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Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests a
a
a
Nahoko Shimizu , Daiki Ishida , Akiko Yamamoto , Misato b
Kuroyanagi & Yoshimitsu Kuroyanagi
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a
R&D Center for Artificial Skin, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan b
Plastic and Aesthetic Surgery, School of Medicine, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan Published online: 24 Jun 2014.
To cite this article: Nahoko Shimizu, Daiki Ishida, Akiko Yamamoto, Misato Kuroyanagi & Yoshimitsu Kuroyanagi (2014) Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests, Journal of Biomaterials Science, Polymer Edition, 25:12, 1278-1291, DOI: 10.1080/09205063.2014.929427 To link to this article: http://dx.doi.org/10.1080/09205063.2014.929427
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 12, 1278–1291, http://dx.doi.org/10.1080/09205063.2014.929427
Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests Nahoko Shimizua, Daiki Ishidaa, Akiko Yamamotoa, Misato Kuroyanagib and Yoshimitsu Kuroyanagia*
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a
R&D Center for Artificial Skin, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan; bPlastic and Aesthetic Surgery, School of Medicine, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan (Received 27 February 2014; accepted 27 May 2014) This study aimed to develop a novel wound dressing composed of hyaluronic acid (HA) spongy sheet containing bioactive components. The wound dressing prepared by the freeze-drying method has a two-layered structure: an upper layer composed of cross-linked high-molecular-weight HA (HMW-HA) and a lower layer composed of low-molecular-weight HA (LMW-HA) containing arginine (Arg), magnesium ascorbyl phosphate (vitamin C derivative: VC), and epidermal growth factor (EGF) (referred to as EGF-dressing). A wound dressing containing only Arg and VC was prepared in a similar manner (referred to as EGF-free-dressing). The potential of each wound dressing was evaluated in animal tests using Sprague Dawley (SD) rats and diabetic mice. In the first experiment, each wound dressing was applied to a full-thickness skin defect in the abdominal region of SD rats. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available non-woven alginate wound dressing (Alg-dressing) was used in a control group. Both EGF-free-dressing and EGFdressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. In particular, EGF-dressing promoted re-epithelialization. In the second experiment, each wound dressing was applied to a full-thickness skin defect in the dorsal region of diabetic mice. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available Alg-dressing was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. These findings indicate that EGF-free-dressing and EGF-dressing have the potential for more effective wound healing when compared with Alg-dressing. In particular, EGF-dressing has a higher potential for wound healing when compared with EGF-free-dressing. Keywords: wound dressing; hyaluronic acid; epidermal growth factor; epithelialization; wound healing
1. Introduction Wound healing processes are impaired in chronic wounds, such as diabetic ulcers and pressure ulcers, in which the wound environments are unfavorable and normal *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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cell responses are attenuated. To improve the wound condition, various wound dressing and growth factor products have been developed, and are commercially available. For example, epidermal growth factor (EGF) products have been used in both China and Korea, and basic fibroblast growth factor (bFGF) products have been used in Japan. Prior to clinical use, EGF and bFGF freeze-dried products must be dissolved in the accompanying solution, must be preserved in a refrigerator, and must be used within a specified time period. Aqueous solutions containing growth factor are then sprayed onto the wound, after which a top dressing is placed as a covering material. However, some problems with this approach have been noted. For example, the aqueous solution is partially absorbed into the top dressing. In addition, growth factor needs to be applied daily, as free-growth factor dissolved in water is biodegraded by proteases present in the chronic wound. Furthermore, it has been suggested that the benzalkonium chloride, present in the solution, accompanying the bFGF product is cytotoxic to keratinocytes and fibroblasts.[1,2] In order to improve the troublesome daily application for commercially available EGF and bFGF products, we have developed several types of wound dressings composed of hyaluronic acid (HA) and collagen (Col) containing EGF or bFGF. HA has been identified as a major extracellular matrix component. HA is an important biomaterial for wound healing activities.[3–5] Because of its unique hygroscopic, rheological, and viscoelastic properties, HA creates an excellent wound-healing environment. Highmolecular-weight HA (HMW-HA) provides an excellent wound-healing environment, while low-molecular-weight HA (LMW-HA) induces angiogenesis following its degradation.[6–8] Col plays a pivotal role in wound healing. Col and Col-derived peptides act as a chemoattractants for fibroblasts in vitro and may have a similar activity in vivo.[9] The first version was a two-layered structure: an upper layer composed of crosslinked HMW-HA and a lower layer composed of LMW-HA containing arginine (Arg) and EGF.[10,11] These previous studies demonstrated a dose dependence of Arg on wound healing and a potential of EGF to facilitate wound healing in animal experiments. This wound dressing was prepared by freeze-drying for three times. A crosslinked HMW-HA spongy sheet was prepared by freeze-drying an HMW-HA aqueous solution containing cross-linking agent (the first step), followed by rinsing to remove free cross-linking agent, and then by freeze-drying (the second step). The resulting spongy sheet was immersed into a LMW-HA aqueous solution containing Arg and EGF, followed by freeze-drying (the third step). In order to simplify this manufacturing process, the present study was designed to prepare a two-layered wound dressing by freeze-drying for one time. The second version was a mono-layered structure composed of HMW-HA, LMWHA, and collagen (Col) containing Arg, magnesium ascorbyl phosphate (vitamin C derivative: VC), and EGF.[12–16] These previous studies demonstrated the combined effect of VC and EGF on wound healing. Based on results in our previous studies,[10–16] the present study aimed to develop a two-layered wound dressing containing Arg, VC, and EGF by freeze-drying for one time. The results obtained in our previous in vitro study demonstrated that this wound dressing has the potential to enhance fibroblast cytokine production in a sustained manner.[17] In the present study, the potential of this wound dressing was investigated in animal tests using Sprague Dawley (SD) rats and diabetic mice.
