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Triphala Incorporated Collagen Sponge—A Smart Biomaterial for Infected Dermal Wound Healing
Journal of Surgical Research 158, 162–170 (2010) doi:10.1016/j.jss.2008.07.006
Triphala Incorporated Collagen Sponge—A Smart Biomaterial for Infected Dermal Wound Healing Muthusamy Senthil Kumar, Ph.D.,* Shanmugam Kirubanandan, M.Tech.,† Ramasamy Sripriya, Ph.D,* and Praveen Kumar Sehgal, Ph.D*,1 *Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai, India; and †Centre for Biotechnology, Anna University, Adyar, Chennai, India Submitted for publication May 21, 2008
Background. Wound infection is a major problem in the medical community since many types of wounds are more prone to microbial contamination leading to infection. Triphala (a traditional ayurvedic herbal formulation) incorporated collagen sponge was investigated for its healing potential on infected dermal wound in albino rats. Materials and Methods. Methanol extract of triphala was prepared and analyzed for the presence of catechin by high-pressure liquid chromatography analysis. Collagen sponge was prepared by incorporating triphala into collagen sponge. The triphala incorporated collagen was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, and water uptake analysis. Infected wound was dressed with triphala incorporated collagen sponge. Wound reduction rate, collagen content, and matrix metalloproteinases in the granulation tissue, histology, and Fourier transform electron microscopy analysis were done to obtain the healing pattern. Results. High-pressure liquid chromatography analysis showed the presence of (-)epigallocatechin gallate. FT-IR spectroscopy study revealed the interaction of polyphenols with the collagen. Triphala incorporated collagen sponge has shown to increase thermal stability and water uptake capability, faster wound closure, improved tissue regeneration, collagen content at the wound site, and supporting histopathological parameters pertaining to wound healing. Matrix metalloproteinases expression was correlated well with reduction in the inflammatory phase, thus confirming efficacy of the dressing.
1
To whom correspondence and reprint requests should be addressed at Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai-600 020, India. E-mail: [email protected].
0022-4804/10 $36.00 Ó 2010 Elsevier Inc. All rights reserved.
Conclusions. Better healing efficacy of triphala incorporated collagen sponge may provides a scientific rationale for the use of this dressing as an effective wound cover in the management of infected dermal wound. Ó 2010 Elsevier Inc. All rights reserved. Key Words: collagen sponge; drug delivery; triphala; wound infection.
INTRODUCTION
From 1970s, the processing technology of collagen has been improved, and new medical-grade collagen products are successfully launched in the market [1]. Currently, collagen scaffolds are employed in tissue engineering such as cartilage, bone, nerve, and skin as a support for cell mobility, infiltration, proliferation, and differentiation [2–4]. Collagen in the form of sponge is useful in the treatment of different wounds, such as pressure sores, donor sites, leg ulcers, and decubitus ulcers, as it adheres well to wet wounds, absorbs large quantities of tissue exudates, preserve a moist environment, and encourages the formation of new granulation tissue and epithelium on the wound [5, 6]. Collagen itself may not facilitate the healing of an infected wound because it is protein in nature and bacteria can use it as a substrate. At this stage, the imbalance between host resistance and bacterial growth leads to infection on the wound, and bacterial infection impairs healing through several mechanisms. In severe wound infection, systemic administration of drugs may lead to insufficient drug concentration reaching the site of infection, drug associated side effect, and systemic toxicity. This has been overcome successfully by topical application of drugs, and collagen dressings with
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antibiotics have also been developed to control infection [7–9]. Development of bacterial resistance to antibiotics continues, and the use of new antimicrobial agents is increasing [10]. Triphala, a compound formulation of the herb Terminalia chebula, Phyllanthus emblica, and Terminalia bellerica has been described in the Ayurveda (ancient Indian system of medicine) as a ‘‘tridoshic rasayan,’’ having balancing and rejuvenating effects on the 3 constitutional elements that govern human life [11]. Triphala is 1 of the important antioxidant rich rasayana drugs that has been reported to treat chronic ulcers [12]. Triphala and/or its constituents have been reported to possess antibacterial, antifungal, antiviral, and antiallergic activities [13–16]. The potential use of triphala on infected full-thickness wound healing was described in our earlier study [17]. In this article, triphala extract was incorporated in collagen sponge and its efficacy was evaluated in healing of infected dermal wounds.
MATERIALS AND METHODS
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echin (C), (–)epicatechin (EC), and(–)epigallocatechin gallate (EGCG; Sigma, St Louis, MO)].
Preparation of Collagen Sponge Type I collagen was extracted from bovine Achilles tendon using the procedure reported earlier [20]. In brief, clean Achilles tendons were minced below 25 C and washed with cold distilled water. The minced tissue was subjected to subsequent chemical and enzyme treatment and pure collagen was extracted. 1% wt/vol aqueous solution of pure Type I collagen (in 0.1% 12N HCl, pH 2.5) was prepared and agitated with 0.1% vol/vol nonionic wetting agent. The frothy mass was poured into Teflon trays and allowed to air-dry [21].
Collagen Sponge with Triphala Triphala extract was redissolved in methanol and incorporated in to the collagen sponge at the concentration of 50 mg/cm2. The sponge was allowed to dry in laminar airflow chamber.
Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra were recorded in a Thermo Nicolet avatar 320 FTIR spectrometer (Nicolet Instrument Con., Madison, WI) from collagen sponge dressing (CD) and triphala incorporated collagen sponge dressing (TD). All spectra were recorded from 400 to 4000 cm1 with a resolution of 4 cm1.
Preparation of Alcohol Extract of Triphala
Differential Scanning Calorimetry (DSC)
Methanol extract of triphala (IMPCOPS Ltd., Chennai, India) was prepared as described earlier [18].
DSC of the soluble collagen sponge and triphala incorporated collagen sponge were recorded using differential scanning calorimeter (DSC 204). Samples were sealed in aluminium pan and an empty pan was used as a reference. The heating rate 1 K (Kelvin) per min and temperature range between –5 C and 200 C in an N2 atmosphere were maintained.
