Application of a PDGF-containing novel gel for cutaneous wound healing

Life Sciences 87 (2010) 1–8

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Application of a PDGF-containing novel gel for cutaneous wound healing R. Judith a, M. Nithya a, C. Rose a,⁎, A.B. Mandal b a b

Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai – 600 020, Tamil Nadu, India Chemical Science Division, Central Leather Research Institute, Adyar, Chennai – 600 020, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 16 December 2009 Accepted 29 April 2010 Keywords: Collagen–chitosan–PDGF Wound healing Monomeric collagen Provisional matrix Biomimetic Antioxidants

a b s t r a c t Aim: The objective of this study was to develop an acellular provisional matrix to mimic the extracellular matrix (ECM) of a wound site, and to evaluate its potential for in vivo dermal regeneration. Key points: This study evaluates the potential of a monomeric collagen–chitosan gel containing plateletderived growth factor to enhance healing and reduce the time of wound closure. Main methods: In the present study, we developed a novel composite consisting of the combination of collagen, chitosan and platelet-derived growth factor (PDGF). This biodegradable Molecular gel matrix was used as a biomimetic surface for cell attachment to promote healing of excision wounds (1.5 cm long × 1.5 cm width). The rate of wound contraction and the concentrations of collagen, hexosamine, uronic acid and growth factors in the granulation tissue were determined. The amount of antioxidants and lipid peroxide in the treated tissues was elevated compared to the control tissue; in particular, tissue from the group treated with the collagen–chitosan–PDGF matrix had remarkable increase in these parameters. Additionally, the histology and ultrastructure of re-epithelialized tissues at the wound site were recorded. Significance: This study reveals that the monomeric nature of biological molecules in association with PDGF exerted simultaneous biomimetic and chemotactic effects and promoted wound healing. © 2010 Elsevier Inc. All rights reserved.

Introduction The repair of injured tissue occurs in an overlapping sequence of events, which involves the inflammation, proliferation and migration of different cell types (Sidhu et al. 1999). The extent to which these events occur depends upon the production of an amorphous gel-like extracellular matrix (ECM) termed the provisional matrix. This matrix contributes to the formation of granulation tissue by producing a scaffold or conduit for the migration and activation of fibroblasts. Endogenous ECM is composed of collagen nanofibers that vary in diameter (50–500 nm) (Chen and Peter 2004). It is beneficial to mimic the size of endogenous ECM to influence the morphology and function of cells on the matrix scaffold (Teixeira et al. 2003), which is composed of synthetic and natural polymers. Among the natural polymers, chitosan is of great interest because it has optimal physical, biological and biodegradable properties and is used in wound dressings (Ueno et al. 2001.). Currently, chitosan is being researched for potential use in medical and pharmaceutical applications. This is because it has appealing intrinsic properties. Indeed, chitosan is known for its biocompatibility and is used for topical ocular tissue applications (Felt et al. 1999), tissue implantation (Patashnik et al. 1997) and tissue injection (Song et al. 2001). Moreover, chitosan is metabolized by human enzymes, such as lysozyme, and is biodegrad⁎ Corresponding author. Department of Biotechnology Central Leather Research Institute, Adyar, Chennai – 600 020 India. Tel.: +91 44 24430273; fax: +91 24911589. E-mail address: [email protected] (C. Rose). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.05.003

able (Muzarelli 1997). In addition, chitosan acts as a penetration enhancer by opening epithelial tight junctions (Kotze et al. 1999). Due to a positive charge at physiological pH, chitosan is also bioadhesive, which increases its retention at the site of application (He et al. 1998; Calvo et al. 1997). Finally, chitosan is profuse in nature, and its production is of low cost and is ecologically friendly. Collagen is another well-established compatible biopolymer that is used in wound healing. Collagen is a potentially useful biomaterial since it is a major constituent of connective tissue. It has been processed into a variety of physical forms, including mini-pellets, tablets and nanoparticles, for use as a drug and gene delivery vehicle, injectable suspension for soft tissue augmentation, matrices for wound dressings and as a construct for engineered blood vessel, heart valve, tendon and skin substitutes (Lee et al. 2001; Huang et al. 2001; Fratzl 2003; Nithya et al. 2003). Several other laboratories have studied the role of collagen in the process of wound healing (Kato and Silver 1990; Pachence et al. 1987; Grant et al. 2001). In addition to collagen or chitosan, the role of growth factors in the wound healing process has received considerable attention. In vitro studies have demonstrated that polypeptide growth factors promote cell proliferation, cell differentiation, motility and matrix synthesis by binding to cell surface mediators. Inflammatory cells, keratinocytes and fibroblasts in the wound area release a variety of growth factors, such as nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF) and platelet-derived growth factor (PDGF). PDGF is an important biochemical mediator of wound healing and is released by platelets,

