MSC-4381

Biomimetic silk fibroin and xanthan gum blended hydrogels for connective tissue regeneration

Prasanna Kumar Byram, Krishna Chaitanaya, Anwesha Barik,Manish Kaushal, Santanu Dhara, Nishant Chakravorty

Abstract

Combination of naturally occurring materials instead of chemically synthesized products has always been an attractive proposition in the field of tissue engineering. In this study, silk fibroin (SF) and xanthan gum(XG) were physically crosslinked to form biocompatible hydrogels. SF/XG hydrogels were prepared using ultrasonication, which induces β-sheets from random coils in SF solution and allows entrapment of heated XG chains homogeneously in the SF network. It is a novel way of blending SF and XG polymers which avoids the usage of chemical crosslinkers. SF/XG blended solutions were used at different ratios for the hydrogel formation. Scanning electron microscopy (SEM) and micro-computed tomography (MCT) were used for morphological analysis of the interconnected network and porosity of the scaffolds, respectively. Rheological studies were performed to understand the changes in mechanical properties due to the incorporation of XG into SF hydrogels. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of SF and XG moieties in the blend scaffolds. Additionally, thermal Analysis (TGA & DSC) established the homogenous mixture and presence of XG in the SF network without any phase separation. Furthermore, the MTT assay demonstrates the cytocompatibility of scaffolds using L929 fibroblast cells. Thus, fabricated SF/XG scaffolds could mimic natural cartilage ECM by exhibiting enhanced water swelling capacity and suitable porosity along with its cytocompatible studies, indicating its potential application in soft tissue engineering.