Determination of Catechins The preliminary purification of catechins from the crude extract was achieved using counter-current chromatography with water/ chloroform (1:1, vol/vol) and water/ethyl acetate (1:1, vol/vol) solvent systems [19]. Catechin compounds that migrated into the ethyl acetate layer were collected and dried in a rotary evaporator. The crude catechins (0.5 g) were applied onto a chromatographic column with Sephadex LH-20 (Sigma, St. Louis, MO) equilibrated with methanol. The column dimensions were 1.6 3 90 cm and the flow rate was 1.2 mL/min. The fractions (8 mL) were collected and analysis of elutes from each tube was performed by measuring their absorbance at 280 nm. The contents were pooled and evaporated in a rotary evaporator. The dried extract dissolved in high-pressure liquid chromatography (HPLC) grade methanol was used for HPLC analysis. The HPLC system consisted of a Waters (Milford, MA) pump equipped with UV-VIS detector, model Shimadzu SPD 10A (Shimadzu, Kyoto, Japan), a rheodyne sample injector with a 20 mL sample loop. The column was a Spherisorb-ODS2 RP-18 column (25 cm long 3 4.6 mm i.d. and 5 mm particle diameter; Waters) equipped with a guard column (1.0 cm long 3 4.6 mm i.d., Varian). Detection was carried out by measurement of UV absorbance at 280 nm. The mobile phase was composed of water/ acetonitrile/methanol/ethyl acetate/glacial acetic acid (89:6:1:3:1 vol/vol/vol/vol/vol). Before the use, the mobile phase was filtered through a G5 sintered glass filter and then degassed for 30 min in an ultrasonic bath. Using sample injector 20 mL of sample was injected to the sample injection port. The mobile phase flow rate started at 0.7 mL/min and was maintained for 15 min, and then linearly increased to 1.2 mL/min in 1 min. This flow rate was maintained for 8 min, and then decreased to 0.7 mL/min. All chromatographic analyses were performed at 19 6 2 C. Chromatographic peaks in the samples were identified by comparing their retention time and UV spectrum with those of the reference standards [(þ)cat-
Water Uptake Study Maximum water uptake was determined by soaking the CD and sponge TD in phosphate buffer saline (pH 7.2) and monitoring the weight. The wet dressings were removed from the buffer and blotted with filter paper to remove excess water, and immediately weighed. Maximum water uptake was calculated from the equation, W ¼ (Mt - Mo)/Mo, where Mt represents the maximum weight and Mo, the original weight of the dressings.
In Vitro Antibacterial Activity Antibacterial efficacy of triphala incorporated collagen sponge (11 mm diameter) was tested on Mueller-Hinton agar for Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Mueller-Hinton agar with 5% sheep blood for Streptococcus pyogenes ATCC 12204 following Kirby-Bauer disk diffusion test [22].
In Vivo Wound Healing Animal Model Healing of full-thickness infected dermal wound was analyzed in male Wister albino rats as reported earlier with the Institute’s Ethical Committee approval and Guidelines (466/01/a/CPCDEA). Animals were divided into 3 groups as follows; Group 1: wound covered with sterile gauze dressing (GD), Group 2: wound covered with CD alone, and Group 3: wound covered with TD. The granulated tissues were excised on d 4, 8, 12, and 16 using sterile scissors and forceps from each group (n ¼ 6). The animals were rehabilitated post-experimentation.
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Wound Healing Rate The percentage of wound closure was calculated as follows by using the initial and final area drawn on glass slides during the experiments [23]: wound area on d 0 e wound area on d n % of wound closure ¼ -------------------------------------3100 wound area on d 0 n ¼ number of d ðd 4; 8; 12; and 16Þ
conjugated monoclonal antibody. In brief, freshly excised granulation tissues were embedded in embedding medium (OCT, Tissuetek; Sakura Finetek Inc., Torrance, CA) and frozen. Serial transverse sections around the granulation tissues were sectioned at 10 mm using a cryostat (JUNG CM 3000; Leica Microsystems GmbH, Wetzlas, Germany). The cryostat sections were picked up on poly-L-lysine coated slides and fixed with 2% paraformaldehyde. Nonspecific blocking sites were blocked using blocking solution (2% goat serum and 3% bovine serum albumin in Dulbecco’s modified phosphate buffered saline) for 10 min at 20 C. Then the sections were treated with fluorescein isothiocyanate-conjugated mouse anti-rat CD11b (Integrin aM chain, Mac-1 a chain) monoclonal antibody (BD Biosciences, San Jose, CA), mounted and observed under confocal microscope (Leica TCS SP5).
Bacteriological Examination of Granulated Tissue The excised granulated tissue (10 mg) was placed in 10 mL of sterile saline, vortexed for a few min, and the total bacterial count was analyzed.
Biochemical Analysis
Statistical Analysis Statistical evaluations were performed using SPSS software (version 7.5; SPSS Inc., Chicago, IL) and the data were expressed as mean 6 SD. The difference between groups was analyzed using 1way analysis of variance. The a-level was set at 0.01.
Five mg of defatted, dry granulation tissue was used to estimate the amount of hydroxyproline [24] and hexosamine [25].
Assay for Matrix Metalloproteinase (MMP) and MMP9 The d 4 granulation tissues of was analyzed for the presence of MMP8 and MMP9 by immunoblot analysis [26]. After electrophoretic separation of tissue proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were electro-transferred onto a nitrocellulose membrane. The membrane was blocked in blocking buffer containing 0.05% Tween-20 and 5% bovine serum albumin (Sigma) for 4 h, followed by incubation in rat anti-MMP8 and antiMMP9 primary antibodies. After 3 washings with Tris-buffered saline containing 0.5% Tween 20, the nitrocellulose membrane was incubated in alkaline phosphate conjugated secondary antibody followed by washing with buffer, and the bands were visualized using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (NBT/BCIP) as a substrate.
RESULTS Determination of Catechins
The column chromatography of the extract using Sephadex LH-20 column significantly increases the degree of purity. The peak corresponding to EGCG was identified based on comparisons of chromatographic retention time and UV-Vis absorbance spectra of triphala extract with the authentic standards in HPLC analysis. Figure 1 depicted a chromatogram of a standard mixture of catechins (A) and EGCG (B) with 11-min retention time.
Histological Analysis FTIR Granulated tissues collected at different intervals were transferred to 10% neutral buffered formalin for 24 h at 4 C. The formalin fixed tissues were dehydrated through grades of alcohol and cleared in xylene and then embedded in paraffin wax (58–60 m.p.). The molds were labeled and stored until use. Five to 7 mm deparaffinized sections were stained with haematoxylin following counterstained with eosin. Masson’s trichrome staining was done at d 8 and d 12 to observe collagen deposition in the granulated tissue [27].
Transmission Electron Microscopy The tissue specimens collected on d 12 experimental rats were fixed in 3% glutaraldehyde, buffered with 0.1 M sodium carbonate, pH 8.0, containing 0.2 M sucrose. Post-fixation was performed with 1% osmium tetroxide in the same buffer for 2 h at 4 C. Embedding was done in a mixture based on Epon 812, after dehydration in ethanol. Ultra-thin sections were post-stained with 2% aqueous uranyl acetate and lead acetate. Then the specimens were examined in a JEOL JEM-1010 electron microscope (JOEL, Tokyo, Japan) at 60 kV and photographed.
Immunohistochemistry The tissue sections from d 4 were used to analyze the presence of infiltrated neutrophils by staining with fluorescein isothiocyanate-
In Fig. 2, curve A shows the characteristics peaks of amide A (3350 cm1), amide B (3080 cm1), amide I (1650 cm1), and amide II (1550 cm1) band pattern in plain collagen sponge. Triphala incorporated collagen sponge (curve B) shows a slight shift in the amide band I, and the band around 3300 cm1 appears to be broadened.
