(J. Taiwan Agric. Res.) 61(1):1 11 (2012) 1 2,3 2,3 2 2,3 4 5,6 2012 61:1 11 (Tremella fuciformis) - (p > 0.05) (Kal-PC) 10 14 (Tp-nonW) 14 (Alg-NC) (p < 0.05)Tp-nonW1014 Alg-NC20%49.2% 14Kal-PC5.8Tp-nonW 5.4Alg-NC4.6 (p > 0.05) (Tp-nonW) (chitin) (chitosan) (alginic acid) (hyaluronic acid) (glycosaminoglycans) (chondroitin sulfate) 1. 261110121 2. 3. 4. 5. 6. debbie@fthes-tari.gov.tw(07)7315590
2 61 1 (Doillon & Silver 1986; Gao et al. 1992; Gilchrist & Martin 1983; Kirker et al. 2002; Liu et al. 2010; Murakami et al. 2010; Wang et al. 2006; Wittayaareekul & Prahsarm 2006; Zou et al. 2009) (Tremella fuciformis) (Zhao et al. 1982) (Ukai et al. 1983) (Yang et al. 2007) (hydrogel) (sponge) Kaltostat, Kal-PCConvaTec (Calcium/Sodium Alginate = 82, http://www.dressings.org/dressings/ kaltosta.html) (sodium alginate nonwoven, Alg-NC) (tremella polysaccharides nonwoven, Tp-nonW, / = 12) (SD) 8300 g3 5 22 25 50 70% 12/12 (Yu et al. 2005) 182% Isoflurane (Halocarbon Laboratories, South Carolina, USA) (Benchtop Small Animal Anesthesia Unit, MSS 003, England) 1A ( 12 cm 6 cm) 2 cm 2 cm 70% 2 cm 2 cm (full thickness)4 5 mm (Roh et al. 2006) Rimadyl (Carprofen, Pfizer, NY, USA) 5 mg/kg1 3 0.9% Kaltostat (MicroporeTM, 3M, St. Paul, MN, USA) (ELATEX, Alcare, Tokyo, Japan) 371014 (1) 371014 13 (1A)
3 (charge coupled device, CCD) (1B) (Lieca, Q500MC, Nussloch, Germany) (%) = [ (1C) (2 cm 2 cm) (1D) ] 100 14 2% Isoflurane 0.5 cm 2.5 cm (Roh et al. 2006) 10% 1 2 μmh & E Masson s trichrome (MT) (Liu et al. 1996) (Opticphot-2, Nikon, Tokyo, Japan) Altavilla et al. (2001, 2005) A 1. (A) 3 (B D) (%) = [ ( C) (2 cm 2 cm) ( D)] 100 Fig. 1. Measurement of residual wound area of rat skin after covered Alg-NC (negative control) for 3 days. (A) A photograph showing the wounded area; (B D) Photographs of wound residual area taken by CCD camera. Note the wound residual area (%) was calculated by the area of wound (C) and divided by total excised area (2 cm 2 cm) (D) and then multiplied with 100.
4 61 1 1. Table 1. Time and procedure of treatment of wound dressing on rats Treatment Alg-NC (Negative control) Kal-PC (Positive control) Tp-nonW (Tremella-polysaccharides) Time of changing Type of wound dressing wound dressing Sodium alginate nonwoven One time at 3, 7, 10 and 14 days Kaltostat dressing One time at 3, 7, 10 and 14 days Tremella polysaccharides nonwoven dressing One time at 3, 7, 10 and 14 days Procedure of application 1. Wound cleaning with 0.9% sterile saline 2. Covering with Sodium alginate nonwoven dressing (2 cm 2 cm) 3. Covering with gauze (2 inch 2 ) 1. Wound cleaning with 0.9% sterile saline for 2. Covering with Kaltostat dressing (2 cm 2 cm) 3. Covering with gauze (2 inch 2 ) 1. Wound cleaning with 0.9% sterile saline 2. Covering with Tremella polysaccharides nonwoven dressing (2 cm 2 cm) 3. Covering with gauze (2 inch 2 ) (re-epithelialization) (granulation) (angiogenesis) 0 40 (, none)1 (+, slight) 