Isabgol–silk fibroin 3D composite scaffolds as an effective dermal substitute for cutaneous wound healing in rats

Abstract

Composite 3D scaffolds using a natural, carbohydrate polymer, isabgol (Isab) and silk fibroin (SF) were prepared by a freeze drying method. 2 wt% solutions of both Isab and SF were blended in four different ratios to obtain the 3D scaffolds. ATR-FTIR results showed the presence of amide bonds and β sheet conformation in Isab/SF scaffolds. SEM micrographs showed the fibrous foam like architecture in the Isab/SF blend. Scaffolds showed sufficient porosity and absorbed higher amounts of PBS solution, enhancing the gas and nutrient supply to the cells. Subsequently, scaffolds showed significant in vitro biodegradation. In vitro cytocompatibility test in NIH 3T3 fibroblast cells showed better viability, proliferation and cell attachment for the Isab/SF 75/25 scaffolds compared to the control. Among all the scaffolds, Isab/SF 75/25 showed a faster wound contraction rate (p < 0.001) and reduced period of epithelialization (16 days) compared to the control (23 days). Shrinkage temperature and collagen content was also increased significantly (p < 0.001) in Isab/SF 75/25 scaffold treated rats compared to the control. Histopathology results strongly demonstrate higher cellular infiltration, increased fibroblasts, dense collagen fibers, and neovascularization in Isab/SF 75/25 treated rats, compared to other treatments. Overall, these results strongly authenticate that the Isab/SF 75/25 3D composite scaffolds enhanced the fluid uptake ability with enough fibroblast attachment and good viability. Also, it accelerated tissue regeneration during wound healing in rats without addition of any bioactive molecules.

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1. Introduction

Wound healing is a fundamental response to tissue damage and tissue repair is a complex physiological process that occurs sequentially. There are four distinct inter-related phases: haemostasis, inflammation, proliferation and remodelling.¹ Hemostasis (blood clotting) is followed by inflammation and then the proliferative phase, which is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization and wound contraction. Finally, the tissue remodeling phase begins with the replacement of granulation tissue with scar tissue formation.²

Infection is the main causes for the poor wound management.³ Development of ideal wound dressing material is a main theme to avoid pathogenic contamination and spread of wound exudate.⁴ Wound management depends on dressings with enough wound exudate absorption property and uncontaminated environment to start its healing process earlier.⁵

Wound healing and tissue engineering process requires an ingenious biomaterial with promising ability to repair the damaged tissues and organs.⁶ Also, vascularization property of these scaffolds is critical to improve the complex tissue and organ regeneration.⁷

Isabgol (Isab), commonly known as psyllium husk, is a natural carbohydrate polymer. It is an important gel forming fiber, universally recognized for its variety of medicinal properties.⁸ Isabgol is made up of hemicelluloses, highly branched acidic arabinoxylan and xylan backbone with both (1–4) and (1–3) linkages. Xylan backbone is mostly substituted with arabinose, xylose and aldobioronic acid at 0–2 and 0–3 positions, alternatively. Therefore, this residue is recognized as galactopyransyluronic acid.⁸ Gelling ability of this isabgol polymer can be useful to absorb large quantities of wound exudates to elude contamination.⁵

Previously, isabgol has been used as a gel forming agent in constipation, diarrhea, and alternative gelling agent in culture media, insulin delivery, drug delivery and anti-diabetic applications.^8–13 In an earlier study, isabgol and silver carbonate were added to alginate fibers to develop wound dressing materials. As isabgol has high water retention ability, the isabgol–alginate composite material showed increased fluid absorption. As the fibers were loaded with silver carbonate, enhanced anti-microbial property was also observed.⁵ However, there are no reports that have extensively studied the potential of isabgol as the primary material in wound healing applications.

Silk fibroin (SF), a protein polymer, has been widely used as a cell supportive biomaterial for variety of biomedical applications.^14–16 Due to its unique biocompatibility, biodegradability and less inflammatory reactions, silk has been used as an attractive protein for skin, bone, cartilage, nerve tissue engineering and wound healing.^17–21 Incorporation of silk in degradable scaffolds also helps in improving the vascularization property of the tissue engineering constructs.²²

Trisodium trimetaphosphate (STMP), a non-toxic substance that is used as an effective crosslinking agent in food industry,²³ to crosslink the isabgol/silk fibroin scaffolds. Here, we have fabricated isabgol and silk fibroin (Isab/SF) microporous scaffolds via freeze drying method to improve the cell attachment, and vascularization during wound healing. Morphology, stability, degradation, cell viability, cell attachment and in vivo wound healing efficacy of these Isab/SF scaffolds have been characterized and compared with controls.

