Handling and managing bleeding wounds using tissue adhesive hydrogel: a comparative assessment on two different hydrogels

Abstract

The present study explores the preparation and a comparative assessment on the physical, mechanical and biological properties of two different tissue adhesive hydrogels (TAHs) for the management of bleeding wounds. TAHs were prepared through tethering of di-/tri-hydroxyl phenolics to a gelatin backbone and subjected to oxidation. It has been observed that all the physical, mechanical and biological properties are at significant levels upon tri-hydroxyl phenolic functionalization rather than di-hydroxyl phenolic functionalization. The considerable hemostatic activity and adhesive strength of the hydrogel upon functionalization with phenolic acids controls the blood loss and supports the early healing of bleeding wounds. In conclusion, functionalization with tri-hydroxyl phenolics imparts hemostatic, adhesive strength and tissue approximation properties of gelatin, which solves most of the problems associated with the management of bleeding wounds.

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1. Introduction

A clear understanding of the reaction mechanism of mussel adhesive mimics suggests that catechol-functionalized proteins, carbohydrates, and synthetic polymers, upon oxidation can perform as hemostatic tissue adhesive agents.^1–4 Based on this, further exploration on the functionalization of proteins using mono and dihydroxy phenolic acids has been initiated to obtain a hemostatic tissue adhesive gel. According to Jacob et al.⁵ an increase in the polyol content of the benzene ring-functionalized polymeric backbone improves the crosslinking density and thereby enhances the physical, mechanical and biological properties of the material. However, no literature guaranteed the use of phenolics of tri-hydroxyl or polyol functionalized polymer as a hemostatic tissue adhesive cum wound healing material.

With respect to wound and its management, the continuous research inputs by various researchers fetched numerous biomaterials in the form of 2D and 3D systems.^2,3 Apart from dermal wounds, internal injuries to the organ need high attention. Bleeding is the major problem associated with the management of internal wounds and demand hemostatic agents.^6,7 Accordingly, various hemostatic agents are available in the market and employed to arrest bleeding.^8–10 However, the demand is not only restricted to the styptic agent but also to the material which should provide the support in the form of adhesive and accelerate the tissue approximation. Though suturing does the effective job, the time consumption, pain and the need for skilled surgeons are the challenges in front.^10,11 For the management of bleeding wounds (external/internal) any biological materials instigating hemostasis followed by tissue approximation and wound healing would consider as the material of interest. Numbers of polymeric materials of natural,^12,13 synthetic¹⁴ and or composites^15,16 as biomaterials are considered as a savior in the life of the human.^17,18 Still the vast amount of research inputs on biomaterials is required to meet the various challenges like bleeding.⁸ On considering the importance of gelatin and its compatibility in the field of biomaterials research, in the present study, we have chosen gelatin as a backbone polymer of interest.

Concerning phenolic acids, protocatechuic acid (PA) and gallic acids (GA) are an endogenous plant phenolics found abundantly in gallnuts, grapes, tea, wines, hops and oak bark. Structurally they differ from each other by possessing an additional hydroxyl group in gallic acid.¹⁹ The presence of di and tri-hydroxyl functional group in protocatechuic acid and gallic acid in the meta and para position of the benzoic acid imparts several functional properties like antioxidant, anti-inflammatory, anti-viral and anti-cancerous activities.¹⁹ Because of these potential functionalities both the phenolic acids are widely used in foods, drugs, and cosmetics.

Hence, to disclose the role of polyphenol functionalized polymer in mimicking the mussel adhesive protein, the present study has been taken up to prepare the tissue adhesive hydrogel (TAH) using tri and di hydroxyl phenolic acids engineered gelatin. Further, a comparative assessment made on the physical, mechanical, biological and functional (hemostatic, adhesive and wound healing) properties of TAHs of both di and tri-hydroxyl phenolics functionalization under in vitro and in vivo conditions for exploration as a suitable material of interest.

