Immunoarchitectural characterization of a human skin model reconstructed in vitro / Caracterização morfológica e imunoistoquímica de um modelo de pele humana reconstruída in vitro

CONTEXT AND OBJECTIVE: Over the last few years, different models for human skin equivalent reconstructed in vitro (HSERIV) have been reported for clinical usage and applications in research for the pharmaceutical industry. Before release for routine use as human skin replacements, HSERIV models need to be tested regarding their similarity with in vivo skin, using morphological (architectural) and immunohistochemical (functional) analyses. A model for HSERIV has been developed in our hospital, and our aim here was to further characterize its immunoarchitectural features by comparing them with human skin, before it can be tested for clinical use, e.g. for severe burns or wounds, whenever ancillary methods are not indicated. DESIGN AND SETTING: Experimental laboratory study, in the Skin Cell Culture Laboratory, School of Medical Sciences, Universidade Estadual de Campinas. METHODS: Histological sections were stained with hematoxylin-eosin, Masson's trichrome for collagen fibers, periodic acid-Schiff reagent for basement membrane and glycogen, Weigert-Van Gieson for elastic fibers and Fontana-Masson for melanocytes. Immunohistochemistry was used to localize cytokeratins (broad spectrum of molecular weight, AE1/AE3), high molecular weight cytokeratins (34βE12), low molecular weight cytokeratins (35βH11), cytokeratins 7 and 20, vimentin, S-100 protein (for melanocytic and dendritic cells), CD68 (KP1, histiocytes) and CD34 (QBend, endothelium). RESULTS: Histology revealed satisfactory similarity between HSERIV and in vivo skin. Immunohistochemical analysis on HSERIV demonstrated that the marker pattern was similar to what is generally present in human skin in vivo. CONCLUSION: HSERIV is morphologically and functionally compatible with human skin observed in vivo.

CONTEXTO E OBJETIVO: Nos últimos anos, diferentes modelos de pele humana reconstruída in vitro (PHRIV) foram descritos para uso clínico e aplicações em pesquisa na indústria farmacêutica. Antes de serem liberados para uso rotineiro como substitutos de pele humana, os modelos de PHRIV necessitam de testes (estudos) comparativos com a pele humana in vivo, por meio de análises morfológica (arquitetural) e imunoistoquímica (funcional). O objetivo deste trabalho é estudar as características imunoistoquímicas de um modelo de PHRIV desenvolvido em nosso serviço, comparando-as com a pele humana, para que esse modelo de PHRIV possa vir a ser testado clinicamente em casos de queimaduras e ulcerações de pele nos quais métodos tradicionais de tratamento não estejam indicados. TIPO DE ESTUDO E LOCAL: Estudo experimental laboratorial realizado no Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas (FCM/Unicamp), Campinas, São Paulo, Brasil. MÉTODOS: Cortes histológicos foram corados com hematoxilina-eosina, tricrômio de Masson para fibras colágenas, ácido periódico-reagente de Schiff para membrana basal e glicogênio, Weigert-Van Gieson para fibras elásticas e Fontana-Masson para melanócitos. Estudo imunoistoquímico foi realizado para identificar citoqueratinas de amplo espectro de pesos moleculares (AE1/AE3), citoqueratinas de alto peso molecular (34βE12), citoqueratinas de baixo peso molecular (35βH11), citoqueratinas 7 e 20, vimentina, proteína S-100 (para melanócitos e células dendríticas), CD68 (KP1, histiócitos) e CD34 (QBend, endotélio). RESULTADOS: A histologia revelou similaridade satisfatória entre PHRIV e a pele in vivo. O estudo imunoistoquímico da PHRIV demonstrou padrão semelhante de marcadores usualmente presentes na pele humana in vivo. CONCLUSÃO: A PHRIV estudada é morfológica e funcionalmente compatível com a pele humana observada in vivo.

The fundamentals of tissue engineering: new scaffolds

The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.

Quality evaluation analysis of bioengineered human skin

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.