A comparative review of human milk banking and national tissue banking programs.

This paper explores the legislative and operational commonalities and differences in Medical Products of Human Origin (MPHO) programs, including blood, hematopoietic cells, tissues and reproductive cells and human milk banking. The analysis includes ethical principles in donation and utilization, policies and legislation, public awareness and education, registries, guidelines in donor selection, safety and quality assurance, operational models and funding, infrastructure and human resources and biovigilance and evaluation of outcomes. Unlike other MPHO, the need for donor human milk (DHM) may be greatly reduced, that is, by ensuring optimal support for maternal lactation and breastfeeding. This should not be lost in the drive for wider and improved service provision. Nevertheless, increased overall demand for DHM is expected as a result of forthcoming international recommendations and also its increased use as the first-choice supplement to a mother's own milk both within and beyond preterm, low-birthweight and sick infant populations. Insight into current human milk banking highlights differences and gaps in practices that can benefit from further exploration and harmonization. Strong similarities with the ethical and operational principles underpinning donation and processing of the diverse MPHO suggest that legislating human milk banks within similar MPHO frameworks may bring additional safety and facilitate improved product quality. Moreover, that MPHO-inspired models operating within attainable regulatory requirements may contribute to sustainable human milk banking activity and growth.

Developing global guidance on human milk banking.

Donor human milk is recommended by the World Health Organization both for its advantageous nutritional and biological properties when mother's own milk is not available and for its recognized support for lactation and breastfeeding when used appropriately. An increasing number of human milk banks are being established around the world, especially in low- and middle-income countries, to facilitate the collection, processing and distribution of donor human milk. In contrast to other medical products of human origin, however, there are no minimum quality, safety and ethical standards for donor human milk and no coordinating global body to inform national policies. We present the key issues impeding progress in human milk banking, including the lack of clear definitions or registries of products; issues around regulation, quality and safety; and ethical concerns about commercialization and potential exploitation of women. Recognizing that progress in human milk banking is limited by a lack of comparable evidence, we recommend further research in this field to fill the knowledge gaps and provide evidence-based guidance. We also highlight the need for optimal support for mothers to provide their own breastmilk and establish breastfeeding as soon as and wherever possible after birth.

Lorsque la mère est dans l'impossibilité d'allaiter, l'Organisation mondiale de la Santé recommande d'opter pour le lait humain provenant de donneuses, tant pour ses propriétés nutritionnelles et biologiques que pour la contribution avérée qu'il apporte à la lactation et à l'allaitement quand il est utilisé à bon escient. Un nombre croissant de banques de lait humain s'établissent dans le monde entier, en particulier dans les pays à faible et moyen revenu, afin de faciliter la collecte, le traitement et la distribution de lait humain provenant de donneuses. Cependant, contrairement à d'autres produits médicaux d'origine humaine, il n'existe aucune norme minimale de qualité, de sécurité et d'éthique en la matière, et aucun organe de coordination global n'a été créé pour guider les politiques nationales. Dans le présent document, nous évoquons les principaux obstacles à la progression des banques de lait humain, notamment l'absence de définition claire ou de registre de produits; les problèmes relatifs à la réglementation, la qualité et la sécurité; ainsi que les questions éthiques entourant la commercialisation et l'exploitation potentielle des femmes. Jugeant cette progression limitée par le manque de données comparables, nous encourageons à mener d'autres recherches dans ce domaine pour combler les lacunes et fournir des orientations fondées sur des preuves. Nous soulignons également la nécessité d'offrir un soutien optimal aux mères afin qu'elles puissent produire leur propre lait et allaiter autant que possible immédiatement après la naissance.

La Organización Mundial de la Salud recomienda la leche humana donada tanto por sus ventajosas propiedades nutricionales y biológicas cuando no se dispone de la propia leche materna como por su reconocido apoyo a la lactancia y al amamantamiento cuando se utiliza de manera adecuada. Cada vez se crean más bancos de leche humana en todo el mundo, sobre todo en los países de ingresos bajos y medios, para facilitar la recogida, el procesamiento y la distribución de leche humana donada. Sin embargo, a diferencia de lo que ocurre con otros productos médicos de origen humano, no existen estándares mínimos de calidad, seguridad y ética para la leche humana donada ni un organismo mundial de coordinación que sirva de base a las políticas nacionales. En este documento se exponen los principales problemas que impiden el progreso de los bancos de leche humana, como la falta de definiciones claras o de registros de productos; los problemas relacionados con la regulación, la calidad y la seguridad; y las preocupaciones éticas sobre la comercialización y la posible explotación de las mujeres. Dado que el progreso de los bancos de leche humana se ve limitado por la falta de evidencias comparables, se recomienda seguir investigando en este campo para compensar los vacíos de conocimiento y proporcionar una guía asistencial. Asimismo, se destaca la necesidad de apoyar al máximo a las madres para que se provean de su propia leche materna y establezcan la lactancia materna tan pronto y siempre que sea posible después del nacimiento.

