.464±0.0215−61.6±7.89CNC-CHI-GEL572±13.740.488±0.025516.9±1.96


## 3.2. FT-IR Spectra

FT-IR spectral analysis was performed to study the alterations in the functional groups of the hydrogels that would have occurred due to cross-linking of CNC. The FT-IR spectra of CNC were found to have absorption bands at wave numbers at 3334 cm-1, 2898 cm-1, 1654 cm-1, 1427 cm-1, 1315 cm-1, 1150 cm-1,1030 cm-1, and 897 cm-1, which can be attributed to O-H, C-H, COO-, O-H bending, C-O stretching, CO-O-CO stretching, and C-H bending, respectively (Figure 2). The spectral data of CNC were in accordance with the spectral data of CNC extracted from rice husk [3]. The absorption band of CNC at a wave number at 1710 cm-1 can be attributed to stretching of carbonyl groups (C=O) resulting from the TEMPO reaction [22]. The characteristic absorption peaks of CNC-CHI-GEL and CHI-GEL at wavenumber≈1170cm−1 can be assigned to the free amine group (-NH2) of the chitosan molecule, whereas on the other hand, the absorption peak of CNC-ALG-GEL and ALG-GEL wavenumber≈1450cm−1 could be related to the carboxylic group (-COOH) of alginate [5]. In line with these reports, our findings correlated well with the results of zeta potential analyses as reported earlier. The pattern of absorption bands of all the hydrogels was in accordance with the FT-IR spectra of fish gelatin [17]. It can be noted that the appearance of a broad absorption band at wavenumber≈1625cm−1 of all hydrogel samples is an indicator of the successful formation of a complex between carboxylic groups of CNC and amino groups of biopolymers. Furthermore, it is stated that the intensity of the peak at region ~1625 cm-1 of all hydrogel samples represents the successful cross-linking of the gelatin chain with the biopolymer hydrogels by overlapping the amide group of the gelatin molecule [3]. In another study by Wang et al., it was mentioned that the broad absorption peaks in the region (3200 cm-1–2900 cm-1) of the spectrum of hydrogels illustrate the strong O-H bond interaction between the biopolymer-gelatin backbone and CNC molecules [16]. The absorption bands of CNC at regions ~1425 cm-1 and ~1225 cm-1 were shifted to different frequencies in hydrogel samples after reinforcement, and this emphasizes the formation complex inter- and intrahydrogen bonding between CNC and polymer matrix [23]. The FT-IR results of the present study clearly indicated that the reinforcement of CNC has not altered the network structure of biopolymer molecules.Figure 2
FT-IR spectra of CNC and hydrogels.


## 3.3. SEM Morphology

The morphology and microstructures of CNC and hydrogels were depicted in Figure3. The morphology of CNC was found to have roughness with fractured surface. It is well known that the morphology of nanocrystals depends upon the severity of hydrolysis used for the reaction [4]. In the present study, the formic acid hydrolysis method was used for the preparation of CNC. The SEM micrographs of ALG-GEL, CNC-ALG-GEL, CHI-GEL, and CNC-CHI-GEL appeared to be smooth and also showed porous structures compared to the micrograph of CNC, which indicated the blend miscibility, high homogeneity, and in situ dissolution of biopolymers to form blend mixtures [22]. The impact of smoothness and porosity was high in CNC-ALG-GEL and CNC-CHI-GEL hydrogels as compared to ALG-GEL and CHI-GEL. It is noticed that certain factors such as hydrogen bonding and complexity of CNC with biopolymers define the network structure formation of hydrogel membranes [24]. The uniform distribution of pores in the CNC-ALG-GEL and CNC-CHI-GEL hydrogels reflected the embedded nature of CNC which resulted from strong interfacial adhesion between the CNC and the biopolymer matrix. Technically, the roughness and smoothness of hydrogel membranes depend upon the concentration of CNC added to hydrogels [9]. In the present study, the interpenetrated networks in the hydrogel membranes due to the reinforcement of CNC were a good indication of superior mechanical properties and good stability against degradation, which correlated well with an earlier report [16]. Moreover, the hydrogels with these characteristics also support other features such as easy injectability and cost-effectiveness which are more suitable for tissue engineering and regenerative medicine applications [1].Figure 3
SEM of CNC and hydrogels.


