Chitosan-alginate membranes accelerate wound healing ^ nio Moretti Andrade,2 Guilherme Ferreira Caetano,1 Marco Andrey Cipriani Frade,2 Thiago Anto 2 3 ^ 3 ~ Marcel Nani Leite, Cecilia Zorzi Bueno, Angela Maria Moraes, Joao Tadeu Ribeiro-Paes4 1
~ o Paulo (USP), Sa ~o Paulo, Biotecnology Post Graduate Program, Institute of Biomedical Sciences, University of Sa ~o Paulo, Brazil Sa 2 ~o Preto School of Medicine, University of Sa ~o Paulo Department of Internal Medicine, Division of Dermatology, Ribeira ~o Preto, Sa ~o Paulo, Brazil (USP), Ribeira 3 Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, State University of ~ o Paulo, Brazil Campinas, Campinas, Sa 4 ~o Paulo State University (UNESP), Assis, Sa ~ o Paulo, Brazil Department of Biology, Sa Received 27 March 2014; revised 27 June 2014; accepted 1 September 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33277 Abstract: The purpose of this study was to evaluate the efficacy of chitosan-alginate membrane to accelerate wound healing in experimental cutaneous wounds. Two wounds were performed in Wistar rats by punching (1.5 cm diameter), treated with membranes moistened with saline solution (CAM group) or with saline only (SL group). After 2, 7, 14, and 21 days of surgery, five rats of each group were euthanized and reepithelialization was evaluated. The wounds/scars were harvested for histological, flow cytometry, neutrophil infiltrate, and hydroxyproline analysis. CAM group presented higher inflammatory cells recruitment as compared to SL group on 2nd day. On the 7th day, CAM group showed higher CD11b1 level and lower of neutrophils than SL group. The CAM group presented higher
CD41 cells influx than SL group on 2nd day, but it decreased during the follow up and became lower on 14th and 21st days. Higher fibroplasia was noticed on days 7 and 14 as well as higher collagenesis on 21st in the CAM group in comparison to SL group. CAM group showed faster reepithelialization on 7th day than SL group, although similar in other days. In conclusion, chitosan-alginate membrane modulated the inflammatory phase, stimulated fibroplasia and collagenesis, accelerating C 2014 Wiley Periodicals, Inc. J wound healing process in rats. V Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: wound healing, chitosan, alginate, membrane, collagen
How to cite this article: Caetano GF, Frade MA, Andrade TA, Leite MN, Zorzi CB, Moraes AM, Ribeiro-Paes JT. 2014. Chitosanalginate membranes accelerate wound healing. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Wound healing is a dynamic process, directed towards restoring the structure of the injured tissue.1,2 It may be divided in four overlapping phases: inflammation, coagulation, tissue formation, and tissue remodeling.2,3 The process begins immediately after the injury with the release of platelet granules, important in the clotting cascade. Simultaneously, cytokines and growth factors are released, which are important in the recruitment of inflammatory cells to the wound, an essential cellular influx for local debridement (inflammatory phase), formation of granulation tissue (angiogenesis), proliferation of fibroblasts, formation of extracellular matrix and collagen, re-epithelialization, and finally tissue remodeling.4,5 About six million people around the world suffer with wound healing disorders and impaired wounds, experimenting decreased life quality and serious consequences.1 Studies about natural therapeutic healing substances of easy
access to the population have gained importance in recent years because of the long treatment time and the high medical care costs associated to the therapy of extensive or longterm wounds. Earlier studies have assessed some stimulators of the wound healing process.6,7 Chitosan and alginate are well known for their healing properties. Chitosan, a cationic polymer obtained from chitin, is biologically renewable, biodegradable, biocompatible, and non-antigenic. It is able to accelerate the wound healing process enhancing functions of inflammatory cells, macrophages, and fibroblasts. Alginate, an anionic hydrophilic polymer, is also biocompatible and biodegradable under normal physiological conditions. It is able to maintain a physiologically moist microenvironment that promotes the formation of granulation tissue.8–10 Complex structures simultaneously composed of chitosan and alginate offer the advantages of both materials and can be tailored for several biomedical applications, such as
Correspondence to: J.T. Ribeiro-Paes; e-mail: [email protected] Contract grant sponsors: CAPES, FAEPA-HCFMRP-USP, FAPESP
C 2014 WILEY PERIODICALS, INC. V
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FIGURE 1. (a) Chitosan-alginate membrane dressing. Surgical procedure: (b) animal in the ventral position to determine the wounds position; (c) animal in lateral position for surgical procedure on the dorsum cervical region; (d) Wounds and samples of skin tissue excised. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
wound dressings, forming a cross-linked polyelectrolyte complex (PEC).10,11 Compared with the isolated compounds, the PEC changes its tendency of swelling, improves their structural strength and mechanical stability. There has been considerable interest in the use of chitosan–alginate PECs as dressings for skin wounds and also in soft tissue engineering as scaffolds to provide adequate three-dimensional biological support that enables the migration, proliferation, and ideal organization of cells in the physiological microenvironment.7,12,13 Despite several reports in the literature focus the biological properties of isolated chitosan and alginate, there are yet few reports about the use of chitosan-alginate PECs as membranes in in vivo studies of cutaneous wound healing.14–16 Therefore, the objective of this study was to evaluate the efficacy of chitosan-alginate PEC membrane on cutaneous wound healing in Wistar rats.
MATERIAL AND METHODS
Chitosan-alginate membrane The membranes [Figure 1(a)] were prepared and characterized based on adaptations of the procedure described by Rodrigues et al.17 and Bueno and Moraes18 The main reagents used were chitosan from shrimp shells and low viscosity sodium alginate from Sigma-Aldrich (United States), calcium chloride dihydrate and sodium hydroxide from Merck (Germany) and glacial acetic acid from Synth (Brazil). The water used was deionized in a Milli-Q system. The chitosan used in this work had a deacetylation degree of 95.24%, measured by titration according to Jiang et al.,19 and a molecular weight of 1.26 3 106 g/ mol, determined by viscosimetry (using a capillary viscometer Ostwald-Cannon-Fenske, size 200) and using the
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constants of the Mark-Houwink-Sakurada equation from Canella; Garcia.20 The procedure of preparation consisted in the slow and controlled addition of 90 mL of a chitosan solution (at 1.0% w/v in acetic acid at 2.0% v/v) to 180 mL of a sodium alginate solution (at 0.5% w/v) at 200 mL/h, 500 rpm, and 25 C. The suspension was homogenized at 1000 rpm during 10 min. NaOH aqueous solution at 1M was added to the suspension to increase the pH to 5.3 and the same stirring rate was maintained for 10 min. Then, 3.6 mL of a 2% (w/ v) CaCl2 aqueous solution were added to crosslink the alginate carboxyl groups that were not bounded to the chitosan amino groups. The suspension was stirred for another 10 min. The final mixture was deaerated under vacuum for 120 min and transferred to polystyrene Petri dishes (15 cm in diameter). The membranes were obtained by casting at 37 C. After drying, the membranes were immersed in 150 mL of 2% (w/v) CaCl2 for 30 min for further crosslinking. Afterwards, the membranes were washed twice in 200 mL of deionized water (30 min), once in 250 mL of 10 mM Hepes buffer (30 min), and once in 500 mL of deionized water (30 min). Finally, the membranes were dried at 37 C for 6 h. Some important physicochemical characteristics of the membranes are summarized on Table I.
