RSC Advances View Article Online PAPER Published on 10 November 2014 Downloaded by test on 22/01/2016 14:48:10 Cite this: RSC Adv., 2014, 4, 65081 View Journal | View Issue Flexible, micro-porous chitosan–gelatin hydrogel/ nanofibrin composite bandages for treating burn wounds P T Sudheesh Kumar,† G Praveen,† Mincy Raj,† K P Chennazhi and R Jayakumar* We developed chitosan–gelatin hydrogel/nanofibrin ternary composite bandages for the treatment of burn wounds and characterized the material by SEM The spherical nanofibrin moieties (229 Æ nm in size) were prepared using an emulsification method and were distributed within the chitosan–gelatin matrix The presence of the fibrin component within the matrix was confirmed by SEM and phosphotungstic acidhematoxylin staining The swelling, biodegradation, porosity, whole-blood clotting, platelet activation, cell viability, cell attachment and cell infiltration properties of the nanocomposite bandages were evaluated The nanocomposite bandages were flexible, degradable and showed enhanced blood clotting and platelet activity compared with control samples The nanocomposite bandages showed adequate swelling ability when immersed in water and phosphate-buffered saline Cell viability studies on normal human dermal fibroblast and human umbilical cord vein endothelial cells proved the non-toxic nature of the composite bandages Cell attachment and infiltration studies showed that the human dermal Received 8th October 2014 Accepted 10th November 2014 fibroblast and human umbilical cord vein endothelial cells attached to the bandage Enhanced collagen deposition and re-epithelialization with the formation of intact mature epidermis was noted in the animal DOI: 10.1039/c4ra11969j groups treated with the nanocomposite bandages compared with the experimental controls These www.rsc.org/advances results show that these ternary nanocomposite bandages are ideal candidates for burn wound dressings Introduction Burn injuries can be a serious health issue as a result of a lack of proper drugs, long-term disability, prolonged hospitalization, loss of body extremities and even death Burn wounds are usually caused by the thermal exposure of the body surface and damage to the skin.1 Inappropriate wound care can delay the healing process, which involves complex mechanisms such as coagulation, inammation, matrix synthesis and deposition, angiogenesis, broplasia, re-epithelization, contraction and remodelling.2–4 Burn wounds are usually characterized by membrane destabilization and protein coagulation They are associated with energy depletion and hypoxia at the cellular level, which leads to extensive tissue necrosis.1 Burn wounds need to be treated based on the severity of the injury Various formulations such as ointments and wound dressings have been developed for the treatment of severe burn wounds However, when ointments or creams are used, their frequent reapplication and washing of the wound region are oen painful to the patient.5–7 To avoid this discomfort, we developed Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi-682041, India E-mail: rjayakumar@aims.amrita.edu; jayakumar77@yahoo com; Fax: +91 484 2802020; Tel: +91 484 2801234 † Authors contributed equally This journal is © The Royal Society of Chemistry 2014 a novel hydrogel-based chitosan–gelatin bandage incorporating nanobrin for burn wounds This composite material is simple to handle and more durable than current dressings during application An ideal dressing should maintain a moist environment at the wound interface, remove excess exudate from the wound surface and allow gaseous exchange.8,9 Chitosan is a typical marine polysaccharide obtained from the exoskeleton of invertebrates.10 Chitosan is the primary derivative of chitin and it has many properties that make it an ideal material for biomedical applications, including biocompatibility, biodegradability, antimicrobial activity and bloodclotting potential.9–11 Chitosan provides a non-protein matrix for three-dimensional tissue growth and activates macrophages for tumoricidal activity and the production of interleukin-1, which, in turn, stimulates cell adhesion and proliferation Chitosan is a haemostat, which helps with natural blood clotting, and blocks nerve endings, which reduces pain.11–15 Gelatin is a biocompatible polymer and is used for many biomedical applications, including wound dressings, and has haemostatic potential.15 The haemostatic action is based on platelet activation at the