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tissue repair and regeneration program ■ 60 Musk Ave, Kelvin Grove, QLD, 4059, Australia ■ e: [email protected] ■ t: +61 7 3138 6185 Human Skin Equivalent Model Overview The human skin equivalent model: an alternative to animal testing The Human Skin Equivalent (HSE) model is a 3-dimensional live cell skin model with the potential to accurately investigate the effects that products may have when used on the skin of a customer. The model is a cost effective, viable alternative to animal testing and has applications in the investigation of products such as cosmetics, cosmeceuticals, suncreams, wound dressings and wound therapies. Background Skin, the largest organ in the body, performs many important roles including protection against physical and chemical insults, regulation of temperature and fluid loss, and immunity and sensory roles. Once damaged, skin cannot perform these normal tasks and numerous complications such as infection or fluid loss can occur1. To restore these roles, the body needs to close the wound as quickly as possible while retaining functionality and integrity. Our current understanding of the biological processes underlying wound closure, along with maintenance of skin function and integrity, is limited. This lack of fundamental knowledge has arisen in part from the less than ideal in vitro and ex vivo models available to facilitate skin research. For example, 2-dimensional (2D) in vitro cell culture studies do not accurately reflect the complex interactions that occur between the multiple cells present in the 3D in vivo skin environment. Similarly, in vivo studies in rodents and other small animals do not translate well to the human situation due to differences in anatomical structure, abundance of hair, and the fact these species are “loose-skinned” and heal wounds by contracture. In contrast, human skin heals via re-epithelialisation. The development of improved models is therefore critical to advancing our understanding of human skin repair, regeneration and maintenance processes2. Our laboratory has been investigating the utility of 3D human skin equivalent (HSEs) models as these appear to offer great promise and importantly, clinical relevance, for advancing the ex vivo study of human skin3. HSEs are created by culturing skin keratinocytes at the air-liquid interface on top of a dermal scaffold4. The dermal scaffold may consist of a de-epidermised dermis (DED), or a fibroblast-populated dermal substitute, such as a collagen matrix or inert filter5-10. The model that we have established in our laboratory, the DED HSE, is often considered more physiologically relevant as it uses a human acellular dermis to create a new multilayered epidermis11 and retains the basement membrane, which has been previously shown to be critical for keratinocyte attachment in vitro12. Our DED HSE model is seeded with keratinocytes and can be customised to include other cell types such as fibroblasts. The structural integrity of this processed DED is also very similar to native skin, therefore studies of the absorption of heat, light or chemicals through the epidermis and into the dermis experience the same physical obstacles/path as native skin13. Importantly, HSEs have been shown to closely resemble human skin in vivo morphologically and biochemically14-16. Therefore, HSEs have been adopted for a variety of purposes including: clinical skin grafts; modelling physiological processes in skin, i.e. wound healing; phototoxicity; toxicity; absorption; drug transfer; irritancy; and, metabolic studies of topically applied products15-17. 2 In Australia, no HSE model is readily obtainable for use by researchers. Although some HSEs have been made commercially available in overseas markets, such as EpiSkin® (L’Oreal, Paris) and EpiDermTM (MatTek, Massachusetts), international transport logistics, quarantine issues and affordability, make them almost impossible for Australians researchers to obtain14. This presents quite a challenge as HSEs are becoming increasingly important to consumer, cosmetic and pharmaceutical companies worldwide as the European Union (EU) regulation (76/768/EEC, Feb. 2003) prohibits the use of animal or animal-derived substances for the development and testing of consumer, cosmetic and pharmaceutical ingredients. This regulation will apply to all products imported into EU countries from 2009. This presents an imperative to eliminate the use of these substances for testing or inclusion in products for this and other international markets. To meet this challenge an effective and reliable skin model has to be available to researchers and to industry in general, here in Australia. As is described herein, our research has been directed at developing and validating a reproducible HSE model for use in a range of studies examining wound healing and new wound therapeutics. This is an area of healthcare that is in desperate need of innovation, as current management of chronic wounds such as diabetic and venous ulcers, as well as bed sores and skin tears, are far from ideal and are yet to adopt modern biotechnological approaches. The HSE model we have developed is an important tool to help elucidate the molecular events involved in