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2. Materials and methods 2.1. Experimental materials
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HA powder (Bio Sodium Hyaluronate HA 20; molecular weight of 2000 kDa) was purchased from Shiseido (Tokyo, Japan). Ethylene glycol diglycidyl ether (EX810) was purchased from Nagasekasei (Osaka, Japan). Recombinant human EGF was purchased from Shanghai Haohai Biological Technology (Shanghai, China). Arg was purchased from Junsei Chemical (Tokyo, Japan). VC was purchased from Showa Denko (Tokyo, Japan). Alginate wound dressing (Sorbsan; Alg-dressing) was purchased from ALCARE (Tokyo, Japan). Alg-dressing was employed as a comparative dressing, because it was composed of polysaccharide. Polyurethane film dressing (Bioclusive) and Elastic tape (Elastikon) were purchased from Johnson & Johnson (New Jersey, USA).
2.2. Preparation of wound dressing composed of HA spongy sheet The spongy sheet was prepared by method described in our previous article.[17] Briefly, HA powder (10 g) was dissolved in 1000 mL of distilled water (DW) to give a HMW-HA (molecular weight: 2000 kDa) solution. This HA solution was adjusted to pH 4.0 using 10% diluted HCl. EX810 (0.5 g) dissolved in 2 mL of DW was added drop-wise to the HMW-HA solution with vigorous stirring for 1 h. Another portion of HA powder (10 g) was dissolved in 1000 mL of DW, and was then autoclaved at 120 °C for 1 h to obtain partially hydrolyzed LMW-HA (molecular weight: 150 kDa) solution. Arg (1 g) dissolved in 20 mL of DW, and VC (1 g) dissolved in 20 mL of DW were added to the LMW-HA solution, and this was adjusted to pH 10.5 using 2 N NaOH. This mixed solution was divided into aliquots of 520 mL, and EGF (1000 μg) dissolved in 10 mL of DW was added to the LMW-HA solution containing Arg and VC (520 mL). This solution was designated EGF-containing solution. For EGF-free solution, 10 mL of DW was added to the LMW-HA solution containing Arg and VC (520 mL). Twenty milliliters of cross-linked HMW-HA solution (pH 4.0) was poured into a tray (5 cm × 8 cm), onto which 21 mL of EGF-free solution or EGF-containing solution (pH 10.5) was poured. Each combined solution was refrigerated at 4 °C for 2 h, followed by freezing at −85 °C and freeze-drying to obtain two types of wound dressing, referred to as EGF-free-dressing (Group I) or EGF-dressing (Group II). EGF-dressing was designed to contain EGF at a concentration of 1 μg/cm2 (expressed as μg/cm2 for convenience instead of μg/area of 1 cm2). Each wound dressing was packed in a bag and kept in a dry sterilizer at 110 °C for 1 h. Surface and cross-sectional views were obtained using a scanning electron microscope.
2.3. First animal test: surgical wound in SD rats The animal experiments were conducted in a similar manner as described previously.[12] Briefly, the abdominal region of SD rats (male, 8 weeks of age) was shaved and a 30-mm diameter region was surgically excised under anesthesia. EGF-free-dressing (Group I) or EGF-dressing (Group II) was then applied to the fullthickness skin defect. In the control group, a commercially available Alg-dressing was applied in a similar manner. A commercially available polyurethane film dressing was
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placed on each wound dressing as a covering material. Sterilized gauze was placed over the polyurethane film dressing and fixed with elastic tape. Wound conditions were evaluated based on macroscopic and histological appearances after 1 week. In another experiment, wound conditions were evaluated macroscopically after 1 week, followed by covering again with each dressing in a similar manner, and were then evaluated based on macroscopic and histological appearances after another 1 week (2 weeks in total). The wound size was measured by tracing out a wound surface area on a polyethylene film, followed by using imaging software (Scion Image; Scion Corporation).
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2.4. Second animal test: surgical wound in diabetic mice The animal experiments were conducted in a similar manner as described previously.[13] Briefly, the dorsal region of genetically type II diabetic BKS.Cg− + Leprdb/+ Leprdb (db/db) mice (male, 10 weeks of age) were shaved and a region with a 1.5 cm × 2.0 cm was surgically excised in the dorsal region on each side of the spine under anesthesia. EGF-free-dressing or EGF-dressing was applied to a full-thickness skin defect. In the control group, a commercially available Alg-dressing was applied in a similar manner. A commercially available polyurethane film dressing was placed on each wound dressing as a covering material. Sterilized gauze was placed over the polyurethane film dressing and fixed with elastic tape. Evaluations were conducted in a similar manner as in the first experiment. This animal study was conducted in compliance with the Animal Study Committee of School of Allied Health Sciences, Kitasato University. 2.5. Histological evaluation of epithelialization and angiogenesis Biopsy specimens were fixed in 10% formalin neutral buffer solution, embedded in paraffin, sectioned at 3 μm, and mounted onto slides. Serial sections were deparaffinized and stained with hematoxylin and eosin (HE). Specimens were then observed by light microscopy. The extent of epithelialization from the wound margin in the cranial and caudal directions was measured using a scale in photographs of HE-stained samples. In the first experiment, the extent of angiogenesis was measured by tracing out the cross-sectional area of blood vessels found in a region measuring 1000 μm × 500 μm on photographs of HE-stained samples, followed by using imaging software (Scion Image; Scion Corporation). Histological observation was conducted in the middle of the cranial and caudal wound margins. In the second experiment, the extent of angiogenesis in a region measuring 1000 μm × 1000 μm was assessed in a similar manner as described above. Histological observation was conducted at two sites: in the middle of the cranial wound margin and in the center, as well as in the middle of the caudal wound margin and in the center. 2.6. Statistical evaluation Data are expressed as means ± SEM. Statistical analysis was performed using the Tukey–Kramer test.
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3. Results 3.1. Preparation of wound dressings composed of HA spongy sheet Macroscopic view and scanning electron microphotograph of the EGF-free-dressing are shown in Figure 1. The spongy sheet showed a unique cross-sectional profile displaying a two-layered structure with vertical holes.