Xeno-free expansion of adult keratinocytes for clinical application: the use of human-derived feeder cells and serum.

Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green's methods; however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f; a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.

Artificial dermal templates: A comparative study of NovoSorb™ Biodegradable Temporising Matrix (BTM) and Integra(®) Dermal Regeneration Template (DRT).

BACKGROUND: Artificial dermal templates play an important role in physiologic wound closure after injury. In addition to contributing to stable, durable and flexible wound closure, they provide a scaffold for tissue repair. Several dermal templates are commercially available, with animal-derived Integra(®) dermal regeneration template perhaps the most widely used. NovoSorb™ Biodegradable Temporising Matrix (BTM) is a fully synthetic alternative that eliminates any risk of cross-species residual antigenicity. In this study, we aimed to compare early response after application of NovoSorb™ BTM with Integra(®) in terms of temporary wound closure, host cell infiltration, neovascularisation and collagen deposition in a mouse model. METHODS: Twenty athymic nude mice received full-thickness skin excision followed by grafting of the dermal template (n=10 NovoSorb™ BTM, n=10 Integra(®)), with the grafts excised and assessed after two weeks. RESULTS: All twenty mice achieved temporary wound closure with no evidence of wound contracture. Microscopically, all twenty grafts became infiltrated with host cells along the entire length of the template, with NovoSorb™ BTM demonstrating a particular abundance of host inflammatory cells. Evidence of new collagen deposition and neovascularisation was observed in both templates, with NovoSorb™ BTM demonstrating a more extensive vascular network at this time point. However, a greater inflammatory response was also observed in the NovoSorb™ BTM grafts at this time point. CONCLUSIONS: In this study, NovoSorb™ BTM demonstrates favourable properties as a dermal template, but further investigation is required to assess the significance of the differing inflammatory and vascular response to its implantation compared with Integra(®).

Use of Clotted Human Plasma and Aprotinin in Skin Tissue Engineering: A Novel Approach to Engineering Composite Skin on a Porous Scaffold.

Tissue-engineered composite skin is a promising therapy for the treatment of chronic and acute wounds, including burns. Providing the wound bed with a dermal scaffold populated by autologous dermal and epidermal cellular components can further entice host cell infiltration and vascularization to achieve permanent wound closure in a single stage. However, the high porosity and the lack of a supportive basement membrane in most commercially available dermal scaffolds hinders organized keratinocyte proliferation and stratification in vitro and may delay re-epithelization in vivo. The objective of this study was to develop a method to enable the in vitro production of a human skin equivalent (HSE) that included a porous scaffold and dermal and epidermal cells expanded ex vivo, with the potential to be used for definitive treatment of skin defects in a single procedure. A collagen-glycosaminoglycan dermal scaffold (Integra(®)) was populated with adult fibroblasts. A near-normal skin architecture was achieved by the addition of coagulated human plasma to the fibroblast-populated scaffold before seeding cultured keratinocytes. This resulted in reducing scaffold pore size and improving contact surfaces. Skin architecture and basement membrane formation was further improved by the addition of aprotinin (a serine protease inhibitor) to the culture media to inhibit premature clot digestion. Histological assessment of the novel HSE revealed expression of keratin 14 and keratin 10 similar to native skin, with a multilayered neoepidermis morphologically comparable to human skin. Furthermore, deposition of collagen IV and laminin-511 were detected by immunofluorescence, indicating the formation of a continuous basement membrane at the dermal-epidermal junction. The proposed method was efficient in producing an in vitro near native HSE using the chosen off-the-shelf porous scaffold (Integra). The same principles and promising outcomes should be applicable to other biodegradable porous scaffolds, combined with autologous cells, for use in wound treatment.