DSC
The differential scanning calorimetry thermograms for plain and triphala incorporated collagen sponges are given in Fig. 3A and B. The denaturation temperature (TD) of the triphala incorporated collagen sponge was 87.8 C, whereas plain collagen sponge was 72.1 C. It could also be seen that the enthalpy of the endotherm peak has increased for the triphala incorporated collagen sponge (DH 594.1 J/g) compared with plain collagen sponge (DH 157.5 J/g).
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FIG. 1. Chromatogram of standard catechins (C ¼ catechin, EC: epicatechin, EGCG ¼ epigallocatechin gallate) (A) and the presence of EGCG in the triphala extract (B).
Water Uptake Study
The incorporation of triphala into the collagen sponge increased the uptake of water up to 7.17 6 0.95 (mean 6 SD) times compared with collagen sponge, where the uptake of water is around 0.94 6 0.09 (mean 6 SD).
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FIG 3. Differential scanning calorimetry thermogram of collagen sponge (A) and triphala incorporated collagen sponge (B).
In Vitro Antibacterial Activity
Clear zone of inhibition was found against Staphylococcus aureus (19 6 2 mm), Pseudomonas aeruginosa (21 6 2 mm), and b-hemolytic streptococci (16 6 1 mm) in the disc diffusion assay. Wound Healing Rate
Significant wound closure rate was observed in triphala incorporated collagen sponge treated group compared to other groups (Fig. 4). More than 95% wound
FIG 2. FTIR spectra of collagen sponge (A) and triphala incorporated collagen sponge (B).
FIG 4. Rate of wound closure by sterile gauze dressing (GD), collagen sponge dressing (CD), and triphala incorporated collagen dressing (TD) on different days of healing. (n ¼ 6). Results are presented as mean 6 SD; statistically significant data are given as *P < 0.01.
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FIG. 5. Graph shows total bacterial count present in the granulation tissues of different groups. GD ¼ sterile gauze dressing, CD ¼ collagen sponge dressing, TD ¼ triphala incorporated collagen dressing on different days of healing (n ¼ 6). Results are presented as mean 6 SD.
closure was observed in triphala incorporated collagen sponge treated rats on d 12, whereas other groups showed below 75% wound closure rate. Bacteriological Examination
The triphala incorporated collagen sponge treated group showed significant reduction of bacterial population on the infected wound site compared with the other groups (Fig. 5). The bacterial count was decreased to 2 3 103 on d 4 and further diminished on subsequent days whereas the control groups showed critical level of bacterial count up to d 8.
FIG. 7. Bar graph representing changes in the hexosamine levels between gauze dressing (GD), collagen sponge dressing (CD), and triphala incorporated collagen dressing (TD) at various days of healing (n ¼ 6). Results are presented as mean 6 SD; statistically significant data are given as *P < 0.01.
during the healing period. However, remarkable (P < 0.01) difference was observed between triphala incorporated collagen sponge treated group compared with other groups. Assay for MMP8 and MMP9
Immunoblot analysis showed changes in the level of MMP 8 and 9 during early wound healing (d 4) in the experimental groups. Elevated level of both MMPs was
Biochemical Analysis
Figure 6 shows the hydroxyproline content in granulation tissues of experimental rats. Significant increase in the hydroxyproline content was observed in triphala incorporated collagen sponge treated rats compared with other groups. Collagen sponge dressing treated group also showed high hydroxyproline content compared with gauze dressed rats. Hexosamine content of granulation tissues are given in Fig. 7. There is a gradual decrease of hexosamine content in all the groups
FIG. 6. Comparison of total hydroxyproline levels between gauze dressing (GD), collagen sponge dressing (CD), and triphala incorporated collagen dressing (TD) at various days of healing (n ¼ 6). Results are presented as mean 6 SD; statistically significant data are given as *P < 0.01.
FIG. 8. Immunoblot analysis shows the expression of MMP 8 and MMP 9 on different groups on d 4. (GD ¼ gauze dressing, CD ¼ collagen sponge dressing, TD ¼ triphala incorporated collagen dressing. (Color version of Figure is available online.)
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found in the gauze dressing and collagen sponge treated rats compared with triphala incorporated collagen sponge treated rats (Fig. 8). Histological Analysis
Increased cellular infiltration was observed from haematoxylin and eosin staining in both gauze dressing and collagen sponge treated groups until d 8 whereas triphala incorporated collagen sponge treated group showed marked infiltration of inflammatory cells on d 4, and dermal and epidermal regeneration with blood vessel formation on d 8 onwards. Massive deposition of collagen was observed in triphala incorporated collagen sponge treated group compared with other groups, which showed diffused collagen bundles. Transmission Electron Microscopy
Transmission electron microscopic photographs have shown the arrangement of collagen in experimental
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groups. Collagen fibers from triphala incorporated collagen sponge treated specimens appeared more packed and presumably better aligned. In the granulation tissue of other groups, collagen content was reduced, and collagen fibers were sparse, immature, loosely packed, and irregularly arranged (Fig. 9). Immunohistochemistry
Significant reduction of neutrophils was observed in the granulation tissue of triphala incorporated collagen sponge treated group whereas gauze dressing and collagen sponge dressing treated groups showed a high number of neutrophil population (Fig. 10). DISCUSSION
Collagen dressing offers great potential as a matrix for localized drug delivery. As a fully biodegradable, restorable, and haemostatic in nature, collagen has more advantages in wound healing over synthetic
FIG 9. Transmission electron microscopy analysis showing the arrangement of collagen bundles in the granulation tissues of experimental groups on d 12. (A) Gauze dressing (GD), (B) collagen sponge dressing (CD), (C) triphala incorporated collagen sponge dressing (TD).
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FIG. 10. Confocal microscopy showing the infiltration of neutrophils in the granulation tissues of experimental groups on d 4. (A) Gauze dressing (GD), (B) collagen sponge dressing (CD), (C) triphala incorporated collagen sponge dressing (TD). A–C are at 2003 magnification. (Color version of Figure is available online.)
biomaterials [2, 28]. With regard to the infected wound, effective wound bed preparation depends on the management of microbial balance as well as the treatment of infection. The continual presence of a bacterial infection stimulates the host immune defenses leading to the chronic production of inflammatory mediators, such as prostaglandin E2 and thromboxane. Neutrophils continue to migrate into the wound, releasing cytotoxic enzymes and free oxygen radicals [29]. Treating infected wounds will help to reduce the bacterial burden and hence remove 1 of the barriers to healing. Topical application of triphala incorporated collagen sponge might be an efficient therapy to destroy microbial population by the release of triphala and booster healing by the collagen. The synergic antibacterial activity of EGCG with blactams against MRSA has been reported earlier, and the presence of EGCG in triphala may help for its antibacterial activity, which controls infection [30]. IR absorption at A1240/A1454 cm1 has shown the integrity
of triple helix configurations in both triphala incorporated and plain collagen. Maintaining the triple helical structure in collagen facilitate the interaction of wide range of receptors in the regenerative cells. The shift in the amide I band in triphala incorporated collagen sponge might be due to hydrogen bond interactions between collagen and polyphenols in triphala. The broadening of band around 3300 cm1 corresponding to the OH stretching frequency of the carboxylic acid group present in the polyphenols. These hydroxyl groups can form a hydrogen bond with the side chain groups of polar amino acids like lysine, arginine, aspartic acid, and glutamic acid. The other amino acids such as serine and threonine can also be involved in the hydrogen bond formation with the polyphenolic molecules in triphala. All these amino acid residues can act as hydrogen bond donors and acceptors. Since these polyphenolics have several hydroxyl and carboxyl groups, they can form hydrogen bonds at multiple points, imparting additional stability to the fiber matrix [31].
