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In Situ Forming and Rutin-Releasing Chitosan Hydrogels As Injectable Dressings for Dermal Wound Healing Ngoc Quyen Tran, Yoon Ki Joung, Eugene Lih, and Ki Dong Park* Department of Molecular Science and Technology, Ajou University, 5 Wonchon, Yeoungtong, Suwon 443-749, Republic of Korea ABSTRACT: An in situ gel-forming system composed of rutinand tyramine-conjugated chitosan derivatives, horseradish peroxidase (HRP), and hydrogen peroxide (H2O2) was prepared and applied to dermal wound repair. Rutin was employed to enhance production and accumulation of extracellular matrix in the healing process. In vitro study demonstrates that released rutin significantly enhanced cell proliferation as compared with media without rutin. In vivo wound healing study was performed by injecting hydrogels on rat dorsal wounds with a diameter of 8 mm for 14 days. Histological results demonstrated that rutin-conjugated hydrogel exhibited enhancement of wound healing as compared with treatments with PBS, hydrogel without rutin, and a commercialized wound dressing (Duoderm). More specifically, rutin-conjugated hydrogels induced better defined formation of neo-epithelium and thicker granulation, which is closer to the original epithelial tissue. As a result, this study suggests that the in situ gel-forming system can be a promising injectable gel-type wound dressing.
’ INTRODUCTION Wound healing process is a series of cellular and biochemical activities composed of several overlapping phases containing inflammation, new tissue formation, and remodeling.1 In the process, the growth of fibroblasts significantly contributes to fill a wound bed by producing collagen and fibronectin that form a new extracellular matrix (ECM), resulting in contracting and healing of the wound.24 To stimulate the healing of wounds, we have investigated numerous natural and synthetic materials and used them as a bioactive dressing in which bioactive molecules are delivered in wound or are constructed from materials having endogenous activity.58 Recent studies have shown that the use of hydrogel as biomaterials for the wound healing is promising. The hydrogel films were moist and hydrated, in which bioactive substances could be delivered to the wound. Moreover, these films not only absorb exudate but also prevent the loss of evaporative water and the wound dehydration that disturbs an ideal environment to stimulate the wound healing process.9,10 The hydrogels would be more desirable if they formed in situ, in which the polymer solution is directly injected into the injury site and fills an irregular space and subsequently forms hydrogel.1012 These hydrogels are in situ formed via either physical crosslinking using hydrophobic interactions, electrostatic interactions, and stereocomplexation or chemical cross-linking such as photopolymerization, Schiff-base reaction, and Michael-type addition, which generally called for physical and chemical hydrogels, respectively.13,14 Although in situ forming hydrogels have been suggested as ideal injectable biomaterials, there have been unsolved problems, such as weak mechanical strength and rapid dissolution of gel network and cytotoxicity.1517 Recently, an interesting approach using an enzyme-catalyzed reaction to prepare hydrogels was suggested; phenol groups containing polymers r 2011 American Chemical Society
were cross-linked in the presence of hydrogen peroxide (H2O2) and horseradish peroxidase (HRP).18,19 This approach enables a hydrogel not only to form under a mild condition in a short period of time but also to enhance the mechanical property. It is known that chitosan has many biologically beneficial properties for wound dressing, such as biocompatibility, biodegradability, tissue-adhesive property, and hemostatic and antiinfective activity.20,21 Chitosan stimulates the macrophage to produce growth factors that have positive effects on ECM production.22,23 However, a rigid crystalline structure of chitosan makes it hard to be dissolved in water, which has partially retarded its potential for the application.2,24 Therefore, watersoluble chitosan-based hydrogels as wound dressing have been widely studied.2527 Rutin is one of the most commonly found flavonol glycosides that exhibits multiple pharmacological activities, such as antioxidative and cytoprotective effects2830 and wound healing.3133 These properties of rutin have been commercially utilized as an antioxidative drug for healing wound. Studies indicated that rutin significantly increased fibroblast proliferation and collagen production in vitro.34,35 The disadvantage of rutin is its poor solubility in aqueous media and low absorption after oral administration that lead to its poor bioavailability.36 Conjugation of rutin to water-soluble chitosan derivative has been expected to increase rutin bioavailability as released in injury site. This is very significant because the process of wound healing can be accelerated if the healing agents are more soluble and interactive to the wound beds. Received: March 10, 2011 Revised: May 12, 2011 Published: May 19, 2011 2872
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Figure 1. Synthetic schemes of polymers: (a) NPCPEGTA, (b) CPT, and (c) RCPT.