Clinical application and viability of cryopreserved cadaveric skin allografts in severe burn: a retrospective analysis.

INTRODUCTION: Cadaveric cutaneous allografts are used in burns surgery both as a temporary bio-dressing and occasionally as definitive management of partial thickness burns. Nonetheless, limitations in the understanding of the biology of these grafts have meant that their role in burns surgery continues to be controversial. METHODS: A review of all patients suffering 20% or greater total body surface area (TBSA) burns over an eight year period that received cadaveric allografts were identified. To investigate whether tissue viability plays a role in engraftment success, five samples of cryopreserved cadaveric cutaneous allograft processed at the Donor Tissue Bank of Victoria (DTBV) were submitted to our laboratory for viability analysis using two methods of Trypan Blue Exclusion and tetrazolium salt (MTT) assays. RESULTS: During the study period, 36 patients received cadaveric allograft at our institution. The average total burn surface area (TBSA) for this group of patients was 40% and all patients received cadaveric skin as a temporizing measure prior to definitive grafting. Cadaveric allograft was used in complicated cases such as wound contamination, where synthetic dressings had failed. Viability tests showed fewer than 30% viability in processed allografts when compared to fresh skin following the thawing process. However, the skin structure in the frozen allografts was histologically well preserved. CONCLUSION: Cryopreserved cutaneous cadaveric allograft has a positive and definite role as an adjunct to conventional dressing and grafting where available, particularly in patients with large TBSA burns. The low viability of cryopreserved specimens processed at DTBV suggests that cell viability in cadaveric allograft may not be essential for its clinical function as a wound dressing or even as permanent dermal substitute.

Estabelecimento de protocolo de glicerolação de membranas amnióticas para uso como curativo biológico / Establishing protocol glicerolação of amniotic membranes for use as a biological dressing

Introdução: Pesquisadores têm procurado explorar várias alternativas terapêuticas (substitutos dérmicos), biológicas ou sintéticas, capazes de assegurar condições ideais ao leito da ferida que favoreça o processo de cicatrização. Uma opção de substituto dérmico menos oneroso é o uso de membranas amnióticas. Os curativos constituídos de âmnion formam uma barreira protetora contra as bactérias ambientais, aceleram a reepitelização das lesões e diminuem a dor local. Objetivo: Estabelecer protocolo de processamento de membranas amnióticas em altas concentrações de glicerol. Método: Foram obtidas três amostras de membranas amnióticas, que preenchiam os critérios de inclusão e que a gestantes concordaram em ceder o material para pesquisa. Resultados: Os exames de cultura do material no momento da captação demonstraram ausência de crescimento bacteriano ou de fungos. As sorologias das pacientes foram todas negativas. Conclusão: Neste trabalho, buscamos estabelecer um protocolo de conservação de membranas amnióticas baseado na glicerolação, pois se trata de um método de baixo custo, relativamente simples e de fácil estocagem do material. Este método apresenta como desvantagem a sua alta toxicidade celular, resultando em destruição das células do tecido, porém preserva a integridade estrutural tecidual, conforme demonstrado nos resultados macroscópicos e microscópicos obtidos neste estudo.

Introduction: Researchers have attempted to explore various alternative therapies (dermal substitutes), biological or synthetic, capable of providing ideal conditions to the wound bed to promote the healing process. An option of dermal substitute less costly is the use of amniotic membranes. Dressings consist of amnion forms a protective barrier against environmental bacteria, accelerate reepithelialization of lesions and reduce local pain. Objective: To establish protocol processing of membranes in high concentrations of glycerol. Methods: Three samples were obtained from amniotic membranes who met the inclusion criteria and that the women agreed to donate the material for research. Results: The examinations of material culture at the time of capture showed no bacterial or fungal growth. The serology of the patients was all negative. Conclusion: In this study, we establish a protocol for the conservation of membranes based on glycerol because it is a low-cost, relatively simple and easy storage of the material. This method presents the disadvantage of its high cell toxicity, resulting in destruction of tissue cells, but preserves the structural integrity of tissue as shown in the microscopic and macroscopic results of this study.

Results of the clinical donor case and quality system case workshops of the European Association of Tissue Banks annual meeting 2009.

