Antibacterial performance and in vivo diabetic wound healing of ...

Materials Science and Engineering C 69 (2016) 1183–1191

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Antibacterial performance and in vivo diabetic wound healing of curcumin loaded gum tragacanth/poly(ε-caprolactone) electrospun nanofibers Marziyeh Ranjbar Mohammadi a,⁎, Shahram Rabbani b, S. Hajir Bahrami c, M.T. Joghataei d, F. Moayer e a

Textile Group, Engineering Department, University of Bonab, Bonab, Iran Tehran Heart Center, Tehran University of Medical Sciences, Iran Textile engineering Department, Amirkabir University of Technology, Tehran, Iran d Cellular and Molecular Research Center, Iran University of Medical Science, Tehran, Iran e Department of Pathobiology, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran b c

a r t i c l e

i n f o

Article history: Received 17 June 2016 Received in revised form 23 July 2016 Accepted 12 August 2016 Available online 13 August 2016 Keywords: Gum tragacanth Poly(caprolactone) Curcumin Diabetic wound healing Nanofibrous scaffolds

a b s t r a c t In this study we describe the potential of electrospun curcumin-loaded poly(ε-caprolactone) (PCL)/gum tragacanth (GT) (PCL/GT/Cur) nanofibers for wound healing in diabetic rats. These scaffolds with antibacterial property against methicillin resistant Staphylococcus aureus as gram positive bacteria and extended spectrum β lactamase as gram negative bacteria were applied in two forms of acellular and cell-seeded for assessing their capability in healing full thickness wound on the dorsum of rats. After 15 days, pathological study showed that the application of GT/PCL/Cur nanofibers caused markedly fast wound closure with well-formed granulation tissue dominated by fibroblast proliferation, collagen deposition, complete early regenerated epithelial layer and formation of sweat glands and hair follicles. No such appendage formation was observed in the untreated controls during this duration. Masson's trichrome staining confirmed the increased presence of collagen in the dermis of the nanofiber treated wounds on day 5 and 15, while the control wounds were largely devoid of collagen on day 5 and exhibited less collagen amount on day 15. Quantification analysis of scaffolds on day 5 confirmed that, tissue engineered scaffolds with increased amount of angiogenesis number, granulation tissue area (μ2), fibroblast number, and decreased epithelial gap (μ) can be more effective compared to GT/PCL/Cur nanofibers. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Diabetes is a complex metabolic disorder that severely has affected more than 382 million people worldwide in 2013 and is predicted to rise to 366 million in 2030 [1]. The diabetic ulcer is characterized by a dysfunction in each step of wound healing, from coagulation and hemostasis to inflammation, proliferation, and remodeling. This kind of ulcers may results in infection with a high risk of sepsis and eventually limb amputation [2–3]. Over the last several years many new wound healing products have become available to help diabetic wound healing by reduction the time of wound closure, and improving limb salvage rates. However, nowadays several novel types of wound healing products are available all over the world to prevent and treat complicated diabetic wounds that may have previously led to partial or complete limb loss wounds [4]. Bioengineered alternative tissue products might be used for healing the wounds [5–6]. Natural and synthetic skin grafts have been used for healing of the same wounds. However, most of them are ⁎ Corresponding author at: Velayat Highway, University of Bonab, P.O. Box: 5551761167, Bonab, Iran. E-mail address: [email protected] (M.R. Mohammadi).

http://dx.doi.org/10.1016/j.msec.2016.08.032 0928-4931/© 2016 Elsevier B.V. All rights reserved.