Animals Protocols involving the use of animals were performed in accordance with the ethical guidelines of the Brazilian College of Animal Experimentation (COBEA) and approved by the Ethics Committee on Animal Experimentation (CETEA), Ribeir~ao Preto School of Medicine, University of S~ao Paulo
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
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TABLE I. Physicochemical Characteristics of the ChitosanAlginate PEC Membranes Property Thickness of dry membranes (mm) Thickness of wet membranes (after 24 hours at 37 C in water) (mm) Absorption capacity of saline solution (after 24 hours at 37 C) (g/g) Stability (mass loss) in saline solution (after one week at 37 C) (%) Tensile strength (MPa) Elongation at break (%)
Value 6 Standard Deviation 72 6 8 723 6 109 35 6 5 28 6 3 22 6 4 3,2 6 0,4
(FMRP-USP), registry number 085/2011. A total of 40 adult male Wistar rats (180g) from the Central Animal Facility of the School of Medicine from Ribeir~ao Preto, SP, Brazil, Department of Clinical Medicine, were housed in collective cages for 7 days before surgical procedure. During the study, they were transferred to individual cages maintained at room temperature (22 C), in light controlled conditions (12 h–12 h light and dark cycles) and received a solid diet R (Tortuga, S~ and water supplemented with VitagoldV ao Paul, Brazil) ad libitum. Surgical procedure and groups The animals were anesthetized by intraperitoneal injection of a 2.5% solution of tri-bromoethanol (100 lL/10g; Sigma, USA). The back of the animals were shaved and, after asepsis with 70% ethanol, two circular full-thickness wounds were made on the dorsum cervical region with a sterile histological punch (1.5 cm diameter) with thickness comprising all the skin layers. The excised skin of each animal was saved for subsequent histological and biochemical assays, representing samples from the initial day (day 0) without treatment [Figure 1(b-d)]. The groups, with 20 animals each one, were separated according to the wound treatment: in the CAM group, both wounds in each animal were treated with chitosan-alginate membranes (circular shape with 2.0 cm diameter) previously moistened with 0.9% saline solution; in the SL group, the wounds were treated only with 0.9% saline solution (150 mL each one). The wounds of all animals of both groups were covered with gauze and tape changed daily after hydration with 0.9% saline solution (100 mL) until the 7th day of follow up. The chitosan-alginate membranes were removed only after animal euthanasia. Harvesting of the material for study Five animals of each group were euthanized (n 5 10 wounds) on the 2nd, 7th, 14th, and 21st post-operative days with an overdose of anesthetic. Both wounds of each rat were photographed separately with a digital camera (Sony DSC-W320) on basic mode (no flash, no zoom) using a metal apparatus where the camera was fixed 30 cm from the base composed by two rulers to evaluate the wound healing rate.19,20 Circular samples of wounds/scars were
collected from the animals using the same punch (1.5 cm diameter) on the 2nd, 7th, 14th, and 21st post-operative days. Samples of each animal were collected for histological study, biochemical assays (myeloperoxidase, hydroxyproline) and flow cytometry. Determination of wound healing rate The area of the wounds were calculated using the ImageJ software to analyze the re-epithelialization by the wound healing rate (WHR) according to Eq. (1).21,22 WHR5ðInitial area2Final areaÞ=Initial area
(1)
The initial area corresponds to the day of the surgery (day 0) and the final area corresponds to the day of euthanasia (day 2, 7, 14, or 21). Histological processing Samples stored in histological cassettes and fixed with buffered formaldehyde solution at 3.7% (pH 7.4) for 24 h were processed for histological analysis. The specimens were sectioned in 5.0 lm-thick histological slides and stained with Hematoxylin and Eosin (HE) for the evaluation of inflammatory infiltrate, blood vessels and fibroblasts. The histological slides used to the analysis of collagenesis were stained with Gomori’s trichrome. Histomorphometrical analysis of inflammatory infiltrate, angiogenesis, and fibroplasia by image analysis. Histological sections stained with HE were visualized in a R microscope equipped with a LEICA LEICA DM 4000BV R DFCV 280 camera (Leica Microsystems, Germany). The R Leica Appliimages were captured using the software LASV cation Suite. Eight fields per slide of each rat of both groups were analyzed at 400x magnification (four fields from epidermis-upper dermis and four fields of the lower dermis-hypodermis) for inflammatory infiltrate and fibroplasia quantification.23,24 Images of fields at 100x magnification in the epidermis-dermis-hypodermis direction were evaluated for angiogenesis quantification. The Cell Counter ImageJ software was used to count inflammatory cells, fibroblasts, and blood vessels and the data were reported as the average number of inflammatory cells, fibroblasts, and blood vessels of five samples of each sampling period to both groups. Histomorphometrical analysis of collagenesis by image analysis. Collagenesis was measured in histological sections stained with Gomori’s trichrome. Six images per slide were captured at 100x magnification (three fields from epidermis-upper dermis and three fields from lower dermishypodermis). The Colour Deconvolution ImageJ software was used to evaluate the percentage of blue staining (collagen) in the image area. This software recognizes the image colors and decomposes them in three basic colors: blue (collagen), red, and purple. The morphometric analysis, corresponding to the blue color, was measured as the percentage of the total pixels in each image using the “Threshold
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FIGURE 2. Evaluation of treated wounds. (a) Re-epithelialization of wounds treated with chitosan-alginate membrane (CAM) and saline (SL) by wound healing rate in 2, 7, 14 and 21 post-operative days. (b) Clinical follow up of both groups for 2, 7, 14 and 21 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
color” (ImageJ software), as described by Leite et al.25 Data were also reported as the pixel average of five samples of each sampling period to both groups. Myeloperoxidase assay The accumulation of polymorphonuclear cells (PMNs) in tissues, predominantly neutrophils, was measured through the myeloperoxidase (MPO) assay, as described by Souza et al.26 to evaluate the neutrophil infiltrate of the inflammatory phase of samples to both groups preserved in Eppendorf flasks with ice-cold buffer (0.1M NaCl, 20 mM NaPO4, 15 mM NaEDTA, pH 4.7) at 280 C. The absorbance was measured R 190 Absorbwith a 96-well microplate reader (SpectraMaxV ance Plate Reader, Molecular Devices) at 450 nm. The results were reported as the total number of PMN cells x 103/mg tissue by comparing to a standard curve. Flow cytometry The following types of cells from wound and scar samples were analyzed: CD41 (T helper lymphocytes), CD81 (cytotoxic T lymphocytes and NK cells) and CD11b1 (macrophages). The samples were cut and digested with collagenase I (GIBCO-Invitrogen) for 1 hour. Cells were incubated at room temperature with 2.0 lL of monoclonal antibodies directly conjugated to the fluorochrome fluorescein isothiocyanate (FITC) or phycoerythrin (PE) (eBioscience - San Diego, CA, USA). Double markings were used for the analysis of lymphocyte subpopulations. All incubations were performed in the dark to avoid loss of fluorescence. Samples were analyzed immediately on FACSort flow cytometer (BD Bioscience - San Diego, USA). About 10,000 events per sample were acquired and the analyses were performed using the CellQuest software (BD Bioscience San Diego, CA, USA).
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Hydroxyproline content (HyP) Collagenesis was also measured using a biochemical assay by determination of hydroxyproline, with minor modifications of the procedures related in the literature.27,28 Samples were incubated at 60 C in open micro tubes for 15 h to obtain the dry weight, cut using a Polytron tissue homogenizer with 6N hydrochloric acid solution (100 lL per milligram of dry tissue) and incubated for 4 hours at 130 C to promote acid hydrolysis. Afterwards, the pH was adjusted to 7.0 at room temperature. Hydroxyproline standard solutions were prepared at concentrations from 1.0 to 100 lg/ mL and 10 lL of standard and samples were transferred to the wells of a 96-well microplate. Then, 90 mL of 0.056M chloramine-T solution and 100 mL of Ehrlich’s reagent were added to each well. The absorbance values were measured at 550 nm and compared to those of the standard curve to determine the concentration of hydroxyproline in the samples. Statistical analysis Statistical variations between both groups at each sampling time were determined using the Student’s t-test, while variations in the same group during the days were analyzed using One-way ANOVA with a55%. Bonferroni test was used to identify statistically significant differences between the mean values determined for each group using the GraphPad Prism 5 software. RESULTS
Wound healing rate (WHR) Re-epithelialization was determined through the WHR, analyzing the wounds area calculated by the ImageJ software. Comparing re-epithelialization on the 2nd day, both groups
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
ORIGINAL RESEARCH REPORT
FIGURE 3. (a) Photomicrography of the wounded area of both groups during follow-up (cellularity in wound tissue) stained with hematoxylineosin. (b) Quantitative analysis of inflammatory infiltrate (histomorphometry) on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(CAM and SL) showed no evidence of difference. On the 7th day, CAM-treated wounds showed higher WHR as compared to SL group (p < 0.001). From the 14th day on, most wounds from both groups were completely healed, although the CAM group had presented better quality of scar tissue [Figure 2(a,b)]. An important fact observed was the transparent appearance of membranes, which facilitated the visualization of the healing process. While the wound healed, the edges of the membranes were peeled away the skin spontaneously until fall by themselves, when the wound was completely healed. Infection was not observed during follow-up in CAM group. Inflammatory phase Histologically, the analysis of the samples revealed significantly increasing of cellularity in the CAM group on 2nd and 7th days in all histological samples analyzed, even in the lowest layer, as compared to the SL group [Figure 3(a)]. Quantitative analysis of inflammatory infiltrate on the 2nd day showed that the CAM group presented higher cellularity than the SL group (p 5 0.013). On the 7th day, the CAM group reduced significantly the inflammatory infiltrate (p < 0.05) whereas in those of SL group the same reduction was not
observed, and the same cellularity level was maintained. After the 7th day, both groups had a constant reduction of the inflammatory infiltrate, as presented in Figure 3(b). The evaluation of neutrophil participation in the inflammatory process by myeloperoxidase assay showed high levels of this enzyme on the 2nd day for both groups. On 7th day, CAM-treated wounds showed an important significant reduction of neutrophil infiltration as compared to the 2nd
FIGURE 4. Tissue neutrophil accumulation determined by MPO levels (biochemical assay) on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days.