The European Association of Tissue Banks (EATB) Donor Case Workshop and Quality System Case workshop are forums held within the program of the EATB Annual Congress. These workshops offer an opportunity to discuss and evaluate approaches taken to challenging situations, regarding donor selection and quality issues, and strengthen the professional tissue banking and regulatory networks across Europe. This report reflects some of the discussion at the congress workshops and also subsequent correspondence between the various individuals who submitted cases for discussion. The cases presented to the workshops demonstrate that the findings, their interpretation, deducted actions and preventive measures in tissue banks are not predictable. The varied responses and lack of consensus corroborate this and clearly indicate that operating procedures cannot comprehensively cover or prepare for all eventualities. For many of the issues raised there is a lack of information in the published literature. The workshops actively engage participants, representing a wide array of international expertise, in an informal, secure and enjoyable setting, which facilitates learning from peers and provides potential solutions to those submitting cases. By publishing a summary of the discussions, we hope to reach a wider audience and to stimulate individuals to undertake full literature reviews or research on some of the discussed subjects.

The importance of ethic in the field of human tissue banking.

A tissue bank is accountable before the community in fulfilling the expectations of tissue donors, their families and recipients. The expected output from the altruistic donation is that safe and high quality human tissue grafts will be provided for the medical treatment of patients. Thus, undertakings of tissue banks have to be not only authorised and audited by national competent health care authorities, but also comply with a strong ethical code, a code of practices and ethical principles. Ethical practice in the field of tissue banking requires the setting of principles, the identification of possible deviations and the establishment of mechanisms that will detect and hinder abuses that may occur during the procurement, processing and distribution of human tissues for transplantation. The opinions and suggestions manifested by the authors in this paper may not be necessarily a reflection of those within the institutions or community they are linked to.

Role of keratinocytes in wound contraction: an impact assessment using a model of collagen matrix populated with fibroblasts / Papel do queratinócito na contração da ferida: avaliação de impacto usando um modelo de matriz de colágeno povoada por fibroblastos

BACKGROUND: The possible participation of keratinocytes in wound remodeling has been widely studied. This study investigated the impact of keratinocytes in wound contraction. METHODS: Murine type I collagen gels populated by human fibroblasts and seeded with human keratinocytes on the surface to form a dermo-epidermal equivalent were used as the study group. Collagen gels populated by only fibroblasts were used as the control group. The criteria for the preparation and storage of gels were similar for both groups. RESULTS: An evident and statistically significant increase in gel contraction was observed in samples populated by keratinocytes compared to the control group. CONCLUSIONS: These results suggest that keratinocytes not only modulate fibroblast proliferation but also play an active role in wound contraction per se. Further research on the mechanisms involved in the communication pathways between cells and between cells and the matrix shall be assessed from the perspective of keratinocyte participation in wound healing and pathologic scarring.

INTRODUÇÃO: A eventual participação de queratinócitos na remodelagem da ferida tem sido estudada há muito tempo. Este trabalho investigou o impacto dos queratinócitos na contração da ferida. MÉTODO: Foi utilizado gel de colágeno tipo I murino povoado por fibroblastos humanos com queratinócitos humanos semeado na superfície (grupo estudo), formando um equivalente dermoepidérmico. Géis de colágeno povoado apenas por fibroblastos foram utilizados como grupo controle. Os critérios de confecção e armazenagem dos géis foram iguais para ambos os grupos. RESULTADOS: Houve aumento evidente e estatisticamente significante na contração de gel das amostras povoadas por queratinócitos, em comparação ao grupo controle. CONCLUSÕES: Esses resultados sugerem que os queratinócitos não só podem modular a proliferação de fibroblastos, mas também, por si só, desempenhar papel ativo na contração da ferida. Novas investigações sobre mecanismos envolvidos nas vias de comunicação entre células e entre célula e matriz devem ser avaliadas sob o ponto de vista de participação dos queratinócitos na cicatrização de feridas e formação de cicatrizes patológicas.

Skin bank development and critical incident response.

The Donor Tissue Bank of Victoria (DTBV), situated in Melbourne, Australia developed a skin banking program in 1994. It remains Australia's only operational skin bank, processing cryopreserved human cadaveric skin for the treatment of burns. The demand for allograft skin in Australia has steadily increased since the development of the program. The bank has been involved in the provision of skin for a number of critical incidences or disasters both in Australia and overseas. Demand always exceeds supply, and in the absence of other local skin banks, the DTBV has needed to develop strategies to enable increased provision of allograft skin nationally.

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate.

