European Journal of Pharmacology 731 (2014) 8–19

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Chitosan-based copper nanocomposite accelerates healing in excision wound model in rats Anu Gopal, Vinay Kant, Anu Gopalakrishnan, Surendra K. Tandan, Dinesh Kumar n Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122, Uttar Pradesh, India

art ic l e i nf o

a b s t r a c t

Article history: Received 20 August 2013 Received in revised form 19 February 2014 Accepted 28 February 2014 Available online 13 March 2014

Copper possesses efficacy in wound healing which is a complex phenomenon involving various cells, cytokines and growth factors. Copper nanoparticles modulate cells, cytokines and growth factors involved in wound healing in a better way than copper ions. Chitosan has been shown to be beneficial in healing because of its antibacterial, antifungal, biocompatible and biodegradable polymeric nature. In the present study, chitosan-based copper nanocomposite (CCNC) was prepared by mixing chitosan and copper nanoparticles. CCNC was applied topically to evaluate its wound healing potential and to study its effects on some important components of healing process in open excision wound model in adult Wistar rats. Significant increase in wound contraction was observed in the CCNC-treated rats. The up-regulation of vascular endothelial growth factor (VEGF) and transforming growth factor-beta1(TGF-β1) by CCNCtreatment revealed its role in facilitating angiogenesis, fibroblast proliferation and collagen deposition. The tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were significantly decreased and increased, respectively, in CCNC-treated rats. Histological evaluation showed more fibroblast proliferation, collagen deposition and intact re-epithelialization in CCNC-treated rats. Immunohistochemistry of CD31 revealed marked increase in angiogenesis. Thus, we concluded that chitosan-based copper nanocomposite efficiently enhanced cutaneous wound healing by modulation of various cells, cytokines and growth factors during different phases of healing process. & 2014 Elsevier B.V. All rights reserved.

Keywords: Copper Chitosan Wound healing VEGF TGF-β1 TNF-α

1. Introduction Copper is an essential trace element for humans and animals and the ancients recognized copper as an essential healing mineral. It facilitates the activity of several enzymes (Borkow and Gabbay, 2009) and provides a role in the development and maintenance of the cardiovascular system, the skeletal system, and the structure and functions of the nervous system. It is evidenced that copper has potent antibacterial properties and is an essential element in many wound-healing-related processes (Borkow and Gabbay, 2005). The emergence of nanoscience and nanotechnology in the last decade presents opportunities for exploring the effects of copper nanoparticles in wound healing. Chitosan, a linear polysaccharide of chitin, has been proposed as a biomaterial because of its apparent satisfactory biocompatibility (Peluso et al., 1994). It is biocompatible and biodegradable and possesses antimicrobial, and wound healing properties which can be synergistically combined with metals like Cu2 þ and Ag2 þ thereby enhancing its antimicrobial and wound healing effects

n

Corresponding author. Fax: þ91 581 230 3284. E-mail addresses: [email protected], [email protected] (D. Kumar).

http://dx.doi.org/10.1016/j.ejphar.2014.02.033 0014-2999/& 2014 Elsevier B.V. All rights reserved.

(Leonida et al., 2011). Various composites based on chitosan such as, silver nanocomposite have been reported with good healing activity (Chambers et al., 2007; Fredriksson et al., 2009). Wound healing involves four temporally overlapping phases i.e. hemostasis, inflammation, proliferation and remodeling (Donald and Zachary, 2011). Despite some recent advances in understanding basic principles of wound healing, it continues to cause significant morbidity and mortality (Fine and Mustoe, 2006). A large variety of treatment modalities are available for the wounds, including application of antibiotics, occlusive layers, bandages, poultices, mechanical devices that reduce evaporation of water and others. However, all of these modalities have one drawback in common; they all help wound healing by supporting the body mechanisms to heal the wound. Unfortunately, this passive wound healing process proves inadequate for some obstinate/recalcitrant wounds or when immunity or other body functions are compromised. To tide over such situations, a treatment modality is desired that speeds up the healing by actively regenerating the skin (dermis and epidermis). A recent study suggests that chitosan and its nanoparticles can inhibit skin aging and facilitates the extracellular matrix (ECM) in remodeling phase of wound healing (Leonida et al., 2011). The composites prepared using metal nanoparticles and polymers could find better utilization due to the enhanced antimicrobial

A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

and wound healing activities (Cioffi et al., 2005). In one pilot study in our laboratory, chitosan-based copper nanocomposite (CCNC) has shown highly significant wound contraction and rapid hair coat regeneration effects in excision wounds in rats. Considering the potentials of chitosan and prospective of nanoparticles of copper in wound healing, the present study was pursued to evaluate wound healing activity and to explain healing mechanism of chitosan-based copper nanocomposite in excision wounds in rats.