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3.2. First animal test: surgical wound in SD rats The macroscopic appearance of the wound area before and after application of each wound dressing at 1 week and 2 weeks is shown in Figure 2. In the case of Alg-dressing, non-woven alginate was not biodegraded within 1 week on the wound surface. A residual alginate was incorporated into granulation tissue and appeared to suppress granulation tissue formation. In contrast, the lower spongy layer of both EGFfree-dressing and EGF-dressing appears to be biodegraded successfully within 1 week and residual material, i.e. the upper spongy layer of cross-linked HMW-HA was not incorporated into granulation tissue. The upper spongy layer of these wound dressings appeared to provide a proper moist environment with a wound surface and thereby facilitating wound healing. Both EGF-free-dressing and EGF-dressing improved wound conditions more successfully and decreased wound size more effectively at 1 week and 2 weeks as compared with Alg-dressing (Figure 3). In particular, EGF-dressing appeared to substantially decrease wound size associated with well epithelialization. The histological appearance of the wound margin at 1 week and 2 weeks after application of each wound dressing is shown in Figure 4. EGF-free-dressing and
Figure 1. Scanning electron microphotographs of EGF-free-dressing: (a) upper layer, (b) lower layer, and (c) cross-section. Scale bar = 1 mm.
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Figure 2. Macroscopic appearance of the wound surface before and after 1 week and 2 weeks of treatment.
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EGF-dressing facilitated epithelialization more effectively at 2 weeks as compared with Alg-dressing. In particular, EGF-dressing appeared to substantially facilitate epithelialization (Figure 5).
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Figure 4. Histological appearance of the wound margin after 1 week and 2 weeks of treatment. Arrows indicate the distance of epithelialization from the wound margin. Scale bar = 250 μm. 10 1w
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The histological appearance of the granulation tissue at 1 week and 2 weeks after application of each wound dressing is shown in Figure 6. In the case of Alg-dressing, residual alginate appeared to suppress granulation tissue formation at 1 week and 2
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Figure 6. Histological appearance of granulation tissue formation in the wound area after 1 week and 2 weeks of treatment. Arrows indicate residual alginate, Scale bar = 250 μm.
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Figure 7. Extent of angiogenesis evaluated by measuring the total cross-sectional area of blood vessels found in a region of granulation tissue after 1 week and 2 weeks of treatment. * p < 0.05 vs. Alg-dressing after 1 week, ††p < 0.01 vs. Alg-dressing after 2 weeks.
weeks. In contrast, EGF-free-dressing and EGF-dressing appeared to successfully facilitate granulation tissue formation. EGF-free-dressing and EGF-dressing facilitated angiogenesis more effectively than Alg-dressing. EGF-dressing facilitated angiogenesis more successfully as compared with EGF-free-dressing (Figure 7). 3.3. Second animal test: surgical wound in diabetic mice The macroscopic appearance of the wound area before and after application of each wound dressing at 1 week and 2 weeks is shown in Figure 8. Both EGF-free-dressing
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Figure 8. Macroscopic appearance of the wound surface before and after 1 week and 2 weeks of treatment.
and EGF-dressing improved wound condition more successfully and decreased wound size more effectively at 2 weeks after application, as compared with Alg-dressing (Figure 9).
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Figure 9. Evaluation of wound size after 1 week and 2 weeks of treatment. * p < 0.05 vs. Alg-dressing after 2 weeks.
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The histological appearance of the wound margin at 1 week and 2 weeks after application of each wound dressing is shown in Figure 10. EGF-free-dressing and EGF-dressing facilitated epithelialization at 2 weeks as compared with Alg-dressing. In particular, EGF-dressing appeared to facilitate epithelialization (Figure 11). The histological appearance of the granulation tissue at 1 week and 2 weeks after application of each wound dressing is shown in Figure 12. Both EGF-free-dressing and EGF-dressing appeared to facilitate granulation tissue formation at 2 weeks as compared
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Figure 10. Histological appearance of the wound margin after 1 week and 2 weeks of treatment. Arrows indicate the distance of epithelialization from the wound margin. Scale bar = 250 μm.
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Figure 12. Histological appearance of granulation tissue formation in the wound area after 1 week and 2 weeks of treatment. Scale bar = 250 μm.
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Figure 13. Extent of angiogenesis evaluated by measuring total cross-sectional area of blood vessels found in a region of granulation tissue after 1 week and 2 weeks of treatment.
