Accelerated Healing of Full-Thickness Wounds by Genipin-Crosslinked Silk Sericin/PVA Scaffolds
Pornanong Aramwit a Tippawan Siritienthong a Teerapol Srichana c
Juthamas Ratanavaraporn b
a Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences and b Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok , and c Department of Pharmaceutical Technology and Drug Delivery System Excellence Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkla , Thailand
ithelialization compared with the control scaffolds without sericin but lower numbers of macrophages and multinucle-ated giant cells. These results indicate that the delivery of sericin from the novel genipin-crosslinked scaffolds effi-ciently healed the wound. Therefore, these genipin-cross-linked sericin/PVA scaffolds represent a promising candidate for the accelerated healing of full-thickness wounds.
Wound dressing can achieve a functionally and aes-thetically satisfactory recovery by providing the optimal microenvironment to the wound [Ramakrishnan and Jayaraman, 1997; Gore and Akolekar, 2003; Middelkoop
Silk sericin has recently been studied for its advantageous biological properties, including its ability to promote wound healing. This study developed a delivery system to acceler-ate the healing of full-thickness wounds. Three-dimensional scaffolds were fabricated from poly(vinyl alcohol) (PVA), glycerin (as a plasticizer) and genipin (as a crosslinking agent), with or without sericin. The physical and biological proper-ties of the genipin-crosslinked sericin/PVA scaffolds were in-vestigated and compared with those of scaffolds without sericin. The genipin-crosslinked sericin/PVA scaffolds exhib-ited a higher compressive modulus and greater swelling in water than the scaffolds without sericin. Sericin also exhib-ited controlled release from the scaffolds. The genipin-cross-linked sericin/PVA scaffolds promoted the attachment and proliferation of L929 mouse fibroblasts. After application to full-thickness rat wounds, the wounds treated with genipin-crosslinked sericin/PVA scaffolds showed a significantly greater reduction in wound size, collagen formation and ep-
Accepted after revision: October 30, 2012 Published online: January 8, 2013
Dr. Pornanong Aramwit Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences Chulalongkorn University PhayaThai Road, Phatumwan, Bangkok 10330 (Thailand) E-Mail aramwit @ gmail.com
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et al., 2004] or by delivering bioactive molecules to accel-erate wound healing. Despite the availability of numer-ous types of biocompatible dressings, there is still a high demand for the development of more effective and af-fordable solutions. The wound healing process involves three overlapping phases: (1) inflammation, (2) prolifera-tion and differentiation including granulation and tissue formation and (3) matrix formation and remodeling [Singer and Clark, 1999; Falanga, 2005]; the ideal wound dressing should be able to support the requirements of each phase. Nevertheless, few of the wound dressings that have been developed thus far are able to fully meet these requirements [Ng and Chua, 2002]. For example, meshed weaved cotton gauze dressings have been effectively ap-plied for the debridement of heavily contaminated exuda-tion and necrosis wounds for long periods of time; how-ever, they are ineffective in stimulating the healing cas-cade [Meng et al., 2009]. Wound dressings made from natural polysaccharides, such as calcium alginate, chitin and chitosan, and synthetic wound dressings, such as sil-icone gel, are good candidates for the inflammation and debridement phases of healing. However, they provide little benefit with regard to epithelialization and granula-tion tissue formation [Cuttle et al., 2006; Gibran et al., 2007]. Many researchers have investigated the potential biomedical applications of the silk protein sericin, which is derived from the silkworm cocoon. We previously found that sericin could activate collagen production in wounds [Aramwit and Sangcakul, 2007], which subse-quently induced epithelialization. Furthermore, sericin has been reported to exhibit distinctive biological prop-erties, including the promotion of attachment and prolif-eration of human skin fibroblasts, osteoblasts and kerati-nocytes [Terada et al., 2002; Sasaki et al., 2005; Tsubouchi et al., 2005; Dash et al., 2008; Zhang et al., 2008]. These properties contribute to the excellent suitability of sericin as a wound dressing material. However, the form of seri-cin delivery still needs further development.
Scaffolds are widely used as wound dressings because they provide a template and physical support to guide the proliferation and differentiation of fibroblasts and kera-tinocytes in the regenerated tissue [Agrawal and Ray, 2001]; they also act as a carrier for the delivery of bioac-tive substances [Okabayashi et al., 2009]. Scaffolds can be fabricated using either natural or synthetic materials [Ba-baeijandaghi et al., 2010; Steinstraesser et al., 2010]. In recent years, scaffolds produced from combinations of natural and synthetic materials have received increasing attention [Mandal et al., 2011; Kundu et al., 2012]. We previously combined sericin, poly(vinyl alcohol) (PVA),
glycerin (as a plasticizer) and genipin (as a crosslinking agent) to fabricate scaffolds with good physical properties [Aramwit et al., 2010b; Siritientong et al., 2011]. Based on the results of this previous study, the strong and stable genipin-crosslinked scaffolds appeared to mechanically support the wound and control the release of sericin to accelerate wound healing.
