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Fenugreek Incorporated Silk Fibroin NanofibersA Potential Antioxidant Scaffold for Enhanced Wound Healing Sowmya Selvaraj and Nishter Nishad Fathima* Chemical Laboratory, Council of Scientific and Industrial Research- Central Leather Research Institute, Adyar, Chennai 600020, India
ABSTRACT: Free radicals are generated by various biochemical pathways in the living system, causing severe oxidative damage to the biomolecules leading to adverse disease conditions. Hence, there is an increasing interest in antioxidant studies for preventing the effects of these free radicals. Herein, we propose a novel electrospun scaffold with antioxidant properties that can be used as wound healing material. Fenugreek, a natural antioxidant incorporated silk fibroin nanofiber, was prepared in four different ratios by the co-electrospinning method. The biocompatibility of the nanofibers and its antioxidant activity were evaluated through 3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay, respectively. The experimental observations indicate that the incorporation of fenugreek increases the thermal and mechanical properties of silk fibroin nanofibers. DPPH assay proves that the antioxidant property is enhanced with increasing concentration of fenugreek in nanofiber mats, and the Swiss albino 3T6 fibroblasts show better proliferation on the nanofibrous scaffolds. Further, the wound healing efficiency of fenugreek incorporated silk fibroin nanofibrous scaffolds was evaluated using full thickness excisional wounds in rat model. Wound healing was accelerated in silk fibroin−fenugreek nanofibers treated wounds with complete re-epithelialization and enhanced collagen deposition. The present study validates the use of fenugreek incorporated silk fibroin nanofiber mats as antioxidant scaffolds in wound healing applications. KEYWORDS: silk fibroin, fenugreek, nanofibers, scaffold and wound healing olaemic activity,7 and anti-ulcerogenic effects.8 Fenugreek seeds show potential antioxidant properties and reduce the H2O2 induced oxidative damage in normal and diabetic erythrocytes.9,10 Ivy and co-workers have used fenugreek as an additive in the preparation of collagen based biomaterials where fenugreek is found to alter the physicochemical properties of collagen.11 Fenugreek absolute obtained from the seeds of the plant Trigonella foenum graecum contains mainly trigonelline, naringenin, nicotinic acid, quercetin, and saponins.9,12 Fenugreek absolute can be used to enhance of the antioxidant properties of biomaterials, which could further increase their wound healing potential. Silk fibroin is a fibrous protein with 5263 amino acid residues obtained from the cocoons of silk worm Bombyx mori, which is composed of 45.9% glycine, 30.3% alanine, 12.1% serine, 5.3%
1. INTRODUCTION Antioxidants play an important role in wound treatment and management. Wound healing consists of three major phases, namely, inflammation phase, cellular proliferation, and remodeling phase.1 During the inflammation phase, at the wound site neutrophils release proteases and reactive oxygen species against the invading microbes. However, the presence of increased amounts of reactive oxygen species could hamper the wound healing process by causing severe damage to the cells like fibroblasts which secretes collagen and glycosaminoglycans for wound repair. Antioxidants reduce the adverse effects of wounds by removing the reactive oxygen species generated during the inflammation phase.2−4 Many of the plant compounds possess a high level of antioxidant properties, which could play a major role in the wound healing process. Fenugreek or methi is one of the oldest medicinal plants, which belong to the family Leguminosae. It is mainly used in food preparations because of its strong flavor and aroma. Fenugreek seeds exhibit hypoglycemic effect,5,6 hypocholester© 2017 American Chemical Society
Received: December 23, 2016 Accepted: January 26, 2017 Published: January 26, 2017 5916
DOI: 10.1021/acsami.6b16306 ACS Appl. Mater. Interfaces 2017, 9, 5916−5926
Research Article
ACS Applied Materials & Interfaces