expensive, require extensive care and do not recover full skin functionalities [7]. The wound healing patch requires to have wound healing properties such as essential swelling capability for absorbing excess exudates and oxygen permeability for respiring and quick delivery of drug to eliminate infections without any side effect [8]. The clinical need to develop novel and cheap methods of treatment to improve the healing of diabetic ulcers with similarity to extra-cellular matrix (ECM) is critical. The special topographical properties of electrospun nanofibers with high specific surface area and a porous structure, simulate the diameter of collagen fibrils (50 to 500 nm) of ECM in natural tissues. Electrospinning has already been shown to provide an alternative fabrication methodology to generate thin, continuous and flexible nanostructure fibers for application in drug delivery systems that depends on different parameters such as molecular, process and technical variables [9–10]. Gum tragacanth has an ancient history and is widely used in Chinese and Iranian folk medicine. Gum tragacanth (GT) is a natural combination of polysaccharides and alkaline minerals connected with small proportions of protein and little amounts of starch and cellulosic material [11] that is extracted from species of Astragalus plant in arid parts of the Middle East. There are several medicinal uses for tragacanth, either alone or in combination with other herbs for the

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healing of diabetes mellitus, cancers, and constipation [12–13]. Interesting biological properties of GT and low cost resulted in using this biopolymer in the form of nanofiber for application in wound healing [14–16], periodontal defect regeneration [17], drug delivery applications and peripheral nerve regeneration [18]. Poly(ε-caprolactone) is a biodegradable, biocompatible, less expensive and stable polymer in ambient conditions. On the other hand, curcumin, an active ingredient of turmeric, is a naturally occurring poly-phenolic compound with a broad range of favorable biological functions including anti-cancer, anti-oxidant, anti-infective, angiogenic, nerve healing properties and anti-inflammatory activities with innate antimicrobial characteristic [19]. The low bioavailability and in vivo stability of curcumin require the development of suitable carrier vehicles such as polymer nanofibers as efficient carriers for drug delivery and wound dressings [20,21]. For overcoming the low bioavailability and in vivo stability of curcumin, we produced PCL/GT as curcumin carrier with suitable physical, mechanical and drug delivery. These fabricated biocompatible nanofibers promoted cell proliferation and enhanced cell attachment [22]. The new concept of this paper is investigation about the antibacterial characteristic and potential of cellular and acellular PCL/GT/Cur nanofibers as a delivery vehicle of curcumin for application in wound healing in rats with diabetic disorders. 2. Experimental and characterization 2.1. Materials Poly(ε-caprolactone) (Mw = 80 kDa) was purchased from SigmaAldrich. Gum tragacanth (GT) was obtained from the stems of floccosus species of Astragalus bushes. Curcumin (Cur) extracted from turmeric was bought from Shahid Beheshti University, acetic acid and other chemical materials were purchased from Merck Company. 2.2. Electrospinning process 7% GT solution and 20% PCL solution (acetic acid (90% (V/V)) as solvent) were blended in 2:1 PCL/GT mass ratio, after preparation this solution, 3% curcumin based on the solid content in the polymer blend solutions were added to it and stirred for 30 min. PCL/GT/Cur blend nanofibrous webs were prepared by electrospinning technique as mentioned in our previous work [22]. Morphological investigations were carried out using SEM micrographs taken by scanning electron microscopy (SEM, XL30-SFEG, FEI Philips) after sputter coating with gold (JEOL JFC-1200 fine coater, Japan) at accelerating voltage of 15 kV. 2.3. Curcumin release from electrospun nanofibers The UV absorbance of released curcumin from PCL/GT/Cur nanofiber was calculated at λmax = 426 nm and converted to the curcumin concentration, according to the calibration curve of curcumin in the same media. Release of curcumin was measured using a UV/VIS instrument (Shimadzu 3600, UV-VIS-NIR Spectrophotometer). After weighting PCL/GT/Cur nanofiber, the nanofiber was immersed in a bath containing release medium (0.5% Tween 80 in phosphate-buffered saline, pH 7.4), then was kept in shaking incubator at 37 °C and 150 rpm. The cumulative release of TCH against release time was further shown.

