Dissertationen zum Thema „Skin equivalent“

Acute skin irritation is the reversible inflammatory response of the epidermis to a topically applied irritant substance. A tissue engineered model of the epidermis is used to test chemicals. The degree of development of the model needs to be carefully judged in order to get the correct proportion of proliferating through to differentiated phenotypes for normal function. This judgement typically necessitates over sensitive models with an underdeveloped barrier functionality, as opposed to an insensitive model due to terminal differentiation and low numbers of basal keratinocytes. It has been reported that the lack of proliferative epidermal cells in cultures may be due to the absence of fibroblasts. Paracrine signalling in response to potential irritants is required for propagating an acute inflammatory response. The aim of this thesis is to develop a skin model using a Three Dimensional scaffold that accurately mimics the micro-environment at the DEJ, for supporting keratinocyte and fibroblast self-organisation. We hypothesise that it takes a full thickness skin model with a complete cascade of inflammatory stimuli and cytokine signalling to provide a real indication of irritation. Initial studies focused on Alvetex® (Reinnervate Ltd.), a highly porous polystyrene scaffold, with the aim of developing a skin model using the immortalised cell line HaCaT (human adult low calcium high temperature) keratinocytes or NhKs, in co-culture with dermal fibroblasts. Skin models using electrospun biodegradable polymer scaffolds made of Poly L-lactide (PLLA) and a Poly L-lactide/Polyhydroxybutyrate-co-hydroxyvalerate/Poly L-lactide (PLLA/PHBV/PLLA) composites were then developed. Issues with achieving epidermal-dermal separation in the Alvetex® scaffold due to keratinocyte entrapment lead to an Alvetex®-PHBV Bilayer. Concentration of the SDS needed to illicit an irritant response was deduced at 2D to be 0.1-0.15mM, 3D submerged to be 0.33-0.5mM and for 3D air-liquid models were at best unaffected by 8mM SDS with a Bilayer scaffold of PHBV-PLLA.

Skin is the largest, and arguably, the most important organ of the body. It is a complex and multi-dimensional tissue, thus making it essentially impossible to fully model in vitro in conventional 2-dimensional culture systems. In view of this, rodents or pigs are utilised to study wound healing therapeutics or to investigate the biological effects of treatments on skin. However, there are many differences between the wound healing processes in rodents compared to humans (contraction vs. re-epithelialisation) and there are also ethical issues associated with animal testing for scientific research. Therefore, the development of skin equivalent (HSE) models from surgical discard human skin has become an important area of research. The studies in this thesis compare, for the first time, native human skin and the epidermogenesis process in a HSE model. The HSE was reported to be a comparable model for human skin in terms of expression and localisation of key epidermal cell markers. This validated HSE model was utilised to study the potential wound healing therapeutic, hyperbaric oxygen (HBO) therapy. There is a significant body of evidence suggesting that lack of cutaneous oxygen results in and potentiates the chronic, non-healing wound environment. Although the evidence is anecdotal, HBO therapy has displayed positive effects on re-oxygenation of chronic wounds and the clinical outcomes suggest that HBO treatment may be beneficial. Therefore, the HSE was subjected to a daily clinical HBO regime and assessed in terms of keratinocyte migration, proliferation, differentiation and epidermal thickening. HBO treatment was observed to increase epidermal thickness, in particular stratum corneum thickening, but it did not alter the expression or localisation of standard epidermal cell markers. In order to elucidate the mechanistic changes occurring in response to HBO treatment in the HSE model, gene microarrays were performed, followed by qRT-PCR of select genes which were differentially regulated in response to HBO treatment. The biological diversity of the HSEs created from individual skin donors, however, overrode the differences in gene expression between treatment groups. Network analysis of functional changes in the HSE model revealed general trends consistent with normal skin growth and maturation. As a more robust and longer term study of these molecular changes, protein localisation and expression was investigated in sections from the HSEs undergoing epidermogenesis in response to HBO treatment. These proteins were CDCP1, Metallothionein, Kallikrein (KLK) 1 and KLK7 and early growth response 1. While the protein expression within the HSE models exposed to HBO treatment were not consistent in all HSEs derived from all skin donors, this is the first study to detect and compare both KLK1 and CDCP1 protein expression in both a HSE model and native human skin. Furthermore, this is the first study to provide such an in depth analysis of the effect of HBO treatment on a HSE model. The data presented in this thesis, demonstrates high levels of variation between individuals and their response to HBO treatment, consistent with the clinical variation that is currently observed.

Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability.
NextBioForm

Melanoma is a fatal form of skin cancer which progresses in an orchestrated pattern in human skin. Characterising these phases of melanoma in vitro can provide key insights into mechanisms of the disease progression. In this thesis, we investigate how in vitro three-dimensional (3D) model assays that recapitulate human skin can be used to identify key features underlying melanoma progression. In particular, we construct a 3D melanoma skin equivalent model using melanoma cells from the early and late phase of the disease. We further quantify melanoma cell migration, proliferation, invasion, as well as melanoma nest formation.

This thesis describes the use of 2- and 3-dimensional cell-based models for studying how skin cells respond to ultraviolet radiation. These methods were used to investigate skin damage and repair after exposure to radiation in the context of skin cancer development. Interactions between different skin cell types were demonstrated as being significant in protecting against ultraviolet radiation-induced skin damage. This has important implications in understanding how skin cancers occur, as well as in the development of new strategies to prevent and treat them.

The ultraviolet (UV) spectrum has been known to cause damage to skin in varying degrees. UVB radiation (290-320 nm) in particular, has been proven to be highly mutagenic and carcinogenic in many animal experiments compared to either UVA or UVC. The alarming rate of increase in skin cancer incidence has prompted many investigators to pursue other alternatives to sunscreens including changes in lifestyle habits and dietary consumption in order to boost our efforts in tackling this widespread disease. Previous studies employing confocal reflectance, MTT assay and histology suggest that milk phospholipids may possess protective properties against UVB-mediated damage but the molecular mechanism for this effect remains unclear. This study aims to evaluate changes in cell morphology, apoptosis and p21 expression in tissue engineered epidermis to increase our understanding of the mechanisms behind the potential protective effects of milk phospholipids against UV-induced photodamage. Human skin tissue equivalents were incubated in either 1% milk phospholipid solution or maintenance media then exposed to 120 mJ/cm2 dose of 300 nm UVB after 24 hours. The upregulation of p21 protein in response to DNA damage was measured with Western blot and immunofluorescence microscopy and markers for apoptosis and hyperplasia were examined 24 hours after irradiation. Results revealed that p21 levels and the amount of apoptotic markers such as fragmented DNA and nuclear condensation were significantly reduced in UV-exposed tissues pre-incubated with milk phospholipids compared to levels seen in both the positive control and UV-exposed skin tissue not incubated with milk phospholipids. This decrease in p21 expression may imply a reduction in DNA damage 24 hours after UV exposure or a decrease in acquired photodamage at the outset. Milk phospholipid incubation however, induced an increase in epidermal thickening with or without UV exposure, which may imply induction of a protective mechanism to enhance the barrier properties of skin.