## 3.4. X-Ray Diffraction (XRD) Pattern

As shown in Figure4, the CNC showed three cellulose characteristic peaks at 2θ=14.2°, 16.4°, and 22.5°, where the prominent characteristic sharp peak at 2θ=22.5° attributing to (200) plane reflection of crystal lattice of alpha-cellulose and hemicelluloses [25]. The peak at 2θ=20° of CHI-GEL indicates the semiamorphous nature of chitosan, whereas the peak at 2θ=19.9° of ALG-GEL indicates the amorphous and crystalline nature of alginate [5, 18]. The characteristic peak of CNC at 2θ=22° appeared in the CNC-ALG-GEL and CNC-CHI-GEL which indicated the presence of CNC in the hydrogel membrane matrix. However, this characteristic peak was slightly shifted, and the disparity in crystallinity can be due to intermolecular interactions. It was noticed that the intensity of the peak at 2θ=22° of CNC-ALG-GEL was lesser as compared to that of CNC-CHI-GEL, which could be due to the difference in the percolation phenomenon of CNC in contact with alginate [8]. The intensity of the peak at 2θ=22° of CNC-ALG-GEL was found to be increased as compared to that of CNC-CHI-GEL, due to the transcrystallization effect of nanocrystals. Furthermore, it can also be stated that the interaction between the CNC and the biopolymer matrix was one of the probable reasons for the increment of crystallinity [9]. However, XRD data in the present study revealed that the reinforcement of CNC does not change the structural uniformity of the hydrogel-based biopolymer matrix by improving the molecular ordering and mechanical properties as well as the amorphous nature of the polymer matrix.Figure 4
XRD of CNC and hydrogels.