2 (++, moderate)3 (+++, complete) 4 (++++, thick granulation) (2) 3 (Alg-NC) (Kal-PC) (Tp-nonW) 312.76 g ± 15.25 g317.56 g ± 11.21 g311.52 g ± 11.89 g 7323.04 g ± 14.96 g 328.84 g ± 11.89 g327.60 g ± 20.88 g10 327.44 g ± 17.24 g330.96 g ± 16.14 g328.65 g ± 15.61 g14 341.28 g ± 19.09 g347.36 g ± 9.83 g 336.43 g ± 18.83 g (p > 0.05) 37101423 (2A C ) Alg-NCTp-nonW Kal-PC Kal-PCTp-nonW 7 (2D F) (granulation tissue) Kal-PC10 (2G I) Kal-PC (2H) Tp-nonW (2I) 14 (2J L) Kal-PC (2K) Tp-nonW (2L) Alg-NCKal-PC Alg-NCKal-PC Tp-nonW3710 1433
5 2. Table 2. Criteria for estimation of wound healing on rats (Based on reports of Altavilla et al. 2001, 2005) Score Re-epithelialization Granulation of tissue Angiogenesis 0, Absent Inflammatory tissue Absence of angiogenesis, inflammation, and hemorrhage 1, + Little epidermal and dermal organization 2, ++ Moderate epidermal and dermal organization 3, +++ Complete remodeling of epidermis and dermis Thin granular layer Moderate granulation layer Thick granulation layer Altered angiogenesis (1 2 vessels per site) characterized by a high degree of edema, hemorrhage, and occasional congestion and thrombosis Few newly formed capillary vessels (3 6 per site), moderate degree of edema and hemorrhage. Occasional congestion and inter vascular fibrin deposition; absence of thrombosis Newly formed capillary vessels (7 10 per site), moderate degree of perivascular and interstitial edema and congestion. Absence of thrombosis and hemorrhage 4, ++++ Very thick granulation layer Newly formed and well- structured capillary vessels (10 per site) vertically disposed toward the epithelium and at the wound margins. Slight degree of perivascular edema (residue area) 65.9% ± 8.7%68.2% ± 10.3%72.7% ± 3.3%7 41.0% ± 13.0%30.7% ± 5.8%44.8% ± 6.3% 10 27.5% ± 7.1%18.9% ± 2.8% 22.0% ± 6.3%Kal-PCAlg-NC (p < 0.05)14 19.7% ± 5.7% 6.7% ± 2.8%10.0% ± 2.7%Kal-PC Tp-nonWAlg-NC (p < 0.05) 33 (healing area) 34.1% ± 8.7%31.8% ± 10.3%27.3% ± 3.3%7 59.0% ± 13.0%69.3% ± 5.8%55.2% ± 6.3%10 72.5% ± 7.1%81.1% ± 2.8%78.0% ± 6.3% Kal-PCAlg-NC (p < 0.05)14 80.3% ± 5.7%93.3% ± 2.8%90.0% ± 2.7%Kal-PCTp-nonWAlg-NC (p < 0.05) 14 (3) Ag-NCKal-PC Tp-nonW1.6 ± 0.51.8 ± 0.4 1.8 ± 0.4 1.8 ± 0.42.6 ± 0.9 2.2 ± 0.4 1.2 ± 0.41.4 ± 0.5 1.4 ± 0.5 4.6 ± 0.55.8 ± 0.55.4 ± 1.0 4 (p > 0.05) Kal-PCTp-nonW (3/5) Alg-NC (structure remodeling)
6 61 1 2. 3710 14 3 (A) (Alg-NC)(B) (Kal-PC)(C) (Kal-PC)7(D) (E) (F) 10 (G) (H) (I) 14 (J) (K) (L) Fig. 2. Naked eye observation of healing of surgical wounds of rat skin covered with wound dressing for 3, 7, 10 and 14 days. Left column (A, D, G, J): Rats were treated with Alg-NC (Negative control) for 3 (A), 7 (D), 10 (G) and 14 (J) days. Middle column (B, E, H, K): Rats were treated with Kal-PC (Positive control) for 3 (B), 7 (E), 10 (H) and 14 (K) days. Right column (C, F, I, L): Rats were treated with Tp-nonW (Tremella polysaccharide dressing) for 3 (C), 7 (F), 10 (I) and 14 (L) days.
