Presencia y Expresión del Receptor ACE2 (Target de SARS-CoV-2) en Tejidos Humanos y Cavidad Oral. Posibles Rutas de Infección en Órganos Orales / Presence and Expression of ACE2 Receptor (Target of SARS-CoV-2) in Human Tissues and Oral Cavity. Possible Routes Infection in Oral Organs

RESUMEN: Un nuevo coronavirus (SARS-CoV-2) ha sido reconocido como el agente etiológico de una misteriosa neumonía originada en Wuhan, China. La OMS ha nombrado a la nueva enfermedad como COVID-19 y, además, la ha declarado pandemia. Taxonómicamente, SARS-CoV-2 pertenece al género de los betacoronavirus junto con SARS-CoV y MERS-CoV. SARS-CoV-2 utiliza la enzima convertidora de la angiotensina 2 (ACE2) como el receptor objetivo para el ingreso en una célula huésped. La expresión de ACE2 en células de tejidos humanos podría indicar un potencial riesgo de reconocimiento por parte del virus y, por ende, ser susceptibles a la infección. Mediante algunas técnicas de laboratorio y de bioinformática, se ha visto una alta presencia de ACE2 en células epiteliales alveolares tipo II de pulmón y en enterocitos del intestino delgado. En la cavidad oral, se ha podido identificar la presencia de ACE2, principalmente, en células epiteliale s de glándulas salivales y células epiteliales de la lengua. Además, se ha reportado la manifestación de algunos síntomas, como sequedad bucal y ambligeustia, los que podrían estar relacionadas con una infección de SARS-CoV-2 en estos órganos. Sin embargo, son necesarios mayores estudios que evidencien esta situación.

ABSTRACT: A novel coronavirus (SARS-CoV-2) has been recognized as a etiologic agent of a mysterious pneumonia originating in Wuhan, China. WHO has named the new disease as COVID-19 and, in addition, has declared it a pandemic. Taxonomically, SARS-CoV-2 belongs to the betacoronavirus genus along with SARS-CoV and MERS-CoV. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the target receptor for entry into a host cell. The expression of ACE2 in cells of human tissues could indicate a potential risk of recognition by the virus and, therefore, be susceptible to infection. Through some laboratory and bioinformatics techniques, high presence of ACE2 has been seen in type II alveolar epithelial cells of the lung and enterocytes of the small intestine. In oral cavity, mainly presence of ACE2 has been identified in epithelial cells of salivary glands and epithelial cells of tongue. In addition, manifestation of some symptoms, such as dry mouth and amblygeustia, have been reported, which could be related to a SARS-CoV-2 infection in these organs. However, further studies are needed to prove this situation.

Toxicity evaluation of water extract of tissue-cultured Taraxacum formosanum by acute, subacute administration, and Ames test

BACKGROUND: Taraxacum species (commonly known as dandelion) used as herbal medicine have been reported to exhibit an antiproliferative effect on hepatoma cells and antitumor activity in non-small-cell lung cancer cells. Although several investigations have demonstrated the safety of Taraxacum officinale, the safety of tissue-cultured plants of T. formosanum has not been assessed so far. Therefore, the present study examines the safety of the water extract of the entire plant of tissue cultured T. formosanum based on acute and subacute toxicity tests in rats, as well as the Ames tests. RESULTS: No death or toxicity symptoms were observed in the acute and subacute tests. The results of the acute test revealed that the LD50 (50% of lethal dose) value of the T. formosanum water extract for rats exceeded 5 g/kg bw. No abnormal changes in the body weight, weekly food consumption, organ weight, or hematological, biochemical, and morphological parameters were observed in the subacute toxicity test. Thus, the no observed adverse effect level (NOAEL) of T. formosanum water extract was estimated to be higher than 2.0 g/kg. Finally, the results of the Ames test revealed that T. formosanum water extract was not genotoxic at any tested concentration to any of five Salmonella strains. CONCLUSIONS: The water extract of tissue-cultured T. formosanum was non-toxic to rats in acute and subacute tests and exhibited no genotoxicity to five Salmonella strains.