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An increase in thermal stability could be related to the increase in the number of cross-links because they decrease the entropy of transition [32]. An increase in the thermal stability and endotherm peak (DH) indicates that cross-linking has occurred between triphala and collagen, which requires more energy to undergo the helix-coil transition. Increase in the water uptake capability of triphala incorporated collagen sponge helps to absorb more wound fluids by the dressing, which may provide an environment rich in white blood cells, enzymes, cytokines, and growth factors beneficial to wound healing [33]. Sato and coworkers reported gallic acid and ethyl gallate in T. chebula Retz (a constituent of triphala) have shown antibacterial activity of ethanol extracts against both methicillin-resistant and -sensitive S. aureus and other bacteria [34]. Antibacterial activity of triphala against these primary wound pathogens helps to enhance the healing potential of infected wound. Collagen biomaterials attract many cell types, primarily fibroblasts that play a major role in extracellular matrix production; it has also been found that the production of collagen was increased when fibroblasts were bound to collagen implants [35, 36]. The slow rate of wound closure found not only in the rats treated with sterile gauze but also in rats treated with collagen sponge dressing might be attributable to the presence of microorganisms and their metabolites, which impair healing. The failure of skin grafts in wound management occurs when the load is more than 105 organisms/g tissue, which provides further evidence that bacterial load impairs repair [37]. This study has demonstrated that high levels of bacteria can be successfully inhibited by the triphala incorporated collagen sponge on infected wound. Collagen is a major protein of the extracellular matrix and is the component that ultimately contributes to wound strength. Collagen breakdown from both triphala incorporated collagen sponge and collagen sponge may liberate available amino acids for the synthesis of new collagen molecules at the wound site, denoted by the increased level of hydroxyproline content in these groups. Hexosamine acts as a ground substratum for the synthesis of extra cellular matrix; significant decrease in the hexosamine content was associated with concomitant increase in the extra cellular matrix synthesis in triphala incorporated collagen sponge treated group. Significant reduction of MMP9 and MMP8 in triphala incorporated collagen sponge treated group supports the reduction of inflammatory phase. Significant reduction of inflammatory cells in the early phase of healing in triphala incorporated collagen sponge treated group indirectly shows the reduction of bacterial population, thereby enhancing healing. Early dermal and epidermal regeneration with blood vessel formation in triphala incorporated
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collagen sponge treated group confirmed the effect of this dressing on cellular proliferation, granulation tissue formation, and epithelialization. Well-formed collagen bundles in triphala incorporated collagen sponge treated group have shown efficacy of the dressing on fibroblast proliferation and collagen deposition. As more fibroblasts reach the wound site, more collagen deposition occurs by cell division. The mature collagen bundle formation in the triphala incorporated collagen sponge treated group on transmission electron microscopy analysis reflects the fibroblast proliferation on d 12. On the other hand, in gauze dressing and collagen dressing treated groups, less immature and degraded fibril assembly indicate fibroblast proliferation was not up to the mark and more degradation compared with synthesis. Cross linking of collagen and remodeling of connective tissue results in the acquisition of wound strength. Hydroxylation of proline and lysine in ribosomes occurs during collagen chain synthesis, where ascorbic acid is used as a cofactor in converting proline residues into hydroxyproline [38]. Ascorbic acid found in the Phyllanthus emblica (a constituent of triphala) helps to increase collagen deposition and tensile strength in triphala incorporated collagen sponge treated group during wound repair. A central feature of the innate reaction is recruitment and activation of neutrophils at the site of infection to eradicate pathogens [39]. CD11b–CD18 is the ligand for the endothelial cell expressed intercellular adhesion molecule 1, and this interaction causes the tight adhesion that is necessary for transmigration of neutrophils into the surrounding tissue [40]. Clinically, increased neutrophil CD11b expression has been demonstrated after major trauma and in patients with sepsis [41]. Result from the immunohistochemistry analysis correlates with these previous investigations, and massive reduction in the CD11bþ neutrophils on the initial phase of healing might be due to the reduction of bacterial population in triphala incorporated collagen sponge treated group. In conclusion, the above results suggest that the application of triphala incorporated collagen sponge on infected wound not only reduces the risk of infection but also improves healing, and can be used as an effective biomaterial to promote infected wound healing. ACKNOWLEDGMENTS The authors are grateful to Dr. A. B. Mandal, Director, CLRI, Chennai, for his kind permission to publish this work. MSK and RS gratefully acknowledge the financial assistance in the form of fellowships received from CSIR, Government of India.