2. Experimental section

Tethering of phenolic acids to gelatin

PEG (Protocatechuic acid Engineered Gelatin) and GEG (Gallic acid Engineered Gelatin) samples were prepared according to the procedures summarized by Thirupathi Kumara Raja et al.³ In brief, to 0.1 M GA (Gallic acid) or PA (Protocatechuic acid) dissolved in 25 mL of 0.1 M MES buffer (pH 5.5) containing EDC and NHS, twice the concentration of GA/PA respectively. The reaction mixture was stirred for 45 min at 25 °C and then mixed with the gelatin solution in HEPES buffer (pH 7.0). The whole reaction mixture was kept at stirring for overnight, and the resultant solution was alcohol precipitated and dissolved in water then freeze dried and stored at 4 °C.

Characterization studies on PEG and GEG

UV-visible, proton NMR, and CD spectroscopic analyzes were performed to confirm the tethering of PA and GA to the gelatin backbone, ¹H NMR spectra of gelatin, PEG, GEG, PA, and GA were recorded using JEOL ECA-500 FT NMR spectrometer. All the samples were dissolved in 1 mL D₂O individually, and ¹H-spectra were recorded at 45 °C (for better solubility) without suppressing the water signal. UV-visible spectra for gelatin, PA, GA, PEG and GEG samples were recorded using UV-vis-2450 (Shimadzu, Japan). In brief, samples (1 mg) were dissolved separately in 1 mL of phosphate buffer (pH 6.5). The spectrum was recorded in the wavelength region of 200 to 600 nm by keeping the buffer as a blank.

Circular Dichroism (CD) measurements of the individual protein as well as engineered protein samples were performed using Jasco J715 spectropolarimeter at 25 °C. In triplicates, the spectrums were measured and averaged, in the wavelength region of 190 and 300 nm.²⁰

The change in the apparent viscosity, surface charge and contact angle of gelatin, PEG, and GEG samples were measured according to the procedures summarized by Raja et al. (2014).²

Preparation of TAHs

Curing rate—tube inversion assay. The curing rate has been calculated from the time taken for the transformation of the solution form of an engineered gelatin to a gel state upon oxidation. In brief, 2 mL of 8, 10 and 12% (w/v), GEG and PEG solutions were taken separately in a 5 mL capacity glass vials and treated with sodium meta periodate. The time taken for gel formation was measured and the gels obtained were thus named as GEG-gel and PEG-gel respective to the substrates gallic acid engineered gelatin and protocatechuic acid engineered gelatin.

Rheological studies TAHs. PEG-gel and GEG-gel prepared at different concentrations (8, 10 & 12% (w/v)) were analyzed for the mechanical strength using oscillatory Anton Paar Rheometer MCR-301. The change in storage (G′) and loss modulus (G′′) of the gel at various frequencies were calculated. Experiments were performed in triplicate, and the average has been plotted. Based on the rubber elastic theory, the average mesh size (ξ) was calculated using the following formula²¹

ξ = (G′N_A/RT)^−1/3

The average molecular weight (M_c) between the crosslinks was also calculated from the following formula;

M_c = (cρRT/G′),

where G′, is the storage modulus, N_A, is Avogadro constant, R, is the molar gas constant, ρ, is the density of water and T, is the temperature (K).

Determination of adhesive strength TAHs: lap shear test. According to ASTM F2255-03 bonding strength of the fabricated adhesive product was determined using bovine skin model.²² Lap shear test was performed for both PEG-gel and GEG-gel with a dimension of 3 × 2 cm (L × B). To one end of the dermal side of the skin the sample was applied and above that another dermal layer has been placed and allowed to cure. To hold the tissues together, to the adhered layer a load of 100 g cm⁻² for 60 min was given. Later the tissue was processed for lap shear testing. Adhesive strength has been calculated based on the force required to detach the two different tissue surfaces.

Surface morphology of TAHs. The freeze dried PEG-gel and GEG-gel (at 10% w/v) were subjected to surface and cross-sectional morphology. The freeze dried sample were placed on the double adhesive carbon tape and gold coated, and the samples were subjected to scanning electron microscopy.