In the current study, the physical and biological prop-erties of genipin-crosslinked sericin/PVA scaffolds were evaluated and compared with those of the genipin-cross-linked PVA scaffolds without sericin, which were used as control scaffolds. The in vitro release of sericin and the attachment and proliferation of L929 mouse fibroblasts cultured in the scaffolds were investigated. To apply these scaffolds as wound dressings, a systematic in vivo study was performed to evaluate their efficacy in wound heal-ing. The scaffolds were tested in full-thickness wounds in the dorsal skin of Sprague-Dawley rats. The wound heal-ing was evaluated in terms of inflammatory response, the reduction of the wound area, the production of collagen and epithelialization.
Materials and Methods
Materials Fresh bivoltine white-shell cocoons of Bombyx mori produced
in a controlled environment were kindly supplied by Chul Thai Silk Co. Ltd. (Wang Chomphu, Thailand). PVA (MW 77,000–82,000) was purchased from Ajax Finechem (Seven Hills, N.S.W., Australia). Genipin, a natural crosslinking agent extracted from gardenia fruit, was obtained from Wako Pure Chemical Indus-tries Ltd. (Osaka, Japan). Glycerin and other chemicals were ana-lytical grade and used without further purification.
Preparation of Genipin-Crosslinked Sericin/PVA Scaffolds The silkworm cocoons were cut into small pieces and the ser-
icin was extracted using a high-temperature and -pressure de-gumming technique [Lee et al., 2003]. Briefly, the silkworm co-coons were placed in deionized water and autoclaved at 120 ° C for 60 min. After filtration through a filter paper to remove fibroin fibers, the sericin solution was concentrated until the desired con-centration was achieved (approximately 7 wt%, measured by the BCA Protein Assay Reagent; Pierce, Rockford, Ill., USA).
PVA was dissolved at 80 ° C with constant stirring for 4 h until it was completely dissolved at 50 mg/ml. Genipin was dissolved in ethyl alcohol to obtain a 20 wt% solution. The sericin (3 wt%), PVA (2 wt%), glycerin (1 wt%) and genipin solutions (0.025 wt%) were mixed at room temperature for 30 min. The mixture was then poured into a petri dish, frozen at –20 ° C and subjected to lyophilization (Heto LL 3000 lyophilizer; Allerod, Denmark) for 72 h. Then, the scaffold was sterilized by gamma irradiation [Siri-tientong et al., 2011], yielding ‘genipin-crosslinked sericin/PVA scaffolds’. These were prepared via the same procedure without sericin addition to serve as control scaffolds.
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Physical and Chemical Characterization of the Scaffolds Morphological Observation. The scaffolds were cross-sec-
tioned, mounted onto aluminum stubs and sputter coated with gold to a thickness of 10–20 nm. Observations were performed on a scanning electron microscope (JSM-5 800LV; JEOL, Tokyo, Ja-pan) at 15 keV. The pore size of the scaffolds was measured using a method modified from Kang et al. [1999]. The major and minor diameters of each pore were measured using a stereo microscope equipped with an optical micrometer. The pore size was calcu-lated as the geometric mean of the major and minor diameters. At least 100 pores were assessed and the values are reported as the mean 8 standard deviation.
Mechanical Test. A compression test was performed on the scaffolds (15 mm in diameter and 8 mm in thickness) using a uni-versal testing machine (No. 4301; Instron, Norwood, Mass., USA) at a constant compression rate of 1 mm/min. The compressive modulus of the scaffolds was determined from the slope of the compressive stress-strain curves in the strain range of 5–30%. All experiments were performed in triplicate.
Swelling Test. A swelling test was carried out according to the method of Mandal et al. [2009] with a slight modification. Briefly, the lyophilized scaffolds were accurately weighed in the dry state and then immersed in 10 ml of deionized water. After 24 h, the scaffolds were carefully removed from the water and weighed in the swollen state. The experiments were performed in triplicate under the same conditions. The equilibrium swelling of the scaf-folds was calculated from the following equation:
Water swelling ratio = [( W t – W 0 )/ W 0 ] ! 100,
where W 0 and W t represent the weights of the dry and swollen scaffolds, respectively.
In vitro Test of Sericin Release from the Scaffolds The sericin/PVA scaffolds were placed in phosphate-buffered
saline solution (PBS, pH 7.4) at 37 ° C with continuous stirring in a closed container. The PBS solutions (1.5 ml) were collected at different time points and the amount of sericin released into the solution was measured using a BCA protein assay kit (Pierce). The absorbance was measured at 562 nm and the amount of sericin released was determined from a standard curve prepared using different concentrations of bovine serum albumin. All experi-ments were performed in triplicate.
In vitro Attachment and Proliferation Tests with L929 Cells L929 mouse fibroblast cells were seeded on the sterilized scaf-
folds at a density of 2.5 ! 10 5 cells/scaffold and cultured in Dul-becco’s Modified Eagle Medium supplemented with 10 vol% fetal bovine serum and 100 U/ml penicillin/streptomycin at 37 ° C and 5 vol% CO 2 . The number of cells attached after 6 h and the num-bers of cells present after 1, 3, 5 and 7 days of culture were quanti-fied using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide assay. All experiments were per-formed in triplicate.
In vivo Study Using the Full-Thickness Wound Model Animals. The animal use in this study was approved by the
Mahidol University Animal Care and Use Committee (MU-ACUC), Bangkok, Thailand. All experimental procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animals, 1996, implemented by the National Labora-
tory Animal Center of Mahidol University, Bangkok, Thailand. Twenty-four 8-week-old male Sprague-Dawley rats (weight 250 8 5 g) were used for this experiment. The rats were fed with a stan-dard diet and housed individually under controlled temperature (22–23 ° C).