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23. Wall SJ, Bevan D, Thomas DW, et al. Differential expression of matrix metalloproteinases during impaired wound healing of the diabetes mouse. J Invest Dermatol 2002;119:91. 24. Neuman RE, Logan MA. The determination of hydroxyproline. J Biol Chem 1950;184:299. 25. Adamsons RJ, Musco F, Enquist IF. The relationship of collagen content to wound strength in normal and scorbutic animals. Surg Gynecol Obstet 1964;119:323. 26. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350. 27. McManus JGA, Mowry RW. Staining methods: Histological and histochemical. New York: Harper and Row, 1964:8. 28. Panduranga RK. Recent developments of collagen-based materials for medical applications and drug delivery. J Biomater Sci Polym Ed 1995;7:623. 29. Laato M, Niinikoski J, Lundberg C, et al. Inflammatory reaction in blood flow and experimental wounds inoculated with Staphylococcus aureus. Eur Surg Res 1988;20:33. 30. Zhao WH, Hu ZQ, Okubo S, et al. Mechanism of synergy between epigallocatechin gallate and ß-lactams against methicillinresistant Staphylococcus aureus. Antimicrob Agents Chemother 2001;45:1737. 31. Madhan B, Subramanian V, Raghava Rao J, et al. Stabilization of collagen using plant polyphenol: Role of catechin. Int J Biol Macromol 2005;37:47. 32. Privalov PL, Tiktopulo EI. Thermal conformational transformation of tropocollagen. I. Calorimetric study. Biopolymers 1970; 9:127. 33. Jones V, Harding K. Chronic wound care. In: Krasner DL, Rodeheaver GT, Sibbald RG, Eds. A Clinical Source Book for Healthcare Professionals. Wayne, PA: HMP Communications, 2001:245–52. 34. Sato Y, Oketani H, Singyouchi K, et al. Extraction and purification of effective antimicrobial constituents of Terminalia chebula Retz against methicillin-resistance Staphylococcus aureus. Biol Pharm Bull 1997;20:401. 35. Anselme K, Bacques C, Charriere G, et al. Tissue reaction to subcutaneous implantation of a collagen sponge. A histological, ultrastructural, and immunological study. J Biomed Mater Res 1990;24:689. 36. Postlethwaite AE, Seyer JM, Kang AH. Chemotactic attraction of human fibroblasts to Type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci USA 1978;75:871. 37. Robson MC, Krizek TJ. Predicting skin graft survival. J Trauma 1973;13:213. 38. Phillips C, Wenstrup RJ. Biosynthetic and genetic disorders of collagen. In: Cohen K, Diegelman RF, Winblad WJ, Eds. Wound healing, biochemical and clinical aspects. Philadelphia: Saunders, 1992:252–261. 39. Witto-Sassat V, Rieu P, Descamps-Latscha B, et al. Neutrophils: Molecules, functions and pathophysiological aspects. Lab Invest 2000;80:617. 40. Mizgerd JP, Meek BB, Kutkoski GJ, et al. Selectins and neutrophil traffic: Margination and Streptococcus pneumoniae-induced emigration in murine lungs. J Exp Med 1996;184:639. 41. Botha AJ, Moore FA, Moore EE, et al. Base deficit after major trauma directly relates to neutrophil CD11b expression: A proposed mechanism of shock-induced organ injury. Intensive Care Med 1997;23:504.
Triphala Incorporated Collagen Sponge—A Smart Biomaterial for Infected Dermal Wound Healing Muthusamy Senthil Kumar, Ph.D.,* Shanmugam Kirubanandan, M.Tech.,† Ramasamy Sripriya, Ph.D,* and Praveen Kumar Sehgal, Ph.D*,1 *Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai, India; and †Centre for Biotechnology, Anna University, Adyar, Chennai, India Submitted for publication May 21, 2008
Background. Wound infection is a major problem in the medical community since many types of wounds are more prone to microbial contamination leading to infection. Triphala (a traditional ayurvedic herbal formulation) incorporated collagen sponge was investigated for its healing potential on infected dermal wound in albino rats. Materials and Methods. Methanol extract of triphala was prepared and analyzed for the presence of catechin by high-pressure liquid chromatography analysis. Collagen sponge was prepared by incorporating triphala into collagen sponge. The triphala incorporated collagen was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, and water uptake analysis. Infected wound was dressed with triphala incorporated collagen sponge. Wound reduction rate, collagen content, and matrix metalloproteinases in the granulation tissue, histology, and Fourier transform electron microscopy analysis were done to obtain the healing pattern. Results. High-pressure liquid chromatography analysis showed the presence of (-)epigallocatechin gallate. FT-IR spectroscopy study revealed the interaction of polyphenols with the collagen. Triphala incorporated collagen sponge has shown to increase thermal stability and water uptake capability, faster wound closure, improved tissue regeneration, collagen content at the wound site, and supporting histopathological parameters pertaining to wound healing. Matrix metalloproteinases expression was correlated well with reduction in the inflammatory phase, thus confirming efficacy of the dressing.
1
To whom correspondence and reprint requests should be addressed at Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai-600 020, India. E-mail: [email protected].
0022-4804/10 $36.00 Ó 2010 Elsevier Inc. All rights reserved.
Conclusions. Better healing efficacy of triphala incorporated collagen sponge may provides a scientific rationale for the use of this dressing as an effective wound cover in the management of infected dermal wound. Ó 2010 Elsevier Inc. All rights reserved. Key Words: collagen sponge; drug delivery; triphala; wound infection.
INTRODUCTION
From 1970s, the processing technology of collagen has been improved, and new medical-grade collagen products are successfully launched in the market [1]. Currently, collagen scaffolds are employed in tissue engineering such as cartilage, bone, nerve, and skin as a support for cell mobility, infiltration, proliferation, and differentiation [2–4]. Collagen in the form of sponge is useful in the treatment of different wounds, such as pressure sores, donor sites, leg ulcers, and decubitus ulcers, as it adheres well to wet wounds, absorbs large quantities of tissue exudates, preserve a moist environment, and encourages the formation of new granulation tissue and epithelium on the wound [5, 6]. Collagen itself may not facilitate the healing of an infected wound because it is protein in nature and bacteria can use it as a substrate. At this stage, the imbalance between host resistance and bacterial growth leads to infection on the wound, and bacterial infection impairs healing through several mechanisms. In severe wound infection, systemic administration of drugs may lead to insufficient drug concentration reaching the site of infection, drug associated side effect, and systemic toxicity. This has been overcome successfully by topical application of drugs, and collagen dressings with
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antibiotics have also been developed to control infection [7–9]. Development of bacterial resistance to antibiotics continues, and the use of new antimicrobial agents is increasing [10]. Triphala, a compound formulation of the herb Terminalia chebula, Phyllanthus emblica, and Terminalia bellerica has been described in the Ayurveda (ancient Indian system of medicine) as a ‘‘tridoshic rasayan,’’ having balancing and rejuvenating effects on the 3 constitutional elements that govern human life [11]. Triphala is 1 of the important antioxidant rich rasayana drugs that has been reported to treat chronic ulcers [12]. Triphala and/or its constituents have been reported to possess antibacterial, antifungal, antiviral, and antiallergic activities [13–16]. The potential use of triphala on infected full-thickness wound healing was described in our earlier study [17]. In this article, triphala extract was incorporated in collagen sponge and its efficacy was evaluated in healing of infected dermal wounds.
MATERIALS AND METHODS
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echin (C), (–)epicatechin (EC), and(–)epigallocatechin gallate (EGCG; Sigma, St Louis, MO)].
Preparation of Collagen Sponge Type I collagen was extracted from bovine Achilles tendon using the procedure reported earlier [20]. In brief, clean Achilles tendons were minced below 25 C and washed with cold distilled water. The minced tissue was subjected to subsequent chemical and enzyme treatment and pure collagen was extracted. 1% wt/vol aqueous solution of pure Type I collagen (in 0.1% 12N HCl, pH 2.5) was prepared and agitated with 0.1% vol/vol nonionic wetting agent. The frothy mass was poured into Teflon trays and allowed to air-dry [21].
Collagen Sponge with Triphala Triphala extract was redissolved in methanol and incorporated in to the collagen sponge at the concentration of 50 mg/cm2. The sponge was allowed to dry in laminar airflow chamber.
Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra were recorded in a Thermo Nicolet avatar 320 FTIR spectrometer (Nicolet Instrument Con., Madison, WI) from collagen sponge dressing (CD) and triphala incorporated collagen sponge dressing (TD). All spectra were recorded from 400 to 4000 cm1 with a resolution of 4 cm1.