with Alg-dressing. In addition, EGF-free-dressing and EGF-dressing facilitated angiogenesis more effectively than Alg-dressing. EGF-dressing facilitated angiogenesis as compared with EGF-free-dressing (Figure 13). 4. Discussion The choice of materials is essential for an ideal wound dressing. In addition, the design of structure is also essential for an ideal wound dressing. Both EGF-free-dressing and EGF-dressing are designed to have a two-layered structure: an upper layer composed of cross-linked HMW-HA and a lower layer composed of non-cross-linked LMW-HA containing bioactive components. The upper spongy layer was designed to provide a
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highly hydrated environment on the wound surface, while the lower spongy layer was designed to promote angiogenesis. Our previous study demonstrated that this type of wound dressing has a potential to facilitate wound healing in animal experiments.[10,11] However, our original manufacturing process was complicated. The present study was designed to prepare a two-layered wound dressing containing Arg, VC, and EGF by freeze-drying for one time. In practice, EGF-dressing was prepared through a unique procedure. Cross-linked HMW-HA solution (pH 4.0) was poured into a tray, onto which LMW-HA solution containing EGF, Arg, and VC (pH 10.5) was poured, and this was refrigerated at 4 °C and frozen at − 85 °C, followed by freeze-drying to obtain a two-layered spongy sheet. Cross-linking agent can react with the carboxyl group of HA under acidic pH conditions, but cannot react with the anionic charged carboxyl group of HA under basic pH conditions.[17] This property is advantageous to establish a simplified manufacturing process. In addition, LMW-HA solution having a lower viscosity can be poured onto HMW-HA solution having a higher viscosity, and thereby a two-layered solution was maintained. This property is also advantageous to establish a simplified manufacturing process. EGF is known to be a potent stimulator of cell proliferation.[18,19] It is known that EGF is beneficial in wound healing because of its effects on proliferation of keratinocytes, fibroblasts, and vascular endothelial cells, thus, facilitating the formation of granulation tissue and re-epithelialization. In addition, EGF can stimulate fibroblasts to synthesize an increased amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF).[17] VEGF and HGF are potent cytokines for the promotion of wound angiogenesis. Recent research has demonstrated that simultaneous administration of VEGF and HGF synergistically promotes new blood vessel formation as compared with the administration of each factor alone.[20] In addition, HGF is considered to be one of the key cytokines involved in epithelialization in addition to angiogenesis.[21] Thus, EGF is a promising component for wound dressings. Arg and Arg-derived nitric oxide stimulate vasodilatation, and also enhance keratinocytes and endothelial cell proliferation.[22–24] In our previous studies, we demonstrated the effects of Arg on wound healing in an animal study using rats.[10] VC stimulates collagen synthesis by fibroblasts, and also has antioxidant effects. Furthermore, HGF production by fibroblasts is markedly stimulated by the synergistic effects of EGF and vitamin C in vitro.[16,25] Therefore, Arg and VC are also promising components for wound dressings. Based on the results of our previous studies, the present study focused on the potential of EGF-dressing to promote wound healing. Prior to the animal study, an in vitro study was conducted using a wound surface model in order to evaluate the sustained potential of EGF-dressing.[17] Human fibroblast-embedded collagen gel sheet (referred to as cultured dermal substitute: CDS) was elevated to the air and culture medium interface to create a wound surface model, on which each wound dressing was placed, followed by culture for 3 days (first period), after which dressings were placed on another CDS for a further 3-day culture period (second period). EGF-dressing enhanced the production of VEGF and HGF by fibroblasts within CDS during the first and second periods, as compared with EGF-free dressing. These results suggest that EGF can be maintained partially in the hydrated upper layer of wound dressing composed of cross-linked HMW-HA. The result in our previous in vitro study [17] demonstrated that a lower layer of free LMW-HA released from an upper layer of cross-linked HMW-HA in a wound surface model. In case of in vitro experiment, the upper layer maintained an original
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spongy structure. On the contrary, this upper layer failed to maintain an original spongy structure, but showed jelly-like residue. This finding suggests that this upper layer would likely be biodegraded partially on a wound surface in animal experiment. The hydrated upper layer was not incorporated into granulation tissue and is thus capable of providing a moist environment on the wound area. In addition to covering the wound surface, this upper layer appeared to hold some of the dissolved LMW-HA and bioactive components, thereby enhancing wound healing in a sustained fashion. 5. Conclusion The animal study demonstrated that EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with a commercially available Alg-dressing. EGFdressing has a particularly high potential for wound healing as compared with EGFfree-dressing. EGF-dressing has excellent potential when applied at intervals of 1 week, but not when applied daily. These findings indicate that EGF-dressing can improve the troublesome daily application for commercially available EGF product. References [1] Kubo M, Moriguchi T. Attached lysate with Fiblast Spray and benzalkonium chloride were cytotoxic to normal human keratinocytes and fibroblasts cultured on type I collagen. Jpn. J. Dermatol. 2011;121:1607–1620. [2] Damour O, Hua SZ, Lasne F, Villain M, Rousselle P, Collombel C. Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes. Burns. 1992;18:479–485. [3] Laurent TC, Fraser JR. Hyaluronan. FASEB. 1992;6:2397–2404. [4] Chen WY, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair Regen. 1999;7:79–89. [5] Pardue EL, Ibrahim S, Ramamurthi A. Role of hyaluronan in angiogenesis and its utility to angiogenic tissue engineering. Organogenesis. 2008;4:203–214. [6] West DC, Hampson IN, Arnold F, Kumar S. Angiogenesis induced by degradation products of hyaluronic acid. Science. 1985;228:1324–1326. [7] Sattar A, Rooney P, Kumar S, Pye D, West DC, Scott I, Ledger P. Application of angiogenic oligosaccharides of hyaluronan increases blood vessel numbers in rat skin. J. Invest. Dermatol. 1994;103:576–579. [8] Lees VC, Fan TP, West DC. Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides of hyaluronan. Lab Invest. 1995;73:259–266. [9] 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;75:871–875. [10] Matsumoto Y, Arai K, Momose H, Kuroyanagi Y. Development of a wound dressing composed of a hyaluronic acid sponge containing arginine. J. Biomater. Sci. Polym. Ed. 2009;20:993–1004. [11] Matsumoto Y, Kuroyanagi Y. Development of a wound dressing composed of hyaluronic acid sponge containing arginine and epidermal growth factor. J. Biomater. Sci. Polym. Ed. 2010;21:715–726. [12] Kondo S, Kuroyanagi Y. Development of a wound dressing composed of hyaluronic acid and collagen sponge with epidermal growth factor. J. Biomater. Sci. Polym. Ed. 2012;23:629–643. [13] Kondo S, Niiyama H, Yu A, Kuroyanagi Y. Evaluation of a wound dressing composed of hyaluronic acid and collagen sponge containing epidermal growth factor in diabetic mice. J. Biomater. Sci. Polym. Ed. 2012;23:1729–1740.
