CHARACTERIZATION AND APPLICATION OF COLLAGEN-POLYMER BASED COMPOSITE AS WOUND HEALING MATERIAL

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1 th ICAMS International Conference on Advanced Materials and Systems CHARACTERIZATION AND APPLICATION OF COLLAGEN-POLYMER BASED COMPOSITE AS WOUND HEALING MATERIAL DENİZ GÜRLER, EYLEM KILIÇ Uşak University Department of Leather Engineering, 64200, Uşak, Turkey, deniz.celik@usak.edu.tr, eylem.kilic@usak.edu.tr Enzymatically derived collagen hydrolysate powder which is well-known with its good compatibility with skin and mucous membrane and thermoplastic polyurethane based synthetic polymer were used for the preparation of composite films as wound healing material. Prepared composite films were characterized and images of materials were taken using a scanning electron microscope. The wound healing pattern of collagen-polymer grafts were investigated in rabbits and additional preliminary clinical trials were conducted on diabetic foot ulcers to test the applicability of materials. Key words: collagen hydrolysate, collagen-polymer composites, woundhealing material. INTRODUCTION Skin replacement is a procedure to reconstitute dermis in case of extensive damage and loss of skin. The most frequently used methods to replace skin loss include covering the wound site with autografts, homografts and heterografts. Autografts may not be available due of scarcity of donor sites. Homografts and heterografts are very expensive, difficult to obtain, and difficult to store for prolonged periods (Travis et al 1987).These difficulties might be easily avoided by developing inexpensive biological or artificial skin substitutes. For this purpose both artificial and natural polymers such as collagen have been used. Collagen and hydrolyzed collagen which are derived from animal by-products and skin are natural substrates for cellular attachment, growth and differentiation, and promotes cellular proliferation and differentiation which are important parameters for wound dressing materials (Ruszczak 2003). This study aimed at developing and investigating the applicability of a new collagen based material as wound dressing material. Animal experiments and preliminary clinical experiments were conducted on patients who have diabetic foot ulcers. MATERIALS AND METHOD Animal skin derived collagen hydrolysate, Nutrilon Powder-I was obtained from Cognis Chemicals Company. Polyurethane based synthetic polymer at biomedical grade, Pellethane A was purchased from Dow Company. N,Ndimethylformamide is used as solvent. In order to obtain a proper film, various amounts of collagen hydrolysate 0.5, 1.0, 2 g and 0.5, 1.0, 2.0, 2.5g of polymer were used. Prior to film preparation, polymer was dissolved in N,N-dimethyl diformamide (Pişkin 1987).Mixing ratio of solvent and polymer-collagen is an important parameter, therefore 0.08g polymer and collagen hydrolysate was dissolved in 1 ml of solvent considering the previous studies (Tuncel 1997). The film preparation procedure is presented in Figure 1.

2 Characterization and Application of Collagen-Polymer Based Composite as Wound Healing Material COLLAGEN HYDROLYSATE POLYMER Dissolution in N-N Dimethylformamide Dissolution in N-N Dimethylformamide COLLAGEN-POLYMER MIXTURE Spreading over 10x20cm glass plates obtaining 30µ thickness and left for 15min for drying f Washing with distilled water Soaking in glycerine-water bath for 10 min. Drying in vacuum oven for 20min. UV sterilization overnight Characterization of collagen/polymer film Figure 1. Flow diagram of the collagen-polymer film preparation procedure RESULTS AND DISCUSSION The optimum collagen:polymer mixing ratio which enables the best proper film formation was determined as 0.5:1 and 1:1. These materials are called as A type and B type respectively, and further studies were conducted using these two types of collagenpolymer films (Figure 2). Figure 2. A and B type collagen-polymer films Characterization of Collagen-Polymer Films A and B type materials were characterized by determination of ph, thickness, tensile strength and elongation at break, colour and water vapour permeability properties. The experiments were performed in triplicate and the results are presented in Table 1 by the mean values.

3 th ICAMS International Conference on Advanced Materials and Systems Table 1. Properties of collagen-polymer films ph Thickness Tensile strength Elongation Colour (L, a, b) Water vapour permeability A type B type mm 8.69 dan/cm2 463% 91.68, -0.84, mg/1000mm2 24h mm 6.08 dan/cm2 426% 93.55, -0.78, mg/1000mm2 24h The thickness of collagen films were measured following the spreading of collagenpolymer mixture over glass plates. Thickness values of collagen films are comparative with the previous studies conducted on collagen based biomaterials (Oran 1999, Lee et al., 2001).Higher tensile strength and elongation properties of A type collagen films reveals that A type has higher mechanical strength and elastic in comparison to B type material..spectrophotometric colour measurements of collagen-polymer films revealed that increasing collagen amount does not affect the colour of material and both films have white colour. A type material has better water vapour permeability property, mainly due to its higher porosity. Scanning Electron Microscopy (SEM) SEM micrographs of A and B type collagen-polymer films were taken at x35, x1000, and x3500 magnifications (Figure 3). B type material had the smallest average pore diameter of 0,62μm and lower porosity. Decrease in polymer concentration increased porosity and resulted in larger pores at A type film, which might provide better attachment of cells during woundhealing and improve recovery time (Vasita 2006). Figure 3. SEM micrographs of A and B material with x35, x1000 and x3500 magnifications