Preparation of Alcohol Extract of Triphala
Differential Scanning Calorimetry (DSC)
Methanol extract of triphala (IMPCOPS Ltd., Chennai, India) was prepared as described earlier [18].
DSC of the soluble collagen sponge and triphala incorporated collagen sponge were recorded using differential scanning calorimeter (DSC 204). Samples were sealed in aluminium pan and an empty pan was used as a reference. The heating rate 1 K (Kelvin) per min and temperature range between –5 C and 200 C in an N2 atmosphere were maintained.
Determination of Catechins The preliminary purification of catechins from the crude extract was achieved using counter-current chromatography with water/ chloroform (1:1, vol/vol) and water/ethyl acetate (1:1, vol/vol) solvent systems [19]. Catechin compounds that migrated into the ethyl acetate layer were collected and dried in a rotary evaporator. The crude catechins (0.5 g) were applied onto a chromatographic column with Sephadex LH-20 (Sigma, St. Louis, MO) equilibrated with methanol. The column dimensions were 1.6 3 90 cm and the flow rate was 1.2 mL/min. The fractions (8 mL) were collected and analysis of elutes from each tube was performed by measuring their absorbance at 280 nm. The contents were pooled and evaporated in a rotary evaporator. The dried extract dissolved in high-pressure liquid chromatography (HPLC) grade methanol was used for HPLC analysis. The HPLC system consisted of a Waters (Milford, MA) pump equipped with UV-VIS detector, model Shimadzu SPD 10A (Shimadzu, Kyoto, Japan), a rheodyne sample injector with a 20 mL sample loop. The column was a Spherisorb-ODS2 RP-18 column (25 cm long 3 4.6 mm i.d. and 5 mm particle diameter; Waters) equipped with a guard column (1.0 cm long 3 4.6 mm i.d., Varian). Detection was carried out by measurement of UV absorbance at 280 nm. The mobile phase was composed of water/ acetonitrile/methanol/ethyl acetate/glacial acetic acid (89:6:1:3:1 vol/vol/vol/vol/vol). Before the use, the mobile phase was filtered through a G5 sintered glass filter and then degassed for 30 min in an ultrasonic bath. Using sample injector 20 mL of sample was injected to the sample injection port. The mobile phase flow rate started at 0.7 mL/min and was maintained for 15 min, and then linearly increased to 1.2 mL/min in 1 min. This flow rate was maintained for 8 min, and then decreased to 0.7 mL/min. All chromatographic analyses were performed at 19 6 2 C. Chromatographic peaks in the samples were identified by comparing their retention time and UV spectrum with those of the reference standards [(þ)cat-
Water Uptake Study Maximum water uptake was determined by soaking the CD and sponge TD in phosphate buffer saline (pH 7.2) and monitoring the weight. The wet dressings were removed from the buffer and blotted with filter paper to remove excess water, and immediately weighed. Maximum water uptake was calculated from the equation, W ¼ (Mt - Mo)/Mo, where Mt represents the maximum weight and Mo, the original weight of the dressings.
In Vitro Antibacterial Activity Antibacterial efficacy of triphala incorporated collagen sponge (11 mm diameter) was tested on Mueller-Hinton agar for Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Mueller-Hinton agar with 5% sheep blood for Streptococcus pyogenes ATCC 12204 following Kirby-Bauer disk diffusion test [22].
In Vivo Wound Healing Animal Model Healing of full-thickness infected dermal wound was analyzed in male Wister albino rats as reported earlier with the Institute’s Ethical Committee approval and Guidelines (466/01/a/CPCDEA). Animals were divided into 3 groups as follows; Group 1: wound covered with sterile gauze dressing (GD), Group 2: wound covered with CD alone, and Group 3: wound covered with TD. The granulated tissues were excised on d 4, 8, 12, and 16 using sterile scissors and forceps from each group (n ¼ 6). The animals were rehabilitated post-experimentation.
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Wound Healing Rate The percentage of wound closure was calculated as follows by using the initial and final area drawn on glass slides during the experiments [23]: wound area on d 0 e wound area on d n % of wound closure ¼ -------------------------------------3100 wound area on d 0 n ¼ number of d ðd 4; 8; 12; and 16Þ
conjugated monoclonal antibody. In brief, freshly excised granulation tissues were embedded in embedding medium (OCT, Tissuetek; Sakura Finetek Inc., Torrance, CA) and frozen. Serial transverse sections around the granulation tissues were sectioned at 10 mm using a cryostat (JUNG CM 3000; Leica Microsystems GmbH, Wetzlas, Germany). The cryostat sections were picked up on poly-L-lysine coated slides and fixed with 2% paraformaldehyde. Nonspecific blocking sites were blocked using blocking solution (2% goat serum and 3% bovine serum albumin in Dulbecco’s modified phosphate buffered saline) for 10 min at 20 C. Then the sections were treated with fluorescein isothiocyanate-conjugated mouse anti-rat CD11b (Integrin aM chain, Mac-1 a chain) monoclonal antibody (BD Biosciences, San Jose, CA), mounted and observed under confocal microscope (Leica TCS SP5).
Bacteriological Examination of Granulated Tissue The excised granulated tissue (10 mg) was placed in 10 mL of sterile saline, vortexed for a few min, and the total bacterial count was analyzed.
Biochemical Analysis
Statistical Analysis Statistical evaluations were performed using SPSS software (version 7.5; SPSS Inc., Chicago, IL) and the data were expressed as mean 6 SD. The difference between groups was analyzed using 1way analysis of variance. The a-level was set at 0.01.
Five mg of defatted, dry granulation tissue was used to estimate the amount of hydroxyproline [24] and hexosamine [25].
Assay for Matrix Metalloproteinase (MMP) and MMP9 The d 4 granulation tissues of was analyzed for the presence of MMP8 and MMP9 by immunoblot analysis [26]. After electrophoretic separation of tissue proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were electro-transferred onto a nitrocellulose membrane. The membrane was blocked in blocking buffer containing 0.05% Tween-20 and 5% bovine serum albumin (Sigma) for 4 h, followed by incubation in rat anti-MMP8 and antiMMP9 primary antibodies. After 3 washings with Tris-buffered saline containing 0.5% Tween 20, the nitrocellulose membrane was incubated in alkaline phosphate conjugated secondary antibody followed by washing with buffer, and the bands were visualized using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (NBT/BCIP) as a substrate.
RESULTS Determination of Catechins
The column chromatography of the extract using Sephadex LH-20 column significantly increases the degree of purity. The peak corresponding to EGCG was identified based on comparisons of chromatographic retention time and UV-Vis absorbance spectra of triphala extract with the authentic standards in HPLC analysis. Figure 1 depicted a chromatogram of a standard mixture of catechins (A) and EGCG (B) with 11-min retention time.