Full-Thickness Wound Model. The rats were anesthetized with an intramuscular injection of Zoletil � (tiletamine hydrochloride, 30 mg/kg body weight; Virbac, Milperra, N.S.W., Australia) and Baytril � (enrofloxacin, 10 mg/kg body weight; Bayer, Germany), along with an antibacterial agent. After anesthesia, all animals were transferred to a warm environment to achieve a constant body temperature. The hair over the dorsum was shaved and the skin around the wounding area was scrubbed with povidone io-dine and a 70 vol% ethanol solution. Application fields were out-lined with a marking pen just prior to skin excision. Two full-thickness skin wounds (1.5 ! 1.5 cm) were prepared on each rat by excision on the dorsum of each rat under aseptic surgery. The genipin-crosslinked PVA and sericin/PVA scaffolds were applied to the left- and right-side wounds, respectively. The wounds were wrapped with Transpore TM medical tape (3M, Saint Paul, Minn., USA) to keep the scaffolds in place and then covered with Co-ban TM (self-adherent elastic wrap; 3M) to secure the dressings. The pain reducer carprofen was subcutaneously injected into all rats at 5 mg/kg body weight postoperatively once daily for 5 days. The body weights were measured and skin irritation was moni-tored daily.
Macroscopic Examination. The wounds were photographed every 2 days after surgery using a stereomicroscope fitted with a camera (Carl Zeiss, Germany) and a Moticcam 2300 � at 1,024 ! 768 pixels. The contours of the wounds were traced on days 3, 7, 14 and 21 using a Visitrak � grid, a transparent graph sheet, which was then analyzed by the Visitrak digital system (a wound mea-surement system manufactured by Smith & Nephew, London, UK). Three well-trained investigators used the Visitrak grid to assess the contours of each wound, and the mean values were re-ported. The rate of wound closure was expressed as a percentage of wound size reduction, which was calculated as follows:
Percentage of wound size reduction (%) = [( A 0 – A t )/ A 0 ] ! 100,
where A 0 and A t represent the wound area at the initial time point and subsequent time points, respectively [Noorjahan and Sastry, 2004].
Histological Examination and Morphometric Analysis. Six rats were sacrificed at 3, 7, 14 and 21 days after surgery, and the wound tissues, including the wound bed and healthy skin sur-rounding the wound, were collected for histological examina-tion. The wounds were bisected in the caudocranial direction and the central portion of the underlying tissue was taken and fixed in 10 vol% neutral-buffered formalin at room temperature for48 h. The fixed tissues were dehydrated and infiltrated with stan-dard tissue processing solutions. Each tissue sample was embed-ded in a paraffin block and thin sections (5 � m) were prepared. At least three sections were randomly taken from each tissue sample. The tissue sections were then mounted on glass slides for hematoxylin-eosin (HE) and Sirius red staining. Digital images were captured from each section under an Olympus BX 50 light microscope and analyzed with Motic Images Plus 2.0 ML. The numbers of giant cells were counted from 6 randomly selected fields of the HE-stained sections from each sample. The vascular density was determined by counting the blood vessels from 10
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randomly selected fields of the HE-stained sections from each sample ( ! 400 magnification). The organization and maturation of collagen bundles were assessed by observing the Sirius red-stained sections under a polarized light microscope [Giattina et al., 2006]. From each sample, color images of 640 ! 480 pixel resolution (at ! 400) were acquired and captured along 1.5 cm of the wounded skin area as a serial photograph divided into three areas. The collagen content in the tissues was then analyzed using image analysis according to the method reported by Kaczmarek et al. [2004]. Briefly, color images of each wound were first ad-justed by altering the color of an area not of interest to white and then replacing that area with the color mode. The adjusted im-ages were then converted to gray-scale images and enhanced by the median filter. The area of positive reaction was estimated by the number of black pixels and was determined as the percentage of black pixels per field in the binary image. All morphometric analyses were performed blindly.
Immunohistochemistry. The tissue sections were mounted on SuperFrost Plus slides (Gerhard Menzel, Braunschweig, Germa-ny) for immunohistochemical staining of macrophage class M1 (CD40), macrophage class M2 (CD168) and type III collagen. Immunohistochemical staining was performed for macro-phages and type III collagen, and heat-induced antigen retrieval with citrate buffer at pH 6 was used to unmask the antigens. Af-ter the sections had been cooled in tap water the endogenous peroxidase was quenched with 1 vol% hydrogen peroxide in methanol. The sections were blocked with 10 vol% normal goat serum (Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA) after washing with PBS. The sections were then incubated with mouse anti-rat macrophage class HIS 36 (1: 800, Santa Cruz Bio-technology Inc.) at room temperature for 30 min, with rabbit anti-rat macrophage class M1 and M2 (1: 100; Santa Cruz Bio-technology Inc.) at room temperature for 60 min, or with rabbit anti-rat type III collagen (1: 80; Millipore, Billerica, Mass., USA) at 4 ° C overnight. The sections were washed and then incubated with biotinylated goat anti-mouse antibody (1: 200; Santa Cruz Biotechnology Inc.) for macrophage staining or with goat anti-rabbit antibody (Dako, Denmark) for type III collagen staining for 30 min. The slides were washed and incubated with an avi-din-biotin peroxidase complex (Santa Cruz Biotechnology Inc.) or EnVision G/2 system/AP (Dako) at room temperature for 30 min for macrophages and type III collagen, respectively. Macro-phages were visualized with diaminobenzidine (Santa Cruz Bio-technology Inc.), while type III collagen was visualized with liq-uid permanent red (LPR; Dako). The slides were counterstained with HE before permanent mounting with Vectamount (Vec-tor Laboratories Inc., Burlingame, Calif., USA). The number of macrophages was determined by counting the positive-stained macrophages from 10 randomly selected fields from the immu-nohistochemically stained sections from each sample ( ! 400 magnification).