Follicle Viability after Vitrification of Bovine Ovarian Tissue / Viabilidade folicular após a vitrificação de tecido ovariano bovino

Abstract Purpose The present study aimed to evaluate the impact of vitrification on the viability of follicles using a three-dimensional (3D) in vitro culture. Methods Bovine ovarian tissue samples (n = 5) obtained from slaughterhouses were utilized. The cortex was cut into small fragments of 2 x 3 x 0.5 mm using a tissue slicer. From these fragments, secondary follicles were first isolated by mechanical and enzymatic methods, then encapsulated in alginate gel and individually cultured for 20 days. Additional fragments of the same ovarian tissue were vitrified in a solution containing 25% glycerol and 25% ethylene glycol. After warming, the follicles underwent the same follicular isolation process that was performed for the fresh follicles. Results A total of 61 follicles were isolated, 51 from fresh ovarian tissue, and 10 from vitrified tissue. After the culture, the vitrified and fresh follicles showed 20% and 43.1% survival rates respectively (p = 0.290),with no significant differences. At the end of the culture, therewere no significant differences in follicular diameter between the vitrified (422.93 ± 85.05 μm) and fresh (412.99 ± 102.55 μm) groups (p = 0.725). Fresh follicles showed higher mean rate of antrum formation when compared with vitrified follicles (47.1% and 20.0% respectively), but without significant difference (p = 0.167). Conclusions The follicles were able to develop, grow and form antrum in the 3D system after vitrification, despite the lower results obtained with the fresh tissue.

Resumo Objetivo O presente estudo teve como objetivo avaliar o impacto da vitrificação na viabilidade dos folículos utilizando a cultura in vitro tridimensional (3D). Métodos Foi utilizado tecido ovariano bovino (n = 5) obtido de abatedouros. O córtex foi cortado em pequenos fragmentos de 2 x 3 x 0,5 mm, utilizando o tissue slicer e a partir destes fragmentos foram isolados folículos secundários por meio de método enzimático e mecânico, encapsulados em gel de alginato e cultivados individualmente durante 20 dias. Outros fragmentos do mesmo tecido ovariano foram vitrificados em solução contendo 25% de glicerol e 25% de etilenoglicol. Após aquecimento, os folículos passaram pelo mesmo processo de isolamento folicular realizado a fresco. Resultados Foram isolados 61 folículos, sendo 51 originários de tecido ovariano a fresco, e 10 de tecido vitrificado. Após a cultura, os folículos vitrificados apresentaram taxa de sobrevida de 20%, e o grupo a fresco apresentou taxa de 43,1% (p = 0,290). O diâmetro folicular ao final da cultura também não apresentou diferença significativa entre o grupo vitrificado (422,93 ± 85,05 μm) e a fresco (412,99 ± 102,55 μm) (p = 0,725). Os folículos a fresco apresentarammaior taxa média de formação de antro do que os folículos vitrificados (47,1% e 20,0%, respectivamente), mas sem diferença significativa (p = 0,167). Conclusões Os folículos foram capazes de se desenvolver, crescer e formar antro em sistema 3D após a vitrificação.

Reseña actualizada de ingeniería tisular en disciplinas odontológicas / An updated review of tissue engineering in dental disciplines

La ingeniería tisular (IT) ha sido considerada un campo interdisciplinario, aplicando los principios de ciencias de ingeniería y biología para el desarrollo de sustitutos biológicos que restauran, mantienen o mejoran la función tisular. Se basa en el entendimiento de losprincipios del crecimiento tisular, y su aplicación, para producir reemplazo de tejidos para uso clínico. Se consideran determinantes para el éxito de la IT las Stem Cells (células madre), morfógenos y las “scaffolds” (constructos). Poner en práctica dicha disciplina requiere elempleo de estrategias terapéuticas biológicas que apuntan a reemplazar, reparar, mantener y/o mejorar la función tisular. El objetivo de este trabajo, ha sido realizar una actualización sobre nuevos conocimientos emergentes de las últimas publicaciones científicas realizadas en el ámbito de esta nueva disciplina. Para ello se realizó una exhaustiva búsqueda de información en la base de datos de Pubmed. Actualmente, la IT concentra sus esfuerzos en lograr la regeneración de tejidos dentarios y para dentarios, así como en lograr la obtención de una pieza dental completa. Su avance clínico ha sido notable. Se han reportado artículos publicados que ya evidencian su aplicación en periodoncia, cirugía, implantología, rehabilitación oral y endodoncia. Si bien, estrategias de la IT ya se utilizan clínicamente en odontología, su rápido desarrollo se convierte entonces en un gran desafío e incógnita tanto para quienes ejercen la profesión en la actualidad, como para aquellos que se encuentran en plena formación académica. Tomar conocimiento de logros y avances resulta entonces fundamental, ya que podría convertirse en un futuro próximo, en una herramienta de uso habitual.

