Abstract
This study explored the feasibility of development of solubilized amniotic membrane extract (AME) as a potential wound healing substrate with improved efficacy. Bovine amniotic membrane was extracted using a mixture of acetic acid and 2-mercaptopropionic acid under sonication, which was followed by the frozen, and then lyophilized processes. The effects of AME on cell migration and growth properties were evaluated from 0 to 24 h of post injury using primary human foreskin fibroblast monolayer culture with one line scratch as an in vitro wound model. Its wound healing efficacy and scar preventive effects were investigated using whole thickness biopsy punch (8 mm) wound model obtained from rabbit ear. Intra dermal injections of AME fluid (10 μl of 1.2 μg/μl) on four wound sites were performed at 1 h pre injury, post 1, 2 and 3 day. The processes and levels of re-epithelialization and dermal regeneration were examined through histological assessment with H–E staining. In cell migration study conducted at 24 h post injury, AME (1.7 μg/ml) treated cells significantly increased wound closure with 54.9 % compared to control. Histological image analysis on AME treated wound sites at 36 days post injury showed properly developed epidermal basal cell layers and weave-like dermal collagen bundles, whereas those of untreated control skin showed over-proliferation of epidermis and aggregated collagen bundles with defected dermal regeneration. The results of this study verified the feasibility of dermal injections of freeze dried AME as a potential wound healing substrate which can promote epidermal and dermal regeneration, while avoiding undesirable hyper-proliferation of damaged tissue.
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References
Bose, B. 1979. Burn wound dressing with human amniotic membrane. Annals of the Royal College of Surgeons of England 61: 444–447.
Cho Lee, A.R., H. Lim, J. Lee, and K.C. Park. 2005. Reversal of silver sulfadiazine impaired wound healing by epidermal growth factor. Biomaterials 26: 4670–4676.
Davis, J.W. 1910. Skin transplantation with a review of 550 cases at the Johns Hopkins hospital. The Johns Hopkins Medical Journal 15: 307.
Fetterolf, D.E., and R.J. Snyder. 2012. Scientific and clinical support for the use of dehydrated amniotic membrane in wound management. Wounds 24(10): 299–307.
Gajiwala, K., and A.L. Gajiwala. 2004. Evaluation of lyophilized, gamma-irradiated amnion as a biological dressing. Cell and Tissue Banking 5: 73–80.
Hunumanthappa, M.B., S. Gopinathan, and D. Gurupirasad Rai. 2012. Amniotic membrane dressing versus conventional dressing in lower limb varicose ulcer: A prospective comparative study. International Journal of Biological and Medical Research 3: 1616–1620.
Kang, M., Park, H., and A.R. Cho Lee. (2012). Suppression of CTGF and α-SMA in cultured human dermal fibroblast by bovine amniotic membrane extracts and its implication in scar-less wound healing. Proceed. Int.Conference of Kor.Soc.Pharm.Sci.Tec. 153–154.
Kloeters, O., A. Tandara, and T.A. Mustoe. 2007. Hypertrophic scar model in the rabbit ear: A reproducible model for studying scar tissue behavior with new observation on silicone gel sheeting for scar reduction. Wound Repair and Regeneration 18: S40–S45.
Kim, S.C., and A.R. Cho Lee. 2010. Preparation of reproducible and responsive scar model and histology analysis. Journal of Pharmaceutical Investigation 40(1): 45–49.
Koizumi, N.J., T.J. Inatomi, and C.J. Sotozono. 2000. Growth factor mRNA and protein in preserved human amniotic membrane. Current Eye Research 20: 173–177.
Krummel, T.M., J.M. Nelson, and R.F. Diegelmann. 1987. Fetal response to injury in the rabbit. Journal of Pediatric Surgery 22: 640–644.
Larson, B.J., M.T. Langaker, and H.P. Lorenz. 2010. Scarless fetal wound healing: A basic science review. Plastic and Reconstructive Surgery 126: 1172–1180.
Lee, J., A.R. Cho Lee, and J.H. Lee. (2012). Method for extracting component of amnion membrane by solubilization of amnion membranes and extract thereof. Patent No. 10-1187423, Korea .
McCauley, R.L., Y.Y. Li, B. Poole, M.J. Evans, M.C. Robson, J.P. Heggers, and D.N. Herndon. 1992. Differential inhibition of human basal keratinocyte growth to silver sulfadiazine and mafenide acetate. Journal of Surgical Research 52(3): 276–285.
Moon, H., H. Yong, and A.R. Cho Lee. 2012. Optimum scratch assay condition to evaluate connective tissue growth factor expression for anti-scar therapy. Archives of Pharmacal Research 35: 383–388.
Nakamura, T., M. Yoshitami, H. Rigby, N.J. Fullwood, W. Ito, T. Inatomi, C. Sotosomo, T. Nakamura, Y. Shimiz, and S. Kinoshita. 2004. Sterilized, freeze-dried amniotic membrane: A useful substrate for ocular surface reconstruction. Investigative Ophthalmology and Visual Science 45: 93–99.
Ozgenel, G.Y. 2002. The influence of human amniotic fluid on the potential of rabbit ear perichondrial flaps to form cartilage tissue. British Journal of Plastic Surgery 55: 246–250.
Piscatelli, S.J., B.M. Michaels, P. Gregory, R.W. Jennings, M.T. Longaker, M.R. Harrison, and J.W. Siebert. 1994. Fetal fibroblast contraction of collagen matrices in vitro: The effects of epidermal growth factor and transforming growth factor-b. Annals of Plastic Surgery 33: 38–45.
Riau, A.K., R.W. Beuerman, L.S. Lim, and J.S. Mehta. 2010. Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction. Biomaterials 31: 216–225.
Russo, A., P. Bonci, and P. Bonci. 2012. The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane. Cell Tissue Bank 13: 353–361.
Schwartzfarb, E., and R.S. Kisner. 2012. Understanding scarring: Scarless fetal wound healing as a model. The Journal of Investigative Dermatology 132: 260.
Tseng, S.C.G. 2001. Amniotic membrane transplantation for ocular surface reconstruction. Bioscience Reports 21: 481–489.
Woo, H., M. Kim, O. Kweon, D. Kim, T. Nam, and J. Kim. 2001. Effects of amniotic membrane on epithelial wound healing and stromal remodeling after excimer laser keratectomy in rabbit cornea. British Journal of Ophthalmology 85: 345–349.
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This work was supported in part by the research grant from Duksung Women’s University. We thank to Prof. Kyung Chan Park at department of dermatology, Seoul National University for providing primary human fibroblasts.
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Kang, M., Choi, S. & Cho Lee, AR. Effect of freeze dried bovine amniotic membrane extract on full thickness wound healing. Arch. Pharm. Res. 36, 472–478 (2013). https://doi.org/10.1007/s12272-013-0079-5
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DOI: https://doi.org/10.1007/s12272-013-0079-5