point of contact of blood with the gelatin, which activates the coagulation cascade.16,17 As a result of its gelation properties, it can act as a binding agent and stops the ow of blood into blood vessels by constricting the vessels.17,18 Clinical and in vivo studies have shown that gelatin is effective in wound healing.15–18 A topical application of gelatin RSC Adv., 2014, 4, 65081–65087 | 65081 View Article Online Published on 10 November 2014 Downloaded by test on 22/01/2016 14:48:10 RSC Advances to skin has proved to be effective in accelerating wound healing.19,20 Hydrogels can minimize hypoxia, which is a major problem in patients with burns, by providing a moist environment on the surface of the wound.21,22 The haemostatic potential of both chitosan and gelatin provide better healing by initiating the blood-clotting cascade Fibrin is an insoluble protein involved in the blood-clotting cascade Fibrin is developed in blood from a soluble protein called brinogen Fibrin is formed aer the thrombin-mediated cleavage of brinopeptide A from the alpha chains and brinopeptide B from the beta chains of brinogen This results in subsequent conformational changes and the exposure of polymerization sites, generating the brin monomer, which has a tendency to self-associate and form insoluble brin.23–29 Nanobrin has a high surface to volume ratio that enhances cell attachment, cell migration and cell proliferation.30 Other benets of nanobrin in the wound-healing process have been reported previously.31–34 We aimed to develop a chitosan–gelatin hydrogel formulation with nanobrin for use as bandages in the treatment of burn wounds These bandages are expected to protect wounds from further infection and to provide a better healing environment In vivo studies were carried out and evaluated histopathologically Experimental details (a) Materials Chitosan (MW 150 kDa, degree of deacetylation, 85%) was purchased from Koyo Chemical Ltd, Japan Minimum essential medium, thrombin and gelatin were purchased from SigmaAldrich Fibrinogen was purchased from Himedia (India) Acetic acid, sodium hydroxide and hen lysozyme were purchased from Qualigens (India) 40 ,6-Diamidino-2-phenylindole (DAPI), Alamar Blue, trypsin–EDTA, fetal bovine serum (FBS) and tetramethyl rhodamine iso-thiocyanate were obtained from Gibco, Invitrogen Corporation The chemicals were all used without any further purication (b) Fabrication of chitosan–gelatin hydrogel/nanobrin ternary composite bandages The chitosan hydrogel was prepared as reported previously.35 Chitosan solution was prepared by dissolving g of chitosan in 1% acetic acid solution at room temperature The solution was then ltered to remove any undissolved particles Chitosan hydrogel was prepared by increasing the pH of the chitosan solution to neutral by the addition of a 1% NaOH solution Unbound water was removed by centrifugation to obtain the chitosan hydrogel The gelatin solution was prepared by dissolving g of gelatin in 100 mL of water followed by mild heating at 50 C for The nanobrin was synthesized by a previously reported method and the prepared nanoparticles were suspended in distilled water and the probe sonicated for 20 min.30 The nanobrin suspensions were mixed with a : concentration of chitosan hydrogel and gelatin solution The mixture was vigorously stirred for h to obtain composite 65082 | RSC Adv., 2014, 4, 65081–65087 Paper bandages with 2% nanobrin incorporated into the chitosan– gelatin hydrogel Stirring encouraged the homogeneous distribution of the nanobrin and gelatin onto the chitosan hydrogel This suspension was poured into a Teon mould and stored overnight at À20 C The frozen samples were then lyophilized for 24 h (Martin Christ, Germany) to obtain the composite bandages (c) Characterization of composite bandages The lyophilized samples of chitosan bandages (CBs), chitosan– brin bandages (CFBs), chitosan–gelatin bandages (CGBs) and chitosan–gelatin hydrogel/nanobrin ternary composite bandages (CFGBs) were examined by SEM to determine their structural morphology (d) Swelling studies The swelling study was carried out in both PBS and distilled water The pH of the PBS solution was maintained at 7.4 Bandages of bare chitosan, CGB, CFB and CFGB were cut into small pieces and immersed in mL of the water and PBS solutions The samples were incubated at 37 C and, at denite time intervals, the samples were taken out of the falcon tube, gently blotted with lter paper and the wet weight recorded The swelling