Study on biopharmaceutical properties of active fraction from Xiangfu Siwu Decoction by composite phospholipid liposome-based artificial skin membrane / 中草药

Objective: To evaluate the feasibility of the application of composite phospholipid liposome-based artificial skin membrane (CPLASM) for measuring biopharmaceutical properties of active fraction extracted from Xiangfu Siwu Decoction (XSD) via transdermal administration. Methods: The HPLC method was established for the determination of active ingredients (ferulic acid, tetrahydrocolumbamine, tetrahydropalmatine, senkyunolide I, and senkyunolide H) in active fraction of XSD. The oil/water partition coefficient values of these active ingredients were measured. The CPLASM was prepared to determine the in vitro permeability parameters of active fraction of XSD. The obtained parameters were further compared with those obtained by porcine ear skin. Results: The oil-water partition coefficients of ferulic acid, tetrahydrocolumbamine, tetrahydropalmatine, senkyunolide I, and senkyunolide H in active fraction of XSD (pH 5.5) were measured to be 1.220 ± 0.280, 0.670 ± 0.085, 0.920 ± 0.110, 1.040 ± 0.092, 1.030 ± 0.093 (n = 3), respectively. The permeability parameters of the active ingredients through porcine ear skin and CPLASM indicated a significant correlation (r > 0.9). Compared with the classical oil-water partition coefficients, the 6 h- cumulative permeation ratio of five active ingredients through CPLASM can be characterized as effectively predicting permeability parameters through porcine ear skin. Conclusion: The biopharmaceutical properties of active fractions from Chinese material medica can be effectively characterized by the application of CPLASM for the determination of in vitro permeability parameters of active ingredients.

Idea and method of regularity knowledge of Chinese materia medica on essential oils as potential penetration enhancers based on drug property characteristics / 中草药

Penetration enhancers (PE) were usually applied as the optimum strategy to improve the percutaneous absorption of active components from pharmaceutical preparations. Essential oils (EOs) were a kind of PE with satisfactory characteristics. Only 34 of among more than 300 EOs are reported to be used as PE. That is to say, about 90% EOs have not been applied until now and EOs possess great potential to act as PE. However, the research of EOs from Chinese materia medica (CMM) mainly followed the strategy of chemical PE. A large amount of studies were carried out to evaluate the transdermal penetration enhancing efficacy and pharmacological activity, resulting in the limited efficiency. EOs of CMM were also the key material basis of CMM pungent flavor. In the theory of traditional Chinese medicines (TCM), it was accepted that the pungent flavor could open the striae and interstice. The results of literature investigation revealed that the penetration enhancing efficacy of EOs directly related with drug property characteristics of CMM. Therefore, in the present paper, it was proposed that a rule existed between the penetration enhancing efficacy of EOs and drug property characteristics of CMM. A series of studies were then designed to reveal the rule with the aid of data analysis. The resultant rule will help to find the effective PE from various EOs. Moreover, to facilitate the high-throughput screening of EOs, liposomal artificial skin membrane was prepared and applied to the evaluation of in vitro penetration enhancing efficacy of EOs with optimization of the evaluation parameters. Finally, the results of the study will provide a novel strategy for the research of EOs as PE and the research of the application of TCM theory to the modern pharmaceutical study. In addition, the application of microemulsion technique will be the future trends of EOs as PE to solve the stability problems.

Advances in research on lipid artificial skin membrane / 中国药学杂志

The pre-design and formulation optimization of transdermal drug delivery preparations require a large amount of animal or human skin samples for percutaneous absorption experiments in vitro. However, the use of animal and human skins is more and more restricted and its reproducibility and quality can not be guaranteed, which stimulated the development of artificial skin model. Lipid artificial skin membrane is a model using the lipid composition of the skin to mimic the skin barrier function. It can be used as a tool for high-throughput screening of drugs. Because of its adjustable and flexible properties, it can be applied for different research purposes. In this paper, the composition, preparation, application and characterization of common lipid artificial skin membranes are reviewed, and the prospect is also discussed.