Histological Analysis FTIR Granulated tissues collected at different intervals were transferred to 10% neutral buffered formalin for 24 h at 4 C. The formalin fixed tissues were dehydrated through grades of alcohol and cleared in xylene and then embedded in paraffin wax (58–60 m.p.). The molds were labeled and stored until use. Five to 7 mm deparaffinized sections were stained with haematoxylin following counterstained with eosin. Masson’s trichrome staining was done at d 8 and d 12 to observe collagen deposition in the granulated tissue [27].
Transmission Electron Microscopy The tissue specimens collected on d 12 experimental rats were fixed in 3% glutaraldehyde, buffered with 0.1 M sodium carbonate, pH 8.0, containing 0.2 M sucrose. Post-fixation was performed with 1% osmium tetroxide in the same buffer for 2 h at 4 C. Embedding was done in a mixture based on Epon 812, after dehydration in ethanol. Ultra-thin sections were post-stained with 2% aqueous uranyl acetate and lead acetate. Then the specimens were examined in a JEOL JEM-1010 electron microscope (JOEL, Tokyo, Japan) at 60 kV and photographed.
Immunohistochemistry The tissue sections from d 4 were used to analyze the presence of infiltrated neutrophils by staining with fluorescein isothiocyanate-
In Fig. 2, curve A shows the characteristics peaks of amide A (3350 cm1), amide B (3080 cm1), amide I (1650 cm1), and amide II (1550 cm1) band pattern in plain collagen sponge. Triphala incorporated collagen sponge (curve B) shows a slight shift in the amide band I, and the band around 3300 cm1 appears to be broadened.
DSC
The differential scanning calorimetry thermograms for plain and triphala incorporated collagen sponges are given in Fig. 3A and B. The denaturation temperature (TD) of the triphala incorporated collagen sponge was 87.8 C, whereas plain collagen sponge was 72.1 C. It could also be seen that the enthalpy of the endotherm peak has increased for the triphala incorporated collagen sponge (DH 594.1 J/g) compared with plain collagen sponge (DH 157.5 J/g).
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FIG. 1. Chromatogram of standard catechins (C ¼ catechin, EC: epicatechin, EGCG ¼ epigallocatechin gallate) (A) and the presence of EGCG in the triphala extract (B).
Water Uptake Study
The incorporation of triphala into the collagen sponge increased the uptake of water up to 7.17 6 0.95 (mean 6 SD) times compared with collagen sponge, where the uptake of water is around 0.94 6 0.09 (mean 6 SD).
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FIG 3. Differential scanning calorimetry thermogram of collagen sponge (A) and triphala incorporated collagen sponge (B).
In Vitro Antibacterial Activity
Clear zone of inhibition was found against Staphylococcus aureus (19 6 2 mm), Pseudomonas aeruginosa (21 6 2 mm), and b-hemolytic streptococci (16 6 1 mm) in the disc diffusion assay. Wound Healing Rate
Significant wound closure rate was observed in triphala incorporated collagen sponge treated group compared to other groups (Fig. 4). More than 95% wound
FIG 2. FTIR spectra of collagen sponge (A) and triphala incorporated collagen sponge (B).
FIG 4. Rate of wound closure by sterile gauze dressing (GD), collagen sponge dressing (CD), and triphala incorporated collagen dressing (TD) on different days of healing. (n ¼ 6). Results are presented as mean 6 SD; statistically significant data are given as *P < 0.01.
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FIG. 5. Graph shows total bacterial count present in the granulation tissues of different groups. GD ¼ sterile gauze dressing, CD ¼ collagen sponge dressing, TD ¼ triphala incorporated collagen dressing on different days of healing (n ¼ 6). Results are presented as mean 6 SD.
closure was observed in triphala incorporated collagen sponge treated rats on d 12, whereas other groups showed below 75% wound closure rate. Bacteriological Examination
The triphala incorporated collagen sponge treated group showed significant reduction of bacterial population on the infected wound site compared with the other groups (Fig. 5). The bacterial count was decreased to 2 3 103 on d 4 and further diminished on subsequent days whereas the control groups showed critical level of bacterial count up to d 8.
FIG. 7. Bar graph representing changes in the hexosamine levels between gauze dressing (GD), collagen sponge dressing (CD), and triphala incorporated collagen dressing (TD) at various days of healing (n ¼ 6). Results are presented as mean 6 SD; statistically significant data are given as *P < 0.01.
during the healing period. However, remarkable (P < 0.01) difference was observed between triphala incorporated collagen sponge treated group compared with other groups. Assay for MMP8 and MMP9
Immunoblot analysis showed changes in the level of MMP 8 and 9 during early wound healing (d 4) in the experimental groups. Elevated level of both MMPs was
Biochemical Analysis
Figure 6 shows the hydroxyproline content in granulation tissues of experimental rats. Significant increase in the hydroxyproline content was observed in triphala incorporated collagen sponge treated rats compared with other groups. Collagen sponge dressing treated group also showed high hydroxyproline content compared with gauze dressed rats. Hexosamine content of granulation tissues are given in Fig. 7. There is a gradual decrease of hexosamine content in all the groups
FIG. 6. Comparison of total hydroxyproline levels between gauze dressing (GD), collagen sponge dressing (CD), and triphala incorporated collagen dressing (TD) at various days of healing (n ¼ 6). Results are presented as mean 6 SD; statistically significant data are given as *P < 0.01.
FIG. 8. Immunoblot analysis shows the expression of MMP 8 and MMP 9 on different groups on d 4. (GD ¼ gauze dressing, CD ¼ collagen sponge dressing, TD ¼ triphala incorporated collagen dressing. (Color version of Figure is available online.)
KUMAR ET AL.: TRIPHALA INCORPORATED COLLAGEN SPONGE
found in the gauze dressing and collagen sponge treated rats compared with triphala incorporated collagen sponge treated rats (Fig. 8). Histological Analysis
Increased cellular infiltration was observed from haematoxylin and eosin staining in both gauze dressing and collagen sponge treated groups until d 8 whereas triphala incorporated collagen sponge treated group showed marked infiltration of inflammatory cells on d 4, and dermal and epidermal regeneration with blood vessel formation on d 8 onwards. Massive deposition of collagen was observed in triphala incorporated collagen sponge treated group compared with other groups, which showed diffused collagen bundles. Transmission Electron Microscopy
Transmission electron microscopic photographs have shown the arrangement of collagen in experimental
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groups. Collagen fibers from triphala incorporated collagen sponge treated specimens appeared more packed and presumably better aligned. In the granulation tissue of other groups, collagen content was reduced, and collagen fibers were sparse, immature, loosely packed, and irregularly arranged (Fig. 9). Immunohistochemistry
Significant reduction of neutrophils was observed in the granulation tissue of triphala incorporated collagen sponge treated group whereas gauze dressing and collagen sponge dressing treated groups showed a high number of neutrophil population (Fig. 10). DISCUSSION
Collagen dressing offers great potential as a matrix for localized drug delivery. As a fully biodegradable, restorable, and haemostatic in nature, collagen has more advantages in wound healing over synthetic
FIG 9. Transmission electron microscopy analysis showing the arrangement of collagen bundles in the granulation tissues of experimental groups on d 12. (A) Gauze dressing (GD), (B) collagen sponge dressing (CD), (C) triphala incorporated collagen sponge dressing (TD).