2.5. Characterization of Silk Fibroin and Silk Fibroin− Fenugreek Nanofibers. Fiber morphologies of the electrospun samples were observed using scanning electron microscope (Phenom G2 Pro) at an acceleration voltage of 5 kV. SEM images were analyzed using ImageJ software to determine the fiber diameters. The average fiber diameter and standard deviations were calculated from 100 random measurements per image. ATR-FTIR spectra of as-spun and ethanol treated nanofibers were obtained from the Jasco FTIR spectrophotometer in the region of 400−4000 cm−1. Crystalline nature of the nanofibers was analyzed by Rigaku Miniflex II desktop X-ray diffractometer with Cu Kα radiation (λ = 1.540562 Å). Thermal properties of electrospun silk and fenugreek incorporated nanofibers were studied using differential scanning calorimeter Netzsch-DSC 204 F1 phoenix. Samples were placed in standard aluminium pans and heated from 25 to 300 °C at a heating rate of 10 °C/min. Tensile properties of the nanofiber mats were analyzed using Instron 3345 universal tensile tester. A segment of electrospun mats with the size of 10 mm width and 50 mm length was clamped at its ends for testing at a stroke rate of 5 mm/min with 40 mm gauge length. Porosity measurements were done using Porous material Inc. automated humid air porometer-HCFP-1100 AE. 2.6. In Vitro Release Studies. The release profile of fenugreek from the silk fibroin nanofiber matrix was investigated in PBS at pH 7.4. Standards were prepared by dissolving known amount of fenugreek in solvent. Standard graphs were plotted using the absorbance at 270 nm which is specific for trigonelline and nicotinic acid. Fenugreek loaded nanofiber mats (5 mg) were incubated with 5 mL of PBS at 37 °C for the period of 24 h. At specific time intervals, 1 mL of solution was withdrawn from the release medium and the same was replaced with fresh medium. The relative amount of fenugreek was determined using Shimadzu UV−vis spectrophotometer 1800 at the wavelength of 270 nm by comparing the standard plot. 2.7. Antioxidant Activity Assay. Antioxidant properties of the electrospun mats were assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with respect to fenugreek concentration and time.24,25 5 mg of the electrospun mats with different fenugreek concentration was added to 3.0 mL of 100 μM DPPH solution in methanol. The reaction mixture was then incubated in the dark for 30 min followed by the wavelength scanning, which was performed using Shimadzu UV−vis spectrophotometer 1800. DPPH degradation was calculated using the following formula.
tyrosine, 1.8% valine, and 0.25% tryptophan with the molecular weight of 391 kDa.13 It has several advantages like good biocompatibility, good oxygen and water vapor permeability, minimal inflammatory reactions, and slow biodegradability.14,15 Silk fibroin promotes collagen synthesis and re-epithelialization via rapid proliferation of epithelial cells.16 Silk fibroin has been used in a variety of forms such as sponges, hydrogels, films, fibers, and microspheres.17,18 Among these materials, nanofibers have some unique characteristics like high surface to volume ratio, superior mechanical properties, and flexibility in functionalities.19 At present, electrospinning has been used for the preparation of nanofibers since it is cost-effective, simple, and versatile where electrostatic force is applied to produce nanofibers from polymer solutions.20,21 In this study, we have fabricated fenugreek seed extract incorporated silk fibroin nanofibers by electrospinning using HFIP. Morphological, spectroscopic, and calorimetric analyses were carried out to investigate the influence of fenugreek on the silk fibroin nanofibers. Antioxidant property was analyzed through DPPH assay whereas biocompatibility of the scaffolds was evaluated by MTT assay using Swiss albino mouse 3T6 fibroblast cell lines. The in vivo wound healing efficiency of the fenugreek incorporated silk fibroin nanofibers was evaluated using full thickness excisional wound model. The objective of the present study is to develop antioxidant enriched scaffolds for wound healing applications.