2. Materials and method

2.1. Materials

Isabgol (psyllium husk) was procured from the pharmacy in the name of Sat-Isabgol. Silk cocoons were purchased from Tamilnadu co-operative silk producers' federation Ltd., Kancheepuram (TANSILK). Trisodium trimetaphosphate was purchased from Sigma Chemical Company, St. Louis, USA. Sodium hydroxide was purchased from SRL, India. All other reagents used for the experiments were high analytical grade.

2.2. Methods

2.2.1. Silk fibroin extraction. Silk fibroin was extracted from silk cocoons (Bombyx mori) by the standard protocol.²⁴ Briefly, silk cocoons were cut into small pieces and boiled in a 0.02 M sodium carbonate aqueous solution for 30 minutes. Then, the fibroin material was washed thoroughly in deionized water and permitted to dry overnight at room temperature. The dehydrated material was then dissolved in a 9.3 M aqueous solution of lithium bromide at 60 °C for 4 h. Excessive lithium bromide was removed from the solution by dialysis for 48 h and then centrifuged to remove the impurities as pellets. Supernatant contains 8–10 wt% silk fibroin in water. Then, the solution was lyophilized and kept at room temperature until further use.

2.3. Fabrication of the scaffolds

Isab/SF scaffolds were prepared by freeze drying method using STMP crosslinking. Briefly, Isab/SF scaffolds were prepared using 2 wt% solutions of isabgol (in 2 wt% lactic acid) and silk fibroin solution via STMP cross-linking.²⁵ Various proportions of these solutions have been taken and mixed with 750 μL of 15% (w/v) STMP and 250 μL 30% (w/v) NaOH for cross-linking. Then, this mixture was continuously vortexed for 1 h. The homogenized isabgol–silk fibroin mixture was transferred to a Teflon mold and kept at −20 °C for 12 h. The frozen samples were lyophilized for 24 h (Christ Alpha 1-2 LD Freeze Dryer, UK) to obtain 3D microporous Isab/SF scaffolds. The scaffolds were labeled as pure isabgol (Isab 100), 75% isabgol + 25% silk fibroin (Isab/SF 75/25), 50% isabgol + 50% silk fibroin (Isab/SF 50/50) and 25% isabgol + 75% silk fibroin (Isab/SF 25/75). These scaffolds were washed three times in deionized water and PBS solution to remove the excess of NaOH. Then, the scaffolds were stored at room temperature for the further characterization, in vitro and in vivo studies (Fig. 1a).

Fig. 1 (a) Digital images of Isab 100, Isab/SF 75/25, Isab/SF 50/50 and Isab/SF 25/75 composite scaffolds. Isab/SF composite scaffolds were fabricated using STMP via freeze drying method (scale bar 2 mm). (b) ATR-FTIR spectra of pure Isab 100, Isab/SF 75/25, Isab/SF 50/50 and Isab/SF 25/75 composite scaffolds showing the spectral characteristics.

2.4. Characterization of the scaffolds

2.4.1. ATR-FTIR spectroscopic analysis. The prepared Isab/SF scaffolds were analyzed using Perkin Elmer ATR-FTIR spectrometer from the range of 400–4000 cm⁻¹ with an average number 4 scans and 2 cm⁻¹ of resolution.

2.4.2. SEM analysis. The morphological characteristic feature of the Isab/SF scaffolds was analyzed in Quanta 200 scanning electron microscopy. The samples were mounted on aluminum stubs and the sections were examined under low vacuum with low voltage. Then, the photomicrographs were taken for the analysis.

2.4.3. Porosity. The porosity was measured by liquid displacement technique.²⁶ Briefly, hexane was used as displacement liquid since it infuses through the scaffolds without swelling or shrinking the scaffold matrix. The scaffolds were immersed in hexane until it was saturated. The weight of the scaffolds were noted before (dry weight, W_d) and after immersing in hexane for 30 min. The weights of the scaffolds in hexane were recorded as W₁. The liquid on the surface of the scaffolds were removed by filter paper after taking out of hexane. The weight of the wet scaffolds was recorded as W_w. The porosity of the Isab/SF scaffolds was calculated using the formula given below. All the tests were performed in triplicates and the values are expressed as mean ± standard deviation (n = 3).