Cell adherence and cell proliferation properties of TAHs. Engineered gelatin gels were assessed for cell adherence and proliferation property using NIH 3T3 cell lines (the details on the cell line maintenance were given in the ESI file S1†). The study was carried out using PEG-gel and GEG-gel pre-coated culture plates, and the uncoated wells were taken as control. The control wells were free from the sample coating. Sterilization of culture plates was carried out using 70% alcohol for 30 min and UV treatment for 1 hour, further, the plates were washed with sterile PBS. Cells were seeded at the concentration of 3 × 10⁴ cells per well and incubated in the growth medium. At regular intervals (6, 12, 24 and 48 h) the samples were observed for cell adherence, viability, and proliferation property. Using MTT assay and live cell tracker assay the samples were analyzed for cell adherence and proliferation.

In vivo studies-maintenance of animals. All experiments were performed in compliance with the relevant laws and institutional guidelines. All experiments were carried out upon getting approval from the institutional ethical committee (vide no. 466/01a/CPCSEA–IAEC no. 08/02/2011b, 02/2014a). During experimentation, the animals were anesthetized with ketamine/xylazine solution (80–100 mg kg⁻¹ : 10–12 mg kg⁻¹ body weight) intraperitoneally. The wound area was sterilized using povidone iodine solution before preceding the experiments.

In vivo hemostasis. Concerning the ethical committee approval and suggestions hemostasis experiments were carried out only with GEG-gel and in vivo rat liver hemorrhaging model has been performed. According to the procedures reported by Choi et al.,⁷ abdominal surgery was carried out on the rat to expose its liver. The single lobe of the liver was placed on the filter paper, and liver hemorrhage was initiated by puncturing the liver with 21 G needle and left unattended for three minutes, the oozing blood was absorbed into the filter paper. For the experimental groups, bleeding was arrested by applying 250 μL of GEG/PEG solution and oxidized on site to form a gel. After one hour the filter paper, which absorbed the blood, was weighed and compared to the control (without GEG-gel treatment).

In vivo tissue approximation and wound healing studies. In vivo wound, healing property of GEG-gel was assessed by excision wound model. Eighteen albino rats with an average weight of 200 ± 25 g were segregated into three groups (Group I control; Group II GEG-gel treated; Group III fibrin sealant treated) and six animals in each group. A 4 cm² area (∼2.5 cm diameter) full thickness excision wound (circular) was made on the dorsal side of the rat. About 1 mL of GEG sample was applied to the wound site along with periodate (<50 μL) for in situ gelation. The positive control group receives 1 mL of fibrin sealant. The animals were observed periodically for 4, 8, 12, 16 and 21 days. The wound margin/area was traced on a transparent paper having a millimeter scale, and the reduction in wound size was measured planimetrically. The period of re-epithelialization was calculated as the number of days required for the wounds to heal completely without any raw wound left behind. For wound breaking strength, the tensile power of the healed tissues was measured.

Statistical analysis. All the experiments were performed in triplicate (n = 3) unless otherwise specified and the results were presented as mean ± SD. All the values were analyzed using the standard student t-test and the ‘p’ values smaller or equal to 0.05 were used as the criterion for a statistically significant difference.

3. Results and discussion

Tethering of phenolic acids to protein

Protocatechuic acid (di-hydroxy phenolic) (PA) and gallic acid (trihydroxy-phenolic) (GA) were tailored to the gelatin backbone through EDC/NHS chemistry. Briefly, the free –COOH of PA and GA was esterified with water soluble carbodiimide and stabilized with NHS, according to the procedure followed by Kuijpers et al.²³ pH optimization for esterification carried out based on the pK_a values of PA and GA. The esterified PA and GA were engineered with gelatin via a ε-NH₂ group of a lysine residue and N-terminal amines (Scheme 1) under optimized pH conditions, to prevent the auto-oxidation of PA and GA.

Scheme 1 Overall schematic representation of engineering the protein gelatin. The figures represent the reaction mechanism involved in the preparation of engineered gelatin using EDC/NHS chemistry. The tailored phenolic acid mimics the mussel adhesive proteins. Upon oxidation the engineered protein undergoes inter and intramolecular crosslinking with free amines (–NH₂) of gelatin in proximal and the reaction is mediated by (a) biaryl coupling; (b) Michaels addition reaction and (c) Schiff base formation.