Desenvolvimento de epiderme humana reconstruída (RHE) como plataforma de testes in vitro para irritação, sensibilização, dermatite atópica e fotoimunossupressão / Development of a Reconstructed Human Epidermis (RHE) as platform to in vitro assays: irritation, sensitization, atopic dermatitis and photoimmunosuppression

O desenvolvimento de novos modelos de pele e novas metodologias in vitro segue uma tendência mundial na busca pela redução ou substituição de testes em animais. Nesse contexto, kits de epiderme humana reconstruída (RHE) apresentam-se como uma plataforma promissoras para essa proposta e, alguns modelos encontram-se validados para ensaios de irritação e corrosão cutânea in vitro. Entretanto, em países como o Brasil, enfrentam-se questões alfandegárias e perda do material por perecibilidade, dificultando e até impedindo, a importação desses kits para utilização por parte das indústrias e laboratórios nacionais. Em contrapartida, o desenvolvimento de um modelo de RHE apresenta-se como um avanço tecnológico e ganho de autonomia para esses países. Assim, no capítulo 1 explorou-se o desenvolvimento de um modelo nacional de RHE (USP-RHE) que atendesse às exigências internacionais descritas no guia OECD 439. O modelo desenvolvido apresentou uma epiderme bem diferenciada e atendeu aos parâmetros de qualidade (histologia, viabilidade e função barreira) bem como da funcionalidade, a qual é expressa na capacidade de distinção entre irritantes e não irritantes, apresentando 85,7% de especificidade, 100% sensibilidade e 92,3% de acurácia quando comparada com a classificação in vivo obtida pelo ensaio do linfonodo local (LLNA). No capítulo 2, células monocíticas THP-1 em monocamada foram capazes de distinguir entre agentes sensibilizantes e não sensibilizantes por meio da expressão de CD86, CD54 e liberação de IL-8. Após a obtenção de RHE e THP-1 funcionais, um cross-talking foi estabelecido gerando uma RHE imunocompetente. A RHEI distinguiu satisfatoriamente entre agentes sensibilizantes e não sensibilizantes por meio da expressão de CD86 e CD54 na membrana das células THP-1. A liberação de IL-8 também foi avaliada na RHEI, mas, não demonstrou ser um bom indicador para a avaliação de sensibilização, ao contrário de IL-1α, que distinguiu satisfatoriamente agentes sensibilizantes de não-sensibilizantes, mas não foi capaz de hierarquizá-los. No capítulo 3, avaliou-se o papel de interleucinas do tipo Th2 e da depleção de colesterol na membrana plasmática no desenvolvimento de características morfológicas e moleculares da dermatite atópica (DA) in vitro em um modelo de RHE. Os resultados demonstram que o uso de IL-4, IL-13 e IL-25 em combinação com a depleção de colesterol na membrana plasmática mimetiza in vitro, as principais características da DA. No capítulo 4, buscou-se avaliar os efeitos imunossupressores da radiação ultravioleta na RHEI. Os ensaios foram realizados em diferentes períodos de exposição, entretanto, não foi possível observar tais efeitos. Os resultados justificam-se pela ausência da liberação de IL-10 pelo RHE imunocompetente, por exemplo, e demonstram uma limitação do RHE imunocompetente para avaliações de inativação da reposta imune. Neste trabalho, concluímos que foi possível obter uma RHE competitiva, similar aos modelos internacionais validados e que pode ser utilizada como plataforma para ensaios de irritação e sensibilização cutânea, além de ser uma plataforma para estudos da dermatite atópica. No modelo é possível estudar a ativação do sistema imune, o que o torna promissor como uma plataforma para avaliação de resposta imunológica in vitro. Conclui-se, portanto, que os objetivos foram amplamente atendidos além de oferecermos um protocolo de livre acesso para reprodução por outros laboratórios e um modelo para validação futura

