Potential benefits of using chitosan and silk fibroin topical hydrogel for managing wound healing and coagulation

Abstract

Background & Objectives

The intricate process of wound healing involves replacing the cellular or tissue structure that has been destroyed. In recent years various wound dressings were launched but reported several limitations. The topical gel preparations are intended for certain skin wound conditions for local action. Chitosan-based hemostatic materials are the most effective in halting acute hemorrhage, and naturally occurring silk fibroin is widely utilized for tissue regeneration. So, this study was conducted to evaluate the potential of chitosan hydrogel(CHI-HYD) and chitosan silk fibroin hydrogel (CHI-SF-HYD) on blood clotting and wound healing.

Methods

Hydrogel was prepared using various concentrations of silk fibroin with guar gum as a gelling agent. The optimized formulations were evaluated for visual appearance, Fourier transforms infrared spectroscopy (FT-IR), pH, spreadability, viscosity, antimicrobial activity, HR-TEM analysis, ex vivo skin permeation, skin irritation, stability studies, and in vivo studies by using adult male Wistar albino rats.

Results

Based on the outcome of FT-IR, no chemical interaction between the components was noticed. The developed hydrogels exhibited a viscosity of 79.2 ± 4.2 Pa.s (CHI-HYD), 79.8 ± 3.8 Pa.s (CHI-SF-HYD), and pH of 5.87 ± 0.2 (CHI-HYD), 5.96 ± 0.1 (CHI-SF-HYD). The prepared hydrogels were sterile and non-irritant to the skin. The in vivo study outcomes show that the CHI-SF-HYD treated group has significantly shortened the span of tissue reformation than other groups. This demonstrated that the CHI-SF-HYD could consequently accelerate the regeneration of the damaged area.

Interpretation & Conclusion

Overall, the positive outcomes revealed improved blood coagulation and re-epithelialization. This indicates that the CHI-SF-HYD could be used to develop novel wound-healing devices.

1. Introduction

Cell proliferation occurs due to the stimulation of growth factors that will help tissue repair by involving various changes, such as the multiplication of parenchymal cells and the production of an extracellular matrix (Shaw and Martin, 2009, Eming et al., 2007). The interruption in the wound healing mechanism occurs due to some modifications in the process of coagulation. The thrombin and fibrin formations are induced when the sub-endothelium of injured blood vessels and capillaries are accumulated by platelets, leading to the wound's healing by coagulation (Gowda et al., 2023a, Hoffman et al., 2006).

Further, the fibrin network establishes a framework for the subsequent process, which also balances out the underlying platelet plug (Drew et al., 2001). In this manner, the underlying fibrin is used as a framework by fibroblasts that come in contact with the injury bed. Angiogenesis will develop new blood vessels (Hadjipanayiet al., 2015). Throughout this process, the keratinocytes will move and multiply over the injury site to restore the skin. (Piipponen et al., 2020, McDonald et al., 2007).

Many formulations are available for wound healing, such as ointments, creams, lotions, etc. However, the hydrogel formulation is gaining attention nowadays due to its advantages over others, such as bioadhesion, biocompatibility, better spreadability with sustained drug-releasing ability, etc. Hydrogels are a three-dimensional, cross-linked network of polymers, and water penetrates these networks, causing swelling and giving the hydrogel a soft and rubbery consistency but will not dissolve (Monica and Gautami, 2014).

Chitosan-based hemostatic materials are the most effective in halting acute hemorrhages. It is a larger molecular weight copolymer that bears glucosamine radicals and acetyl glucosamine in its structure that forms viscous polyelectrolytes upon dissolution in inorganic and organic acids. Chitosan is widely used for its various advantages, such as biodegradability and biocompatibility. Recently many studies have also shown its ability to act as an anti-bacterial and bind to LDL to convert them into HDL, which further helps to reduce the risks associated with hyperlipidemia. It was also found that it can be a good procoagulant (Hattori et al., 2017). Chitosan adheres to the erythrocytes and encourages platelet adhesion, activation, and aggregation at the injured site. It leads to the activation of coagulation factors and achieves hemostasis (Stricker-Krongrad et al., 2018).