In this study, we suggest rutin-conjugated chitosan-based hydrogels as a type of injectable wound dressing for dermal wounds. This study is expected to introduce for the first time a promising injectable-formed wound dressing based on chitosan with enhanced efficacy for wound healing. The enzymatic crosslinking will enable chitosan to form rapidly a hydrogel and adhere stably to a wound site for a desired period of time. The conjugation of rutin will certainly facilitate the acceleration of wound healing process due to the controlled release in addition to the bioactivity of chitosan for wound healing. Several physicochemical characterizations will demonstrate the adjustable properties of the hydrogels due to the enzyme-triggering system, such as gelation, degradation, and rutin release. In vitro and in vivo studies will practically show enhanced healing efficacy of the hydrogel for dermal wound.
’ EXPERIMENTAL SECTION Materials. Chitosan (low molecular weight ∼70 000 Da, 7585% deacetylation), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), succinic anhydride (Sa), triethylamine (TEA), p-nitrophenyl chloroformate (NPC), tyramine (TA), H2O2, and HRP (type VI, 298 purpurogallin unit/mg solid) were purchased from Aldrich. Rutin were
purchased from Acros Organics. Poly (ethylene glycol) (PEG, Mw 1500 g/mol) was obtained from Polyscience. All other chemicals and solvents were used without further purification.
Synthesis of Tyramine-poly(ethylene glycol)-p-nitrophenyl Carbonate Ester (NPCPEGTA). NPCPEGTA was prepared through two steps. First, two end groups of PEG were activated with NPC. Second, NPC-activated PEG was partially substituted by tyramine (Figure 1). In brief, dried PEG (10 g, 13.33 mmol of OH) was dissolved in methylene chloride (MC, 50 mL) and kept in ice bath. Then, TEA (1.47 g, 14.55 mmol) and NPC (2.93 g, 14.55 mmol) were added to the solution. The mixed solution was stirred overnight under a nitrogen atmosphere. The reaction mixture was precipitated in excess diethyl ether, filtered, and dried in vacuo to obtain a totally NPCactivated PEG. DS (1H NMR): 1.98 of 2 hydroxyl groups. Yield: 9.90 g (83%). 1H NMR (CDCl3, δ): 3.60 (s, CH2CH2, PEG), 7.38 and 8.22 (d, CHdCH, NPC). Subsequently, TA solution (0.78 g, 5.70 mmol in DMF) was dropped in 30 mL of DMF solution containing the NPC-activated PEG (9 g, 9.84 mmol of NPC) with stirring. After 12 h of reaction, the solution was precipitated in excess diethyl ether, filtered, and dried in vacuo to obtain NPCPEGTA: DS (1H NMR) = 55% of TA, yield = 8.38 g (93%). 1H NMR (CDCl3, δ): 2.68 and 3.35 (m, CH2CH2, TA), 3.62 (s, CH2CH2, PEG), 6.78 and 6.98 (d, CHdCH, TA), 7.38 and 8.22 (d, CHdCH, NPC). 2873
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Biomacromolecules Synthesis of Hemisuccinate Rutin (Ru-Sa). Rutin (5 g, 7.5 mmol) was dissolved in 50 mL of DMF with stirring. TEA (0.83 g, 8.25 mmol) and succinic anhydride (0.825 g, 8.25 mmol) were subsequently added to the DMF solution under a nitrogen atmosphere. After being maintained for 12 h at room temperature, the reaction mixture was precipitated in diethyl ether, filtered, and dried in vacuo to obtain RhSu: substitution degree (1H NMR) = 1.05 mol/mol of rutin, yield =5.08 g (93%). 1H NMR (DMSO-d6, δ): 1.02 (m, proton of CH3, rhamnosyl), 2.50 (m, CH2CH2, succinate), 3.053.74 (m, H, rhamnoglucosyl), 6.16 (s, H6), 6.38 (s, H8), 6.80 (d, H50 ), 7.54 (m, H20 and H60 ). Synthesis of Rutin-Conjugated Chitosan-poly(ethylene glycol)-tyramine (RCPT). RCPT was prepared in two steps to give carbamate and amide linkages between free amine groups of chitosan and NPCPEGTA and RuSa, respectively. In the first step, chitosan (1.6 g) was dissolved in hydrochloride acid solution (pH 3.5, 240 mL) in a round-bottomed flask; then, the pH was adjusted to 5. The solution of NPCPEGTA (3 g in distilled water) was added to the chitosan solution. The mixture was stirred at room temperature for 12 h. To remove a small amount of remained NPC from the mixture, we added 0.5 g TA and stirred for 12 h. The solution was dialyzed against 0.2 M NaCl aqueous solution and deionized (DI) water (molecular weight cutoff (MWCO) = 8000). The dialyzed solution was lyophilized to obtain CPT. The DS was eight units per one hundred glucosamines (1H NMR and UVvis). 