The development of new in vitro skin models and new methodologies follows a global trend in search for reductions or replacement of animal testing. In this context, Reconstructed Human Epidermis kits (RHE) are presented as a promising platform in the search for alternative methods to animal use, and some models are validated for skin irritation and corrosion in vitro tests. However, in countries such as Brazil, who face customs issues and loss of material due to perishability, making it challenging and even compromising the importation of these kits for use by industries and laboratories. In contrast, the development of an RHE model is presented as a technological breakthrough and gain of autonomy for these countries. Thus, in Chapter 1 we explored the development of a national model of RHE (USP-RHE) that meet international requirements described in OECD TG 439. The developed model presented a well-differentiated epidermis and met the quality parameters, for instance, histology, viability, and barrier function as well as the functionality expressed in the capacity of screening between irritants and nonirritants, with 85.7 % of specificity, 100 % of sensitivity and 91.7% of accuracy in comparision to in vivo UN GHS classification from Local limph node assay (LLNA). In chapter 2, monocytic THP-1 cell line, as monolayers, were able to distinguish between sensitizers and non-sensitizers by expression of CD86, CD54, and IL-8 release. In this model, functional RHE and THP-1 were used in a cross-talking, and thus an immunocompetent RHE (RHEI) was generated. The RHEI has distinguished satisfactorily between sensitizers and non-sensitizers through CD86 and CD54 expression that was larger and more sensitive in this model. The release of IL-8 was also evaluated in RHEI, however, did not demonstrate to be a good parameter for this evaluation, unlike IL-1α, which satisfactorily distinguished sensitizers from non-sensitizers, but was not able to hierarchize them. In chapter 3, we evaluated the role of Th2-related cytokines and plasma membrane cholesterol depletion (CD) in the development of atopic dermatitis (AD) morphological and molecular characteristics in an in vitro model of RHE. The results showed that combination of IL-4, IL-13 and IL-25 in combination with CD can reproduce the major features of AD in vitro. In Chapter 4, we sought to evaluate the ultraviolet radiation-induced immunosuppressive effects in RHE. The tests were performed at different times. However, it was not possible to observe such effects. The results are justified by the absence of IL-10 release by RHEI, for example, and show a limitation of RHEI for rating inactivation of the immune response. In this work, we conclude that it was possible to obtain a competitive RHE similar to the validated international models that can be used as a platform for irritation and skin sensitization tests, besides being a platform for the study of atopic dermatitis. Using this model is possible to explore the activation of immune system, which makes it promising as a platform for the evaluation of immune response in vitro. We conclude, therefore, that the objectives have been met as well as it is offering an open source protocol for breeding by other laboratories, thus offering the RHE model developed here for future validation tests

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

Scalded Skin of Rat Treated by Using Fibrin Glue Combined with Allogeneic Bone Marrow Mesenchymal Stem Cells

BACKGROUND: It is difficult to achieve satisfactory results with the traditional treatment of large-area skin defects and deep burns. OBJECTIVE: To test the treatment effect of an active dressing film made of a mixture of fibrin glue and bone marrow mesenchymal stem cells (BMSCs) for repairing burn wounds on the skin of rats. METHODS: Two scald wounds were made on the back of each rat. A total of 30 scald wounds were randomly divided into 3 groups, with 10 wounds in each group. In the experimental treatment group, the scald wounds were covered with the fibrin glue and BMSC mixture. The wounds of the experimental control group were covered with fibrin glue only. No intervention was administered to the blank control group. Thirty days after treatment, pathological sections were cut from the scalded local tissues of all rats from the 3 groups and observed with a microscope. RESULTS: The speed of scald wound healing in the experimental treatment group was faster than the other 2 groups. In the experimental treatment group, histopathological analysis revealed that the sebaceous glands showed obviously proliferous at the edge of the new tissue and gradually extended to the deep dermal layer of the new tissue. CONCLUSION: BMSCs may have an active role in promoting skin tissue repair and generating skin appendages. Allogeneic BMSCs mixed with fibrin glue can contribute to the quick formation of a film-like gel over the scald wounds, which might be of significance for emergency treatment and skin-grafting operations.