Silk is a natural protein polymer extracted from mulberry silk by removing the outer silk sericin. USFDA also approves it for wide medical applications (Nasrine et al., 2022). The silk may produce health benefits associated with fibroin (Altman et al., 2003). Alongside this, silk-fibroin involves multiple advantages such as biocompatibility, biodegradability, low immunogenicity, etc. Thus, it is widely utilized as a medicine for tissue engineering and regenerative medicine, for example, cartilage, cornea, and bone repair. Also, its hemostatic ability could be a potent biomaterial for skin-repairing activity (Chen and Liu, 2016, Gil et al., 2013). The studies also indicated that SF is non-harmful under acute dermal toxicity (Padol et al., 2011).

Here, we have developed chitosan hydrogels (CHI-HYD) and chitosan silk fibroin hydrogels (CHI-SF-HYD) as novel drug delivery systems for effective wound healing treatment. Further evaluated for coagulation and wound healing properties to determine the potential of combinational polymers in tissue regeneration.

2. Methods

2.1. Materials

The supplier of chitosan was Yarrow Chem Products in Mumbai. We received a sample of silk fibroin as a gift from Sericare in Bangalore. The supplier of guar gum was Loba Chemicals in Mumbai. Finally, acetic acid was bought from Hyderabad's Avra Synthesis Pvt ltd. Without additional purification, analytical-grade chemicals were utilized for all other substances.

2.2. Procurement and maintenance of animals

The Institutional Animal Ethics Committee, Yenepoya University, Deralakatte, Mangalore, approved the study protocol (YU/IAEC/15/2019, dated 09/11/2019). Animals were procured from Liveon Biolabs, Tumakuru, Karnataka (1610/ROBiBt/S/2012/CPCSEA). Healthy male Wistar albino rats weighing 150–200 g were used in the study. They were housed in ventilated cages in maintained conditions (25˚C, 12 h light and dark cycles), having access to regular rat pellets and water ad libitum. All the animal experiments were performed following the guidelines of the Committee for Control and Supervision of Experiments on Animals (CPCSEA).

2.3. Formulation of chitosan hydrogels and chitosan silk-fibroin topical hydrogels

2.3.1. Chitosan hydrogels (CHI-HYD)

Overnight soaked chitosan in 40 mL of water and 1 mL acetic acid was stirred well using a digital magnetic stirrer at 250 rpm for one hour. Then guar gum was added in small aliquots with continuous stirring to avoid the formation of lumps (500–600 rpm). Then 0.1 g of sodium benzoate was added and continued stirring for another 30 min.

2.3.2. Chitosan silk fibroin hydrogel (CHI-SF-HYD)

To prepare CHI-SF-HYD, the same steps were followed until the addition of guar gum (Table 1). Then silk-fibroin (0.3 g) was added slowly with continuous stirring. Then sodium benzoate 0.1 g was added and stirred for another 30 min (Misal et al., 2012).

Table 1.

Composition of hydrogels.

INGREDIENTS	FORMULATION CODE
	CHI-HYD	CHI-SF-HYD
Chitosan	0.5 g	0.5 g
Acetic acid (1 %)	1 mL	1 mL
Guar gum	0.5 g	0.5 g
Silk-fibroin	–	0.3 g
Sodium benzoate	0.1 g	0.1 g
Deionized water	q.s	q.s

2.4. Evaluation

2.4.1. FT-IR spectroscopy

FT-IR studies were performed using CHI, SF, guar gum (GG), and CHI-SF HYD formulation with an FT-IR spectrophotometer (Shimadzu, Japan). IR spectral analyses were used to examine the interaction between the API and pharmacological excipients by looking for any change in the peaks. The sample was put immediately beneath the probe, to which it was securely fastened. The search was then scanned in the 4000–500 cm⁻¹ wavenumber range. Functional groups at each wavenumber were identified from the collected IR spectra (cm⁻¹) (KumirskaJ et al., 2010, Negrea et al., 2015, Sanjana and Ahmeda, 2021a).

