Evaluation of treatment response to autologous transplantation of noncultured melanocyte/keratinocyte cell suspension in patients with stable vitiligo

Abstract Background: Vitiligo is a chronic disease characterized by the appearance of achromic macules caused by melanocyte destruction. Surgical treatments with melanocyte transplantation can be used for stable vitiligo cases. Objectives: To evaluate treatment response to the autologous transplantation of noncultured epidermal cell suspension in patients with stable vitiligo. Methods: Case series study in patients with stable vitiligo submitted to noncultured epidermal cell suspension transplantation and evaluated at least once, between 3 and 6 months after the procedure, to observe repigmentation and possible adverse effects. The maximum follow-up period for some patients was 24 months. Results: Of the 20 patients who underwent 24 procedures, 25% showed an excellent rate of repigmentation, 50% good repigmentation, 15% regular, and 10% poor response. The best results were observed in face and neck lesions, while the worst in extremity lesions (88% and 33% of satisfactory responses, respectively). Patients with segmental vitiligo had a better response (84%) compared to non-segmental ones (63%). As side effects were observed hyperpigmentation of the treated area and the appearance of Koebner phenomenon in the donor area. Study limitations: Some limitations of the study included the small number of patients, a subjective evaluation, and the lack of long-term follow-up on the results. CONCLUSION: Noncultured epidermal cell suspension transplantation is efficient and well tolerated for stable vitiligo treatment, especially for segmental vitiligo on the face and neck.

Bioingeniería de tejidos: cultivo de queratinocitos humanos en el tratamiento de la epidermólisis bullosa / Treatment of epidermolysis bullosa with allogeneic human cultured queratinocytes

Background: Epidermolysis bullosa (EB) or "crystal skin" is a group of inherited disorders that affect the protein that forms the anchor between dermis and epidermis, producing blister injuries. Case report: We report a four years old boy with junctional EB and lesions in 80 percent of the body lasting 48 months. His right lower limb was treated with allogeneic human cultured queratinocytes during five weeks. After the treatment period, a re-epithelization of 90 percent of the intervened limb was observed. Its diameter increased from 23 to 27 cm, the wound assessment scale score decreased from 30 to 13 and the visual analogue pain assessment score decreased from eight to two. Therefore allogeneic human cultured queratinocytes are a novel therapeutic alternative for EB.

Introducción: La epidermólisis Bullosa (EB) o "piel de cristal" es un grupo de trastornos hereditarios, el cual afecta las proteínas que forman la unión dermo-epidérmica de piel y mucosas, lo que lleva a la formación de lesiones ampollares. Objetivo: Comunicar la primera intervención con cultivos de queratinocitos humanos alogénicos (CQHA) en el tratamiento de la EB, en un centro de salud familiar de Chile el año 2013. Metodología: Se presenta el caso de un paciente de 4 años, quien presentaba lesiones en el 80 por ciento del cuerpo de 48 meses de evolución. Se realizó una intervención de la extremidad inferior derecha con el 90 por ciento comprometido con CQHA durante 5 sem con el fin de lograr la reepitelización y formación de piel indemne. Resultados: Posterior a las 5 sem del tratamiento se logró reepitelización del 90 por ciento de la extremidad intervenida, incremento del diámetro de la pierna de 23 a 27 cm., en la escala de valoración de heridas se reduce de 30 a 13 puntos y en la escala de valoración análoga del dolor de 8 a 2 puntos. Conclusión: Se presenta una alternativa terapéutica para pacientes con EB.

Non-cultured melanocyte/keratinocyte transplantation for the treatment of stable vitiligo on the face: report of two cases / Transplante de suspensao celular de melanocitos/queratinocitos para o tratamento de vitiligo estavel na face: relato de dois casos

There are many alternatives to treat vitiligo, including surgical procedures, which are recommended for patients resistant to other therapies. The melanocyte/keratinocyte transplantation consists in the separation of epidermal cells obtained from a donor site and spreading these cells on the depigmented and dermabraded recipient area. Two patients were submitted to transplantation, showing more than 70% repigmentation in the treated areas after four months, both with excellent degree of satisfaction. The method requires some laboratory skills, but represents a simple and safe procedure.

Existem várias alternativas para o tratamento do vitiligo, incluindo procedimentos cirúrgicos, que são indicados para pacientes refratários aos outros tipos de tratamento. O transplante de suspensão celular de melanócitos/queratinócitos consiste na separação de células da epiderme obtidas de área doadora, e aplicação destas células na área receptora despigmentada, após dermoabrasão. Dois pacientes com vitiligo estável foram submetidos ao transplante de suspensão de melanócitos/queratinócitos, apresentando repigmentação acima de 70% nas áreas tratadas após quatro meses, ambos com excelente grau de satisfação. O método requer alguma habilidade laboratorial, mas representa um procedimento simples e seguro.