Porosity (%) = (W_w − W_d)/(W_w − W₁) × 100

2.4.4. PBS solution absorption assay. The water sorption abilities of the Isab/SF scaffolds were determined by soaking the scaffolds in phosphate buffer saline (PBS), pH 7.4 at 37 °C for 1 h.²⁷ The water absorption ratio is defined as the ratio of weight increase (W_w − W_d) to the initial weight (W_d).

W = [(W_w − W_d)/W_d] × 100

where, W_w represents the wet weight of the scaffolds immersed in the PBS solution for 1 h to allow the samples to achieve equilibrium and W_d is the initial weight of the dry scaffolds. The wet weight of the scaffolds (W_w) was determined by first gently blotted with filter paper to remove water that was adhered on the surface and immediately weighed. The values are expressed as the mean ± standard deviation (n = 3).

2.4.5. In vitro biodegradation. The in vitro biodegradation of the scaffolds was tested in PBS solution, pH 7.4 at 37 °C. Briefly, scaffolds were equally weighed, soaked in PBS solution, pH 7.4 and incubated at 37 °C for 12 days with shaking speed of 100 rpm. Scaffolds were removed at regular interval and rinsed thoroughly with deionized water to remove ions adsorbed on the surface and lyophilized. The dry weight of the each scaffold was weighed as W_t and initial weight as W_i. The biodegradation of scaffolds was calculated using the formula.²⁸

Biodegradation (%) = (W_i − W_t)/W_i × 100

2.5. Cytocompatibility of the Isab/SF scaffolds

2.5.1. MTT assay. The percentage cell viability of the scaffolds was evaluated using NIH 3T3 cells.^29,30 Cells were cultured in DMEM with 10% FBS supplemented with penicillin (100 units per mL), streptomycin (100 μg mL⁻¹) and amphotericin B (0.25 μg mL⁻¹) at 37 °C, humidified with 5% CO₂. The cells cultured in an empty well were used as a positive control and scaffolds without cells as a negative control. Prior to the analysis, the scaffolds were sterilized in UV light for an hour. Sterile scaffolds were placed in 12-well culture plate containing 1 × 10⁴ cells. Experiment was performed in triplicate and 1 mL of cells was added in culture plates. The cells were then incubated in a humidified atmosphere of 5% CO₂ at 37 °C and the medium was changed every day. The quantitative evaluation of cell viability and proliferation was performed at 24 and 48 h using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay.

2.5.2. DAPI staining. DAPI (4′,6′-di-amidino-2-phenyl-indol) stain was used to identify the live and dead NIH 3T3 cells that were attached on the scaffolds.³¹ Briefly, NIH/3T3 cells were seeded on scaffold materials using 24 well tissue culture plates with a control (empty well) 1 × 10⁴ cells per well and left overnight for the cell attachment. After 24 h, medium was removed from the cells and rinsed three times with PBS. Then, the cells were fixed for 10 min in 3.7% formaldehyde. Formaldehyde was removed by rinsing the cells thrice in PBS. The cells were permeabilised by incubating in 0.2% Triton X-100 for 5 min. Triton X was thoroughly removed in PBS wash. DAPI stock solution (5 mg mL⁻¹) was diluted to 1 : 5000 in PBS. Cells were then incubated for 5 min in DAPI labeling solution at room temperature. Later, labeling solution was removed using PBS wash. The nuclei were counted in 10 randomly chosen fields of vision (FOV) for all the scaffold materials at a magnification of ×4 using an inverse fluorescence microscope. The digital images were taken at an excitation wavelength of 359 nm.

2.5.3. Cell attachment study. The NIH 3T3 cells were seeded on the porous scaffolds and cultured for 48 h in a 24-well plate at a concentration of 1 × 10⁴ cells per well. After 48 h of incubation, the scaffolds were rinsed with PBS solution and fixed with 2.5% glutaraldehyde for 1 h. Then, the samples were thoroughly washed with PBS and sequentially dehydrated in a graded-ethanol series (50%, 70%, 95% and 100%) and lyophilized. The cell attachment in scaffolds was examined by scanning electron microscopy (SEM) and images have been taken.^28,32

2.6. In vivo wound healing study

2.6.1. Animals grouping and excision wound creation. Healthy male Wistar rats (150–180 g) were used for the in vivo wound healing experiment. The rats were housed in wire topped cages with sterilized rice husk as bed material under controlled conditions of light/dark cycle (12 : 12 hours) and temperature at 29–31 °C. Rats were fed with commercial rat feed (pellets) and water ad libitum. All the procedures were followed according to the provisions of the Institutional Animal Care and Use Committee. A formal approval from the Institutional Animal Ethical Committee (IAEC) was also obtained (IAEC no. 01/2015(b)/21.09.2015).