2.4.2. Thermal analysis

To clarify any interactions between chitosan and the other polymers utilized in the study, thermal analysis using differential scanning calorimetry (DSC) was conducted. Shimadzu's DSC-60 Plus, made in Japan, was used for DSC. The instrument was built with components from the Japanese Shimadzu Corporation, including a thermal analyzer (TA 60), a flow controller (FCL 60), a calorimeter (DSC 60), and operational software (TA-60 WS). The samples were placed in a sealed aluminum pan and heated from 30 to 300 °C at a 15 °C/min scanning rate while under nitrogen flow (30 mL/min). DSC thermograms were then determined for test samples (Narayana et al., 2022a, Mudgil and Khatkar, 2012).

2.4.3. Organoleptic properties

Tests were done on the hydrogel formulations for appearance, texture, etc. Visual observation was used to evaluate the hydrogel's appearance. The texture was evaluated by pressing a small amount of the prepared gels between the thumb and index finger. To assess the surface, the formulation’s uniformity and the presence of large particles were considered (Chen et al., 2016).

2.4.4. Spreadability test

Applying the gel on a flat surface and looking for grainy hydrogel properties can help determine spreadability. For example, on a glass plate, the 0.5 g of produced gel was spread over a 2 cm diameter circle that had been marked. Then, half the weight of the gel was allowed to rest on the upper glass plate for five minutes. The method was carried out three times, and the circle’s diameter after the gels had been disseminated was measured (Sanjana et al., 2021b, Al-SuwayehSA et al., 2014).

2.4.5. Determination of pH and viscosity

The formulation's pH was measured using a digital pH meter after the weighed amount of gel (1 g) was dissolved in 25 mL of distilled water. The pH analysis was carried out in triplicates for both formulations (Kumar et al., 2018). The viscosity of the gel formulations was then measured using a Brookfield viscometer at a temperature of 25 °C and spindle number 7 at 100 rpm (Sanjana et al., 2022c, Monica and Gautami, 2014, Misal et al., 2012).

2.5. Drug content analysis

Chitosan distribution in both gels was determined using a drug content analysis. With deionized water, the gels were thinned out. The gels and deionized water solutions were vortexed for five minutes to achieve uniform mixing. The mixes were then centrifuged for 20 min at 1000 rpm. Each gel formulation's drug content was assessed using UV–vis spectrophotometry (SHIMADZU 1900, Japan) (ChundranNK and Rubianti, 2015, Drew et al., 2001, Dubey and Prabhu, 2014, El-KasedReham, 2016, Eming et al., 2007, Fong Yen et al., 2015).

2.6. Anti-bacterial activity

Using the cup plate method, an agar diffusion test was used to measure the anti-bacterial activity. The substance permitted diffusion across a dense agar media. Then, the nutritional agar medium was made and sterilized for 15 min at a pressure of 15 lb/sq inch. Then prepared agar with bacterial suspension was poured into control and test Petri dishes and were incubated for 24 h. In the test plate, when the inoculated agar was solidified, cups were made using a sterile borer and then filled with prepared formulation and incubated for 24 h. The zone of inhibition, a measure of anti-bacterial action, was discovered after 24 h (Mandal et al., 2012, Anbarasan et al., 2019).

2.7. Sterility testing

A different thioglycolate medium and a soybean casein digest media were used to assess the hydrogel sterility. Both the positive and negative control test was conducted. The microbes Bacillus subtilis and Candida albicans were employed. In every instance, incubation was done, and growth was observed (Singh et al., 2010, Narayana and Ahmed, 2022b).