study in PBS was carried out for up to 12 days and that in water was performed for 48 h The swelling ability was evaluated by the formula:35 DS ¼ WW À WD  100 WD where DS is the degree of swelling and WW and WD represent the wet and dry weights of the bandages, respectively (e) Biodegradation studies The dry weight was determined in triplicate for each 0.5 cm  0.5 cm lyophilized sample Each sample was immersed in mL of the PBS–lysozyme solution and incubated at 37 C At denite time intervals the samples were removed and washed with PBS The study was carried out for up to weeks The samples were freeze-dried and the dry weight recorded The degradation was calculated by the formula:35 Degradationð%Þ ¼ Wi À Wt  100 Wt where Wi is the initial weight and Wt is the dry weight of the bandage aer lyophilization (f) Evaluation of porosity Cylinder-shaped CBs, CGBs, CFBs and CFGBs were prepared and the height and diameter of the bandages determined in triplicate using a vernier caliper to calculate the volume The bandages were immersed in absolute ethanol until saturated The weights of the bandages before and aer immersion in alcohol were recorded The porosity (P) was calculated using the formula:35 This journal is © The Royal Society of Chemistry 2014 View Article Online Paper RSC Advances P¼ W2 ÀW1 Â100 rV1 in the equation, W1 and W2 indicate the weight of bandages before and aer immersion in alcohol, respectively V1 is the volume before immersion in alcohol; r is a constant (density of alcohol) Published on 10 November 2014 Downloaded by test on 22/01/2016 14:48:10 (g) Whole-blood clotting Blood clotting was studied by drawing blood from the ulnar vein of human patients and anticoagulating with acidic citrate dextrose (20 mM citric acid, 110 mM sodium citrate, mM dextrose) at a ratio of : v/v Bare chitosan and Kaltostat were used as a control and all samples were in triplicate Citrated whole blood was dispensed onto the bandages and 10 mL of 0.2 M CaCl2 solution were added to initiate the clotting process The samples were incubated at 37 C for 15 By adding mL of water, the red blood cells that were not trapped on the bandages were haemolysed and washed out The absorbance of the supernatant was measured at 540 nm using a plate reader (BioTek PowerWave XS) to determine the blood-clotting ability.33 (h) Platelet activation studies Platelet-rich plasma was isolated from blood samples by centrifugation at 2500 rev minÀ1 for A 100 mL aliquot of platelet-rich plasma was added to 10 mg pieces of bandage and incubated at 37 C for 20 Bare chitosan and Kaltostat were used as controls The bandages were then washed with PBS solution and xed using a 0.1% glutaraldehyde solution The bandages were dried, xed on aluminium stubs and sputtercoated with gold for SEM imaging.33 (i) Cell viability using Alamar Blue assay An Alamar Blue assay was used to evaluate the cell viability of the prepared CFGBs on human dermal broblast (HDF) cells and human umbilical cord vein endothelial cells (HUVECs).30,32 The bandages were cut into small pieces and were sterilized by ethylene oxide gas The cell seeding density was  104 cells each for the HDF cells and HUVECs and they were incubated for up to 48 h The assay was performed by adding Alamar Blue into the plates containing the sterilized bandage materials and the cells The optical density was measured at 570 nm, with 620 nm set as the reference wavelength using a micro-plate spectrophotometer (Biotek PowerWave XS) (j) Cell adhesion and proliferation studies The cell morphologies of the HUVECs within the bandages were observed using SEM.30 The cells were seeded onto the CFGBs at a concentration of  105 cells per well Aer 24 and 48 h of incubation, the bandages were rinsed with PBS and xed with 2.5% gluteraldehyde for h The samples were thoroughly washed with PBS, dehydrated through a series of graded ethanol solutions and then air-dried Aer sputtering with gold in a vacuum, the samples were examined by SEM This journal is © The Royal Society of Chemistry 2014 For DAPI staining, the HUVECs were seeded onto the CFGBs and the bandages were xed with 4% paraformaldehyde for 20 DAPI is a uorescent stain that preferentially stains the nucleus of cells.32 Aer adding 0.5% Triton X-100 in PBS and incubating for min, the bandages were treated with 1% FBS and washed with PBS The cell-seeded bandages were stained with 50 mL of DAPI (1 : 30 dilution with PBS) The bandages were then incubated in darkness for and viewed under a uorescent