The rats were divided into five groups, each group comprising five rats:

Group I: control rats, dressed with gauze;

Group II: dressed with Isab 100 scaffold;

Group III: dressed with Isab/SF 75/25 scaffold;

Group IV: dressed with Isab/SF 50/50 scaffold and

Group V: dressed with Isab/SF 25/75 scaffold.

Rats were anaesthetized by a single intraperitoneal injection of thiopentone (50 mg kg⁻¹ body weight) dissolved in sterile distilled water. A 2 cm² (4 cm) full thickness open excision wound was made on the back of the rat as reported in our earlier experiments.³³ The control rats were treated with normal gauze dressing and the other group of rats treated with Isab/SF scaffold dressing in every four day interval, until the wounds healed completely. The rate of wound contraction was traced and photographed on regular interval till completion. Granulation tissues formed on day 8 was removed and used for the collagen estimation.

2.6.2. Wound contraction and epithelialization. Wound contraction was measured as a percentage of the original wound size which was determined by tracing the wound area on to a transparent graph sheet and measuring the surface area planimetrically. Percentage wound contraction can be measured by the following formula.³⁴
where, n = number of days (day 4, 8, 12 and 16).

A decrease in epithelialization period is considered as an important factor as it denotes the number of days taken for complete closure of the wounds.

2.6.3. Estimation of collagen. The collagen content in granulation tissue samples was determined spectrophotometrically at 557 nm using Woessner method (1961) in terms of hydroxy proline.³⁵ Briefly, tissue samples were defatted in chloroform : methanol (2 : 1 v/v) and frozen in acetone. Weighed tissue samples were first hydrolyzed in 6 N HCl for 18 h at 110 °C, evaporated to dryness, and then made up with a known volume of double distilled water. From this, an aliquot was taken to estimate the amount hydroxy proline in tissues. Collagen content was calculated by the following formula.³⁶

Collagen = hydroxyproline × 7.46

2.6.4. Shrinkage temperature. The shrinkage temperatures of the granulation tissues were also measured on day 8 to check the strength of the newly formed collagen. The strength of the granulation tissue normally depends on the crosslinking of collagen, which is evaluated by measuring the shrinkage temperature.³⁷ Shrinkage meter was used to determine the shrinkage temperature of the granulation tissue. Briefly, tissue samples were taken and placed in cavity slide and the cavity was filled with 100 μL of water. Then the slide was kept at the heating microscope stage which was connected with thermometer. The temperature was gradually increased by heating. The temperature at which the sample starts shrinking was noted as the shrinkage temperature of the sample.

2.7. Histopathology

The granulation tissues removed on day 8 were used for the histopathological analysis. Tissue samples were fixed in 10% formalin–saline, dehydrated through graded alcohol series, cleared in xylene and embedded in paraffin wax (melting point 56 °C). Tissue sections of 5 μm thicknesses were used for staining with haematoxylin & eosin (H&E) and Masson's trichrome. The stained tissue sections were examined under light microscope for the histological signs.

2.8. Statistical analysis

Statistical analyses were performed using Graph Pad Prism (version 5.0; Graph Pad software Inc. San Diego CA, California, USA). All the values are expressed as mean ± standard deviation and results obtained were analyzed using Student's t-test. The values of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered as statistically significant.