## 3.5. Thermal Characteristics

DTG/DTA curves of hydrogel samples at 20°C/min heating rate under inert atmosphere are depicted in Figures5(a) and 5(b). The thermal characteristic data illustrated that all the hydrogel samples have shown three major degradation steps. Thermal degradation of polymers includes dehydration followed by decomposition along with breakage of intra- and interlinkages in the structure of polymers [26]. The first degradation step of CNC was shown between 50°C and 300°C, which might be due to the loss of water bound to the polymer. The second degradation step was in the range of 300–360°C, and the maximum degradation temperature at this point was found to be 343°C. The final and third degradation step was found to be approximately at 450°C. The first degradation steps of hydrogels ALG-GEL, CHI-GEL, CNC-ALG-GEL, and CNC-CHI-GEL were found to be 82°C, 67°C, 75°C, and 68°C, respectively. The maximum degradation temperatures of hydrogels during the second step were observed to be 256°C, 274°C, 319°C and 336°C, respectively. The maximum degradation temperature of hydrogels hardly changes with the incorporation of CNC in the biopolymer matrix. It indicated that the CNC retained the thermal stability of hydrogels due to the strong interactions between CNC and biopolymers [27]. The significant weight loss of CHI-GEL and CNC-CHI-GEL hydrogels at the 200-400°C temperature range can be attributed to the depolymerization of chitosan through deacetylation and cleavage of glycosidic linkages via dehydration and deamination, whereas in the case of ALG-GEL and CNC-ALG-GEL, it could be due to chain scission and ring opening reactions of alginate [9]. Maximum weight loss was observed to be in the second degradation step which could be attributed to degradation and dislocation of backbone linkages as well as functional groups of biopolymers [26]. The final and third degradation steps of hydrogels ALG-GEL and CHI-GEL were found to be at 472°C and 619°C, respectively, whereas for CNC-ALG-GEL and CNC-CHI-GEL hydrogels, they were at 441°C and 512°C, respectively. It can be observed that the reinforcement of CNC to the hydrogels has reduced the degradation temperature. This might be due to the decomposition of CNC derivatives that indicated the carbonization of polymeric biomaterial [28]. This difference in the degradation temperature and weight loss between the hydrogels might be due to the difference in the formation of hydrogen bonding between the CNC and molecules of biopolymers, and cross-linkers [29].Figure 5
(a) DTA curves of hydrogels (A) CNC, (B) ALG-GEL, (C) CHI-GEL, (D) CNC-ALG-GEL, and (E) CNC-CHI-GEL at 20°C/min heating rate. (b) DTG curves of hydrogels (A) CNC, (B) ALG-GEL, (C) CHI-GEL, (D) CNC-ALG-GEL, and (E) CNC-CHI-GEL at 20°C/min heating rate.
(a)(b)Figures5(a) and 5(b) and Table 2 illustrate the To (onset temperature), Tm (melting temperature), and ∆H (enthalpy) of hydrogels. To, Tm, and ∆H (enthalpy) of polymeric blends usually represent the kinetic hindrances that take place during crystallization and melting. CNC showed a Tm value of 370°C that corresponds to the decomposition of glycosyl units and the formation of carbonaceous residues [30]. The CNC-CHI-GEL hydrogels reinforced with CNC were found to have a high Tm value compared to that of CHI-GEL, whereas CNC-ALG-GEL was marginally lesser than ALG-GEL. In a previous study by Rescignano et al., it was noted that the Tm values of chitosan and alginate biomaterials incorporated with CNC were found to be 289°C and 317°C, respectively [20]. In the present study, the Tm values of chitosan and alginate hydrogels reinforced with CNC were observed to be 359°C and 417°C, respectively. The disparity in Tm values might be due to the formation of intermolecular hydrogen interactions between chitosan, alginate, and CNC at elevated temperature. As the Tm value goes beyond the elevated temperature, the biopolymer gets the tendency to donate hydrogen bonds and results in the production of more -OH groups, and these groups facilitate more interaction and influence the melting temperature of biopolymeric materials [8]. Moreover, chemically modified CNC incorporated in biopolymers influence the Tm, due to strong interactions between chemically modified CNC and polymer matrices which could be originated from Tm difference [6]. Notably, the chemically modified CNC do not have any impact on the To of biopolymers [1]. The other resultant exothermic peaks of hydrogel membranes might be due to the decomposition of alginate as well as the breakdown of intermolecular structure of gelatin [31]. It is demonstrated from the present data that the biomaterial-based hydrogels reinforced with cellulose nanocrystals showed better thermal stability and miscibility as revealed by thermal gravity analysis.Table 2
Thermal characteristics of CNC and hydrogels.
SamplesTo (°C)Tm (°C)ΔH (mJ/mg)CNC313.2±14.86370.4±16.82−618±23ALG-GEL230.4±11.24486.7±17.80−5675±209CHI-GEL237.7±12.45340.9±29.87−889±236CNC-ALG-GEL237.8±13.56471.1±14.30−4992±215CNC-CHI-GEL246.9±10.41359.9±13.74−707±55