Epithelialization Evaluation. The analysis was performed on color images of 640 ! 480 pixel resolution acquired with a light microscope (BX51, Olympus � ), a stereoscope (Stemi 2000-C, ZEISS � ) and a digital camera (Moticam 1000, Moticam � ) with an image analysis program (ImageJ, NIH). Each set of measurements was standardized by a calibrated scale within the image analysis program. The extent of epithelialization was determined from HE-stained sections. The length of the epithelial tongue was de-termined as the distance that the neoepithelium had migrated
from the margin of the wound (epithelial tip) as defined by the presence of hair follicles in nonwounded skin. The width of the gap between the epithelial tips was also measured [Eming et al., 2007].
Statistical Analysis All quantitative data are reported as the mean 8 SD. For the
physical characterization data including pore size, compressive modulus and water swelling ratio, the statistical significance of the differences between two sample groups was determined by an unpaired Student’s t test. For the data from the in vitro and in vivo studies, all treatment groups at all time points were compared by a repeated measures ANOVA, and the differences between groups at different time points were compared by a post hoc test. The data with equal variances were analyzed by a Bonferroni test, while the data with unequal variances were analyzed by Dunnett’s T3. p ! 0.05 was considered to be significant.
Results
Morphological and Chemical Characteristics of the Scaffolds The genipin-crosslinked PVA and sericin/PVA scaf-
folds formed stable matrices exhibiting the pale blue col-or of genipin. Figure 1 shows the cross-sectional struc-ture of the scaffolds. Both scaffolds had porous structures with highly interconnected pores but different pore ar-rangements. The average pore sizes of genipin-cross-linked PVA and sericin/PVA scaffolds were approximate-ly 32.67 8 13.57 and 45.69 8 15.38 � m, respectively.
Physical Properties of the Scaffolds Table 1 presents the physical properties of genipin-
crosslinked PVA and sericin/PVA scaffolds. The com-pressive modulus of genipin-crosslinked sericin/PVA scaffolds (172.1 8 41.7 Pa) was approximately 7-fold higher than that of the scaffolds without sericin (25.6 8 8.7 Pa). The equilibrium water swelling ratio of genipin-crosslinked sericin/PVA scaffolds was 51.9 8 8.6, while that of the scaffolds without sericin was 38.9 8 12.3.
Table 1. P hysical properties of genipin-crosslinked PVA and seri-cin/PVA scaffolds
* p < 0.05, significant against the value of genipin-crosslinked PVA scaffold without sericin.
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Release Profile of Sericin from the Scaffolds Figure 2 shows the in vitro release profile of sericin
from the scaffolds. A burst release of sericin from the scaffolds was observed after the initial 12 h; thereafter, sericin was released at a constant rate. The percentage of sericin released from the scaffold after 72 h was approxi-mately 4 wt% of the initial incorporated amount.
In vitro Attachment and Proliferation of L929 Cells Cultured in the Scaffolds Figure 3 a shows the number of cells present in geni-
pin-crosslinked PVA and sericin/PVA scaffolds over time. After 6 h of culture, the number of cells attached on
the genipin-crosslinked sericin/PVA scaffolds was signif-icantly higher than that of the scaffolds without sericin. Significantly more cells were observed in the genipin-crosslinked sericin/PVA scaffolds throughout the culture period than in the scaffolds without sericin. Figure 3 b shows the morphology of the cells after 3 days of culture in the genipin-crosslinked sericin/PVA scaffolds. The cells were uniformly distributed throughout the scaf-folds.
Wound Size Reduction Figure 4 a shows the appearance of the wounds at 14
days after surgery. Treatment with the genipin-cross-
Fig. 1. Scanning electron microscope cross-section of genipin-crosslinked PVA and sericin/PVA scaffolds. Scale bar = 50 � m.
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Fig. 2. In vitro release profile of sericin from the genipin-crosslinked sericin/PVA scaffolds after incubation in PBS (pH 7.4) at 37 ° C, as assessed by a BCA protein assay kit.