2. EXPERIMENTAL SECTION 2.1. Materials. Bombyx mori cocoons were obtained from department of sericulture, Tamilnadu. Fenugreek absolute, 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2-diphenyl-1-picrylhydrazyl, fluorescein diacetate, and 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide were purchased from Sigma-Aldrich. Lithium bromide and sodium carbonate were provided from Hi-Media. Dulbeccos modified eagle medium (DMEM) and fetal bovine serum were purchased from ThermoFisher Scientific. Swiss 3T6 mouse fibroblast cells were provided from National Centre for Cell Sciences, Pune. All chemicals were used as received without further purification. 2.2. Silk Fibroin Extraction. Pupae removed Bombyx mori cocoons were boiled in 0.02 M Na2CO3 solution for 30 min to remove sericin and washed three times with ultrapure water. The resulting silk threads were dried overnight. Ten grams of silk thread was dissolved in 9.3 M LiBr solution at 60 °C for 5 h. The silk fibroin solution was dialyzed extensively against deionized water for 3 days. The water was changed at every 6 h interval. After dialysis, the solution was lyophilized to obtain silk fibroin sponges.17 2.3. Electrospinning of Fenugreek Incorporated Silk Fibroin Nanofibers. Silk fibroin sponges were dissolved in 1,1,1,3,3,3hexafluoro-2-propanol (HFIP) to obtain 3% (w/v) silk fibroin solution. A series of silk fibroin−fenugreek solutions (1:0.1, 1:0.2, 1:0.5, and 1:1 w/w) were prepared by mixing fenugreek with silk fibroin solution at predetermined levels. The solutions were stirred at room temperature for 3 h. Silk fibroin and silk fibroin−fenugreek solutions were electrospun using ESPIN−NANO electrospinning apparatus. The solutions were loaded in 5 mL syringe with 24G needle. The applied voltage was fixed as 25 kV and the distance between tip to the collector was maintained as 10 cm. The solution was injected from the syringe at the flow rate of 0.5 mL/h. All electrospinning experiments were carried out at 25 °C. 2.4. Chemical Treatment of Silk Fibroin and Silk Fibroin− Fenugreek Electrospun Mats. Silk fibroin and silk fibroin− fenugreek nanofiber mats were treated with ethanol vapor in order to improve the stability in water and to induce the structural changes from silk I (random coil) to silk II (β sheet). The mats were treated with 75% ethanol vapor at 25 °C for 3 days and dried under vacuum at room temperature for 1 day.22,23
Percentage of DPPH scavenging =
AB − A S × 100 AB
where AB is Absorbance of blank at 517 nm and AS is Absorbance of sample at 517 nm. For time dependent assay, 5 mg of nanofibrous mat was suspended in 1 mL of PBS for different time intervals and the suspension was incubated with 1 mL of 100 μM DPPH solution for 30 min after which the absorbance was measured at 517 nm. 2.8. Cell Culture. A mouse fibroblast cell line 3T6-Swiss albino was cultured in Dulbeccos modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic in an atmosphere of 5% CO2 and 37 °C. The nanofibers were coated on the coverslips and placed on to 24 well cell culture plates. The cells were detached by trypsin-EDTA from the culture flask and were seeded on the nanofibers at a density of 1.5 × 104 cells per well. 2.9. Biocompatibility Assay. The biocompatibility of the nanofibers was evaluated using MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) at different culture periods up to 72 h in triplicates.26 MTT solution was added to each well and was incubated for 3 h. The formazan complex was dissolved in dimethyl sulfoxide and the optical density was measured with microplate reader at the wavelength of 570 nm. Percentage of cell viability was calculated from the optical density of samples and control. 2.10. Live Cell Staining and SEM Analysis. Viability of the Swiss albino 3T6-fibroblast cells on the nanofiber scaffold was assessed through fluorescein diacetate staining. FDA solution was prepared with 10 μg/mL concentration. After the removal of cell culture medium, the scaffolds with cells were washed with phosphate buffer saline (1×) and 5917