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macrophages, endothelial cells and fibroblasts (Heldin and Westermark 1990). Among these cells, platelets are known to be a rich source of PDGF, and several investigators have used platelets for healing wounds (Carter et al. 2003; Crovetti et al. 2004). Since PDGF is known to improve dermal regeneration, promote local protein and collagen synthesis and cause endothelial migration or angiogenesis, we incorporated PDGF into a collagen–chitosan system and assessed the effects of these biomolecules on the wound healing process.

Preparation of PDGF–containing collagen–chitosan gel PDGF (0.2 µg) was dissolved in 1 ml of 10 mM acetic acid. The stock solution was transferred into plastic vials and stored at −20 °C. A PDGF–containing collagen–chitosan gel was prepared by mixing 100 mg collagen and 100 mg chitosan with 5 µl of a 10 mM acetic acid solution that contained 1.25 ng PDGF. Wound assessment by planimetry

Materials and methods Animals Female albino rats were purchased from Sri Ramachandra Medical College and Hospital, Chennai, India. Animals were maintained individually in metabolic cages under hygienic conditions, and they were fed with commercial balanced diet and water ad libitum. All of the animal protocols used in this study were approved by the Institutional Animal Ethics Committee (Reg. No. 466/ 01/a/CPCSEA). Chemicals Thiobarbituric acid (TBA) [T5500], catalase (CAT) [C3556], glutathione reductase [G3664], 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) [D8130], glutathione (GSH) [G4251], 3, 3′-diaminobenzidine (DAB) [D4418], bathophenanthroline [133159], platelet-derived growth factor (PDGF) [P3201], monoclonal anti-PDGF antibody purified from mouse [P6101], nerve growth factor (NGF) [N2513], monoclonal anti-NGF antibody purified from mouse [N3279] and bovine serum albumin (BSA) [A964] were purchased from Sigma (St. Louis, MO, USA). Secondary antibodies and other fine chemicals were of analytical grade and were obtained from chemical companies. Wound creation and treatment Fur on the back of rats was shaved while the animals were under mild ether anesthesia. Skin tissue measuring 1.5 cm long × 1.5 cm width was removed surgically to create an excision wound, on the back using sterile surgical tools. The wound of the animals were treated for 7 days according to the treatment protocol given in Table 1, and the wound healing was monitored. Extraction and preparation of collagen A 2% (w/v) solution of collagen from marine catfish (Rose et al. 1988) was prepared using 0.05 M acetic acid and subjected to gamma irradiation (1000 rad for 10 min) to suppress antigenicity and to sterilize. Preparation of chitosan samples One hundred milligrams of chitosan was prepared from crab shell chitin according to the method of Okafor (1965). This was dissolved in 5 ml of 0.05 M acetic acid and subjected to gamma irradiation (1000 rad) for 10 min to suppress antigenicity and to sterilize. Table 1 Wound treatment protocol. Group

Treatment

Group I (control)

0.1 M phosphate buffer saline (pH 7.4) was topically applied to the wounds once a day. Wounds were treated with PDGF-incorporated chitosan at a concentration of 1.25 ng/1.5 mg/cm2 once a day. Wounds were treated with PDGF–incorporated collagen– chitosan at a concentration of 1.25 ng/1.5 mg/cm2 once a day.