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

Medical devices which cross the skin, such as drivelines that power artificial hearts, create what are essentially chronic wounds and are a major source of life-threatening infection. This research aimed to understand how novel 3-dimensional skin models that closely resemble native human skin, can be used to investigate novel driveline surfaces for their ability to increase skin integration and thus reduce bacterial infections. Using these models, it was shown that porous surfaces increase skin integration but may also exacerbate bacterial infection if microgaps between the tissue and the driveline are present.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

L’objectif de cet projet est la conception, réalisation et caractérisation d’un actionneur / capteur piézoélectrique piézorésonant destiné à la mesure du vieillissement de la peau humaine. L’étude présentée est le fruit d’une collaboration entre le groupe de recherche de l'Electrodynamique du INP-ENSEEIHT (Toulouse), LAPLACE Laboratoire de Recherche et l'École Polytechnique de Gdask, Département Génie Electrique et Automatique. Un concept d’actionneur / capteur pour la caractérisation des propriétés mécaniques des tissus mous a été présenté. Un actionneur piézoélectrique résonant, appelé "unimorphe" a été choisi parmi les différentes structures piézoélectriques fondées sur le cahier des charges. L'innovation du projet réside dans l'intégration de la méthode d'indentation dynamique en utilisant un unimorphe comme dispositif d'indentation. Ceci permet l'utilisation d'un certain nombre de propriétés électromécaniques favorables des transducteurs piézo-électriques. Ce mémoire est divisé en 7 chapitres. Le chapitre 1 présente la thèse et ses objectifs. Le chapitre 2 présente le phénomène piézoélectrique et les applications piézoélectriques dans les domaines de la médecine et de la bio ingénierie. Le chapitre 3 décrit le cahier des charges pour le transducteur développé. Le choix du transducteur unimorphe est ainsi justifié. Le chapitre 4 présente une description analytique du transducteur unimorphe, y compris les calculs de déformations statiques, la description du circuit équivalent de Mason, et la description des conditions de contact entre la sonde d'indentation et les matériaux testés. Le chapitre 5 contient l'analyse numérique du transducteur unimorphe en utilisant le modèle virtuel MEF. Les résultats de simulations statiques et modales sont décrits par deux géométries considérées du transducteur. Le chapitre 6 décrit le processus de vérification expérimentale des modèles analytiques et numériques développés pour le transducteur unimorphe. Enfin, le dernier chapitre comprend des conclusions générales concernant les résultats de recherche obtenus, ainsi que les travaux futurs possibles. Afin de vérifier la thèse d'un cycle complet de recherche a été effectuée, qui a couvert: étude analytique, l'analyse numérique (simulations MEF), réalisation de prototype, et la vérification expérimentale des actionneurs / capteurs piézoélectriques considérés
The main goal of the dissertation was following: preparation of a new concept, implementation and analysis of the piezoelectric resonant sensor/actuator for measuring the aging process of human skin. The research work has been carried out in the framework of cooperation between the INP-ENSEEIHT-LAPLACE, Toulouse, France, and at the Gdansk University of Technology, Faculty of Electrical and Control Engineering, Research Group of Power Electronics and Electrical Machines, Gdask, Poland. A concept of transducer for the characterization of mechanical properties of soft tissues was presented. The piezoelectric resonant, bending transducer, referred to as “unimorph transducer” was chosen from different topologies of piezoelectric benders based on the fulfillment of the stated requirements. The innovation of the project lies in the integration of the dynamic indentation method by using a unimorph as an indentation device. This allows the use of a number of attractive electromechanical properties of piezoelectric transducers. The thesis is divided into seven chapters. Chapter 1 states the thesis and goals of the dissertation. Chapter 2 presents piezoelectric phenomenon and piezoelectric applications in the fields of medicine and bioengineering. Chapter 3 describes the requirements for the developed transducer. The choice of unimorph transducer is justified. Chapter 4 presents an analytical description of the unimorph transducer, including the calculations of static deformations, equivalent circuit description, and description of the contact conditions between the transducer and the tested materials. Chapter 5 contains the numerical analysis of the unimorph transducer using FEM virtual model. Results of static and modal simulations are described for two considered geometries of the transducer. Chapter 6 describes the experimental verification process of analytic and numerical models developed for unimorph transducer. The final chapter includes general conclusions concerning obtained research results and achievements, as well as possible future works. In order to verify the proposition of the thesis a full research cycle was carried out, that covered: analytical study, numerical analysis (FEM simulations), prototype realization, and experimental verification of the considered (developed) piezoelectric sensor/actuator structures

La peau et son vieillissement sont un enjeu de santé publique. Les modèles expérimentaux disponibles pour l'étude du vieillissement cutané restent perfectibles. Dans ce contexte, nos objectifs étaient simultanément d'utiliser les modèles de peaux reconstruites (PR) développés dans notre laboratoire afin i) de mieux comprendre les mécanismes du vieillissement cutané, ii) de démontrer l'efficacité et le mécanisme d'action du sélénium comme « anti âge » et enfin iii) de les faire évoluer en utilisant le support poreux ou auto-assemblé avec des fibroblastes du même donneur prélevés à des âges différents. Ainsi, le modèle de PR cultivé sur une longue période a montré une surexpression du microARN miR30-a par RT qPCR dans les PR « âgées » avec une altération de la fonction barrière mesurée par la perte insensible en eau et une perturbation de la différenciation terminale (baisse d'expression de la loricrine et de l'involucrine). Avec le même modèle in vitro, nos résultats démontrent que la supplementation en sélénium retarde la sénescence des kératinocytes souches. Cette efficacité passe non pas par un effet antioxydant comme attendu mais par l'activation de leur adhésion à la lame basale, qui participe à les conserver souche et donc à préserver le renouvellement épidermique. Enfin, nous avons eu la chance exceptionnelle de préparer des PR avec des fibroblastes provenant d'un donneur unique prélevé à 36 et 72 ans. Les résultats immunohistologiques montrent que l'âge induit une augmentation de l'expression de l'élastine et de la fibrilline ainsi que leur co-expression. L'augmentation de LTBP1 et aSMA suggère que cette augmentation inattendue est due à une dérégulation de la voie TGF-ß et une différenciation des fibroblastes en myofibroblastes. En conclusion l'utilisation de différents modèles de PR a permis d'explorer les mécanismes conduisant au vieillissement cutané et de démontrer l'efficacité du sélénium comme anti âge
Skin and its aging is a public health issue. In vitro skin models available for the study aging remain perfectible. In this context, our objectives were simultaneously to use skin equivalent (SE) developed in our laboratory i) to better understand mechanisms of skin aging, ii) to demonstrate the effectiveness of selenium as “anti-aging” and finally iii) to improve SE using the porous or scaffold free model with fibroblasts from the same donor at different ages. Thus, the model of SE mimicking senescence showed an overexpression of microRNA miR30-a by RT qPCR in old SE with an alteration of the barrier function measured by the transepidermal water loss and a deficiency of epidermal terminal differentiation (decreased expression of loricrin and involucrin). With the same SE model, our results demonstrate that selenium supplementation delays the senescence of keratinocytes stem cells. This effectiveness does not involve antioxidant effect as expected but the activation of their adhesion to the basement membrane, which participates in preserving stemness and epidermal renewal. Finally, we had the opportunity to prepare SE with fibroblasts from a single donor at 36 and 72 years old. The histological results show that age induces an increase in the expression of elastin and fibrillin as well as their co-expression. The increase of LTBP1 and aSMA suggests that this unexpected increase is due to deregulation of the TGF-ß pathway and fibroblasts differentiation into myofibroblasts. In conclusion, the use of different models of SE helps us to explore some mechanisms leading to skin aging and to demonstrate the efficacy of selenium as “anti-aging”