amount of the diluted culture was then plated on Mueller Hinton Agar plates and incubated again for 24 h. After incubation the number of colonies grown in each plate was counted. All the steps were done in aseptic conditions. PCL nanofibers were used as control. %antibacterial¼ðB‐T=TÞ100 where, T = cfu*/ml of the test sample, C = cfu/ml; concentration of colony of bacteria, B = blank sample, *cfu: colony-forming unit. 2.5. Cell seeded scaffolds Mesenchyme stem cells (MSc) were cultured on to PCL/GT/Cur scaffolds. The cells were washed and cultured in DMEM supplemented with 10% fetal bovine serum and glucose (4.5 g/l) in 5% CO2 in a 37 °C incubator. Cells were seeded at a density of 4000 cells/cm2 on PCL/GT/Cur scaffolds and kept at 37 °C in a humidified CO2 incubator for 3 days before applying on the excisional wounds. 2.6. In vivo study All of the in vivo experiments were confirmed appropriately by the ethical committee of Tehran University Heart Center and animals received human care according to the ‘Guide for the care and use of laboratory animals published by the US National Institute of Health (publication no. 85-23 revised 1996). Twelve adult male Sprague Dawley rats weighing 250 ± 20 g were used for this study. All of the animals were kept separately in plastic cages for adaptation one week prior to study in the temperature and humidity of 22 °C ± 2 °C and 50–60%, respectively under the supervision of a licensed veterinarian. The animals were anesthetized with single intra-peritoneal injections of 50 mg/kg ketamine (Sigma, St Louis, MO, USA) + 5 mg/kg xylazine and intraperitoneally injected with 100 ml of streptozotocin (60 mg/ kg) in sterilized PBS (pH 7.4) to gain diabetic mellitus-like symptoms. After passing 3–4 days, the blood glucose level was measured by Accu-Chek Active (Roche Diagnostics GmbH, Germany). It should be noted that the blood glucose levels were recorded for all rats before doing any treatment too. Animals at blood glucose levels of ≥250 mg/ dL were attended as diabetic [23]. For doing in vivo tests, acellular scaffolds and cell-seeded scaffolds were applied on the test wounds. Three time points were selected to sacrifice the animals, that is, on days 5, 10 and 15. For each animal four round excisional wounds (10.0 mm in diameter) were created (two above wounds were covered with acellular scaffold and two below wounds were wrapped with cellular scaffolds). The animals were anesthetized with intra- peritoneal injections of 50 mg/kg ketamine and 5 mg/kg xylazine. After anesthesia, a sterile, template with a diameter of 10.0 mm was placed on the dorsum of each rat and a full thickness wound was made by excising the skin [24]. The open wound area was documented using a digital camera on days 0, 10, and 15 and the areas of wounds in each group were analyzed using Image J software. Wound closure was expressed as percentage closure of the original wound and was calculated using the following formula [25]. Fig. 1 shows the procedures of creating full thickness wounds (Fig. 1a and b) and putting scaffolds on wounds (Fig. 1c). Wound closure¼

2.4. Antibacterial properties In vitro antibacterial activity of PCL/GT/Cur nanofibers by broth dilution method was investigated against methicillin resistant Staphylococcus aureus (MRSA) as gram positive bacteria and extended spectrum β lactamase (ESBL) as gram negative bacteria. In brief, several dilutions were prepared in nutrient broth. The media was then inoculated with freshly prepared bacterial suspension. After incubation of inoculated tubes at 37 °C for 24 h, the cultures were diluted various times. An

wound area day 0−wound area ðday 5; 10 and 15Þ wound area day 0100 ð1Þ

2.7. Histology Prepared full thickness of the skin was excised and fixed in 10% formaldehyde for pathologic investigations. The tissue sections were embedded in paraffin and then cut into 5 mm thick sections and placed on glass microscope slides. The samples exposed routine histological


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Fig. 1. The process of creating wounds on dorsum of rats, (a) marking the place of wounds, (b) wounds after creation, (c) the wounds wrapped with scaffolds.

procedures for hematoxylin and eosin (H&E) staining or were stained with Masson's trichrome stain (MTS) [26–27]. The slides were dehydrated, mounted in Richard-Allan Cytoseal-60 mounting medium and imaged with a Nikon Microphot-FX with a Leica DFC310 FX camera using Leica Application Suite 4.3.0.