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linked sericin/PVA scaffolds substantially reduced the wound size compared with the scaffolds without sericin. A new epithelial layer was clearly formed over the wound treated with the genipin-crosslinked sericin/PVA scaf-folds, while the original margin of the wound remained. Figure 4 b shows the average wound size reduction after treatment with the two scaffolds. The size of wounds treated with the genipin-crosslinked sericin/PVA scaf-
folds was substantially reduced beginning 3 days after surgery, whereas the size of the control wounds did not change significantly until 7 days after surgery. Wounds treated with the genipin-crosslinked sericin/PVA scaf-folds showed a significantly higher percentage of wound size reduction (smaller wound size) than those treated with the scaffolds without sericin throughout the treat-ment period (p ! 0.05). The genipin-crosslinked sericin/
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Fig. 3. a The number of cells attached initially (6 h) and present throughout the culture period (1, 3, 5 and 7 days) in the genipin-crosslinked PVA ( _ ) and sericin/PVA scaffolds ( + ), where * p ! 0.05 indicates a significant difference with genipin-crosslinked
PVA scaffolds without sericin at the corresponding time. b Mor-phology of cells in the genipin-crosslinked sericin/PVA scaffolds after 3 days. Scale bar = 100 � m.
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Fig. 4. a Gross images of wounds treated with genipin-crosslinked PVA (left) and sericin/PVA scaffolds (right) for 14 days after sur-gery. b Percentage of average wound size reduction after treat-ment with genipin-crosslinked PVA ( + ) and sericin/PVA scaf-
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PVA scaffolds healed the wounds almost completely within 14 days, while the healing of the control wounds was delayed.
Inflammation and Vascularization Figure 5 a, b shows the histological appearances of
macrophages (brown color) and giant cells (purple) pres-ent in the wounds of both treatments at 7 days after sur-gery. More macrophages and giant cells were observed in the wounds treated with the scaffold without sericin. Fig-
ure 3 c shows the numbers of macrophages, giant cells and blood vessels in the wounds subjected to both treatments at 3, 7, 14 and 21 days after surgery. The wounds treated with the genipin-crosslinked sericin/PVA scaffolds ex-hibited fewer macrophage cells throughout the treatment period compared with the control wounds. A significant difference in the number of macrophages was observed between the two treatments at 7 days after surgery. Simi-larly, wounds treated with the genipin-crosslinked seri-cin/PVA scaffolds held significantly fewer multinucleat-
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Fig. 5. a Immunohistochemically stained images of macrophages (positively stained; brownish color) in the wounds treated with genipin-crosslinked PVA (left) and sericin/PVA scaffolds (right) for 7 daysafter surgery. Scale bar = 10 � m. b HE-stained images of multinucleated giant cells in the wounds treated with genipin-crosslinked PVA (left) and sericin/PVA scaffolds (right) for 7 days after surgery. Scale bar = 30 � m. c The numbers of mac-rophages, giant cells and blood vessels in the wounds treated with genipin-cross-linked PVA and sericin/PVA scaffolds for 3, 7, 14 and 21 days, where * p ! 0.05 indi-cates a significant difference with genipin-crosslinked PVA scaffolds without sericin at the same time.
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ed giant cells than the control wounds at 14 and 21 days after surgery. On the other hand, a high microvessel den-sity was observed in wounds subjected to both treatments at the early stages of tissue granulation (3 and 7 days after surgery). At 21 days after surgery, the wounds treated with the genipin-crosslinked sericin/PVA scaffolds had a significantly lower vascular density than the control wounds.
Figure 6 a shows the histological appearance of CD40+ (M1) and CD163+ (M2) macrophages present in the wounds of both treatments at 14 days after surgery. More CD40+ (M1) macrophages were observed in the wounds treated with genipin-crosslinked PVA scaffolds without sericin, while the wounds treated with genipin-cross-linked sericin/PVA scaffolds contained more CD163+ (M2) macrophages. Figure 6 b shows the number of CD40+ (M1) and CD163+ (M2) macrophages present in
the wounds of both treatments at 3, 7, 14 and 21 days after surgery. The number of CD40+ (M1) macrophages in wounds treated with genipin-crosslinked sericin/PVA scaffolds initially seemed to be high, but after more treat-ment time this number became significantly lower than that of wounds treated with genipin-crosslinked PVA scaffolds. However, CD163+ (M2) macrophages were present in wounds subjected to both treatments in similar numbers at 3 and 7 days after surgery. The number of CD163+ (M2) macrophages in wounds treated with genipin-crosslinked sericin/PVA scaffolds was signifi-cantly higher than that of genipin-crosslinked PVA scaf-folds at 14 and 21 days after surgery.
Collagen Formation Figure 7 a shows the histological appearance of type
III collagen in both treatment groups at 3 days after sur-
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Fig. 6. a Immunohistochemically stained images of CD40+ (M1) macrophages (positive stained; brownish color) and CD163+ (M2) macrophages (positive stained; reddish color) in the wounds treated with genipin-crosslinked PVA (left) and sericin/PVA scaf-folds (right) for 14 days after surgery. b Number of CD40+ (M1) and CD163+ (M2) macrophages in the wounds treated with genipin-crosslinked PVA ( + ) and sericin/PVA scaffolds ( _ ) for 3, 7, 14 and 21 days, where * p ! 0.05 indicates a significant dif-ference with genipin-crosslinked PVA scaffolds without sericin at the same time.