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Facepe
Dentre os diversos procedimentos intervencionistas com fins terapêuticos, a quimioembolização hepática tem se destacado por ser de alta complexidade e resultar em altas doses de radiação aos pacientes e à equipe médica. Em alguns casos, o paciente requer várias sessões para tratar a mesma lesão, o que aumenta a probabilidade de ocorrência de lesões na pele e/ou efeitos estocásticos. Embora seja uma técnica altamente utilizada no Brasil, a quimioembolização não tem sido alvo de estudos dosimétricos. Este estudo apresenta os resultados da avaliação dosimétrica realizada na equipe médica e pacientes durante 109 procedimentos de quimioembolização hepática, realizados em seis serviços de hemodinâmica na cidade de Recife, Pernambuco. Os procedimentos foram realizados utilizando equipamentos de angiografia digital de diferentes fabricantes e tecnologias. A dosimetria dos pacientes foi caracterizada através das estimativas da máxima dose na pele (MDP), do produto kerma ar-área (PKA) e do kerma ar de referência (Ka,r). A MDP foi estimada a partir da utilização de filmes radiocrômicos do tipo Gafchromic XR RV3. Para avaliar o risco de efeitos estocásticos, foi estimada a dose absorvida em órgãos a partir de simulações Monte Carlo utilizando fantomas antropomórficos femininos e masculinos da serie FASH e MASH. Os resultados da dosimetria com filme radiocrômico mostraram valores da MDP variando de 180 a 5650 mGy; sendo que 40% dos pacientes apresentaram valores de dose na entrada da pele que ultrapassaram o limiar de dose para ocorrência de eritema transitório, que é de 2 Gy. O estudo das correlações entre a MDP, PKA e Ka,r mostrou que o Ka,r pode ser utilizado para avaliar a possibilidade de ocorrência de reações tissulares na pele dos pacientes submetidos a procedimentos de quimioembolização. Os resultados das simulações mostraram que alguns órgãos internos dos pacientes podem receber doses entre 500 mGy e 1 Gy. A dosimetria ocupacional foi realizada utilizando dosímetros termoluminescentes e dispositivos eletrônicos pessoais distribuídos em varias regiões do corpo dos profissionais. Os resultados mostraram que, com apenas uma quimioembolização por semana, o médico principal pode ultrapassar o limite anual de 20 mSv para o cristalino quando não são utilizados dispositivos de radioproteção como óculos ou telas de acrílico plumbíferos. O valor mais alto de equivalente de dose pessoal Hp(d) por procedimento medido no corpo do médico principal foi 5135,3 μSv no pé esquerdo. A ausência da cortina plumbífera durante a realização dos procedimentos é uma explicação para os valores altos registrados. Os valores médios de dose efetiva por procedimento para o médico principal, médico auxiliar e anestesista numa das instituições acompanhadas foram: 13 μSv, 6,1 μSv e 13,7 μSv, respectivamente. Estes resultados mostram que os níveis de exposição recebidos pelo anestesista em procedimentos de quimioembolização podem ser superiores aos do médico principal. Os resultados da dosimetria ocupacional com dosimetros eletrônicos mostraram que estes dispositivos podem ser utilizados de forma complementar na estimativa da dose ocupacional no cristalino em procedimentos de quimioembolização hepática. Nas seis instituições avaliadas observou-se uma alta variabilidade nos valores de dose no paciente e equipe médica, devido, principalmente, ao desempenho dos equipamentos, complexidade dos procedimentos, características físicas dos pacientes e experiência dos médicos.
Among interventional procedures, hepatic chemoembolization has been recognized as a complex procedure where high radiation doses to patients and medical staff are delivered. In some cases the patient has to endure several sessions to treat the same lesion, which increases even more the probability of skin injuries or stochastic effects. In Brazil, chemoembolization is widely used; however few dosimetric studies have been done so far. This study presents dosimetric results for medical staff and patients based on 109 hepatic chemoembolization procedures conducted in six hemodynamic departments in Recife, Pernambuco. The procedures were performed using digital angiography equipments from different manufacturers, using different technologies. Patient dosimetry comprised the measurement of the maximum skin dose (MSD), air kerma-area product (PKA) and reference air kerma (Ka,r). The MSD was measured using radiochromic films of type Gafchromic XR RV3. To assess the risk of stochastic effects, organ absorbed doses were calculated by Monte Carlo simulations using female and male anthropometric phantoms of the FASH and MASH series. MSDs between 180 and 5650 mGy were found based on the radiochromic film measurements. 40% of the patients monitored with radiochromic films received MSDs above the 2 Gy threshold for transient skin erythema. The findings of this study showed that the Ka,r can be used for risk estimates of tissue reactions in patients undergoing chemoembolization procedures. The Monte Carlo simulations showed that patients may receive organ doses between 500 mGy and 1 Gy. Occupational dosimetry was performed using thermoluminescent dosimeters and personal electronic devices distributed over various regions of the physician’s body. The results showed that the main operator could reach the annual limit of 20 mSv for the equivalent dose in the lens of the eyes with just one procedure per week if the radiation shields such as the ceiling suspended screen and goggles are not used. The highest values of personal dose equivalent Hp(d), measured in the body of the main operator was 5135.3 μSv in the left foot. Lack of table curtains explains the registered high values. Mean effective doses for the main operator, the auxiliary physician and the anesthesiologist in one of the institutions were 13 μSv, 6.1 μSv e 13.7 μSv, respectively. These results show that occupational doses received by the anesthesiologist in chemoembolization procedures may be higher than those received by the main operator. The results of the occupational dosimetry using electronic dosimeters showed that these devices can be used in a complementary way to estimate the occupational eye lens doses in hepatic chemoembolization procedures. High variability of radiation doses to patients and medical staff was observed among the six medical institutions, mainly because of the performance of X-ray equipments, complexity of the procedures, physical characteristics of the patients and the physician´s experience

