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Development Of Dried Allogeneic Human Cultured Epidermis As A Wound Dressing: Evaluation Of Healing Acceleration In Murine And Porcine Skin Defects
Takashi Nakano, MD1, Michiharu Sakamoto, MD, PhD1, Itaru Tsuge, MD, PhD1, Hiroki Yamanaka, MD, PhD1, Yasuhiro Katayama, MD, PhD1, Yoshihiro Shimizu, MS2, Yoshika Note, MS2, Masukazu Inoie, MS2, Akishige Hokugo, MD, PhD3, Naoki Morimoto, MD, PhD1.
1Graduate School of Medicine, Kyoto University, Kyoto City, Kyoto, Japan, 2Japan Tissue Engineering, Co., Ltd., Gamagori, Aichi, Japan, 3Regenerative Bioengineering and Repair Laboratory, Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, CA, USA.

PURPOSE: Cryopreserved allogeneic cultured epidermis (CE) has been used for treating second-degree burn wounds and diabetic foot ulcers; however, the need for cryopreservation limits its usability. We have previously reported that CE accelerates wound healing irrespective of its viability, and hypothesized that dried CEs act as an effective wound dressing. In this study, we investigated the efficacy of dried human cultured epidermis in accelerated wound healing using diabetic murine full-thickness skin defects and porcine split-thickness skin defects.METHODS: CE was prepared from human donor skin using Greens technique, with some modifications. Then, dried CE was prepared by drying fresh CE sheet under controlled temperature and humidity conditions until all water was completely removed. Cryopreserved CE was prepared at -80 C with a cryopreservation medium containing 10% dimethyl sulfoxide for more than 1 day. We investigated the morphology and physical properties of dried and cryopreserved CEs, including breaking strength, modulus of elasticity, water permeability, and levels of various active cytokines and chemokines (EGF, b-FGF, IFN-γ, IL-1a, IL-1b, IL-6, IL-8, MMP-1, MMP-9, PDGF, TGF-α, TNF-α, and VEGF). Furthermore, the acceleration of wound healing by dried CE was elucidated in vivo. Two symmetrical full-thickness skin defects 8 mm in diameter, were created on both sides of the back of each diabetic mouse. Each wound was covered with the cryopreserved or dried CEs (1.5 1.5 cm2). The wound area and neo-epithelial length were compared on days 4, 7, and 14 after application. In the porcine experiment, a male Clawn miniature pig was used. Using an electric dermatome, split-thickness skin wounds (8 9 cm2 in size, 1.5 mm in depth) were made at four locations on the back. Dried and cryopreserved CEs were applied to the wound, and the epithelialization ratios at 4 and 7 days after implantation were evaluated.RESULTS: Hematoxylin-eosin staining, immunostaining for basement membrane, and electron microscopy showed that the morphologies of dried CE and cryopreserved CE were comparable (consisting of 4-5 layers of keratinocytes, including a monolayer of basal cells), and that the membrane structure was not damaged. The breaking strength, levels of various active cytokines and chemokines, and moisture permeability in dried CE were also comparable to those in cryopreserved CE. In the diabetic murine experiment, dried CE significantly reduced the wound area on days 4 and 7 (p<0.05) and increased the epithelium length on days 4 and 7 (p<0.01). Dried CE also significantly increased the epithelialization ratio in the porcine donor site model (on days 4: 15.0 12.6% and 7: 88.0 10.0%), similar to that observed for cryopreserved CE (on days 4: 29.7 14.6% and 7: 67.9 20.7%).CONCLUSION: Dried CE has similar morphology, physical properties, and wound-healing effects as cryopreserved CE and can be a physiological and versatile wound dressing that can be stored for a long time at room temperature and used immediately off the shelf.


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