Sustitutos dérmicos definidos / Dermal substitutes defined

Son muchas las patologías que producen defectos de piel. El método de elección para la cobertura cutánea de estos defectos es el injerto de piel parcial, sin embargo, en algunos casos sus resultados no son adecuados. Los sustitutos dérmicos son una alternativa de cobertura cutánea, que existen en la actualidad y que permiten obtener mejores resultados funcionales y estéticos. Existen muchos sustitutos dérmicos en el mercado, cada uno con distintas características, beneficios y complicaciones. Esta revisión logra agrupar los aspectos más relevantes de los sustitutos dérmicos para poder tener una base teórica sobre estos productos que son de gran ayuda para poder tratar a pacientes con distintas patologías.

Many are the pathologies that cause skin defects. The gold standard for skin coverage of these defects is the partial skin graft, but in some cases the results are not appropiate. Dermal substitutes are an alternative for skin coverage that exists today and allow get better functional and aesthetic results. There are many dermal substitutes in the pharmaceutical industry, each one with different characteristics, benefits and complications. This review brings together the most important aspects of dermal substitutes to have a theoretical background on these products that are helpful to treat patients with different pathologies.

Contractura axilar por quemadura tratada con Integra® / Axillary burn contracture treated with Integra®

Introduction: Severe axillary burn is an unusual accident that frequently evolves to contracture generating important cosmetic and functional deficiencies. Contracture scars in this region are difficult to treat because of the anatomic characteristics of the area that has multiple power vectors. Functional restoration has to be one of the main goals in the management of burns in the axilla and flaps have shown high rate of morbidity. Integra® provides satisfactory elasticity and dermal resistance which results in positive functional results. Objective: Analyze the results of the use of Integra® in axillary burn contracture scars at a specialized Burns Center. Materials and Methods: There were 4 patients who underwent reconstructive surgery using Integra® for axillary burn contractures between January 2002 and March 2006. Follow-up was divided into perioperative and late. Early follow-up checked general post-operative evolution and late follow-up was focused on functionality and patient independence evaluated using Barthel's index of daily living activities. Results: There were 3 males and 1 female, average age 27 (18-41) with a minimum follow up of 9 months. There were no perioperative complications and good or very good range of motion results. Conclusions: Our results are similar to artificial skin substitutes used in other anatomical regions.

Las quemaduras axilares severas son un accidente infrecuente que evolucionan a la retracción generando deficiencias cosméticas y funcionales. Estas cicatrices son difíciles de tratar por las características anatómicas del área, donde la corrección de un vector de movimiento puede alterar otro. Objetivo: Mostrar nuestros resultados utilizando el sustituto cutáneo Integra® en el tratamiento de cicatrices retráctiles axilares por quemadura. Pacientes y Métodos: Se recolectaron antecedentes médicos y fotográficos de pacientes portadores de cicatrices retráctiles axilares por quemadura entre enero de 2002 y marzo de 2006 en el Hospital del Trabajador de Santiago. Se evaluó pre y postoperatoriamente a los pacientes en forma subjetiva por fisiatra y con el Test de Barthel. Resultados: Se incluyeron 4 pacientes en el estudio (3 mujeres y 1 hombre), edad media 27 años (18-41). Todas las quemaduras fueron producidas por fuego. Índice de Barthel preoperatorio fue de 87,5 (levemente dependiente para las actividades de la vida diaria) y rango de movimiento moderadamente afectado. En el postoperatorio los pacientes fueron catalogados como independientes según el Test de Barthel y el rango de movimiento fue descrito como bueno o muy bueno por el fisiatra. No hubo complicaciones peri operatorias, Integra® prendió adecuadamente en todos los casos. Los pacientes fueron seguidos en promedio 16 meses (9-22). Conclusiones: Los sustitutos dérmicos han sido usados para la corrección de cicatrices de quemaduras con buenos resultados, pero no hay reportes en la axila. El pequeño número de pacientes que presentamos tienen un excelente resultado funcional, lo que nos estimula a seguir trabajando en este rumbo.