2.8. Blood clotting test

A lancet was used to puncture the rat tail vein, and the blood was then allowed to run continuously. The timer was started concurrently with the pricking. Blood drops were blotted every 30 s-time intervals on one strip at a time. The same procedure was repeated for the remaining two strips with tested compounds, and blood clotting time was measured. All operations were carried out with anesthesia (Parasuraman et al., 2010).

2.9. Morphological analysis using high resolution-transmission electron microscopy (HR-TEM) evaluation

With the aid of an HR-TEM analyzer (JEOL, JEM-2100 electron microscope; Tokyo, Japan), the shape of a particle and its size distribution was analyzed. The sample was placed over carbon coated fine mesh grid (2 0 0), dried at room temperature, and analyzed (Alencastre et al., 2006).

2.10. Skin irritation study

The adult rats were placed in the cage. Water and regular feed for rats were given ad libitum. The room temperature was maintained at 22 ± 3 ⁰C with 30 – 70 % RH. The artificial lighting was set up to last for 12 h every day, from 8 am to 8 pm. The rat’s hair on the back (3 cm²) was cleanly shaved 24 h before the test. The final gel formulations (gel base, CHI-HYD, and CHI-SF-HYD) were evenly applied to the shaved area (2 cm²). Skin reactivity at the application site was observed and graded once daily at 1, 24, 48, 72 h, 7, and 10 days, as appropriate (Alencastre et al., 2006).

2.11. Ex vivo permeation in rat skin

Franz diffusion cell was used to study the ex vivo diffusion of hydrogels using 7.4 pH phosphate buffer as the receptor media. A membrane for dialysis was made from rat abdomen skin. The stratum corneum side of the skin was in close touch with the formulation's release interface within the donor cell. The rat's skin was covered with a weight of 1 mL of gel, which was then gently dipped into 100 mL fresh receptor fluid media while still being repeatedly stirred. At 37 °C, the temperature was maintained. After each sample withdrawal, the same quantity of media was replaced in the receptor compartment to keep the sink condition. The collected sample read spectrophotometrically at 247 nm (Alencastre et al., 2006).

2.12. In vivo studies

Healthy adult Wistar albino male rats weighing 150–200 g were chosen for the investigation. The institutional animal ethics committee of Yenepoya (Deemed to be University) approved the use of animals in research. Intravenous ketamine was used to anesthetize the rats before and during the making of the wounds. Using a razor, the hairs around the dorsal midline region were removed. After that, a 2 cm diameter was determined and marked. Around the circle that has been drawn, an excision is conducted using a scalpel (1 cm). Three groups of six rats each were typically formed. Group I: Topical application of base hydrogel (base hydrogel contains other than chitosan and silk fibroin) negative control. Group II: Topical application of CHI-HYD. Group III: Topical application of CHI-SF-HYD. Then animals were observed to recover from wounds (Aiyalu et al., 2016, Zhang, n.d., Nayeem et al., 2021). The wound area was recorded by using transparent paper (tracing paper) on the wound. Then the percentage of wound contraction was calculated using the below-mentioned formula. The data collected were statistically analyzed using GraphPad Prism version 6.01 (GraphPad Software, La Jolla, California, USA), and data were expressed as mean ± SD. Statistical significance was determined by applying variance analysis (one-way ANOVA) followed by Tukey's multiple comparisons tests. Statistical significance was accepted at the 95 % confidence level (p < 0.05) (Rajoo et al., 2021).

$$\mathrm{N}\mathrm{t}\mathrm{h}\mathrm{d}\mathrm{a}\mathrm{y}=\frac{\mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{n}0\mathrm{d}\mathrm{a}\mathrm{y}-\mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{n}\mathrm{N}\mathrm{t}\mathrm{h}\mathrm{d}\mathrm{a}\mathrm{y}}{\mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{n}0\mathrm{d}\mathrm{a}\mathrm{y}}\ast 100$$

2.13. Stability studies

The primary goal of stability testing is to prove that the prepared formulation's efficacy is maintained over its shelf life. According to ICH recommendations, stability investigation for the topical hydrogel formulations (25 ± 2 °C, and 60 ± 5 %, RH) was carried out in a humidity chamber (LABON INSTRUMENTS). Samples were collected at the beginning, 1st, 2nd, and 3rd respective months to assess the visual appearance, spreadability, pH, and viscosity. Any modifications to the evaluation parameters that were reported. Tests were run in triplicate, and the mean and standard deviation of the observed data were recorded (Ahamed et al., 2011).