2. Materials and methods 2.1. Chemicals Copper nanoparticles (with approximate size 50 nm; Fig. 1) and Chitosan (deacetylation degree 79%) were purchased from Sigma, USA. Glacial acetic acid was purchased from SRL, India. The polyclonal antibodies for vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) and Platelet endothelial cell adhesion molecule-1 (PECAM-1) for CD-31 were procured from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and ELISA kits for IL-10 and TNF-α were obtained from Komabiotech Ltd. USA. The chemicals for SDS-PAGE and Western blotting were obtained from Amresco, USA. All other chemicals and reagents were of analytical grade. 2.2. Preparation of CCNC Chitosan colloidal solution (10%) was prepared by slowly adding chitosan powder in 1% acetic acid solution in normal saline followed by continuous stirring for 8 h. The solution was then filtered using a clean muslin cloth. The chitosan-based copper nanocomposite (CCNC) (0.3%) was prepared by slowly adding copper nanoparticles, pre-dispersed in little ethanol to the chitosan colloidal solution under continuous stirring followed by sonication for 5 min. The final concentration of acetic acid in the solutions and the formulation was kept at r0.1% for topical application. 2.3. Animals Healthy adult male Wistar rats (140–160 g) were procured from the Laboratory Animal Resource Section, Indian Veterinary Research Institute, Izatnagar (U.P.), India. The experimental protocols were approved by the Institute Animal Ethics Committee and conform to the guidelines for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH

9

Publication No. 85-23, revised 1996). Ad libitum feed and water were provided to the rats throughout the experiment. 2.4. Wound creation The rats were anesthetized with intraperitoneal (i.p.) injection of pentobarbitone sodium (40 mg/kg). The dorsal skin of the animals was shaved and cleaned with 70% ethanol and a fullthickness open excision type skin wound (E400 mm2) was created. After recovery from anesthesia, they were housed individually in properly disinfected cages. The animals were equally and randomly divided into three groups of 20 each i.e. Group I (control or r0.1% acetic acid-treated), group II (1% chitosantreated) and group III (0.3% CCNC-treated). The solutions and the formulation were applied topically on the wound area once daily for 14 days. 2.5. Photography of wounds and measurement of percentage wound contraction Photograph of each wound was taken on days 0, 3, 7, 11 and 14. Margins of the wounds were traced on a transparent paper by a fine tip permanent marker. The area (in square millimeters) within the boundaries of each tracing was determined planimetrically. The wound area on 0 day of each animal was measured at predetermined time interval starting at 3 h post wounding and subsequent measurement of wound area from both the groups was taken on days 3, 7, 11 and 14 post-wounding. The results of wound measurements on various days were expressed as per cent wound contraction. The values were expressed as per cent values of the 0 day measurements and were calculated by Wilson's formula as follows: % wound contraction ¼

0 day wound area  unhealed wound  100 0 day wound area

2.6. Collection of tissue After the measurement of wound area, the animals were killed on days 3, 7, 11 and 14 with an overdose of diethyl ether and the granulation/healing tissue was carefully collected. The tissue was immediately divided into three pieces. One piece was stored in RNA later at -20 1C for Real Time PCR. The second piece was snap frozen in liquid nitrogen and tissue homogenate was prepared in ice-cold lysis buffer (100 mg tissue in 500 ml lysis buffer: a buffer with 500 μl Triton X-100, 250 μl of aprotinin, 50 μl of diluted phenyl methane sulfonyl fluoride and 50 μl of leupeptin in 50 ml of PBS) with the help of motor homogenizer at 4 1C. The homogenates were then incubated at 4 1C for 30 min and centrifuged at 12,000 rpm for 10 min at 4 1C. The supernatant was aliquoted and stored at  80 1C till further processing for Western blotting and ELISA assay. The third piece was immediately preserved in 10% neutral buffer formalin for histopathological observations such as, hematoxylin and eosin staining, picrosirius red staining for collagen type-I and type-III and immunohistochemical labeling of CD31. 2.7. mRNA expression studies

Fig. 1. Transmission electron micrograph of copper nanoparticles showing size (50 nm).

Total RNA was isolated using the standard method described by Amresco, USA with Ribozol TM RNA extraction reagents. cDNA synthesis was carried out from total RNA using cDNA synthesis kit (FERMENTAS) as per standard protocol. The polymerase chain reaction was standardized for each gene using cDNA from granulation/ healing tissue (Sambrook and Russell, 2001). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the house keeping

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A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

Table 1 Description of primers used in this study. S. no.

Gene

Primer sequences

Product size

Annealing temperature

Accession number

1.

GAPDH

223

55 1C

GU214026.1

2.

IL-10

161

60 1C

NM012854.2

3.

TNF- α

153

60 1C

NM012675.3

4.

TGF-ß1

246

62 1C

NM021578.2

5.