1H NMR (D2O, δ): 1.86 (s, COCH3, chitosan), 3.62 (s, CH2CH2, PEG), 6.68 and 7.00 (d, CHdCH, TA). The amount of obtained CPT was 2.38 g (yield = 84%). In the second step, CPT (1.8 g) was dissolved in DI water (200 mL). RuSa (0.43 g, 0.62 mmol) and EDC (120 mg, 0.62 mmol in DI water) were added to the CPT solution with stirring. The mixture was stirred at room temperature for 24 h. The solution was dialyzed against DI water for 3 days. The dialyzed solution was lyophilized to obtain final product of RCPT. The DS was four units per one hundred glucosamines (UVvis). 1H NMR (D2O, δ): 1.02 (m, proton of CH3, rhamnosyl of rutin), 1.86 (s, COCH3, chitosan), 2.252.60 (m, CH2CH2, succinate), 3.62 (s, CH2CH2, PEG), 6.68 and 7.00 (d, CHdCH, TA). The obtained RCPT was 1.96 g (yield = 88%). Polymer Characterizations. We determined the structure and composition of NPCPEGTA, RuSa, CPT, and RCPT by using an NMR-400 apparatus (Varian, 400 MHz) and an ultraviolet visible (UVvis) spectrophotometer (JASCO V-570). The DS of tyramine was measured at a wavelength of 275 nm. Rutin conjugation and release were confirmed at a wavelength of 357 nm. Preparation of Hydrogels. All hydrogels (1 mL) were prepared in vials at room temperature. RCPT (12 mg) was dissolved in PBS solution (pH 7.4, 888 μL) and then equally separated into two parts. The PBS solutions of HRP (50 μL of 0.0032 mg/mL stock solution) and H2O2 (50 μL of 0.0032 wt % stock solution) were separately added to each part. In situ gel formation of RCPT could occur easily by mixing the polymer solutions. The final concentration of RCPT in hydrogel was 1.2 wt %. The time in which it takes for the formation of gel (denoted by gelation time) was determined using a vial tilting method. The gelation time was determined by regarding the gel state when the solution does not flow for 1 min after inverting a vial. The CPT hydrogel was prepared in the same manner as RCPT, in which CPT (14 mg) was dissolved in a PBS solution (pH 7.4, 886 μL) and followed by separately adding HRP (50 μL of 0.0032 mg/mL stock solution) and H2O2 (50 μL of 0.0032 wt % stock solution). The final concentration of the CPT solution was 1.4 wt %. Morphology of Dehydrated Hydrogels. The microporous structure of the dehydrated hydrogels was imaged by SEM. For this study, the hydrogel disk was prepared using a Teflon mold at different concentrations of HRP and H2O2 (0.0032 mg/mL and 0.0032 wt % and 0.0128 mg/mL and 0.0128 wt %, respectively) and washed in distillated
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water for 3 h. After washing, the hydrogel specimens were freeze-dried under vacuum for 3 days. The samples were cross-sectioned and sputtercoated with gold. The morphology of the specimens was observed by SEM (JEOL, JSM-6380). In Vitro Degradation Test. The RCPT hydrogel was prepared in a vial with known weight (wi) according to the above-mentioned procedure. The samples were subsequently incubated in 2 mL of PBS solution (pH 7.4) at 37 °C. The hydrogels were weighed to determine the gel weight (wt) at regular time intervals. After hydrogels were weighed, a fresh PBS solution was supplemented to the samples. The weight ratio of the hydrogels (wt/wi) was plotted as a function of incubation time. All experiments were carried out with each sample of hydrogels in triplicate. In Vitro Rutin Release Test. The RCPT hydrogels (1 mL) and rutin-encapsulated CPT gel (750 μg of rutin in CPT 1 mL) were prepared in vials according to the above-mentioned procedure. The samples were subsequently incubated in 4 mL of PBS solution (pH 7.4) at 37 °C. At a predetermined time interval, 1 mL of the incubated solutions was drawn to determine the amount of rutin, and a fresh PBS solution