Mecanismo de ação do 4-nerolidilcatecol na indução da morte celular e contenção da invasão em linhagens de melanoma humano e modelo de pele artificial / Mechanism of action of 4nerolidylcathecol: induction of apoptosis via ROS accumulation and inhibition of invasion in melanoma and skin reconstructs model

O melanoma é a forma mais mortal de câncer de pele, origina-se de células produtoras de pigmentos, os melanócitos. Esses podem ser cutâneos ou não-cutâneos (encontrados no revestimento da membrana coróide do olho, nas meninges, e nos tratos gastrintestinal e geniturinário). O aumento da incidência de melanomas malignos nas últimas décadas, e sua alta taxa de mortalidade e grande resistência a maior parte das terapias, tem sido um enorme desafio para a comunidade científica. Particularmente, a falta de habilidade de indução à morte por apoptose em resposta à quimioterapia e outros estímulos externos permitem uma vantagem seletiva para progressão tumoral, formação de metástase e resistência à terapia em melanomas. O estresse oxidativo e espécies reativas de oxigênio (EROs) vêm sendo, há muito tempo, reconhecidos como importantes desencadeadores e moduladores da apoptose. Porém o exato papel do estresse oxidativo no processo apoptótico ainda é uma questão de debate. Antioxidantes tendem a possuir propriedades regulatórias de tradução de sinais que devem ou não estar ligadas as suas capacidades de inativar oxidantes. Porém em certas condições, um forte ambiente oxidante onde há falta de suporte para regenerar (reduzir) antioxidantes oxidados, permite que alguns antioxidantes assumam características de um pró-oxidante. Foi demonstrada a capacidade citotóxica de um potente antioxidante, 4-nerolidilcatecol (4-NC), extraído da planta Pothomorphe umbellata L. Miq, sobre linhagens tumorais de melanoma e sobre fibroblastos humanos normais. Esse composto foi capaz de induzir a parada do ciclo celular em G1, bem como diminuir a atividade de MMPs e em outras linhagens de melanoma foi capaz de induzir a morte celular por apoptose. Estudos subseqüentes mostraram que o mecanismo de ação deste composto inicia-se com a formação e acúmulo de EROs, além da inibição da enzima catalase. O 4-NC foi capaz de induzir a morte por apoptose via mitocondrial, aumentando os níveis das...

Melanoma is the most agressive form of skin cancer, it arises from the pigment-producing cells, melanocytes. These may be cutaneous or non-cutaneous (found in the lining membrane of the eye choroid, the meninges, and gastrointestinal and genitourinary tracts). The increased incidence of malignant melanomas in recent decades, its high mortality rate and high resistance to most therapies has been a major challenge to the scientific community. It's particularly difficult to induce cell death by apoptosis in response to chemotherapy and other external stimuli, which may provide a selective advantage for tumor progression, metastasis formation and resistance to therapy in melanoma. Oxidative stress and reactive oxygen species (ROS) have been recognized for a long time as important triggers and modulators of apoptosis, but the exact role of oxidative stress in the apoptotic process is still a matter of discussion. Antioxidants tend to possess properties to regulate transduction signals that may not be related to their ability to inactivate oxidants. Under certain conditions, in a strong oxidizing environment where there is lack of support to regenerate (reduce) oxidized antioxidants, some antioxidants can assume characteristics of pro-oxidant. The 4-nerolidylcatechol (4-NC) is a potent antioxidant that is extracted from the plant Pothomorphe umbellata L. Miq. Its citotoxic potential has been demonstrated on melanoma tumor cell lines and on normal human fibroblasts. This compound was able to induce cell cycle arrest in G1, decrease the activity of MMPs and cell death by apoptosis. Subsequent studies showed that the mechanism of action of this compound starts with the formation and accumulation of ROS, and inhibition of the enzyme catalase. The 4-NC was able to induce apoptosis via mitochondria, increasing the levels of p53, Noxa, Mcl1, cleaving Bax and Bid and inducing cleavage of caspases 3 and 9. Furthermore, in a model of artificial skin containing melanoma 4-NC...