Characterization studies

To confirm the integration of GA and PA to the gelatin backbone, UV, proton NMR and CD spectroscopic measurements were made. Results from the UV-visible spectra of PA and GA displayed maximum absorbance at 260 and 270 nm (Fig. 1a). However, gelatin does not show any significant peak at this wavelength. However, the tailored samples (PEG and GEG) displayed a new absorbance peak at 260 nm and 270 nm, which substantiates the integration/functionalization.

Fig. 1 (a) UV-visible spectra of gelatin, PA (Protocatechuic acid), GA (Gallic acid), PEG (Protocatechuic acid Engineered Gelatin) and GEG (Gallic acid Engineered Gelatin) showcasing the modification in gelatin (b) CD spectrum of gelatin, before and after oxidation of PEG and GEG (c) inverted tube assay displays the phase transformation of PEG and GEG solution determined at several concretions. Darkening of the reaction mixtures portrays the increase in the number of available phenolics at higher concentration (d) rate of curing of PEG and GEG at different concentrations upon oxidation with a mild oxidizing agent (all the values are the mean ± SD of three measurements, * indicates statistical significance, P < 0.01).

¹H NMR spectrum was recorded for the entire sample (ESI File Fig. S1†). ¹H NMR spectrum of PA and GA displayed chemical shifts at δ 7.4 (d, H4) and δ 7.1 (s, H2) respectively. The ¹H NMR spectrum of gelatin and the functionalized gelatin displayed broad chemical shifts from 7.2 to 7.4 corresponds to residual aromatic amino acid present in the protein backbone. Whereas the integrated samples PEG and GEG shown a significant change in the chemical shift at 7.4, and 7.1 ppm which corroborates with the spectrum of PA and GA.

To elucidate the change in the secondary structure of the gelatin before and after tailoring of PA and GA, we performed the Circular dichroism (CD) analysis (Fig. 1b). Gelatin alone exhibit a single negative minimum band intensity at 200 nm reasoned to the unordered structure. Upon conjugation of PA/GA to the gelatin backbone, a decrease in band intensity at 200 nm for PEG samples, and an increase in band intensity for GEG samples suggest that the tailoring of PA and GA to the gelatin backbone increases the denaturation and unordered structure of the protein. Further, when PEG and GEG samples subjected to oxidation, a change in the spectral intensity observed might be reasoned to the crosslinking of the protein.

Table 1 depicts the physical characteristics (viscosity, contact angle, and surface charge) of the integrated samples. The viscosity of the integrated samples showed 45 and 69% decrease respective to PA and GA on comparing with gelatin. The difference in the percentage decrease reasoned to the poly-ol content (hydroxyl groups) of gelatin backbone upon conjugation. In brief, the ortho and para directing hydroxyl groups of PA and the presence of three hydroxyl groups in the meta and para position of GA, involved in the hydrogen bonding interaction with water and thereby decreases the viscosity.

Table 1 Physical characterization of gelatin, PEG (Protocatechuic acid Engineered Gelatin) and GEG (Gallic acid Engineered Gelatin)

Sample	Viscosity (cP)	Surface charge (meV)	Contact angle (θ°)
Gelatin	35.2 ± 0.8	7.8 ± 0.7	82.7 ± 2.3
PEG	19.2 ± 1.2	6.4 ± 0.9	66.25 ± 0.8
GEG	10.9 ± 0.7	5.8 ± 0.9	36.15 ± 1.6

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Similarly, we observed a reduction in surface charge (18.0 and 35.6%) measured in terms of zeta potential in the integrated samples in comparison with gelatin alone. During integration, the esterified PA and GA conjugate to the primary NH₂ group of the gelatin and thereby decreases the number of free amines, which are all responsible for the positive charge of the protein. Similar results have been observed during the conjugation of thiols to the gelatin backbone.²⁴

Contact angle measurements, in general, reveal the wettability of the material. In the present study, contact angle measurements made for the integrated samples suggest that the angle of wettability as (θ°) 82.6 ± 2.3, 66.2 ± 0.8, and 36.15 ± 1.6 for gelatin, PEG, and GEG samples respectively. A significant deviation in the theta value of 90° infers that integration of either PA or GA increases the wettability at considerable level due to the presence of hydroxyl groups. Further, a difference in the contact angle between PA and GA attributed to the variation in the solvent activity coefficient of PA and GA. The solvent activity coefficient of PA and GA was found to be 0.11 and 0.002 respectively, and higher the solvent activity lower is the solubility and vice versa.²⁵ Xi Yang et al.,⁴ made similar observations in the dopamine-coupled human gelatin samples.