## 3.6. Rheological Study

Different sorts of physicodynamic or computer rheological models such as Herschel–Bulkley, Newtonian, Bingham, Casson, and Power law were usually used to present the flow profile of blends and hydrogels to describe shear stress and shear behaviour. The Herschel–Bulkley rheological model is mostly preferred due to its accuracy. In the present study, the rheological Herschel–Bulkley model was used to analyse the experimental result data (shear stress and viscosity-shear rate) of hydrogel solutions (Table3).The yield stress (Ʈo), flow behaviour index (ƞ), and consistency coefficient (k) were also calculated using the software provided with the rheometer. The yield stress (Ʈo) represents the interaction potential of colloidal particles in the biopolymer and also plays an important role in demonstrating the macrolattice structure of biopolymers [32]. The rheological Herschel–Bulkley model exhibited yield stress values for hydrogel samples which were studied (Table 3). The Ʈo and consistency coefficient (k) values of ALG-GEL and CHI-GEL were found to have higher values as compared to those of CNC-ALG-GEL and CNC-CHI-GEL hydrogels, respectively. It has been stated that the method of analysis and experimental condition influences the yield stress [33]. Several research studies have conflated views on the yield stress which could be linked with the transition between liquid and solid states and/or two same fluids with different viscosities [34].Table 3
Herschel–Bulkley parameters for hydrogels.
HydrogelsƮokƞ’R2CNC0.030.741.380.99ALG-GEL0.141.401.040.99CNC-ALG-GEL0.040.961.260.99CHI-GEL0.0820.50.800.99CNC-CHI-GEL0.0611.71.060.99Ʈo: yield stress; k: consistency coefficient; ƞ: flow behavior index; R2: regression coefficient.The ALG-GEL exhibited shear thickening and Newtonian behaviour, whereas the CHI-GEL exhibited shear thinning and pseudoplastic behaviour, which represents an irreversible structural breakdown (Figure6). The Newtonian behaviour of ALG-GEL at low shear rates was characterized by shear rate-independent viscosity. The shear thinning behaviour of CHI-GEL indicates the disrupted intermolecular junctions with a slower rate of reformations, which tends to drop in viscosity at a lower shear rate [35]. As the shear rate increases, the viscosity of CNC-ALG-GEL and CNC-CHI-GEL hydrogels tends to decrease. This might be due to the distributed and weak nature of the hydrogel network, and this leads to release of the entrapped liquid which resists the flow behaviour, thus inducing the lower viscosity [36]. This nonlinear relationship represents the higher viscosity of hydrogels which is a strong indication on the impact of cross-linking [22]. The viscosity of a shear thinning liquid has an inversely proportional relationship with the shear rate, whereas a shear thickening liquid has a directly proportional relationship between viscosity and the shear rate [37]. Pseudoplastic nature is important for topical formulations that have viscous behaviour under static conditions; nevertheless, this viscous behaviour tends to decrease at a higher shear rate which results in better spreadability and improves the drug permeation of specific substances upon topical application [11]. By the reinforcement of CNC to hydrogels, the viscosity vs. shear rate curve of CNC-ALG-GEL and CNC-CHI-GEL hydrogels has transformed to a plateau shape, due to the breaking down of CNC domains by shearing force [38]. With regard to the flow behaviour index, ƞ=1 for a Newtonian fluid and ƞ<1 for a shear thinning fluid or rheofluid, whereas ƞ>1 for shear thickening or rheothickening fluid. In the present study, ƞ values of CNC-ALG-GEL and CNC-CHI-GEL hydrogels indicated a Newtonian and shear thickening nature. This phenomenon of transition represents the network structure formed by CNC within alginate, chitosan, and gelatin which was constructed by strong hydrogen interactions and eventually maintains the viscosity [39, 40]. However, the increase in ƞ values of CNC-ALG-GEL and CNC-CHI-GEL hydrogels was also an indication of the shear thickening nature [2]. The biomaterial-based hydrogels reinforced with CNC showed a dependent behaviour which results from rheological characterization.Figure 6
Flow properties (shear stress vs. shear rate and viscosity vs. shear rate) of hydrogels. (a) CNC, (b) ALG-GEL, (c) CHI-GEL, (d) CNC-ALG-GEL, and (e)CNC-CHI-GEL.


## 3.7.In Vitro Degradation

The ALG-GEL and CHI-GEL hydrogels were found to have 50% of weight loss from day 1 to day 7 (Figure7). This illustrates that the majority of gelatin content in the respective hydrogel samples were susceptible to enzymatic degradation. In the hydrogel samples (CNC-ALG-GEL and CNC-CHI-GEL), 15% of weight loss were significantly observed from day 1 to day 7. In a study by Wang et al. [16], the hydrogels were prepared using alginate and gelatin biopolymers; moreover, the hydrogels without CNC were found to have 50% weight loss, and the hydrogels with CNC have significantly improved resistance to biodegradation. This improved resistance to enzymatic degradation might be due to homogenous dispersion and hydrophobicity [16]. It was believed that the CNC might have helped in creating the interpenetrated matrix between biomaterials alginate, chitosan, and gelatin due to cross-linking and can be expected to distribute uniformly by hydrogen bonding and hydrophobic interactions. Wen et al. [41] developed interpenetrated network (IPN) hydrogels with 25% of weight loss observed within 2 h of enzymatic degradation. They stated that the biomaterials blended with gelatin tend to have superior mechanical characteristics which resulted from interpenetrated matrix, and this provides good stability against degradation [41]. Furthermore, the crystallinity and hydrophilicity are important factors that influence the degradation rate. In addition, the surface hydrophilicity tends to cause the invasion of the degradation medium and water into the amorphous regions of CNC [42]. In this viewpoint of degradation, the biomaterial-based hydrogels reinforced with CNC are mostly recommended for bone and tissue engineering applications.Figure 7
In vitro degradation of CNCs and hydrogels.