Evaluation of the Various Artificial Skin Substitutes Implanted onto Nude Mice

PURPOSE: The purpose of this study is to evaluate the remodeling process of the various skin substitutes in 4th and 6th weeks following the transplantation when transplanted onto nude mice. METHODS: Three types of artificial skin substitutes, such as PLGA scaffold with keratinocyte sheets(group 1), acellular human dermis(Surederm(TM)) and keratinocyte sheet(group 2), bioengineered skin(Neoderm(TM))(group 3), were applied to the wound on nude mice. All mice were killed in 2, 4 weeks and/or 6 weeks after grafting and tissue samples were harvested from the back of mice. The changes in wound size, degree of angiogenesis, formation of basement membrane and epidermis, density of collagen fibers and neural restoration were examined. RESULTS: There was no significant changes in wound size among the three groups. However, the size of wound decreased in the non-substituted group due to contracture. Degree of angiogenesis and systhesis of collagen or neurofilaments were mostly increased in bioengineered skin(Neoderm(TM))(group 3), followed by acellular human dermis(Surederm(TM)) and keratinocyte sheet(group 2), PLGA scaffold with keratinocyte sheets (group 1). However, group 3 and group 2 showed similar thickness of basement membrane and epidermis. CONCLUSION: We found that degree of angiogenesis, formation of basement membrane and skin appendages, density of collagen fibers and neurofilaments can be the categories to evaluate the success of artificial skin substitution in early stages.

The Effects of Adipose Tissue-Derived Mesenchymal Stem Cells on the Formation ofEpidermis and Basement Membrane in Artificial Skin Models / 대한피부과학회지

BACKGROUND: There is an increasing need for making a more ideal artificial skin model. OBJECTIVE: To evaluate the effects of adipose tissue-derived mesenchymal stem cells (ATMSC) on the formation of epidermis and basement membrane in artificial skin models. METHODS: ATMSC were isolated from lipo-aspirated fat tissues, and their phenotypes were confirmed by cell surface markers. Three kinds of artificial skin models were made using three different dermal substitutes. The dermal substitutes in the three models contained fibroblasts only, fibroblasts together with ATMSC or ATMSC only. The formation of epidermis and basement membrane was evaluated by immunohistochemical stains and transmission electron microscopy. RESULTS: Among the three models, the model with both fibroblasts and ATMSC in the dermal substitute showed the most excellent formation of epidermis and, especially, basement membrane. In this model, the basement membrane proteins, laminin and type IV collagen, were expressed most apparently at the dermo-epidermal junction and, lamina lucida, lamina densa and anchoring fibrils were most evidently observed under transmission electron microscopy. Whereas, the model with only ATMSC did not show keratin 1 expression, suggesting that the 'skin-type' stratified squamous epithelium was not formed well. CONCLUSION: ATMSC together with fibroblasts can be used effectively in constructing artificial skin models.

Advances in Applications of Bacterial Cellulose in Biomedical Materials / 中国生物工程杂志

Bacterial cellulose (BC) is a natural polymer that has bioactivity, biodegradability and biocompatibility. It displays unique physical, chemical and mechanical properties including high crystallinity, high water holding capacity, nanofibre-network structure, high tensile strength and elastic modulus. Due to its unusual material properties, BC has recently become a kind of attractive biomedical material in the international research.Describes BC's properties, study history and its applications as biomedical materials, especially gives emphasis to introduce the applications of BC on scaffold tissue engineering, artificial blood vessels, artificial skin and the treatment of skin wound, as well as the present study status.