Preparation of PEG/GEG-gel

Tissue adhesive hydrogel (PEG-gel/GEG-gel) of di and tri-hydroxyl phenolics functionalized gelatin was prepared in the presence of oxidizing agent at 2% (w/v). Optimization of the concentration of PEG or GEG made based on the time taken for the transformation of the solution to gel state in the presence of the oxidizing agent. Scheme 1 represents the possible cross-linking mechanisms involved in the hydrogel transformation. The curing time, i.e., the time was taken by the engineered protein solution to transform into a hydrogel was calculated by tube inversion assay as shown in Fig. 1c. It has been observed that the gelation time decreases with an increase in the concentration of the engineered protein. However, a significant difference in the gelation time was found between PEG and GEG samples. At 10% concentration, gelation of GEG sample was within five seconds, whereas PEG samples took more than 50 s (Fig. 1d). The difference in gelation could be reasoned to the nature of the phenolics tethered to gelatin. The number of hydroxyl groups in GA and PA also determines the effective intermolecular interactions. According to Jacob et al.⁵ an increase in the poly-ol content of the aromatic acid increases the crosslinking density of proteins. Hence, the gelation time of GEG is much lower than the PEG.

Rheological characterization of TAH

The phase transition observed in the present study was further confirmed by rheological analysis. The rheological analysis was performed at 37 °C with 10% strain (since 10% strain falls within the linear viscoelastic region) and by varying the oscillatory frequency. The storage/elastic (G′) and loss/viscous modulus (G′′) were calculated. When the concentration of engineered gelatin varied from 8 to 12%, a significant increase in storage modulus was observed (Fig. 2a). The observed storage modulus of both the PEG-gel and GEG-gel was comparatively higher (1460 Pa) than the hydrogel obtained from DOPA-modified gelatin (10 Pa) upon cross-linking with tyrosinase and Fe³⁺.⁷

Fig. 2 (a) Rheological spectra of PEG-gel (Protocatechuic acid Engineered Gelatin-gel) and GEG-gel (Gallic acid Engineered Gelatin-gel) obtained at various concentrations. Values represent the storage modulus of the gel studied under parallel plate geometry. (b) Lap-shear stress showcases the adhesive strength of PEG-gel and GEG-gel studied at different concentrations under in vitro conditions (* indicates statistical significance, P < 0.01). (c) Scanning electron microscopic image demonstrates the cross-sectional morphology of PEG-gel and GEG-gel prepared at 10% (w/v) concentration. The scale bar measures 300 μm.

Affine network model has been adopted to establish the relationship between the concentration of engineered protein, gel strength, and network structures. The mesh size and the average molecular weight between the crosslinks were calculated based on the peak storage and loss modulus of the engineered gelatin gel. The results (ESI file, Table S1†) obtained for PEG-gel emphasizes that the mesh size has a direct relationship with the concentration of PEG. When the engineered protein concentration increases, a proportionate decrease in mesh size was observed. However, in the case of GEG-gel, there was no relationship in mesh size respectively to the concentration, and this could be reasoned to the increased cross-linking density and gel strength observed even at a lower concentration of GEG.