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FIGURE 6. Blood vessels histomorphometry of wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days.
Lesions of the CAM group showed a significantly higher stimulus to recruit CD11b1 macrophages on the 7th day (p < 0.05) when compared to its 2nd day and to the lesions of the SL group (p 5 0.037). After the 7th day, both groups had no difference regarding the recruitment of CD11b1 macrophages [Figure 5(c)].
Angiogenesis Quantitative analysis of blood vessels in the histological sections showed no difference between both groups during the follow-up period. However, on 7th day both groups presented significant increase of the number of blood vessels, characterized by granulation tissue (Figure 6), as also shown on Figure 3(a), remaining with high density on 14th and 21st days.
FIGURE 5. Tissue lymphocytes and macrophage accumulation determined by flow cytometry on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days. (a) T helper lymphocytes CD41; (b) T cytotoxic lymphocytes CD81; (c) Macrophages CD11b1.
day (p < 0.05) and also to the SL group (p 5 0.004). At day 14 and 21, both groups had no difference, decreasing the MPO levels (Figure 4). The distribution of other inflammatory cells was also evaluated by flow cytometry. CAM-treated wounds presented higher number of CD41 T helper lymphocytes subpopulation than the SL group on the 2nd day (p 5 0.001) and this stimulus decreased on day 7 (p < 0.05), becoming similar to the levels observed in the SL group. However, in the 14th and 21st days, SL presented an increasing of the CD41 cells as compared to CAM (p < 0.05) [Figure 5(a)]. On the 2nd day, the SL group presented more CD81 T cytotoxic lymphocytes than the CAM group (p 5 0.012). On day 7, both groups showed similar numbers. On the 14th day, the CD81 subpopulation cells reduced dramatically in both groups, increasing again in day 21 [Figure 5(b)], but with no evidence of statistical difference.
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Fibroblast proliferation and collagenesis Qualitative histological analysis of wounds/scars showed greater stimulation of fibroplasia on the 7th, 14th, and 21st days for both groups with a high degree of differentiation. On the 21st day, fibroblasts, particularly in the CAM group, were well-organized in the newly formed tissue, arranged in parallel [Figure 3(a)]. On the 7th day, both groups had significant proliferation of fibroblasts, but the CAM group showed higher number of fibroblasts than the wounds in the SL group (p 5 0.027) until the 14th day (p 5 0.008). On the 21st day, CAM reduced this stimulus (p < 0.05), becoming similar to the SL group
FIGURE 7. Quantitative analysis of fibroblast proliferation (histomorphometry) on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days.
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
ORIGINAL RESEARCH REPORT
FIGURE 8. New collagen accumulation on wound treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 postoperative days. (a) Photomicrography of the ulcerated area of both groups during follow-up stained with Gomori’s trichrome. (b) Collagen content measured by digital densitometry in percentage of area. (c) Determination of wound hydroxyproline content as an indicator of collagen levels in mg of hydroxyproline/mg of dry wound tissue. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
that remained with no significant fibroblast variation throughout the period from the 7th day on (Figure 7). The Gomori’s trichrome assay showed that the CAMtreated wounds had more organized and dense collagen fibers as compared to the lesions of the SL group [Figure 8(a)]. On the 21st day, the CAM group showed higher percentage of collagen in the histological slides than the SL group (p 5 0.021) as shown in Figure 8(b). The results of the hydroxyproline assay showed that both groups presented collagen increasing during the follow-up period. The lesions of the CAM group showed faster and higher
collagenesis than SL on 2nd day (p 5 0.004). On the other hand, on the 14th day, the SL group showed higher level of hydroxyproline than those of the CAM group (p 5 0.001), a finding that, interestingly, changed on day 21st when the wounds of the CAM group reached higher hydroxyproline level than those of the SL group (p 5 0.024), as presented in Figure 8(c). DISCUSSION
The wound healing rate of CAM group was higher at day 7 than that of the SL group [Figure 2(a)], as also described by
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Wang et al.29 for membranes prepared using a different procedure. Wound closure involves a complex orchestrated interaction of different cell types, mainly neutrophils, leukocytes, macrophages, fibroblasts, and keratinocytes.3,30 Thus, the rapid rate of wound closure observed in this study might be attributed to the increased proliferation and migration of the several types of cells into the injured tissue. Cellular recruitment to the injured tissue during the inflammatory phase is important to remove debris, dead cells, and necrotic tissue. Neutrophils are attracted to the wound by chemotactic gradients, releasing enzymes, such as myeloperoxidase for the digestion of cellular debris and contributing to the death of bacteria by phagocytosis. Our data showed an intense inflammatory infiltrate in the beginning of the healing process (day 2), higher in the CAM group than in the SL group. The recruitment of neutrophils was similar between both groups at the 2nd day according to the myeloperoxidase level. Therefore, these data suggest that the major inflammatory infiltrate observed in the CAMtreated wounds on 2nd day was possibly due to CD41 lymphocytes, considering the extremely reduced number of CD81 cells and similar CD11b1 cells in both groups. The role of these cells has not been elucidated yet, but their presence as important regulators of the complex series of events that lead to wound healing has been suggested.31 If the inflammatory stimulus is persistent, high concentrations of proteolytic enzymes are produced, which stimulates oxidative stress, impairing the healing process.3,32 On the 7th post-operative day, the significant reduction of inflammatory infiltrate in the CAM group followed by the decrease of neutrophils and CD41 cells might indicate that the chitosanalginate membrane better regulated the inflammatory stimulus as compared to the SL group. Moreover, chitosan-alginate membrane stimulated the recruitment of CD11b1 macrophages, an important cell type that play a crucial role in the healing process because it continues the action of neutrophils, act as regulatory cells, as growth factors reservoir and releasing several types of metalloproteinase and collagenase to break the remaining extracellular content, the transition to the next phase of healing.31,33 Although the inflammatory infiltrates were similar on the 7th day for both groups, the cell types present were not the same. The wounds in the SL group did not achieve such regulation, keeping up with high-dose neutrophils in the same period, what may explain the delayed healing activity of this group. The initial dense inflammatory infiltrate observed in the CAM group on the 2nd day seemed to have an important role to the debridement of the wound. The regulatory activity observed on the 7th day in the wounds treated with chitosan-alginate and the earlier appearance of macrophages in the CAM group might have contributed to the advanced stage of the wound healing process, corroborating with the literature that has showed that depolymerization of chitosan stimulates functional macrophage recruitment to the injured tissue.9 Although the wounds on 14th day were already reepithelialization to both treatments, there were differences in
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cellularity (CD41) and collagen accumulation, also and particularly on 21st day, once wounds of SL group showed higher number of CD41 cells than the wounds of CAM group. The wounds were still in an inflammatory state and these inflammatory cells can produce proteolytic enzymes, which stimulates oxidative stress, impairing the healing process in the dermis layer, hampering the tissue formation and tissue remodeling in SL group, different from CAM group as observed with the higher collagen accumulation. It was also noticed that on day 21st, the scar tissue quality was much better in the case of the CAM group lesions (Figure 2). The modulated inflammatory infiltrate in the CAM group was followed by proliferation and migration of fibroblasts, higher on the 7th and 14th days than in SL-treated group. These events are important to the tissue formation phase, new collagen synthesis and subsequent remodeling. Diegelmann et al.34 analyzed the effects of a subcutaneous implant of a polyvinyl alcohol sponge treated with chitosan in rats and demonstrated a regulating ability of chitosan in the healing process compared to implants without chitosan. The authors observed significant cellular reduction and great collagenesis in animals treated with chitosan from the 7th to the 14th days, while the control group showed larger cellularity during the whole period. Our study is partially consistent with these data, but the appearance of fibroblasts, which was not reported by Diegelmann et al.,34 would be responsible for higher collagenesis along the wound healing period. A large number of macrophages remained longer in the tissue of animals that received chitosan, suggesting a potent immune-protective role of these cells by modulating the migration activity,35 subsequent fibroplasia and epithelialization, resulting in rapid and effective healing process according to the stimulation observed by chitosan-alginate membrane. The importance of angiogenesis is related to the intense need of nutrients and oxygen during the healing process, mainly during the inflammatory phase, because cells are in a high-energy demand stage due to intense activity and cell mitosis, thus promoting granulation tissue formation.36,37 As expected, increased angiogenesis was observed on the 7th day, but no evidence of difference between the groups was observed. Although chitosan is able to enhance the formation of new blood vessels by stimulating the production of cytokines and growth factors38,39 that, in addition to fibroblasts proliferation, stimulates angiogenesis by activating endothelial cells, in the histological study reported by Dai et al.40 poor presence of blood vessels was detected in animals treated with a chitosan-alginate porous dressing (spongeshape). Our data, as well as reported by Dai et al.,40 no difference related to angiogenesis was detected by histological analysis. Therefore, other methods become necessary to be applied for a better study of angiogenesis. Collagen is the major structural component of granulation tissue, strengthening the extracellular matrix. The amino-acid proline is an important component of the