Group II (chitosan– PDGF) Group III (collagen– chitosan–PDGF)

The size of the wound in the control and experimental rats was measured periodically using a transparent graph sheet, and the rate of epithelialization was calculated and is expressed as percent contraction (Morgan et al. 1994). Biochemical analysis The concentration of collagen and hexosamine in the granulation tissues was estimated using the methods of Woessner (1961) and Elson and Morgan (1933), respectively. Measurement of uronic acid in the tissues was performed according to the method of Schiller et al. (1961) and analyzed by the method of Bitter and Muir (1962). Protein concentration was measured using the method of Lowry et al. (1951). Determination of skin lipid peroxides (TBARS) The concentration of lipid peroxidation products, such as malonaldehyde, was determined in skin tissues by thiobarbituric acid (TBA) reaction as described by Draper and Hadley (1990). Briefly, 2 ml of supernatant was added to a 0.5 ml of the skin homogenate and mixed with 2 ml TCA. After mixing, the solution was centrifuged at 4000 rpm for 20 min. Two milliliters of the resulting supernatant was mixed with 2 ml of the TBA reagent. The reagent blank and standards (5–20 nmol) were treated similarly. The contents were then heated for 20 min in a boiling water bath and the absorbance was measured at 532 nm after the tubes had cooled to room temperature. Lipid peroxide content is expressed as nanomoles of MDA/mg protein. Measurement of the concentration of enzymatic and non-enzymatic antioxidants in skin The concentration of reduced glutathione was determined by the method of Moron (1979). One milliliter of supernatant from the skin homogenate was precipitated with 5% TCA and centrifuged at 10,000 rpm or 10 min. One-half milliliter of supernatant was then added to 0.5 ml of phosphate buffer (pH 8.0). Dithiobis-nitrobenzoic acid (DTNB) reagent (2.0 ml) was then added. Absorbance was measured at 412 nm using a Shimadzu UV-160A UV–visible spectrophotometer. A series of standards were similarly measured. The amount of glutathione is expressed as μg/mg protein. Ascorbic acid concentration was measured by the method of Omaye et al. (1971). Aliquots of the supernatant from the tissue homogenate were taken, precipitated by adding ice-cold TCA and centrifuged for 20 min at 3500 ×g. Dinitrophenyl hydrazine–thiourea–copper sulphate (DTC) reagent, 0.2 ml, was added to 1.0 ml of the supernatant solution and incubated for 3 h at 37 °C. 1.5 ml icecold 65% H2SO4 was added, mixed well and allowed to incubate at room temperature for 30 min. Absorbance was measured at 520 nm. Samples containing ascorbic acid were similarly prepared. The amount of ascorbic acid is expressed as μg/mg protein. The method of Beers and Seizer (1952) was used to determine catalase activity (CAT). Three milliliters of reaction mixture containing 1.9 ml phosphate buffer (0.05 M, pH 7.0) and 1.0 ml hydrogen peroxide (5.0 mM) was added to 0.1 ml diluted enzyme (skin homogenate). Activity was measured as the change in OD at 240 nm over 3 min at 30 s intervals.

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Superoxide dismutase (SOD) activity was measured according to the method of Misra and Fridovich (1972). This method is based on the SOD-dependent inhibition of adrenochrome production by epinephrine. Adrenochrome is produced in a reaction mixture containing 0.2 ml EDTA (0.6 mM), 0.4 ml sodium carbonate (0.25 M) and 0.2 ml epinephrine (3.0 mM) at a final volume of 2.5 ml. Adrenochrome absorbance was measured at 470 nm. The production of adrenochrome from epinephrine was inhibited by the addition of the SOD. The activity of glutathione peroxidase (GPX) was determined using the method of Rotruck et al. (1973). The reaction mixture contained 0.4 ml buffer (pH 7.0), 0.1 ml sodium azide and 0.2 ml reduced glutathione (0.1 ml H2O2 and distilled water were added to a final volume of 2.0 ml). Enzyme was added and the reaction was incubated at 37 °C for 3 min. The reaction was quenched by the addition of TCA. To determine the residual glutathione content, the supernatant was removed by centrifugation and 3.0 ml disodium hydrogen phosphate and 1.0 ml DTNB reagent was added. Absorbance was measured at 412 nm in a Shimadzu UV-160A UV–visible spectrophotometer. The blank consisted of disodium hydrogen phosphate and 1.0 ml DTNB reagent. In addition, aliquots of standards were taken and treated similarly. GPX activity is expressed as μg of glutathione consumed/min/mg protein.