was added to the samples. The amount of released rutin was quantified using the UVvis spectrometer at a wavelength of 357 nm.39 In Vitro Cell Study. The cytotoxicity of hydrogels was tested according to a protocol from Yang and Unger.27,37 This method was applied to our study to extrude the interference due to the presence of rutin in RCPT hydrogel in MTT assay.38 An extract from hydrogel was obtained by adding hydrogel fragments to distilled water at a concentration of 0.4 g/mL and was incubated at 37 °C for 24 h. The extract was sterilized by filtration (filter diameter = 220 nm). The sterilized extract was added to an equal volume of double-concentrated RPMI-1640 culture media (Gibco) containing 6 vol % newborn calf serum (Gibco) to get 100% hydrogel extract. L929 mouse fibroblast suspension (100 μL) was seeded on a 96-well culture plate at the number of 5000 cells per well. The plate was incubated for 12 h at 37 °C/5% CO2. Then, the media was discarded and replaced with 100 μL of 100% extract. The RPMI-1640 culture media with 3 vol % newborn calf serum was used as negative control and RPMI-1640 culture media (3 vol % new-born calf serum) with 0.7 vol % acrylamide was used as positive control. Samples and controls were tested in sextuplicate. The plates were incubated for 1, 3, and 5 days. At the end of the culture time, the extract-contained culture media was removed and washed with the RPMI-1640 culture media. We added 10 μL of MTT solution (5 mg/mL) to each well. After 4 h of incubation, 100 μL of 10 wt % SDS in 0.01 mol L1 HCl was added to each well and incubated overnight. The optical density (OD) of each well was measured by using a microplate reader at a wavelength of 570 nm, with a reference wavelength of 655 nm. Finally, the cell viability of hydrogel extracts compared with the controls was calculated. In Vivo Wound Healing Study. Five male SpragueDawley rats weighing 250300 g (Dooyeol Biotech) were used for the experiment. These rats were anesthetized with a mixture (0.2 mL/kg of body weight) of Zoletil 50 (50 mg/mL; Virbac Laboratories, Carros, France) and xylazine hydrochloride (Bayer, Ansan, Korea) (zoletil/xylazine 1:2). Hair on the dorsal side was shaved, and the skin was cleaned with 70% ethanol. On each rat was created four incisions (8 mm biopsy punch; Acuderm). These incisions were filled with sterilized RCPT and CPT hydrogels, Duoderm (ConvaTec), and PBS. The Duoderm is an adherent dressing indicated for the management of exuding wounds and promotion of granulation that was fabricated from a fruit pectin derivative. The concentrations of CPT and RCPT hydrogels were, respectively, 1.4 and 1.2 wt %, which we mixed to form in situ gels by using a double syringe. On the basis of the initial swelling of RCPT hydrogels, the gel in this study was prepared with concentrations of HRP and H2O2 to be 0.0032 mg/mL and 0.0032 wt %, respectively, which enables us to manage effectively exudation from wound. These filled incisions on rats were covered with plastic sheet. After 14 days of post 2874
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wounding, animals were euthanized by inhalant anesthetic. The skin including the wound was removed and fixed in 10% formaldehyde solution. The fixed tissues were bisected and embedded center-face down in liquid paraffin-contained molds. Tissue-embedded paraffin was solidified and then released out of the mold. The tissue-embedded paraffin was sectioned in 4 μm to obtain many thin layers at the center of the wound. The sections were positioned on glass slides for staining with hematoxylin and eosin (H&E). The thickness of granulation formation per photomicrograph was evaluated.8 The animal experiments were approved and done with the guidelines of Medical School, Ajou University, Suwon, South Korea. Statistical Analysis. Data were analyzed by a Student’s t test. P values less than 0.05 or 0.005 were considered to be statistically significant. All values are expressed as the mean ( standard deviation (SD).