Skin substitutes: a brief review of types and clinical applications

Replacing skin defects has witnessed several developments over the centuries. It started with the introduction of skin grafting by Reverdin in 1871, Since then, varieties of skin grafting techniques have been used successfully. Despite being clinically useful, skin grafts have many limitations including the availability of the donor site especially in circumstances of extensive skin loss, immune rejection in allogenic skin grafts, pain, scarring, slow healing and infection[1,2] For these reasons, scientist have worked hard to find skin substitutes to replace skin defects without the need for a "natural" skin graft. These materials which are used to cover skin defects are called [Skin substitutes]. This article briefly discusses the common types of skin substitutes and their clinical uses

Thermolysin in human cultured keratinocyte isolation

BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

INTRODUÇÃO: No tratamento do paciente grande queimado, onde se usa lâminas de epiderme cultivadas, o principal problema é o tempo necessário para sua produção. O isolamento tradicional de queratinócitos utiliza normalmente tripsina. No presente estudo, foi utilizada uma modificação do método de isolamento tradicional, que poderia produzir uma maior pureza e um maior número de colônias formadas a partir da população de células epidérmicas isoladas. OBJETIVO: Comparar a ação da tripsina e da termolisina no isolamento de queratinócitos usando pele de prepúcio de récem-nascidos. MÉTODOS: Essa metodologia utilizou a termolisina, que realiza a separação seletiva entre a epiderme e a derme. Após essa separação, a epiderme foi submetida à ação da tripsina para a obtenção da suspensão celular. RESULTADOS: Comparado ao método convencional, os experimentos mostraram que no grupo da termolisina mostrou facilidade para a separação entre a epiderme e derme, não houve contaminação por fibroblastos e produziu um maior número de colônias formadas, com diferença estatística significante. CONCLUSÃO: O número de colônias no grupo termolisina foi significantemente maior que no grupo tripsina.

Experimental model of cultured skin graft

Um dos modelos animais mais utilizados de auto-enxertia de queratinócitos cultivados é baseado em xeno-enxerto de queratinócitos humanos em rato atímico, um receptor imunologicamente neutro que atua como carreador biológico. Muitos fatos podem ser estudados nesse modelo que acontecem após o transplante sem os aspectos éticos do estudo clínico. A proposição do modelo experimental esta relacionada a sequência do transplante de pele parcial ou total como auto-enxerto ou xeno-enxerto, cultivado ou não, no dorso do rato atímico. O modelo apresenta a possibilidade do estudo in vivo do animal atímico, quando o estudo in vivo em anima nobili não é considerado ético. Isso permite a avaliação do xeno-enxerto de células humanas normais ou modificadas geneticamente modificadas cultivadas e a associação de células cultivadas e substitutes dérmicos, de enxertos compostos e de auto-enxerto.

Tissue-engineered human living skin substitutes: development and clinical application

The skin acts as a barrier to exogenous substances, pathogens, and trauma. Skin defects caused by burns, venous ulcer, diabetic ulcer, or acute injury occasionally induce life-threatening situations. Tissue engineering provides an alternative for autologous or allogeneic tissue transplantation, which is required because of donor site limitations and the risks of transmitting infection. Currently, skin substitutes are made of only extracellular matrix, mainly cells, or combination of cells and matrices. New biotechnological approaches have led to the development of the skin equivalent, the closest match yet to native human skin in terms of histological and functional properties. This review article focuses upon the development of the in vitro and in vivo epidermis and dermis and their clinical applications.

Análisis de los medios de transporte y cultivo de piel para auto o aloinjerto humano / Analysis of transport and culture skin medium for human auto or aloinjert

Las condiciones óptimas para la conservación y transporte de piel humana destinada al cultivo de queratinocitos para autotransplante son el motivo de este estudio. Se simularon condiciones de transporte, colocando muestras de piel humana tanto joven (prepucios de niños, n=305) como maduras (piel de mastectomías, n=616) en cinco diferentes medios: Medio de crecimiento de queratinocitos (KGM, n=276), Medio de crecimiento 3T3 (3T3GM, n=220), Solución Ringer (SR, n=174), Solución Salina Isotónica (SS, n=128) y Solución Buffer Fosfato (PBS, n=123), a 21ºC y 4ºC respectivamente, por 1,24 y 72 horas, al cabo de las cuales se prepararon cultivos de queratinocitos a partir de dichas muestras. Se determinó tanto la Eficacia Formadora de Colonias (EFC) como el porcentaje de células muertas. El medio óptimo para el transporte y/o preservación de piel humana es el KGM, a 4ºC y hasta por 72 horas expresado por los valores significativamente más altos de EFC=0,67 por ciento (p<0,001) al compararlo a los restantes medios. PBS y SS mostraron la EFC más baja (0,28 y 0,26 por ciento respectivamente, p<0,001). La prueba de viabilidad celular con azul tripán, luego de la preservación de la piel, no se correlacionó con los valores de la EFC (r=-0,05; p>0,05), siendo éste último el método más confiable y adecuado para evaluar el potencial formador de colonias de las células provenientes de las muestras de piel. Los queratinocitos provenientes de piel de donantes jóvenes (prepucio de niños) tienen un potencial mayor de formar colonias (EFC=0,70) que la piel de adultos (de mastectomía) (EFC=0,38; p<0,001). Estos resultados caracterizan las condiciones óptimas de un potencial sistema de envío de muestras de piel destinadas a la preparación de injertos de queratinocitos para pacientes hospitalizados a distancia del laboratorio de cultivo