microscope (Olympus-BX-51) (k) In vivo evaluation of wound healing The evaluation of wound healing in animals was approved by the Institutional Animal Ethical Committee, Amrita Institute of Medical Sciences and Research Center, Cochin, India SpragueDawley rats were kept under standard laboratory conditions The rats were randomly divided into ve groups of six rats On the day of burn creation, the rats were anaesthetized by an intramuscular injection of 35.0 mg per kg ketamine and 5.0 mg per kg xylazine The dorsal area of the rats was depilated and cleaned with Wokadine The burn was created using a copper bar with an area of 1.5 cm2 The copper bar was heated to 150 C using electric hot-plate and the temperature was monitored with a thermometer.38 The heated copper bar was placed on the depilated area for 15 s and the rats were housed individually On day aer burn creation, skin was removed surgically from the burnt area The wounds were then covered with the composite bandage materials and a secondary dressing This was sutured to ensure that the dressing materials were intact The rats were then housed individually Wound closure was noted each week by taking photographs and marking the wound area using a graph sheet Wounds treated with Gelspon were used as positive controls and bare wounds were used as negative controls Aer and weeks the wound tissue was excised for histological and collagen deposition analyses according to previously reported protocols.38,39 (l) Statistical analysis All the experiments were performed in triplicate and Student's t-test was performed to determine the statistical signicance p < 0.05 was considered statistically signicant Results and discussion Fig 1a shows the average hydrodynamic particle size distribution and SEM representation of the nanobrin component with an average particle size of 229 Æ nm and an average zeta potential of À34 mV Fig 1b shows representative photographs of the prepared composite bandage; the exibility of the bandage is clearly evident Fig 1c shows SEM images of the CGBs and CFGBs The SEM images show that all the bandages were porous enough to absorb the excess exudate and that the pore size was in the range 200–300 mm The porous structure of the CFGBs is retained even aer the incorporation of the nanobrin and gelatin The swelling study in PBS was carried out up to day 12 The swelling ratio analysis revealed that the swelling ability of the RSC Adv., 2014, 4, 65081–65087 | 65083 View Article Online Published on 10 November 2014 Downloaded by test on 22/01/2016 14:48:10 RSC Advances (a) Hydrodynamic size distribution of nanofibrin component and SEM image; (b) photographs of CFGBs; and (c) SEM images of composite bandages Fig bandages increased aer the incorporation of gelatin compared with bare chitosan; this is because the incorporation of gelatin can enhance the porosity by disrupting the porous structure There was no signicant difference in the swelling ratio of the CFGBs compared with the CGBs and CFBs The swelling study in water was carried out for 48 h All the bandages showed a swelling ratio from 10 to 15 and there was no difference in the swelling ratio aer the incorporation of gelatin and brin compared with the bare CBs The presence of nanobrin or gelatin had no impact on the swelling ratio of the composite bandages This might be because the concentration of nanobrin used was low (2% of the total bandage weight) The gelatin may have become incorporated within the chitosan polymers to form a well-integrated matrix The composite bandages therefore had the same swelling ratio even aer the addition of nanobrin and gelatin The degradation rate of the bandages in the in vitro biodegradation studies was greater in week than in week The CGBs showed a maximum degradation rate of around 65% The presence of gelatin may have reduced the strength of the chitosan matrix making it more susceptible to biodegradation than the chitosan control and bandages with incorporated nanobrin The gelling properties of gelatin also had an impact on the degradation prole The gelatin may have been easily degraded aer swelling However, the incorporation of brin reduced the degradation rate of the CFGBs In these samples, the pores of the bandages were occupied by brin nanoparticles that provided strength to the bandages This may be the reason for the reduced amount of degradation aer the incorporation of brin An alcohol displacement method was used to evaluate the porosity of the CFGBs.35 Aer the incorporation