VEGF

F: 50 -AACTTTGGCATTGTGGAAGG-30 R:50 -ACACATTGGGGGTAGGAACA-30 F:50 -CCTGCTCTTACTGGCTGGAG-30 R: 50 -TGTCCAGCTGGTCCTTCTTT-30 F:50 GGCCACCACGCTCTTTCTGTCA-30 R:50 -TGGGCTACGGGCTTGTCACTC-30 F:50 -AAGTGGATCCACGAG CCC AA-30 R:50 - GCTGCACTTGCAGGAGCGCA-30 F:50 GCCAGCACATAGGAGAGATGAG-30 R: 50 ACCGCCTTGGCTTGTCAC-30

234

62 1C

NM031836.2

gene. After validation of primer annealing temperature, the cDNA of target gene along with reference gene was studied for mRNA expression by Real Time PCR (Stratagene Q-Cycler) and analyzed using Mx 3000P software with 2  QuantiTect SYBR Green PCR Master Mix, Qiagen. To assess the specificity of the amplified product, dissociation curve was generated at temperature 55 1C through 95 1C. The optimum annealing temperatures as determined by PCR for the respective gene using the specific primers were as follows, 60 1C for IL-10 and TNF-α subunit, 62 1C for VEGF and TGF-β1 and 55 1C for GAPDH (Table 1). The relative change in gene expression was studied using the method previously described (Livak and Schmitten, 2001). 2.8. SDS-PAGE and western blot analysis SDS-PAGE was performed by subjecting equal protein concentrations under reducing conditions on 12–15% polyacrylamide followed by electrophoretic transfer to polyvinylidene difluoride (PVDF) membrane at 15 V for 75 min by using semidry Genei Blot Transfer apparatus. The membranes were blocked in 3% BSA in PBS-T for 1 h at 37 1C followed by overnight incubation at 4 1C with the following antibodies: goat polyclonal IgG-EG-VEGF antibody (cat. sc-30343; 1:300, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), goat polyclonal IgG-TGF-β1 (cat. sc-31609; 1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and goat polyclonal IgG-GAPDH (cat. sc20356; 1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) separately. After washing with phosphate buffered saline with Tween 20 (PBS-T), blots were incubated with secondary antibody; HRPconjugated chicken anti-goat IgG (cat. sc-2953; 1:1000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37 1C for 1–2 h. Following successive washes, the blots were developed using the 3,30 -Diaminobenzidine (DAB) system (GeNei, Bangalore Genei, India). The blots were subsequently scanned and band intensity was quantified by densitometry software (Image J, NIH). The Western blot data for VEGF and TGF-β1 were corrected for corresponding GAPDH values and the results were expressed as normalized protein levels. A minimum of three such blots were performed for every protein analyzed. 2.9. ELISA assay for TNF-α and IL-10 ELISA assays of the lysate for TNF-α and IL-10 were conducted as per the manufacturer's instructions (cat. K0331196; 96 well Komabiotech Inc., Seoul, Korea for TNF-α and cat. K0332134 96 well Komabiotech Inc., Seoul, Korea for IL-10). 2.10. Hematoxylin and eosin staining The granulation tissue/healing fixed in 10% neutral buffer formalin was embedded in paraffin and 5 mm thick tissue sections were obtained and stained with H&E as per standard method. The stained sections were visualized under light microscope (Olympus CX31, Tokyo, Japan) at magnification  100 and  400. The

histopathology properties of granulation/healing tissue sections were blindly scored by an experienced pathologist unknown to experimental groups. Ten random fields from different sections in each group were examined. Scoring was done according to the method of Greenhalgh et al. (1990). Briefly, scoring for each field of H&E stained section was done from 1 to 12. Score (i) 1 to 3 was given to none to minimal cell accumulation and granulation tissue (ii) 4 to 6 to thin immature granulation tissue that is dominated by inflammatory cells but has few fibroblasts, capillaries, or collagen deposition and minimal epithelial migration, (iii) 7 to 9 to moderately thick granulation tissue can range from being dominated by inflammatory cells to more fibroblasts and collagen deposition, extensive neovascularization, epithelium can range from minimal to moderate migration and (iv) 10 to 12 to thick, vascular granulation tissue dominated by fibroblasts and extensive collagen deposition and epithelium partially to completely covering the wound. 2.11. Immunohistochemistry for labeling of CD31 (PECAM-1) Angiogenesis in wound tissues on different days of healing was confirmed by immunohistochemistry with endothelial cell marker CD31. The tissue section (5-μm-thick paraffin sections) were deparaffinized and rehydrated in graded alcohols and immunohistochemical detection of CD31 was performed. Antigen retrieval was performed by treating the slides in sodium citrate buffer (10 m M, pH 6.0) in a microwave oven for 10–12 min and cooled at room temperature for 20 min. Blocking of endogenous peroxidase was done by incubating in 3% hydrogen peroxide in a humidified chamber for 20 min at 37 1C. Non-specific bindings were blocked by incubating the tissue sections with 4% goat serum for 45 min. The overnight incubation of the sections was done at approximately 4 1C with monoclonal mouse antirat CD31 antibody (NB100-64796-Novus Biologicals, USA with 1:50 dilution). After washing, the sections were then incubated with horseradish peroxidase (HRP)-conjugated goat anti mouse IgG (sc2005; Santa Cruz Santa Cruz Biotechnology Inc., USA, 1:70 dilution) for 45 min at 37 1C. Slides were then treated with aminoethylcarbazole (AEC) chromogen substrate (AEC Staining Kit; Sigma-Aldrich) for 10– 15 min for the color reaction. The development of red-brown color indicating positive reaction and the reactions were stopped by washing the slides in distilled water. Then the sections were counterstained with Gill No. 3 hematoxylin (Sigma) for 15 s and washed in distilled water. Microphotographs of 10 random high power fields (  400) were taken to determine the microvessel density (MVD) per image using semi-automated computerized Image J software (National Institutes of Health, USA). 2.12. Picrosirius red staining for collagen To evaluate thick and thin collagen in healing wound, sections were stained with picrosirius red (direct red 80 from sigma Aldrich, USA) by modified picrosirius procedure (Dayan et al., 1989)