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
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collagen fiber and hydroxyproline is used as a biochemical marker for collagen tissue, as a positive indication of the progression of healing.41,42 The presence of organized collagen fibers observed in CAM-treated wounds added to the increased collagen content observed by both methods, hydroxyproline and Gomori’s trichrome assay, suggests the contribution of the chitosan–alginate PEC to fibroblasts proliferation and synthesis of extracellular matrix during wound healing, as also proposed by Dai et al.40 and Ara ujo et al.43 Collagenesis, assessed by HyP measurement, increased continuously until the end of the process in the CAM group in addition to the predominance of fibroblasts. However, a higher collagenesis was observed on the 14th post-operative day for the SL group. These results suggest stimulus at different times for collagen production. Further studies are needed on the types of collagens formed, since the scar quality seems to be better in the CAM group. Thus, the appearance of denser and more organized collagen fibers suggest an important role of the biomaterial in the wound bed at all stages of healing, corroborating the literature. According to Wang et al.,29 chitosan-alginate PEC can facilitate remodeling of the scar tissue, increasing the rate of collagen synthesis and compaction of the new fiber and the appearance of mature fibroblasts, as observed in the CAM group. Moreover, chitosan-alginate membrane has stimulated and regulated all phases of wound healing process and our data suggest the use of chitosan-alginate membrane for the treatment of skin ulcers beneficially influences all phases of wound healing. Furthermore, collagen synthesis and contraction of the wound modulated by the chitosan-alginate membrane might have resulted in the rapid injury closure.29,38,44 Although the proposed model does not consider chronic wounds, the data presented herein are very important in clinical practices to treat chronic ulcers when significant inflammatory stimulus is required to change the initial status of the ulcers from senescence to granulation tissue (angiogenesis, fibroplasia and collagenesis) and also to reepithelialization, resulting in faster healing with a better quality scar. The treatment of acute and chronic wounds is a pressing need in many facets of medicine. Traditional wound dressings (e.g., gauze) have provided a good barrier function, keeping the wound dry by allowing evaporation of wound exudates while preventing entry of pathogen into the wound.45 In contrast, modern dressings mainly based on alginate, provide a moist wound environment and facilitate wound healing,46 as observed in our data. Alginate dressings in the dry form absorb wound fluid to re-gel, and the gels can supply water to a dry wound, maintaining a physiologically moist microenvironment and minimizing bacterial infection at the wound site. These functions can also promote granulation tissue formation, rapid epithelialization, and healing.47 It is important to point, according to Wang et al.29 and Dai et al.,40 and also observed in the present work, there was adherence of gauze fibers on the new tissue of dry wounded area in SL group and it became difficult the dressing change process, resulting in loss of new tissue and minor injuries to the wound. This fact was not observed in CAM-treated wounds, since there was no need to change
the chitosan-alginate membrane with no adherence to the wound site, which completely isolated the injury from the gauze or external environment, an important advantage in the overall wound healing process. CONCLUSION
Our results showed that the chitosan-alginate membrane modulated the inflammatory phase, stimulated the proliferation of fibroblasts and also collagenesis, accelerating the cutaneous wound healing in the earlier phases and improving the quality of the scar tissue. Furthermore, the chitosanalginate membrane showed to be a non-adherent to wound, dispensing daily changes and avoiding secondary damage and injury on newly-formed tissue. In this context, the chitosan-alginate PEC as wound dressing is indeed a promising formulation for application as a new therapeutic option, a potentially interesting for the treatment of tissue injuries and impaired wounds. ACKNOWLEDGMENTS
The authors acknowledge Ms. Carolina Caliari for all effort in the flow cytometry assays, Dr. Marcio Fronza for all support in the biochemical assays, and Kleber Loureiro for all support in the histological methods. REFERENCES 1. Ranzato E, Martinotti S, Burlando B. Wound healing properties of jojoba liquid wax: An in vitro study. J Ethnopharmacol 2011;134: 443–449. 2. Menke MN, Menke NB, Boardman CH, Diegelmann RF. Biologic therapeutics and molecular profiling to optimize wound healing. Gynecol Oncol 2008;111:S87–S91. 3. Stroncek JD, Bell N, Reichert WM. Instructional powerpoint presentations for cutaneous wound healing and tissue response to sutures. J Biomed Mater Res A 2009;90:1230–1238. 4. Velnar T, Bailey T, Smrkolj V. The wound healing process: An overview of the cellular and molecular mechanisms. J Int Med Res 2009;37:1528–1542. 5. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature 2008;453:314–321. 6. Moura LIF, Dias AMA, Carvalho E, Sousa HC. Recent advances on the development of wound dressings for diabetic foot ulcer treatment—A review. Acta Biomater 2013;9:7093–7114. 7. Mulder G, Wallin K, Tenenhaus M. Regenerative materials that facilitate wound healing. Clin Plastic Surg 2012;39:249–267. 8. Hein S, Wang K, Stevens WF, Kjems J. Chitosan composites for biomedical applications: Status, challenges and perspectives. Mater Sci Technol 2008;24:1053–1061. 9. Paul W, Sharma CP. Chitosan and alginate wound dressings: A short review. Trends in Biomater Artif Org 2004;18:18–23. 10. Sæther HV, Holme HK, Maurstad G, Smidsrïd O, Stokke BT. Polyelectrolyte complex formation using alginate and chitosan. Carbohydr Polym 2008;74:813–821. 11. Li X, Xie H, Lin J, Xie W, Ma X. Characterization and biodegradation of chitosan–alginate polyelectrolyte complexes. Polym Degrad Stab 2009;94:1–6. 12. Lim SM, Song DK, Oh SH, Lee-Yoon DS. In vitro and in vivo degradation behavior of acetylated chitosan porous beads. J Biomater Sci Polym Ed 2008;19:453–466. 13. Clark RAF, Ghosh K, Tonnesen MG. Tissue engineering for cutaneous wounds. J Invest Dermatol 2007;127:1018–1029. 14. Pei HN, Chen XG, Li Y, Zhou HY. Characterization and ornidazole release in vitro of a novel composite film prepared with chitosan/ poly(vinyl alcohol)/alginate. J Biomed Mater Res A 2008;85:566–572. 15. Wang L, Khor E, Lim LY. Chitosan-alginate-CaCl2 system for membrane coat application. J Pharm Sci 2001;90:1134–1142.
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16. Takka S, Gurel A. Evaluation of chitosan/alginate beads using experimental design: Formulation and in vitro characterization. AAPS PharmSciTech 2010;11:460–466. 17. Rodrigues AP, Sanchez EMS, Costa AC, Moraes AM. The influence of preparation conditions on the characteristics of chitosanalginate dressings for skin lesions. J Appl Polym Sci 2008;109: 2703–2710. 18. Bueno CZ, Moraes AM. Development of porous lamellar chitosanalginate membranes: Effect of different surfactants on biomaterial properties. J Appl Polym Sci 2011;122:624–631. 19. Jiang X, Chen L, Zhong W. A new linear potentiometric titration method for the determination of deacetylation degree of chitosan. Carbohydr Polym 2003;54:457–463. ~o de quitosana por cro20. Canella AMNC, Garcia RB. Caracterizac¸a ~o em gel – influe ^ncia do me todo de prematografia de permeac¸a ~o e do solvente. Quımica Nova 2001;24:13–17. parac¸a 21. Santana LA, Alves JM, Andrade TAM, Kajiwara JK, Garcia SB, Gomes FG, Frade MAC. Clinical and immunohistopathological aspects of venous ulcers treatment by Low-Intensity Pulsed Ultrasound (LIPUS). Ultrasonics 2013;53:870–879. 22. Minatel DG, Frade MAC, Franca S, Enwemeka CS. Phototherapy promotes healing of chronic diabetic leg ulcers that failed to respond to other therapies. Lasers Surg Med 2009;41:433–441. 23. Andrade TA, Iyer A, Das PK, Foss NT, Garcia SB, Coutinho-Netto ~o-Jr AA, Frade MAC. The inflammatory stimulus of a natJ, Jorda ural latex biomembrane improves healing in mice. Brazil J Med Biol Res 2011;44:1036–1047. ~o 24. Gonc¸alves RO, Silva EM, Marques TC, Lopes-Filho GJ. Avaliac¸a geno na fascia transversalis e na histoquımica quantitativa do cola sculo reto abdominal em doentes com bainha anterior do mu rnia inguinal. Revista Cole gio Basileiro Cir 2003;3:286–292. he ~o-Junior AA, Andrade TAM, Masson DS, Frade 25. Leite SN, Jorda MAC. Experimental models of malnutrition and its effect on skin trophism. An Bras Dermatol 2011;86:681–8. 26. Souza DG, Cassali GD, Poole S, Teixeira MM. Effects of inhibition of PDE4 and TNF-alpha on local and remote injuries following ischaemia and reperfusion injury. Br J Pharmacol 2001;134:985– 994. 27. Reddy GK, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 1996;29:225– 229. 28. Reddy AS, Kalyani S, Kumar NS, Boddu VM, Krishnaiah A. Dehydration of 1,4-dioxane by pervaporation using crosslinked calcium alginate-chitosan blend membranes. Polym Bull 2008;61:779–790. 29. Wang L, Khor E, Wee A, Lim LY. Chitosan-alginate PEC membrane as wound dressing: assessment of incisional wound healing. J Biomed Mater Res 2002;63:610–618. 30. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008;16:585–601.
