Fibers and Polymers 2015, Vol.16, No.5, 1020-1030 DOI 10.1007/s12221-015-1020-y
ISSN 1229-9197 (print version) ISSN 1875-0052 (electronic version)
Flexible Silk Fibroin Films for Wound Dressing Chandra Mohan Srivastava, Roli Purwar*, Rekha Kannaujia1, and Deepak Sharma2 Department of Applied Chemistry and Polymer Technology, Delhi Technological University, Delhi 110042, India 1 Department of Botany, University of Lucknow, Lucknow 226007, India 2 Department of Pharmaceutics, Central Drug Research Institute, Lucknow 226031, India (Received January 5, 2015; Revised March 9, 2015; Accepted March 17, 2015) Abstract: In the present study, an effort has been made to create dextrose incorporated (5-15 % w/w) flexible silk fibroin films for wound dressing applications. The flexibility of silk fibroin films increases with increase in dextrose content. The elongation at break properties of dextrose modified silk fibroin (DMSF) films increases from 3.2 % to 40 % with increase in dextrose content. The glass transition temperature (Tg) of the films decreases from 176 oC to 155 oC with increase in dextrose content. This shows that dextrose is acting as plasticizer for silk fibroin films. The structural and morphological properties of dextrose modified silk films (DMSF) are characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) scanning electron microscopy (SEM) and atomic force microscopy (AFM). FTIR and XRD studies show that the dextrose content does not affect the crystalline structure of silk fibroin films. The surface roughness of the films also increases with increases in dextrose content in DMSF films. The addition of dextrose enhances the swelling and hydrophilicity of silk fibroin films. The adherence, proliferation and viability of L929 fibroblast cells cultured on DMSF films indicate that it has ability to support cell growth and proliferation as compared to SF film. The 15 % DMSF film showed significantly higher mass loss than SF film after 50 days of incubation in Protease XIV. Further, the data presented here constitute strong evidence that dextrose modified film has the great potential to be utilized as dermal wound dressing material. Keywords: Silk fibroin, Wound dressing materials, Hydrophilicity, Cytocompatibility, Biodegradation
being employed as tissue scaffolds for skin reconstruction [5,6]. Silk has been used in textile industries for centuries. The silk protein from silkworm Bombyx mori contains two fibroin proteins fibers held together by glue like protein called sericin. When sericin is presented to a body, it is detected as an antigenic factor by T-cells and causes immunologic reactions. Therefore, sericin is needed to be removed from cocoon fibers by a process called degumming. These degummed Bombyx mori silk fibroin exhibit unique properties as biomaterials. Fibroin was reported to be a perfect substrate for the proliferation and adhesion of large variety of cells. Silk fibroin has found diverse applications in the biomedical field, which can be attributed to its high tensile strength, controlled biodegradability, haemostatic properties, noninflammatory characteristic [7-10]. To develop an ideal skin substitute, the performance of regenerated membranes from silk fibroin and its blends was examined by several researchers. Studies evaluated the biocompatibility of the membrane with fibroblasts and endothelial cells, which are the most important tissue repairing cells in healing. It was found that regenerated silk fibroin membrane does not show any toxicity [11] or genotoxicity [12]. Regenerated silk fibroin materials in the form of films, nano-fibrous membrane and woven textile have been employed as wound dressing for skin regeneration. Liu et al. [13] have explored the feasibility of using regenerated silk fibroin membrane to construct artificial skin substitute for wound healing. They observed that silk fibroin film does not have an adverse influence on the growth and biofunction of fibroblast and vascular endothelial cells. It also does not interfere with the secretion of angiogenesis
Introduction The skin forms a self renewing and self repairing interface between the body and the environment. It provides an effective barrier against microbial invasion and has properties that can protect against mechanical, chemical, osmotic, thermal and photo damage [1]. Skin is composed of three structural layers, the epidermis, dermis and subcutis. Trauma to skin can be categorized into several degrees. The least damaging trauma relates to damage done at the epidermis layer and wound healing takes place via re-epithelialization. More serious trauma can lead to partial or complete damage of epidermis, dermis and subcutis layer of skin [2]. Wounds that extend partially through the dermis are capable of regeneration, but unfortunately the body can not heal deep dermal injuries adequately. With burn injuries arising from fire, accidents, terrorist attacks and an aging population (for chronic wound), there will always be a continual demand for skin regeneration products [3]. Many skin substitutes such as xenografts, allografts and autografts have been employed for wound healing. However, these approaches have many disadvantages such as higher cost, limited availability of skin grafts in severely burned patients, problems of disease transmission and immune response [2,4]. One strategy for dealing with serious skin damage is to develop tissue engineered skin substitute. Various synthetic and natural polymers such as chitosan, silk, collagen, poly(glycolic acid), poly(L-Lactic acid) and poly(lactic acid-co-glycolic acid) are currently *Corresponding author:
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growth factors such as VEGF, Ang-1, FGF2 and PDGF. Inpanya et al. [14] have studied the Aloe vera gel blended silk fibroin films and compared to aloe free fibroin films; the blended film enhanced the attachment and proliferation of skin fibroblasts. Vasconcelos et al. [15] have developed the silk fibroin and elastin scaffolds for treatment of burn wounds. Silk fibroin/keratin films incorporating a synthetic inhibitor of elastase to control the higher levels of this enzyme produced in a chronic wound environment [16]. Silk fibroin/alginate sponge demonstrates a higher healing effect than both components acting alone [17]. Karahaliloglu et al. [18] have modified the surface of silk fibroin film to obtain a biologically inspired nano-featured surface morphology, which enhanced the adhesion and proliferation of human epidermal keratinocytes and dermal fibroblasts. Regenerated silk fibroin films are prepared by dissolving silk fibroin in suitable solvent, using solution casting method. These regenerated silk fibroin (SF) films contain α-helix or random coil structure and do not have enough intramolecular bonds to form stable structure [19]. Such films are water soluble and having low mechanical properties. To enhance the mechanical properties, films are crystallized in organic solvents such as methanol, in order to induce β-sheet structure. However, with methanol treatment silk fibroin films become brittle in the dry state and not suitable for wound dressing, particularly for load bearing application [20]. Therefore, there is a need to develop flexible fibroin films with good mechanical and biodegradable properties. The flexibility of the SF films can be improved by blending with natural polymers such as hyaluronic acid [21], chitosan [22] and alginate [23]. Plasticizers such as sorbitol, glycerol, glucose and PEG are commonly added to protein based films to enhance their flexibility [24-27]. The flexibility of silk fibroin films has been improved by incorporating glycerol [24] and glucose [25]. In present study, an attempt has been made to develop flexible silk fibroin films by incorporating dextrose. The mechanical, thermal and structural properties of dextrose modified films are characterized to understand the interaction between the silk fibroin and dextrose. The morphology, hydrophilicity, water absorption, biodegradation and cytocompatibility of flexible silk fibroin are also studied.
Experimental Materials and Methods Multivoltine silk cocoons of Bombyx mori silkworm were purchased from silk development department, Kalpipara, Bahraich, India. All other chemicals used in this study were purchased from Merck, India. Distilled water was used throughout this study. Preparation of Dextrose Modified Silk Fibroin Films Degumming of silk cocoons was carried out according to
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standard procedures [28,29]. The Bombyx mori cocoons were cut in small pieces, vacuum dried, weighed, and placed in 1 liter boiling solution of Na2CO3 (0.02 M) for 1 hour to remove sericin. Subsequently, the silk fibers were rinsed three times in 1 liter hot water (90 oC) for 20 min and dried overnight. Degummed silk fibroin fibers were dissolved in a 2 % CaCl2/formic acid solution (w/v) at room temperature by continuous stirring. The final concentration of silk fibroin solution was kept ~6 wt%. Different silk fibroin solutions were prepared by incorporating 0 to 15 % (w/w) dextrose. The solutions were poured onto polystyrene plates and dried at room temperature for formation of films. In order to induce crystallization, these air dried films were immersed in 80 % (v/v) methanol solution for 30 minutes. Mechanical Properties The tensile strength and elongation at break properties of the films were determined using Universal Testing Machine (Instron 3369) according to ASTM D 882-02. The preconditioned test samples (10×2.5 cm) were mounted between grips of the machine with span length 50 mm and pulled at cross head speed of 50 mm/min. Average tensile properties of five specimens were measured. Thermal Properties The thermal analysis of silk films was performed by Differential Scanning Calorimetry (DSC) instrument (TA Instruments Q100 DSC) under a dry nitrogen gas flow of 50 ml/min. The silk film of approximately 5 mg was filled in aluminium pan and heated at 2 oC/min from 0 oC to 400 oC. The dynamic mechanical thermal analysis of silk films was carried out on a DM 8000 dynamic mechanical analyzer (Perkin Elmer) by using film testing fixture. Specimens of 40×10 mm were run at tensile mode at a frequency of 1 Hz with 0.125 % of strain and 3 oC/min of heating rate. The tan δ of specimens was determined as the function of temperature from 25-250 oC. Morphological Characterizations The surface morphology of SF and DMSF films was observed with a scanning electron microscope (SEM) (Hitachi S-3700N) at a voltage of 15 kV. Specimens were sputtercoated with gold before mounting on SEM machine. Roughness measurements of SF and DMSF films were performed with atomic force microscope (Park) in contact mode with a tip mounted on V shaped silicon nitride cantilever with spring constant 40 N/m. Air dried SF and DMSF films were used for AFM measurement. All the measurements were carried out at room temperature with a scan area 2×2 μm. The roughness values were calculated as the rootmean-square (rms) deviation and arithmetic average height (Ra). The rms values represent the standard deviation of the height values within a given area and allows the surface roughness to be determined by statistical methods, whereas