PURPOSE: In order to circumvent several difficulties that have been met in the routine use of the in vitro keratinocyte cultures using the standard procedure described by Rheinwald and Green, and obtain a more resilient and the least possible immunogeneic skin substitute for a future clinical application, this work studied a new keratinocyte culture system, which envisages the utilization of a fibrin substrate in association with high densities of human keratinocytes. METHODS: Through light and transmission electron microscopy and immunohistochemical assays, long-term proliferative and differentiative characteristics of keratinocytes cultured onto a fibrin gel under immerse and air-liquid interface culture conditions were evaluated. RESULTS: Despite the absence of a dermal substitute, the results demonstrated that the proposed composite was constituted of a transparent and elastic fibrin film covered by a well-attached, multistratified epithelium with morphological characteristics that resemble human epidermis, including the neoformation, albeit incomplete, of the basement membrane. CONCLUSIONS: Increased mechanical resistance due to the presence of an easy handling substrate, the delivery of nonclonfluent keratinocytes as well as the removal of animal-derived cells from the culture system suggest its potential use for future transplantation purposes.

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate / Avaliação ultraestrutural do crescimento e da diferenciação de queratinócitos sobre um substrato de fibrina

PURPOSE: In order to circumvent several difficulties that have been met in the routine use of the in vitro keratinocyte cultures using the standard procedure described by Rheinwald and Green, and obtain a more resilient and the least possible immunogeneic skin substitute for a future clinical application, this work studied a new keratinocyte culture system, which envisages the utilization of a fibrin substrate in association with high densities of human keratinocytes. METHODS: Through light and transmission electron microscopy and immunohistochemical assays, long-term proliferative and differentiative characteristics of keratinocytes cultured onto a fibrin gel under immerse and air-liquid interface culture conditions were evaluated. RESULTS: Despite the absence of a dermal substitute, the results demonstrated that the proposed composite was constituted of a transparent and elastic fibrin film covered by a well-attached, multistratified epithelium with morphological characteristics that resemble human epidermis, including the neoformation, albeit incomplete, of the basement membrane. CONCLUSIONS: Increased mechanical resistance due to the presence of an easy handling substrate, the delivery of nonclonfluent keratinocytes as well as the removal of animal-derived cells from the culture system suggest its potential use for future transplantation purposes.

OBJETIVO: Com o intuito de contornar diversas dificuldades encontradas no uso rotineiro de queratinócitos cultivados in vitro pela técnica descrita por Rheinwald e Green, e obter um substituto cutâneo mais resistente e o menos imunogênico possível para futuras aplicações clínicas, este trabalho avaliou um novo sistema de cultura de queratinócitos que prevê a utilização de um substrato de fibrina em associação com queratinócitos humanos em alta densidade. MÉTODOS: Através de microscopia óptica e eletrônica e análise imunohistoquímica, foram avaliadas as características proliferativas e de diferenciação em longo prazo de queratinócitos cultivados em condição imersa e na interface ar-líquido. RESULTADOS: Apesar da ausência de um substituto dérmico, foi demonstrado que o composto proposto constituiu-se de um substrato de fibrina transparente e elástico coberto por epitélio multi-estratificado, bem aderido, com características morfológicas semelhantes à epiderme humana, incluindo a neo-formação, embora incompleta, da membrana basal. CONCLUSÕES: A maior resistência mecânica com a presença de um substrato de fácil manuseio, a possível liberação de queratinócitos não-confluentes, e a remoção de células com origem animal dos sistemas de cultura sugerem que o composto proposto neste estudo apresenta grande potencial para uso clínico futuro.

Estabelecimento de protocolo de glicerolização de membranas amnióticas para uso como curativo biológico / Establishment of amniotic membranes glycerolization protocol for use as biological dressing