of gelatin, the bandages had a porosity in the range 60–70% This was significantly different from the porosity of bare chitosan and brinincorporated CBs The incorporation of gelatin may have altered the porous structure of the bandages, resulting in the high porosity of the gelatin-incorporated bandages There was no signicant change in the porosity aer the incorporation of 65084 | RSC Adv., 2014, 4, 65081–65087 Paper nanobrin compared with the gelatin bandages Therefore the bandages are capable of absorbing large volumes of exudate from the wound surface and may enhance the distribution of nutrients, provide gaseous exchange and thereby promote wound healing The haemostatic potential of the CFGBs was assessed Fig 2a shows representative photographs of blood clotting on the CFGBs The lower OD value indicates high blood clotting The CFGBs showed an enhanced blood-clotting ability compared with the Kaltostat and blank samples (Fig 2b) The chitosan control, CGB and CFGB samples showed the same level of blood-clotting potential as the controls Blood-clotting data for the chitosan control and the chitosan–nanobrin composite have been reported previously.31 The presence of brin and gelatin did not alter the haemostatic ability of chitosan This may be due to the availability of some positive charges on the chitosan matrix that were not decreased in the presence of nanobrin or gelatin The presence of brin and the positive charges of the chitosan trigger the blood-clotting cascade Blood clotting was also initiated by the activation of platelets by the composite bandages The activated platelets form a mesh with brinogen and other factors, which is eventually converted to a blood clot Platelet activation was analysed by SEM Platelet activation was observed on the CFGBs (Fig 2c) The data corresponding to the CBs, CFBs and Kaltostat have been reported previously.31 The activated platelets showed some deformation in their shapes and spread over all the bandage surface The interaction between the cationic chitosan and negatively charged blood results in greater blood clotting and therefore platelet activation.36–39 Cell viability analysis using HDF cells (Fig 3a) and HUVECs (Fig 3b) did not show any toxicity at 24 and 48 h CFGBs with 2% nanobrin showed 100% viability of HUVECs at both 24 and 48 h These results conrmed the cytocompatibility of the prepared CFGBs SEM was used for cell attachment studies on the bandages to determine the cytocompatibility of the bandages The SEM images showed that HUVECs were attached to the CFGBs and began to spread on the bandages aer 24 h of incubation The attached cells started proliferating aer 48 h of incubation, as conrmed by DAPI staining The images show that more cells Fig Blood-clotting analysis (a) Photographs of a piece of CFGB, blood on the CFGB and clotted blood on the CFGB (b) Whole-blood clotting evaluation of bandages (c) SEM image of platelet activation on the composite bandage This journal is © The Royal Society of Chemistry 2014 View Article Online Published on 10 November 2014 Downloaded by test on 22/01/2016 14:48:10 Paper Fig RSC Advances Cell viability of bandages using (a) HDF cells and (b) HUVECs Fig Comparison of cellular attachment of HUVECs on CFGBs as seen by SEM (upper panels) and subsequent cell proliferation visualized through DAPI nuclear staining (lower panels) Fig were attached on the CFGBs containing 2% nanobrin Fig shows the cell attachment and proliferation of HUVECs aer 24 and 48 h Fig shows closure of the wound area aer 1, 2, and weeks of treatment The wounds treated with the CFGB showed faster healing aer weeks than the wounds treated with the other bandages The rate of healing was further enhanced aer weeks for the CFGB group The rate of healing was 80% aer weeks and the healing was completed at week for the wounds treated with the CFGBs (Fig 6) The presence of gelatin improved the healing rate, perhaps as a result of the migration and proliferation of keratinocytes and broblasts on the wound site.40 Matrix-assisted cell migration and the enhanced deposition of collagen is a related phenomenon that is promoted by the cumulative action of the inherent growth factors in the system and the effect of brindegraded products from the matrices Fig shows the haematoxylin–eosin stained images of the wound tissue excised aer and weeks The wounds treated with the CFGBs showed intact re-epithelialization and enhanced formation of epidermis Aer weeks the reepithelialization was complete in