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Determination of PDGF and NGF by ELISA The PDGF concentration in skin tissue samples was determined using a monoclonal human anti-PDGF antibody according to the method of Harrison and Cramer (1994). Briefly, protein-binding, sterile, polystyrene microtiter plates were coated with 50 mM carbonate buffer (pH 9.6) containing 2 μg monoclonal anti-PDGF IgG (100 μl/well) and incubated overnight at 4 °C. The non-specific binding of antibodies was blocked with 1% fetal calf serum for 1 h at 4 °C. Each well was then incubated for 12 h at 4 °C with 100 μl of sera samples. Additionally, coated wells without PDGF were used in each assay. Monoclonal anti-PDGF antibodies (2 μg) in 100 μl PBS containing 1% fetal calf serum and 0.05% Tween-20 was added (100 μl per well) and incubated for 12 h at 4 °C. This was followed by incubation with 100 μl of an affinity purified biotinylated goat anti-rat IgG in PBS containing 1% fetal calf serum for 12 h at 4 °C. The antibody complex was quantified using 100 μl of a peroxidase-conjugated streptavidin solution in PBS containing 1% fetal calf serum. Finally, a substrate solution containing 0.4% o-phenylene diamine dihydrochloride in 50 mM citrate-phosphate buffer and 0.03% H2O2 was added and incubated for 30 min. The optical density was read at 492 nm using an ELISA reader (Kontron SLT 210). Similarly, the NGF concentration was determined using a human anti-NGF antibody according to the method Matsuda et al. (1998).

Histolopathology Tissue from the wound site of individual rats was removed immediately after animal euthanization. These specimens were fixed in 10% formalin, dehydrated by a graded alcohol series, cleared in xylene and embedded in paraffin wax (melting point = 56 °C). Serial 5-μm sections were cut and stained with Haematoxylin and Eosin (H&E). The sections were examined under a light microscope.

Statistical analysis Data are expressed as the mean ± S.D. A Student's t-test was performed in order to compare the mean values of the various parameters between the experimental and control groups. Results

Transmission electron microscopy (TEM) Tissue epithelialization and the rate of wound contraction The healed skin of both the control and experimental rats were taken and fixed in a 3% glutaraldehyde solution, which was buffered with a 0.1 M sodium cacodylate solution (pH 8.0) and that contained 0.2 M sucrose. Post fixation was then conducted using 1% osmium tetroxide in the same buffer for 2 h at 4 °C. After dehydration in ethanol, embedding was performed in an Epon 812-based mixture. Ultra-thin sections were post-stained with 2% aqueous uranyl acetate and lead citrate. Sections were examined by a Phillips 201C electron microscope at 60 KV and photographed.

Macroscopic evaluation of the wounds revealed that collagen– chitosan–PDGF (CCP)-treated groups required a period of only 10 days for complete epithelialization to occur. In the control- and collagen–PDGF-treated animals complete healing was achieved after 20 and 14 days, respectively. The rate of healing was faster and more consistent in animals that received CCP treatment. Periodical observation of the wounds during the healing process determined that the greatest amount of wound contraction was in the CCP-treated animals. The chitosan–PDGF-treated wounds did undergo contraction

Immunohistochemical examination Samples of re-epithelialized tissue and the unwounded tissue were gently flattened on a piece of thick filter paper and were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 h at 4 °C. Tissues were then embedded in paraffin and 4-μm sections were cut. The sections were placed on gelatin-coated glass slides. Tissues were then deparaffined, rehydrated and washed in PBS (pH 7.4). After pretreatment with PBS supplemented with 0.3% hydrogen peroxide and 0.1% sodium azide for 10 min (to inhibit endogenous peroxidases), the preparations were washed in PBS twice and incubated with blocking medium (10% normal goat serum in PBS) for 10 min. Monoclonal anti-PDGF antibodies diluted 1:4000 in PBS supplemented with 1% BSA were applied to the tissues and incubated overnight at 4 °C. After two washes with PBS, the tissue samples were incubated with peroxidase-conjugated goat anti-rabbit IgG antibodies diluted 1:100 in PBS for 30 min. Visualization of the reaction products was performed with 0.2 mg/ml 3′,3′-diaminobenzidine tetrahydrochloride in PBS supplemented with 0.003% hydrogen peroxide. The tissues were then counterstained with Haematoxylin and Eosin after immunoreaction.