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[14] Yu A, Matsuda Y, Takeda A, Uchinuma E, Kuroyaangi Y. Effect of EGF and bFGF on fibroblast proliferation and angiogenic cytokine production from cultured dermal substitutes. J. Biomater. Sci. Polym. Ed. 2012;23:1315–1324. [15] Yu A, Niiyama H, Kondo S, Yamamoto A, Suzuki R, Kuroyanagi Y. Wound dressing composed of hyaluronic acid and collagen containing EGF or bFGF: comparative culture study. J. Biomater. Sci. Polym. Ed. 2013;24:1015–1026. [16] Niiyama H, Kuroyanagi Y. Development of novel wound dressing composed of hyaluronic acid and collagen sponge containing epidermal growth factor and vitamin C derivative. J. Artif. Organs. 2014;17:81–87. [17] Yamamoto A, Shimizu N, Kuroyanagi Y. Potential of wound dressing composed of hyaluronic acid containing epidermal growth factor to enhance cytokine production by fibroblasts. J. Artif. Organs. 2013;16:489–494. [18] Carpenter G, Cohen S. Human epidermal growth factor and the proliferation of human fibroblasts. J. Cell. Physiol. 1976;88:227–237. [19] Carpenter G, Cohen S. Epidermal growth factor. Annu. Rev. Biochem. 1979;48:193–216. [20] Xin X, Yang S, Ingle G, Zlot C, Rangell L, Kowalski J, Schwall R, Ferrara N, Gerritsen ME. Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. Am. J. Pathol. 2001;158:1111–1120. [21] Conway K, Price P, Harding KG, Jiang WG. The molecular and clinical impact of hepatocyte growth factor, its receptor, activators, and inhibitors in wound healing. Wound Repair Regen. 2006;14:2–10. [22] Witte MB, Barbul A. Role of nitric oxide in wound repair. Am. J. Surg. 2002;183:406–412. [23] Witte MB, Barbul A. Arginine physiology and its implication for wound healing. Wound Repair Regen. 2003;11:419–423. [24] Curran JN, Winter DC, Bouchier-Hayes D. Biological fate and clinical implications of arginine metabolism in tissue healing. Wound Repair Regen. 2006;14:376–386. [25] Wu YL, Gohda E, Iwao M, Matsunaga T, Nagao T, Takebe T, Yamamoto I. Stimulation of hepatocyte growth factor production by ascorbic acid and its stable 2-glucoside. Growth Horm. IGF Res. 1998;8:421–428.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests a
a
a
Nahoko Shimizu , Daiki Ishida , Akiko Yamamoto , Misato b
Kuroyanagi & Yoshimitsu Kuroyanagi
a
a
R&D Center for Artificial Skin, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan b
Plastic and Aesthetic Surgery, School of Medicine, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan Published online: 24 Jun 2014.
To cite this article: Nahoko Shimizu, Daiki Ishida, Akiko Yamamoto, Misato Kuroyanagi & Yoshimitsu Kuroyanagi (2014) Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests, Journal of Biomaterials Science, Polymer Edition, 25:12, 1278-1291, DOI: 10.1080/09205063.2014.929427 To link to this article: http://dx.doi.org/10.1080/09205063.2014.929427
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 12, 1278–1291, http://dx.doi.org/10.1080/09205063.2014.929427
Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests Nahoko Shimizua, Daiki Ishidaa, Akiko Yamamotoa, Misato Kuroyanagib and Yoshimitsu Kuroyanagia*
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a
R&D Center for Artificial Skin, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan; bPlastic and Aesthetic Surgery, School of Medicine, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan (Received 27 February 2014; accepted 27 May 2014) This study aimed to develop a novel wound dressing composed of hyaluronic acid (HA) spongy sheet containing bioactive components. The wound dressing prepared by the freeze-drying method has a two-layered structure: an upper layer composed of cross-linked high-molecular-weight HA (HMW-HA) and a lower layer composed of low-molecular-weight HA (LMW-HA) containing arginine (Arg), magnesium ascorbyl phosphate (vitamin C derivative: VC), and epidermal growth factor (EGF) (referred to as EGF-dressing). A wound dressing containing only Arg and VC was prepared in a similar manner (referred to as EGF-free-dressing). The potential of each wound dressing was evaluated in animal tests using Sprague Dawley (SD) rats and diabetic mice. In the first experiment, each wound dressing was applied to a full-thickness skin defect in the abdominal region of SD rats. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available non-woven alginate wound dressing (Alg-dressing) was used in a control group. Both EGF-free-dressing and EGFdressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. In particular, EGF-dressing promoted re-epithelialization. In the second experiment, each wound dressing was applied to a full-thickness skin defect in the dorsal region of diabetic mice. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available Alg-dressing was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. These findings indicate that EGF-free-dressing and EGF-dressing have the potential for more effective wound healing when compared with Alg-dressing. In particular, EGF-dressing has a higher potential for wound healing when compared with EGF-free-dressing. Keywords: wound dressing; hyaluronic acid; epidermal growth factor; epithelialization; wound healing
1. Introduction Wound healing processes are impaired in chronic wounds, such as diabetic ulcers and pressure ulcers, in which the wound environments are unfavorable and normal *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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cell responses are attenuated. To improve the wound condition, various wound dressing and growth factor products have been developed, and are commercially available. For example, epidermal growth factor (EGF) products have been used in both China and Korea, and basic fibroblast growth factor (bFGF) products have been used in Japan. Prior to clinical use, EGF and bFGF freeze-dried products must be dissolved in the accompanying solution, must be preserved in a refrigerator, and must be used within a specified time period. Aqueous solutions containing growth factor are then sprayed onto the wound, after which a top dressing is placed as a covering material. However, some problems with this approach have been noted. For example, the aqueous solution is partially absorbed into the top dressing. In addition, growth factor needs to be applied daily, as free-growth factor dissolved in water is biodegraded by proteases present in the chronic wound. Furthermore, it has been suggested that the benzalkonium chloride, present in the solution, accompanying the bFGF product is cytotoxic to keratinocytes and fibroblasts.[1,2] In order to improve the troublesome daily application for commercially available EGF and bFGF products, we have developed several types of wound dressings composed of hyaluronic acid (HA) and collagen (Col) containing EGF or bFGF. HA has been identified as a major extracellular matrix component. HA is an important biomaterial for wound healing activities.[3–5] Because of its unique hygroscopic, rheological, and viscoelastic properties, HA creates an excellent wound-healing environment. Highmolecular-weight HA (HMW-HA) provides an excellent wound-healing environment, while low-molecular-weight HA (LMW-HA) induces angiogenesis following its degradation.[6–8] Col plays a pivotal role in wound healing. Col and Col-derived peptides act as a chemoattractants for fibroblasts in vitro and may have a similar activity in vivo.[9] The first version was a two-layered structure: an upper layer composed of crosslinked HMW-HA and a lower layer composed of LMW-HA containing arginine (Arg) and EGF.[10,11] These previous studies demonstrated a dose dependence of Arg on wound healing and a potential of EGF to facilitate wound healing in animal experiments. This wound dressing was prepared by freeze-drying for three times. A crosslinked HMW-HA spongy sheet was prepared by freeze-drying an HMW-HA aqueous solution containing cross-linking agent (the first step), followed by rinsing to remove free cross-linking agent, and then by freeze-drying (the second step). The resulting spongy sheet was immersed into a LMW-HA aqueous solution containing Arg and EGF, followed by freeze-drying (the third step). In order to simplify this manufacturing process, the present study was designed to prepare a two-layered wound dressing by freeze-drying for one time. The second version was a mono-layered structure composed of HMW-HA, LMWHA, and collagen (Col) containing Arg, magnesium ascorbyl phosphate (vitamin C derivative: VC), and EGF.[12–16] These previous studies demonstrated the combined effect of VC and EGF on wound healing. Based on results in our previous studies,[10–16] the present study aimed to develop a two-layered wound dressing containing Arg, VC, and EGF by freeze-drying for one time. The results obtained in our previous in vitro study demonstrated that this wound dressing has the potential to enhance fibroblast cytokine production in a sustained manner.[17] In the present study, the potential of this wound dressing was investigated in animal tests using Sprague Dawley (SD) rats and diabetic mice.