Tissue engineering (TE) is considered an interdisciplinary field, and applies principles of engineering and biology to develop biologicalsubstitutes that restore, maintain, or improve tissue function. It is based on the application of the principles of tissue growth to producetissue replacement for clinical use. Stem cells, morphogens, and scaffolds are determinant to the success of TE. Implementation of TErequires the use of biological therapeutic strategies aiming to replace, repair, maintain, and/or improve tissue function. The objective ofthe present work was to perform an update of new knowledge presented in recent scientific publications in this field. For this purpose, weconducted an extensive search for information on Pubmed. At present, TE focuses on achieving regeneration of dental and para-dentaltissues, as well as on obtaining a whole tooth. There have been outstanding clinical advances in this field. There are reports showingsuccessful application of TE in periodontics, surgery, implantology, oral rehabilitation and endodontics. Although TE strategies arealready used in dentistry, their rapid development poses a great challenge both to current practitioners and to those who are in the midst oftheir academic training. Gaining an awareness of the achievements and advances in TE is therefore essential, since it could become widelyapplied in the near future.

In vitro culture of immature embryos of Cinnamomum tamala Nees.— The role of different factors.

Seed characteristics and in vitro culture of C. tamala embryos were studied. Embryos desiccated below 50% (fresh weight) exhibited poor morphogenetic response in vitro and confirmed the recalcitrant nature of seeds. The immature embryos of various developmental ages (4-16 week after flowering, WAF) were cultured on different strengths of MS medium. Morphogenesis responses were recorded after 10 days of culture. The best culture responses were achieved from the immature embryos of 12 WAF on MS medium with sucrose (3%, w/v), polyvinyl pyrollidone (100 mg L-1) and benzyl adenine (12 µM). Under optimum condition ~60% explants responded; and ~7.3 shoots buds developed per explants after 35 days of culture initiation. The shoot buds could be converted into micro-shoots on MS medium with sucrose (3%) and kinetin (3 µM). About 5.3 micro-shoots/shoot buds sprouted per sub-culture. The micro-shoots were rooted by maintaining them on MS medium with α-naphthalene acetic acid (3 µM) where within 6-8 wk of culture ~8-10 roots developed. The rooted plantlets were acclimatized in vitro before they were transferred to community potting mix and maintained in the poly-shade ca 75% shading. The transplants registered ~70% survival after two months of transfer.

Reguladores de crescimento vegetal no cultivo in vitro de Mentha x Piperita L / Plant growth regulators on in vitro culture of Mentha x piperita L

A micropropagação é uma técnica muitas vezes indicada para a multiplicação em larga escala de plantas com propriedades medicinais. Dentre elas, destaca-se a hortelã-pimenta (Mentha x Piperita L.), cujo óleo essencial é utilizado no tratamento de transtornos digestivos e respiratórios. Para otimizar o protocolo de micropropagação dessa espécie são necessários estudos, principalmente quanto à suplementação do meio de cultura para garantir a produção massal in vitro e posterior extração do óleo essencial. Nesse contexto, objetivou-se avaliar o efeito de concentrações e combinações de reguladores de crescimento vegetal na morfogênese in vitro de hortelã-pimenta. Segmentos nodais provenientes de plântulas estabelecidas in vitro foram utilizados como fonte de explante e inoculados em meio de cultura MS suplementado com 0; 2,0 e 4,0 mg L-1 de BAP (6-benzilaminopurina), 0; 0,5 e 1,5 mg L-1 de ANA (ácido naftaleno-acético) e 0; 0,5 e 1,0 mg L-1 de GA3 (ácido giberélico). O delineamento experimental adotado foi inteiramente casualizado, com os tratamentos distribuídos em esquema fatorial 3x3x3 com oito repetições. Concluiu-se que o BAP favoreceu a sobrevivência de segmentos nodais de M. x Piperita inoculados in vitro e, quando combinado ao GA3, promoveu a brotação dos explantes. Essas características, no entanto, não foram estimuladas pela adição de ANA ao meio de cultura. Conclui-se que após a multiplicação dos brotos in vitro estes devem ser transferidos para meio sem reguladores para seu desenvolvimento. Apesar dos efeitos benéficos do BAP na organogênese de M. x Piperita, elevadas concentrações deste regulador de crescimento vegetal promoveram a formação de calos.

