3.1. Characterization of Ch-SF hydrogel
In order to overcome the long-term cytotoxicity of metal nanoparticles in tissue engineering applications, hydrogels were incorporated with metal nanoparticles which are efficient with electrical and biological characteristics of biological tissues that require the conductivity [26,27]. In recent years, the blends of natural polymers have been attractive considerably significant due to their potential in replacing synthetic polymers in various applications in addition of being renewable resources, non-toxic, inexpensive and leave biodegradable waste [28,29]. Among natural polymers, chitosan and its blends have received special interest due to its versatility and suitability for a numerous number of applications. Further, the chitosan properties have been improved by blending with synthetic and naturally occurring macromolecules [30–34]. In this aspect, the hydrogel polymer was prepared by gold loaded with chitosan-silk fibroin complex which are well known as the conductive hydrogel.
FT-IR spectroscopy was used to find out the functional groups present in Chitosan (Ch) and Chitosan/Silk fibroin (Ch-SF) hydrogel (Figure 1). The IR spectrum of the Ch displayed main absorption peaks at 3481, 2930, 1745, 1702, 1212 and 693 cm-1 respectively. Whereas, the IR spectrum of Ch-SF had shown major absorption peaks at 3468, 3042, 1842, 1740, 1496 and 1164 cm-1 respectively. The observed broad absorption peaks at 3400 to 3500 cm-1 was due to the stretching vibration of −NH2 and −OH groups. The band at 3042 cm-1 found out the vibrational stretches of methylene present in the Ch. The band at 1700 to 1900 cm-1 was due to the absorption of the peptide backbones of N–H bending vibration of amides I, II, and III, respectively present in Ch and Ch-SF hydrogels . The peak differences in the 1900 to 1200 cm-1 have confirmed the differentiation pattern of two hydrogels. The overlapped absorption bands, intensity and differences indicated the intermolecular H bond interaction and conjugation of Ch with SF . Further, the structures of the hydrogel Ch and Ch-SF conjugates were confirmed by 1H NMR (Figure 1). The spectrum of Ch-SF exhibited new signals at δ 4.14, δ 4.21 and δ 3.31 when compared with Ch spectrum which indicated the existence of the conjugated protons of Ch-SF.
3.2 Development of Gold loaded Ch-SF hydrogel
Gold nanoparticle formation was evidently observed with the pink-red color formation in the reaction mixture containing HAuCl4∙3H2O with the Ch-SF. UV-Vis spectral analysis has confirmed the presence of AuNPs by the absorbance peak at 530 nm. The comparative analysis of newly prepared [email protected] hydrogel and the same after 30 days were depicted in Figure 2. Both the spectra were found to be almost same which confirms the stability of the synthesized hydrogel. Varieties of metal nanoparticles have been used in the preparation of nano-scaffold hydrogels in the field of biomaterials including gold  and silver . Since metal nanoparticles possess the desired electrical conductivity, magnetic properties, and antibacterial properties. The nano-scaffold hydrogels along with metal nanoparticles are widely used in conductive scaffolds [39–41].
3.3 Mechanical properties of [email protected] hydrogel
Preparation of injectable hydrogel is a challenging task due to immediate gelation properties of hydrogels. The slow gelation rate leads to the formation intermediate reactions before the gelling which is the desired property . The gelation property of Ch, Ch-SF and [email protected] has shown in Figure 2. The gelation time of [email protected] was comparatively less than the time taken for Ch and Ch-SF hydrogels. The gelation time of [email protected] hydrogel was 4 ± 0.33 min and the time of Ch and Ch-SF hydrogels were 9 ± 0.33 and 7 ± 0.33 min, respectively. This gelation time of gold incorporated hydrogel [email protected] shows the stronger intermolecular interactions between Ch and SF hydrogels. Recent study also documented that Chitosan conjugated SF hydrogels forms gelling property approximately 4 min . Moreover, temperature played a significant role in gelation property. At initial testing temperature (4 °C) 4 min, the gelation was not observed. Beyond 24 °C, the viscosity was increased and temperature increment favored to the gelation after 28 °C. The clear gel was formed at 37 °C and 4 min (Figure 2). The rheological measurement was conducted by a method of dynamic viscoelastic as a function of temperature. The temperature dependence of the elastic (storage) modulus (Gʹ) and viscous (loss) modulus (Gʹʹ) were recorded between 20 - 50 °C and the results were displayed in Fig. 2. The Gʹ represents the measure of the deformation of stored energy during a shear process or