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Recreated skin

Artificial model tests efficacy of new compounds for pharmaceutical and cosmetic products

EDUARDO CESARArtificial skin made from material discarded during plastic surgeryEDUARDO CESAR

Artificial skin identical to human skin, which reproduces the same biological tissues and can be used to evaluate the toxicity and efficacy of new compounds for pharmaceutical products, was developed by researchers from the School of Pharmaceutical Sciences at the University of São Paulo. Cells taken from the skin of donors who had undergone corrective plastic surgery are the raw material used to produce artificial skin in the lab. “We developed a model that mimics the entire epidermis and dermis of the human skin”, says professor Silvya Stuchi Maria-Engler, coordinator of the research project.

The epidermis, the outermost layer of the skin, is obtained by means of a culture of keratinocytes, the cells that synthesize keratin and are responsible for protection, and a culture of melanocytes, the cells that produce the skin’s melanin and pigment. The dermis, the layer below the epidermis, is reconstituted from a culture of human fibroblasts – which produce fibers and synthesize collagen and elastin – cultivated in collagen gel. These cell structures have growth and morphology characteristics that closely resemble the human skin. This enhances the uniformity and reproducibility of medication and cosmetics testing.

“Our model will meet a market need”, says Silvya. In the case of cosmetics, for example, cosmetics companies send the active principles of new products to be tested abroad, because since early 2009, a European Community guideline establishes that no cosmetic product can be tested on lab animals to evaluate its safety and efficacy. To substitute animals, tests ensuring the safety and efficacy of new active principles and formulas have to be conducted on in vitro models, with isolated cells, or preferably on biomimetic models, such as the one that simulates human skin.

New molecules
Apart from the cosmetic industry, the pharmaceutical industry could also stop conducting some tests on animals by using skin biomimetic systems. Potential pharmaceutical components for treatment of conditions such as melanoma, a type of aggressive skin cancer, can be tested with the model developed at USP. Although melanoma is not rampant in Brazil, its mortality rate is high. The chances of healing are high in its early stages, but when it is found at an advanced stage, patient survival rates are very low, because the tumor is highly resistant to the chemotherapy compounds used in treatment. “Our studies focus on the search for new molecules that can attack this tumor cell, and try to subvert the effects of resistance to medication”, says Silvya.

Some artificial skin models are available in the United States and Europep, produced by the Episkin and SkinEthic companies, subsidiaries of the French cosmetics company L’Oréal, and by MatTek, a company from the state of Massachusetts. However, there are difficulties related to transportation and importing, because this is live material and, as such, is perishable. “The skin we developed is identical to the one produced abroad”, says Silvya. The USP model uses human cells that come from primary cultures, whose proliferating power helps differentiate the layers of the in vitro epidermis. This will allow custom-made kits to be produced in line with a client’s needs, because one can reproduce the entire skin or just the dermis or epidermis.

The products available on the international market use established cell lines. Although the manipulation of these products is easier because they do not depend on donors, they are unable to split into multiple layers, in the manner of primary cells. “In our product, the transformations are minimized because of the short period of cultivation time in the lab”, says Silvya. The customized kits, for example, can include melanocytes, usually absent in the products found on the market. If the objective is to evaluate the effects of new molecules for skin pigmentation, melanocytes that reflect the different ethnic groups can be incorporated into the cell culture to produce artificial pigmented skin. To evaluate anti-melanoma chemotherapy medication, the skin can be produced with the melanoma simulating the tumor invasion process in vitro.

CARLA ABDO BROHEM/USPCell structures of artificial skin, augmented 20xCARLA ABDO BROHEM/USP

One of the future possibilities for the artificial skin developed at USP is its use in corrective surgery on burn patients or patients with chronic lesions, a trend that has been growing abroad. In the United States, a group of researchers led by professor James McGuire, from Philadelphia’s Temple University, has successfully used artificial skin for the treatment of chronic lesions in diabetic patients. In New Zealand, the trend is to incorporate substances such as honey and particles of silver – which have antiseptic and antibacterial properties – to grafts made from collagen.