Introdução: Pesquisadores têm procurado explorar várias alternativas terapêuticas, biológicas ou sintéticas, capazes de assegurar condições ideais ao leito da ferida, que favoreçam o processo de cicatrização. Uma opção menos onerosa é o uso de membranas amnióticas. Os curativos constituídos de âmnion formam uma barreira protetora contra as bactérias ambientais, aceleram a reepitelização das lesões e diminuem a dor local. Objetivo: O objetivo deste trabalho foi estabelecer protocolo de processamento de membranas amnióticas em altas concentrações de glicerol. Método: Foram obtidas 3 amostras de membranas amnióticas, que preenchiam os critérios de inclusão e que as gestantes concordaram em ceder o material para pesquisa. Resultados: Os exames de cultura do material no momento da captação mostravam ausência de crescimento bacteriano ou de fungos. As sorologias das pacientes eram todas negativas. Conclusão: Nesse trabalho, buscamos estabelecer um protocolo de conservação de membranas amnióticas baseado na glicerolização, pois se trata de um método de baixo custo, relativamente simples e de fácil estocagem do material. Apresenta como desvantagem a sua alta toxicidade celular, resultando em destruição das células do tecido, porém preserva a integridade estrutural tecidual, conforme demonstrado em nossos resultados macroscópicos e microscópicos.

Background: Researchers have attempted to explore various alternative therapies, biological or synthetic, capable of providing ideal conditions to the wound bed to promote the healing process. An option less costly is the use of amniotic membranes. Dressings consist of amnion forms a protective barrier against environmental bacteria, accelerate reepithelialization of lesions and reduce local pain. Objective: The aim of this study was to establish protocol processing of membranes in high concentrations of glycerol. Methods: Three samples were obtained from amniotic membranes who met the inclusion criteria and that the pregnant agreed to donate the material for research. Results: The examinations of material culture at the time of capture showed no bacterial or fungal growth. The serology of the patients was all negative. Conclusion: In this paper, we established a protocol for the conservation of membranes based on glycerol because it is a low-cost, relatively simple and easy storage of the material. Presents the disadvantage of its high cell toxicity, resulting in destruction of tissue cells, but preserves the structural integrity of tissue as shown in our results the microscopic and macroscopic.

Pentoxifylline modifies three-dimensional collagen lattice model contraction and expression of collagen types I and III by human fibroblasts derived from post-burn hypertrophic scars and from normal skin.

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF - 1mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL's and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p<0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL.

Tratamento de cicatrizes hipocrômicas pós-queimadura com transplante de melanócitos cultivados in vitro / Transplant of cultivated melanocytes for the treatment of hypochromic

Introdução: As cicatrizes hipocrômicas pós-queimadura são um problema frequente, quecausa aos pacientes constrangimento social e sofrimento psicológico, uma vez que podemser bastante evidentes, graças aos contrastes de cores de pele que surgem ao seu redor (diferentestons de pele entre a pele normal e a área vizinha à cicatriz - normalmente hipercrômica,e a cicatriz pós-queimadura, hipocrômica). As opções de tratamentos atuais para talproblema não são muito eficientes; estão hoje disponíveis: a maquiagem – que apresenta oinconveniente de sair com o passar das horas, ou a tatuagem, que muitas vezes não acompanhao tom da pele normal. Método: Neste trabalho, utilizamos epitélios de queratinócitosautólogos cultivados in vitro como veículo de transporte de melanócitos funcionantes parao tratamento dessas áreas cicatriciais hipocrômicas resultantes de queimaduras. Estes epitélioscultivados serviram como instrumento para a repopulação definitiva destas áreas comcélulas produtoras de pigmento melânico. O método empregado incluiu o cultivo in vitro deepitélios autólogos constituídos por queratinócitos e melanócitos funcionantes, a abrasãosuperficial das áreas a serem tratadas, objetivando a remoção de epiderme hipocrômica, eo transplante dos enxertos autólogos cultivados. Resultados: Os resultados obtidos com aintegração destes epitélios cultivados, ou seja, a restauração e a repigmentação das áreashipocrômicas, foram avaliados por meio do uso de tabelas colorimétricas e biópsias, que demonstrarama repigmentação das cicatrizes e a persistência dos melanócitos transplantados.

Background: Post-burn hypochromic scars can be the subject of great personal sufferingto their victims. Due to the color contrast between the normal and affected skins, they arevisible lesions that embarrass the injured subjects and limit their social performance. Theavailable treatment options in the present days are insufficient, and tend to evolve withpoor results. Methods: We present in this paper the use of in vitro cultivated keratinocyteepithelium as a vehicle for the transplantation of working melanocytes to repopulate thesehypochromic areas in four patients. Results: The results were evaluated with the use of colortables and skin biopsies that allowed us to clearly demonstrate the partial repigmentationof the scar areas and the integration of the transplanted melanocytes. The repigmented skinaverage after 3 and 6 months of the procedure was of 30.52% and 51.66%, respectively. Ifwe consider the low-morbidity of the procedure, these results are already very encouraging,but we strongly believe that technical refinements could allow even better results.