elastic response of materials. Gʹʹ denoted the measure of the dissipated energy during the shear process as heat or the viscous response of the materials. While the Gʹ was lower than Gʹʹ at the initiating stage of gelation, it was a viscous liquid. Meanwhile, Gʹ was higher than Gʹʹ it’s become an elastic solid . Our study results showed that the elastic modulus (Gʹ) value of the Ch was 60 Pa at 50 °C while Gʹ value of the Ch-SF and [email protected] Ch-SF were 20 and 12.5 Pa at 50 °C, respectively. Hence, the Ch hydrogels had a stronger elastic gel than the Ch-SF and [email protected] Ch-SF hydrogels. This incidence due to the electrostatic interactions between the hydroxyl/amine groups of SF and Ch molecules resulting in the charge density of the Ch molecules was decreased. Additionally, the incorporation of SF into the hydrogels might be decrease interaction between the ammonium group of Ch and phosphate group of BGP. Therefore, the addition of SF into the Ch hydrogels did not promote their mechanical properties. Moreover, the critical gel transition temperature (Tgel) was tested. The Tgel of ch hydrogels was 40 °C while Tgel of the Ch-SF and [email protected] hydrogels were 36 and 32-34 °C, respectively. These results confirmed that the incorporation of SF enhances the gelation through increasing entanglements and intermolecular interactions. The mechanical properties of Ch, Ch-SF and [email protected] hydrogel such as compression force, compression strength, tensile force, tensile strength and elongation was reported in Table 1. These mechanical properties possessed sufficient strength to retain its structure and shape during gel formation.
3.4 Structural analysis
XRD analysis of Ch-SF and [email protected] hydrogels demonstrated that the diffractions from (111), (200), (220) and (311) were due to the formation of nanocrystalline gold. As shown in Figure 3A, the absence of peaks in Ch-SF and the presence of peaks in [email protected] confirmed the incorporation of gold in Ch-SF hydrogel. FT-IR analysis was carried out to investigate the changes of functional groups in the [email protected] Figure 3B shows the FT-IR spectrum of Ch-SF and [email protected] hydrogels. The [email protected] shows the bands at 3461, 2947, 1612, 1560, 1309, 1047 and 645 cm-1 respectively. On the contrary, Ch-SF showed absorption band peaks at 3367, 2961, 1621, 1321, 1284, 1257, 1128, 1049 and 649 cm-1 respectively. The differences in peaks in the region 1300 to 1100 cm-1 was due to the formation of intermolecular hydrogen bond interaction cross links which confirmed the gelling property.
3.5 Morphology of [email protected] hydrogel
The micro morphologies of Ch, SF, Ch-SF and [email protected] hydrogels were analyzed using scanning electron microscope (Figure 4). The cross-sectional hydrogels were prepared and observed which has a pore sizes ranging from 50–300 μm with inter connected structures. In the previous studies, it has been shown that silk fibroin hydrogel with 90–250 μm pore sizes had provided the better atmosphere for adhesion and proliferation of chondrocytes . In addition, the porous structure all hydrogels Ch, SF, Ch-SF and [email protected] could be promote the passage of water or biomolecules into the hydrogel which is helpful for any drug molecules to diffuse through hydrogels . The EDAX spectrum of [email protected] hydrogel had illustrated the characteristic Au peaks (Figure 4). The elements Au, C, N and O were documented with different composition in Ch-SF and [email protected] hydrogels.
3.6 Weight loss of hydrogel
The conformational changes of Ch-SF and [email protected] hydrogels were analyzed by TGA thermograms (Figure 5A). TGA was carried out to find the function of percentage weight loss against temperature. 10 % of decomposition was observed in the temperature range from 175 to 185 °C and after increasing temperatures favored decomposition of both samples. 50 % weight loss was observed for Ch-SF at 238 °C and [email protected] at 392 °C. The initial decomposition temperature was found to be higher for [email protected] (232°C) than Ch-SF (181 °C) hydrogel. It was reported that, the decomposition initiation was due to the dehydration and loss of volatile molecules and further stage decomposition was due to depolymerization reactions . The final decomposition temperature of [email protected] at 505 °C showed better thermal stability of the crosslinked hydrogel.
The weight loss of hydrogel was evaluated in PBS medium (pH 7.4) up to 30 days (Figure 6A). The results showed that the weight loss of all hydrogels favored with increasing submersion period. Comparatively [email protected] is stable than Ch-SF hydrogel and the submersion analysis confirmed that more than 65% of hydrogel was stable at 30 days.