Introduction

It allows entrapment of heated XG chains homogeneously in the SF network. It is a novel way of blending SF and XG polymers which avoids the usage of chemical crosslinkers. SF/XG blended solutions were used at different ratios for the hydrogel formation. Scanning electron microscopy (SEM) and micro-computed tomography (MCT) were used for morphological analysis of the interconnected network and porosity of the scaffolds, respectively. Rheological studies were performed to understand the changes in mechanical properties due to the incorporation of XG into SF hydrogels. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of SF and XG moieties in the blend scaffolds. Additionally, thermal Analysis (TGA & DSC) established the homogenous mixture and presence of XG in the SF network without any phase separation. Furthermore, the MTT assay demonstrates the cytocompatibility of scaffolds using L929 fibroblast cells. Thus, fabricated SF/XG scaffolds could mimic natural cartilage ECM by exhibiting enhanced water swelling capacity and suitable porosity along with its cytocompatible studies, indicating its potential application in soft tissue engineering. binding tissues together and protects various tissues and organs of the body. While most of the pathologies related to connective tissue disorders are largely considered to be associated with either genetic factors or ageing or trauma; however, all of these factors are known to contribute to tissue dysfunction to different extents[1]. Abnormality in structure and function of the connective tissue is believed to be a result of both mechanical and biological events destabilising the balance between degradation and synthesis of extracellular matrix (ECM)[2]. Majority of the clinical complications associated with cartilaginous tissue are due to the absence of blood vessels that leads to its poor regeneration capability and this has led the researchers to study on the biomaterials available for the native connective tissue replacement[3]. The armoury of cutting edge tools and techniques of tissue engineering and regenerative medicine have provided us with methods to develop novel cell-laden hydrogels which can mimic the structure and biochemical composition of native tissue[4]. Hydrogels have been significantly used in tissue engineering scaffolds since they can provide a soft tissue -like environment for enhancing the nutrients delivery, efficient exchange of gases, metabolites transport, cell attachment, proliferation and migration through the elastic hydrogel network[5].
A hydrogel is a three-dimensional (3D) material consisting of hydrophilic polymeric networks with high water absorption capability [6]. Hydrogels with tunable mechanical properties, biocompatibility, minimal inflammatory response and high permeability for oxygen and nutrients have garnered special attention in tissue engineering with their prospects in the development of promising biomaterials [7]. Although hydrogels are well known for their excellent biochemical and biological properties, they often suffer from poor mechanical strength, stiffness and solubility. Therefore to overcome these limitations, they are crosslinked by either physical or chemical methods [8]. Chemical crosslinking in hydrogels is a profoundly adaptable approach which is known to exhibit enhanced mechanical properties, stability and degradation response [9]. However, chemical crosslinking methods like free radical polymerisation, Schiff base formation, enzyme induced crosslinking, Michael type addition, oxime formation, etc. [10] often use or lead to the production of toxic substances which may lead to harmful tissue responses like inflammation, immunomodulation and tissue damage [11]. Further, chemical crosslinking often leads to the loss of beneficial properties of the individual components. In contrast, physical crosslinking techniques like entangled chains, hydrophobic interactions, hydrogen bonding, and crystallite formation [10], do not lead to any permanent bond formation, but instead they are sufficient to form insoluble hydrogels. Therefore the usage of a physical crosslinker is often advantageous over chemical crosslinker, since it aids in biological safety and avoiding cytotoxicity [11]. Physically crosslinked hydrogels are thus considered attractive materials for biomedical applications.
In general, many synthetic and natural biomaterials are available in nature which has their own importance and relevance in terms of structure and function. Among these, biomaterials like collagen, hyaluronic acid, gelatin and chitosan have been used extensively in repair/ regeneration of the native connective tissues [3]. Even though these biomaterials are used frequently, many of them have some limitation or the other like immunogenicity through the presentation of telopeptides [12], low mechanical properties[13], fast degradation[14], limited solubility at physiology pH[15] etc., impeding their potential usage in tissue regeneration. Besides, a special focus has been attributed to silk fibroin (SF) owing to its unique features like versatility, biodegradability, biocompatibility and tunable mechanical properties that makes it a successful material in tissue engineering [16]. The Food and Drug Administration (FDA) has approved silk fibroin scaffolds for soft tissue regeneration for a long time now [17][18]. Bombyx mori SF contains two proteins: (i) fibroin (70-80wt%), a structural protein and (ii) sericin (20-30wt%), a water-soluble glue, which coheres the fibroin fibres together [19]. The fibroin consists of heavy chain (H-370kDa) and light chain (L-25kDa) attached together by disulfide bonds and forms H-L complex. The high molecular weight heavy chains of fibroin consist of alternative hydrophilic and hydrophobic oligopeptides. The amino acid composition of fibroin consists of 18 types of amino acids including glycine, alanine, serine and tyrosine repeats which self-assemble into β-sheet conformation upon gelation [20][21]. This property has been exploited to fabricate fibroin into various forms: such as hydrogels [22], nanofibres [23], films [24], electrospun fibres [25] etc. and used in various in vitro and in vivo applications in the field of tissue engineering.
Xanthan gum (XG) is a heteropolysaccharide which is produced from Xanthomonas campestris bacteria through submerged aerobic fermentation[26][27]. XG was discovered at the National Centre for Agriculture Utilisation, the United States Department of Agriculture in 1950 [28]. The molecular weight of XG ranges from 2×106 – 2x107Da [29]. It consists of the main chain with repeating units of D-glucose connected by β1-4 linkage and a side chain of Dmannose and D-glucuronic acid[27]. XG’s main chain has a helical structure which coils around a side chain through hydrogen bonding[27]. Additionally, it exhibits various characteristic properties such as stability under a wide range of temperatures and pH levels, a high degree of pseudoplastic behaviour, high viscosity at low concentrations, and compatibility with metallic salts [27]. XG is used in several healthcare applications such as thickened fluids in dysphagia management [30], as a mucoadhesive for buccal mucosa [31], in the form of eye-drops for dry eyes [32], as agents for delivery of omega-e PUFA [33], etc. XG is similar to hyaluronic acid (HA) in rheology and viscosity, however it is more stable than HA[20][27]. Moreover, XG is known to support cartilage regeneration by inhibiting chondrocytes apoptosis, matrix metalloproteinase-1, -3 protein expressions and protecting the cartilage[20][34]. It has been approved by the FDA due to its biocompatible, bioadhesive, and wound healing properties which allow its multifaceted usage in various medical and pharmaceutical applications[28]. Unfortunately, owing to its poor physical strength, XG by itself cannot provide a suitable platform for connective tissue regeneration.
Generally, the extracellular matrix (ECM) is majorly composed of collagen and proteoglycans (one of the components is hyaluronic acid) which connects together to form a structurally stable composite, contributing to mechanical support of the tissue[2][35]. The alterations in the composition and structure of the ECM can be related to the abnormality in the connective tissue artefacts[36]. To mimic the microenvironment of ECM, we have chosen a silk fibroin(SF) and xanthan gum(XG) as biomaterials in this study. SF is a well-known biocompatible polymer with good mechanical properties and is known to mimic collagen in its function. [37]. On the other hand, XG is known to have similar viscoelasticity and more stable than hyaluronic acid in its function[20][27].
In the present study, we developed a novel physical crosslinking method for blending silk fibroin and xanthan gum biopolymers by means of ultrasonication as an alternative to chemical crosslinking. The technique induces the β-sheet formation of silk protein and entraps heated XG chains in the SF network. Furthermore, different proportions of SF and XG in SF/XG scaffolds have been characterized by rheology, SEM, micro CT, thermal analysis and cytocompatibility. These scaffolds offer stable and tunable systems along with cytocompatible properties to be useful in various regenerative medicine and soft tissue engineering applications. It is expected that such a combination will have the favourable properties of both SF and XG which will be beneficial for connective tissue regeneration.

2. Materials and methods 2.1 Materials

Silkworm Bombyx mori cocoons were acquired from a local silk farm near IIT Kharagpur. Lithium bromide (LiBr), Xanthan gum and Thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich (USA). Sodium carbonate (Na2CO3) and Dimethyl sulfoxide (DMSO) were purchased from Merck (USA). Dialysis membrane (MWCO 3.5K), Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), and Trypsin–EDTA were purchased from Thermofisher (USA). L929 cells were obtained from NCCS Pune, India.

2.2 Preparation of Aqueous Silk Fibroin Solution

Silk fibroin was extracted from the cocoons of Bombyx mori as described in the previous studies [38]. Briefly, silk cocoons (5 grams) were cut into small pieces and boiled at 100 °C for 30 min in 2 L of deionized water by adding 0.02 M sodium carbonate (Na2CO3) to remove sericin. Further, these degummed cocoons were rinsed thoroughly with deionized water for 20 min three times to remove sericin completely. Then the fibroin was allowed to dry at 37 °C for overnight. The dried fibroin was dissolved in 9.3M lithium bromide (LiBr) for 4 h at 60 °C and dialysis against deionized water in 3500 MWCO dialysis membrane for 48 h. To remove any impurities, the solution was centrifuged at 9000 rpm for 20 min at 4°C. The final silk fibroin concentration obtained was 8% w/v, which was diluted to 2% w/v with deionized water for further studies.