3. Results and discussion

3.1. Characterization of the Isab/SF scaffolds

3.1.1. ATR-FTIR analysis. ATR-FTIR spectroscopy was used to analyze the structural characteristic results of the Isab/SF scaffolds (Fig. 1b). A broad absorption band observed at 3230 cm⁻¹ is due to OH stretching of alcohols for pure isabgol scaffold (Isab 100).³⁸ This peak is shifted to 3263 cm⁻¹, 3265 cm⁻¹ and 3270 cm⁻¹ for the Isab/SF composite scaffolds. Shifting of bands indicates that the significant interactions between isabgol and silk fibroin. A peak at 2930 cm⁻¹ is attributed to the CH stretching of alkanes, which is also slightly shifted for the Isab/SF scaffolds. C–O–C stretching was observed at 1078 cm⁻¹ for all the scaffolds. ATR-FTIR results showing the shift in many stretching bands suggested that the interaction of isabgol and silk. The peaks at 878, 776 and 619 cm⁻¹ are recognized due to the polymer backbone bending.³⁸ The general characteristic absorption bands of the Isab/SF composite scaffolds are observed around 1560–1587 cm⁻¹ for amide I (C=O stretching vibration), 1416–1420 cm⁻¹ for amide II (bending vibrations of the N–H bond) and 1308–1314 cm⁻¹ for the amide III (N–H deformation and C–N stretching vibrations). These peaks are characteristic of the β sheet conformation in silk.³⁹

3.1.2. SEM analysis. Morphology and internal microporous architecture (cross sectional view) of the Isab/SF composite scaffolds was examined using scanning electron microscopy. Internal cross sectional views with high magnification were observed and given in Fig. 2. A rough and irregularly interconnected polyhedral shape was observed for the Isab 100 scaffolds. A flower like architecture with branched stems was seen in Isab/SF scaffolds. Roughness was slightly increased while increasing the concentration of silk fibroin (Isab/SF 25/75).

Fig. 2 SEM micrographs showing the cross sectional morphology of (a) Isab 100, (b) Isab/SF 75/25, (c) Isab/SF 50/50 and (d) Isab/SF 25/75 3D scaffolds.

3.1.3. Porosity. Porosity of the prepared scaffolds was studied using liquid displacement technique and depicted in Fig. 3a. Hexane is used as a non-solvent, able to penetrate through the scaffolds without causing shrinkage or swelling unlike ethanol. Among various methods of the 3D porous scaffold preparation, freezing lyophilization method provides a significantly high porosity in scaffolds. Isab 100 scaffolds showed 94% porosity, whereas scaffolds with SF exhibited around 79, 70 and 50% for Isab/SF 75/25, 50/50 and 25/75, respectively. SF introduction reduced the formation of pores slightly due its fibrous protein nature. The presence of β sheet random coil increases the stiffness and water insolubility to Isab/SF scaffolds. However, an earlier study shows that silk fibroin can enhance vascularization, which will help in wound healing.²²

Fig. 3 (a) Porosity measurement, (b) phosphate buffer solution absorption assay, (c) in vitro biodegradation. Values are expressed as mean ± SD (n = 3) and the level of significance is expressed as *p < 0.05, **p < 0.01 and ***p < 0.001 respectively, compared to the control.

3.1.4. PBS solution absorption ability. The fluid uptake, particularly PBS absorption ability was determined and showed in Fig. 3b. This property is considered as a marker to evaluate the wound exudate absorption ability of the scaffolds.⁵ Therefore, PBS is commonly used as a pseudo exudate solution. Results revealed that 503–783% of PBS uptake ability was observed for the Isab 100 and Isab/SF composite scaffolds. Scaffolds with enough porosity and exudate absorption capacity are crucial for the dermis regeneration process. This property attributes the scaffolds as an ideal dermal substitute enhances the cell activity.²⁷ Polymer and porous nature of the scaffolds are the central factors for the exudate absorption property.

3.1.5. In vitro biodegradation. Biodegradation is an essential characteristic feature for any biomaterial. We had determined the degradation ability of scaffolds in PBS solution (pH 7.4 at 37 °C) and the results were given in Fig. 3c. Isab/SF scaffolds showed significant weight loss at predetermined time interval and suggests that these materials are biodegradable. Results showed that Isab/SF composite scaffolds showed significant weight loss at predetermined time interval compared to Isab 100 scaffolds (control). Among the different combinations of these Isab/SF scaffolds, Isab/SF 75/25 showed well-ordered biodegradation (41–78%) from day 4 to 12.

