Flexible, micro-porous chitosan–gelatin hydrogel/nanofibrin composite bandages for treating burn wounds

Abstract

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 ± 3 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 acid-hematoxylin 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 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 groups treated with the nanocomposite bandages compared with the experimental controls. These results show that these ternary nanocomposite bandages are ideal candidates for burn wound dressings.

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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, inflammation, matrix synthesis and deposition, angiogenesis, fibroplasia, 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 often painful to the patient.^5–7 To avoid this discomfort, we developed a novel hydrogel-based chitosan–gelatin bandage incorporating nanofibrin 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 blood-clotting 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 flow 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 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 fibrinogen. Fibrin is formed after the thrombin-mediated cleavage of fibrinopeptide A from the alpha chains and fibrinopeptide B from the beta chains of fibrinogen. This results in subsequent conformational changes and the exposure of polymerization sites, generating the fibrin monomer, which has a tendency to self-associate and form insoluble fibrin.^23–29 Nanofibrin has a high surface to volume ratio that enhances cell attachment, cell migration and cell proliferation.³⁰ Other benefits of nanofibrin in the wound-healing process have been reported previously.^31–34

We aimed to develop a chitosan–gelatin hydrogel formulation with nanofibrin 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 Sigma-Aldrich. Fibrinogen was purchased from Himedia (India). Acetic acid, sodium hydroxide and hen lysozyme were purchased from Qualigens (India). 4′,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 purification.

(b) Fabrication of chitosan–gelatin hydrogel/nanofibrin ternary composite bandages

The chitosan hydrogel was prepared as reported previously.³⁵ Chitosan solution was prepared by dissolving 2 g of chitosan in 1% acetic acid solution at room temperature. The solution was then filtered 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 1 g of gelatin in 100 mL of water followed by mild heating at 50 °C for 1 min. The nanofibrin 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 nanofibrin suspensions were mixed with a 1 : 1 concentration of chitosan hydrogel and gelatin solution. The mixture was vigorously stirred for 1 h to obtain composite bandages with 2% nanofibrin incorporated into the chitosan–gelatin hydrogel. Stirring encouraged the homogeneous distribution of the nanofibrin and gelatin onto the chitosan hydrogel. This suspension was poured into a Teflon mould and stored overnight at −20 °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–fibrin bandages (CFBs), chitosan–gelatin bandages (CGBs) and chitosan–gelatin hydrogel/nanofibrin 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 5 mL of the water and PBS solutions. The samples were incubated at 37 °C and, at definite time intervals, the samples were taken out of the falcon tube, gently blotted with filter 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
where DS is the degree of swelling and W_W and W_D 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 5 mL of the PBS–lysozyme solution and incubated at 37 °C. At definite time intervals the samples were removed and washed with PBS. The study was carried out for up to 2 weeks. The samples were freeze-dried and the dry weight recorded. The degradation was calculated by the formula:³⁵
where W_i is the initial weight and W_t is the dry weight of the bandage after 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 after immersion in alcohol were recorded. The porosity (P) was calculated using the formula:³⁵
in the equation, W₁ and W₂ indicate the weight of bandages before and after immersion in alcohol, respectively. V₁ is the volume before immersion in alcohol; ρ is a constant (density of alcohol).

(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, 5 mM dextrose) at a ratio of 8 : 2 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 μL of 0.2 M CaCl₂ solution were added to initiate the clotting process. The samples were incubated at 37 °C for 15 min. By adding 2 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 min⁻¹ for 5 min. A 100 μL aliquot of platelet-rich plasma was added to 10 mg pieces of bandage and incubated at 37 °C for 20 min. Bare chitosan and Kaltostat were used as controls. The bandages were then washed with PBS solution and fixed using a 0.1% glutaraldehyde solution. The bandages were dried, fixed on aluminium stubs and sputter-coated 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 fibroblast (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 5 × 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 1 × 10⁵ cells per well. After 24 and 48 h of incubation, the bandages were rinsed with PBS and fixed with 2.5% gluteraldehyde for 1 h. The samples were thoroughly washed with PBS, dehydrated through a series of graded ethanol solutions and then air-dried. After sputtering with gold in a vacuum, the samples were examined by SEM.

For DAPI staining, the HUVECs were seeded onto the CFGBs and the bandages were fixed with 4% paraformaldehyde for 20 min. DAPI is a fluorescent stain that preferentially stains the nucleus of cells.³² After adding 0.5% Triton X-100 in PBS and incubating for 5 min, the bandages were treated with 1% FBS and washed with PBS. The cell-seeded bandages were stained with 50 μL of DAPI (1 : 30 dilution with PBS). The bandages were then incubated in darkness for 5 min and viewed under a fluorescent 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. Sprague-Dawley rats were kept under standard laboratory conditions. The rats were randomly divided into five 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 3 after 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. After 2 and 4 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 significance. p < 0.05 was considered statistically significant.