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2. Materials and methods 2.1. Experimental materials
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HA powder (Bio Sodium Hyaluronate HA 20; molecular weight of 2000 kDa) was purchased from Shiseido (Tokyo, Japan). Ethylene glycol diglycidyl ether (EX810) was purchased from Nagasekasei (Osaka, Japan). Recombinant human EGF was purchased from Shanghai Haohai Biological Technology (Shanghai, China). Arg was purchased from Junsei Chemical (Tokyo, Japan). VC was purchased from Showa Denko (Tokyo, Japan). Alginate wound dressing (Sorbsan; Alg-dressing) was purchased from ALCARE (Tokyo, Japan). Alg-dressing was employed as a comparative dressing, because it was composed of polysaccharide. Polyurethane film dressing (Bioclusive) and Elastic tape (Elastikon) were purchased from Johnson & Johnson (New Jersey, USA).
2.2. Preparation of wound dressing composed of HA spongy sheet The spongy sheet was prepared by method described in our previous article.[17] Briefly, HA powder (10 g) was dissolved in 1000 mL of distilled water (DW) to give a HMW-HA (molecular weight: 2000 kDa) solution. This HA solution was adjusted to pH 4.0 using 10% diluted HCl. EX810 (0.5 g) dissolved in 2 mL of DW was added drop-wise to the HMW-HA solution with vigorous stirring for 1 h. Another portion of HA powder (10 g) was dissolved in 1000 mL of DW, and was then autoclaved at 120 °C for 1 h to obtain partially hydrolyzed LMW-HA (molecular weight: 150 kDa) solution. Arg (1 g) dissolved in 20 mL of DW, and VC (1 g) dissolved in 20 mL of DW were added to the LMW-HA solution, and this was adjusted to pH 10.5 using 2 N NaOH. This mixed solution was divided into aliquots of 520 mL, and EGF (1000 μg) dissolved in 10 mL of DW was added to the LMW-HA solution containing Arg and VC (520 mL). This solution was designated EGF-containing solution. For EGF-free solution, 10 mL of DW was added to the LMW-HA solution containing Arg and VC (520 mL). Twenty milliliters of cross-linked HMW-HA solution (pH 4.0) was poured into a tray (5 cm × 8 cm), onto which 21 mL of EGF-free solution or EGF-containing solution (pH 10.5) was poured. Each combined solution was refrigerated at 4 °C for 2 h, followed by freezing at −85 °C and freeze-drying to obtain two types of wound dressing, referred to as EGF-free-dressing (Group I) or EGF-dressing (Group II). EGF-dressing was designed to contain EGF at a concentration of 1 μg/cm2 (expressed as μg/cm2 for convenience instead of μg/area of 1 cm2). Each wound dressing was packed in a bag and kept in a dry sterilizer at 110 °C for 1 h. Surface and cross-sectional views were obtained using a scanning electron microscope.
2.3. First animal test: surgical wound in SD rats The animal experiments were conducted in a similar manner as described previously.[12] Briefly, the abdominal region of SD rats (male, 8 weeks of age) was shaved and a 30-mm diameter region was surgically excised under anesthesia. EGF-free-dressing (Group I) or EGF-dressing (Group II) was then applied to the fullthickness skin defect. In the control group, a commercially available Alg-dressing was applied in a similar manner. A commercially available polyurethane film dressing was
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placed on each wound dressing as a covering material. Sterilized gauze was placed over the polyurethane film dressing and fixed with elastic tape. Wound conditions were evaluated based on macroscopic and histological appearances after 1 week. In another experiment, wound conditions were evaluated macroscopically after 1 week, followed by covering again with each dressing in a similar manner, and were then evaluated based on macroscopic and histological appearances after another 1 week (2 weeks in total). The wound size was measured by tracing out a wound surface area on a polyethylene film, followed by using imaging software (Scion Image; Scion Corporation).