wound healing, as well as for evaluating potential wound dressings that could help improve wound healing outcomes. The current prevalence of chronic wounds in Australia is estimated to be between 1.3% of the population/year and cost the Australian community upwards of AU$500 million per annum to treat 17,18 . These wounds are a major cause of grief and concern for affected individuals and also contribute significantly to their overall diminished quality of life 19. Moreover, these wounds are particularly relevant to rural and indigenous communities, where prevalence of chronic diseases, such as Type II Diabetes, is significantly above the rest of the population. While our own research is directed at improving wound healing, ultimately we anticipate that our leading research and the ongoing development of the HSE through the inclusion of additional cell types important to other processes in skin will facilitate not only our own ongoing research, but also that by the Australian biomedical research community. Indeed, the longer term goal is to establish a cost-recovery service that skin researchers across Australia can use and access, thereby facilitating internationally-pioneering research, improving pre-clinical data and minimising the use of animals for testing. Results to Date Recent ex vivo work in our laboratory suggests that the HSE model exhibits many of the characteristics found in human skin. This has been demonstrated by the research team in a number of different applications. For example, following modifications to a published de-epidermised dermis HSE model12,20, we validated the HSE employed in our laboratory using a number of epidermal cell markers, namely keratins 1/10/11, keratin 6, keratin 14 and a basement membrane marker, collagen IV21. In addition, following creation of the stratified epithelium, we have been able to create burn injuries in the HSE and then observe subsequent in-growth of cells into the damaged tissue. The healing of the burn in the HSE was assessed using a metabolic assay and histological analysis at days 0, 2, 4 and 6 post-injury. We have further used this model to quantitatively assess the potential of a novel wound therapy22 that is currently in clinical trials. In addition to testing therapeutics, we have also used the HSE model to evaluate biological mechanisms underpinning current wound treatments. For example, we have employed the HSE model to study the effects of daily 90-minute hyperbaric oxygen (HBO) treatments (for up to 5 days) on the reconstruction of an epidermis23. The influence of HBO on the anatomy of the newly formed epidermis was evaluated, as well as the expression of epidermal protein markers of proliferation, differentiation, and remodeling of the basement membrane. This study revealed that HBO stimulates the enhanced formation of an epidermis in the HSE model23; this in turn providing promising preliminary evidence that HBO therapy improves wound healing outcomes. The 3D HSE model has also been used to determine the efficacy of the 3D expansion of skin cells24. In this project animal-derived feeder cells were eliminated from the standard culture of skin cells by seeding the keratinocytes onto microspheres in suspension culture. After culture, the cell-laden microspheres were inoculated onto the 3D HSE 3 model to evaluate the migration of skin cells onto the dermal scaffold and to evaluate the structure of the viable epidermis that subsequently formed. This study demonstrated that normal human skin cells could be expanded on microspheres in the absence of feeder cells and retained their ability to form an epidermis24. Most recently, we have also refined the media used to culture the HSE so that we have removed all undefined or animal-derived products, including fetal calf serum, a product commonly used in the culture of mammalian cells25. In addition, we have used the HSE model to examine the effects of novel wound healing agents and dermal replacements through both partial thickness and full thickness lesions, respectively. HSE models were created using the previously described method20 and wounds were created using skin biopsy punches. The wounds were then exposed to biomimetic hydrogels designed to enhance healing. Functional assays demonstrated that the hydrogel treatments significantly enhanced epidermal reconstitution of the wounds over 7 days culture in vitro, while histological analysis revealed that that the fibroblasts and keratinocytes migrate into the hydrogel-implanted wound area, leading to closure over a 10-day period (unpublished data). Our research team has also used the HSE model to test biocompatibility of novel wound dressings under development in other R&D projects26. In particular, functionalised hydrogel dressings designed for chronic ulcer applications have been applied to the HSE model to assess any potential toxicity in this ex vivo model. Therefore, these HSE wound models appear to provide a highly relevant tool to assess potential wound healing agents in vitro and ex vivo. About the model Innovation Our laboratory has reported for the first time in Australia the development of an ex vivo 3D HSE20, as well as data demonstrating that the model can be cultured in a defined animal-product free medium25. Significantly, the