A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

and stained sections were viewed under polarized light. According to the birefringence pattern, collagen types were differentiated as (i) thick and denser collagen showing orange to red color and (ii) thinner collagen fibers showing yellow to green (Vodovotz et al., 1993; Rizzoni et al., 2005). 2.13. Statistical analysis Results are expressed as mean 7S.E.M. with n equal to number of animals. Data were analyzed between acetic acid and CCNC and between chitosan and CCNC by one-way ANOVA followed by Dunnet's test and two-way ANOVA followed with Bonferroni's multiple comparison test. A value of P o0.05 was considered to be statistically significant.

3. Results 3.1. Effect of topical application of acetic acid, chitosan and CCNC on wound closure (wound contraction) in rats The representative photographs of the wound area of all the three groups of rats on days 0, 3, 7, 11 and 14 are given in Fig. 2A. The per cent increase in the wound contraction was found to

Day 0

Day 3

11

increase in a time-dependent manner in all the groups (Fig. 2B). Wound contraction was markedly higher in CCNC- and chitosantreated rats, as compared to control on different days of postwounding. Amongst the three groups, the CCNC-treated wounds showed fast contraction throughout the study period. Also CCNCtreated groups significant wound contraction on days 3, 7, 11, and 14 compared to chitosan and control treated groups.

3.2. Effects of topical application of acetic acid, chitosan and CCNC on expression of VEGF, TGF-β1, in granulation/healing tissue of excision wounds of rats The expression of VEGF was significantly (Po0.01) upregulated in CCNC-treated group on day 3 and 7 post wounding compared to chitosan treated and control groups (Fig. 3A). The expression in chitosan group was also significantly higher on days 3 as compared to control group. The TGF-β1 mRNA was also found to be significantly upregulated in CCNC-treated group in comparison to chitosantreated and control group on day 3 and 7 (Fig. 3B). The same was reduced on day 11 and 14. The mRNA expression of TGF-β1 in chitosan-treated group was also up-regulated on days 3, 7 and 14 in comparison to control.

Day 7

Day 11

Day 14

Acetic acid

Chitosan

CCNC

Wound contraction

90

Wound contraction (%)

** **

Acetic acid Chitosan CCNC

100

** *

80 70

** **

60 50

** **

40 30 20

**

**

10 0 3

7

11

14

Days Fig. 2. (A) Representative photographs showing wound closure of acetic acid (  0.1%)-, chitosan- and CCNC-treated rats on days 0, 3, 7, 11 and 14. (B) Effects of topical application of acetic acid (  0.1%), chitosan and CCNC on per cent wound closure on different days in rats (wound contraction, %). n¼ 5 rats; n P o0.05; nn Po 0.01.

** **

15

Fold change in TGF β1 mRNA expression

A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

Fold change in VEGF mRNA expression

12

VEGF mRNA Acetic acid Chitosan CCNC

** **

12

9

6

*

3

0 3

7

11

TGFβ1 mRNA

10.0

7.5

** *

5.0

2.5

0.0

14

3

7

Day 3 CH

Day 7 CCNC

11

14

Days

Days

AA

Acetic acid Chitosan CCNC

** **

AA

CH

Day 11 CCNC

AA

Day 14

CH

CCNC

AA

CH

CCNC

GAPDH

VEGF TGF-β1

TGF-β1 protein expression

VEGF protein expression 3.5

Acetic acid Chitosan CCNC

** 2

** **

** *

1

** ** Acetic acid Chitosan CCNC

3.0 Normalized TGF-β1 protein levels

Normalized VEGF protein levels

3

2.5 2.0

** *

1.5

*

1.0 0.5

0

0.0 3

7

11

14

Days

3

7

11

14

Days

Fig. 3. Effects of topical application of acetic acid (  0.1%), chitosan and CCNC on mRNA expression of VEGF (A) and TGF-β1 (B). (C) Western blot analysis of GAPDH, TGF-β1 and VEGF in granulation tissue of acetic acid (  0.1%)-, chitosan- and CCNC-treated rats on days 3, 7, 11 and 14. Effects of topical application of acetic acid (  0.1%), chitosan and CCNC on TGF-β1 (D) and VEGF (E) protein expression on different days in granulation/healing tissue of excision wounds in rats. n ¼5; n P o0.05; nn P o 0.01.