Ethical approval

The animal study protocol was approved by the Institutional Ethics Committee of the Institutional Review Board (or Ethics Committee) of Yenepoya (Deemed to be University) with approval number YU-IAEC (YU/IAEC/15/2019) dated 09/11/2019.

3. Results

3.1. Organoleptic properties

The prepared CHI-HYD and CHI-SF-HYDs were thick and opaque. The texture was smooth, and no phase separation was observed. The pH of prepared hydrogels was found to be within the acceptable range. The developed topical hydrogel’s viscosity was 79.2 ± 4.2 Pa.s for CHI-HYD and 79.8 ± 3.8 Pa.s for CHI-SF HYD. All results are displayed in Table 2.

Table 2.

Organoleptic properties of CHI-HYD and CHI-SF-HYD.

Formulation code	Visual appearance	Texture	pH	Viscosity (Pa.s)
CHI-HYD	Thick, opaque	Smooth	5.87 ± 0.2	79.2 ± 4.2
CHI-SF-HYD	Thick, opaque	Smooth	5.96 ± 0.1	79.8 ± 3.8

*Results are shown as mean ± SD (n = 3).

3.2. Ft-IR studies

The interactions between the components were studied by performing FT-IR spectroscopy. The characteristic absorption bands in the CHI, SF, and GG were listed. The same peaks were observed in the formulation spectra, confirming that no interaction between the components and formulation was compatible. The range is shown in Fig. 1, and the interpretation of functional groups is displayed in Table 3.

Fig. 1.

FT-IR spectrum of(a) CHI, (b) SF, (c) GG, (d) Formulation.

Table 3.

Interpretation of FT-IR spectra.

Sample	Absorption cm−1	Classified by group	Class of compound
CHI	3300–2500	Strong, broad O—H stretching	Carboxylic acid
	3000–2800	Strong, broad N—H stretching	Amine salt
	1620–1610	Strong C C stretching	α, β unsaturated ketone
	1550–1500	Strong N—O stretching	Nitro compound
	1500–1300	Medium C—H bending	Alkane methylene, methyl group
	1075–1020	Strong S O stretching	Sulfoxide group
	900–800	C C bending	Alkene
	750 ± 20	Strong C—H bending	Monosubstituted
SF	3300–2500	Strong broad O—H stretching	Carboxylic acid
	1618	N—H bending	Amine
	1550–1500	Strong N—O stretching	Nitro compound
	1250–1020	Medium C—N stretching	Amine
	840–650	C C bending	Alkene
GG	3300–2500	Strong broad O—H stretching	Carboxylic acid
	1670–1600	Weak C C stretching	Alkene
	1390–1310	Medium O—H bending	Phenol
	1000–650	Strong C C bending	Alkene

3.3. Thermal analysis

To detect thermal stability or formulation incompatibility between the excipients DSC study has been carried out. DSC thermograms of CHI, SF, and the physical mixture of these were depicted in Fig. 2. The thermogram of CHI showed a strong sharp endothermic at 295.91 °C, close to the CHI melting point. At 231.06 °C, SF displayed an exothermic peak. The thermogram of the physical mixture showed the same peaks, and it was confirmed that the formulation was thermally stable.

Fig. 2.

Shows the DSC thermogram of (a) CHI, (b) SF, and (c) physical mixture.