model has also shown promise as a technology platform for the study of epithelial cellular events and for evaluating new dermal wound healing therapies22,26,27. In addition, we have extended the use of this HSE model to a number of applied projects. Instead of simply focussing on molecular and cellular events, we have made practical advances that have enabled us to use this technology in preclinical testing. Some examples of this include our studies examining the biocompatibility of novel biomaterials26, as well as injuring/burning the model and assessing novel growth factors complexes that encourage re-epithelialisation and healing22. Moreover, our current studies that involve “wounding” of the HSE model allow us to test novel wound healing agents that may improve healing times and healing outcomes for the thousands of patients who suffer chronic ulcers each year. Furthermore, we have recently performed a small pilot study examining the use of this model in assessing the effects of UV irradiation on human skin (unpublished data); again, the HSE model appears to hold significant promise in this field of research. UV irradiation is a major cause of skin cancer in Australia, a disease affecting thousands of Australians each year. Therefore, continuation of the current research with the HSE will provide us with further information on the mechanisms of UV damage in the skin, which in turn will lead to improved prevention and treatment strategies. Thus our past and current research directed at further development and refinement of the HSE is paving the way for future research, both by ourselves and others, without the use of animals as preliminary testing agents. In addition, knowledge generated through the development of this HSE model is also likely to improve current techniques for culturing keratinocytes and/or HSEs for clinical transplant purposes, since the HSE model itself has the potential to be used clinically as grafts for fullthickness wounds. Importantly, in view of the EU regulation prohibiting the sale of products testing on animals from 2009, this technology will also benefit Australian biomedical, device, pharmaceutical and cosmetics companies selling into European markets through the use of the model for developing and testing consumer products. Rigour of the research program and scholarly excellence The research described herein exemplifies the ideals of scientific rigour and scholarly excellence, as shown through its publication in several leading journals. The pilot study demonstrating that this HSE model could be established in Australia was published in Primary Intention (recently renamed as Wound Practice and Research), the quarterly journal of the Australian Wound Management Association. Dawson et al. (2006) were able to demonstrate that keratinocytes grown in a serum-free 4 culture media were able to retain their ability to form an epidermis when cultured on this HSE model (Transplantation, JIF: 3.972, Ranked 3 (of 19) in Transplantation, ISSN: 0041-1337). In addition, Kairuz et al. (2007) showed that the HSE was an excellent model to investigate the molecular responses of primary human keratinocytes to hyperbaric oxygen therapy (Wound Repair and Regeneration, JIF: 2.230, Ranked 10 (of 39) in Dermatology, ISSN: 1067-1927). This model was also able to be used in an initial toxicity study of novel hydrogels for wound dressing applications, as published in Biomaterials (JIF: 5.196, Ranked 1 (of 14) in Materials Science and Biomaterials, ISSN: 0142-9612). Furthermore, the 3D culture of primary human keratinocytes on microcarriers was examined by analysing their ability to reform an epidermis on the HSE model (Journal of Biomedical Materials Research Part A, JIF: 2.497, Ranked 4 (of 42) in Biomedical Engineering, ISSN: 00219304). Importantly, the HSE model has also been correlated with in vivo wound healing data demonstrating the efficacy of a novel wound healing agent (Journal of Investigative Dermatology, JIF: 4.535, Ranked 1 (of 39) in Dermatology, ISSN: 0022-202X). Most recently, our research group has shown that keratinocytes grown serum-free can also be transplanted on to this HSE model and maintained in serum-free culture, thereby removing any potential animal products (Tissue Engineering, JIF: 3.725, ranked 23 (of 140) in Biotechnology and Applied Microbiology, ISSN: 10763279). Therefore, this research clearly demonstrates strong scientific integrity and has passed intense scrutiny from several international external peer reviewers. Future research is directed at extending the model to include a number of cells types and in situ 2-photon imaging to investigate the structural integrity of the model. In situ 2-photon imaging can examine cellular morphology and transdermal delivery of agents non-destructively. When applied to the HSE model, it could be used to investigate the structural integrity of the model and track topical therapies traversing through the epidermis, e.g. amphiphilic silicone oligomers for hypertrophic scar remediation. By increasing the number of different cell types contained within the model, we hypothesise that the model will be even more relevant to the in vivo situation. This will involve incorporating additional cell types such as