ESBL bacteria which they are dangerous pathogens causing nosocomial infections in hospitals. Results of antibacterial activity of the nanofibers are shown in Fig. 3. PCL/GT/Cur nanofibers were 99.9% antibacterial against MRSA and 85.14% against ESBL. The results showed that this nanofiber is very promising scaffold for antibacterial applications.

2.8. Statistical analysis

3.3. Macroscopic results of samples

All presented data are reported as mean ± standard deviation (SD). Statistical analysis was done using one-way analysis of variance (ANOVA), followed by Turkey post hoc test for multiple comparisons, and significance was shown at p ≤ 0.05.

Diabetes complications contain nerve harms (neuropathy) and poor blood flow. These difficulties make the feet vulnerable to skin ulcers and their healing is so slow. Neuropathy causes loss of feeling in the feet, decreasing body ability to feel pain, and not detecting an injury or irritation. Medical nanofibers were developed to treat chronic ulcers like diabetes in short time [29]. In our previous work curcumin loaded PCL/GT nanofibers were produced. In this paper, animals with diabetic symptoms were subjected to wound-healing treatments with the PCL/ GT/Cur nanofibers and controls. Quicker wound closure facilitates the biological event of healing by joining the wound edges. Fig. 4 displays a representative on an animal from each group (curcumin loaded PCL/ GT nanofibers) and control on days five, ten, and fifteen after treatment. The results exhibited that wound closing happens faster than control wounds after the same time points. Gum tragacanth is able to contract myofibroblasts, for a faster closure of the wound [30]. It seems that, active components of tragacanth gum, such as bassorin and tragacanthin [31], may contribute to the healing effects of tragacanth mucilage. Hydrolysis of tragacanthin into arabinose and glucoronic acid may results in coagulation of surface proteins, and inhibit wound infection that causes a faster wound healing [32]. Fig. 6 plots the changes in the areas of the wounds after various periods of healing. The areas of the wounds wrapped with cellular and acellular PCL/GT/Cur nanofibers closed completely on day 15, but for control sample the wound area decreased to 20.96 ± 1.35%. Electrospun curcumin-eluting nanofibers

3. Results and discussion 3.1. The morphology of electrospun nanofibers The SEM micrograph of nanofibrous webs is shown in Fig. 2a. Nanofibrous web with bead-less morphology is electrospun from PCL/ GT/Cur blend solution [22]. Fig. 2b exhibits the morphology of stem cells under optical microscopy. Cells were attached and spread well on nanofibrous scaffold (Fig. 2b). 3.2. Antibacterial properties of nanofibers Several kinds of nanofibers and nanoparticles with antibacterial properties have been produced for application in biomedical fields. For this aim antibacterial materials such as chitosan, silver and drugs were used in nanofibers formulation [28]. For thousands of years, nature has presented a source of medicinal agents for different applications. In this paper curcumin with antibacterial property was loaded in PCL/ GT nanofibers. Fabricated nanofibers were analyzed against MRSA and

Fig. 2. (a) The morphology of the PCL/GT/Cur nanofibers, (b) The confluency of cells on the scaffolds by optical microscopy.