## 3.8. Cytotoxicity

Thein vitro cytotoxicity of CNC and hydrogel membranes was investigated using the L929 cell line by MTT assay, and absorbance was read at 570 nm to evaluate cell viability [43]. In this method, the viability of the L929 fibroblast cell line was observed after 24 h of incubation. According to GB/T 16886.5-2003 (ISO 10993-5:1999), the biomaterial-based hydrogel membranes with cell viability more than 75% can be considered as noncytotoxic and are recommended for biomedical applications. The cell viabilities of ALG-GEL and CHI-GEL hydrogels were significantly higher than those of CNC (p<0.05). The CNC-ALG-GEL and CNC-CHI-GEL hydrogel membranes reinforced with CNC had 96% and 97% cell viabilities, respectively, which were significantly higher than CNC, ALG-GEL, and CHI-GEL hydrogels (p<0.05) (Figures 8(a) and 8(b)).Figure 8
(a) Cell viability assay (different letters indicate significant difference;p<0.05). (b) Fluorescence microscope images of CNC and hydrogel membranes at ×10 magnification.
(a)(b)The alginate and chitosan bionanocomposite membranes reinforced with CNC enhanced the viability ofNIH-3T3 fibroblasts [16, 24]. In an another study by Shaheen et al., the chitosan-alginate-hydroxyapatite scaffolds fabricated with CNC were found to have 100% cell viability and promising cell growth and cell adherence [5]. In a previous study of our lab, promising biocompatibility characteristics of polyvinyl alcohol/starch polymer were proven [44]. It was noticed that the mechanical properties and zwitterion polymer network provide the suitable environment for cell growth and proliferation [23]. Interaction of viable cells with the substratum matrix of hydrogel membranes reinforced with CNC via physicobiochemical and molecular interactions and surface topological parameters has an essential role in the cytocompatibility of biomaterial membranes such as cell adhesion, differentiation, proliferation, and migration [5]. Furthermore, the pore size of hydrogel membranes is mostly controlled by the zeta potential which enables the transportation of necessary bioactive molecules and migration of cells within the matrix of hydrogel membranes. In the present study, the zeta potential values of CNC-ALG-GEL, and CNC-CHI-GEL were quite different from those of ALG-GEL and CHI-GEL which might be due to the reinforcement of CNC. Surface charge modification by TEMPO was also most likely a probable reason for changes in zeta potential values. It was noted from a previous study that the CNC and gelatin promote the cell growth by forming mimetics of native bone tissue [45].


## 4. Conclusion

In the present study, biopolymer-based hydrogels reinforced with CNC were synthesized. Electrostatic chemical attractions, cross-linking of zinc ions, and intermolecular interactions were involved between CNC and alginate, chitosan, and gelatin used for the hydrogel preparation. These biopolymer-based hydrogel membranes were characterized by desirable physicochemical and morphological properties. Surface charge modification and the reinforcement of CNC enhanced the crystallinity and stability against degradation. Cytotoxicity studies carried out withL929 cell lines have confirmed its biocompatible nature for applications in tissue engineering, drug delivery, and biomedical fields. Also, hydrogels reinforced with CNC enabled effective cell adhesion, growth, and proliferation as evidenced in our study. However, further experimental studies need to be carried out for the applicability of these hydrogel membranes reinforced with CNC as a supportive biomaterial membrane in the treatment of burn wounds, skin implantations, in vivo drug delivery, and tissue transplantation.



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