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FIG. 10. Confocal microscopy showing the infiltration of neutrophils in the granulation tissues of experimental groups on d 4. (A) Gauze dressing (GD), (B) collagen sponge dressing (CD), (C) triphala incorporated collagen sponge dressing (TD). A–C are at 2003 magnification. (Color version of Figure is available online.)
biomaterials [2, 28]. With regard to the infected wound, effective wound bed preparation depends on the management of microbial balance as well as the treatment of infection. The continual presence of a bacterial infection stimulates the host immune defenses leading to the chronic production of inflammatory mediators, such as prostaglandin E2 and thromboxane. Neutrophils continue to migrate into the wound, releasing cytotoxic enzymes and free oxygen radicals [29]. Treating infected wounds will help to reduce the bacterial burden and hence remove 1 of the barriers to healing. Topical application of triphala incorporated collagen sponge might be an efficient therapy to destroy microbial population by the release of triphala and booster healing by the collagen. The synergic antibacterial activity of EGCG with blactams against MRSA has been reported earlier, and the presence of EGCG in triphala may help for its antibacterial activity, which controls infection [30]. IR absorption at A1240/A1454 cm1 has shown the integrity
of triple helix configurations in both triphala incorporated and plain collagen. Maintaining the triple helical structure in collagen facilitate the interaction of wide range of receptors in the regenerative cells. The shift in the amide I band in triphala incorporated collagen sponge might be due to hydrogen bond interactions between collagen and polyphenols in triphala. The broadening of band around 3300 cm1 corresponding to the OH stretching frequency of the carboxylic acid group present in the polyphenols. These hydroxyl groups can form a hydrogen bond with the side chain groups of polar amino acids like lysine, arginine, aspartic acid, and glutamic acid. The other amino acids such as serine and threonine can also be involved in the hydrogen bond formation with the polyphenolic molecules in triphala. All these amino acid residues can act as hydrogen bond donors and acceptors. Since these polyphenolics have several hydroxyl and carboxyl groups, they can form hydrogen bonds at multiple points, imparting additional stability to the fiber matrix [31].
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An increase in thermal stability could be related to the increase in the number of cross-links because they decrease the entropy of transition [32]. An increase in the thermal stability and endotherm peak (DH) indicates that cross-linking has occurred between triphala and collagen, which requires more energy to undergo the helix-coil transition. Increase in the water uptake capability of triphala incorporated collagen sponge helps to absorb more wound fluids by the dressing, which may provide an environment rich in white blood cells, enzymes, cytokines, and growth factors beneficial to wound healing [33]. Sato and coworkers reported gallic acid and ethyl gallate in T. chebula Retz (a constituent of triphala) have shown antibacterial activity of ethanol extracts against both methicillin-resistant and -sensitive S. aureus and other bacteria [34]. Antibacterial activity of triphala against these primary wound pathogens helps to enhance the healing potential of infected wound. Collagen biomaterials attract many cell types, primarily fibroblasts that play a major role in extracellular matrix production; it has also been found that the production of collagen was increased when fibroblasts were bound to collagen implants [35, 36]. The slow rate of wound closure found not only in the rats treated with sterile gauze but also in rats treated with collagen sponge dressing might be attributable to the presence of microorganisms and their metabolites, which impair healing. The failure of skin grafts in wound management occurs when the load is more than 105 organisms/g tissue, which provides further evidence that bacterial load impairs repair [37]. This study has demonstrated that high levels of bacteria can be successfully inhibited by the triphala incorporated collagen sponge on infected wound. Collagen is a major protein of the extracellular matrix and is the component that ultimately contributes to wound strength. Collagen breakdown from both triphala incorporated collagen sponge and collagen sponge may liberate available amino acids for the synthesis of new collagen molecules at the wound site, denoted by the increased level of hydroxyproline content in these groups. Hexosamine acts as a ground substratum for the synthesis of extra cellular matrix; significant decrease in the hexosamine content was associated with concomitant increase in the extra cellular matrix synthesis in triphala incorporated collagen sponge treated group. Significant reduction of MMP9 and MMP8 in triphala incorporated collagen sponge treated group supports the reduction of inflammatory phase. Significant reduction of inflammatory cells in the early phase of healing in triphala incorporated collagen sponge treated group indirectly shows the reduction of bacterial population, thereby enhancing healing. Early dermal and epidermal regeneration with blood vessel formation in triphala incorporated
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collagen sponge treated group confirmed the effect of this dressing on cellular proliferation, granulation tissue formation, and epithelialization. Well-formed collagen bundles in triphala incorporated collagen sponge treated group have shown efficacy of the dressing on fibroblast proliferation and collagen deposition. As more fibroblasts reach the wound site, more collagen deposition occurs by cell division. The mature collagen bundle formation in the triphala incorporated collagen sponge treated group on transmission electron microscopy analysis reflects the fibroblast proliferation on d 12. On the other hand, in gauze dressing and collagen dressing treated groups, less immature and degraded fibril assembly indicate fibroblast proliferation was not up to the mark and more degradation compared with synthesis. Cross linking of collagen and remodeling of connective tissue results in the acquisition of wound strength. Hydroxylation of proline and lysine in ribosomes occurs during collagen chain synthesis, where ascorbic acid is used as a cofactor in converting proline residues into hydroxyproline [38]. Ascorbic acid found in the Phyllanthus emblica (a constituent of triphala) helps to increase collagen deposition and tensile strength in triphala incorporated collagen sponge treated group during wound repair. A central feature of the innate reaction is recruitment and activation of neutrophils at the site of infection to eradicate pathogens [39]. CD11b–CD18 is the ligand for the endothelial cell expressed intercellular adhesion molecule 1, and this interaction causes the tight adhesion that is necessary for transmigration of neutrophils into the surrounding tissue [40]. Clinically, increased neutrophil CD11b expression has been demonstrated after major trauma and in patients with sepsis [41]. Result from the immunohistochemistry analysis correlates with these previous investigations, and massive reduction in the CD11bþ neutrophils on the initial phase of healing might be due to the reduction of bacterial population in triphala incorporated collagen sponge treated group. In conclusion, the above results suggest that the application of triphala incorporated collagen sponge on infected wound not only reduces the risk of infection but also improves healing, and can be used as an effective biomaterial to promote infected wound healing. ACKNOWLEDGMENTS The authors are grateful to Dr. A. B. Mandal, Director, CLRI, Chennai, for his kind permission to publish this work. MSK and RS gratefully acknowledge the financial assistance in the form of fellowships received from CSIR, Government of India.
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