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31. Park JE, Barbul A. Understanding the role of immune regulation in wound healing. Am J Surg 2004;187:11S–16S. 32. Doughty DB, Sparks-Defriese B. Wound healing physiology. In: Bryant RA, Nix DP. Acute and Chronic Wounds. Current Management Concepts, 4th ed. Missouri: Elsevier Mosby; 2012. pp 63–82. 33. Guo S, Dipietro LA. Factor affecting wound healing. J Dental Res 2010;89:219–229. 34. Diegelmann RF, Dunn JD, Lindbald WJ, Cohen IK. Analysis of the effects of chitosan on inflammation, angiogenesis, fibroplasia, and collagen deposition in polyvinyl alcohol sponge implants in rat wounds. Wound Repair Regen 1996;4:48–52. 35. Kosaka T, Kaneko Y, Nakada Y, Matsuura A, Tanaka S. Effect of chitosan implantation on activation of canine macrophages and polymorphonuclear cells after surgical stress. J Veterinary Med Sci 1996;58:963–967. 36. Bates DO, Jones OP. The role of vascular endotelial growth fator in wound healing. Int J Low Extrem Wounds 2003;2:107–120. 37. Schreml S, Szeimies RM, Prantl L, Landthaler M, Babilas P. Wound healing in the 21st century. J Am Acad Dermatol 2010;63: 866–881. 38. Muzzarelli RAA. Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohydr Polym 2009;76:167– 182. 39. Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosan—A versatile semi-synthetic polymer in biomedical applications. Prog Polym Sci 2011;3:981–1014. 40. Dai M, Zheng X, Xu X, Kong X, Li X, Guo G, Luo F, Zhao X, Quanwei Y, Qian Z. Chitosan-Alginate Sponge: Preparation and application in curcumin delivery for dermal wound healing in rat. J Biomed Biotechnol 2009;2009:1–8. 41. Kokane DD, More RY, Kale MB, Nehete MN, Mehendale PC, Gadgoli CH. Evaluation of wound healing activity of root of Mimosa pudica. J Ethnopharmacol 2009;124:311–315. 42. Nayak BS, Kanhai J, Milne DM, Pereira LP, Swanston WH. Experimental evaluation of ethanolic extract of Carapa guianensis L. leaf for its wound healing activity using three wound models. Evid Based Complement Alternat Med (eCAM) 2011;2011:1–7. ~es A, Mosqueira VCF, Carneiro CM, jo LU, Grabe-Guimara 43. Arau Silva-Barcellos NM. Profile of wound healing process induced by allantoin. Acta Cir Bras 2010;25:460–466. 44. Khan TA, Peh KK. A preliminary investigation of chitosan film as dressing for punch biopsy wounds in rats. J Pharm Pharm Sci 2003;6:20–26. 45. Boateng JS, Matthews KH, Stevens HNE, Eccleston GM. Wound healing dressings and drug delivery systems: A review. J Pharm Sci 2008;97:2892–923. 46. Queen D, Orsted H, Sanada H, Sussman G. A dressing history. Int Wound J 2004;1:59–77. 47. Lee KY, Mooney DJ. Alginate: Properties and biomedical applications. Prog Polym Sci 2012;37:106–126.
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
~ o Paulo (USP), Sa ~o Paulo, Biotecnology Post Graduate Program, Institute of Biomedical Sciences, University of Sa ~o Paulo, Brazil Sa 2 ~o Preto School of Medicine, University of Sa ~o Paulo Department of Internal Medicine, Division of Dermatology, Ribeira ~o Preto, Sa ~o Paulo, Brazil (USP), Ribeira 3 Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, State University of ~ o Paulo, Brazil Campinas, Campinas, Sa 4 ~o Paulo State University (UNESP), Assis, Sa ~ o Paulo, Brazil Department of Biology, Sa Received 27 March 2014; revised 27 June 2014; accepted 1 September 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33277 Abstract: The purpose of this study was to evaluate the efficacy of chitosan-alginate membrane to accelerate wound healing in experimental cutaneous wounds. Two wounds were performed in Wistar rats by punching (1.5 cm diameter), treated with membranes moistened with saline solution (CAM group) or with saline only (SL group). After 2, 7, 14, and 21 days of surgery, five rats of each group were euthanized and reepithelialization was evaluated. The wounds/scars were harvested for histological, flow cytometry, neutrophil infiltrate, and hydroxyproline analysis. CAM group presented higher inflammatory cells recruitment as compared to SL group on 2nd day. On the 7th day, CAM group showed higher CD11b1 level and lower of neutrophils than SL group. The CAM group presented higher
CD41 cells influx than SL group on 2nd day, but it decreased during the follow up and became lower on 14th and 21st days. Higher fibroplasia was noticed on days 7 and 14 as well as higher collagenesis on 21st in the CAM group in comparison to SL group. CAM group showed faster reepithelialization on 7th day than SL group, although similar in other days. In conclusion, chitosan-alginate membrane modulated the inflammatory phase, stimulated fibroplasia and collagenesis, accelerating C 2014 Wiley Periodicals, Inc. J wound healing process in rats. V Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: wound healing, chitosan, alginate, membrane, collagen
How to cite this article: Caetano GF, Frade MA, Andrade TA, Leite MN, Zorzi CB, Moraes AM, Ribeiro-Paes JT. 2014. Chitosanalginate membranes accelerate wound healing. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Wound healing is a dynamic process, directed towards restoring the structure of the injured tissue.1,2 It may be divided in four overlapping phases: inflammation, coagulation, tissue formation, and tissue remodeling.2,3 The process begins immediately after the injury with the release of platelet granules, important in the clotting cascade. Simultaneously, cytokines and growth factors are released, which are important in the recruitment of inflammatory cells to the wound, an essential cellular influx for local debridement (inflammatory phase), formation of granulation tissue (angiogenesis), proliferation of fibroblasts, formation of extracellular matrix and collagen, re-epithelialization, and finally tissue remodeling.4,5 About six million people around the world suffer with wound healing disorders and impaired wounds, experimenting decreased life quality and serious consequences.1 Studies about natural therapeutic healing substances of easy
access to the population have gained importance in recent years because of the long treatment time and the high medical care costs associated to the therapy of extensive or longterm wounds. Earlier studies have assessed some stimulators of the wound healing process.6,7 Chitosan and alginate are well known for their healing properties. Chitosan, a cationic polymer obtained from chitin, is biologically renewable, biodegradable, biocompatible, and non-antigenic. It is able to accelerate the wound healing process enhancing functions of inflammatory cells, macrophages, and fibroblasts. Alginate, an anionic hydrophilic polymer, is also biocompatible and biodegradable under normal physiological conditions. It is able to maintain a physiologically moist microenvironment that promotes the formation of granulation tissue.8–10 Complex structures simultaneously composed of chitosan and alginate offer the advantages of both materials and can be tailored for several biomedical applications, such as
Correspondence to: J.T. Ribeiro-Paes; e-mail: [email protected] Contract grant sponsors: CAPES, FAEPA-HCFMRP-USP, FAPESP
C 2014 WILEY PERIODICALS, INC. V
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FIGURE 1. (a) Chitosan-alginate membrane dressing. Surgical procedure: (b) animal in the ventral position to determine the wounds position; (c) animal in lateral position for surgical procedure on the dorsum cervical region; (d) Wounds and samples of skin tissue excised. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
wound dressings, forming a cross-linked polyelectrolyte complex (PEC).10,11 Compared with the isolated compounds, the PEC changes its tendency of swelling, improves their structural strength and mechanical stability. There has been considerable interest in the use of chitosan–alginate PECs as dressings for skin wounds and also in soft tissue engineering as scaffolds to provide adequate three-dimensional biological support that enables the migration, proliferation, and ideal organization of cells in the physiological microenvironment.7,12,13 Despite several reports in the literature focus the biological properties of isolated chitosan and alginate, there are yet few reports about the use of chitosan-alginate PECs as membranes in in vivo studies of cutaneous wound healing.14–16 Therefore, the objective of this study was to evaluate the efficacy of chitosan-alginate PEC membrane on cutaneous wound healing in Wistar rats.