An Developmental Study of Artificial Skin Using the Alginate Dermal Substrate: Preliminary Report

Alginate, a polymer of guluronic and mannuronic acid, is used as a scaffolding material in biomedical applications. The research was to produce highly-purified alginate from seaweeds and to evaluate the efficacy of alginate as dermal substrate. Our alginate purification method showed a production rate as high as 25%. The purified alginate contained little polyphenol contents and endotoxin, proteins. For study of wound healing, full thickness skin defects were made on the dorsal area of the animal models. And then alginate, fibroblast-growth-factor mixed alginate, alginate-collagen complex, vaseline gauze as control were applied on the wound, respectively, and were evaluated grossly and histopathologically. For biocompatibility test, alginate and alginate-collagen complex discs were implanted on the back of Sprague-Dawly rats. Four weeks after implantation, the animals were examined immunologically against alginate and collagen. Alginate and FGF-mixed alginate, alginate-collagen complex group showed statistically higher percentage of wound contraction and wound healing than control group(p<0.05). Alginate-collagen complex group and FGF-mixed alginate group showed statistically higher percentage of wound healing than alginate group. The experiment of biocompatibility and immunologic reaction against impanted alginate or collagen needs more investigation. Highly-purified alginate from seaweeds by our purification method, showed the effect of wound healing, and addition of FGF or collagen increases the alginate's wound healing effect. It shows the possibility of alginate as a dermal substrate.

Application of Human Dermal Fibroblast and Keratinocyte on Allogenic Dermis(AlloDerm(R))

PURPOSE: Large skin defect by various causes, should be covered by autologous skin graft. But, the donor site of autologous skin graft is limited and leaves permanent donor scar and contracture. There have been our trial to engineer artificial skin using allogenic dermis (AlloDerm) with basement membrane. METHODS: Dermal and epidermal layer were separated by immersing in dipase solution for 30 minutes, and the separated layers were treated with 0.05% trypsin for 10 minutes. And then each layer was cultivated to fibroblasts and keratinocytes on a culture medium. Fibroblasts were first penetrated into basement membrane of allogenic dermis facing down, then allogenic dermis was flipped over to face up and keratinocytes were transplanted to allogenic dermis. RESULTS: Observing artificial skin fabricated in vitro, we found following: 1) The artificial skin opened in air for 5 days formed epidermal layer. In dermal layer, fibroblast was distributed evenly among all. 2) The artificial skin opened in air for 30 days formed thicker and thicker, and it formed basement membrane, spinous and granular layers. PAS stain to confirm existence of basement membrane showed positive reaction. 3) Cytokeratin 10 stain to confirm the formation of epidermal layer showed positive reaction. 4) The formation of thick keratin, lamellar body and desmosome similar to human skin were observed in result of an electron micrograph. CONCLUSION: As a result of research, the structure seen in normal skin such as rete ridge, is found in reproduced artificial skin. This type of artificial skin can be used as a useful model for investigating skin disease and for clinical application also.

Distribution Patterns of Involucrin in the Stratum Corneum of the Normal and Psoriatic Artificial Skins / 대한해부학회지

Cornified envelope is highly insoluble structure formed beneath the plasma membrane during terminal differentiation of keratinocytes and is stabilized by cross linking of various proteins, including involucrin, loricrin, and cornifin. Psoriasis is a chronic skin disease characterizing inflammatory reaction and hyperproliferation of keratinocyte. There are some differences in involucrin immunolabelling in stratum corneum between normal and psoriasis epidermis. Labelling was convergent to cornified envelope in psoriasis skin but throughout cytoplasm in normal skin. To compare terminal differentiation patterns of normal and psoriasis keratinocytes, we reconstructed normal and psoriatic artificial skin by using primary cultured keratinocytes from normal and psoriasis skin and then performed immunogold labelling for involucrin in stratum corneum. Psoriatic artificial skin had thin and poorly organized corneal layer. Immunogold labelling for involucrin revealed same pattern of that in vivo by showing throughout cytoplasm in lower layer but convergent cornified envelope in upper layer. Compared with psoriatic artificial skin, normal artificial skin had well organized and thick stratum corneum. Involucrin labelling was throughout cytoplasm in most of corneal layer but convergent to cornified envelope in some uppermost cells. Even though some cells show convergent pattern in normal artificial skin, absolute number of this pattern was no lesser than in artificial psoriatic skin because of normal artificial skin had thick stratum corneum. This result showed there was no difference in involucrin distribution in terminal differentiation of normal and psoriasis keratinocytes in organotypic culture model. It is concluded that although well organized multiple corneal layers are formed in normal artificial skin, they can not reach to full maturation of cornified envelope, and difference of involucrin localization in cornified envelope of psoriasis epidermis is related with not peculiarities of the cells but rapid growing in vivo.