endothelial cells, e.g. microvascular endothelial cells (MVECs), inflammatory cells, (Langerhans’ cells) and other epidermal cells, (e.g. melanocytes). Impact The HSE model is an important scientific technology that enables the use of animals in scientific research to be replaced, reduced and refined. This aligns well with the Code of practice for the care and use of animals for scientific purposes, version 7, published by the National Health and Medical Research Council. The principle of the 3Rs, Replacement, Reduction and Refinement are all enshrined within this code, and are important targets met by this current research. By using this HSE model in initial testing stages, e.g. biocompatibility of novel hydrogels, animals are replaced by this ex vivo model. More importantly, the number of animals later used in in vivo testing can be greatly reduced by analysing different drug concentrations and modes of application in the ex vivo HSE model, instead of on the animal itself. This also leads on to the final principle of “refinement”. The type of animal used and the numbers per treatment can all be refined by the excellent ex vivo data that this model provides, which has already been shown by our research group in a number of diverse applications. References 1. Moulin V, Auger F, et al. Burns. 2000;26:3-12. 2. Gottrup F, Agren M, et al. Wound Repair and Regeneration. 2000;8:83-96. 3. MacNeil S. Nature. 2007;445:874-80. 4. Faller C, Bracher M, et al. Toxicology In Vitro. 2002;16:557-72. 5. Rosdy M, Clauss L. Journal of Investigative Dermatology. 1990;95:409-14. 6. Tinois E, Tiollier J, et al. Experimental Cell Research. 1991;193:310-9. 7. Cannon C, Neal P, et al. Toxicology in Vitro. 1994;8:889-91. 8. Prunieras M, Regnier M, et al. Journal of Investigative Dermatology. 1983;81:28S-33S. 9. Bell E, Parenteau N, et al. Toxicology in Vitro. 1991;5:591-6. 10. Ponec M, Weerheim A, et al. J Lipid Res. 1988;29:949-62. 11. Pianigiani E, Andreassi A, et al. Biomaterials. 1999;20(18):1689-94. 12. Chakrabarty KH, Dawson RA, et al. British Journal of Dermatology. 1999;141(5):811-23. 13. Dawson RA, Goberdhan N, et al. Burns. 1996;22(2):93-100. 14. Poumay Y, Dupont F, et al. Archives of Dermatological Research. 2004;296:203-11. 5 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Ponec M, Boelsma E, et al. Skin Pharmacology. 2002;15:4-17. Welss T, Basketter D, et al. Toxicology in Vitro. 2004;18:231-43. Gruen RL, Chang S, et al. Australia and New Zealand Journal of Surgery. 1996;66:171-4. Baker SR, Stacey MC. Australia and New Zealand Journal of Surgery. 1994;64:258-61. MacLellan DG. Australian Prescriber. 2000;23:6-9. Dawson RA, Upton Z, et al. Transplantation. 2006;81:1668-76. Topping G, Malda J, et al. Primary Intention. 2006;14(1):14-21. Upton Z, Cuttle L, et al. Journal of Investigative Dermatology. In press. Kairuz E, Upton Z, et al. Wound Repair and Regeneration. 2007;15(2):266-74. Borg DJ, Dawson R, et al. Journal of Biomedical Materials Research Part A. In press. Richards S, Leavesley DI, et al. Tissue Engineering. 2008;In press. Rayment EA, Dargaville TR, et al. Biomaterials. 2008;29(12):1785-95. Topping G, Malda J, et al. Primary Intention. 2006;14(1):14-21. Relevant Publications by our Research Team Richards, S., Leavesley, D.I. & Upton, Z. (2008) Development of Defined Media for the Serum-Free Expansion of Primary Keratinocytes and Human Embryonic Stem Cells., Tissue Engineering., 2008 July 11; [Epub ahead of print] (JIF: 3.725, ranked 23 (of 140) in Biotechnology and Applied Microbiology, ISSN: 1076-3279) Borg, D.J., Dawson, R.A., Leavesley, D.I., Upton, Z. & Malda, J. (2008) Functional and phentoptyic characterization of keratinocytes expanded using microcarrier culture. Journal of Biomedical Materials Research, Part A., 2008 Feb 19; [Epub ahead of print] (JIF: 2.497, Ranked 4 (of 42) in Biomedical Engineering, ISSN: 0021-9304) Rayment E. A., Dargaville T.R., Shooter G.K., George G.A. & Upton, Z. (2008) Attenuation of protease activity in chronic wound fluid (CFW) with bisphosphonate-functionalised hydrogels. Biomaterials, 29(12):1785-95. (JIF:5.196, Ranked 1 (of 14) in Materials Science and Biomaterials, ISSN:0142-9612) Upton, Z., Cuttle, L., Noble, A., Kempf, M., Topping, G., Hayes, M., Malda, J., Xie, Y., Mill, J., Leavesley, D., Kravchuk, O., Harkin, D.G. & Kimble, R. (2008) Vitronectin:growth factor complexes hold potential as a wound therapy approach. Journal of Investigative Dermatology, 128(6); 1535-44, (JIF: 4.535, Ranked 1 (of 39) in Dermatology, ISSN: 0022-202X) Kairuz, E., Upton, Z., Dawson, R. & Malda, J. (2007) Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents. Wound Repair and Regeneration, 15:266-274. (JIF: 2.230, Ranked 10 (of 39) in Dermatology, ISSN: 1067-1927) Dawson, R.A., Upton, Z., Malda, J. & Harkin, D.G. (2006) IGF-I used in conjunction with IGFBP-5, EGF and vitronectin supports rapid preparation of cultured skin transplants under serum-free conditions. Transplantation, 81: 1668-1676. (JIF: 3.972, Ranked 3 (of 19) in Transplantation, ISSN: 0041-1337) Topping, G., Malda, J., Dawson, R.A., & Upton, Z. (2006) Development and characterization of human skin equivalents and their potential application as a burn wound model. Primary Intention, 14: 14-21. (ISSN: 1323-2495)