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gery. The extent of type III collagen (as shown by the in-tensity of the red color; immunostained with LPR) pro-duced in the wounds treated with the genipin-cross-linked sericin/PVA scaffolds was higher than that of the control wounds. Figure 7 b shows the area fraction of
type III collagen stained with the LPR of wounds of both treatments at 3, 7, 14 and 21 days after surgery. The area fraction of type III collagen in both wounds gradually increased throughout the treatment period. The wounds treated with genipin-crosslinked sericin/PVA scaffolds
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Fig. 7. a Immunohistochemically stained images of type III col-lagen formation in the wounds treated with genipin-crosslinked PVA and sericin/PVA scaffolds for 3 days after surgery, stained with LPR. b Area fraction of type III collagen in the wounds treat-
ed with genipin-crosslinked PVA ( + ) and sericin/PVA scaffolds ( _ ) for 3, 7, 14 and 21 days, where * p ! 0.05 indicates a significant difference with genipin-crosslinked PVA scaffolds without seri-cin at the same time.
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Fig. 8. a Histologically stained images of total tissue collagen for-mation in the wounds treated with genipin-crosslinked PVA (left) and sericin/PVA scaffolds (right) for 3 and 7 days after surgery, stained with Sirius red. Scale bar = 300 � m. b Area fraction of total tissue collagen in the wounds treated with genipin-cross-
linked PVA ( + ) and sericin/PVA scaffolds ( _ ) for 3, 7, 14 and 21 days, where * p ! 0.05 indicates a significant difference with the values of genipin-crosslinked PVA scaffolds without sericin at the same time.
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had a significantly higher type III collagen fraction than the control wounds at 7, 14 and 21 days after surgery(p ! 0.05).
Figure 8 a shows the histological appearance of the to-tal tissue collagen in wounds of both treatment groups at 3 and 7 days after surgery. The extent of total tissue col-lagen (as shown by the intensity of the red color; stained with Sirius red) produced in the wounds treated with the genipin-crosslinked sericin/PVA scaffolds was higher than that of the control wounds. Figure 8 b shows the area fraction of total tissue collagen stained with Sirius red of wounds of both treatment groups at 3, 7, 14 and 21 days after surgery. Corresponding to the formation of type III collagen, the area fraction of total tissue collagen in both wounds increased throughout the treatment period, and the wounds treated with genipin-crosslinked sericin/PVA scaffolds exhibited a significantly higher total tissue collagen fraction than the control wounds at 7 and 14 days after surgery (p ! 0.05).
Epithelialization Figure 9 a shows the histological appearances of epi-
thelial tips in wounds of both treatment groups at 14 days after surgery. The wounds treated with genipin-cross-linked sericin/PVA scaffolds had shorter distances be-
tween epithelial tips than the control wounds. Figure 9 b shows the quantitative distances between the epithelial tips in wounds of both treatment groups throughout the treatment period. The wounds treated with genipin-crosslinked sericin/PVA scaffolds showed significantly shorter distances between epithelial tips than the control wounds at 7 and 14 days after surgery.
Figure 10 a shows the histological appearances of epi-thelial tongue wounds of both treatments at 7 days after surgery. The length of the epithelial tongue in wounds treated with the genipin-crosslinked sericin/PVA scaf-folds was longer than that of the control wounds. Fig-ure 10 b shows the length of epithelial tongue in wounds of both treatments at 3 and 7 days after surgery. The wounds treated with genipin-crosslinked sericin/PVA scaffolds exhibit a significantly longer epithelial tongue than the control wounds at 7 days after surgery.
Discussion
Silk sericin has biological properties that are advanta-geous in promoting wound healing [Aramwit et al., 2009]. In this study, we hypothesized that an appropriate sericin delivery system could accelerate the healing of
Fig. 9. a HE-stained images of epithelial tips of the wounds treated with genipin-crosslinked PVA (left) and sericin/PVA scaffolds (right) for 14 days after surgery. G = Granulation tissue; D = dermis. b Dis-tance between epithelial tips in the wounds treated with genipin-crosslinked PVA ( + ) and sericin/PVA scaffolds ( _ ) for 3, 7, 14 and 21 days, where * p ! 0.05 indicates a significant difference with genipin-cross-linked PVA scaffolds without sericin at the same time.
a
b
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full-thickness wounds. Many formulations of porous scaffolds fabricated from either natural or synthetic ma-terials have been developed for accelerated wound heal-ing [Agrawal and Ray, 2001; Babaeijandaghi et al., 2010; Steinstraesser et al., 2010]. Our previous study reported that sericin blended with PVA formed a three-dimen-sional scaffold with a fragile structure [Aramwit et al., 2010b]. Glycerin (as a plasticizer) and genipin (as a cross-linking agent) were added to make a strong and stable matrix. The chemical and physical properties of sericin/PVA scaffolds with or without glycerin and genipin at various concentrations were fully characterized [Aram-wit et al., 2010b]. In this study, the physical properties and biological potential for wound healing of the geni-pin-crosslinked sericin/PVA scaffolds were of interest. We first observed the morphology of the scaffolds because the porous structure of the scaffolds was important for cell infiltration and for the transport of nutrients and waste [Dainiak et al., 2010; Hutmacher, 2000]. We found that the genipin-crosslinked sericin/PVA scaffolds ex-hibited a well-arranged porous structure ( fig. 1 ). This structure could result from the crosslinking of sericin by
genipin. Genipin is a naturally occurring crosslinking agent that can crosslink proteins including collagen, gel-atin, silk fibroin and silk sericin in a spontaneous reac-tion [Yoo et al., 2011]. The modification to the scaffold morphology induced by the genipin crosslinking has been reported elsewhere [Jun-Sheng et al., 2009].