The VEGF protein expression was significantly greater in CCNCtreated group on 3rd day compared to control and on 7th day compared to chitosan-treated and control groups (Fig. 3D). The expression in chitosan-treated groups on day 7 was also higher as compared to control. After day 7, the expression started to decline which was more in the CCNC-treated group, which showed a significant reduction on 14th day as compared to control. The TGF-β1 protein expression was significantly greater on day 3 and 7 in CCNC-treated group in comparison to other days indicating its increased level during early phase of wound healing (Fig. 3E). Also on day 14, TGF-β1 protein expression was significantly reduced in CCNC-treated groups as compared to control group. 3.3. Effect of topical application of acetic acid, chitosan and CCNC on TNF-α and IL-10 protein levels in granulation/healing tissue of excision wounds of rats The TNF-α mRNA expression in chitosan-treated wounds was significantly (P o0.01) up-regulated on day 3, when compared to

control group (Fig. 4A). However, in the CCNC-treated wounds, the expression was found to be reduced throughout the experiment as compared to control. IL-10 mRNA expression showed a significant increase, (P o0.01and P o0.05) on days 3 and 7 after treatment with CCNC (Fig. 4B). The TNF-α levels (pg/mg protein) remained lower during the entire experiment in the CCNC-treated group, as compared to chitosan and control groups and the levels were significantly lower on days 3 (P o0.05), 7 (P o0.01) and 11 (P o0.01) in the CCNC-treated group (Fig. 4C). The chitosan-treated wounds revealed a non-significant increase in the TNF-α level on day 3, as compared to control and a significant increase (P o0.01) compared to CCNC-treated group. Thereafter, the levels remained lower in comparison to control group during the entire experiment and a significant (Po0.01) decrease in TNF-α levels was evident on day 11, as compared to control group. The levels of IL-10 (pg/mg protein) in CCNC-treated group revealed significant increase on day 3 (P o0.01) in comparison to chitosan-treated and control rats and on day 7 (P o0.05) in comparison to control (Fig. 4D). On day 3, chitosan-treated group

3

TNFα mRNA

**

Acetic acid Chitosan CCNC

2

1

0 3

7

11

Fold change in IL-10 mRNA expression

Fold change in TNF α mRNA expression

A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

3.0 2.5

IL-10 mRNA Acetic acid Chitosan CCNC

**

2.0

* 1.5 1.0 0.5 0.0

14

3

7

Days

*

TNF-α protein expression

**

Acetic acid Chitosan CCNC

**

2500

800 700

** **

2000

11

14

Days

IL-10 (pg/100 mg tissue)

TNF-α (pg/mg protein)

3000

13

1500 1000 500

** **

IL-10 protein expression

*

Acetic acid Chitosan CCNC

600 500 400 300 200 100 0

0 3

7

11

14

Days

3

7

11

14

Days

Fig. 4. Effects of topical application of acetic acid (  0.1%), chitosan and CCNC on fold changes of TNF-α (A), IL-10 (B) mRNA expression on different days in granulation/ healing tissue of excision wounds in rats. Effects of topical application of acetic acid (  0.1%), chitosan and CCNC on TNF- α (C) and IL-10 (D) protein levels on different days in granulation/healing tissue of excision wounds in rats. n¼ 5; n Po 0.05; nn P o0.01.

showed a non-significant decrease in the IL-10 levels compared to control. (Fig. 4D).

3.4. Histopathological findings 3.4.1. Effect of topical application of acetic acid, chitosan and CCNC on maturity of granulation/healing tissue of excision wound in rats The H&E stained sections of healing wounds of control, chitosan- and CCNC-treated groups on different days are presented in Fig. 5. On day 3 in control group, engorged vessels with few fibroblasts and inflammatory cells were observed. The stained sections in chitosan-treated wound showed more infiltration of inflammatory cells and edematous deposition with few fibroblasts. However, the CCNC-treated group revealed less inflammatory cell infiltration, as compared to chitosan treated group. Also, superficial necrotic tissue along with edematous deposition and few fibroblasts were observed. On day 7, the wound sections of control group still showed presence of inflammatory cells and fibroblast proliferation with few capillaries and mild collagen deposition. In chitosan-treated group, there was severe inflammatory reaction along with some proliferation of fibroblast. In CCNC-treated group, only mild inflammatory reaction with more fibroblast proliferation was observed. On day 11, the sections of control group showed well formed granulation tissue with fibroblasts, collagen deposition and newly forming blood vessels. The observations in chitosan-treated group were similar to control group. However, the CCNC-treated group

showed marked fibroblast proliferation and well formed blood vessels along with collagen deposition. On day 14, sections of wound area of control group showed well formed granulation tissue and collagen deposition, but the epithelial layer was still not formed. However, the chitosan- and CCNC-treated wounds showed formation of complete superficial epithelial layer with well formed granulation tissue. The superficial epithelial layer was better formed in CCNC-treated group, as compared to chitosan-treated group. Also, the collagen fibers were well arranged in CCNC-treated group in comparison to other groups. Histological scoring also found to be higher in CCNCtreated groups on days 11 and 14 (Fig. 5D).