On entend par substitut dermo-épidermique un épiderme reconstruit à la surface d'un derme équivalent composé de fibroblastes cultivés classiquement dans un biomatériau support, souvent à base de collagène poreux ou sous forme de gel. Ce support possède ses propres propriétés biomécaniques, influant sur la réponse biomécanique globale des peaux reconstruites, nous nous sommes donc intéressés à un modèle de peau reconstruite sans support, dans lequel le derme équivalent est « auto-assemblé » par les fibroblastes néosynthétisant leur propre matrice extracellulaire (MEC). Notre premier objectif a été d'optimiser et de caractériser ce modèle auto-assemblé en termes de structure, de reproductibilité et de fonctionnalité. Notre second objectif a été d'adapter aux peaux reconstruites in vitro (PR) des outils traditionnellement utilisés pour des études in vivo, pour explorer leurs propriétés biophysiques et biomécaniques. Ces outils permettent une exploration morphologique à des résolutions différentes avec l'échographie, la tomographie à cohérence optique (OCT) et la microscopie confocale à balayage et une exploration fonctionnelle des propriétés biomécaniques des PR par cutométrie. Ces données biophysiques ont ensuite été analysées par rapport aux résultats en histologie, immunohistologie et microscopie électronique à transmission. La cinétique de culture du modèle auto-assemblé sur un temps prolongé a montré la grande stabilité de l'épiderme et le remodelage continuel de la MEC avec notamment l'augmentation des fibres de collagène et d'élastine. Au temps de culture de référence sélectionné, correspondant à l'obtention de la différenciation terminale de l'épiderme, nous avons démontré la reproductibilité des épaisseurs de l'épiderme et du derme en histologie et en OCT, de la maturité de l'épiderme et de la jonction dermo-épidermique et de l'expression dermique de l'élastine colocalisée avec la fibrilline. Sur le plan fonctionnel, nous avons démontré la fonction barrière de l'épiderme via l'imperméabilité du stratum corneum et des jonctions serrées
A skin equivalent consist of a epidermis reconstructed on the top of a dermis equivalent classically composed of fibroblasts cultured into a biomaterial scaffold which is often a collagen gel or sponge. This scaffold hold its own mechanical properties, influencing the global skin equivalent biomechanical response, so we choose to develop a scaffold-free skin equivalent (SFSE), based on the ability of fibroblasts to synthezise their own extracellular matrix. Our first objective was to optimize and characterize the structure, the reproducibility and functionality of this scaffold-free model. Our second goal was to adapt biophysical and biomechanical tools classically used for in vivo evaluation to in vitro skin equivalents. Their morphology was explored with different resolutions using echography, optical coherence tomography (OCT) and laser scanning microscopy whereas biomechanical functionality was evaluate by a suction test, the cutometry. This biophysical data were compared to more classical histological, immununohistological and transmission electronic microscopy results. The long-term culture of the scaffold-free model showed the good stability of epidermis and the continuous remodelling of MEC with notably an increase of collagen and elastin fibers. We selected a reference culture time, corresponding to the complete terminal differentiation of epidermis. At this culture time, we showed the epidermis and dermis thickness reproducibity in histology and OCT, the constant epidermis and dermo-epidermal junction maturity and the dermal expression of elastin, colocalized with fibrillin. The barrier function of epidermis was also demonstrated via stratum corneum and tight junctions impermeability

The routine use of regenerative medicine products in patients requires cost-effective manufacturing processes and products that meet business and customer needs. The dermal skin substitute lCX-SKN, produced by lntercytex for the treatment of acute wounds, completed Phase I clinical trials in 2007. lCX-SKN is manufactured by seeding neonatal human dermal fibroblasts in a fibrin matrix and culturing for 49 days to form a collagen matrix synthesised by the cells. The results captured by this thesis demonstrate an integrated engineering and biological science approach to improve the current lCX-SKN process model and identify methods for process and product improvement. Measurement of the changes in the biochemical, mechanical and physical properties of lCX-SKN during the 49 day manufacturing period produced an improved four-phase process model describing cell proliferation, matrix compaction, fibrin degradation, collagen synthesis and matrix remodelling. Ultrasound was identified as a scalable form of mechanical stimulation for product improvement particularly as it does not require physical coupling to the constructs. A custom-built ultrasound device was used to investigate the effect of ultrasound on collagen synthesis and mechanical properties. A design of experiments showed that different combinations of ultrasound intensity (0.5-2.5W/cm2 ), duty cycle (5-80%) and duration (5-30minutes) affected the shear storage modulus (G') and collagen content. However, a significant effect on G' only resulted from combinations of duty cycle and duration. Further experiments to improve the properties of the construct, using 0.5W/cm2 intensity, 50% duty cycle and 14 minute duration resulted in a 73% increase in G' primarily through increased collagen deposition. The results showed that further work is required to minimise process variation through control of the input raw materials. Optimisation of the fibrin matrix and diffusion of the culture media were identified as key areas to improve manufacturing cost-effectiveness. Enhanced understanding of the physical and molecular mechanisms by which ultrasound elicits cell responses will enable further optimisation of the ultrasound process for product improvement.

A glicação não enzimática das proteínas é um fator comum para a fisiopatologia de uma série de transtornos relacionados ao envelhecimento e a doenças como o diabetes mellitus (DM). O geração dos produtos de glicação, os AGEs (do inglês: Advanced Glycation End Products) se dá através de reações de glicação da mariz extracelular (MEC) na derme e têm sido apontado como um dos fatores responsáveis pela perda de elasticidade e deficiência de cicatrização da pele. A permeação cutânea de compostos anti-AGE é uma limitação importante para eficiência terapêutica de compostos que devem atingir camadas mais profundas da pele. Modelos de pele reconstruída contendo equivalente dérmico glicado são estruturas tridimensionais geradas in vitro que mimetizam a pele humana e representam um eficiente modelo para o estudo de células e modificações provocadas na MEC no processo de envelhecimento e DM. O modelo 3D de pele reconstruída tem características metabólicas, de permeabilidade e atividade semelhantes à da pele original, potencializando seu papel nas investigações sobre permeabilidade de drogas, toxicidade, irritação, eficácia e segurança de compostos e diferenciação de queratinócitos. Uma série de compostos naturais ou sintéticos inibidores de AGEs têm sido descobertos e apresentados recentemente e podem representar inovação terapêutica no tratamento de modificações causadas pela a formação e acúmulo destes AGEs também na pele. Este estudo avaliou o desenvolvimento da pele reconstruída glicada e posteriormente, a avaliação da eficácia e toxicidade de compostos anti-glicação como aminoguanidina e carnosina em modelo de pele reconstruída glicada. Em perspectiva, este estudo contribuiu para o desenvolvimento de uma nova tecnologia in vitro, a pele reconstruída glicada, que auxiliará a compreensão da biologia da interação célula-MEC mimetizando processos fisiopatológicos importantes como o envelhecimento e o DM.
The Advanced Glycation End Products (AGEs) of proteins is a common factor to the pathophysiology of a number of disorders related to aging and diseases such as diabetes mellitus (DM). The generation of the AGEs products on skin occurs mainly through non-enzymatic glycation reactions of the dermal extracellular matrix and has been touted as one of the factors responsible for loss of elasticity and disability of skin healing. The skin permeation of compounds is an important limitation for therapeutic/cosmetic efficacy of anti-AGE compounds, which must reach the deepest layers of the skin. Reconstructed skin model containing dermal equivalent modified by in vitro glycation is able to mimic the elderly human skin and represent an efficient model for the study of cells interactions and changes in extracellular matrix induced by aging and diabetes. The 3D reconstructed skin model has metabolic characteristics, permeability and activity similar to the original skin, reinforcing its role in drug permeability of investigations toxicity, irritation, safety and efficacy evaluation of compounds and differentiation of keratinocytes. A number of natural or synthetic AGEs inhibitor compounds have been recently discovered and displayed and can represent therapeutic innovation for the treatment of changes caused by the aging of the skin. In this study we performed the development of reconstructed glycated skin model and evaluated the efficacy and toxicity of anti-glycation compounds such as aminoguanidine and carnosine. In perspective, this study has contributed to the development of a new technology in vitro, and for the understanding cell-extracellular matrix interaction during the aging of skin.