Silk fibroin is a class of an enzymatically biodegradable protein polymer composed of hydrolytically stable amide bonds. Hydrolytic enzymes help in the degradation of fibroin significantly.^23,25 Proteolytic enzymes had been employed in PBS to assess the biodegradation property of silk fibroin and protease was found to be very effective. However, biodegradation studies performed using PBS, in the absence of proteolytic enzymes, showed very minimal degradation. These studies displayed that around 5–12% of weight loss occurred in 80–160 days of incubation. These results confirmed that the PBS was ineffective for in vitro biodegradation of silk fibroin after longer incubation periods.^29,40 However, our results showed significant amount of biodegradation in PBS alone without adding any enzyme. This change in degradation behavior is possibly being due to the presence of isabgol in the composite scaffold. This result associates with the biodegradation behavior of the isabgol.⁴¹ Hence, Isab/SF scaffolds are biodegradable due to the presence of highly degradable polysaccharide and can be useful for biomedical applications.

3.2. In vitro cell culture study

3.2.1. Cytotoxicity. Biocompatibility of the prepared Isab/SF scaffolds were tested in NIH 3T3 fibroblast cells and the toxicity was determined by MTT assay. Results showed that Isab/SF 75/25 scaffold showed 80 and 110% of viability compared to control at 24 and 48 h of incubation. Other combinations showed slightly lesser viability at 24 and 48 h incubation. This observation was probably because the cells were adhered firmly in to the scaffolds and were not leached completely at the time of MTT assay. Hence, the reduced viability was observed in MTT assay for the Isab/SF 50/50 and 25/75 scaffolds. However, cell loaded scaffolds were examined under light microscope after 24 h to evaluate the viability, attachment and proliferation of fibroblast cells (Fig. 4a). Microscopical images showed adequate amounts of cell proliferation and adhesion in all the scaffolds compared to positive control (data not shown). However, Isab/SF 75/25 showed higher cell attachment and proliferation compared to other scaffolds at 24 and 48 h. This result correlates with the DAPI staining and suggests that Isab/SF 75/25 blend had ideal morphology to supply sufficient gas, nutrient to the cells to adhere and proliferate. In addition, other Isab/SF scaffolds also showed more number of viable cells (purple color) but there was a difficulty in removing the live cells from the 3D scaffolds while evaluating the MTT assay.

Fig. 4 (a) MTT assay, (b) DAPI nuclei staining using NIH 3T3 fibroblast cells.

3.2.2. DAPI staining. Viability and cell count assay was done using DAPI stain and the nuclei were counted randomly under the fluorescence microscope to assess the toxicity of the Isab and Isab/SF scaffolds (Fig. 4b). The present study shows that significant number of viable cells for Isab/SF composite scaffolds as control (culture plate). This study explores that these Isab/SF scaffolds are biocompatible, nontoxic and can be used as a wound dressing material in in vivo model.

3.2.3. NIH 3T3 fibroblast cell attachment. NIH 3T3 fibroblast was seeded on the Isab/SF scaffolds and incubated for 48 h to examine the cell attachment using SEM analysis. Fig. 5 shows the fibroblast attachment on the control, and Isab/SF scaffolds. SEM micrographs exposed the presence of fibroblast cells on the surface and inner pores of the scaffolds. SF incorporation increased the layer by layer deposition and spindle shaped fibroblast cells in Isab/SF scaffolds. Generally, protein based materials are capable to mimic the microenvironment of the native tissues and ECM properties. Therefore, these biomaterials are ideal to accelerate cell migration, proliferation and attachment during tissue repair.^42,43 Also, blending of silk fibroin with other substances improved the biodegradation, release, cell growth, migration, differentiation and wound healing.^32,42,44

Fig. 5 SEM images showing the fibroblast cell attachment in (a) Isab 100, (b) Isab/SF 75/25, (c) Isab/SF 50/50 and (d) Isab/SF 25/75 3D scaffolds.

3.3. In vivo wound healing experiment

The in vivo results demonstrated that the use of Isab/SF scaffolds reduced the frequent dressing, spread of wound exudate and resulted in accelerated wound healing in dermal wounds. Isab as well as Isab/SF composite scaffolds were firmly adsorbed on the wound surface and degraded slowly at wound surface. Biocompatibility and lower inflammatory response of the SF offer the Isab scaffolds with SF combination as an ideal wound dressing material to enhance the cutaneous wound healing.¹⁴