2.3 Preparation of Xanthan gum Solution

The xanthan gum powder (1 wt%) was dissolved in cold deionized water at room temperature by stirring. Following this dissolved solution was heated at 85°C for 60 min [39][40].

2.4 Preparation of ultrasonication silk /xanthan gum hydrogels

The silk/xanthan gum hydrogels were prepared by adding heated XG to the SF solution in different proportions to make a final volume of 5 ml, as shown in Table 1. The components were allowed to mix thoroughly using magnetic stirring. Subsequently, the blended solutions were ultrasonicated at 55% amplitude for 2 min to prepare the hydrogels [41]. These samples were transferred to -20°C for overnight incubation, followed by lyophilization for 24 hours. Further, to increase the strength and sterilization, scaffolds were treated with 70% (v/v) The porosity of lyophilized silk and silk /xanthan gum scaffolds after ethanol treatment was observed by scanning at 1000 scan slice /sample at accelerating voltage of 90 kV, and a beam current of 40 mA with voxel size (resolution) of 3.8 μm using micro CT ( GE Phoenix V|tome|X).

2.6 Swelling Behaviour

The silk and silk/ xanthan gum hydrogels were lyophilized to form the scaffolds. The dry weights of the scaffolds were recorded, and then the scaffolds were immersed in 1X phosphate-buffered saline (PBS, pH 7.4) at 37 °C. At regular time intervals, PBS was removed from the surface of the scaffolds with blotting paper, and then swollen weight of the hydrogels was measured. Swelling ratio was calculated by using equation (1)[43] : Where Ws is the swollen weight, and Wd is the dry weight of scaffolds. Each data point was obtained by average values from three samples.

2.7 Fourier transform infrared spectroscopy ( FTIR )

The lyophilized powder samples of silk fibroin and blended silk/xanthan gum were characterized using FTIR to confirm the molecular interactions and presence of biopolymers in scaffolds. FTIR was performed for all samples using KBr pelletization technique in the range of 4000-700 cm-1 using Nicolet 6700 FTIR spectrophotometry (Thermo Scientific, USA).

2.8 Rheological

The rheological assessment of hydrogels (20mm diameter and 5mm thickness) were carried out using Anton Paar Rheometer (MCR-302). The stress amplitude sweep and frequency sweep tests of hydrogels were carried out in order to compare the relative viscoelastic nature of hydrogels in terms of dynamic moduli (storage and loss modulus: G′ and G′′ respectively). All the rheological experiments, measurements were conducted at 25°C, using a parallel plate geometry (of diameter 25 mm) maintaining an inter-plate gap of 3.8 mm. In case of stress amplitude sweep test, the sinusoidal stress field (𝜎 = 𝜎0 𝑠𝑖𝑛𝜔𝑡) was imposed, wherein stress amplitude was varied in the range of 10-1-103 Pa at a frequency of 0.1 Hz. The frequency sweep test was carried out in the linear viscoelastic regime, by imposing the oscillatory strain field (𝛾 = 𝛾0 𝑠𝑖𝑛𝜔𝑡, where 𝛾0 is in the linear regime) with a frequency variation in the range of 0.05 to 10 Hz.

2.9 Thermal Analysis

Thermal properties of SF/XG scaffolds were determined by using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA measurements were conducted using Perkin Elmer(USA). The Lyophilized powder samples (5-6mg) were loaded in an alumina pan and heating in a nitrogen atmosphere from 30 °C to 600 °C at a heating rate of 10 °C/min.
DSC measurements were performed by using Pyris Diamond DSC (USA). Calibration of temperature and enthalpy were done using the t zero hermetic aluminium pan. Lyophilized powder samples around 3 – 5 mg were encapsulated in an aluminium pan and heated at the rate of 10 °C/min in the presence of the nitrogen gas flow 30ml/min.

2.10 MTT

In vitro cytotoxicity of hydrogels was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Different concentrations of SF/XG hydrogels were prepared with a fixed dimension of 12 mm diameter and 2 mm thickness. The hydrogels were sterilized by ethanol treatment for 5 h, followed by phosphate buffer saline (PBS) wash. L929 fibroblast cells were seeded on hydrogels at a density 2×104 cells/well and cultured using DMEM media supplemented with 10% FBS. Cytotoxicity of hydrogels at different incubation time intervals of 24, 72 and 120 h was analysed by using an MTT assay. MTT was added at 5 mg/ml concentration to each hydrogel and incubated at 37 °C for 4 h. After 4 h, MTT was replaced with dimethyl sulfoxide (DMSO), and absorbance was measured at 595 nm using Bio-Rad microplate absorbance reader. Each combination of SF/XG hydrogel was set up in triplicates.