7 3. Table 3. Area of residue and healing of each wound dressing covered on surgical wounds of rat skin Duration of treatment (Days) Area (%) Alg-NC z Kal-PC Tp-nonW 3 Residue y 65.9 ± 18.7 w 68.2 ± 10.3 72.7 ± 3.3 Healing x 34.1 ± 18.7 31.8 ± 10.3 27.3 ± 3.3 7 Residue 41.0 ± 13.0 30.7 ± 15.8 44.8 ± 6.3 Healing 59.0 ± 13.0 69.3 ± 15.8 55.2 ± 6.3 10 Residue 27.5 ± 17.1 18.9 ± 12.8* 22.0 ± 6.3 Healing 72.5 ± 17.1 81.1 ± 12.8* 78.0 ± 6.3 14 Residue 19.7 ± 15.7 6.7 ± 12.8* 10.0 ± 2.7* Healing 80.3 ± 15.7 93.3 ± 12.8* 90.0 ± 2.7* z Alg-NC: Negative control; Kal-PC: Positive control; Tp-nonW: Tremella polysaccharide dressing. y Wound residual area (%) was calculated by the area of wound and divided with total excised area (2 cm 2 cm) and then multiplied with 100. x Wound healing area (%) was calculated by the formula: Healing area (%) = (total excised area of wound)/total excised area (2 cm 2 cm) 100. w Data was expressed as Mean ± SD (n = 5). * Significant difference between the control and treated groups at p < 0.05. (mature collagen deposition) (scar) (SD rats) 2 cm 2 cm (full thickness) 371014 (%) (p > 0.05)3710 14 Kal-PC1014 Alg-NC (p < 0.05) Tp-nonW10 Alg-NC80.0%14 Alg-NC50.8% (p < 0.05) Tp-nonW1014 Alg-NC20.0%49.2% (p < 0.05) 14 Kal-PC5.8Tp-nonW5.4 Alg-NC4.6 (p > 0.05) Alg-NC Tp-nonW Kal-PC (3) 12 (Matthew et al. 1993; Odell et al. 1994) (Berry et al. 1996)Tp-nonW Kal-PC
8 61 1 A 3. 14 (A) (B) (Alg-NC)(C) (Kal-PC)(D) (Tp-nonW)Masson s Trichrome staining, 40 Fig. 3. Histopathology of healing granuloma formed on surgical wounds of rat skin after 14 days of covered with wound dressing of Noraml skin (A), Alg-NC (B), Kal-PC (C) or Tp-nonW (D). Magnification: 40. 4. Table 4. Pathological scores of healing of wounded tissues of rats by the treatment of wound dressing Type of wound dressing Pathological score Alg-NC z Kal-PC Tp-nonW Epidermal and dermal regeneration 1.8 ± 0.4 y 2.6 ± 0.9 2.2 ± 0.4 Granulation tissue thickness 1.6 ± 0.5 1.8 ± 0.4 1.8 ± 0.4 Angiogenesis 1.2 ± 0.4 1.4 ± 0.5 1.4 ± 0.5 Mean score 4.6 ± 0.5 5.8 ± 0.5 5.4 ± 1.0 z Alg-NC: Negative control group; Kal-PC: Positive control; Tp-nonW: Tremella polysaccharide dressing. y Data was expressed as Mean ± SD (n = 5). (Panchatcharam et al. 2006; Roh et al. 2006)
9 (%) 10 (Kal-PC) (Alg-NC) (p < 0.05)14 (p < 0.05) (Kaltostat, Kal-PC) > (Tremella polysaccharides nonwoven, Tp-nonW) > (Sodium alginate nonwoven, Alg-NC) (Literature cited) Altavilla, D., A. Saitta, D. Cucinotta, M. Galeano, B. Deodato, M. Colonna, V. Torre, G. Russo, A. Sardella, G. Urna, G. M. Campo, V. Cavallari, G. Squadrito, and F. Squadrito. 2001. Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse. Diabetes 50:667 674. Altavilla, D., M. Galeano, A. Bitto, L. Minutoli, G. Squadrito, P. Seminara, F. S. Venuti, V. Torre, M. Calò, M. Colonna, P. Lo Cascio, G. Giugliano, N. Scuderi, C. Mioni, S. Leone, and F. Squadrito. 2005. Lipid peroxidation inhibition by raxofelast improves angiogenesis and wound healing in experimental burn wound. Shock 24:85 91. Berry, D. P., S. Bale, and K. G. Harding. 1996. Dressings for treating cavity wounds. J. Wound Care 5:10 17. Doillon, C. J. and F. H. Silver. 1986. Collagen-based wound dressing: effects of hyaluronic acid and firponectin on wound healing. Biomaterials 7:3 8. Gao, Z. R., Z. Q. Hao, Y. Li, M. J. Im, and R. J. Spence. 1992. Porcine dermal collagen as a wound dressing for skin donor sites and deep partial skin thickness burns. Burns 18:492 496. Gilchrist, T. and A. M. Martin. 1983. Wound treatment with Sorbsan An alginate fibre dressing. Biomaterials 4:317 320. Kirker, K. R., Y. Luo, J. H. Nielson, J. Shelby, and G. D. Prestwich. 2002. Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing. Biomaterials 23:3661 3671. Liu, C. H., Y. C. He, W. F. Jang, J. P. Jhu, and S. J. Wang. 1996. An Atlas and Manual of Histopathological Staining Methods: Histochemistry. Animal Technology Institute Taiwan (ATIT). Miaoli. 205 pp. (in Chinese) Liu, S. J., Y. C. Kau, C. Y. Chou, J. K. Chen, R. C. Wu, and W. L. Yeh. 2010. Electrospun PLGA/collagen nanofibrous membrane as early-stage wound dressing. J. Membr. Sci. 355:53 59. Matthew, I. R., R. M. Browne, J. W. Frame, and B. G. Millar. 