Estudo in vitro dos efeitos do bloqueador do canal decálcio (verapamil) sobre a biossíntese e contração damatriz extracelular / In vitro study of effects of calcium channel blocker (verapamil) on biosyntheses

Introdução: A adequada formação do tecido de granulação depende do equilíbrio entre abiossíntese e a degradação da matriz extracelular (MEC). A síntese excessiva de colágeno e adeficiente remodelação da MEC podem induzir o aparecimento de lesões dérmicas tipo quelóidese cicatrizes hipertróficas. Método: No presente estudo, utilizamos fibroblastos dérmicoshumanos povoados em gel de colágeno I, tratados ou não pelo bloqueador de canal de cálcio(verapamil). Uma vez no gel de colágeno, a morfologia tridimensional dos fibroblastos foianalisada por microscopia confocal e em cortes corados por hematoxilina & eosina. A atividadeda colagenase (MMP-I) foi determinada por “immunoblot” do meio condicionado. Resultados:Observamos neste estudo maior contração do gel de colágeno povoado por fibroblastoscontrole, quando comparado ao gel povoado por fibroblastos tratados com verapamil. A maiorcontração demonstrada pelos fibroblastos controle foi relacionada à reorientação das fibrascolágenas, à organização dos filamentos de actina e aos níveis de cálcio citosólico. Observamos,também, que o verapamil aumenta a secreção de colagenase. Conclusão: Concluímos que overapamil, ao alterar o metabolismo celular do cálcio, exerce influência sobre a contração daMEC e produção de proteases, com consequente efeito sobre o desenvolvimento de afecçõescicatriciais, como quelóides e cicatrizes hipertróficas.

Background: The appropriate formation of the granulation tissue depends on the balancebetween the biosyntheses and the degradation of the extracellular matrix (EM). The excessivesynthesis of collagen and the deficient degradation of the EM might cause dermal lesions suchas keloids and hypertrophic scars. Methods: In this study we used an experimental modelwhere collagen gels populated by dermal human fibroblast underwent progressive contraction.Fluorescence preparations were studied by confocal microscopy. Collagenase activity (MMPI)was determined in conditioned medium from fibroblasts using immunoblot. Results: Thisstudy showed a greater contraction of the gels populated with control fibroblasts per the timestudied in relation to the gels populated with fibroblasts treated with verapamil. Perhaps thishigher contracting ability is involved with actin organization and levels of cytosolic calcium.We also observed that verapamil increase the secretion of MMP-I. Conclusion: We concludethat cellular calcium metabolism appears to regulate EM production and contraction and thosehypertrophic disorders of wound healing (keloids and hypertrophic scars) may respond totherapy with calcium antagonist drugs (verapamil).

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes.

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Brazil.

Until 2000, efforts into organising tissue banks in Brazil had not progressed far beyond small "in house" tissue storage repositories, usually annexed to Orthopaedic Surgery Services. Despite the professional entrepreneurship of those working as part time tissue bankers in such operations, best practices in tissue banking were not always followed due to the lack of regulatory standards, specialised training, adequate facilities and dedicated personnel. The Skin Bank of the Plastic Surgery Department of the Hospital das Clinicas of Sao Paulo, the single skin bank in Brazil, was not an exception. Since 1956, restricted and unpredictable amounts of skin allografts were stored under refrigeration for short periods under very limited quality controls. As in most "tissue banks" at that time in Brazil, medical and nursing staff worked on a volunteer and informal basis undergoing no specific training. IAEA supported the implementation of the tissue banking program in Brazil through the regional project RLA/7/009 "Quality system for the production of irradiated sterilised grafts" (1998-2000) and through two interregional projects INT/6/049 "Interregional Centre of Excellence in Tissue Banking", during the period 2002-2004 and INT/6/052 "Improving the Quality of Production and Uses of Radiation Sterilised Tissue Grafts", during the period 2002-2004. In 2001-2002, the first two years of operation of the HC-Tissue Bank, 53 skin transplants were carried out instead of the previous 4-5 a year. During this period, 75 individuals donated skin tissue, generating approximately 90,000 cm(2) of skin graft. The IAEA program were of great benefit to Brazilian tissue banking which has evolved from scattered make shift small operations to a well-established, high quality tissue banking scenario.