The physical properties of the scaffolds were then eval-uated to confirm the mechanical function of the scaffolds as wound dressings. The compressive modulus and water swelling ability of the genipin-crosslinked scaffolds were improved by the incorporation of sericin ( table 1 ). In the presence of sericin, the crosslinking interaction within the scaffolds may have been stronger than that of the scaf-folds without sericin, resulting in a higher compressive modulus. On the other hand, the increased water swell-ing ability of the scaffolds could be due to the hydrophil-ic nature of the sericin. The good water swelling ability of the scaffolds would be advantageous in terms of fluid ab-sorption at the wound bed.
Furthermore, we proved that sericin could be released in a sustained manner from genipin-crosslinked scaf-folds, possibly because of the crosslinking by genipin
Fig. 10. a HE-stained images of epithelial tongue in the wounds treated with geni-pin-crosslinked PVA (left) and sericin/PVA scaffolds (right) for 7 days after sur-gery. D = Dermis; E = epidermis; G = gran-ulation tissue; F = fat (adipose tissue); PC = panniculus carnosus. b The length of the epithelial tongue in the wounds treated with genipin-crosslinked PVA ( + ) and sericin/PVA scaffolds ( _ ) for 3, 7, 14 and 21 days, where * p ! 0.05 indicates a sig-nificant difference with genipin-cross-linked PVA scaffolds without sericin at the same time. b
a
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( fig. 2 ). This release behavior was beneficial for the pro-longed healing of wounds by sericin. Based on the bioac-tivity of sericin, genipin-crosslinked sericin/PVA scaf-folds were found to promote the initial attachment and proliferation of L929 cells ( fig. 3 ). This finding fits with the biological functions of sericin itself, as reported else-where [Terada et al., 2002; Sasaki et al., 2005; Tsubouchi et al., 2005; Dash et al., 2008; Zhang et al., 2008]. This bioactive function of sericin would consequently pro-mote wound healing.
To confirm the potential of these genipin-crosslinked sericin/PVA scaffolds as wound dressings, the scaffolds were then tested in full-thickness wounds in the dorsal skin of Sprague-Dawley rats. The healing of full-thickness wounds was accelerated by the genipin-crosslinked seri-cin/PVA scaffolds, more so than the scaffolds without ser-icin, as confirmed by the wound size reduction, collagen formation and epithelialization ( fig. 4–10 ). Collagen is a crucial component and plays an important role in tissue healing by providing tissue strength and an extracellular matrix framework for cell adhesion and migration [Auk-hil, 2000]. Collagen is also one of the key factors in scar formation. A cutaneous scar caused by excessive inflam-mation and immature collagen has a distinctly different collagen pattern compared to normal skin collagen [Kim et al., 2010]. We found here that the genipin-crosslinked sericin/PVA scaffolds induced the production of total tis-sue collagen and type III collagen, the second most abun-dant collagen found in extensible connective tissues such as the skin, lung and the vascular system, in association with type I collagen ( fig. 7 , 8 ). Epithelialization or epider-mal recovery refers to the migration and growth of kera-tinocytes on the neodermis followed by the formation of a complete basement membrane that ensures the struc-tural and mechanical stability of the dermo-epidermal junction. The epithelialization process depends on the self-renewal, proliferation and migration of keratinocytes residing at the basal cell layer. In this study, a greater ex-tent of epithelialization occurred when wound healing was promoted by the genipin-crosslinked sericin/PVA scaffolds, as confirmed by the shorter distance between the epithelial tips and the longer epithelial tongue ( fig. 9 , 10 ). Furthermore, the new epithelial formation was clear-ly observed over the wound treated with the genipin-crosslinked sericin/PVA scaffolds, while the original mar-gin of the wound remained ( fig. 4 a). This reveals that the wound healing induced by the genipin-crosslinked seri-cin/PVA scaffolds was mainly due to the new epitheliali-zation, not the wound contraction.