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Fig. 3. Antibacterial activity against MRSA and ESBL for samples containing Cur (average CFU/ml).

improved the wound healing compared with control samples significantly and they showed complete healing at the surface (p ≤ 0.05) (Fig. 5). 3.4. Microscopic studies Diabetes has been shown to impair immune function and impede wound healing, both of which are critical to survival and recovery from major wound injury [33–34]. Curcumin from Curcuma longa L.

has antioxidant, anti-inflammatory, anti-infective, anti-tumour, angiogenic and nerve healing properties [35]. Excessive glucose deposition in blood results in the damage of peripheral nerves (peripheral neuropathy). To investigate about the efficacy of nanofibrous composites, H&E (Figs. 6, 7) at day 5 and 15, quantification analysis containing amount of angiogenesis number, granulation tissue area (μ2), fibroblast number, and epithelial gap (μ) (Fig. 8) at day 5, 10 and 15 and Masson's Trichrome staining (MTS) at day 5 and 15 (Figs. 10, 11) were performed on sectioned tissue samples of the wounds. Figs. 6 and 7 visualizes

Fig. 4. Wound closure for rat after 5, 10, 15 days. For scaffold wrapped samples two above wounds treated with acellular scaffolds and two lower wounds treated with cellular nanofibers.


Wound area (%)

Control S S-Cell

Post treatment time (Day) Fig. 5. Changes in the areas of the wounds after various periods of healing (Control, S shows treated wounds with acellular scaffold, S-Cell shows treated wound with cellular scaffold).

images obtained as part of the histological analysis. In brief, microscopic results, showed quick healing effects of acellular and cell-seeded scaffolds on full thickness wound compared with control samples in the same time. However, improved granulation, tissue formation and collagen regeneration were observed in wounds that had been treated with cell-seeded scaffolds. These results could be due to two different reasons. First, because of nanofibers innate properties and presence of GT in nanofiber structure which can introduce faster signaling pathway, imitating natural ECM and attract fibroblasts to the derma layer. The second is the incorporation of cells in nanofibrous scaffolds provides the signals needed for tissue formation. Moreover, using from natural biopolymer in scaffold formulation can help to quicker regeneration of wound. The mineral constituents of GT including calcium, magnesium, and potassium may also play a substantial role in the wound healing process. Calcium has a major performance in the normal homeostasis of mammalian skin and serves as a modulator in keratinocyte proliferation and maturation and its existence in epidermal cell migration and regeneration is required [36]. Magnesium might increase motility of fibroblasts and keratinocytes [37]. However, it has been confirmed that GT could be effective on the proliferation and remodeling phases of wound healing [38]. On the other hand sustained releasing curcumin

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from electrospun nanofibers with anti-inflammatory and antibacterial characteristic can accelerate wound healing process. For control samples on day 5 only blood scabs, fibrins, high amount of polymorphonuclear neutrophils (PMNs) and a lot of inflammatory agents were found. Fibrin layer is directly in contact with the wound area and causes slower wound healing process (Fig. 6a1 & a2). There was no evidence of collagen regeneration and epithelialization. Figs. 6b and c show that in scaffold treating wounds, porous nanofibrous webs may absorb fibrin layers on the wounds and enhance the healing process. It is necessary to say that, the inflammation stage in wound healing process occurs shortly after injury. In this stage, mobilization of the components of the immune system remove damaged tissue and bacteria from the wound. It seems that releasing antibacterial curcumin in to injured place and the presence of antibacterial GT in scaffold composition can inhibit bacteria growth, so inflammation phase happens quicker. However, wounds that were treated with acellular and cell-seeded PCL/GT/Cur scaffolds were well integrated into the surrounding skin without any need to suturing and no significant inflammatory response in the wounds and showed better collagenous regeneration compared with controls. In acellular scaffold treated wounds epithelial layer had partially formed. However, the total wound healing factor was weak. Collagen with much fibrils and moderate epithelial layer was formed in excisional wounds that were treated with cell-seeded scaffolds. But granulation tissue appeared slightly thicker for PCL/GT/Cur-cell nanofibers compared to the PCL/GT/Cur mats and this amount for PCL/GT/Cur scaffold is higher than control samples. Moreover, on day 5, full coverage of new epithelium was identified for cellular PCL/GT/Cur scaffold wrapped wounds compared with control and acellular PCL/GT/Cur nanofibers. Scaffold containing wounds exhibited faster re-epithelialization than control samples. On day 15 after surgery, generally, wounds that were treated with scaffolds (with cells or without cells) showed higher grade of healing compared with control samples. Control wound still showed less improvement in healing process. The wounds wrapped with scaffolds almost healed, with newly synthesized fibrous tissue and sparse inflammatory cells in the dermis and subcutis, covered by a reepithelialized epidermis in each case. Complete epidermal on the edge of wound and no skin appendages were visible in acellular scaffold treated wounds (Fig. 7b). In the same time point, wound treated with cell-seeded scaffold showed high amount of collagen bundle with irregular pattern and complete epidermal layer on the edge of wounds and showed more stratum corneum than those in other samples (Fig. 7c).