3.3.1. Percentage wound contraction. The wound contraction rate and contracted digital wound images was given in Fig. 6a and b to authenticate the wound healing ability of the prepared scaffolds. The percentage wound closure rate was measured from day 0 to complete healing and depicted in picture as well as in figure. Control wound showed around 6% of wound contraction on day 4, 13% on day 8, 21% on day 12 and 48% on day 16. Isab 100 showed slightly higher wound closure rate on day 4 (10%), day 8 (18%), day 12 (25%) and day 16 (62%) compared to control wounds. Isab/SF 75/25 composite scaffold showed significantly higher (p < 0.001) wound contraction rate compared to control and all other scaffolds. Wounds treated with Isab/SF 75/25 achieved more than 95% of wound closure on day 16. However, other combinations like Isab/SF 50/50 and 75/25 did not show significant rate of wound contraction. This is probably due to the lack of porosity and absorption capacity like Isab 100 and Isab/SF 75/25 scaffolds. Finally, the higher wound closure rate was found in Isab/SF 75/25 scaffolds treatment with ample wound exudate absorption property, and probably vascularization ability. Wound contraction is a positive process where the tissue matrix contracts to reduce the healing time by producing sufficient granulation tissue to repair the tissue.⁴⁵

Fig. 6 (a) Digital images showing the wound contraction rate, and (b) percentage wound contraction. Values are expressed as mean ± SD (n = 3) and the level of significance is expressed as *p < 0.05, **p < 0.01 and ***p < 0.001 respectively, compared to the control.

3.3.2. Period of epithelialization. Period of epithelialization was studied and given in Fig. 7a. The results showed that control and pure Isab (Isab 100) scaffold treated wounds took around 23 and 20 days, respectively, for the complete healing. However, Isab/SF 75/25 took only 16 days for complete healing. Isab/SF 50/50 (20 days) and Isab/SF 25/75 (21 days) scaffolds did not heal the wounds faster.

Fig. 7 (a) Period of epithelialization (b) collagen content and (c) shrinkage temperature. Values are expressed as mean ± SD (n = 3) and the level of significance is expressed as*p < 0.05, **p < 0.01 and ***p < 0.001 respectively, compared to the control.

3.3.3. Collagen content. Collagen is a major connective tissue protein, providing the structural integrity for the remodeling tissue during wound repair and tissue regeneration. Wound healing occurs based on regulated biosynthesis of collagen, deposition and its subsequent maturation in the extra cellular matrix (ECM).³³ Therefore, collagen is considered as an essential component in the ECM for the active wound healing process.

The amount of collagen content was measured in terms of hydroxyproline in day 8 granulation tissues and depicted in Fig. 7b. Collagen content was significantly increased in Isab/SF scaffolds treated wounds. Isab 100 scaffold treatment showed 62.9% higher collagen content compared to control. Isab/SF 75/25 treatment showed 200% increment in collagen than the control. Also, Isab/SF 50/50 and 25/75 scaffolds treated wounds showed 117% and 112% increased collagen content compared to control.

3.3.4. Shrinkage temperature. Fig. 7c shows the shrinkage temperature measurement of the scaffolds treated granulation tissues. Shrinkage temperature is the strength of newly formed tissue, which is a key factor for measuring the crosslinking of collagen. Stability and strength is based on the amount of hydroxyproline, proline and inter molecular crosslinking of newly formed collagen.⁴⁵ Shrinkage temperature of the Isab/SF 75/25 scaffolds treated granulation tissues was found 72% higher than control tissues. Hydroxyproline has a tendency to form ester links between polypeptide chains through the formation of inter-chain hydrogen bonding between hydroxyl groups of hydroxyproline and carbonyl oxygen of the adjacent peptide. This determines the stability and reactivity of collagen fiber.⁴⁶ This result substantiates that the Isab/SF 75/25 scaffolds treatment not only increased the collagen content, also enhanced the collagen cross-liking significantly. However, other scaffolds includes Isab 100, Isab/SF 50/50 and 25/75 blend also significantly increased the shrinkage temperature compared to control.