2.11 Statistical analysis

The experiments were multi- replicated, and obtained data were reported as mean ± standard deviation (SD)( n=3). The analyses were performed using Graph Pad Prism 5.0 by one-way analysis of variance (ANOVA) and statistically significant data was selected at P<0.05. In the case of MTT, Dunnett post-hoc test was used to compare between different groups by considering 24 h as a control. 3.Results As shown in fig.1, schematic representation of hydrogel fabrication process and their appearance. 3.1 Surface morphological and porosity analysis The microstructure of fabricated SF and XG scaffolds were examined by SEM micrographs, as shown in fig 2. Upon studying the microarchitecture of scaffolds, SF scaffolds were observed to have a porous network of interconnected fibres. In contrast, the blended SF/XG scaffolds exhibited leafy morphology. The morphology appeared to be similar to the increasing concentrations of XG from 10 % to 50 %. Furthermore, the SEM analysis also demonstrated that the individual polymers in the scaffolds were undistinguished from each other, thus confirming uniform blending. The interconnected nature of porous scaffolds is considered necessary for cell attachment, growth, proliferation and exchange of nutrition. The SF/XG blends were thus successfully prepared by freeze-drying technique to achieve interconnected porous architecture. The micro CT images of silk and blended silk/xanthan gum samples have been shown in fig 2. The useful in tissue engineering applications. 3.3 Fourier transform infrared spectroscopy (FTIR ) FTIR spectrum of silk fibroin was analyzed in the range of 400 -4000 cm-1[11][44]. The range of 1500 -1700 cm-1 IR was consigned to peptide backbone, amide I (1700 – 1600 cm-1), amide II (1600 -1500 cm-1 ) and amide III (1350-1200cm-1). Amide I region was observed mainly due to stretching vibration of C=O (>80%) which directly relies upon the protein secondary structure quantitatively. Amide II region formation can be attributed to the out of phase combination of NH bending vibrations and CN stretching. Formation of Amide III region was due to NH bending vibration modes. The CH2 bending band of protein was observed (methylene scissoring mode) at 1437cm-1. The major deformation of CH2, CH3 and minor COO- the symmetric stretching peak was observed at 1405 cm-1. FTIR spectrum of Xanthan gum (XG) alone was observed at the range of 400-4000 cm-1[45]. The IR range 3400-3450 cm-1 for – OH group, 2850 – 2950 cm-1 for C- H (present in CH2, CH3 and CHO), 1700-1600 cm-1 for C=O group (present in ester, aldehyde, ketone and acid), 1430- 1650 cm-1 for C=O (in enols), 1410- 1430 cm-1 for C-H, and 1050- 1100 cm-1 for C- O.
The silk fibroin (SF) spectra were observed at 1640 cm-1 for Amide I, 1517 cm-1 for Amide II and 1240 cm-1 for Amide III, in the same range with respect to XG FTIR spectrum. Increased peak intensities were observed with increased intensity of the amide I, amide II and amide III without any shift in the peaks with an increase of XG concentration. The vibration peaks of glycosidic groups (C-O-C) were also seen to intensify with an increasing concentration of XG content (around 1174 cm-1) since silk fibroin doesn’t contain any significant vibration mode in this region shown in fig 4. This region can be set as an indicator peak of XG component in the SF/XG scaffolds, which has shown no peak shift at different compositions. Similar results were observed in silk fibroin, and hyaluronic acid (polysaccharide) blended polymers using sonication method [11]. These results confirm the blending and presence of silk and xanthan gum in the scaffolds.
The results of the stress amplitude sweep test are shown in fig 5a, wherein the storage and loss modulus has been plotted as a function of stress amplitude for various compositions of hydrogels. For any such curve, first, a storage modulus (G′) plateau has been observed (known as a linear regime) followed by a sharp decrease in G′ value, which signifies solid-like to liquid-like transition, and is known as the yield point (the corresponding stress is considered to yield stress). It is amply clear from fig 5a, that with an increase in the concentration of SF in the SF/ XG hydrogels, the linear viscoelastic regime as well as yield stress, both increase.
The results of the frequency sweep test, as shown in fig 5b, clearly indicates that with the increase in SF content in the hydrogels, the frequency-dependent storage modulus curve shifts upwards to higher magnitude. Both the observations confirm that incorporation of SF leads to increase the β-sheet formation, thereby reduction in chain flexibility. This eventually enhances the modulus of rigidity of the hydrogel. However, the above trends in rheological properties help us to choose an optimal composition of these hydrogels based on the desired mechanical strength of silk fibroin and xanthan gum. porosity. All the scaffolds showed a plateau phase when the temperature was raised to 120°210°C. The second phase of weight loss was observed at between 270°C and 380°C. This may be associated with the breakdown of chain length into small groups and peptide bonds and thus leading to degradation and weight loss of the scaffolds. Ultrasonicated SF showed higher stability than the SF/XG scaffolds. It was observed that SXG2 was found to be more thermally stable than the other fabricated scaffolds, as the molecular interactions of silk and xanthan gum were found to be maximum at this ratio as shown in the first derivate. Above 400°C, the degradation pattern was similar for all the samples. The obtained TGA results indicate that SF/XG were well blended prior to formation of the scaffolds
The DSC thermograms for SF and SF/XG blends powder has been shown in fig 6b with heat flow at the rate of 10°C/min as a function of temperature. DSC curves with endothermic peaks show the degradation temperature of SF and SF/XG blends. The SF, endothermic peak at 290°C related to its thermal degradation temperature. As the xanthan gum concentration was increased, the endothermic peaks were seen to decrease below 290°C, indicating the change in 300 to 6000 C with a heating rate of 100 c/ min in a nitrogen atmosphere . b) DSC curves of silk and silk/xanthan gum scaffolds at the rate of 100 c/ min in the presence of the nitrogen gas flow in 30ml/min.