Fig. 1. Rate of wound contraction as percentage of original wound size in the tissue of control and experimental rats.

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but to a lesser extent. The rate of wound contraction in the control animals was much lower compared to the treated animals (Fig. 1).

Biochemical parameters Figs. 2–4 depict the amount of collagen, hexosamine and uronic acid formed in the granulation tissues of the control and experimental rats after the excision. The collagen content in group III was significantly (p b 0.001) higher on day 4 than that of group II. However, on day 8, the collagen levels did not significantly differ between the treated groups. This clearly indicates that the incorporation of PDGF into the topically applied matrix resulted in inflammation and increased collagen production. At the end of day 4, the collagen concentration increased by 2.6- and 3.2-fold in groups II and III, respectively, compared to the control group. The hexosamine content (Fig. 3) in the granulation tissues gradually decreased from day 4 to day 10, and the opposite trend was observed for collagen synthesis. The rate of decrease in the ground substratum component was much faster in group III, indicating enhancement of collagen remodeling. The uronic acid concentration in the experimental groups increased slightly until day 8. After day 8 the concentration decreased in parallel with the collagen concentration. However, this decrease was not observed in the control animals. The protein concentration was higher in CCPtreated animals in comparison to the other groups (Fig. 5).

Histology Fig. 6a shows the histology of tissue from the control group. In the control group, collagen fibers were loosely packed in an irregular arrangement and undifferentiated keratinocytes were observed under the basal lamina layer. Incomplete epithelialization from the active fibroblasts was observed. Fig. 6b shows the histology of wound tissue from chitosan–PDGF-treated animals. On day 10 after excision, we observed a newly formed epidermis consisting of a stratified squamous epithelial cells and loose connective stroma tissue, which was rich in capillaries. The CCP-treated animals showed a uniform accumulation of collagen fibers in the wound region with prominent thick bundles consisting of a moderate number of fibroblasts. The epidermal layer was continuous, consisting of a well-defined epithelium, and no inflammatory cells were observed in the wound site. The structures of the hair follicles, sudoriferous glands and sebaceous glands were normal. In this group, the healing process was completed within 10 days (Fig. 6c).

Fig. 2. Collagen content in the granulation tissue of control and experimental rats (mg/ 100 mg dry weight).

Fig. 3. Hexosamine content in the granulation tissue of control and experimental rats (mg/100 mg dry weight).

Immunocytochemistry Differences in immunoreactivity at the wound site were observed in the tissue of the control rats and treated rats 10 days after excision (Fig. 7a–c). The rate of wound healing was associated with the number of active cells that produce PDGF. The wound site of the control group exhibited fewer PDGF immunopositive cells (+). In contrast, tissue from animals in group II had a moderate level of immunopositive cells (++). The newly formed epithelial tissue from animals in group III showed intense immunoreactivity (+++) using the human monoclonal anti-PDGF antibody. Electron microscopic studies Collagen The ultra-structural details were observed in the wounds from the control animals by electron microscopy (Fig. 8a). Similar to the histological results, these tissues showed loosely packed collagen fibers in an irregular arrangement. Tissue from the chitosan–PDGFtreated group showed an increase in collagen content compared to the control group. These collagen fibers aggregated into bundles (Fig. 8b). Tissue from the CCP-treated animals had mature collagen bundles, which were organized into a compact structure (Fig. 8c). Fibroblast Fibroblast cells in the epithelial tissues were examined using the TEM. Fig. 8d–f shows fibroblast ultrastructure in the epithelial tissues from groups I–III. Fibroblasts in the tissue from CCP-treated animals

Fig. 4. Uronic acid content in the granulation tissue of control and experimental rats (mg/100 mg dry weight).