’ RESULTS AND DISCUSSION Preparation of RCPT. RCPT was prepared through a scheme, as shown in Figure 1. First, PEG was activated with NPC to obtain NPCPEGNPC, which could be observed in 1H NMR spectrum (Figure 2a). The product was partially substituted with TA. The DS of TA-substituted PEG was calculated to be >55%. As shown in 1H NMR spectrum of Figure 2b, the integral of aromatic protons of TA (δ 6.78 and 6.98) was higher than that of aromatic protons of PNC (δ 7.38 and 8.22). The fed amount and adding order of chemicals could decrease the portion of NPC PEGNPC or TAPEGTA in the product, in which the small portion may cause cross-linking between chitosans and no conjugation to chitosan. Second, NPCPEGTA was conjugated to chitosan via the reaction between amine groups of chitosan and the NPC moiety to obtain CPT. Figure 2c shows the NMR spectrum of CPT presenting aromatic protons of TA and acetyl protons of chitosan (δ 1.86), which was calculated to be 8 units over 100 glucosamines. PEG chains are expected to play roles of improving inherent poor solubility of chitosan and facilitating cross-linking between chitosans as a spacer. Finally, rutin was conjugated to the CPT to obtain RCPT, in which the carboxylic group of hemisuccinate rutin was bound to the amine group of CPT by using EDC. Figure 2d shows the 1H NMR spectrum of RCPT presenting the presence of rhamnosyl protons (δ 1.02) and aromatic protons. The conjugation degree of rutin was determined using UVvis at a wavelength of 357 nm because the esterification of rutin did not change the UV absorbance according to literature.39The obtained RCPT contained ∼10 rutin molecules per 100 glucosamine units, and the amount did not affect the water solubility (data not shown). The conjugation of rutin is expected to enhance bioavailability of rutin that has been limited because of its poor solubility and burst release.39,40 Hydrogel Formation. Hydrogels were prepared via the HRPmediated coupling reaction of phenolic moieties in RCPT, as shown in Figure 3a. In the presence of HRP and H2O2, tyramine moieties at the end of the polymers are coupled to each other via a carboncarbon bond at the ortho positions of phenol groups or via a carbonoxygen bond between the carbon atom at the ortho position of phenol groups and the phenolic oxygen.17,19 At the lower concentrations of catalysts, the hydrogels show a yellow color that is similar to the color of RCPT solution, as shown in Figure 3b. However, when HRP and H2O2 were, respectively, used above 0.0032 mg/mL and 0.0032 wt %, the hydrogels had a brown color. This was attributed to the oxidation
Figure 2. 1H NMR spectra of polymers: (a) NPC-activated PEG, (b) NPCPEGTA, (c) CPT, and (d) RCPT.
of rutin molecules by excess catalysts, In this case, Michael-type or Schiff-base reactions may occur between oxidized phenolic moieties of rutin and amine groups of chitosan. The result indicates that conjugated rutins could be a potential cross-linker. For CPT hydrogel, polymer concentration could form hydrogel to be 1.4 wt %. This supposition is also supported by the result that CPT hydrogel was formed at the lower catalytic concentration than that for RCPT hydrogels. Figure 3c shows the effect of catalytic concentration on gelation time. The gelation times decrease from 103 to 3 s as the HRP (mg/mL) and H2O2 (wt %) concentrations increase from 0.0016 to 0.0128. The higher the concentration of HRP and H2O2, the more decomposition of H2O2 and the phenolic radical production by HRP. Degradation Behavior of RCPT Hydrogel. RCPT hydrogels that were prepared at different catalytic concentrations revealed slightly different degradation profiles, as shown in Figure 4. Increases in gel weight in the early stage of incubation indicate that the water swelling of hydrogels and the gel weight decreased 2875
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Figure 4. Time-dependent weight ratios of hydrogels prepared with different catalyst concentrations (n = 3, mean ( S.D.).