3.6 MTT

Fibroblasts cells, L929, murine fibroblast cell line were seeded on hydrogels and incubated for 24, 72 and 120 h. A response curve was plotted for different time intervals against optical density (OD) at 595 nm shown in fig 7a. All the SF and SF/XG scaffolds showed similar absorbance after 24 h of incubation, thus indicating similar levels of metabolic activity. Further, after 72 h, a similar level of increase in the mitochondrial activity of L929 cells on all the scaffolds. This finding suggests that the porous interconnected network might promote cellular activity and allow better cellular proliferation, leading to cellular crosstalk. Moreover, the proliferation of fibroblasts was evident from the statistically significant increase ((*p ≤

4.Discussion

In the present study, the physical crosslinking technique is used to fabricate the silk fibroin (SF) and xanthan gum (XG) hydrogels. Preferring ultrasonication over the other physical methods helped in entrapment of cells or compounds in the SF network, since, there is a lag time between sonication and onset of gelation of SF[19]. However, XG doesn’t undergo gelation during the sonication process as it is a heteropolysaccharide with a helical chain structure. Thus, the gelation is exclusively controlled by SF. Furthermore, apart from the ultrasonication method, XG cannot be blended homogeneously with other compounds due to its helical nature. In the present work, XG was heated at 85°C for 60 min to undergo structural conformation by means of order-disorder reaction. Also, sonication induces β-sheets from random coils in SF solution[46][47] and allows entrapment of heated XG chains homogeneously in the SF network fig 1.
Using the freeze-drying technique, SF/XG hydrogels were fabricated to be porous in nature with an interconnected network. SEM studies revealed the microarchitecture of SF hydrogel as porous network compared with the other composite hydrogels. Indeed, the SF/XG composite hydrogels exhibited leafy morphology with the interconnected network. The porosity of the SF/XG composite hydrogels was determined by micro-CT analysis. The findings revealed that SF hydrogels were found to have the smallest porosity percentage 53.71± 1.8, whereas the composite hydrogel, i.e., SXG5 had shown higher porosity percentage of 71.48 ± 1.4. The increased porosity of the composite hydrogels may contribute to enhancing the nutrients delivery, efficient exchange of gases, metabolites transport, cell attachment, proliferation and migration[31]. The swelling behaviour of the hydrogels was further explored to understand the uptake of media and to study the retainment of hydrogel shape, which is vital in cell culture/implantation studies. It was observed that blended SF/XG hydrogels have higher swelling nature when compared to the SF hydrogels. The reason behind the higher swelling rate was due to hydrophilic nature and anionic characteristics of XG[48]. The rate of absorption of sterile PBS by hydrogels was found to be higher at the initial 30 min and attained equilibrium within 3 hours. Thus, the results obtained have shown that SXG5 has a higher porosity percentage, better swelling ratio and enhanced cell proliferation (L929 cells) rate when compared with the SF.
FTIR studies have confirmed the presence of SF and XG moieties in the blended hydrogels. Moreover, the vibration peaks of the glycosidic groups (C-O-C) in the hydrogels were observed to be intensified (around 1174 cm-1) with an increase in the concentration of XG percentage, since, the SF doesn’t contain any significant vibration in that region. Similar results were observed in silk fibroin, and hyaluronic acid (polysaccharide) blended polymers using sonication method[6].
Rheological studies were performed to understand the mechanical properties of the hydrogels.
The stress amplitude sweep test shows that Gʹ>Gʺ for all the fabricated hydrogels was mainly exhibiting their elastic nature[49]. With an increase in the concentration of XG percentage in the composite hydrogels, there was a sharp decrease in the G′ value compared with the SF. This implies the transition phase of the hydrogels between solid-like to liquid-like conformation. Moreover, the frequency sweep test shows the frequency-dependent storage modulus curve G′ values decreases with increase in the XG composition in blended scaffolds. Both the observations confirm that incorporation of Silk-fibroin leads to the greater extent of network formation and thus enhances the tensile properties/mechanical strength of the hydrogel.
TGA and DSC analysis were carried out to examine the thermal properties of the hydrogels. The TGA results emphasized on change in the weight percentage of the hydrogel during heating from room temperature to 600°C. Initial weight loss of hydrogels was witnessed around 7 to 10 % because of bound and absorbed free water from the samples. Primarily, the weight loss of SF was found to be around 7 % but with the increase of XG percentage in the composite hydrogel resulted in more weight loss. The derivative thermogravimetry increased with the addition of XG to SF. Also, the stability of the SF was found to be more prominent over the SF/XG hydrogel in the second phase of the weight loss, as shown in fig 6a. It is noteworthy to mention that SXG2 exhibited more thermal stability than the other composite hydrogels. In addition to the TGA thermal results, DSC studies had further showcased the changes in the degradation temperature of the hydrogels with the addition of XG to SF. SF was found to be more thermally stable at 290°C and a gradual increase in XG percentage resulted in decrease of the degradation temperature. Also, the DSC results attempted to highlight the changes in the physical properties of the hydrogels after the interaction of XG with SF. Altogether, TGA and DSC studies confirmed the homogenous mixture and presence of XG in the SF network.
In vitro cytotoxicity analysis was performed to assess the biocompatibility of hydrogels. The MTT assay was performed using L929 fibroblast cells (suggested for use per ISO Standard10993-5) seeded on hydrogels[50]. Uptake of MTT into the live cells will determine the mitochondrial activity/ survival and proliferation of cells. MTT analysis further confirmed that the hydrogels were supportive in the growth of L929 cells. Indeed, it was found that the mitochondrial activity/ proliferation of cells was increased with the exposure time of cells on the hydrogels were increased, thus explaining its biocompatible nature.