Fig. 6. Histological evaluation of wounds treated by PCL/GT/curnanofibers. (a) H&E staining for skin wound samples of control (open wound), (b) PCL/GT/Cur, (c) PCL/GT/Cur/cell; granulation tissue, epithelial regeneration, angiogenesis and collagen fibers were indicated by blue, yellow, red and green arrows, respectively (after 5 days) (a1, b1, c1 100×); (a2, b2, c2 400×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. Histological evaluation of wounds treated by PCL/GT/Cur nanofibers. (a) H&E staining for skin wound samples of control (open wound), (b) PCL/GT/Cur, (c) PCL/GT/Cur/cell; granulation tissue, epithelial regeneration, angiogenesis and collagen fibers were indicated by blue, yellow, red and green arrows, respectively (after 15 days), (a1, b1, c1 100×); (a2, b2, c2 400×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Enhanced epithelial gap closure is one of the most important factors which determine faster wound healing process. The results of figure 8a1, a2 exhibits the maximum amount of epithelial gap for both groups on day5 compared with their amount on day 10 and 15. For cellular GT/ PCL/Cur wrapped wounds, the EG on day 5 was the least amount and

on day 10 its amount reaches zero but the difference of EG on both cellular and acellular samples was not significant. The granulation tissue formation on day 5 was also notably higher for cellular PCL/GT/Cur wrapped wounds (Fig. 8b1, b2). It could be therefore inferred that tragacanth components accelerate the transition from

Fig. 8. Quantification of; (A) epithelial gap (μ) (B) granulation tissue (μ2) (C) angiogenesis number (no.) (D) fibroblast cell (no.)


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Fig. 9. Masson's Trichrome staining images of wounds treated by PCL/GT/Cur mats (a) control, (b) PCL/GT/Cur, (c) PCL/GT/Cur-cell at 5 days of treatment, (a1, b1, c1 100×); (a2, b2, c2 400×).

the inflammation and tissue granulation phases of the wound healing process and enhance mature scar formation and extracellular matrix remodeling which boosts the eventual wound contracture and closure. The findings of our study are consistent with similar wound healing experiments employing various materials including tragacanth [38]. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes [39]. In scaffold treated wounds, angiogenesis increases and well-formed blood vessels with increased micro vessel density are observable on day 5 (Fig. 8c), but on day 10, its content decreased, whereby in control samples increases up to day ten. It seems that curcumin results in angiogenesis hastens in diabetic rats. The same results were reported by other researchers before [40]. On histologic examination of the specimens on day 15, epidermis and dermis layers were formed completely for PCL/GT/Cur.