A pele é o maior órgão do corpo humano e constitui a principal defesa do organismo contra agentes físicos e químicos, sendo também fundamental para evitar a perda de água por dessecação. Formada por três camadas distintas, mas complementares, sendo as duas principais denominadas derme e epiderme, contendo diferentes tipos celulares, como fibroblastos, queratinócitos, melanócitos, células de Merkel e células de Langerhans, sendo que estas últimas desempenham um papel fundamental na hipersensibilidade de contato. Devido à importância da manutenção da pele saudável para a vida humana, existe uma crescente necessidade da elaboração de substitutos de tecidos para o tratamento de feridos e doentes, assim como, há grande demanda de pele para testes químicos das áreas farmacêutica e cosmética. Outro fator de fundamental importância para o desenvolvimento de métodos alternativos in vitro, é a pressão mundial para que estes testes substituam os modelos animais. Esta abordagem vai de encontro aos novos conceitos de substituição, redução e refinamento na utilização de animais em estudos científicos, ditando o futuro da cultura celular e bioengenharia de tecidos. Graças ao grande desenvolvimento do cultivo celular e descoberta de que as células cultivadas podem ser reagrupadas de acordo com o delineamento experimental, se torna possível à criação de equivalentes dermoepidérmicos para estudos in vitro, como por exemplo, testes de cito e fototoxicidade ou avaliação da fase inicial da reação alérgica e processos de sensibilização da pele. Neste caso, se faz necessária a obtenção de grande quantidade de células de Langerhans imaturas. As células de Langerhans (CLs) são células dendríticas imaturas localizadas na epiderme e epitélio superficial que desempenham um papel central na imunidade da pele, agindo como verdadeiras sentinelas capazes de captar antígenos de contato. Desta forma, foram testados quatro diferentes protocolos para extração e criopreservação destas células, sendo ainda analisadas as suas características morfológicas e fenotípicas. Obtivemos resultados não expressivos quanto ao isolamento, pureza e marcação positiva para CD1a no Protocolo 2 (Expansor de Pele). Os Protocolo 1A (Coleta de Sobrenadante) e 3 (Epiderme + Gradiente de Ficoll Paque) ofereceram altos níveis de células marcadas positivamente para CD1a, apresentando a mesma qualidade de marcação. No entanto, o Protocolo 3 forneceu um maior número de células viáveis, e uma maior pureza da amostra, uma vez que só utiliza a epiderme para a obtenção da suspensão de células, o que o coloca como modelo a ser seguido em posteriores experimentos. Os métodos aqui apontados como mais promissores, podem ser reproduzidos em laboratórios de cultura celular convencionais, contribuindo para aumentar a reprodutibilidade e confiabilidade de resultados experimentais relativos às CLs. Da mesma forma, avaliamos a utilização dos equivalentes de pele humana para a realização de testes in vitro de cito e fototoxicidade, os quais podem de fato reduzir a utilização de modelos animais para identificação do perfil tóxico de uma substância ou de formulações mais complexas.
The skin is the largest organ from the human body and constitutes the main protection of the organism against physical and chemical agents and it is also fundamental to avoid water loss by desiccation. Formed by three distinct stratus, yet complementary, being the two main called dermis and epidermis, containing different cell types, as fibroblasts, keratinocytes, melanocytes, Merkel cells and Langerhans cells (LCs), being these latter fundamental in the contact hypersensitivity. Due to the importance of the healthy skin maintenance to the human´s life, there is a growing need of elaboration of skin models to the treatment of injured and diseased, as well as there is a big demand of skin models to chemical tests from the pharmaceutics and cosmetology fields. Another factor of fundamental importance to the development of alternative in vitro methods is the worldwide pressure for these tests to replace animal models. This approach meets new concepts of replacement, reduction and refinement in the use of animals in scientific studies, dictating the future of cell culture and bioengineering of skin models. Thanks to the large development of cell culture and the discovery that the cultured cells can be regrouped according to the experimental delineation, the creation of skin models to in vitro studies is made possible, as for instance, tests of cytotoxicity and phototoxicity or evaluation of the initial phase from the allergic reaction and processes of skin sensitization. In this case, it is necessary the achievement of a large amount of immature Langerhans cells. The LCs are immature dendritic cells located in the epidermis and superficial epithelium that perform a central role in the skin immunity, acting as real sentinels able to collect contact antigens. Accordingly, were tested four different protocols for extraction and cryopreservation of these cells, and further analyzed its morphological and phenotypic features. We obtained no significant results in relation to the isolation, purity and CD1a positive expression in the Protocol 2. The Protocols 1A and 3 offered high levels of CD1a positively marked cells, showing the same expression levels. However, the Protocol 3 provided a bigger number of viable cells and a high purity yield, since it only uses the epidermis to obtain the single cell suspension, which places it as a model to be followed in subsequent experiments. The methods appointed here as the most promising, can be reproduced in conventional cell culture labs, contributing to increase the reproducibility and reliability of experimental results related to the LCs. In the same way, we evaluated the use of the human skin equivalents to the accomplishment of in vitro tests of cyto and phototoxicity, which can in fact reduce the use of animal models to the identification of single substances toxicity or even complex formulations.

The exposure of human skin to ultraviolet-B radiation (UVBR), causes damage in skin cells, which if not repaired may lead to cell death or skin cancer. This research aimed to understand how IGF-I, a growth factor secreted by some skin cells protects adjacent cells from the damaging effects of UVBR. Using 2-dimensional cell co-cultures and 3-dimensional skin models that closely mimic cellular interactions in native skin, the molecular mechanisms of the rescue effects were investigated. The findings may have implications in the development of treatment strategies against sunburns or photodamage.