3.4. Histopathology

Granulation tissues collected at the wound site on day 8 were used for the hematoxylin and eosin (H&E) staining. Collagen formation and deposition was qualitatively assessed using Masson's trichrome staining. Tissue sections were examined under light microscope and the images given in Fig. 8a and b. Fibroblasts are essential to promote re-epithelialization process at the wound site by producing ECM proteins like collagen.⁴⁷ In addition to fibroblasts, macrophages, keratinocytes and epithelial cells are also produced in the early phase of inflammation and playing major role for the wound healing process.⁴⁸ Macrophage plays multifunctional roles during wound repair like host defense, cell proliferation and removal of apoptotic cells from the wound site. Previous studies strongly reveals that the macrophage dysfunction or depletion can lead to poor wound healing.⁴⁸

Fig. 8 (a) Histopathology results showing the hematoxylin and eosin (H&E; 20×) stained granulation tissues on day 8. White arrows indicate the blood vessels formation and black arrows indicate the fibroblast cells. E denotes the epidermis development, D denotes the dermis layer and C denotes the deposition of collagen fibers in treated tissues. Scale bar 100 μm. (b) Masson's trichrome (20×) stained granulation tissues on day 8. C denotes the deposition of collagen fibers in treated tissues. Scale bar 100 μm.

Control tissues showed large number of inflammatory cells on day 8. It clearly emphasizes that the inflammation still continues and not started to regenerate the dermis and epithelial layer. It also shows less number of blood cells, fibroblasts and collagen deposition. Isab 100 scaffold treated rats showed thin epithelial layer, reduced number of inflammatory cells with minimal fibroblasts and blood vessel formation compared to control. This indicates that Isab 100 treatment started the healing process. Isab/SF 75/25 composite scaffold treatment showed high amount of fibroblast as well as the dense collagen fiber deposition (Fig. 8b) on day 8 compared to control. Thick dermis and epithelial layer is also seen with high number of blood vessel formation. This result correlates with increased collagen content, faster wound contraction and period of epithelialization. However, Isab/SF 50/50 and 25/75 treatment showed lesser cellular infiltration with moderate collagen deposition with thin dermis and epithelium compared to Isab/SF 75/25. Increased number of blood vessels was also found in these two scaffolds treated wounds as a result of angiogenesis. Collagen fibers were not observed as seen in the other scaffold treated tissues on day 8. In accordance with the previous findings, these observations confirmed that the SF influences the neovascularization process by activating the stem cell differentiation into endothelial cells.²² A new scaffold, Isab/SF 75/25 offered an ideal condition with enhanced vascularization to regenerate the damaged tissue. Hence, histopathological analysis showed the enhanced neovascularization in Isab/SF composite scaffolds treatment.

4. Conclusion

We used isabgol and SF to fabricate the porous 3D scaffolds with STMP via freeze drying method technique. ATR-FTIR data revealed that the presence of β sheet confirmation in Isab/SF composite scaffolds for the requisite properties. The prepared Isab/SF scaffolds showed porous architecture with irregularly interconnected sponge like networks. A significant rate of biodegradation was also observed for all the scaffolds. MTT assay and DAPI staining showed higher viability of NIH 3T3 cells at 24 h for Isab/SF 75/25 scaffolds compared to control and other scaffolds. However, scanning electron microscopical image analysis confirmed that fibroblast cells were firmly adhered and proliferated to the interior portion of the scaffolds. Results showed that the higher number of cells that were adhered and attached only on the Isab/SF 75/25 scaffolds. An ideal wound dressing material should reduce the frequent wound dressing, infection and inflammation. Subsequently, it should accelerate the cellular activity to heal the wounds faster with minimal scar formation. This SF protein incorporated Isab scaffolds showed better wound exudate absorption ability, degradation, fibroblast cell attachment, enhanced neovascularization and wound contraction. These unique characteristics of this Isab/SF scaffolds promoted the remodelling of complex tissue and deposition of abundant collagen at the wound site. Hence, the period of epithelialization was shorter in Isab/SF 75/25 scaffolds treated wounds. In conclusion, Isab/SF 3D composite scaffolds showed porous architecture, enhanced fluid uptake ability with enough fibroblast attachment and viability on the scaffolds. Also, Isab/SF scaffold dressings significantly improved the rate of wound healing and reduced the epithelialization period. These properties will support for sufficient gas, nutrient transfer, absorb excess wound exudates, wastes to accelerate the tissue regeneration and dermal wound healing applications.

Conflict of interest

Authors declare that there is no conflict of interest.

Acknowledgements

First author thanks Indian Institute of Technology Madras for the Institute Post-doctoral fellowship. Authors acknowledge the Sophisticated Analytical Instrument Facility (SAIF), IITM for characterization of the biomaterial. We acknowledge Prof. Vani Janakiraman and Dr Purva Bhatter for their help in cell culture work. We also thank CSIR-Central Leather Research Institute for the approval to utilize the animal facility in CLRI.

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