3.4.2. Effect of topical application of acetic acid, chitosan and CCNC on angiogenesis in granulation/healing tissue of excision wound in rats CD31 labeled histological sections of CCNC-treated rats showed many newly formed capillaries lined by CD31 positive endothelial cells (brownish color) dispersed within the granulation tissue on days 3, 7 and 11, compared to chitosan-treated and control rats (Fig. 6A). Microvessel density (MVD) of CCNC-treated group was found to be maximum on day 7 and day 11 (Fig. 6B). Chitosantreated group also showed more MVD compared to control on days 7 and 11. On day 14 also, section of CCNC-treated group showed well formed capillary vessels lined by CD31 positive endothelial cells whereas in control rats, ill formed capillary vessels were found to be scattered in immature granulation tissue.

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A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

I

3 Day I

I

C

7 Day

F I F I

B

B

F

B

11 Day

F

I

F

C

F

C

14 Day

B C C

B F

12

*

10

Histological score

** *

Acetic acid Chitosan CCNC

8 6 4 2 0 3

7

11

14

Days

Fig. 5. Histopathological characteristics of cutaneous wound on days 3, 7, 11 and 14 in acetic acid- (A), chitosan- (B) and CCNC-treated (C) groups at  100 (scale bar 200 μm) and  400 (scale bar 50 μm) magnifications. I: inflammatory cells; F: fibroblasts; B: blood vessels; E: epithelial layer; C: collagen. (D) Histological scoring of the wound sections on days 3, 7, 11 and 14 in different groups. n¼ 5; n Po 0.05; nn P o0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4.3. Effect of topical application of acetic acid, chitosan and CCNC on collagen deposition in granulation/healing tissue of excision wound in rats The representative picrosirius red stained sections of wounds of control, chitosan- and CCNC-treated rats on different days are presented in Fig. 7. On day 3, the collagen fibers in the sections of all the three groups exhibited irregular greenish birefringence

under polarized microscope revealing the presence of thin collagen. The greenish birefringence was more intense in the CCNC-treated group, as compared to control and chitosan group. On day 7, the green fibers were started to be replaced by the yellow-red fibers (thick collagen fibers). The proportion of green fibers was more in control and chitosan-treated groups. However, the CCNC-treated group showed more proportion of thick fibers

A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

3 day

7 day

11 day

15

14 day

Acetic acid

Chitosan

CCNC

18

Acetic acid Chitosan CCNC

Microvessel density/ high power field

15 12 9 6 3 0 3

7

11

14

Days Fig. 6. (A) CD31 immunohistochemistry of wound sections of acetic acid-, chitosan- and CCNC-treated groups on day 3, 7, 11 and 14 post-wounding for analysis of angiogenesis (scale bar ¼100 mm and magnification  200). (B) Microvessel density of wound sections on days 3, 7, 11 and 14 in different groups at high power field (  400). n¼ 5; n P o0.05; nn Po 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(reddish orange). Also, the intensity of thick fibers in CCNCtreated group was more pronounced with regular and parallel arrangement. On day 11, the thick fibers (yellow) were predominant in chitosan- and CCNC-treated groups. However, the control group still showed the predominance of thin fibers. On day 14, all the three groups showed the presence of thick mature collagen fibers. The arrangement of these fibers in control and chitosan-treated group was irregular. However, the parallel arrangement and more compactness of the thick mature collagen fibers were evident in the CCNC-treated group.

4. Discussion Wound healing begins with inflammatory response which is followed by proliferation and migration of dermal and epidermal cells and matrix synthesis, in order to fill the wound gap and reestablish the skin barrier (Hackam and Ford, 2002; Harding et al.,

2005). Finally, tissue remodeling and differentiation enable almost full recovery of the skin tissue and restoration of skin aesthetics (Hackam and Ford, 2002; Diegelmann and Evans, 2004). An ideal healing agent should possess the properties such as: it should check the infection, speed up the healing by modulation of cytokines and growth factors, favorable for cell proliferation and matrix deposition, better reepithelialization, less or no scar formation. Integration of nanotechnology into biology has brought to fore metals like copper in the form of nanoparticles as potential topical antimicrobial agents. Copper and its nanoparticles facilitate activity of several enzymes and provide a role in the development and maintenance of various systems in the body. Chitosan due to its apparent satisfactory biocompatibility and ability to improve the ECM remodeling phase of wound healing can be incorporated to wound healing formulations (Leonida et al., 2011). Thus, the nanobiotechnology combined with the knowledge on cellular and subcellular events occurring in wound healing offers great opportunities for improving wound care.

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A. Gopal et al. / European Journal of Pharmacology 731 (2014) 8–19

3 day

7 day

11 day

14 day

Acetic acid

Chitosan

CCNC 100 µm Fig. 7. Collagen fibers in the sections of granulation/healing tissue stained with picrosirius red and visualized under polarized microscope of the rats treated with acetic acid (A), chitosan (B) and CCNC (C) on days 3, 7, 11 and 14 post-wounding. G ¼ green fibers, R¼red fibers and Y ¼yellowish fibers. Bar ¼ 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Proposed mechanisms by which CCNC stimulates angiogenesis and wound healing.