Figure 3. In situ gel formation of RCPT polymer solution in the presence of HRP and H2O2: (a) a scheme showing enzymatic crosslinking of RCPT, (b) photographic images of RCPT hydrogels under different catalytic concentrations, and (c) the effect of catalytic concentration on gelation time. (n = 4, mean ( S.D.).
up to 30 days after reaching the maximum weight. When HRP and H2O2 were used at the lowest concentration (0.0032 mg/mL and 0.0032 wt %, respectively), the hydrogels exhibited the highest swelling ratio and the lowest weight loss. At the medium concentration (0.0064 mg/mL and 0.0064 wt %, respectively), a slightly lower swelling ratio and higher weight loss were measured compared with the lower concentration. At the highest concentration (0.0128 mg/mL and 0.0128 wt %, respectively), the swelling ratio was almost negligible. At much lower concentration (0.0016 mg/mL and 0.0016 wt %, respectively), the formed hydrogel structure was collapsed after 30 min of incubation (data not shown). An increase in catalytic concentration that riggers the gelation of a polymer involves higher cross-link density. In this result, the higher cross-link density seems to decrease the water-swelling rate of hydrogels observed in the early stage of degradation test. The conjugation of relatively
hydrophilic PEG resulted in enhancing poor water solubility of chitosan. Additional conjugation of a small portion of rutin nearly did not affect the water solubility of CPT (data not shown). Water-soluble CPT and RCPT were cross-linked via linkages between TAs of PEG to form a hydrophilic and water-insoluble network structure that is shown as a water-swollen hydrogel macroscopically. Herein, an increase in the degree of cross-link would reduce the capacity of water uptake of cross-linked chains because the increment could result in reducing the porosity of the hydrogel structure that was confirmed by SEM (Figure 5). Although each hydrogel shows different ratios of weight loss, it is hard to assert that the ratio of lost weights caused by degradation is definitely different because hydrogels have different swelling ratios. The ratio of weight loss of all hydrogels was ∼40% of total weight of maximally swollen hydrogels for 30 days. In this study, such a slow degradation behavior of hydrogels is similar to that of other chitosan-based hydrogels, which is attributed to dehydration effect.41 In Vitro Release Profiles of Rutin. It is known that conjugation of a bioactive agent to a polymer suppresses an initial burst of the release.42In this study, rutin was conjugated to CPT. As shown in Figure 6, the release profiles of rutin released from hydrogels are quite different. Free rutin just contained in CPT hydrogel exhibited the fastest release rate. Over 70% of rutins were released within 20 h. Conjugated rutins that were covalently linked to CPT show controlled profiles of release. More interestingly, the release profiles of conjugated rutins show quite a different manner at different catalytic concentrations. At the lower concentration of HRP and H2O2 (0.0032 mg/mL and 0.0032 wt %, respectively), ∼50% of conjugated rutin was released, whereas only ∼10% of conjugated rutin was released at four times higher concentration (0.0128 mg/mL and 0.0128 wt %, respectively). This difference is probably caused by an excess amount of catalysts (four times higher amount). The hemisuccinic group of rutin was conjugated to the amine group of chitosan to form an ester bond that is hydrolyzable in aqueous solution. Excess amount of HRP and H2O2 not only catalyzes the conjugation between tyramines but also oxidizes the polyphenol group of rutin, resulting in the conjugation of rutin to chitosan. Therefore, it is supposed that the higher catalytic concentration lead to extremely low release rate. However, the free rutin group 2876
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Figure 5. Scanning electron micrographs of the inner section of hydrogels were prepared at HRP and H2O2 concentration (A) 0.0032 mg/mL and 0.0032 wt % and (B) 0.0128 mg/mL and 0.0128 wt %.
Figure 6. Cumulative release profiles of rutin from hydrogels prepared with different catalyst concentrations (n = 3, mean ( S.D.)
Figure 8. Photographs of wounds treated with filled with PBS, DuoDerm, CPT hydrogel, and RCPT hydrogel. The surface of covered wounds after 7 days of postwounding was gently removed.