Currently, clinical trials in patients are required by most regulatory authorities for the assessment of bioequivalence of topical products where the drug is not intended for systemic absorption. Hence there is a dire need for suitable methods for the assessment of bioavailability and bioequivalence of such products since clinical safety and efficacy studies are expensive, time-consuming and require very large numbers of patients. Except for topical corticosteroid products where the human skin blanching assay/vasoconstrictor assay has been approved by the US FDA for bioequivalence assessment of those products, no other method has been “officially” approved for use in those investigations. However, a few alternative methods such as tape stripping and microdialysis have been pursued and considered to have the potential for use in ioequivalence/bioavailability studies. The human skin blanching assay was used to assess the bioequivalence of commercially available topical products containing 0.05% clobetasol propionate. Both visual and chromameter data were obtained and a commercially available topical corticosteroid product, Dermovate® cream was used as both the “Test” and the “Reference” product. The results indicated that both visual and chromametric assessments were comparable to each other and that either could be used for the assessment of the bioequivalence of topical products containing clobetasol propionate. The screening procedure was optimized to identify potential “detectors” for inclusion in the bioequivalence studies. This resulted in fewer subjects being required in a bioequivalence pivotal study, still having the necessary power to confirm bioequivalence using the human skin blanching assay. Another objective of this research was to re-visit tape stripping and other possible alternative methods such as dermal microdialysis and to optimize these procedures for bioequivalence assessment of topical formulations where the drug is not intended for systemic absorption. In the past few decades, tape stripping has been used to investigate bioavailability/bioequivalence of various topical formulations. This technique involves the removal of the stratum corneum to assess drug penetration through the skin. A draft FDA guidance for tape stripping was initially published but was subsequently withdrawn due to high variability and poor reproducibility. This research project used an optimized tape stripping procedure to determine bioavailability and establish bioequivalence between three commercially available formulations containing 0.05 % m/m clobetasol propionate. Furthermore, tape stripping was validated by undertaking a study to assess the bioequivalence of a 0.05% topical cream formulation (Dermovate® cream) using the same cream as both the “Test” and “Reference” product, in which bioequivalence was confirmed. The findings highlight the potential of tape stripping as an alternative method for the assessment of bioequivalence of clobetasol propionate formulations and may possibly be extended for use in other topical products. Microdialysis is another useful technique that can assess the penetration of topically applied substances which diffuses through the stratum corneum and into the dermis. Microdialysis has previously been successfully used for in vivo bioavailability and bioequivalence assessments of topical formulations. However, the drugs which were under investigation were all hydrophilic in nature. A major problem with the use of microdialysis for the assessment of lipophilic substances is the binding/adherence of the substance to the membrane and other components of the microdialysis system. As a result, this necessitates the development of a microdialysis system which can be used to assess lipophilic drugs. Intralipid® 20% was investigated and successfully utilized as a perfusate to recover a lipophilic topical corticosteroid, clobetasol propionate, in microdialysis studies. Hence, the bioavailability of clobetasol propionate from an extemporaneous preparation was determined in healthy human volunteers using microdialysis. These findings indicate that in vivo microdialysis can be used to assess lipophilic drug penetration through the skin. A novel approach to investigate drug release from topical formulations containing 0.05% clobetasol propionate using in vitro microdialysis was also undertaken. The in vitro findings were found to be in agreement with the results obtained using tape stripping to assess bioequivalence of the same commercially available products, namely Dermovate® cream, Dovate® Cream and Dermovate® ointment. These results indicate the potential to correlate in vitro with in vivo data for bioequivalence assessment of such topical dosage forms.

Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares.
This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.

博士
國立臺灣大學
醫學工程學研究所
94
Abstract Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. First aim of this study was prepared the feeder gel with optimum fibroblast density and optimum conditioned medium that promoted keratinocyte proliferation without further differentiation for skin equivalent tissue engineering. The optimum cell density in collagen feeder gel for optimum selected medium preparation will be determined by checking the level of keratinocyte growth factor (KGF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) in conventional medium. The results showed that the cell density of 1x105 cells/gel in the feeder gel is better to produce optimum selected medium. The conditioned medium is prepared by mixing the optimum selected medium and MCDB 153 medium together in different ratios for keratinocyte growth. The keratinocyte viability will be measured by MTT assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed the better medium for the keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to co-culture with keratinocyte is also determined as 1x105 cells/gel. Cytokeratin-10 and TUNEL stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocyte should not be cultured in post-confluent condition due to toward undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned medium could effectively promote proliferation, inhibit apoptosis and prevent from differentiation. The combination of conditioned media and feeder gel to culture keratinocyte without external supplements can provide an inexpensive way for keratinocyte proliferation and construct an environment for real-time communication between the two cells. The results conclude that keratinocyte cultivation in feeder gel with modified medium should be feasible in the production of high quality keratinocyte for skin equivalents preparation. The second approach of preparing skin equivalents is regulation of proliferated and differentiated capacity. We investigated Ca2+ effects on the proliferation and differentiation using the primary keratinocytes model. Keratinocytes were incubated in DMEM (containing 1.2 mM Ca2+ concentration) or DK11 medium (containing 0.4 mM Ca2+ concentration) or K medium (containing 0.03 mM Ca2+ concentration). Cell viability was assessed with the MTT assay. Crystal violet assay was evaluated the proliferation rate and colony formation size of keratinocyte. Real-time PCR used to determine the terminal differentiated keratinocyte which expressed Caspase-14. Proliferation assays and real–time PCR were correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes. High Ca2+ concentration was inhibited the cell viability and proliferation rate of keratinocyte. Ca2+ also increased caspases-14 expression, and inhibited cell viability, and cell colony forming efficiency. These results are consistent with Ca2+ induction of the keratinocyte differentiation. Thus, the overall Ca2+ actions connote protective functions for the epidermis that appear to include the triggering or acceleration of the differentiation. At last, we designed a novel culture system included a self-designed 3-D collagen scaffold with different pore size and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-beta1), in the cell-cell and cell-matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air-liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10ng/ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10ng/ml of EGF addition in the air-lifted stage resulted in uneven cell-matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-beta1).

碩士
高雄醫學大學
生理及分子醫學研究所
98
Many methodologies such as enzymatic (mushroom tyrosinase) and monolayer cell culture assays are routinely used to screen novel de-pigmentary agents. These models have disadvantages in terms of physiological and economic relevance. Besides, due to the promotion of animal protection organizations, cosmetic experiments on animals had been forbidden by the European Union since March, 2009. At the present time, there are no models of bioengineered skin that completely replicate the physiological conditions of uninjured skin. A natural product, (-)-N-formylanonaine, was isolated from the leaves of Michelia alba D.C. (Magnolianceae). It was found to inhibit mushroom tyrosinase and was further tested on human epidermal melanocytes (MCs) to be with tyrosinase and melanin reducing activities in human epidermal MCs without apparent cytotoxicity to human cells, superior to the known tyrosinase inhibitors, such as kojic acid and 1-phenyl-2-thiourea (PTU). We fabricated an appropriate scaffold with hyaluronic acid, collagen, and gelatin for three kinds of cells, keratinocytes (KCs), MCs and fibroblasts (FBs) to co-culture on and tested the pore size and swelling ratios of this scaffold. As co-culturing three kinds of cells, the morphology was checked by paraffin section and staining to manufacture the de-pigmentation platform. The de-pigmentation effect of (-)-N-formylanonaine will be tested on our novel established platform of 3D human skin equivalent.