Adhesive strength of TAHs—in vitro assessment

The adhesive power of the engineered gelatin gel was assessed under in vitro lap shear test using bovine skin model (Fig. 2b). Adhesive strength increases with an increase in the concentration of engineered protein from 8 to 10% (w/v). Further increase in the concentration reduces the adhesive strength significantly. On comparing the adhesive strength of PEG-gel and GEG-gel, a significant difference was observed. The maximum adhesive strength of 35.1 kPa was observed with GEG-gel prepared at 10% concentration, and it was only 28.3 kPa for PEG-gel of the same concentration. According to Petrie,²⁶ adhesive strength depends on both the adhesive and the cohesive forces, and in the present study, an increase in the cohesive strength and adhesive strength for GEG-gel reasoned to the optimum cross-linking density between the tissues and the adhesive. Yan et al., reported, that oxidative crosslinking of walleye pollack skin in the presence of gallic acid and rutin suggested that rutin shows higher crosslinking density than gallic acid due to poly hydroxyl groups available to interact with the skin protein.²⁷ Similarly, in the present study, the presence of an additional hydroxyl group in GEG in comparison with PEG can be able to advance the Schiff base formation with the primary amines of tissue proteins and gelatin results in the increased gel strength.

Surface morphology of TAHs

Scanning electron microscopic images of TAHs of PEG and GEG revealed the presence of a well defined three-dimensional matrix with porous structure attributed to the intra and intermolecular cross-linking (Fig. 2c). The porous structure facilitates the water absorption and promotes cell adherence, proliferation and molecular diffusion as evidenced from the in vitro and in vivo studies.

Cell adhesion and proliferation profile of TAHs

The property of cell adherence and proliferation of a biomaterial is essential when in vivo application studies are concerned. In the present study, cell adherence and cell proliferation potential of PEG-gel and GEG-gel were assessed using NIH 3T3 cells. Fig. 3a illustrates the fluorescence image of the cells observed at different time intervals. In the present study, no significant difference in fluorescence intensity in the engineered gelatin gel treated cells compared to the control. Furthermore, no morphological changes were observed in the PEG-gel and GEG-gel treated cells. Results of cell viability and quantification analyzes showed 100% cell viability in both the samples suggested that engineering of protocatechuic acid and gallic acid to the gelatin do not have any negative impact on cell viability (Fig. 3b). According to the reported literature, phenolic acids can effectively reduce the levels of apoptosis and increases the cellular viability. Hence, engineering of the phenolic acids to the gelatin backbone does not provoke any toxicity.^28,29

Fig. 3 (a) Cytocompatibility assessments of the samples (PEG-gel and GEG-gel) through live cell tracker assay using calcein as a fluorescent probe demonstrates the cell adherence and proliferation observed at different time intervals in comparison with control (uncoated wells). The scale bar measures 10 μm. (b) Cytotoxicity assessment of the samples (PEG-gel and GEG-gel) using MTT assay by measuring the absorbance at 570 nm in comparison with control.

In vivo studies

Management of bleeding wound using TAHs. To examine the efficacy of the GEG-gel as an effective hemostatic agent, in the present study, in vivo rat liver hemorrhaging model was used. Effective hemostatic agents should be in the form of liquid and must rapidly solidify in the presence of external triggers/agents or in the presence of blood components. Fig. 4 showed the hemostatic effect of GEG-gel compared to the untreated wound. In situ gelation within 5 seconds arrests the bleeding at an appreciable level. Interestingly, the weight of the blood loaded paper for the untreated animal was 43.4 ± 0.5 mg, whereas it was only 5.0 ± 0.8 mg for GEG-gel treated animal. Similar observations were made for PEG-gel treatments. Irrespective of the type of the sample, the engineered protein solutions transformed to an adhesive gel within 5 seconds and arrested the bleeding accordingly.

Fig. 4 A representative image of the hemostatic activity of GEG-gel (Gallic acid Engineered Gelatin-gel) studied under in vivo rat liver hemorrhaging model (* indicates statistical significance, P < 0.01).

Wet tissues approximation—in vivo open wound healing assay. In clinics, the purpose of wound dressings is to protect the wound from the open atmosphere, to absorb wound exudates and maintain the moisture content. According to Balakrishnan et al.,³⁰ moist environment makes the wound cool and reduces the pain. GEG-gel is composed of several three-dimensionally cross-linked network structure of gelatin (the hydrolyzed matrix protein) which holds water molecules and act as a pseudo extracellular matrix. This structure may facilitate the intrusion of different cells (like fibroblast) and the movement of extracellular fluid within the network structures during the stages of wound healing. A full thickness excision (circle) wound healing model, was performed in albino rats and GEG-gel was applied. Commercially available fibrin sealant was taken as a standard positive control. The reduction in wound area was assessed and compared with the untreated groups. It has been observed that >97% of wound area was closed on day 16 for GEG-gel treated groups, whereas it was only >85% for fibrin sealant and >70% for untreated groups (Fig. 5a and b).