The research project that resulted in the artificial skin began in 2005, when professor Sílvia Berlanga de Moraes Barros, from USP’s School of Pharmaceutical Sciences (FCF/USP) was testing compounds with protective properties against UVB ultraviolet rays, the most damaging to the skin. Silvya Stuchi, at the time a recently hired associate professor at FCF, had been working in this field since her master’s program and therefore had the requisite knowledge and tools to develop an artificial skin model. “The result of my thesis was a structure that closely resembled the dermis, based on the collagen from mouse tendons”, says the researcher.

After concluding her master’s and doctoral programs at Unicamp, Silvya got a grant from FAPESP to go to the United States for a post-doctoral program. She stayed in the United States only for a short while, yet this short stay sufficed for her to learn how to produce artificial blood vessels. Upon her return, she joined the group led by researcher Mari Cleide Sogayar, from USP’s Chemistry Institute. Mari Cleide is working on a project on cell transplants for diabetic patients. Two years later, Silvya passed the entry exam for professors at the School of Pharmaceutical Sciences. As she already knew how to produce dermis, she proposed to professor Sílvia Berlanga to test sunblock compounds on this model, initially. “My subsequent project was to try and reorganize the epithelium, a cohesive, stratified tissue made up of various layers of keratinocytes, to which would be added the melanocytes, thus representing the dermal-epidermal unit resulting in skin”, she says. The major difficulty to be overcome was to obtain two types of specific human cells – the melanocytes and the keratinocytes. “We only had the fibroblasts, which can be purchased from cell banks”, she adds.

The opportunity arose when she was invited to spend a year at the Dermatology Department of the University of Michigan in the United States to work on anti-tumor medication for the skin, a line of research that she had already been working on. “I went there to work with professor María Soengas from Spain, one of the leading academic experts in this field”, says Silvya. At the time, María Soengas, currently the head of the melanoma group at Spain’s CNIO Cancer Research Center, was working with the University of Michigan on research into this kind of tumor. Recently, the Spanish researcher’s group identified a synthetic compound capable of unleashing the self-destruction of melanoma cells; this discovery has opened up the way for the manufacturing of new medications.

NÍVEA OLIVEIRA/USPCulture plate for biomimetic model NÍVEA OLIVEIRA/USP

Brazilian model
During her stay in Michigan, Silvya learned how to isolate and produce a culture of human patients’ keratinocytes and melanocytes from the prepuce skin of newborn babies. “This material has great proliferation capacity”, she says. Silvya, however, went beyond her original objective. She proposed to researcher María Soengas to reproduce artificial skin according to the Brazilian model developed in a project conducted in partnership with researchers Luisa Lina Villa and Enrique Boccardo, from the Ludwig Cancer Research Institute in São Paulo. “Our initial objective was to study new pharmaceutical products for the treatment of melanoma, but we realized that the Brazilian cosmetics industry needed the skin that we had developed to test new active principles”, says Silvya.

In the new pharmaceutical products line of research, professor Sílvia Berlanga’s group had already tested a new molecule on the artificial skin. This molecule had been isolated from a native Brazilian plant with chemotherapy potential. “We put the melanoma in the place of the melanocyte”, explains Silvya. “We observed a regression of the melanoma in vitro when we applied the new molecule.” The related data, which is being prepared for publication, is part of the doctoral thesis of student Carla Brohem.

Some companies have already contacted the research group from USP to establish partnerships, but no agreement has been formalized yet. “We are prepared to conduct tests with the artificial skin, but not systematically or on an industrial scale”. To this end, the model has to be validated according to international standards. So far, the research group from the School of Pharmaceutical Sciences has recreated the artificial skin with cells supplied by the University of Michigan’s cell bank. To meet future demand, the group established a partnership with USP’s Universitário teaching hospital. “We will receive the skin discarded from corrective plastic surgery as soon as this is approved by the ethics committee, as the skin is a human genetic asset”, says the researcher.

The Project
Generation of human artificial skins and invasive melanomas as a platform for pharmacological testing (nº 08/58817-4); Modality Regular Research Awards; Coordinator Silvya Stuchi Maria-Engler – USP; Investment R$ 145,597.39 (FAPESP)