Wound contraction is a dynamic process where cells organize their surrounding tissue matrix to reduce normal healing time by shrinking the amount of ECM that needs to be produced (Jones et al., 2004). In many respects, wound contraction is beneficial as it can significantly reduce healing time because less granulation tissue needs to be produced to replace tissue loss (Calvin, 1998). In

view of the above, measurement of wound contraction is an important tool to ascertain the progress of cutaneous wound healing. In the present study, the significantly higher wound contraction in CCNC-treated rats can be attributed to the increased fibroblast proliferation, increased collagen synthesis and better epithelialization. Although wound area was also significantly

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decreased in chitosan-treated group, the effect was more pronounced in the CCNC-treated group, suggesting increased effect of CCNC in improving cutaneous wound healing with chitosan. In a recent study copper nanoparticles induced sharp decrease in the wound surface area on the first day after application in mice (Rakhmetova et al., 2010). Contraction data during 7–11 days showed that CCNC has role in the proliferative phase also. Various cells, cytokines and growth factors are involved in the different phases of healing. Copper directly or indirectly stimulates many factors and these factors are important for proliferation of keratinocytes and fibroblasts, epithelialization, collagen synthesis, extracellular matrix remodeling and angiogenesis, which result in accelerated wound healing (Borkow et al., 2010). Several studies have shown that chitosan also stimulates cell proliferation and histoarchitectural tissue organization (Jayakumar et al., 2011). Modulation of pro-inflammatory (IL-1, TNF-α, IL-6, etc.) and antiinflammatory (IL-4, IL-10, IL-13, etc.) is yet another important aspect in wound healing. To better understand the mechanisms by which CCNC induces enhanced wound healing, we analyzed various cytokines and growth factors as these can provide a better insight into their role in time-dependent healing. The chitosan-treated rats showed significant increase in TNF-α mRNA expression on third day, which indicates its ability to stimulate monocytes and macrophages to produce TNF-α in the initial phase of healing (Otterlei et al., 1994). The decrease in TNF-α on levels on 3rd, 7th and 11th day in the CCNC-treated rats implies its potential to decrease the inflammatory reaction. It appears that CCNC not only masked the inflammatory response of chitosan, but also caused marked decrease in expression of mRNA and protein of TNF-α on 3rd day post-wounding. Decrease of TNF-α production by copper has been reported in an earlier study also (Canapp et al., 2003). Studies have also reported that subcutaneous administration of copper complexes in animal experimental models showed reduced inflammation (Jackson et al., 2000). Influence of GHK, GGH and their copper complexes on inhibition of TNF-α -dependent IL-6 secretion in fibroblasts reported recently also suggest the anti-inflammatory action of copper (Gruchlik et al., 2012). The significant increase in the mRNA expression and protein levels of IL-10 on the third day in the CCNC-treated further supplement the anti-inflammatory effect of copper. IL-10 decreases the production of pro-inflammatory cytokines (TNF-α and IL-6) by down regulating MHC II (de Waal-Malefyt et al., 1991; Becherel et al., 1995). In agreement of our results copper application also has induced marked increase on the expression of IL-10 (Song et al., 2009). Thus, in the CCNC-treated rats the decreased TNF-α level might be due to the increased expression of IL-10. However, in the chitosantreated group the mRNA expression and protein levels of IL-10 decreased and this might be the reason for increased inflammatory response of chitosan. Formation of new blood vessels, which occurs through a combination of angiogenesis and vasculogenesis, plays a vital role in wound healing. During re-epithelialization phase of the wound healing process, proliferation and migration of keratinocytes increase over the wound bed (Haase et al., 2003). Studies have revealed that neovascularization which occurs during reepithelialization phase is associated with an enhanced expression of VEGF by migrating keratinocytes and with the up-regulation of VEGF receptors on dermal microvessels (Brown et al., 1992). Also the expression of VEGF, a major angiogenic stimulant, is sensitive to copper and the angiogenic potential of copper might be harnessed to accelerate dermal wound contraction and closure (Sen et al., 2002). Copper has been reported to enhance keratinocyte migration by modulation of integrin functions (Tenaud et al., 2000; Rafi et al., 2011). In the present study, the marked expression of VEGF mRNA and protein on 3rd day post-wounding in CCNC-treated rats might be one important cause of increased

