Download Human Skin Equivalent Model

tissue repair and regeneration program
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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.
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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
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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
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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.
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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)