Figure 7. Viability of fibroblasts cultured in extracts from hydrogels containing CPT hydrogel (white) and RCPT hydrogels at different concentrations of HRP and H2O2 (gray, 0.0032 mg/mL and 0.0032 wt %, respectively) and (black, 0.0128 mg/mL and 0.0128 wt %, respectively). The RPMI-1640 media with supplement of new-born calf serum and acrylamide were used as negative and positive controls, respectively (n = 6, mean ( S.D., *P < 0.05 and **P < 0.005).
shows a greatly fast release rate despite the condition of excess catalyst. This can be explained by the fact that the poor solubility of free rutin protected it from oxidative reaction by HRP and H2O2. The sustained release of rutin using a little lower catalytic concentration is likely to enhance the bioavailability of rutin during a long-term period. This fact may be applied to overcome the poor absorption of rutin that is orally administered.36,40
Effect of Rutin on Cell Proliferation. It is reported that rutin enhances fibroblast proliferation at a low concentration and has very low cytotoxicity at high concentration. To verify the availability of the rutin-conjugated hydrogels for wound healing application, it is necessary to evaluate the effect of rutin on cell proliferation and cytotoxicity as well as that of chitosan derivatives. The RCPT hydrogel exhibited a yellow color that interferes with measuring OD; therefore, the cell viability was indirectly tested. Rutin concentration in extract was determined at 88 μg/ mL. Fibroblasts were cultured in extracts from CPT and RCPT hydrogels for 1, 3, and 5 days, respectively, and the cell viability was measured as shown in Figure 7. At 1 day of incubation, the cell viabilities in extracts from CPT and RCPT hydrogels were ∼100% as compared with control. At 3 and 5 days of incubation, cell viabilities in extracts from RCPT hydrogels were higher than those in extracts from CPT hydrogel. Kavanov’s study demonstrates that this result is attributed to the effect of rutin on cell proliferation.35The result of RCPT hydrogels also reveals that the release rate of rutin certainly affects the cell proliferation. As shown in release profiles of rutin, RCPT hydrogel with the higher 2877
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Figure 9. Histology of wounds at 14 days of postwounding covered with DuoDerm and hydrogels or filled with PBS. The left panels show an overview of formed granulation tissues in wound beds with blood vessels (red arrows), extracellular proteins (pink arrows), and granulation layers (black arrows). The right panels show fibroblasts (black triangles).
release rate (HRP and H2O2 is 0.0032 mg/mL and 0.0032 wt %, respectively) showed slightly higher cell viabilities than that with the lower release rate. (HRP and H2O2 are 0.0128 mg/mL and 0.0128 wt %, respectively.) As a result, it is concluded that CPTbased hydrogels are cell-compatible and rutin-conjugated to those facilitates enhancing the proliferation of fibroblast. In Vivo Wound Healing Study in Rats. It has been reported that chitosan improves the healing of wound. Rutin had been commercialized as an orally administrative drug that supports wound healing. Previous studies demonstrated that rutin significantly increases fibroblast proliferation and collagen production. A combination of rutin and chitosan is expected to have many advantages as a potential material for wound healing. For
in vivo study, the RCPT hydrogel was prepared with HRP and H2O2 (0.0032 mg/mL and 0.0032 wt %, respectively), which had exhibited the highest swelling ratio and the most enhanced fibroblast proliferation. A wound model was created on the back of each rat using biopsy punch. Figure 8 shows the appearance of wounds either covered with hydrogels and Duoderm or filled with PBS solution. There was no sign of inflammation or infection formation in materials-covered wounds. RCPT- and Duoderm-covered incisions exhibit the larger contraction as compared with PBS-filled and CPT-covered incisions 14 days post-wounding. The growth of new epidermis extended from margin to center of the wound bed, resulting in reduced depth and area of wound. The epithelization of incisions increased in 2878
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Figure 10. Granulation thickness in the tissue formation that was measured with histological images (n = 3, mean ( SD *P < 0.05).