Results and discussion

Fig. 1a shows the average hydrodynamic particle size distribution and SEM representation of the nanofibrin component with an average particle size of 229 ± 7 nm and an average zeta potential of −34 mV. Fig. 1b shows representative photographs of the prepared composite bandage; the flexibility 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 μm. The porous structure of the CFGBs is retained even after the incorporation of the nanofibrin and gelatin.

Fig. 1 (a) Hydrodynamic size distribution of nanofibrin component and SEM image; (b) photographs of CFGBs; and (c) SEM images of composite bandages.

The swelling study in PBS was carried out up to day 12. The swelling ratio analysis revealed that the swelling ability of the bandages increased after 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 significant 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 after the incorporation of gelatin and fibrin compared with the bare CBs. The presence of nanofibrin or gelatin had no impact on the swelling ratio of the composite bandages. This might be because the concentration of nanofibrin 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 after the addition of nanofibrin and gelatin.

The degradation rate of the bandages in the in vitro biodegradation studies was greater in week 2 than in week 1. 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 nanofibrin. The gelling properties of gelatin also had an impact on the degradation profile. The gelatin may have been easily degraded after swelling. However, the incorporation of fibrin reduced the degradation rate of the CFGBs. In these samples, the pores of the bandages were occupied by fibrin nanoparticles that provided strength to the bandages. This may be the reason for the reduced amount of degradation after the incorporation of fibrin.

An alcohol displacement method was used to evaluate the porosity of the CFGBs.³⁵ After 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 fibrin-incorporated 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 significant change in the porosity after the incorporation of nanofibrin 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–nanofibrin composite have been reported previously.³¹ The presence of fibrin 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 nanofibrin or gelatin. The presence of fibrin 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 fibrinogen and other factors, which is eventually converted to a blood clot.

Fig. 2 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.

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% nanofibrin showed 100% viability of HUVECs at both 24 and 48 h. These results confirmed the cytocompatibility of the prepared CFGBs.

Fig. 3 Cell viability of bandages using (a) HDF cells and (b) HUVECs.

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 after 24 h of incubation. The attached cells started proliferating after 48 h of incubation, as confirmed by DAPI staining. The images show that more cells were attached on the CFGBs containing 2% nanofibrin. Fig. 4 shows the cell attachment and proliferation of HUVECs after 24 and 48 h.

Fig. 4 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. 5 shows closure of the wound area after 1, 2, 3 and 4 weeks of treatment. The wounds treated with the CFGB showed faster healing after 2 weeks than the wounds treated with the other bandages. The rate of healing was further enhanced after 3 weeks for the CFGB group. The rate of healing was 80% after 2 weeks and the healing was completed at week 3 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 fibroblasts 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 fibrin-degraded products from the matrices.

Fig. 5 Photographs of the closure of the burn wound areas.

Fig. 6 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. 7 shows the haematoxylin–eosin stained images of the wound tissue excised after 2 and 4 weeks. The wounds treated with the CFGBs showed intact re-epithelialization and enhanced formation of epidermis. After 4 weeks the re-epithelialization was complete in the groups treated with the composite bandages compared with the other groups. The CFB group also showed improved re-epithelialization. Chitosan assisted the migration of fibroblasts and keratinocytes to the wound site, helping to improve re-epithelialization and collagen deposition.^9,33,37 Fig. 8 shows the Picro-Sirius red stained images of the wound tissue excised after 2 and 4 weeks. The composite bandages showed enhanced collagen deposition on the wound sites compared with the controls. The presence of gelatin, chitosan and fibrin enhanced the migration of keratinocyte and fibroblast 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 wound-healing cascade and serves in situ as a reservoir of growth factors for proliferating cells. The degradation of fibrin 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 nanofibrin component to the matrices.

Fig. 7 Haematoxylin–eosin stained images of wound tissue after 2 and 4 weeks.

Fig. 8 Picro-Sirius red stained images of wound tissue after 2 and 4 weeks.

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 fibrin, 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 flexibility 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 blood-clotting 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 findings 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 financial support under grant BT/PR6758/NNT/28/620/2012 (dated 14-08-2013). P.T. Sudheesh Kumar and G. Praveen acknowledge the Council of Scientific and Industrial Research, India for Senior Research Fellowships. We are also grateful to 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

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Footnote

† Authors contributed equally.

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