A Universal Classification System of Skin Substitutes Inspired by Factorial Design.

The complexity of the dermal layer of skin means that damage to this section can result in permanent impairment of function. Partial or total dermal loss is a feature of deep burns and chronic wounds such as pressure sores or diabetic ulcers. The issues posed by traditional skin grafts have led to substantial research being carried out in the fields of tissue engineering and biomaterials science to develop a vast array of alternative skin substitutes. Given the large number of different materials, manufacturing methods, and techniques for implementation described for artificial skin substitutes, many classification systems have been created to simplify their categorization. Some of these systems are oriented toward clinicians while others toward researchers. However, none address the needs of both groups and none are intuitive. The creation of an effective classification system would be particularly helpful in the regulation, distribution, organization, and selection of skin substitutes. The aim of this review is to examine existing methods of classification of skin substitutes, and to propose a new system that uses an algorithm that is inspired by factorial design. Our system allows multiple factors to be simultaneously investigated or in this case, described, since all skin substitutes possess multiple characteristics: (1) cellularity (acellular or cellular), (2) layering (single layer or bilayer), (3) replaced region (epidermis, dermis, or both), (4) materials used (natural, synthetic, or both), and (5) permanence (temporary or permanent). The factors and levels are combined into an algorithm where all the possible combinations are shown. The multifactorial and palindromic structure of our system should enable all users to quickly understand the makeup of a selected skin substitute, or search for a skin substitute depending on their specific requirements. We feel that our proposed classification can be used by clinicians and biomedical researchers alike, which should be an advantage given the multidisciplinary nature of the tissue engineering field and the science that underpins the development of skin substitutes. We also touch upon some of the state-of-the-art skin substitutes that are commercially available or under development to demonstrate how our new method of classification might work.

How biophysical in vivo testing techniques can be used to characterize full thickness skin equivalents.

BACKGROUND: The reliability of the biophysical properties of skin equivalents (SEs) remains a challenge for medical applications and for product efficacy tests following the European Directive 2003/15/EC2 on the prohibition of animal experiments for cosmetic products. METHODS: We propose to adapt the biophysical in vivo testing techniques to compare full thickness model growth vs. time. The interest in using such techniques lies in possible comparisons between in vivo and in vitro skin as well as monitoring samples over the culture time. RESULTS: High frequency ultrasound technique, optical coherence tomography (OCT), and laser scanning microscopy were used to analyze SEs morphology at days D42 and D60 whereas their microstructure was assessed through transmission electron microscopy and classical histology. A correlation between these observations and mechanical measurements has been proposed so as to underline the consequence of both the development of the dermis elastic fibers and the epidermis differentiation. CONCLUSION: Ultrasounds measurements show a highly homogeneous dermis whereas the OCT technique clearly distinguishes the stratum corneum and the living epidermis. The increase in the thicknesses of these layers as well as the growth in elastin and collagen fibers results in strong modifications of the samples mechanical properties.

Efeito do resveratrol e do 2-Metoxiestradiol em linhagens de melanoma humano em modelos de monocamada e de pele reconstituída / Effects of resveratrol and 2-methoxyestradiol in human melanoma cell lines in monolayer and skin reconstruct models

Os melanomas são o tipo mais mortal de câncer de pele, apesar da baixa incidência, 80% das mortes de câncer de pele são devem-se ao melanoma metastático. Novas abordagens farmacológicas e a busca por novos compostos para a terapêutica do melanoma, em aplicações isolados ou em combinação com outros fármacos é imprescindível. Esta busca ocorre principalmente no campo das terapias de alvos específicos, devido à aquisição de resistência tumoral e recidiva. O resveratrol (RES) é um polifenol com atividade anti-oxidante, e seu efeito anti-tumoral foi mostrado pela indução de morte celular, porém o seu estudo não foi aprofundado pela inviabilidade do uso de altas doses in vitro para observação de efeitos celulares. Outro composto, o 2-methoxiestradiol (2ME) é um metabólito do estrógeno cujo efeitos anti-câncer já foi demonstrado em melanoma, porém sem elucidação das vias de sinalização envolvidas. O efeito em células com resistência adquirida também nunca foram testados. Neste estudo ampliamos o painel de linhagens celulares de melanoma humano, e demonstramos que o 2ME induz morte celular, inibe a proliferação destas células sendo que esta inibição está associada a indução de senescência. Pela primeira vez foi observada a inibição de proliferação pelo 2ME em células com a mutação BRAF V600E resistentes ao vemurafenibe (inibidor de BRAF) e duplo resistentes ao vemurafenibe e trametinibe (inibidor de MEK). A inibição de proliferação foi acompanhada pela modulação de p21Cip1, Ciclina B1, pRb, proteínas envolvidas na regulação do ciclo celular. A exposição prolongada ao 2ME inibiu a formação de colônias em todas as linhagens de melanoma (não resistentes e resistentes), mas não teve o mesmo efeito em fibroblastos primários, mostrando efeito seletivo. Em modelo tridimensional de esferóides, foi observado que as linhagens resistentes (Sk-Mel-28R) e duplo resistentes (Sk- Mel-28RT) são mais invasivas que a parental (Sk-Mel-28). Neste modelo, o 2ME foi capaz de inibir a invasão e viabilidade destas células. No modelo de pele reconstituída, na ausência de tratamento, observa-se invasão das células de melanoma pela derme, porém este fenômeno é diminuído quando as peles são tratadas com 2ME. Estes resultados demonstram que o 2ME é um efetivo agente anti-melanoma, independente de sua resistência