the order of PBS < CPT < Duoderm ≈ RCPT. This fact indicates that materials-covered wounds give the moist environment to accelerate healing wounds. The hydrogels and Duoderm were moist and able to not only absorb exudate but also prevent the loss of evaporative water and wound dehydration, considered as an ideal environment to stimulate the wound healing process.9,10,37 Histology of wounds either covered with hydrogels and Duoderm or filled with PBS on the 14th postoperative day are shown in Figure 9. The surface of materials-covered wounds shows a complete formation of new epithelium, which can be observed in the left panels. More new blood vessels are observed in RCPT hydrogel-covered wound compared with others. The formation of extracellular proteins that mainly contain collagen was increased in the order of PBS < CPT < Duoderm < RCPT. The fibroblasts density can also be observed in the Figure (right panels) in which RCPT hydrogel-covered wound induced the highest fibroblast density. RCPT hydrogel-covered wound also shows the formation of extracellular proteins with the highest density. The greater formation of blood vessels, the well-proliferated fibroblast, and the well-produced collagens in the RCPT hydrogel-covered wound bed contribute to significantly enhanced granulation tissue formation, as shown in Figure 10. The wounds covered with Duoderm exhibited the largest contraction and epithelization that can be explained by its inherent effect. It is known that Duoderm is an adherent dressing effective for the management of exuding wounds and promotion of granulation. In the RCPT hydrogel-covered wound, the effect of chitosan and rutin mainly resulted in the granulation tissue formation with the highest density and high contraction as well as epithelization. Besides several beneficial properties of chitosan for wound healing, rutin-conjugated chitosan can contribute its bioactivities to accelerate the healing, such as cytoprotective effects, stimulation of fibroblast proliferation, and collagen production.2128 It was reported that rutin could significantly stimulate fibroblasts proliferation at a low concentration and there was no cytotoxic effect below 100 μg/mL,35 and fibroblasts were cultured with rutin-stimulated collagen production at a concentration up to 80 μg/mL.34 These effects could be explained by stabilization in collagen confirmation of its sugar moiety and inhibition to collagenolytic enzymes of its flavonol moiety, resulting in an ideal environment for fibroblast proliferation.34,43,44 It was reported that fibroblasts are wellproliferated on the conformation-stabilized collagen.45 Collagenolytic enzymes are matrix metalloproteinases (MMPs) that are
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inhibited by decrement of free zinc concentrations caused by chelating agents such as rutin. By inhibiting MMP, rutin could increase the rate and amount of collagens synthesized by fibroblasts that is necessary for the formation of new ECM in wound bed. In summary, we used “Duoderm” as a commercial product for control sample. The product was verified as an effective wound dressing. The product is an adherent dressing indicated for the management of exuding wounds and promotion of granulation that was fabricated from a fruit pectin derivative. In some issues, the significance of RCPT hydrogels can be suggested as follows: (1) The type of dressing is different: skinadhesive hydrogel (RCPT) and hydrocolloid synthetic polymer patch (DuoDerm). It was demonstrated that gel-type dressing may be more effective than patch-type. (2) Chitosan is known to promote wound repair in a similar mechanism of Duoderm. (3) Moreover, rutin is a bioactive molecule that contributes to enhance cell proliferation, resulting in a faster healing rate. (4) Most of hydrogels are permeable to a solute. In our experiment, the concern could be confirmed by rutin release test. We designed the hydrogels for the purpose of offering a biocompatible interface between hydrogel and wound surface as well as supplying bioactive molecule to promote wound healing.
’ CONCLUSIONS A bioactive hydrogel of rutin-conjugated chitosan was prepared in view of a potential wound dressing with enhanced healing efficacy, injectable solution form, and tissue-adhesive property. Rutin could be released from RCPT hydrogel in a sustained manner to stimulate fibroblast proliferation and collagen production that can have a significant effect on the healing phases. In the rat wound model, the wound covered with RCPT hydrogels exhibited fast contraction of an incision and facilitated the formation of new well-defined ECMs. In summary, a combination of the bioactivity of rutin and chitosan in the injectable hydrogel contributed to improve the healing of dermal wound. ’ AUTHOR INFORMATION Corresponding Author
*Tel: þ82-31-219-1846. Fax: þ82-31-219-1592. E-mail: kdp@ ajou.ac.kr.
’ ACKNOWLEDGMENT This research was supported by grants from the Fundamental R&D Program for Core Technology of Materials funded by the Ministry of Knowledge Economy, Republic of Korea (K0006028), an NRF grant funded by the Korean government (2010-0027776), and the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0028294). ’ REFERENCES (1) Gurtner, G. C.; Werner, S.; Barrandon, Y.; Longaker, M. T. Nature 2008, 453, 314–321. (2) Chen, R. N.; Wang, G. M.; Chen, C. H.; Ho, H. O.; Sheu, M. T. Biomacromolecules 2006, 7, 1058–1064. (3) Ueno, H.; Mori, T.; Fujinaga, T. Adv. Drug Delivery Rev. 2001, 52, 105–115. (4) Joung, Y. K.; Bae, J. W.; Park, K. D. Expert Opin. Drug Delivery 2008, 5, 1173–1184. 2879
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