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Fig. 5. Protein concentration in the tissue of control and experimental rats (mg/100 mg tissue).

were spindle shaped and elongated; the cell body was completely occupied by the nucleus (Fig. 8f). The fibroblasts of group II (Fig. 8e) were similar to those in group III and were unlike those in group I (Fig. 8d).

Fig. 7. Expression of PDGF in skin tissue of control and experimental rats measured by immunocytochemistry (×400). (a) Control tissue — poor immunoreaction (+), (b) chitosan-treated tissue — moderate immunoreaction to the PDGF monoclonal antibody (++), and (c) collagen–chitosan–PDGF-treated tissue — positive immunoreaction (+++).

Mast cell Numerous mast cells with an intact cell membrane were observed in tissue from the CCP-treated animals. These cells contained several homogenous electron-dense granules in the periphery of the nucleus, which had a normal morphology (Fig. 8i). Tissue from the chitosan– PDGF-treated animals contained cells with a single nucleus and dispersed electron-dense granules in the periphery of the cytoplasm. These cells had a similar architecture to the tissue from the CCPtreated animals (Fig. 8h). Tissue from control-treated animals possessed fewer dense granules and contained cytoplasmic vacuoles, which contained flocculent materials (Fig. 8g). Lipid peroxidation (LPO)

Fig. 6. Skin tissue 10 days after excision stained with hematoxylin-eosin (X 400). (a) Control tissue-minimal collagen formation and incomplete epithelialization, (b) chitosan–PDGF-treated tissue — densely packed collagen and incomplete epithelialization, and (c) collagen–chitosan–PDGF-treated tissue — organized collagen bundles with complete epithelialization.

Table 2 depicts the concentration of malondialdehyde (MDA), an end product of lipid peroxidation. The MDA concentration was relatively low in tissues from the treated groups compared to the control group. Within each group, the MDA concentration in tissue from wounded skin was higher than the normal skin due to tissue injury and inflammation.

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Fig. 8. Ultrastructure of the dermal wound (10,000×) showing the arrangement and distribution of collagen fibers, fibroblasts and mast cells, (a) control tissue with loosely packed collagen fibers, (b) chitosan–PDGF-treated tissue with a moderate amount of collagen fibers, (c) collagen–chitosan–PDGF-treated tissue with densely packed collagen fibers, (d) control tissue with inactive fibroblasts, (e) chitosan–PDGF-treated tissue with active fibroblasts, (f) collagen–chitosan–PDGF-treated tissue — wound showing active and healthy fibroblasts, (g) control tissue showing poor architecture, (h) chitosan–PDGF-treated tissue showing normal architecture, (i) collagen–chitosan–PDGF-treated tissue — wound showing normal mast cells with intact granules.

Antioxidants The amount of endogenous antioxidant and antioxidant enzymes in the skin tissues was determined (Table 3). An increase in the concentration of GSH, AA, CAT, SOD and GPX was observed in each of the treated groups and was greatest in the CCP-treated animals.

PDGF and NGF concentration in skin tissues The concentration of PDGF and NGF was measured in the epithelial tissue of the wound site 10 days after excision in order to understand the influence of exogenous PDGF on the cells that produce these growth factors. The PDGF and NGF concentrations are presented in Fig. 9. Tissue from treated animals had a higher concentration of growth factors than the tissue from the control animals, confirming the immunocytochemistry results (Fig. 7a–c). Though treatment of the wound ended on day 7, an increase in PDGF and NFG was observed Table 2 Concentration of TBARS in the tissue of normal and wounded skin (nmol MDA per mg protein). Groups

Normal skin

Wounded skin

Control Chitosan–PDGF Collagen–Chitosan–PDGF

2.63 ± 0.18 1.13 ± 0.13*** 0.81 ± 0.02***

2.81 ± 0.22 1.24 ± 0.02*** 0.86 ± 0.03***

Values are expressed as the mean ± S.D. of six animals. A Student's t-test was performed to compare the mean values of the various parameters between the experimental and control groups (**P b 0.01; ***P b 0.001).