Melanomas are the deadliest type of skin cancer, and in spite of the low incidence, 80% of the skin cancer associated death cases are due to metastatic melanoma. New pharmacological approaches and the search of new compounds for melanoma therapeutics, for monotherapy or combination therapy, are essential. This search occurs mainly in the targeted therapy field because of the melanoma acquisition of resistance to the current treatments. Resveratrol (RES) is a polyphenol with anti-oxidant activity, and its anti-tumor effect has been shown through the induction of cell death. However, the study of this compound has been discontinued in this work due to the impossibility of using high doses in vitro for the observation of cellular effects. Another compound, 2-methoxyestradiol (2ME) is a metabolite from estrogen, and its anti-cancer effects has already been shown in melanoma, but with no elucidation of the signaling pathways involved. Furthermore, the effects in cells with acquired resistance have never been shown. In this study we used a broader panel of human melanoma cell lines and demonstrated that 2ME induces cell death, inhibits proliferation of these cells, and this inhibition is associated with the induction of cell senescence. The inhibition of proliferation caused by 2ME was observed for the first time in BRAF V600E cells that are resistant to Vemurafenib (BRAF inhibitor) and double resistant to Vemurafenib and Trametinib (MEK inhibitor). The proliferation inhibition was related to the modulation of p21Cip1,Cyclin B1 and pRb, which are proteins involved in cell cycle regulation. Long exposure to 2ME in colony formation assay showed the inhibition of colony in all melanoma cell lines (regardless of resistance and mutational status), but not in primary fibroblasts, showing selective effect. In three-dimensional spheroid model, it was observed that the resistant (Sk-Mel- 28R) and double-resistant (Sk-Mel-28RT) cell lines were more invasive than the parental cell line (Sk-Mel-28). In this model, 2ME was able to inhibit cell invasion and cell viability. In the skin reconstruct model, in the absence of treatment, melanoma cell invasion can be observed in the dermis layer. However, after the treatment with 2ME these cell invasion foci are inhibited. Altogether, these effects demonstrate that 2ME is an effective anti-melanoma agent, regardless of resistance

Skin substitutes in dermatosurgery.

Skin substitutes are a growing market since technical advancements have allowed a substantial progress in treating extensive defects of the skin. A variety of skin substitutes with different properties and thus resulting different indications is offered on the market. Important benefits of skin substitutes are their ready availability in almost any quantity and the predictable product properties concerning implantation, incorporation, resorption and long-term outcome. Although, most skin substitutes still need skin grafts at a later date which is disadvantageous. Nevertheless dermal substitutes have reduced the need for thick skin grafts allowing the donor site and the patient to heal faster with fewer surgeries. The use of skin substitutes in dermatologic surgery is widespread and new fields of application are emerging. The variety of artificial skin has definitely changed the reconstructive ladder helping to cover larger defects with less time and effort which is an important issue especially in elderly and multimorbid patients. In the last years a growing number of studies in the literature report the use of artificial skin substitutes to secure a rapid reconstruction with reliable cosmetic and functional results after oncological resections. Furthermore, skin substitutes are used to cover chronic wounds like diabetic foot ulcers or venous leg ulcers to promote healing. Congenital diseases like giant hairy nevi, aplasia cutis congenital or epidermolysis bullosa are conditions in children where skin substitutes play a role. But even in tissue augmentation or in cosmetic surgery skin substitutes come into vogue. The latest advance are cultured autologous or allogenic substitutes some even in combination with alloplastic material. Besides of medical questions that arise from the use of these materials in reconstructive surgery legal and economic aspects have to be taken into account. This article is giving an overview over the most common skin substitutes and their use in dermatosurgery.

Biologic dressing in burns.

Advances in cellular biology and knowledge in wound healing and growth factors have given us a wide variety of choices to attack the problem of the complex burn wound. Split-thickness skin grafting with autograft is at present the standard of care. It, however, is not an ideal substitute and frequently is not available for full-burn coverage. This article will review honey, human amnion, xenograft, allograft, cultured epithelial autograft, and various engineered commercial products for use in the biologic treatment of burn wounds.

Current issues regarding skin substitutes using living cells as industrial materials.

There have been many clinical cases of skin treatment using cultured epidermis, as established by H. Green in 1975. This procedure, famously, made it possible to make artificial skin using living cells. Permanent coverage for injury wounds was achieved with cultured epidermis made from autologous cells. Recently, many types of skin substitutes using living cells have been used clinically. The extent to which cellular skin substitute techniques established through basic research can be incorporated into general medical treatment is a controversial issue at present. Some techniques are at the commercializing stage as medical materials. Cultured epidermis, as described above, cultured dermis, and skin equivalent are already well established. In this article, we will describe the current issues regarding skin substitutes using living cells.

Tissue engineering of skin.

The skin plays a crucial role in protecting the integrity of the body's internal milieu. The loss of this largest organ is incompatible with sustained life. In reconstructive surgery or burn management, substitution of the skin is often necessary. In addition to traditional approaches such as split- or full-thickness skin grafts, tissue flaps and free-tissue transfers, skin bioengineering in vitro or in vivo has been developing over the past decades. It applies the principles and methods of both engineering and life sciences toward the development of substitutes to restore and maintain skin structure and function. Currently, these methods are valuable alternatives or complements to other techniques in reconstructive surgery. This review article deals with the evolution and current approaches to the development of in vitro and in vivo epidermis and dermis.