1993. Tissue response to a haemostatic alginate wound dressing in tooth extraction sockets. Br. J. Oral Maxillofac. Surg. 31:165 169. Murakami, K., H. Aoki, S. Nakamura, S. I. Nakamura, M. Takikawa, M. Hanzawa, S. Kishimoto, H. Hattori, Y. Tanaka, T. Kiyosawa, Y. Sato, and M. Ishihara. 2010. Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings. Biomaterials 31:83 90. Odell, E. W., P. Oasdes, and T. Lombardi. 1994. Symptomatic foreign body reaction to haemostatic alginate. Br. J. Oral Maxillofac. Surg. 32:178 179. Panchatcharam, M., S. Miriyala, V. S. Gayathri, and L. Suguna. 2006. Curcumin improves wound healing by modulating collagen and decreasing reactive oxygen species. Mol. Cell Biochem. 290:87 96. Roh, D. H., S. Y. Kang, J. Y. Kim, Y. B. Kwon, H. U. Kweon, K. G. Lee, Y. H. Park, R. M. Baek, C. Y. Heo, C. Cho, and J. H. Lee. 2006. Wound healing effect of silk fibroin/alginate-blended sponge in full thickness skin defect of rat. J. Mater. Sci. Mater. Med. 17:547 552. Ukai, S., T. Kiho, C. Hara, I. Kuruma, and Y. Tanaka. 1983. Polysaccharides in fungi. XIV. Anti-inflammatory effect of the polysaccharides from the fruit bodies of several fungi. J. Pharmacobiodyn. 6:983 990. (in Japanese with English abstract) Wang, T. W., J. S. Sun, H. C. Wu, Y. H. Tsuang, W. H. Wang, and F. H. Lin. 2006. The effect of gelatin chondroitin sulfate hyaluronic acid skin substitute on wound healing in SCID mice. Biomaterials 27:5689 5697. Wittaya-areekul, A. and C. Prahsarm. 2006. Develop-
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11 Effect of Tremella Polysaccharides Nonwoven Dressing on Healing of Surgical Wounds of Rats 1 Kun-Chao Chen 2,3, Jhaol-Huei Wu 2,3, Chung-Hsin Chen 2, Jiunn-Wang Liao 2,3, Chan-Yi Yang 4, and Shu-Hui Yang 5,6 Abstract Chen, K. C., J. H. Wu, C. H. Chen, J. W. Liao, C. Y. Yang, and S. H. Yang. 2012. Effect of Tremella polysaccharides nonwoven dressing on healing of surgical wounds of rats. J. Taiwan Agric. Res. 61:1 11. Some commercial products used as wound dressing in clinics are made of natural polysaccharides which are biodegradable and biocompatible. Polysaccharides extracted from fruiting bodies of Tremella fuciformis are nature polymers with moisturizing and anti-inflammatory properties. The objective of this study was to determine effects of the Tremella-polysaccharide non-woven product (TpnonW) as wound dressing on healing of surgical wounds of rats. Results showed that wound dressing with Tp-nonW or Kal-PC (a commercial product Kaltostat made of Calcium/Sodium Alginate and used as positive control) significantly (p < 0.05) decreased the wound area of rats. Compared to the treatment of wound dressing of Alg-NC (a commercial product made of Sodium Alginate and used as negative control), the healing area of wounds covered with Tp-nonW for 10 and 14 days increased by 20 and 49.2% respectively. Results of the histopathological study showed that the score of regrowth tissues from wounded skin was 5.8 and 5.4 for the treatment of Kal-PC and Tp-nonW, respectively, compared to the score of 4.6 for the negative control Alg-NC, but the difference among these three treatments was statistically insignificant (p > 0.05). This study suggests that Tremella-polysaccharide non-woven dressing (Tp-nonW) has wound healing properties on rats and it may warrant further studies in human clinic trials. Key words: Tremella fuciformis, Polysaccharides nonwoven, Wound dressing, Animal model. 1. Contribution No. 2611 from Taiwan Agricultural Research Institute (TARI), Council of Agriculture. Accepted: February 1, 2012 2. Research Assistant and Professor, Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan, ROC. 3. Research Assistant and Professor, Animal Disease Diagnostic Center, National Chung Hsing University, Taichung, Taiwan, ROC. 4. Assistant Researcher, Department of Raw Materials and Yarns, Taiwan Textile Research Institute, New Taipei City, Taiwan, ROC. 5. Assistant Researcher, Department of Management and Utilization, Fengshan Tropical Horticultural Experimental Station, TARI, Kaohsiung, Taiwan, ROC. 6. Corresponding author, e-mail: debbie@fthes-tari.gov.tw; Fax: (07)7315590.