Table 3 Concentration of antioxidant enzymes in the skin of control and experimental groups. Antioxidants

Normal skin

Wounded skin

Antioxidant concentrations (μg per mg protein) Glutathione Control Chitosan–PDGF Collagen–Chitosan–PDGF

2.62 ± 0.18 4.84 ± 0.39*** 6.56 ± 0.59***

2.68 ± 0.09 10.46 ± 1.23*** 11.21 ± 1.62***

Ascorbic acid Control Chitosan–PDGF Collagen–Chitosan–PDGF

2.01 ± 0.07 2.57 ± 2.57** 3.06 ± 0.13***

2.12 ± 0.09 2.62 ± 0.23 ** 3.49 ± 0.36***

Catalase Control Chitosan–PDGF Collagen–Chitosan–PDGF

2.02 ± 0.03 2.42 ± 0.22* 3.13 ± 0.26***

2.37 ± 0.04 2.58 ± 0.31** 3.61 ± 0.43***

SOD Control Chitosan–PDGF Collagen–Chitosan–PDG

1.46 ± 0.04 2.03 ± 0.11*** 1.83 ± 0.10***

1.51 ± 0.03 2.29 ± 0.18*** 2.52 ± 0.13***

GPX Control Chitosan–PDGF Collagen–Chitosan–PDG

1.54 ± 0.26 6.83 ± 0.89*** 9.43 ± 1.92***

2.96 ± 0.27 9.26 ± 1.34*** 11.43 ± 2.18***

Values are expressed as the mean ± S.D. for six animals. A Student's t-test was performed to compare the mean values of the various parameters between the experimental and control groups (*P b 0.05; **P b 0.01; ***P b 0.001).

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Fig. 9. PDGF and NGF concentrations as determined by ELISA. The values represent the average of triplicate samples (n = 3).

in the tissue of groups II and III, which received exogenous PDGF. These results are in agreement with the histology and wound contraction data. Discussion This study examines the effects of chitosan–PDGF and collagen– chitosan–PDGF on the wound repair process and provides information on the cellular and biochemical changes that take place during healing in a rat model. Wound epithelialization in CCP-treated animals was complete by 10 days after the excision. This took 20 days to occur in control animals, whereas the chitosan–PDGFtreated group took 14 days for complete healing of a 1.5 × 1.5 cm wound. The concentration of collagen in the epithelialized tissue indicates the effectiveness of wound healing (Grant et al. 2001; Pilcher et al. 1998). In general, the production of granulation tissue within 3–5 days of the excision overlaps with the inflammatory response. Fibroblasts conduct granularization by producing collagen. In the present study, monomeric collagen was used in the CCP-hydrated mix. Therefore, exogenous collagen was readily accepted by the wound surface as part of the provisional matrix. Likewise, the chitosan within the exogenous application consisted of a fully-solubilized gel. Combination of the two biocompatible biopolymers mimics the extracellular matrix. Therefore, this mixture should cause cellular wound healing activities to progress well ahead of the normal process. The free NH2 group of the solubilized chitosan interacts with functional groups on the cell surface and promotes chitosan migration across the wound surface. The CCP, which contains solubilized monomeric collagen and chitosan, readily binds fibronectin, a matrix component, as well as glycosaminoglycans. In the normal healing process, the ECM provides a scaffold upon which the fibroblasts migrate and synthesize collagen. In our study, exogenous collagen supplementation enabled faster migration of cells that are involved in cutaneous wound healing. Since the exogenous collagen is molecular in nature (Rose et al. 1988; Rose and Mandal 1996; Nithya et al. 2003) and supplants endogenous collagen in vivo, it readily integrates with the wound tissue and facilitates the attachment, migration and proliferation of cells on the wound site. Therefore, the use of collagen for treatment of cutaneous wounds, in addition to chitosan and PDGF, enhances the healing process in its early phases. Our findings strongly suggest that the application of PDGF along with collagen–chitosan promotes effective collagen biosynthesis. This is due to the fact that PDGF acts as a chemoattractant for fibroblasts and stimulates fibroblast proliferation as well as the synthesis of glycosaminoglycans (GAGs), proteoglycans and collagen (Martin et al. 1992). PDGF is also a chemotactic for inflammatory cells and influences endothelial vascular smooth muscle cells to stimulate angiogenesis. Therefore, PDGF treatment (1.25 ng in 1.7 mg of gel preparation per cm2 of wound) increases the migration and proliferation of fibroblasts and