MATERIAL AND METHODS
Chitosan-alginate membrane The membranes [Figure 1(a)] were prepared and characterized based on adaptations of the procedure described by Rodrigues et al.17 and Bueno and Moraes18 The main reagents used were chitosan from shrimp shells and low viscosity sodium alginate from Sigma-Aldrich (United States), calcium chloride dihydrate and sodium hydroxide from Merck (Germany) and glacial acetic acid from Synth (Brazil). The water used was deionized in a Milli-Q system. The chitosan used in this work had a deacetylation degree of 95.24%, measured by titration according to Jiang et al.,19 and a molecular weight of 1.26 3 106 g/ mol, determined by viscosimetry (using a capillary viscometer Ostwald-Cannon-Fenske, size 200) and using the
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constants of the Mark-Houwink-Sakurada equation from Canella; Garcia.20 The procedure of preparation consisted in the slow and controlled addition of 90 mL of a chitosan solution (at 1.0% w/v in acetic acid at 2.0% v/v) to 180 mL of a sodium alginate solution (at 0.5% w/v) at 200 mL/h, 500 rpm, and 25 C. The suspension was homogenized at 1000 rpm during 10 min. NaOH aqueous solution at 1M was added to the suspension to increase the pH to 5.3 and the same stirring rate was maintained for 10 min. Then, 3.6 mL of a 2% (w/ v) CaCl2 aqueous solution were added to crosslink the alginate carboxyl groups that were not bounded to the chitosan amino groups. The suspension was stirred for another 10 min. The final mixture was deaerated under vacuum for 120 min and transferred to polystyrene Petri dishes (15 cm in diameter). The membranes were obtained by casting at 37 C. After drying, the membranes were immersed in 150 mL of 2% (w/v) CaCl2 for 30 min for further crosslinking. Afterwards, the membranes were washed twice in 200 mL of deionized water (30 min), once in 250 mL of 10 mM Hepes buffer (30 min), and once in 500 mL of deionized water (30 min). Finally, the membranes were dried at 37 C for 6 h. Some important physicochemical characteristics of the membranes are summarized on Table I.
Animals Protocols involving the use of animals were performed in accordance with the ethical guidelines of the Brazilian College of Animal Experimentation (COBEA) and approved by the Ethics Committee on Animal Experimentation (CETEA), Ribeir~ao Preto School of Medicine, University of S~ao Paulo
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
ORIGINAL RESEARCH REPORT
TABLE I. Physicochemical Characteristics of the ChitosanAlginate PEC Membranes Property Thickness of dry membranes (mm) Thickness of wet membranes (after 24 hours at 37 C in water) (mm) Absorption capacity of saline solution (after 24 hours at 37 C) (g/g) Stability (mass loss) in saline solution (after one week at 37 C) (%) Tensile strength (MPa) Elongation at break (%)
Value 6 Standard Deviation 72 6 8 723 6 109 35 6 5 28 6 3 22 6 4 3,2 6 0,4
(FMRP-USP), registry number 085/2011. A total of 40 adult male Wistar rats (180g) from the Central Animal Facility of the School of Medicine from Ribeir~ao Preto, SP, Brazil, Department of Clinical Medicine, were housed in collective cages for 7 days before surgical procedure. During the study, they were transferred to individual cages maintained at room temperature (22 C), in light controlled conditions (12 h–12 h light and dark cycles) and received a solid diet R (Tortuga, S~ and water supplemented with VitagoldV ao Paul, Brazil) ad libitum. Surgical procedure and groups The animals were anesthetized by intraperitoneal injection of a 2.5% solution of tri-bromoethanol (100 lL/10g; Sigma, USA). The back of the animals were shaved and, after asepsis with 70% ethanol, two circular full-thickness wounds were made on the dorsum cervical region with a sterile histological punch (1.5 cm diameter) with thickness comprising all the skin layers. The excised skin of each animal was saved for subsequent histological and biochemical assays, representing samples from the initial day (day 0) without treatment [Figure 1(b-d)]. The groups, with 20 animals each one, were separated according to the wound treatment: in the CAM group, both wounds in each animal were treated with chitosan-alginate membranes (circular shape with 2.0 cm diameter) previously moistened with 0.9% saline solution; in the SL group, the wounds were treated only with 0.9% saline solution (150 mL each one). The wounds of all animals of both groups were covered with gauze and tape changed daily after hydration with 0.9% saline solution (100 mL) until the 7th day of follow up. The chitosan-alginate membranes were removed only after animal euthanasia. Harvesting of the material for study Five animals of each group were euthanized (n 5 10 wounds) on the 2nd, 7th, 14th, and 21st post-operative days with an overdose of anesthetic. Both wounds of each rat were photographed separately with a digital camera (Sony DSC-W320) on basic mode (no flash, no zoom) using a metal apparatus where the camera was fixed 30 cm from the base composed by two rulers to evaluate the wound healing rate.19,20 Circular samples of wounds/scars were
collected from the animals using the same punch (1.5 cm diameter) on the 2nd, 7th, 14th, and 21st post-operative days. Samples of each animal were collected for histological study, biochemical assays (myeloperoxidase, hydroxyproline) and flow cytometry. Determination of wound healing rate The area of the wounds were calculated using the ImageJ software to analyze the re-epithelialization by the wound healing rate (WHR) according to Eq. (1).21,22 WHR5ðInitial area2Final areaÞ=Initial area
(1)
The initial area corresponds to the day of the surgery (day 0) and the final area corresponds to the day of euthanasia (day 2, 7, 14, or 21). Histological processing Samples stored in histological cassettes and fixed with buffered formaldehyde solution at 3.7% (pH 7.4) for 24 h were processed for histological analysis. The specimens were sectioned in 5.0 lm-thick histological slides and stained with Hematoxylin and Eosin (HE) for the evaluation of inflammatory infiltrate, blood vessels and fibroblasts. The histological slides used to the analysis of collagenesis were stained with Gomori’s trichrome. Histomorphometrical analysis of inflammatory infiltrate, angiogenesis, and fibroplasia by image analysis. Histological sections stained with HE were visualized in a R microscope equipped with a LEICA LEICA DM 4000BV R DFCV 280 camera (Leica Microsystems, Germany). The R Leica Appliimages were captured using the software LASV cation Suite. Eight fields per slide of each rat of both groups were analyzed at 400x magnification (four fields from epidermis-upper dermis and four fields of the lower dermis-hypodermis) for inflammatory infiltrate and fibroplasia quantification.23,24 Images of fields at 100x magnification in the epidermis-dermis-hypodermis direction were evaluated for angiogenesis quantification. The Cell Counter ImageJ software was used to count inflammatory cells, fibroblasts, and blood vessels and the data were reported as the average number of inflammatory cells, fibroblasts, and blood vessels of five samples of each sampling period to both groups. Histomorphometrical analysis of collagenesis by image analysis. Collagenesis was measured in histological sections stained with Gomori’s trichrome. Six images per slide were captured at 100x magnification (three fields from epidermis-upper dermis and three fields from lower dermishypodermis). The Colour Deconvolution ImageJ software was used to evaluate the percentage of blue staining (collagen) in the image area. This software recognizes the image colors and decomposes them in three basic colors: blue (collagen), red, and purple. The morphometric analysis, corresponding to the blue color, was measured as the percentage of the total pixels in each image using the “Threshold
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FIGURE 2. Evaluation of treated wounds. (a) Re-epithelialization of wounds treated with chitosan-alginate membrane (CAM) and saline (SL) by wound healing rate in 2, 7, 14 and 21 post-operative days. (b) Clinical follow up of both groups for 2, 7, 14 and 21 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
color” (ImageJ software), as described by Leite et al.25 Data were also reported as the pixel average of five samples of each sampling period to both groups. Myeloperoxidase assay The accumulation of polymorphonuclear cells (PMNs) in tissues, predominantly neutrophils, was measured through the myeloperoxidase (MPO) assay, as described by Souza et al.26 to evaluate the neutrophil infiltrate of the inflammatory phase of samples to both groups preserved in Eppendorf flasks with ice-cold buffer (0.1M NaCl, 20 mM NaPO4, 15 mM NaEDTA, pH 4.7) at 280 C. The absorbance was measured R 190 Absorbwith a 96-well microplate reader (SpectraMaxV ance Plate Reader, Molecular Devices) at 450 nm. The results were reported as the total number of PMN cells x 103/mg tissue by comparing to a standard curve. Flow cytometry The following types of cells from wound and scar samples were analyzed: CD41 (T helper lymphocytes), CD81 (cytotoxic T lymphocytes and NK cells) and CD11b1 (macrophages). The samples were cut and digested with collagenase I (GIBCO-Invitrogen) for 1 hour. Cells were incubated at room temperature with 2.0 lL of monoclonal antibodies directly conjugated to the fluorochrome fluorescein isothiocyanate (FITC) or phycoerythrin (PE) (eBioscience - San Diego, CA, USA). Double markings were used for the analysis of lymphocyte subpopulations. All incubations were performed in the dark to avoid loss of fluorescence. Samples were analyzed immediately on FACSort flow cytometer (BD Bioscience - San Diego, USA). About 10,000 events per sample were acquired and the analyses were performed using the CellQuest software (BD Bioscience San Diego, CA, USA).