4. Conclusion

In this study, silk fibroin and xanthan gum hydrogels were fabricated with green technology using ultrasonication method. Physicochemical characterization of the hydrogels were performed using various techniques like TGA, DSC and FTIR to confirm the blending ability of silk and xanthan gum polymers. Thus fabricated scaffolds exhibited different architecture, increased porosity with water retention and interconnected networks with increased xanthan gum concentration when compared with the silk fibroin. The rheological studies reveal that incorporation of silk fibroin leads to the greater extent of network formation owing to β-sheet formation and thus enhances the mechanical properties of the hydrogel. Furthermore, nontoxic nature was attributed to silk and silk/xanthan gum hydrogels that supported L929 cells for its viability and enhanced cellular proliferation after 120 h. The mechanical stability, porosity, water swelling capacity and cytocompatibility features of SF/XG scaffolds were established through this work. Several reports have suggested XG has therapeutic activity that can inhibit chondrocytes apoptosis, matrix metalloproteinase-1,-3 protein expression significantly and protects the connective tissue. These studies, along with our work, indicate towards its the potential use of SF/XG scaffolds in repair/regeneration various connective tissues with further validation.

8.References

[1] B. R. Freedman and D. J. Mooney, “Biomaterials to Mimic and Heal Connective Tissues,” Adv. Mater., vol. 31, no. 19, pp. 1–27, 2019, doi: 10.1002/adma.201806695.
[2] A. Manuscript, “NIH Public Access,” vol. 0000000000, pp. 1–8, 2015, doi: 10.1097/IJG.0000000000000108.Biology.
[3] L. Li et al., “Natural hydrogels for cartilage regeneration: Modification, preparation and application,” J. Orthop. Transl., vol. 17, no. 2, pp. 26–41, 2019, doi: 10.1016/j.jot.2018.09.003.
[4] H. Geckil, F. Xu, X. Zhang, S. Moon, and U. Demirci, “Engineering hydrogels as extracellular matrix mimics.Geckil, H., Xu, F., Zhang, X., Moon, S., & Demirci, U. (2010). Engineering hydrogels as extracellular matrix mimics. Nanomedicine (London, England), 5(3), 469–84. http://doi.org/10.2217/nnm.10.12,” Nanomedicine (Lond)., vol. 5, no. 3, pp. 469–84, 2010, doi: 10.2217/nnm.10.12.
[5] † and Roger E Marchant Junmin Zhu1, “NIH Public Access Author Manuscript Expert Rev Med Devices. Author manuscript; available in PMC 2012 July 1. Published in final edited form as: Design properties of hydrogel tissue-engineering scaffolds Expert,” Expert Rev Med Devices, vol. 8, no. 5, pp. 607–626, 2012, doi: 10.1586/erd.11.27.Design.
[6] A. Manuscript, “NIH Public Access,” vol. 8, no. 5, pp. 607–626, 2012, doi: 10.1586/erd.11.27.Design.
[7] K. T. Nguyen and J. L. West, “Photopolymerizable hydrogels for tissue engineering applications,” Biomaterials, vol. 23, no. 22, pp. 4307–4314, 2002, doi: 10.1016/S0142-9612(02)00175-8.
[8] K. M. Salleh et al., “Chemically crosslinked hydrogel and its driving force towards superabsorbent behaviour,” Int. J. Biol. Macromol., vol. 118, pp. 1422–1430, 2018, doi: 10.1016/j.ijbiomac.2018.06.159.
[9] J. Maitra and V. K. Shukla, “Cross-linking in Hydrogels – A Review,” Am. J. Polym. Sci., vol. 4, no. 2, pp. 25–31, 2014, doi: 10.5923/j.ajps.20140402.01.
[10] W. Hu, Z. Wang, and Y. Xiao, “Biomaterials Science Advances in crosslinking strategies of biomedical hydrogels,” pp. 843–855, 2019, doi: 10.1039/c8bm01246f.
[11] X. Hu et al., “Biomaterials from Ultrasonication-Induced Silk Fibroin – Hyaluronic Acid Hydrogels,” pp. 3178–3188, 2010.
[12] J. Glowacki and S. Mizuno, “Collagen scaffolds for tissue engineering.,” Biopolymers, vol. 89, no. 5, pp. 338–344, May 2008, doi: 10.1002/bip.20871.
[13] Q. Xing, K. Yates, C. Vogt, Z. Qian, M. C. Frost, and F. Zhao, “Increasing Mechanical Strength of Gelatin Hydrogels by Divalent Metal Ion Removal,” Sci. Rep., vol. 4, no. 1, p. 4706, 2014, doi: 10.1038/srep04706.