3.7 Mechanical properties
The elasticity of the nanocomposite hydrogel has been recognized as a dominating factor of cell fate in TE . The compression test was performed on fully-formed hydrogels to verify the elastic properties of the Ch-SF and [email protected] hydrogels. The stress-strain curve results showed that incorporation of gold nanoparticle into the Ch-SF hydrogel improved compressive modulus from 14.2 MPa to 26.3 MPa (Fig. 5B). [email protected] hydrogel could withstand compressive forces without breakage than the Ch-SF hydrogel.
3.8 SCA measurements
SCA were used to determine the surface hydrophilicity of Ch-SF and [email protected] hydrogels. The contact angles of of Ch-SF and [email protected] hydrogels are reported in Figure 5D. The contact angle value of 61.8 ± 0.8° was observed for Ch hydrogel and the Ch-SF and [email protected] hydrogels were found to be 87.8 ± 0.8 °and 58.8 ± 0.8°. A recent study shows that the electro-synthesized hydrogel resulted in the contact angle value of 67 ± 4° .
3.9 In vitro drug release studies
The percentage of Au release from [email protected] hydrogels was plotted against time. As shown in Fig. the hydrogel exhibited a pH-responsive releasing behavior which displayed 78.76% Au release after 24 h at pH 5.7. In contrast, the lower level of Au release (33.6%) was recorded at pH 7.4 it may be due to the greater hydrolytic stability of Schiff base linkages. Moreover, the higher Au releasing activity at pH 5.7 attributed to the greater swelling capacity of [email protected] hydrogels at lower pH.
3.10 Electrical conductivity property
The conductivity property of the Ch-SF and [email protected] hydrogels were measured by Cyclic Voltammetry (CV) measurement. The closed CV curves exhibited the capacitive capacity of tested hydrogels and shown in Figure 5C. The capacitive capacity of Ch-SF hydrogel is relatively weak due to the smallest enclosed area in CV curve and the [email protected] hydrogel showed enlarged circles which confirm the stronger capacitance. As shown in Figure 6B, [email protected] hydrogel displayed a conductivity of 0.164 S/m which is higher than the hydrogel without gold nanoparticles. Recently Baei et al.  synthesized a thermosensitive conductive hydrogel by combining AuNPs and chitosan with the conductivity of 0.13 S/m which is reported as closer to native myocardium. This conductivity of 0.164 S/m from [email protected] hydrogel could support the metabolism, viability, migration, and proliferation of myocardial cells.
3.11 In vitro studies
3.11.1 Cytotoxicity analysis
In vitro biocompatibility of implant material is one of the key factors in biological systems to avoid toxic effects. An increasing body of evidence has proved that the incorporation of Ch-SF hydrogels might enhance the biocompatibility of the Au nanoparticle-based hydrogels to cells by promoting cell attachment and proliferation. The biocompatibility of [email protected] on MS cells were presented in Fig. S1 (supplementary data). MS cells treated with the supernatant of [email protected] showed 101.4±2% cell viability over the positive control (3.7±1%) which indicates high biocompatibility of [email protected] hydrogel. Also, there are no significant differences were found between MS cells cultured using normal media and media containing [email protected] hydrogel supernatant. Further, the cytotoxicity assay was carried out by H9c2 cells. The cells were treated with Ch, Ch-SF, [email protected] and mesenchymal stem cells along with [email protected] hydrogel polymer coatings. On day one, the viability of H9c2 cells cultured with all the above hydrogels ranged between ~52 and ~64% (Figure 7A) and the viability were increased on 15th day ranged between ~72 and ~93%. This activity was higher than treatment of MS alone that showed the activity ~83% at 15 days. The cytotoxicity results demonstrated that the hydrogels loaded gold nanoparticles and mesenchymal stem cells exhibited no toxicity against H9c2 cells. These results confirmed that these hydrogels had the potential for use in biomedical applications.
3.11.2 Immunofluorescent staining method
Immunofluorescent staining revealed that H9c2 cells induced by mesenchymal stem cells along with [email protected] hydrogel was strongly positive for beta-myosin heavy chain (Figure 7ii). DAPI stain showed nuclear region and the red stained β-MHC expression confirmed the cardiac differentiation of mesenchymal stem cells. The H9c2 cells were incubated with anti-β-MHC conjugated to Alexa 647 resulting the cells treated with MS cells + [email protected] hydrogel showed more red-stained β-MHC expression which confirm the cardiac differentiation of mesenchymal stem cells. These findings were well matched with the previous studies by Shi et al., .