3.4. Spreadability test

For the good therapeutic efficacy of topical gel formulation, spreadability plays an important role. Fig. 3 displays the spreadability values, i.e., detected diameters within one minute. Results indicated no gritty surface, and it quickly spread over the glass plate.

Fig. 3.

Spreadability values for the CHI-HYD and CHI-SF-HYD *Results are shown as mean ± SD (n = 3).

3.5. Blood clotting test

The obtained results revealed that CHI has hemostatic activity. In addition, the time required for blood clotting was found to be comparatively less in CHI-HYD and CHI-SF-HYD than natural clotting process. The results are shown in Table 4 and Fig. 4.

Table 4.

Blood clotting test for control, CHI-HYD, and CHI-SF-HYD.

	Number of blood drops				Blood coagulation (sec)
	Trials			Mean ± SD
	1	2	3
Control	8	8	7	7.66 ± 0.5	229.8
CHI-HYD	5	6	5	5.33 ± 0.5	159.9
CHI-SF-HYD	4	4	5	4.33 ± 0.5	129.9

*Results are shown as mean ± SD (n = 3).

Fig. 4.

Blood clotting test for Control, CHI-HYD& CHI-SF-HYD.

3.6. Drug content analysis

The percentage of CHI in CHI-HYD was found to be 81 ± 3.5 %, and in CHI-SF-HYD was found to be 83 ± 1.4 %. The obtained outcomes indicated that CHI was distributed uniformly in the gels, and the CHI loss during the formulation of gels was negligible (Table 5).

Table 5.

Drug content data for hydrogels.

Formulation code	Drug Content (%)
CHI-HYD	81 ± 3.5
CHI-SF-HYD	83 ± 1.4

*Results are shown as mean ± SD (n = 3).

3.7. Anti-bacterial activity

The antibacterial activity of the prepared formulations was tested against the most common microorganisms like staphylococcus aureus and Escherichia coli. The gel formulation showed good zones of inhibition. The anti-bacterial activity outcomes are displayed in Table 6 and Fig. 5. The zone of inhibition was found to be between 25 and 28.5 mm.

Table 6.

Anti-bacterial activity.

Microorganism	Zone of inhibition(mm)
	Marketed formulation	CHI-HYD	CHI-SF-HYD
Staphylococcus aureus& Escherichia coli	28.5 ± 1.1	25 ± 0.6	25 ± 0.4

*Results are shown as mean ± SD (n = 3).

Fig. 5.

Antimicrobial activity (a). control, (b). marketed formulation, (c). CHI-HYD, (d). CHI-SF-HYD.

3.8. HR-TEM analysis

The CHI-SF-HYD were found to be spherical, and the porous interconnected well-distributed particles with a smooth and homogenous surface were observed. As depicted in Fig. 6, the particles’ size in the nano range with uniform distribution.

Fig. 6.

HR-TEM analysis of CHI-SF-HYD.

3.9. Test for sterility

The test showed that the medium used was sterile. The results of the test for sterility are shown in Table 7. The results indicated no bacterial growth. Hence, the tested formulation passes the test for sterility.

Table 7.

Test for sterility.

Days	1		2		3			4		5	6		7
	F	S	F	S	F	S	F	S	F	S	F	S	F	S
CHI-HYD	–	–	–	–	–	–	–	–	–	–	–	–	–	–
CHI-SF-HYD	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Positive control	+	+	+	+	+	+	+	+	+	+	+	+	+	+
Negative control	–	–	–	–	–	–	–	–	–	–	–	–	–	–

(+) = growth of microorganism (-) = no growth F = fluid thioglycollate medium.

S = soyabean casein digest medium.

3.10. Skin irritation study

The gel base, CHI-HYD, and CHI-SF-HYDs were identically applied to each rat's shaved area (2 cm²). Any skin reaction at the applied site was individually evaluated and recorded once daily at 1, 24, 48, 72 h, 7, and 10 days. No allergy or swelling was spotted for developed CHI-HYD, CHI-SF-HYDs, and gel base formulations.