[14] and B. S. P. Jonathan Posner, James A. Russell, “基因的改变NIH Public Access,” Bone, vol. 23, no. 1, pp. 1–7, 2008, doi: 10.1038/jid.2014.371.
[15] B. Applications, “marine drugs,” pp. 1819–1846, 2015, doi: 10.3390/md13041819.
[16] G. Cheng et al., “Advanced Silk Fibroin Biomaterials for Cartilage Regeneration,” ACS Biomater. Sci. Eng., vol. 4, no. 8, pp. 2704–2715, 2018, doi: 10.1021/acsbiomaterials.8b00150.
[17] D. S. Musson et al., “ In Vitro Evaluation of a Novel Non-Mulberry Silk Scaffold for Use in Tendon Regeneration ,” Tissue Eng. Part A, vol. 21, no. 9–10, pp. 1539–1551, 2015, doi: 10.1089/ten.tea.2014.0128.
[18] Y. Qi et al., “A review of structure construction of silk fibroin biomaterials from single structures to multi-level structures,” Int. J. Mol. Sci., vol. 18, no. 3, 2017, doi: 10.3390/ijms18030237.
[19] D. N. Rockwood, R. C. Preda, T. Yücel, X. Wang, M. L. Lovett, and D. L. Kaplan, “Materials fabrication from Bombyx mori silk fibroin,” Nat. Protoc., vol. 6, no. 10, pp. 1612–1631, 2011, doi: 10.1038/nprot.2011.379.
[20] Z. Zhang et al., “Functionalization of polyethylene terephthalate MSC-4381 fabrics using nitrogen plasma and silk fibroin/chitosan microspheres,” Appl. Surf. Sci., vol. 495, no. July, p. 143481, 2019, doi: 10.1016/j.apsusc.2019.07.223.
[21] S. K. Samal, D. L. Kaplan, and E. Chiellini, “Ultrasound sonication effects on silk fibroin protein,” Macromol. Mater. Eng., vol. 298, no. 11, pp. 1201–1208, 2013, doi: 10.1002/mame.201200377.
[22] P. Wu et al., “Novel silk fibroin nanoparticles incorporated silk fibroin hydrogel for inhibition of cancer stem cells and tumor growth,” Int. J. Nanomedicine, vol. 13, pp. 5405–5418, 2018, doi: 10.2147/IJN.S166104.
[23] J. Chen et al., “Enhanced physical and biological properties of silk fibroin nanofibers by layer-by-layer deposition of chitosan and rectorite,” J. Colloid Interface Sci., vol. 523, pp. 208–216, 2018, doi: 10.1016/j.jcis.2018.03.093.
[24] C. Cubayachi et al., “Silk fibroin films stabilizes and releases bioactive insulin for the treatment of corneal wounds,” Eur. Polym. J., vol. 118, no. June, pp. 502–513, 2019, doi: 10.1016/j.eurpolymj.2019.06.022.
[25] C. Pignatelli, G. Perotto, M. Nardini, R. Cancedda, M. Mastrogiacomo, and A. Athanassiou, “Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate,” Acta Biomater., vol. 73, pp. 365–376, 2018, doi: 10.1016/j.actbio.2018.04.025.
[26] A. C. Mendes, E. T. Baran, R. C. Pereira, H. S. Azevedo, and R. L. Reis, “Encapsulation and Survival of a Chondrocyte Cell Line within Xanthan Gum Derivative,” Macromol. Biosci., vol. 12, no. 3, pp. 350–359, 2012, doi: 10.1002/mabi.201100304.
[27] G. Han et al., “Preparation of xanthan gum injection and its protective effect on articular cartilage in the development of osteoarthritis,” Carbohydr. Polym., vol. 87, no. 2, pp. 1837–1842, 2012, doi: 10.1016/j.carbpol.2011.10.016.
[28] A. Kumar, K. M. Rao, and S. S. Han, “Application of xanthan gum as polysaccharide in tissue engineering: A review,” Carbohydr. Polym., vol. 180, no. September 2017, pp. 128–144, 2018, doi: 10.1016/j.carbpol.2017.10.009.
[29] H. Shao et al., “Intra-articular injection of xanthan gum: A potential therapy for osteoarthritis,” Adv. Biosci. Biotechnol., vol. 03, no. 04, pp. 449–453, 2012, doi: 10.4236/abb.2012.324063.
[30] L. Rofes, V. Arreola, R. Mukherjee, J. Swanson, and P. Clavé, “The effects of a xanthan gum-based thickener on the swallowing function of patients with dysphagia,” Aliment. Pharmacol. Ther., vol. 39, no. 10, pp. 1169–1179, 2014, doi: 10.1111/apt.12696.
[31] R. R. Shiledar, A. A. Tagalpallewar, and C. R. Kokare, “Formulation and in vitro evaluation of xanthan gum-based bilayered mucoadhesive buccal patches of zolmitriptan,” Carbohydr. Polym., vol. 101, no. 1, pp. 1234–1242, 2014, doi: 10.1016/j.carbpol.2013.10.072.