the groups treated with This journal is © The Royal Society of Chemistry 2014 Photographs of the closure of the burn wound areas Fig Measurement data for wound area closure Group 1, bare wound; 2, chitosan control; 3, chitosan–fibrin; 4, chitosan–fibrin– gelatin; and 5, Gelspon (positive control) Fig Haematoxylin–eosin stained images of wound tissue after and weeks the composite bandages compared with the other groups The CFB group also showed improved re-epithelialization Chitosan assisted the migration of broblasts and keratinocytes to the wound site, helping to improve reepithelialization and collagen deposition.9,33,37 Fig shows the Picro-Sirius red stained images of the wound tissue excised aer and weeks The composite bandages showed enhanced collagen deposition on the wound sites compared with the controls The presence of gelatin, chitosan and RSC Adv., 2014, 4, 65081–65087 | 65085 View Article Online RSC Advances Paper Mr Sajin P Ravi for his help with the SEM analysis We are grateful to the Amrita Centre for Nanosciences and Molecular Medicine for infrastructure support Notes and references Fig Picro-Sirius red stained images of wound tissue after and Published on 10 November 2014 Downloaded by test on 22/01/2016 14:48:10 weeks brin enhanced the migration of keratinocyte and broblast cells to the wound site, which eventually assisted the deposition of collagen on the wound site The collagen deposition improves the intactness and elasticity of skin tissues Fibrin is a biopolymer naturally synthesized during the woundhealing cascade and serves in situ as a reservoir of growth factors for proliferating cells The degradation of brin from the wound site is therefore in synergy with the formation of native tissue The wound-healing process is therefore triggered by the addition of the nanobrin component to the matrices Conclusion Several research approaches have been used to improve the functionality of chitosan-based wound-dressing materials to enhance the regeneration of skin tissue at chronic burn wound sites We fabricated composite bandages of chitosan, gelatin and brin, which were superior to bare chitosan and commercially available Gelspon and Kaltostat bandages The prepared CFGBs were macro-porous, biocompatible and biodegradable and were able to absorb excess exudate from the wound interface They also had adequate exibility and tensile strength The enhanced blood clotting and platelet activation seen with the CFGBs showed their potential as an effective wound-dressing material for patient with bloodclotting disorders In vivo evaluation of the CFGBs in healing burn wounds in Sprague-Dawley rats showed their efficacy compared with control wound-dressing materials The enhanced deposition of collagen and re-epithelialization with the formation of intact mature epidermis was seen in the CFGB-treated animal groups compared with the experimental controls These research ndings suggest that the composite CFGBs prepared in this study support the healing process and regeneration of skin tissue in chronic burn wound sites Acknowledgements The authors are grateful to the Department of Biotechnology, India for nancial support under grant BT/PR6758/NNT/28/620/ 2012 (dated 14-08-2013) P.T Sudheesh Kumar and G Praveen acknowledge the Council of Scientic and Industrial Research, India for Senior Research Fellowships We are also grateful to 65086 | RSC Adv., 2014, 4, 65081–65087 N K Upadhyay, R Kumar, S K Mandotra, R N Meena, M S Siddiqui, R C Sawhney and A Gupta, Food Chem Toxicol., 2009, 47, 46 J Somboonwong, M Kankaisre, B Tantisira and M H Tantisira, BMC Complementary Altern Med., 2012, 12, 103 C Alemdaroglu, Z Degim, N Celebi, F Zor, S Ozturk and D Erdogan, Burns, 2006, 32, 319 R Jayakumar, M Prabaharan, P T S Kumar, S V Nair and H Tamura, Biotechnol Adv., 2011, 29, 322 S Aoyagi, H Onishi and Y Machida, Int J Pharm., 2007, 330, 138 D Hu, Z Zhang, Y Zhang, W Zhang, H Wang, W Cai, X Bai, H Zhu, J Shi and C Tang, Burns, 2012, 38, 702 T Wanga, X Zhub, X Xuea and D Wua, Carbohydr Polym., 2012, 88, 75 J O Kim, Biol Pharm Bull., 2008, 31, 2277 S Y Ong, J Wu, S M Moochhala, M H Tan and J Lu, Biomaterials, 2008, 29, 4323 10 R Jayakumar, M Prabaharan, S V Nair and H Tamura, Biotechnol Adv., 2010, 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Chitosan hydrogel was prepared by increasing the pH of the chitosan solution to neutral by the addition of a 1% NaOH solution Unbound water was removed by centrifugation to obtain the chitosan hydrogel