DOI: 10.1021/acsami.6b16306 ACS Appl. Mater. Interfaces 2017, 9, 5916−5926
Research Article
ACS Applied Materials & Interfaces
Figure 1. (a−e) SEM images of nanofibers and (f−j) normalized histograms of fiber diameters: (a,f) silk fibroin nanofibers, (b,g) silk fibroin− fenugreek nanofibers (1:0.1), (c,h) silk fibroin−fenugreek nanofibers (1:0.2), (d,i) silk fibroin−fenugreek nanofibers (1:0.5), (e,j) silk fibroin− fenugreek nanofibers (1:1). incubated with FDA solution for 30 min. Again, the scaffolds were washed with PBS and the cells were visualized through Leica inverted microscope. For SEM analysis after removing the culture media, the scaffolds were washed with PBS and fixed with 4% para-formaldehyde for 30 min. Then the scaffolds were dehydrated in a graded series of ethanol solutions and dried overnight, after which the scaffolds were examined under SEM at 5 kV accelerating voltage. 2.11. In Vivo Wound Healing Experiments. Healthy male wistar rats weighing 200−230 g were used for excisional wound healing studies. All the experimental procedures were approved by Institutional Animal Ethical Committee (Central Leather Research Institute, India, IAEC No. 11/2016(a)). Animals were anesthetized individually by an intraperitoneal administration of ketamine (35 mg/ kg) and xylazine (5 mg/kg). The dorsal surface of the rats was shaved and disinfected with 70% ethanol, and a full thickness (2 × 2 cm2) excision wound was created by excising the dorsal skin. A total of 18 animals were divided into three groups (six animals per group). Group 1 (control) animals were treated with gauze, whereas group 2 were treated with silk fibroin nanofibers and group 3 were treated with fenugreek incorporated silk fibroin nanofibers (1:1). Scaffolds were further covered with gauze and fixed with bandage to hold the material
on the wound area. On days 4, 8, 12, and 16 post wounding, wounds were photographed and the wound size was traced using a transparent sheet followed by changing the dressing materials. Percentage of wound closure was calculated by the following formula
Cn =
(S0 − Sn) × 100 S0
where Cn is the percentage of wound size reduction on days 4, 8, 12, and 16 post wounding, S0 is the initial wound area, and Sn is the wound area on days 4, 8, 12, and 16 post wounding. Granulation tissues formed on day 8 of wound healing were collected from all the groups. Animals were sacrificed on the 16th day post wounding and the wound site with surrounding skin was surgically excised. 2.12. Histological Analysis. Tissues collected on postoperative days 8 and 16 were fixed with 10% formalin and embedded in paraffin. Sections of 4 μm thickness were prepared and stained with hemotoxylin−eosin (H&E) and Masson’s trichrome stains. Histological sections were observed using Zeiss Axioscope microscope. 5918
DOI: 10.1021/acsami.6b16306 ACS Appl. Mater. Interfaces 2017, 9, 5916−5926
Research Article
ACS Applied Materials & Interfaces
Figure 2. (a−e) SEM images of nanofibers after 75% ethanol vapor treatment: (a) silk fibroin nanofibers, (b) silk fibroin−fenugreek nanofibers (1:0.1), (c) silk fibroin−fenugreek nanofibers (1:0.2), (d) silk fibroin−fenugreek nanofibers (1:0.5), (e) silk fibroin−fenugreek nanofibers (1:1).
Figure 3. ATR-FTIR spectra of fenugreek, as-spun, and ethanol treated nanofibers: (a) fenugreek, (b) as-spun silk nanofibers, (c) ethanol vapor treated silk nanofibers, (d) as-spun silk fibroin−fenugreek nanofibers, (e) ethanol treated silk fibroin−fenugreek nanofibers. 2.13. Statistical Analysis. All the experiments were carried out in triplicate and the data were expressed as average ± standard deviation. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test using OriginPro 8. P values