Dissertação de mestrado em Genética Molecular
Skin is the major organ of the human body and its function is to protect the organism from the environment, constituting a very “impermeable” hydrophobic barrier. This is mainly accomplished by a very specialized avascular external layer – the epidermis followed by the dermis. The epidermis is subdivided in several layers representing different stages of keratinocyte differentiation that is accomplished by their continuous proliferation supported by the dermis, keratinization and death. 2D cell cultures, used worldwide, fail to reproduce the very relevant complexity of in vivo skin microenvironment. Thus, the development of in vitro 3D models that mimic more closely the natural skin architecture is of great relevance. They have become a very important tool as skin research models since the use of animals has been restricted due to ethical issues. The purpose of our work is to develop and validate a noncommercial skin equivalent in our laboratory. In order to accomplish the proposed aim, collagen was extracted from chicken and pig skins using an acid/alkali/enzymatic conjugation method and their performances were compared with commercial calf skin and rat tail collagens. All collagens were characterized by electrophoresis, protein and collagen quantification. After polymerization, the collagens were compared in terms of fibroblast proliferation (BJ-5ta cell line). Optimization experiments were performed. The best pore size of the membrane inserts was defined as 1.0 μm in order to assure the best conditions for fibroblast proliferation. The best fibroblast initial seeding density in the collagen matrices was defined as 6x105 cells/ml. The best collagen concentration for the models development was 3 mg/ml. To validate them, the differentiation of keratinocytes (HaCaT cell line) was evaluated by HE staining and by immunohistochemistry for the expression of keratins 10 and 14 (K10/14 – keratinocyte differentiation markers). For rat and calf collagen matrices, 3 to 5 layers of epidermis were observed and K10 expression was essentially present in the supra-basal layers. K14 had a more uniform expression across the keratinocyte layers, however is less expressed in the outermost layers. Models developed with chicken and pig collagen matrices presented only 1 to 2 discontinuous and disorganized layers. Thus, the rat tail and calf skin collagens proved to be the best choices for the development of a skin equivalent. A better optimization of the collagen extraction protocol is required, and the addition of a biopolymer, such as elastin, to our skin equivalents would also be interesting. Due to the numerous advantages that 3D models present, we believe it is important to continue this work.
A pele é o maior órgão do corpo humano e tem por função proteger o nosso organismo do meio ambiente, constituindo uma barreira hidrofóbica muito “impermeável”. Barreira essa composta por uma camada externa avascular muito especializada - a epiderme seguida da derme. A epiderme está subdividida em diversas camadas que representam diferentes estágios da diferenciação dos queratinócitos que é adquirida pela sua proliferação contínua suportada pela derme, queratinização e morte. As culturas celulares 2D, usadas mundialmente, falham na reprodução da complexidade da pele in vivo. Assim, o desenvolvimento de modelos 3D in vitro que imitam a arquitetura do tecido natural é de grande importância. Tornaram-se pois uma ferramenta importante como modelos de investigação, já que o uso de animais foi restringido devido a questões éticas. O objetivo do nosso trabalho é desenvolver e validar um modelo 3D de pele não comercial no nosso laboratório. De forma a cumprir o objetivo proposto, foi extraído colagénio da pele de galinha e porco usando a conjugação de métodos ácido/básico/enzimático e as suas performances foram comparadas com colagénios comerciais de pele de vitela e cauda de ratazana. Todos os colagénios foram caracterizados através de eletroforese, quantificação de proteína e colagénio. Após polimerização, foram comparados relativamente à proliferação dos fibroblastos (linha celular BJ-5ta). Foram realizadas experiências de otimização. O melhor tamanho de poro do insert foi definido como 1.0 μm de forma a assegurar as melhores condições para a proliferação dos fibroblastos. A melhor densidade celular de fibroblastos a plaquear nas matrizes de colagénio foi definida como 6x105 cél/ml. A concentração do colagénio mais adequada para desenvolver os modelos é 3 mg/ml. Para validá-los, a diferenciação dos queratinócitos (linha celular HaCaT) foi avaliada através da marcação com HE e por imunohistoquimica para a expressão da queratina 10 e 14 (K10/14 – marcadores de diferenciação de queratinócitos). Para matrizes de colagénio de ratazana e vitela, 3 a 5 camadas da epiderme foram observadas onde a expressão da K10 estava essencialmente presente nas camadas supra basais. A K14 teve uma expressão uniforme através das camadas de queratinócitos, contudo está em menor quantidade nas camadas mais externas. Modelos desenvolvidos com matrizes de galinha e porco apresentam apenas 1 a 2 camadas descontínuas e desorganizadas. Assim, os colagénios de cauda de ratazana e pele de vitela provaram ser a melhor escolha para o desenvolvimento de um modelo 3D de pele. Uma melhor otimização do protocolo de extração de colagénio é necessária e a adição de um biopolímero, como a elastina, ao nosso modelo poderá ser interessante. Devido às inúmeras vantagens que os modelos 3D apresentam, acreditamos que é importante continuar este trabalho.

Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2019
A indústria farmacêutica e a indústria cosmética estão entre as que mais investem em pesquisa e desenvolvimento, o que leva ao constante aparecimento de novos fármacos e cosméticos. Todos estes produtos, antes de serem introduzidos no mercado, necessitam de passar por uma avaliação criteriosa relativamente à sua eficácia e toxicidade, pois podem provocar efeitos nocivos no organismo humano. Anteriormente, estas avaliações eram realizadas recorrendo à experimentação animal, no entanto com o passar dos anos desenvolveu-se uma consciencialização relativamente ao uso de animais para este fim, o que levou a que restrições legais fossem criadas (em diversos países de todo o mundo) e por conseguinte a uma utilização muito limitada desta prática. Em substituição deste método começaram a desenvolver-se métodos alternativos que permitissem a reprodutibilidade, quantidade e predictibilidade das avaliações. No que diz respeito aos produtos com aplicação tópica, não só a sua eficácia e toxicidade têm de ser testadas mas também a sua eficiência de penetração. A pele sendo a interface entre o organismo e o seu ambiente externo, constitui uma barreira de proteção contra todas as interferências exteriores e é dividida fundamentalmente em 3 camadas: a hipoderme, a derme e a epiderme, esta última a principal responsável pela função de barreira apresentada por este órgão. A epiderme é a camada mais exterior da pele e é constituída por queratinócitos os quais sofrem um processo continuo de diferenciação originando um tecido estratificado. Para que o produto aplicado topicamente penetre a pele e tenha o efeito desejado, esta camada tem de ser transposta. Hoje em dia já existem diversos modelos in vitro de pele humana para testar estes produtos, alguns contendo apenas a epiderme, outros apenas a derme e outros ainda contendo ambas as camadas (epiderme e derme). Todos estes modelos se assemelham morfologicamente e bioquimicamente à pele in vivo, no entanto ainda não exibem uma adequada propriedade de barreira, evidenciando maiores permeabilidades que a pele nativa. Esta disparidade parece estar relacionada com a camada apical da epiderme - stratum corneum - que integra células mortas e achatadas envoltas numa matriz lipídica, onde se observam variações tanto no perfil lipídico como na sua estruturação. Dado verificar-se uma maior permeabilidade nos tecidos in vitro do que nos tecidos in vivo, sempre que um produto é submetido a avaliações toxicológicas e de eficiência de penetração, extrapolações têm de ser realizadas para fases clínicas o que torna o processo pouco preciso. Surge assim a necessidade de descobrir quais os parâmetros que influenciam a formação da barreira da pele durante a cultura de equivalentes epidérmicos humanos in vitro, de forma a gerar um modelo que mais se assemelhe com a epiderme humana nativa, tanto em termos de morfologia como de propriedade barreira. Assim, este projeto pretendeu produzir equivalentes epidérmicos in vitro sob quatro condições distintas, resultantes da alteração independente de quatro fatores relativamente às condições standard de cultura, de forma a avaliar o efeito dessas alterações nos tecidos produzidos e nas suas propriedades de barreira. Para tal, queratinócitos humanos primários isolados foram cultivados em monocamada, cumprindo as regras básicas de cultura celular, e quando estes se encontraram em 4ª passagem foram semeados em insertos com filtro de policarbonato para reconstrução de epidermes in vitro, as quais foram obtidas ao fim de onze dias de interface ar-liquido. Após obtenção dos tecidos epidérmicos, foram realizadas análises histológicas de forma a avaliar as características morfológicas e ensaios de permeabilidade a três fármacos de diferentes polaridades (cafeína, hidrocortisona e testosterona) em células de difusão de Franz para avaliar as propriedades de barreira. Os resultados das análises histológicas foram avaliados por observação microscópica enquanto que os resultados dos ensaios de permeabilidade foram avaliados por leitura espectrofotométrica, a partir da qual se obteve a quantidade de fármaco permeada pelo tecido ao longo do ensaio, que permitiu calcular o seu fluxo. As quatro alterações testadas, que foram implementadas durante a fase de exposição da cultura à interface ar-líquido, foram: (1) o aumento da disponibilidade de factores de autocrinos e homocrinos; (2) o aumento da disponibilidade de nutrientes via suplementação do meio de cultura com FBS; (3) a diminuição da temperatura de incubação; (4) o aumento da disponibilidade de oxigénio. A alteração relativa aos factores autocrinos e homocrinos resultou da dedução de que as células dispunham de pouco tempo em contacto com os factores de sinalização produzidos por elas. Como tal, primeiramente variou-se o período de renovação dos meios de modo a avaliar qual o período ótimo entre renovações consecutivas e posteriormente cultivou-se tecido epidérmico com um período entre renovações estendido de forma a aumentar a sua exposição aos factores. A alteração relativa à suplementação do meio de cultura com FBS adveio de resultados contraditórios descritos na bibliografia, em que certos estudos descrevem o FBS como sendo benéfico para o desenvolvimento da cultura, enquanto que outros descrevem-no como prejudicial. Como tal, realizou-se uma cultura em que este suplemento foi adicionado, numa concentração de 10%, ao meio de cultura de forma a avaliar os seus efeitos. A alteração da temperatura derivou da constatação de que os queratinócitos que integram a epiderme crescem naturalmente a 32 ̊C, temperatura registada à superfície da pele, contrariamente à maioria das células que crescem à temperatura fisiológica humana (37 ̊C). Como tal, inicialmente avaliou-se o impacto da redução da temperatura, para 32 ̊C, no período de desenvolvimento dos queratinócitos e posteriormente desenvolveu-se uma cultura sob essas mesmas condições de forma a avaliar o efeito da temperatura natural na formação da epiderme. A alteração da disponibilidade de oxigénio surgiu de um resultado proveniente de um estudo realizado anteriormente no laboratório, o qual indiciava que as células estavam sob condições limitantes de oxigénio. Como tal, aumentou-se a superfície de contacto entre o meio e a atmosfera de forma a permitir a solubilização de mais oxigénio no meio que nutre as células. Os resultados obtidos em cada alteração foram comparados com os resultados obtidos em equivalentes epidérmicos humanos produzidos in vitro segundo o protocolo adoptado como standard. Verificou-se que à excepção da cultura suplementada com FBS, todas as alterações geraram tecidos com a propriedade de barreira melhorada, ou seja, com uma permeabilidade mais reduzida. Quando a exposição aos factores autocrinos e homocrinos foi aumentada, assim como quando a temperatura de incubação foi diminuída, foram produzidos tecidos bem diferenciados e com uma espessura média superior à apresentada pelos tecidos epidérmicos cultivados em condições standard. Consequentemente, a permeabilidade destes tecidos à cafeina e à testosterona melhorou significativamente (p < 0.05), contudo não alterou a permeabilidade à hidrocortisona (p > 0.05). Quando se aumentou a disponibilidade de oxigénio produziram-se tecidos bem diferenciados, mas com uma espessura média bastante próxima à apresentada pelos tecidos epidérmicos cultivados em condições standard. Consequentemente, a permeabilidade destes tecidos à cafeina e à hidrocortisona não mostraram alterações significativas (p > 0.05), contudo apresentaram melhorias significativas na permeabilidade à testosterona (p < 0.05). No que diz respeito ao aumento da disponibilidade de nutrientes via suplementação do meio de cultura com FBS, constatou-se que o tecido produzido era pouco diferenciado e estratificado além de pouco compacto. Isto refletiu-se na sua permeabilidade, a qual piorou significativamente relativamente à cafeina e à hidrocortisona (p < 0.05), apesar de não ter alterado significativamente a permeabilidade à testosterona (p > 0.05). Estes resultados parecem promissores para o desenvolvimento de um protocolo otimizado, porém outros testes complementares à análise histológica e aos ensaios de permeabilidade deveriam ser realizados para uma melhor compreensão do impacto das alterações nos tecidos produzidos. Assim, este trabalho contribui para a percepção de como estes parâmetros podem ser variados para produzir um equivalente epidérmico humano in vitro mais semelhante à epiderme humana nativa. No entanto, é claro que mais estudos são necessários, pois os valores de permeabilidade ainda estão longe dos observados in vivo.
The skin is the interface between the organism and the external environment, and its outermost layer - epidermis - is the major responsible for the barrier function of this organ. As such, epidermis is a crucial obstacle to be overcome by any substance intended to penetrate the skin. Cosmetic and pharmaceutical products for topical administration, prior to their placement in the market, are evaluated as to their safety and efficacy as well as penetration efficiency. Previously these tests were performed using animal experimentation. However due to legal demands, this use was discontinued and replaced by in vitro human skin equivalents. Although the in vitro epidermal models currently produced resemble the morphological and biochemical characteristics of the human epidermis, their barrier function is still reduced when compared to in vivo skin, evidencing higher permeability values which require extrapolation to clinical stages. Thus, it is necessary to discover the parameters that influence the skin barrier formation during epidermal tissue culture, in order to generate an in vitro model that better resembles the in vivo human epidermis. This work aimed to produce human epidermal equivalents under four different conditions in order to evaluate the effect of these alterations on the cultured tissue barrier function. The four studied conditions were: increased availability of cellular growth factors; increased availability of nutrients via FBS supplementation; lowered incubation temperature; increased availability of oxygen. Subsequently their morphology was assessed by histological analysis and their permeability by diffusion studies in Franz diffusion cells using three model drugs with different polarities (caffeine, hydrocortisone and testosterone). Out of the four conditions tested, the ones related to cellular growth factors, temperature and oxygen have shown to produce well stratified and differentiated tissues containing all the expected strata. Additionally, these tissues have evidenced statistically significant improvements in reducing permeability. The same was not true for FBS medium supplementation which produced tissues that were poorly consistent and differentiated without a well-defined stratification. These tissues evidenced statistically significant worsening in reducing permeability. This work provides a preliminary insight into how these parameters can be adjusted to produce an in vitro human epidermal equivalent that better resembles the in vivo human epidermis. Although it is clear that further study is needed as permeability values are still far from those observed in vivo.