The in vivo results indicated that the genipin-cross-linked sericin/PVA scaffolds were effective for the treat-ment of full-thickness skin wounds in rats. Silk sericin cream was previously reported to promote wound heal-ing in rats [Aramwit and Sangcakul, 2007]. The mecha-nism for the accelerated healing of full-thickness wounds by the genipin-crosslinked sericin/PVA scaffolds is not yet clearly understood. However, there are several expla-nations for this phenomenon. Sericin has been reported to promote the proliferation of fibroblasts [Terada et al., 2002; Tsubouchi et al., 2005], as was found in this study ( fig. 3 ). When the genipin-crosslinked sericin/PVA scaf-folds were applied to the wound, the proliferation of fibroblasts within the wound produced collagen and ex-tracellular matrix and secreted cytokines and growth factors that subsequently promoted wound healing. Fur-thermore, sericin was found to promote collagen produc-tion within the wounds [Aramwit and Sangcakul, 2007; Aramwit et al., 2010a]. These results indicate that the genipin-crosslinked sericin/PVA scaffolds control the re-lease of sericin to accelerate the long-term healing of wounds. The acceleration of wound healing by the geni-pin-crosslinked sericin/PVA scaffolds could also be ex-plained from the point of view of inflammation and vas-cularization. The inflammation stage involves many cell types and results in the activation of macrophages. The activation of macrophages has fundamental implications for several aspects of wound healing, such as debride-ment, matrix synthesis and angiogenesis [Witte and Bar-bul, 1997]. Ideal healing is characterized by minimal in-flammation and scarless healing [Olutoye et al., 1996]. On microscopic examination, local inflammatory re-sponses were observed in wounds treated with both types of scaffold. However, more macrophages and giant cells were found in the wounds treated with the scaffolds with-out sericin ( fig. 5 ). Helbig et al. [2009] reported that in-flammatory cells are involved in the replacement of the necrotic zones and seem to be crucial for wound healing. However, the number of macrophages and giant cells is related to scar formation [Druecke et al., 2004]. High numbers of macrophages and giant cells are responsible for localized inflammatory reactions that could suppress further stages of wound healing and subsequently lead to scar formation [Druecke et al., 2004; Ratanavaraporn et al., 2012]. Thus, the higher number of macrophages and giant cells in wounds treated with the genipin-cross-linked PVA scaffolds without sericin could delay wound healing compared with the genipin-crosslinked sericin/PVA scaffolds. It is also interesting to note that not all macrophages are necessarily proinflammatory cells for
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the wound healing process. Macrophages can be classi-fied into two broad phenotypic groups: (i) classically ac-tivated or type I macrophages (M1), which are proinflam-matory effectors and (ii) alternatively activated or type II macrophages (M2) that possess anti-inflammatory prop-erties and contribute to constructive tissue remodeling [Khanna et al., 2010; Rodero and Khosrotehrani, 2010]. High numbers of M1 macrophages indicate acute and chronic inflammation that would impair wound healing, while a high number of M2 macrophages would promote a constructive remodeling of damaged tissue [Badylak et al., 2008]. In this study, we found that treatment with genipin-crosslinked sericin/PVA scaffolds activated CD163+ (M2) macrophages in wounds, particularly in the later stages of healing ( fig. 6 ). The high number ofactivated CD163+ (M2) macrophages would accelerate wound healing. In contrast, the genipin-crosslinked PVA scaffolds without sericin were found to promote the acti-vation of CD40+ (M1) macrophages, resulting in delayed healing due to the activated acute and chronic inflamma-tion. Stout [2010] demonstrated that the implantation of PVA sponges resulted in an influx of M1 macrophages. However, silk fibers have been reported to be immuno-logically inert when cultured with RAW 264.7 murine macrophage cells [Panilaitis et al., 2003]. Soluble seri-cin proteins extracted from native silk fibers did not in-duce a significant inflammatory reaction. Therefore, the wounds treated with these genipin-crosslinked sericin/PVA scaffolds exhibited a low inflammatory response, possibly due to the inert property of sericin by which it may switch proinflammatory activated M1 macrophages to constructive remodeling M2 macrophages, possibly resulting in accelerated wound healing.
New blood vessels are also necessary for the wound healing process as they induce the migration of fibro-blasts and subsequent epithelialization [Koga et al., 2007]. Revascularization normally occurs 96 h after injury [Lin-denblatt et al., 2008], which is similar to our results, and shows that the vessel density increased significantly over the initial 7 days ( fig. 5 c). Our results also showed that wounds treated with the genipin-crosslinked sericin/PVA scaffolds generated slightly more new blood vessels compared with the control wounds at 7 and 14 days. This increase in blood flow would be beneficial for the healing by the genipin-crosslinked sericin/PVA scaffolds. Inter-estingly, the control wounds showed an increased num-ber of blood vessels at 21 days. This may be due to the delayed inflammatory reaction because the high num-bers of macrophages and giant cells present in the control wounds were also related to the tissue vascularization
[Tong et al., 2009]. In other words, these findings indicate that the stages of wound healing were accelerated by the genipin-crosslinked sericin/PVA scaffolds compared with the control wounds. Finally, the superior physical properties of the genipin-crosslinked sericin/PVA scaf-folds make them suitable to support wound healing.
Taken together, the genipin-crosslinked sericin/PVA scaffolds were more effective in promoting the healing of full-thickness skin wounds than the control scaffolds without sericin. These results demonstrate the in vivo ap-plication of natural silk sericin, a biocompatible and bio-active matrix, for accelerated wound healing.
Conclusion
Genipin-crosslinked sericin/PVA scaffolds were fabri-cated and their biological potential for healing full-thick-ness wounds was evaluated. The genipin-crosslinked ser-icin/PVA scaffolds promoted wound healing more effec-tively than the genipin-crosslinked PVA scaffolds without sericin, as confirmed by the wound size reduction, the level of inflammatory reactions, collagen formation and epithelialization. The sericin likely accelerated the heal-ing process mainly by promoting the migration and pro-liferation of skin cells and collagen production.
Acknowledgements
This research was mainly supported by the Agricultural Re-search Development Agency and partly supported by the Thai-land Research Fund, Bangkok, Thailand. We extend our thanks to Prof. Subhas Chandra Kundu, Department of Biotechnology, Indian Institute of Technology, India, for his kind advice on the manuscript preparation.
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