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2.4. Second animal test: surgical wound in diabetic mice The animal experiments were conducted in a similar manner as described previously.[13] Briefly, the dorsal region of genetically type II diabetic BKS.Cg− + Leprdb/+ Leprdb (db/db) mice (male, 10 weeks of age) were shaved and a region with a 1.5 cm × 2.0 cm was surgically excised in the dorsal region on each side of the spine under anesthesia. EGF-free-dressing or EGF-dressing was applied to a full-thickness skin defect. In the control group, a commercially available Alg-dressing was applied in a similar manner. A commercially available polyurethane film dressing was placed on each wound dressing as a covering material. Sterilized gauze was placed over the polyurethane film dressing and fixed with elastic tape. Evaluations were conducted in a similar manner as in the first experiment. This animal study was conducted in compliance with the Animal Study Committee of School of Allied Health Sciences, Kitasato University. 2.5. Histological evaluation of epithelialization and angiogenesis Biopsy specimens were fixed in 10% formalin neutral buffer solution, embedded in paraffin, sectioned at 3 μm, and mounted onto slides. Serial sections were deparaffinized and stained with hematoxylin and eosin (HE). Specimens were then observed by light microscopy. The extent of epithelialization from the wound margin in the cranial and caudal directions was measured using a scale in photographs of HE-stained samples. In the first experiment, the extent of angiogenesis was measured by tracing out the cross-sectional area of blood vessels found in a region measuring 1000 μm × 500 μm on photographs of HE-stained samples, followed by using imaging software (Scion Image; Scion Corporation). Histological observation was conducted in the middle of the cranial and caudal wound margins. In the second experiment, the extent of angiogenesis in a region measuring 1000 μm × 1000 μm was assessed in a similar manner as described above. Histological observation was conducted at two sites: in the middle of the cranial wound margin and in the center, as well as in the middle of the caudal wound margin and in the center. 2.6. Statistical evaluation Data are expressed as means ± SEM. Statistical analysis was performed using the Tukey–Kramer test.
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3. Results 3.1. Preparation of wound dressings composed of HA spongy sheet Macroscopic view and scanning electron microphotograph of the EGF-free-dressing are shown in Figure 1. The spongy sheet showed a unique cross-sectional profile displaying a two-layered structure with vertical holes.
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3.2. First animal test: surgical wound in SD rats The macroscopic appearance of the wound area before and after application of each wound dressing at 1 week and 2 weeks is shown in Figure 2. In the case of Alg-dressing, non-woven alginate was not biodegraded within 1 week on the wound surface. A residual alginate was incorporated into granulation tissue and appeared to suppress granulation tissue formation. In contrast, the lower spongy layer of both EGFfree-dressing and EGF-dressing appears to be biodegraded successfully within 1 week and residual material, i.e. the upper spongy layer of cross-linked HMW-HA was not incorporated into granulation tissue. The upper spongy layer of these wound dressings appeared to provide a proper moist environment with a wound surface and thereby facilitating wound healing. Both EGF-free-dressing and EGF-dressing improved wound conditions more successfully and decreased wound size more effectively at 1 week and 2 weeks as compared with Alg-dressing (Figure 3). In particular, EGF-dressing appeared to substantially decrease wound size associated with well epithelialization. The histological appearance of the wound margin at 1 week and 2 weeks after application of each wound dressing is shown in Figure 4. EGF-free-dressing and
Figure 1. Scanning electron microphotographs of EGF-free-dressing: (a) upper layer, (b) lower layer, and (c) cross-section. Scale bar = 1 mm.
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EGF-dressing facilitated epithelialization more effectively at 2 weeks as compared with Alg-dressing. In particular, EGF-dressing appeared to substantially facilitate epithelialization (Figure 5).
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The histological appearance of the granulation tissue at 1 week and 2 weeks after application of each wound dressing is shown in Figure 6. In the case of Alg-dressing, residual alginate appeared to suppress granulation tissue formation at 1 week and 2
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weeks. In contrast, EGF-free-dressing and EGF-dressing appeared to successfully facilitate granulation tissue formation. EGF-free-dressing and EGF-dressing facilitated angiogenesis more effectively than Alg-dressing. EGF-dressing facilitated angiogenesis more successfully as compared with EGF-free-dressing (Figure 7). 3.3. Second animal test: surgical wound in diabetic mice The macroscopic appearance of the wound area before and after application of each wound dressing at 1 week and 2 weeks is shown in Figure 8. Both EGF-free-dressing
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Figure 8. Macroscopic appearance of the wound surface before and after 1 week and 2 weeks of treatment.
and EGF-dressing improved wound condition more successfully and decreased wound size more effectively at 2 weeks after application, as compared with Alg-dressing (Figure 9).
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The histological appearance of the wound margin at 1 week and 2 weeks after application of each wound dressing is shown in Figure 10. EGF-free-dressing and EGF-dressing facilitated epithelialization at 2 weeks as compared with Alg-dressing. In particular, EGF-dressing appeared to facilitate epithelialization (Figure 11). The histological appearance of the granulation tissue at 1 week and 2 weeks after application of each wound dressing is shown in Figure 12. Both EGF-free-dressing and EGF-dressing appeared to facilitate granulation tissue formation at 2 weeks as compared
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Figure 10. Histological appearance of the wound margin after 1 week and 2 weeks of treatment. Arrows indicate the distance of epithelialization from the wound margin. Scale bar = 250 μm.
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Figure 12. Histological appearance of granulation tissue formation in the wound area after 1 week and 2 weeks of treatment. Scale bar = 250 μm.