3.11. Ex vivo permeation in rat skin

Fig. 7 shows the ex vivo permeation of gel in rat skin. The cumulative amount of CHI permeated through CHI solution, CHI-HYD and CHI-SF-HYD were 2.1, 8.4, and 19.5 μg/ cm², respectively. It was confirmed that the permeation capacity of CHI has significantly enhanced SF-containing hydrogel.

Fig. 7.

Ex vivo permeation of Chitosan solution, CHI-HYD, and CHI-SF-HYD in rat skin.

3.12. Invivo studies

Fig. 8 depicts images of the wound for all groups: base HYD treated, CHI-HYD treated, and CHI-SF-HYD treated animals from days 1 to 20. The CHI-SF-HYD-treated group exhibited a fast tissue regeneration process in all the animals tested with wound cavities. Compared to other groups, the CHI-SF-HYD treated groups showed a significant difference in wound closure. Fig. 9 shows the % of wound contraction for various groups on the 5th,10th,15th, and 20th days. For days 5, 10, and 15, the highest % wound contraction was observed for CHI-SF-HYD treated group (33.5, 48.5, and 93.5 %, respectively). The CHI-HYD-treated group showed % wound contraction of 20.66, 35, and 49.25 %, respectively, comparatively lesser than that of the CHI-SF-HYD treated. Interestingly on the 17th day, the % of wound contraction for the CHI-SF-HYD-treated rats was 99.50 %. A highly significant difference in the % of wound contraction was observed for CHI-SF-HYD (p < 0.0001) throughout the experiment period when compared with CHI-HYD and gel base treated group.

Fig. 8.

Representative images of the wound areas of the various rat groups (Base-HYD, CHI-HYD, and CHI-SF-HYD).

Fig. 9.

Percentage of wound contraction Percentage of wound contraction for CHI-SF-HYD treated, CHI-HYD treated, and base-HYD treated rats for days 5, 10, 15, and 20. *Indicates significant differences in wound contraction. ****=p < 0.0001, ***=p < 0.001.

3.13. Stability studies

According to ICH criteria for developed gel formulations, a stability study was conducted to ensure safety and efficacy during storage. The formulations have good quality characteristics. The outcomes of the stability studies disclosed that the developed hydrogels are stable.

4. Discussion

The present study was planned to develop CHI-HYD and CHI-SF-HYD and to determine the potential of combinational polymers in the tissue regeneration process. The studies showed that SF is widely reported for its ability in tissue regeneration and engineering (Chen and Liu, 2016, Gil et al., 2013). The developed CHI-HYD and CHI-SF-HYD were smooth in texture with good spreadability, as shown in Fig. 3. Chen et al. reported that the spreadability values of gels with this range evidenced that easy spreading on the surface by applying a low shear rate (Chen et al., 2006). The IR spectral analyses exhibited that excipients used in the formulation were compatible with each other (Fig. 1), and the DSC analysis curves shown in Fig. 3 proved the thermal stability of hydrogels. Our study found that blood clotting time was lesser when treated with CHI-HYD and CHI-SF-HYD than natural blood clotting (Table 3 and Fig. 4). Radwan et al. reported that the interaction of blood with CHI formulation increases platelet volume, and the distribution width leads to platelet adhesion activation and aggregation. Platelet activation stimulates coagulation factors and blood clots (Radwan-Pragłowska et al., 2019). A good zone of inhibition confirmed the anti-bacterial activity of the developed formulation, but the addition of SF does not significantly improve its anti-bacterial action (Table 6, Fig. 5). Other researchers obtained similar results where it was found that SF alone does not offer any anti-bacterial activity. Still, antibiotics can be released sustainably from SF biomaterials, killing the bacteria growth (El-KasedReham et al., 2016). The HR-TEM results in Fig. 6 showed that the porous interconnected well-distributed particles with a smooth and homogenous surface of particles in CHI-SF-HYD improved the adhesion properties of the gel (Zhang et al., 2009). The skin irritation study showed no allergy or swelling even after ten days of study, signifying that the developed gel base, CHI-HYD, and CHI-SF-HYD formulations were safe to apply for blood clotting and wound healing activity. Due to CHI's excellent biodegradability and biocompatibility, skin irritation or local adverse effects are uncommon. As a result, using CHI reduces the likelihood of developing contact dermatitis compared to conventional anti-bacterial treatments (Zheng et al., 2020, Aiyalu et al., 2016). The test for sterility result is shown in Table 7. The results indicated no bacterial growth.