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Ra values gives average deviation of roughness irregularities from mean line over one sampling length [30,31]. Structural Characterization As mechanical properties of silk is directly related to conformation of fibroin chain (β-sheet and random coil/ helix), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed to assess the secondary conformation of SF and DMSF films. ATR-FTIR measurements were performed using a Thermo Scientific Nicolet 380 Spectrometer, Japan, in reflection mode at 4 cm-1 resolution using 32 scans in the spectral range 4000 to 400 cm-1. The X-ray scans of SF and DMSF films were performed with a Seimens type-F, X-ray diffractometer (Bruker D S Advanced, Germany). The X-ray source was Cu Kα radiation (40 kV, 30 mA and λ=1.5 Å). The samples were mounted on aluminium frames and scanned from 5 to 50 o (2θ) at a speed of 2.0 o/min. Hydrophilicity Measurements Water Absorption (%) The water absorption (%) of SF and DMSF films was calculated with the following equation: Water absorption (%)=(Ws−Wd)/Wd × 100 where Ws is the weight of the swollen samples and Wd is the weight of the dry samples. The weight of the completely dried sample was measured directly. The weight of sample swollen in distilled water at 37 oC for 48 hours was measured after blotting the surface with filter paper. Contact Angle Measurement Hydrophilicity of SF and DMSF films was determined by analyzing water droplet angle formed between liquid/solid interfaces of different films by Sessile drop method. The volume of water droplet was kept at 4 μl. Contact angle value for each sample was taken as the average of three readings obtained at five different places of the sample. Cell Viability Experiments Cell Culture The SF and DMSF films were assessed for their ability to support L929 fibroblast cells purchased from National Center for Cell Science, India, grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10 % fetal bovine serum and 1 % UI ml-1 streptomycin-penicillin (SigmaAldrich). Cell Seeding on Different Matrices SF and DMSF films were cut into specific size (6.4 mm diameter) and sterile. The sterile samples were allowed to dip into DMEM media for 5 hours. The wells were treated with 0.2 % bovine serum albumin (BSA) in sterile phosphatebuffered saline (PBS≈pH 7.4) for 2 hours to prevent adhesion
Chandra Mohan Srivastava et al.
of cells on the vacant glass surface. Excess BSA was removed with sterile PBS (pH 7.4) washes. The films were placed into 96 well plates carefully with the help of forceps. For the cell proliferation study, approximately 20,000 L929 fibroblast cells were seeded per well on SF and DMSF films at 37 oC in a humidified atmosphere containing 5 % CO2, and were monitored at different time-intervals (1, 3 and 5 days). Cell Morphology, Cytocompatibility and Viability The L929 fibroblast cells seeded on different SF and DMSF films in 96-well tissue culture plate were visualized under an Inverted phase contrast microscope (Nikon ECLIPSE TS100) over 5 days. MTT assay was applied to determine the cytocompatibility of regenerated SF and DMSF films with L929 fibroblast cells. The MTT assay is a rapid calorimetric technique, in which yellow MTT is reduced to a purple formazan by mitochondrial dehydrogenase in cells to assess the cell proliferation. After 1, 3 and 5 day incubation in DMEM, the viability of L929 fibroblast cells were assessed. Briefly, L929 cells were washed three times with PBS to remove unattached cells. Then 100 μl serum free medium and 10 μl MTT stock solution (5 mg/ml in DMEM) were added in each well plate and incubated for 3 hours (37 oC) for MTT formazan formation. After 3 hours incubation at 37 oC, the reaction solution was carefully removed from each well and 100 μl dimethyl sulfoxide was added. The plates were gently agitated until the formazan precipitate was dissolved, followed by measurement of OD values by spectrophotometry at 595 nm with an ElX-800 Microelisa reader (Bio-Tek Inc., USA) with DMSO as a blank. Trypan Blue exclusion assay is used to determine the number of viable cells present in 96 well plates with SF and DMSF films. This method is based on the fact that a viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm. In vitro Biodegradation For in vitro biodegradation study SF and DMSF films (20 cm in length) (N=4 per group and time point) were incubated at 37 oC in 3 ml solution of Protease XIV (1 mg/ ml in PBS) (from Streptomyces griseus, Sigma, 3.5 unit/mg activity) and in the PBS as a negative control over 50 days. Each solution contained an approximately equivalent (±2 mg) mass of silk films. Solutions were changed daily. At designated time points, samples were rinsed with distilled water, dried and weight. Statistical Analysis Numerical data are presented as mean±standard deviation. The data were analyzed using standard analysis of variance (ANOVA) techniques and statistical differences were considered at p