Fibroblast cells are one of the most important and effective cells in wound healing process which they reach to the injury place at day 3 of healing. Chronic wounds like diabetic wounds fail to heal because of lack of fibroblast infiltration. Various studies have proven that curcumin can effectively infiltrate fibroblasts. It is also clear that some of the inflammatory cells on day 5 were replaced by fibroblasts and number of fibroblast cells increased significantly on day 10 compared to day 5 for group B samples. Sidhu et al. [41] have reported that myofibroblasts at wound sites could result in diabetic mice healing which thy treated with curcumin, too. One of the important signs of tissue regeneration is the formation of collagen, which is essential for the function and structure of healthy skin. To examine the amount and quality of collagen in treated and untreated wounds, Masson's trichrome staining was performed on tissue

Fig. 10. Masson's Trichrome staining images of wounds treated by PCL/GT/Cur mats (a) control, (b) PCL/GT/Cur, (c) PCL/GT/Cur-cell at 5 days of treatment (a1, b1, c1 100×); (a2, b2, c2 400×)

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Table 1 Blood glucose measurements for control and PCL/GT/Cur wrapped wounds in rats. Sample Control

Time (day)

5 10 15 PCL/GT/cur 5 10 15

Glucose level before surgery

Glucose level after surgery

540 530 505 550 487 525

560 542 510 500 390 420

sections. Figs. 9 and 10 shows histological sections of untreated, cellular and acellular PCL/GT/Cur treated wounds in after 5 and 15 days in which collagen is stained in blue. Collagen is largely missing in the untreated wounds, even after 15 days, by contrast, the PCL/GT/Cur and PCL/GT/ Cur/cell treated wounds, albeit still not completely normal, show significant levels of collagen and more organized collagen alignment. Taken together the histological data clearly indicate that our scaffolds accelerated and enhanced a more natural mode of tissue regeneration in the rat model of full thickness excisional wound healing. 3.5. Blood glucose monitoring Dietary curcumin significantly decreases inflammation and delays or prevents obesity-induced insu1lin resistance and associated complications, including atherosclerosis and immune mediate liver disease. Unfortunately dietary curcumin is poorly absorbed by the digestive system and undergoes glucuronidation and excretion rather than being released into the serum and systemically distributed. A recent study showed that curcumin-treated diabetic rats had lower blood glucose and glycated hemoglobin levels, in association with lower oxidative stress [42]. Furthermore, treatment with curcumin has been shown to reduce reactive oxygen species (ROS) levels in cells that are isolated from diabetic patients [43]. Experimental studies with diabetic animals demonstrated that curcumin supplementation can suppress cataract development [44] and the cross-linking of collagen [45] promotes wound healing [41], and lower blood lipid and glucose levels in a streptozotocin treated diabetic animal model [46]. In our studies, blood glucose measurements for PCL/GT/Cur nanofibers (Table 1) showed that in the samples containing curcumin glucose content decreased significantly after passing determined time points with releasing curcumin. The release rate profiles of curcumin from electrospun PCL/GT/3% Cur scaffold is shown in Fig. 11. This scaffold released about 65% of drug up to a period of 20 days with sustained manner. It seems

Fig. 11. Release of curcumin from different electrospun scaffolds.