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Hydroxyproline content (HyP) Collagenesis was also measured using a biochemical assay by determination of hydroxyproline, with minor modifications of the procedures related in the literature.27,28 Samples were incubated at 60 C in open micro tubes for 15 h to obtain the dry weight, cut using a Polytron tissue homogenizer with 6N hydrochloric acid solution (100 lL per milligram of dry tissue) and incubated for 4 hours at 130 C to promote acid hydrolysis. Afterwards, the pH was adjusted to 7.0 at room temperature. Hydroxyproline standard solutions were prepared at concentrations from 1.0 to 100 lg/ mL and 10 lL of standard and samples were transferred to the wells of a 96-well microplate. Then, 90 mL of 0.056M chloramine-T solution and 100 mL of Ehrlich’s reagent were added to each well. The absorbance values were measured at 550 nm and compared to those of the standard curve to determine the concentration of hydroxyproline in the samples. Statistical analysis Statistical variations between both groups at each sampling time were determined using the Student’s t-test, while variations in the same group during the days were analyzed using One-way ANOVA with a55%. Bonferroni test was used to identify statistically significant differences between the mean values determined for each group using the GraphPad Prism 5 software. RESULTS
Wound healing rate (WHR) Re-epithelialization was determined through the WHR, analyzing the wounds area calculated by the ImageJ software. Comparing re-epithelialization on the 2nd day, both groups
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
ORIGINAL RESEARCH REPORT
FIGURE 3. (a) Photomicrography of the wounded area of both groups during follow-up (cellularity in wound tissue) stained with hematoxylineosin. (b) Quantitative analysis of inflammatory infiltrate (histomorphometry) on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(CAM and SL) showed no evidence of difference. On the 7th day, CAM-treated wounds showed higher WHR as compared to SL group (p < 0.001). From the 14th day on, most wounds from both groups were completely healed, although the CAM group had presented better quality of scar tissue [Figure 2(a,b)]. An important fact observed was the transparent appearance of membranes, which facilitated the visualization of the healing process. While the wound healed, the edges of the membranes were peeled away the skin spontaneously until fall by themselves, when the wound was completely healed. Infection was not observed during follow-up in CAM group. Inflammatory phase Histologically, the analysis of the samples revealed significantly increasing of cellularity in the CAM group on 2nd and 7th days in all histological samples analyzed, even in the lowest layer, as compared to the SL group [Figure 3(a)]. Quantitative analysis of inflammatory infiltrate on the 2nd day showed that the CAM group presented higher cellularity than the SL group (p 5 0.013). On the 7th day, the CAM group reduced significantly the inflammatory infiltrate (p < 0.05) whereas in those of SL group the same reduction was not
observed, and the same cellularity level was maintained. After the 7th day, both groups had a constant reduction of the inflammatory infiltrate, as presented in Figure 3(b). The evaluation of neutrophil participation in the inflammatory process by myeloperoxidase assay showed high levels of this enzyme on the 2nd day for both groups. On 7th day, CAM-treated wounds showed an important significant reduction of neutrophil infiltration as compared to the 2nd
FIGURE 4. Tissue neutrophil accumulation determined by MPO levels (biochemical assay) on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days.
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FIGURE 6. Blood vessels histomorphometry of wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days.
Lesions of the CAM group showed a significantly higher stimulus to recruit CD11b1 macrophages on the 7th day (p < 0.05) when compared to its 2nd day and to the lesions of the SL group (p 5 0.037). After the 7th day, both groups had no difference regarding the recruitment of CD11b1 macrophages [Figure 5(c)].
Angiogenesis Quantitative analysis of blood vessels in the histological sections showed no difference between both groups during the follow-up period. However, on 7th day both groups presented significant increase of the number of blood vessels, characterized by granulation tissue (Figure 6), as also shown on Figure 3(a), remaining with high density on 14th and 21st days.
FIGURE 5. Tissue lymphocytes and macrophage accumulation determined by flow cytometry on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days. (a) T helper lymphocytes CD41; (b) T cytotoxic lymphocytes CD81; (c) Macrophages CD11b1.
day (p < 0.05) and also to the SL group (p 5 0.004). At day 14 and 21, both groups had no difference, decreasing the MPO levels (Figure 4). The distribution of other inflammatory cells was also evaluated by flow cytometry. CAM-treated wounds presented higher number of CD41 T helper lymphocytes subpopulation than the SL group on the 2nd day (p 5 0.001) and this stimulus decreased on day 7 (p < 0.05), becoming similar to the levels observed in the SL group. However, in the 14th and 21st days, SL presented an increasing of the CD41 cells as compared to CAM (p < 0.05) [Figure 5(a)]. On the 2nd day, the SL group presented more CD81 T cytotoxic lymphocytes than the CAM group (p 5 0.012). On day 7, both groups showed similar numbers. On the 14th day, the CD81 subpopulation cells reduced dramatically in both groups, increasing again in day 21 [Figure 5(b)], but with no evidence of statistical difference.
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Fibroblast proliferation and collagenesis Qualitative histological analysis of wounds/scars showed greater stimulation of fibroplasia on the 7th, 14th, and 21st days for both groups with a high degree of differentiation. On the 21st day, fibroblasts, particularly in the CAM group, were well-organized in the newly formed tissue, arranged in parallel [Figure 3(a)]. On the 7th day, both groups had significant proliferation of fibroblasts, but the CAM group showed higher number of fibroblasts than the wounds in the SL group (p 5 0.027) until the 14th day (p 5 0.008). On the 21st day, CAM reduced this stimulus (p < 0.05), becoming similar to the SL group
FIGURE 7. Quantitative analysis of fibroblast proliferation (histomorphometry) on wounds treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 post-operative days.