[32] F. Faraldi et al., “A new eye gel containing sodium hyaluronate and xanthan gum for the management of post-traumatic corneal abrasions,” Clin. Ophthalmol., vol. 6, no. 1, pp. 727–731, 2012, doi: 10.2147/OPTH.S31776.
[33] S. Trombino, S. Serini, R. Cassano, and G. Calviello, “Xanthan gum-based materials for omega-3 PUFA delivery: Preparation, characterization and antineoplastic activity evaluation,” Carbohydr. Polym., vol. 208, no. January, pp. 431–440, 2019, doi: 10.1016/j.carbpol.2019.01.001.
[34] W. Zhang et al., “Lower range of molecular weight of xanthan gum inhibits apoptosis of chondrocytes through MAPK signaling pathways,” Int. J. Biol. Macromol., vol. 130, pp. 79–87, 2019, doi: 10.1016/j.ijbiomac.2019.01.071.
[35] K. Words and C. H. Clark, “Connective Tissues : Matrix,” vol. 79, no. 3, 1999.
[36] T. J. Mckee, G. Perlman, M. Morris, and S. V Komarova, “Extracellular matrix composition of connective tissues : a systematic review and meta-analysis,” Sci. Rep., no. July, pp. 1–15, 2019, doi: 10.1038/s41598-019-46896-0.
[37] T. P. Nguyen, Q. V. Nguyen, V. Nguyen, T. Le, and Q. Van Le, “Silk Fibroin-Based Biomaterials for Biomedical,” pp. 1–25, 2019.
[38] S. Ghosh, “Enhanced Redi ff erentiation of Chondrocytes on Microperiodic Silk/ Gelatin Sca ff olds: Toward Tailor-Made Tissue Engineering,” 2013, doi: 10.1021/bm301193t.
[39] S. K. H. Gulrez, S. Al-Assaf, Y. Fang, G. O. Phillips, and A. P. Gunning, “Revisiting the conformation of xanthan and the effect of industrially relevant treatments,” Carbohydr. Polym., vol. 90, no. 3, pp. 1235–1243, 2012, doi: 10.1016/j.carbpol.2012.06.055.
[40] J. Moffat, V. J. Morris, S. Al-Assaf, and A. P. Gunning, “Visualisation of xanthan conformation by atomic force microscopy,” Carbohydr. Polym., vol. 148, pp. 380– 389, 2016, doi: 10.1016/j.carbpol.2016.04.078.
[41] “US_8722067_B2.pdf.” .
[42] J. Rnjak-Kovacina, T. M. Desrochers, K. A. Burke, and D. L. Kaplan, “The effect of sterilization on silk fibroin biomaterial properties,” Macromol. Biosci., vol. 15, no. 6, pp. 861–874, 2015, doi: 10.1002/mabi.201500013.
[43] Y. P. Singh, N. Bhardwaj, and B. B. Mandal, “Potential of Agarose/Silk Fibroin Blended Hydrogel for in Vitro Cartilage Tissue Engineering,” ACS Appl. Mater. Interfaces, vol. 8, no. 33, pp. 21236–21249, 2016, doi: 10.1021/acsami.6b08285.
[44] X. Hu, D. Kaplan, and P. Cebe, “Dynamic Protein – Water Relationships during -Sheet Formation,” pp. 3939–3948, 2008.
[45] M. A. S. P. N. Hazirah, M. I. N. Isa, and N. M. Sarbon, “Effect of xanthan gum on the physical and mechanical properties of gelatin-carboxymethyl cellulose fi lm blends,” Food Packag. Shelf Life, vol. 9, pp. 55–63, 2016, doi: 10.1016/j.fpsl.2016.05.008.
[46] T. Vu et al., “Comparative study of ultrasonication-induced and naturally selfassembled silk fibroin-wool keratin hydrogel biomaterials,” Int. J. Mol. Sci., vol. 17, no. 9, pp. 1–15, 2016, doi: 10.3390/ijms17091497.
[47] W. Xiao, W. Liu, J. Sun, X. Dan, D. Wei, and H. Fan, “Ultrasonication and Genipin Cross-Linking to Prepare Novel Silk Fibroin–Gelatin Composite Hydrogel,” J. Bioact. Compat. Polym., vol. 27, no. 4, pp. 327–341, Jun. 2012, doi: 10.1177/0883911512448692.
[48] S. Sethi, Saruchi, B. S. Kaith, M. Kaur, N. Sharma, and V. Kumar, “Cross-linked xanthan gum–starch hydrogels as promising materials for controlled drug delivery,” Cellulose, vol. 27, no. 8, pp. 4565–4589, 2020, doi: 10.1007/s10570-020-03082-0.
[49] A. Nanodispersion, F. Cuomo, M. Cofelice, and F. Lopez, “Rheological Characterization of Hydrogels from,” 2019, doi: 10.3390/polym11020259.
[50] O. Gsib et al., “Evaluation of fibrin-based interpenetrating polymer networks as potential biomaterials for tissue engineering,” Nanomaterials, vol. 7, no. 12, pp. 1–21, 2017, doi: 10.3390/nano7120436.