that releasing curcumin with sustained behavior could result in decreased amount of blood glucose levels. 4. Conclusion In this work, antibacterial PCL/GT/Cur membranes that sustainably delivered curcumin to repair diabetic wounds were developed. Nanofibrous membranes released curcumin for about 20 days. Tissue engineered scaffolds were established by MSC cells culture on the scaffolds. Macroscopic investigations were done to evaluate the ability of scaffolds in accelerating of wound closure. Generally, scaffold treated wounds showed smaller wound area compared to control samples, and wounds which were treated with cell-seeded scaffolds showed smaller scabs areas in comparison with ones treated with acellular scaffolds. To study the effect of scaffolds on wound healing process, pathological results were carried out. However, the nanofibrous curcuminloaded PCL/GT membranes had an effect on increasing the collagen content in treating diabetic wounds and an effectively promoter for healing of such wounds in the early stages and accelerated healing process. The resulted effect might be related to nanofibrous structure of the scaffolds that simulate the natural ECM, high biological properties of GT, sustained release of curcumin for 20 days and high physical–mechanical properties of PCL that causes to maintain the stability of scaffolds in front of blood and fibrin. Histo-chemical results showed much better healing performance for scaffolds stem cells followed by acellular scaffolds compared with control samples because of stem cells ability of collagen regeneration and providing the signals needed for tissue building. PCL/GT/Cur nanofibers decreased blood glucose level compared with control samples. In conclusion, application of blend PCL/GT/Cur scaffolds is effective in healing of wounds in the rat models. References [1] K. Sümegi, B. Melegh, A. Maasz, P. Kisfali, J. Bene, B. Melegh, Triglyceride level affecting shared susceptibility genetic variants in type 2 diabetes mellitus, J. Diabetes Metab. S13 (2013) 007. [2] P. Palumbo, L. Melton, C. Haskell, Peripheral vascular disease and diabetes, Diabetes Am. 85 (1985) XV1–XV20. [3] R.E. Pecoraro, G.E. Reiber, E.M. Burgess, Pathways to diabetic limb amputation. Basis for prevention, Diabetes Care 13 (1990) 513–521. [4] S. O'Meara, N. Cullum, M. Majid, T. Sheldon, Systematic reviews of wound care management: (3) antimicrobial agents for chronic wounds; (4) diabetic foot ulceration, Health Technol. Assess. 4 (1999) 1–237. [5] P.J. Kim, M. Heilala, J.S. Steinberg, G.M. Weinraub, Bioengineered alternative tissues and hyperbaric oxygen in lower extremity wound healing, Clin. Podiatr. Med. Surg. 24 (2007) 529–546. [6] J.S. Steinberg, B. Werber, P.J. Kim, Bioengineered alternative tissues for the surgical management of diabetic foot ulceration, in: T. Zagonis (Ed.), Surgical Reconstruction of the Diabetic Foot & Ankle, 1st editionLippincott Williams & Wilkins, Philadelphia 2009, pp. 100–117 (Chapter). [7] S.P. Zhong, Y.Z. Zhang, C.T. Lim, Tissue scaffolds for skin wound healing and dermal reconstruction, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 510–525. [8] K. Kataria, A. Gupta, G. Rath, R.B. Mathur, S.R. Dhakate, In vivo wound healing performance of drug loaded electrospun composite nanofibers transdermal patch, Int. J. Pharm. 469 (2014) 102–110. [9] N. Ketabchi, M. Naghibzadeh, M. Adabi, S.S. Esnaashari, R. Faridi-Majidi, Preparation and optimization of chitosan/polyethylene oxide nanofiber diameter using artificial neural networks, Neural Comput. & Applic. (2016). [10] M.A. Karimi, P. Pourhakkak, M. Adabi, S. Firoozi, M. Adabi, M. Naghibzadeh, Using an artificial neural network for the evaluation of the parameters controlling PVA/chitosan electrospun nanofibers diameter, E-Polymers 15 (2015) 127–138. [11] W.W. Tragacanth, Karaya, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, 1st ed.CRC Press, Boca Raton 2009, pp. 231–246. [12] T. Fleming, PDR for Herbal Medicines, Medical Economics Company, Montvale, New Jersey, 2000 767–768. [13] J.A. Duke, E.S. Ayensu, Medicinal Plants of China, Reference Publications, New York, 1995. [14] M. Ranjbar-Mohammadi, S.H. Bahrami, M.T. Joghataei, Fabrication of novel nanofiber scaffolds from gum tragacanth/poly(vinyl alcohol) for wound dressing application: in vitro evaluation and antibacterial properties, Mater. Sci. Eng. C. Mater. Biol. Appl. 33 (2013) 4935–4943. [15] M. Ranjbar-Mohammadi, S.H. Bahrami, M.T. Joghataei, Development of nanofibrous scaffolds containing gum tragacanth/poly (ε-caprolactone) for application as skin scaffolds, Mater. Sci. Eng. C Mater. Biol. Appl. 48 (2015) 71–79.

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