Hence, the tested hydrogel formulations are considered to pass the test for sterility. Also, the permeation capacity of CHI was significantly increased in CHI-SF-HYD than in CHI-HYD and simple CHI solution, which may be due to the porous interconnected structure of CHI-SF-HYD (Dubey and Prabhu, 2014, Yang et al., 2004, Huang and Brazel, 2003). In vivo animal studies evidenced faster-wound healing with CHI-SF-HYD-treated animals, which can be identified in Fig. 8. This may be due to the high amount of β-sheet patterns present in SF intensifies cell adhesion, proliferation, and migration of cells, hence it boosts the tissue reformation around the wounded area (Wang et al., 2016, Aljady et al., 2000). Also, literature reported that SF's porous and interconnected structure might lead to tissue regeneration and faster wound healing (He et al., 2019). Complete wound recovery (99.50 %) was observed for CHI-SF-HYD on day 17. A highly significant difference in the % of wound contraction was observed for CHI-SF-HYD (p < 0.0001) throughout the experiment period when compared with CHI-HYD and gel base treated group.

The shelf life of the HYDs, shown in Table 8, indicates no change in color, odor, spreadability, pH, and viscosity. The outcomes revealed that the developed HYDs are stable (Massensini et al., 2015, Meng et al., 2014).,

Table 8.

Physical parameters after accelerated stability study of the optimized formulation.

Physical Parameters	0th day		30th day		60th day		90th day
	CH	CSH	CH	CSH	CH	CSH	CH	CSH
Visual appearance	Thick, opaque	Thick, opaque	Thick, opaque	Thick, opaque	Thick, opaque	Thick, opaque	Thick, opaque	Thick, opaque
Spreadability	26 ± 1.2	27 ± 1.4	26 ± 2.1	27 ± 1.7	25 ± 1.5	26.5 ± 2.6	25 ± 1.8	26 ± 2.1
pH	5.87 ± 0.2	5.96 ± 0.1	5.91 ± 0.1	5.92 ± 0.3	5.89 ± 0.2	5.91 ± 0.1	5.88 ± 0.2	5.90 ± 0.4
Viscosity (cps)	79.20 ± 4.2	79.80 ± 3.8	7923 ± 3.5	7975 ± 2.9	7921 ± 1.8	7982 ± 4.5	7922 ± 2.9	7981 ± 3.9

*Results are shown as mean ± SD (n = 3).

The present research has a robust design and positive results, which pave the path for future wound healing treatment. However, limitations of the study could be the small in vivo sample size and lack of microscopic evaluation data for wound tissues.

5. Conclusion

In this study, CHI-HYD formulations alone and together with SF were designed and evaluated for various characteristics. The CHI-HYD and CHI-SF-HYD demonstrated excellent biocompatibility, desired stability, and acceptable physical characteristics. In addition, CHI-SF-HYD remarkably shortened the span of tissue reformation in an in vivo animal model compared with CHI-HYD alone, and the gel base, which demonstrated these developed hydrogels, could consequently accelerate the regeneration of the damaged area. Overall, the positive findings indicate an improved knowledge of the events contributing to blood coagulation and re-epithelialization. This suggests that in the future, the CHI-SF-HYD could be employed to develop novel wound-healing devices to provide compliance for patients and the healthcare sector.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.