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production of connective tissue protein, extracellular matrix and neovascularization during the wound healing process. This has been observed in a previous study (Martin et al. 1992). A significant increase in collagen content within the granulation tissue from experimental groups may be due to increased synthesis of collagen and may correlate with the effectiveness of wound healing (Pilcher et al. 1998). An increased hexosamine and uronic acid concentration (Figs. 3,4) during the early phase of wound healing indicates that PDGF, probably in combination with collagen, indirectly enhances the synthesis of ground substances by directly stimulating the fibroblast proliferation (Dunphy and Udupa 1955). Exogenous PDGF induces the chemotaxis and the production and release of endogenous growth factors at the wound site in tissue from group III (Fig. 9). During the process of PDGF-induced chemotaxis, activated inflammatory cells and fibroblasts stimulate the production of endogenous PDGF and NGF in the re-epithelialized tissue. Therefore, a small increase in NGF and a marked increase in the PDGF concentration occurred in the wounds that were exogenous PDGF in comparison to control tissue. It has been suggested that PDGF recruits additional macrophages into the wound, which produce endogenous growth factors, such as TGF-β, and increase collagen synthesis (Pierce et al. 1989). Exogenous PDGF treatment, in addition to collagen and/or chitosan, should stimulate NGF production in the same manner. In general, cutaneous wounds cause anatomical and functional damage of the peripheral sensory neurons within the skin. NGF stimulates neuron regeneration, an important step in the remodeling of wounded tissue (Matsuda et al. 1998), and promotes cutaneous wound healing (Matsuda et al. 1998). PDGF-induced NGF production was observed in this study. Increased PDGF level concentration was observed in tissues from groups II and III. This increase was observed even toward the end of healing stage, indicating that the number of healthy cells, such as macrophages, endothelial cells and fibroblasts, increased. This result is also reflected in the immunocytochemical results (Fig. 7b, c). Active fibroblasts were detected by histology and electron microscopy in tissue from the CCP-treated group, possibly underlying the increase in collagen synthesis at the wound site (Figs. 6 and 8). Importantly, the histological features of the epithelial tissue from the CCP-treated wounds confirm the accelerated epidermal differentiation and production of an organized, dense collagen matrix without a prolonged inflammatory response (Nigra et al. 1972). Lipid peroxidation (LPO) results from reactive oxygen species (ROS)induced oxidative stress, and the extent of LPO is indicated by the MDA concentration. Oxidative stress is induced by the production and release of ROS by inflammatory cells when they destroy the contaminating bacteria by phagocytosis in the early phase of inflammation (Gate et al. 1999). The MDA concentration was reduced in the CCP-treated group in comparison to the other groups, possibly due to the reduction of lipid peroxidation by collagen–PDGF. This may prevent the degeneration of the cell walls of mast cells and fibroblasts, which was observed by TEM. Fibroblasts, a connective tissue cell type, are responsible for collagen deposition, which is necessary to repair tissue (Ross 1969). The amount of mast cells is another indicator of wound healing. These cells release granules filled with enzymes, histamine and other active amines, molecules that indicate inflammation (Artuc et al. 1999). The active amines released from the mast cells cause surrounding vessels to become leaky and allow mononuclear cells to access the injury area (Fig. 8). Measurement of lipid peroxidation revealed that CCP treatment had significant antioxidant activity, which would prevent oxidative damage and promote the healing process. Conclusion In addition to enhancing the cellular activities by PDGF treatment, the wound bed preparation using CCP improved wound healing. Implementing a PDGF-containing wound bed preparation using CCP optimizes healthy tissue granulation and improves the efficiency of

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therapy. Ultimately, the time to wound closure is reduced. The CCP system could also be successfully implemented in non-healing wounds. Conflict of interest statement There was none declared.

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