4 Characterization and Application of Collagen-Polymer Based Composite as Wound Healing Material Animal Experiments and Preliminary Clinical Trials Figure 4 shows the rabbit skin grafting experimental procedures. Eight New Zealand rabbits were selected for the study. Animals were anesthetized and open wounds (1.5x3.0cm) were made under aseptic conditions on lateral chest and abdominal barrier of rabbits (Fig 4a, b). Following the excision of the skin, the grafts cut into the same dimensions were placed on the wound bed (Fig 4c) and sutured to skin by surgical sutures (Fig 4d). The grafted areas were covered with sterile gauze and bandaged. In control group only antibiotic pomade was used. Figure 4. Application of collagen-polymer films on wound defects Wound healing patterns of implanted A and B type collagen-polymer grafts were investigated at 7th, 14th and 21st days and images are shown in Figure 5. Control Day 7 B type A type Control B type A type Day 14 Control B type A type Day 21 Figure 5. Progression of wound healing at 7th, 14th and 21st days With respect to macroscopic observations on wound healing periods of collagenpolymer films, it was found that the wound implanted with collagen-polymer films healed faster than the control wounds. At the end of the 21st day it was observed that the wound coated with A type material was completely recovered, defect covered with B type was almost recovered and the control defect was not recovered and exhibits excessive granulated defect form. Collagen-based substrates induce less wound contraction than occurs in an untreated control wound (Ophof et al. 2003). Because of evidence that prepared collagen grafts can promote wound healing, preliminary clinical trials were undertaken at Department of Endocrinology and Metabolism Diseases of Ege University to evaluate its effectiveness on four patients who have diabetic foot ulcers (Figure 6).

5 th ICAMS International Conference on Advanced Materials and Systems Patient I Week 1 Week 2 Patient II Week 0 Week 3 Week 1 Week 12 Patient III Patient IV Week 2 Week 11 Figure 6. Preliminary clinical trials on diabetic foot ulcers

6 Characterization and Application of Collagen-Polymer Based Composite as Wound Healing Material In clinical trials, it was observed that collagen graft improved the wound healing process. However because of the wound localization and compressive forces applied to the feet, graft of patient II teared up in course of walking. Therefore, further trials should be under taken to modify and to improve the mechanical strength of grafts, and additional clinical studies should be performed with improved collagen-polymer films. CONCLUSION In the present study two types of collagen-polymer films were developed. In animal experiments, application of collagen-polymer films on wound sites prevented the wound contraction and deposition of excess granulation tissue and complete recovery was observed at the end of the 21st. Collagen grafts promoted wound healing in preliminary clinical trials, however their mechanical properties should be improved in case of foot wound applications. REFERENCES Lee, C. et al. (2001), Biomedical applications of collagen, International Journal of Pharmaceutics, 19, 221(1-2), Lee, C.H. et al. (2001), Biomedical applications of collagen, International Journal of Pharmaceutics, 221, Ophof, R. et al. (2003), Histologic evaluation of skin-derived and collagen-based substrates implanted in palatal wounds, Wound Repair and Regeneration, 12(5), Oran, E. (1999), Collagen based biomaterials, Hacettepe University, Deparment of Bioengineering, Master thesis, 1-33, Ankara. Pişkin, E. (1987), PolimerTeknolojisine Giriş, İnkilapKitabevi, İstanbul. Ruszczak, Z. (2003), Effect of collagen matrices on dermal wound healing, Adv Drug Deliv Rev., 28, 55(12), Travis, J.H., Harney, J.H. and Thornton, J.W. (1987), Modified Collagen Membrane as a Skin Substitute: Preliminary Studies, J.Biomedical. Mer. Res., 9, Tuncel, A. et al. (1997), Nondegradable ve Biyodegradable Yara ve Yanık Örtü Materyalleri, Sağlık Bilimleri AraştırmaGrubu, Ankara. Vasita, R. and Katti, D.S. (2006), Nanofibers and their applications in tissue engineering, Int J Nanomedicine, 1(1),