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with Alg-dressing. In addition, EGF-free-dressing and EGF-dressing facilitated angiogenesis more effectively than Alg-dressing. EGF-dressing facilitated angiogenesis as compared with EGF-free-dressing (Figure 13). 4. Discussion The choice of materials is essential for an ideal wound dressing. In addition, the design of structure is also essential for an ideal wound dressing. Both EGF-free-dressing and EGF-dressing are designed to have a two-layered structure: an upper layer composed of cross-linked HMW-HA and a lower layer composed of non-cross-linked LMW-HA containing bioactive components. The upper spongy layer was designed to provide a
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highly hydrated environment on the wound surface, while the lower spongy layer was designed to promote angiogenesis. Our previous study demonstrated that this type of wound dressing has a potential to facilitate wound healing in animal experiments.[10,11] However, our original manufacturing process was complicated. The present study was designed to prepare a two-layered wound dressing containing Arg, VC, and EGF by freeze-drying for one time. In practice, EGF-dressing was prepared through a unique procedure. Cross-linked HMW-HA solution (pH 4.0) was poured into a tray, onto which LMW-HA solution containing EGF, Arg, and VC (pH 10.5) was poured, and this was refrigerated at 4 °C and frozen at − 85 °C, followed by freeze-drying to obtain a two-layered spongy sheet. Cross-linking agent can react with the carboxyl group of HA under acidic pH conditions, but cannot react with the anionic charged carboxyl group of HA under basic pH conditions.[17] This property is advantageous to establish a simplified manufacturing process. In addition, LMW-HA solution having a lower viscosity can be poured onto HMW-HA solution having a higher viscosity, and thereby a two-layered solution was maintained. This property is also advantageous to establish a simplified manufacturing process. EGF is known to be a potent stimulator of cell proliferation.[18,19] It is known that EGF is beneficial in wound healing because of its effects on proliferation of keratinocytes, fibroblasts, and vascular endothelial cells, thus, facilitating the formation of granulation tissue and re-epithelialization. In addition, EGF can stimulate fibroblasts to synthesize an increased amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF).[17] VEGF and HGF are potent cytokines for the promotion of wound angiogenesis. Recent research has demonstrated that simultaneous administration of VEGF and HGF synergistically promotes new blood vessel formation as compared with the administration of each factor alone.[20] In addition, HGF is considered to be one of the key cytokines involved in epithelialization in addition to angiogenesis.[21] Thus, EGF is a promising component for wound dressings. Arg and Arg-derived nitric oxide stimulate vasodilatation, and also enhance keratinocytes and endothelial cell proliferation.[22–24] In our previous studies, we demonstrated the effects of Arg on wound healing in an animal study using rats.[10] VC stimulates collagen synthesis by fibroblasts, and also has antioxidant effects. Furthermore, HGF production by fibroblasts is markedly stimulated by the synergistic effects of EGF and vitamin C in vitro.[16,25] Therefore, Arg and VC are also promising components for wound dressings. Based on the results of our previous studies, the present study focused on the potential of EGF-dressing to promote wound healing. Prior to the animal study, an in vitro study was conducted using a wound surface model in order to evaluate the sustained potential of EGF-dressing.[17] Human fibroblast-embedded collagen gel sheet (referred to as cultured dermal substitute: CDS) was elevated to the air and culture medium interface to create a wound surface model, on which each wound dressing was placed, followed by culture for 3 days (first period), after which dressings were placed on another CDS for a further 3-day culture period (second period). EGF-dressing enhanced the production of VEGF and HGF by fibroblasts within CDS during the first and second periods, as compared with EGF-free dressing. These results suggest that EGF can be maintained partially in the hydrated upper layer of wound dressing composed of cross-linked HMW-HA. The result in our previous in vitro study [17] demonstrated that a lower layer of free LMW-HA released from an upper layer of cross-linked HMW-HA in a wound surface model. In case of in vitro experiment, the upper layer maintained an original
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spongy structure. On the contrary, this upper layer failed to maintain an original spongy structure, but showed jelly-like residue. This finding suggests that this upper layer would likely be biodegraded partially on a wound surface in animal experiment. The hydrated upper layer was not incorporated into granulation tissue and is thus capable of providing a moist environment on the wound area. In addition to covering the wound surface, this upper layer appeared to hold some of the dissolved LMW-HA and bioactive components, thereby enhancing wound healing in a sustained fashion. 5. Conclusion The animal study demonstrated that EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with a commercially available Alg-dressing. EGFdressing has a particularly high potential for wound healing as compared with EGFfree-dressing. EGF-dressing has excellent potential when applied at intervals of 1 week, but not when applied daily. These findings indicate that EGF-dressing can improve the troublesome daily application for commercially available EGF product. References [1] Kubo M, Moriguchi T. Attached lysate with Fiblast Spray and benzalkonium chloride were cytotoxic to normal human keratinocytes and fibroblasts cultured on type I collagen. Jpn. J. Dermatol. 2011;121:1607–1620. [2] Damour O, Hua SZ, Lasne F, Villain M, Rousselle P, Collombel C. Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes. Burns. 1992;18:479–485. [3] Laurent TC, Fraser JR. Hyaluronan. FASEB. 1992;6:2397–2404. [4] Chen WY, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair Regen. 1999;7:79–89. [5] Pardue EL, Ibrahim S, Ramamurthi A. Role of hyaluronan in angiogenesis and its utility to angiogenic tissue engineering. Organogenesis. 2008;4:203–214. [6] West DC, Hampson IN, Arnold F, Kumar S. Angiogenesis induced by degradation products of hyaluronic acid. Science. 1985;228:1324–1326. [7] Sattar A, Rooney P, Kumar S, Pye D, West DC, Scott I, Ledger P. Application of angiogenic oligosaccharides of hyaluronan increases blood vessel numbers in rat skin. J. Invest. Dermatol. 1994;103:576–579. [8] Lees VC, Fan TP, West DC. Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides of hyaluronan. Lab Invest. 1995;73:259–266. [9] 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;75:871–875. [10] Matsumoto Y, Arai K, Momose H, Kuroyanagi Y. Development of a wound dressing composed of a hyaluronic acid sponge containing arginine. J. Biomater. Sci. Polym. Ed. 2009;20:993–1004. [11] Matsumoto Y, Kuroyanagi Y. Development of a wound dressing composed of hyaluronic acid sponge containing arginine and epidermal growth factor. J. Biomater. Sci. Polym. Ed. 2010;21:715–726. [12] Kondo S, Kuroyanagi Y. Development of a wound dressing composed of hyaluronic acid and collagen sponge with epidermal growth factor. J. Biomater. Sci. Polym. Ed. 2012;23:629–643. [13] Kondo S, Niiyama H, Yu A, Kuroyanagi Y. Evaluation of a wound dressing composed of hyaluronic acid and collagen sponge containing epidermal growth factor in diabetic mice. J. Biomater. Sci. Polym. Ed. 2012;23:1729–1740.
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