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
ORIGINAL RESEARCH REPORT
FIGURE 8. New collagen accumulation on wound treated with chitosan-alginate membrane (CAM) and saline (SL) in 2, 7, 14, and 21 postoperative days. (a) Photomicrography of the ulcerated area of both groups during follow-up stained with Gomori’s trichrome. (b) Collagen content measured by digital densitometry in percentage of area. (c) Determination of wound hydroxyproline content as an indicator of collagen levels in mg of hydroxyproline/mg of dry wound tissue. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
that remained with no significant fibroblast variation throughout the period from the 7th day on (Figure 7). The Gomori’s trichrome assay showed that the CAMtreated wounds had more organized and dense collagen fibers as compared to the lesions of the SL group [Figure 8(a)]. On the 21st day, the CAM group showed higher percentage of collagen in the histological slides than the SL group (p 5 0.021) as shown in Figure 8(b). The results of the hydroxyproline assay showed that both groups presented collagen increasing during the follow-up period. The lesions of the CAM group showed faster and higher
collagenesis than SL on 2nd day (p 5 0.004). On the other hand, on the 14th day, the SL group showed higher level of hydroxyproline than those of the CAM group (p 5 0.001), a finding that, interestingly, changed on day 21st when the wounds of the CAM group reached higher hydroxyproline level than those of the SL group (p 5 0.024), as presented in Figure 8(c). DISCUSSION
The wound healing rate of CAM group was higher at day 7 than that of the SL group [Figure 2(a)], as also described by
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Wang et al.29 for membranes prepared using a different procedure. Wound closure involves a complex orchestrated interaction of different cell types, mainly neutrophils, leukocytes, macrophages, fibroblasts, and keratinocytes.3,30 Thus, the rapid rate of wound closure observed in this study might be attributed to the increased proliferation and migration of the several types of cells into the injured tissue. Cellular recruitment to the injured tissue during the inflammatory phase is important to remove debris, dead cells, and necrotic tissue. Neutrophils are attracted to the wound by chemotactic gradients, releasing enzymes, such as myeloperoxidase for the digestion of cellular debris and contributing to the death of bacteria by phagocytosis. Our data showed an intense inflammatory infiltrate in the beginning of the healing process (day 2), higher in the CAM group than in the SL group. The recruitment of neutrophils was similar between both groups at the 2nd day according to the myeloperoxidase level. Therefore, these data suggest that the major inflammatory infiltrate observed in the CAMtreated wounds on 2nd day was possibly due to CD41 lymphocytes, considering the extremely reduced number of CD81 cells and similar CD11b1 cells in both groups. The role of these cells has not been elucidated yet, but their presence as important regulators of the complex series of events that lead to wound healing has been suggested.31 If the inflammatory stimulus is persistent, high concentrations of proteolytic enzymes are produced, which stimulates oxidative stress, impairing the healing process.3,32 On the 7th post-operative day, the significant reduction of inflammatory infiltrate in the CAM group followed by the decrease of neutrophils and CD41 cells might indicate that the chitosanalginate membrane better regulated the inflammatory stimulus as compared to the SL group. Moreover, chitosan-alginate membrane stimulated the recruitment of CD11b1 macrophages, an important cell type that play a crucial role in the healing process because it continues the action of neutrophils, act as regulatory cells, as growth factors reservoir and releasing several types of metalloproteinase and collagenase to break the remaining extracellular content, the transition to the next phase of healing.31,33 Although the inflammatory infiltrates were similar on the 7th day for both groups, the cell types present were not the same. The wounds in the SL group did not achieve such regulation, keeping up with high-dose neutrophils in the same period, what may explain the delayed healing activity of this group. The initial dense inflammatory infiltrate observed in the CAM group on the 2nd day seemed to have an important role to the debridement of the wound. The regulatory activity observed on the 7th day in the wounds treated with chitosan-alginate and the earlier appearance of macrophages in the CAM group might have contributed to the advanced stage of the wound healing process, corroborating with the literature that has showed that depolymerization of chitosan stimulates functional macrophage recruitment to the injured tissue.9 Although the wounds on 14th day were already reepithelialization to both treatments, there were differences in
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cellularity (CD41) and collagen accumulation, also and particularly on 21st day, once wounds of SL group showed higher number of CD41 cells than the wounds of CAM group. The wounds were still in an inflammatory state and these inflammatory cells can produce proteolytic enzymes, which stimulates oxidative stress, impairing the healing process in the dermis layer, hampering the tissue formation and tissue remodeling in SL group, different from CAM group as observed with the higher collagen accumulation. It was also noticed that on day 21st, the scar tissue quality was much better in the case of the CAM group lesions (Figure 2). The modulated inflammatory infiltrate in the CAM group was followed by proliferation and migration of fibroblasts, higher on the 7th and 14th days than in SL-treated group. These events are important to the tissue formation phase, new collagen synthesis and subsequent remodeling. Diegelmann et al.34 analyzed the effects of a subcutaneous implant of a polyvinyl alcohol sponge treated with chitosan in rats and demonstrated a regulating ability of chitosan in the healing process compared to implants without chitosan. The authors observed significant cellular reduction and great collagenesis in animals treated with chitosan from the 7th to the 14th days, while the control group showed larger cellularity during the whole period. Our study is partially consistent with these data, but the appearance of fibroblasts, which was not reported by Diegelmann et al.,34 would be responsible for higher collagenesis along the wound healing period. A large number of macrophages remained longer in the tissue of animals that received chitosan, suggesting a potent immune-protective role of these cells by modulating the migration activity,35 subsequent fibroplasia and epithelialization, resulting in rapid and effective healing process according to the stimulation observed by chitosan-alginate membrane. The importance of angiogenesis is related to the intense need of nutrients and oxygen during the healing process, mainly during the inflammatory phase, because cells are in a high-energy demand stage due to intense activity and cell mitosis, thus promoting granulation tissue formation.36,37 As expected, increased angiogenesis was observed on the 7th day, but no evidence of difference between the groups was observed. Although chitosan is able to enhance the formation of new blood vessels by stimulating the production of cytokines and growth factors38,39 that, in addition to fibroblasts proliferation, stimulates angiogenesis by activating endothelial cells, in the histological study reported by Dai et al.40 poor presence of blood vessels was detected in animals treated with a chitosan-alginate porous dressing (spongeshape). Our data, as well as reported by Dai et al.,40 no difference related to angiogenesis was detected by histological analysis. Therefore, other methods become necessary to be applied for a better study of angiogenesis. Collagen is the major structural component of granulation tissue, strengthening the extracellular matrix. The amino-acid proline is an important component of the
CHITOSAN-ALGINATE MEMBRANES ACCELERATE WOUND HEALING
ORIGINAL RESEARCH REPORT
collagen fiber and hydroxyproline is used as a biochemical marker for collagen tissue, as a positive indication of the progression of healing.41,42 The presence of organized collagen fibers observed in CAM-treated wounds added to the increased collagen content observed by both methods, hydroxyproline and Gomori’s trichrome assay, suggests the contribution of the chitosan–alginate PEC to fibroblasts proliferation and synthesis of extracellular matrix during wound healing, as also proposed by Dai et al.40 and Ara ujo et al.43 Collagenesis, assessed by HyP measurement, increased continuously until the end of the process in the CAM group in addition to the predominance of fibroblasts. However, a higher collagenesis was observed on the 14th post-operative day for the SL group. These results suggest stimulus at different times for collagen production. Further studies are needed on the types of collagens formed, since the scar quality seems to be better in the CAM group. Thus, the appearance of denser and more organized collagen fibers suggest an important role of the biomaterial in the wound bed at all stages of healing, corroborating the literature. According to Wang et al.,29 chitosan-alginate PEC can facilitate remodeling of the scar tissue, increasing the rate of collagen synthesis and compaction of the new fiber and the appearance of mature fibroblasts, as observed in the CAM group. Moreover, chitosan-alginate membrane has stimulated and regulated all phases of wound healing process and our data suggest the use of chitosan-alginate membrane for the treatment of skin ulcers beneficially influences all phases of wound healing. Furthermore, collagen synthesis and contraction of the wound modulated by the chitosan-alginate membrane might have resulted in the rapid injury closure.29,38,44 Although the proposed model does not consider chronic wounds, the data presented herein are very important in clinical practices to treat chronic ulcers when significant inflammatory stimulus is required to change the initial status of the ulcers from senescence to granulation tissue (angiogenesis, fibroplasia and collagenesis) and also to reepithelialization, resulting in faster healing with a better quality scar. The treatment of acute and chronic wounds is a pressing need in many facets of medicine. Traditional wound dressings (e.g., gauze) have provided a good barrier function, keeping the wound dry by allowing evaporation of wound exudates while preventing entry of pathogen into the wound.45 In contrast, modern dressings mainly based on alginate, provide a moist wound environment and facilitate wound healing,46 as observed in our data. Alginate dressings in the dry form absorb wound fluid to re-gel, and the gels can supply water to a dry wound, maintaining a physiologically moist microenvironment and minimizing bacterial infection at the wound site. These functions can also promote granulation tissue formation, rapid epithelialization, and healing.47 It is important to point, according to Wang et al.29 and Dai et al.,40 and also observed in the present work, there was adherence of gauze fibers on the new tissue of dry wounded area in SL group and it became difficult the dressing change process, resulting in loss of new tissue and minor injuries to the wound. This fact was not observed in CAM-treated wounds, since there was no need to change
the chitosan-alginate membrane with no adherence to the wound site, which completely isolated the injury from the gauze or external environment, an important advantage in the overall wound healing process. CONCLUSION
Our results showed that the chitosan-alginate membrane modulated the inflammatory phase, stimulated the proliferation of fibroblasts and also collagenesis, accelerating the cutaneous wound healing in the earlier phases and improving the quality of the scar tissue. Furthermore, the chitosanalginate membrane showed to be a non-adherent to wound, dispensing daily changes and avoiding secondary damage and injury on newly-formed tissue. In this context, the chitosan-alginate PEC as wound dressing is indeed a promising formulation for application as a new therapeutic option, a potentially interesting for the treatment of tissue injuries and impaired wounds. ACKNOWLEDGMENTS
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