Expression Patterns of Cytokeratin and Involucrin in the Epidermis of the Artificial Skin Reconstructed by Cultured Cells / 체질인류학회지

To investigate differentiation and growth process of keratinocytes in organotypic cultured skin, we carried out immunohistochemical studies for cytokeratin (CK) 10, 14, 16, 17 and involucrin in the cultured skin. In normal skin CK14 and CK10 were detected in the basal and all suprabasal layer, respectively, whereas in artificial skin CK14 was detected up to the middle of spinous layer but CK10 expressed from the middle of spinous layer. The detection of involucrin in normal skin was from the upper spinous layer but found from lower spinous layer in the artificial skin. Both CK16 and CK17 did not expressed in in vivo skin but expressed weakly in the spinous layer of artificial skin. It is therefore concluded that the characteristics of basal cell were maintained in the several, lower layers of the sartificial skin. The growth and differentiation steps of the skin were similar to those of in vivo although differences were seen in the expression level.

Development of Psoriasis Model in Vitro / 대한해부학회지

Psoriasis is a disease caused by hyperproliferation of keratinocytes. Pathogenesis of psoriasis is still unclear, but many reports suggest that psoriatic keratinocytes themselves may have some factors of pathogenesis. The author developed artificial psoriatic skin by culturing keratinocytes of psoriasis skin over collagen lattice which was constructed with collagen and normal fibroblasts. After the keratinocytes had grown to full layers of stratification, expression patterns of differentiation marks and ultrastructural changes were investigated by immunohistochemistry and electron microscope. The results were very similar to those of psoriasis skin in vivo as follows. Cytokeratin (CK)10, marker of initiation of differentiation of keratinocytes, was expressed in the spinous layer. CK14, marker of basal cells of stratified squamous epithelium, was expressed in the basal and spinous layer. CK16 and CK17, markers of fast turnover of squamous epithelium, were expressed in the spinous layer. Involucrin, marker of terminal differentiation of squamous epithelium, was expressed weakely over the lower spinous layer. In immuno electron microscopical study, involucrin was expressed but confined to cornified cell envelops in the horney layer. Mitochondria, rER and ribosomes were abundant in the basal layer. They continued to appear in the upper spinous layer but intermediate filaments were scarce. Keratohyalin granules were visible in some parts of the granular layer zone, but the granules were smaller and fewer. In the horney layer, cells were thicker than normal and there were many lipid droplets within the cells. Intercellular spaces were enlarged at the basal layer but disappeared in the upper spinous layer. In these results, non systematic expression of differentiation markers and ultrastructural changes suggest that psoriasis is a disease caused by hyperproliferation of keratinocytes concurrent with unstable maturation and degeneration. Artificial psoriatic skin, in exclusion of systemic or dermal effects, showed very similar results with psoriasis skin in vitro. So it was concluded that psoriasis keratinocytes had some factors of pathogenesis and this kind of model on artificial psoriatic skin can be used for further studying of psoriasis.

Reconstruction of Artificial Skin In Vitro / 체질인류학회지

Artificial skin can be employed as a useful model for investigating skin disease and clinical application such as burn. To evaluate this, the skin was reconstructed by culturing keratinocytes in airfluid interface over the collagen lattice and ultrastructures was also investigated by electron microscope. Epidermis showed more than 10 layers including basal, spinous, granular and honey layers but basement membrane and anchoring fibrils were not formed at the dermoepidermal junction. Desmosomes were formed at cell junctions but reduced in number. Intermediate filaments were scarce and fine, especially at the spinosal layer. Keratohyalin granules were smaller and fewer than those of in vivo. Parakeratosis was observed at the honey layer. It was, therefore, concluded that the artificial skin could be a valuable experimental model although the differentiation process of keratinocytes was somewhat unstable.