3.12 In vivo studies
3.12.1 Evaluation of apoptosis by TUNEL-DAPI staining
The histochemical analysis was performed with Masson's-trichrome staining to view nuclear, collagenous and cytoplasmic region of infarcted and treated heart sections. Figure 8 shows the micrographs of the infarcted area of blue collagen which showed a severe infarcted myocardium. The MI hearts treated with MS cells showed little improved heart tissue which showed myofibers with central nuclei indicated that the cardiac muscle fibers are regenerating. Similarly, the treatment of MS cells loaded Ch-SF showed the cardiac muscle fibers regeneration characteristics. However, the MS cells + [email protected] hydrogel treated sections showed much improves fibers and the collagens were stained less than the other treated group. These findings were confirmed the regenerated tissues. The quantitative image data in Figure 8 showed 26 ± 3% fibrosis area of infarcted tissue. The infarcted tissue treated with MS cells + Ch-SF hydrogel showed 7 ± 2% fibrosis. However, the lowest fibrosis area was recorded in MS cells + [email protected] hydrogel (9.3± 3.5%) which is the protective effect of electroactive nanoparticles loaded Ch-SF hydrogel along with MS cells. In order to find the apoptotic cells in the hearts after MI, TUNEL-DAPI co-staining was used to evaluated the tissue sections. The infarcted tissues showed significant apoptosis as noted by increased TUNEL-DAPI positive cells. However, treatment with MS cells + [email protected] hydrogel effectively prevented induced apoptosis than the cells treated with MS cells alone and the combination of MS cell and Ch-SF (Figure 8). The quantitative image data showed 41 ± 3% of apoptotic cells in the saline-treated infarcted tissues and the same was improved upon treatment with MS cells (37.6± 3%) and MS+Ch-SF (33.3± 2%). However, the superior activity was noted in MS cells+ [email protected] hydrogel treated tissue which showed 23 ± 2 % of apoptotic cells.
3.12.2 Improvement and restoration of myocardial damage by β-MHC and Cx43
To evaluate the process of cardiomyogenesis, immunohistochemical staining of Cx43 was carried out which served as cardiac-specific marker. As shown in Fig. 8, the infarcted tissues treated with saline showed the least amount of Cx43 expression than the untreated tissue (Normal). In contrast, the infarcted tissue treated with MS cells alone showed positive Cx43 cells expression. Among the treatments, the highest expression of Cx43 positive cells were observed in MS cells + [email protected] hydrogel treatment group, suggesting a large restoration of the myocardial damage after MI. This gold incorporated Ch-SF hydrogel might have provided electromechanical signals in the infarcted myocardial tissue. The quantitative image data of Cx43-DAPI ratio showed the expression pattern of Cx43 in MS cells + [email protected] hydrogel treated group was higher than the other treatments. This confirmed that injectable hydrogels, conductivity and mesenchymal stem cells had a constructive effect on the improvement and restoration of cardiac function after MI. Although the native cardiac cells do not have proper regeneration capacity following the myocardial infarction, the implanted MS cells transferred via hydrogels were found to secrete new tissues which are confirmed through β-MHC and Cx43 cardiac markers. In parallel with the above results, a relationship between expressions of cardiac-specific markers and electro-conductive elements such as gold within the hydrogel scaffolds has been previously reported .
Myocardium engineering studies has been reported 4–6 weeks as a proper time frame for degradation of hydrogels and in our study 4 weeks treated SD rats are analysed for the protective role of MI . In addition, the prepared [email protected] hydrogel resembled native myocardium mechanics in terms of providing electro conductivity and biocompatible tissue microenvironment [53,54] which could have increased the propensity of MS cells to become cardiomyocytes .
3.12.2 Biocompatibility analysis
Further, in vivo biocompatibility test was carried out by examining the hematological parameters (HGB and RBC), liver enzymes (AST and ALT), and histomorphological changes after Ch-SF, MS cells loaded with Ch-SF hydrogels and MS cells loaded with [email protected] hydrogels treatment. At the time of the experiment, it was demonstrated that there were no significant differences in weight between the control and treated groups, i.e., the prepared hydrogels did not cause any changes in the weight of the rat (Fig. 9A ). Both AST and ALT are liver enzymes used to evaluate liver function. Increased levels of AST and ALT are indicators of liver damage . In this study, it was found that the levels of AST and ALT in the hydrogel-injected groups were similar to those in control, meaning that the hydrogels treatment did not cause liver damage in rats (Fig. 9B and C). Similarly, hematological parameters such as HGP and RBC were found at a similar level to the control group (Fig. 9D and E). Furthermore, histomorphological examination did not show any internal injury or infectious lesions, showing that it was normal (Fig. 9F). From the biocompatibility results of this study, it is demonstrated that the prepared hydrogels did not show any side effects during the treatment period.