Fig. 5 (a) Comparative assessment of healing pattern of full thickness circle shaped wound (4 cm²) concerning control (untreated), GEG-gel treated, and fibrin sealant treated groups on different experimental days 0, 4, 8, 12, 16 and 21 in albino rats (n = 6). (b) Reduction in wound area measured planimetrically and compared with the all the treatments. (c) The tensile strength of the healed wound of untreated, GEG-gel and standard fibrin glue treated wound (* indicates statistical significance, P < 0.01).

Similarly, GEG-gel treated wound shows a significant increase in the tensile strength of the healed skin. The tensile strength values measured were 1350, 740 and 1050 kPa respectively for GEG-gel treated, untreated, fibrin sealant treated wound respectively (Fig. 5c). The overall assessment suggests that GEG-gel promotes wound healing at an appreciable level.

Comparisons on salient features of phenolic acid tethered gelatin. Table S2 (ESI file†) depicts the overall comparative assessment made for the phenolic acids engineered protein hydrogel. Though the present study emphasized gallic acid and protocatechuic acid engineered gelatin, our previous results on caffeic acid (CEG)^3,31 has also been considered for a better understanding of the benefits of phenolic acids tethered gelatin hydrogel. It has been observed that all the three phenolic acids well tethered with gelatin after activation with EDS-NHS. The number of free hydroxyl groups and the molecular structure of the phenolic acids tethered to the gelatin backbone determine the various physical and mechanical properties. The curing time, i.e., less than 4 s shown by GEG reasoned to the presence of the three hydroxyl groups. When comparing all the characteristic features such as gel strength, adhesive strength, swelling ratio, hemostatic, adhesive and wound healing properties, GEG samples ranks first followed by di-hydroxy phenolic acid functionalized samples (PEG and CEG). Reports of Temdee et al., and Mehanna et al. also corroborates well on the increased cross-linking density of gallic acid observed from the present study.^32,33

4. Conclusions

The present study explores preparation, characterization and application of tissue adhesive hydrogel (TAH) for the management of bleeding wounds. TAH samples were prepared using phenolic acid functionalized gelatin upon oxidation. Two different phenolic acids (di-hydroxyl and tri-hydroxyl) were taken for the study and comparative assessment of physical, mechanical and biological properties made following the standard protocols and procedures. The concentration of the engineered protein and the number of hydroxyl groups determines the time taken for the formation of an adhesive hydrogel. From all the experimental analyzes it has been concluded that tissue adhesive hydrogel prepared from the tri-hydroxy phenolics shows maximal benefit on comparing with di-hydroxyl engineered gelatin. It has been observed that (i) in situ transformation of solution form to gel state abruptly controls the bleeding at the time of application; (ii) the formation of quinone moieties offered the adhesive property (interaction with tissue proteins); and (iii) the hydrophilic, antioxidant and the biocompatibility nature of the gel provide the moist environment and enhances the synthesis of required ground substances and finally accelerate the healing of open wound. Thus, the present study emphasizes the management of bleeding wounds using tissue adhesive hydrogel prepared from phenolic acids engineered protein.

Acknowledgements

The authors thank DBT, New Delhi for funding this project in the form of TATA Innovation Fellowship. The first author thanks, Council of Scientific and Industrial Research (CSIR, New Delhi) for the Senior Research Fellowship. The second author thanks CSIR, New Delhi for the financial assistance, in the form of Research Associate-ship. Thanks are also to Mr. R. Mohan, SDDC, CSIR-CLRI, for his help with the mechanical testing of the samples. Thanks are also to Mr. V. Elango, Senior Technician, Animal House facility, CSIR-CLRI, Adyar, Chennai for his help and support during the animal experiments.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00284f

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