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population of keratinocytes at the wound edges. Copper ions are also involved in the activity of several transcription factors (via HIF-1 and proline hydroxylase) and can bind to cell membrane releasing complex, facilitating release of VEGF and other growth factors from respective cells (Martin et al., 2005; Rajalingam et al., 2005; Feng et al., 2009). Thus, the enhanced expression of VEGF in CCNC-treated groups might be due to the angiogenic potential of copper. TGF-β is important in angiogenesis and wound healing (Baum and Arpey, 2005). TGF-β1 can be both inhibitory and stimulatory and is a potent chemo-attractant for monocytes, macrophages, lymphocytes, neutrophils and fibroblasts and stimulates the release of diverse cytokines (e.g. IL-l, IL-6, TNF-α and basic fibroblast growth factor (bFGF) from these cells (Roberts and Sporn, 1990). TGF-β1 auto induces its own expression and the expression of other TGF-β isoforms and thus, amplifying its effects. Studies have reported that TGF-β induces angiogenesis in vitro with a threshold pattern and this effect is secondary to its chemoattractant role (Fajardo et al., 1996). TGF-β1 is also an important regulator of the ECM, stimulating fibroplasia and collagen deposition, inhibiting ECM degrading proteases and up-regulating the synthesis of protease inhibitors. In the present study, CCNC-induced significant increase in the expression of mRNA and protein of TGF-β1 on 3rd and 7th day might be attributed to better wound healing by stimulating chemotaxis, angiogenesis and fibroblast proliferation. In an earlier study, copper oxide wound dressings increased the TGF-β1 levels by 33 fold (Borkow et al., 2010). Further, it has also been suggested that copper has skin regeneration potential through the stimulation of ECM proteins, TGF-β1, VEGF and inhibition of oxidative stress effects at physiological concentrations (Philips et al., 2012). Also chitosan mediated stimulation of macrophages can upregulate the TGF- β1 levels (Ueno et al., 2001). Thus the combination of chitosan with copper nanoparticles might have enhanced the TGF- β1 upregulatory effect of CCNC. Histological evaluation involves morphometric evaluation of inflammation, angiogenesis, fibroplasias, wound contraction, maturity of granulation tissue and re-epithelialization. In the present study, the more and less infiltration of inflammatory cells in chitosan- and CCNC-treated groups again strengthened the inflammatory and anti-inflammatory nature of chitosan and CCNC, respectively. The evidently early presence of fibroblasts in CCNCtreated group suggests that copper might stimulate migration and proliferation of fibroblasts. Fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. In comparison to other groups, the abundant presence of greenish thin collagen fibers in CCNC-treated group was found in the early days of healing and later on these fibers were also rapidly replaced by compact, parallel arranged thick fibers. Prolyl 4hydroxylases and lysyl oxidase which are copper dependent enzymes, significantly contribute in collagen synthesis. Further, collagen synthesis and deposition might have been indirectly enhanced due to an efficient vascularization in CCNC-treated rats. Moreover, as there was augmented expression of VEGF and TGF-β1 in the present study, so, the vascularization would also improve. Therefore, copper indirectly favours the collagen deposition via up-regulating these growth factors. Moreover, the TGF-β1 has pivotal role in collagen synthesis (Bastiaansen-Jenniskens et al., 2008). TGF-β family proteins are known to stimulate collagen and fibronectin formation in a variety of fibroblast cell lines (Roberts et al., 1986; Steed, 1997). The high expression of TGF-β1 by CCNC can stimulate the fibroblasts to produce a dense collagen network. Both keloids and hypertrophic scars express aberrantly elevated levels of TGF-β1 (Ladin et al., 1995). Therefore, decreased TGF-β1 in the final phase of wound repair can reduce scar formation and improve normal wound closure. Thus, decreased mRNA and

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protein expression of TGF-β1 in the final phase of healing in CCNCtreated group reveals the well organized healing in this group. Also, the complete well formed superficial epithelial layer in CCNC-treated group further substantiates the orderly and efficient healing. CD31 (PECAM-1), a member of the immunoglobulin superfamily, is a 130-kDa transmembrane glycoprotein is found on the surface of platelets, monocytes, neutrophils, and some types of T-cells, and makes up a large portion of intercellular junctions between endothelial cells. It is likely involved in leukocyte migration, angiogenesis, and integrin activation and used primarily to demonstrate the presence of endothelial cells in histological tissue sections. This has been recognized for its angiogenic role and can help to evaluate the degree of angiogenesis in the healing tissue (De Lisser et al., 1997; Matsumura et al., 1997; Zhou et al., 1999). In our study, CCNC treatment increased MVD on day 7 and 11 as compared to other groups. Copper seems to be necessary for endothelial cell activation as it stimulates their proliferation and migration. It stimulates proliferation of human endothelial cells directly in vitro (Hu, 1998) and it is also considered as a cofactor for many angiogenic mediators (Patstone and Maher, 1996; Simeon et al., 2000). Thus increased angiogenesis by CCNC treatment supports the rapid healing in CCNC-treated group compared to control and chitosan-treated groups. Chitosan group also showed better angiogenesis compared to control confirming angiogenic potential of chitosan also. The illustration in Fig. 8 depicts possible healing mechanism of CCNC.

5. Conclusions Thus, it is clear that CCNC involves in the regulation of multiple events that are central to the process of healing. From the present study, it can be concluded that incorporation of nanocopper in the chitosan forms an appropriate combination, which efficiently promote different phases of cutaneous wound healing by systematic modulation of different cytokines and growth factors on different days. It might be tried in chronic non-healing diabetic wounds and ulcers, as it possesses pro-angiogenic potential.

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