Micropropagation is a technique used for the large-scale production of medicinal plants. Among them, peppermint (Mentha x piperita L.) may be mentioned because of the pharmacological importance of its essential oil, which is used on the treatment of digestive and respiratory disorders. Studies are needed in order to optimize the micropropagation protocol of this species, especially concerning the culture medium, to ensure the in vitro mass clonal production and to enable the future extraction of the plant essential oil. Therefore, this work aimed to evaluate the effects of concentrations and combinations of different plant growth regulators on the in vitro morphogenesis of peppermint. Nodal segments from plantlets already established in vitro were used as explants and inoculated on MS medium supplemented with 0, 2.0 and 4.0 mg L-1 of BAP (6-benzylaminopurine), 0, 0.5 and 1.5 mg L-1 of NAA (naphthalene acetic acid) and 0; 0.5 and 1.0 mg L-1 of GA3 (gibberellic acid). The experiment was in a completely randomized design, set up as a 3x3x3 factorial design with eight replicates. We concluded that BAP increases the survival rate of in vitro inoculated nodal segments of M. x piperita. In addition, its combination with GA3 stimulates explants shooting. Those aspects, however, are not promoted by the addition of NAA into the culture medium. Also, the results indicate that, after in vitro multiplication, peppermint shoots must be transferred to another medium without plant growth regulators for shoot elongation. High concentrations of BAP promote calli induction, despite having beneficial effects on the organogenesis of M. piperita.

Variation in bioactive principles of Artemisia amygdalina Decne. in wild and tissue culture regenerants

Wild and tissue culture raised regenerants of Artemisia amygdalina, a critically endangered and endemic plant of Kashmir and North West Frontier Provinces of Pakistan were screened for the amount of bioactive principles and in particular antimalarial compound artemesinin. Phytochemical screening of extracts revealed the presence of terpenes, alkaloids, phenolics, tannins [polyphenolics], cardiac glycosides and steroids in wild [aerial, inflorescence] and tissue culture regenerants [in vitro grown plant, callus and green house acclimatized plants]. HPLC of Artemisia amygdalina revealed the presence of artemesinin in petroleum ether extracts of wild aerial part, tissue culture raised plant and green house acclimatized plants. Acetonitrile and water in 70:30 ratios at flow rate of 1ml/min was standardised as mobile phase. Retention time for standard chromatogram was 6.7. Wild inflorescences and callus does not produce artemesinin. This is the first report of phytochemical screening and artemesinin estimation of wild and tissue culture raised regenerants of Artemisia amygdalina

Aplicações da cultura de tecidos em plantas medicinais / Applications of tissue culture in medicinal plants

Esta revisão tem por objetivo levantar dados de literatura sobre o histórico e a situação atual das técnicas de cultura de tecidos em plantas medicinais. Para tanto, foi realizada uma revisão de publicações do período de 1976 a 2009. A cultura de tecidos é muito utilizada em pesquisas envolvendo plantas medicinais, com destaque para a técnica de micropropagação. A aplicação das técnicas de cultura de tecidos em plantas medicinais tem como perspectivas a obtenção de germoplasma competitivo e adaptado a diversos métodos de cultivo, escolha de novas espécies que servirão como fonte de compostos biologicamente ativos e aprimoramento da produção de fitofármacos, a fim de assegurar exploração sustentável destas espécies.

The aim of this literature review is to conduct a survey concerning the history and current situation of tissue culture techniques in medicinal plants. Therefore, a review was done considering the period from 1976 to 2009. Tissue culture is widely applied in medicinal plants researches, especially micropropagation. The perspectives of tissue culture techniques in medicinal plants are related to the development of competitive germoplasm adapted to diverse methods of cultivation, the election of new species that will serve as source of biological active composts, and the improvement of phytochemicals production, in order to assure sustainable exploration of these species.

Citocininas na multiplicação in vitro de hortelã-pimenta (Mentha x Piperita L. ) / Cytokinins on in vitro multiplication of peppermint (Mentha x Piperita L. )

O hortelã-pimenta é um hibrido triplo, Mentha x piperita, utilizado de forma medicinal no tratamento de náuseas, cólicas gastrointestinais, flatulências, cálculos biliares, icterícia, ansiedade, expectoração e expulsão de vermes intestinais. Na micropropagação, o BAP (6-benzilaminopurina) e KIN (cinetina) têm sido as fontes de citocininas mais empregadas. O objetivo deste trabalho foi avaliar o efeito de reguladores vegetais na multiplicação in vitro de hortelã-pimenta. Explantes constituídos de segmentos nodais provenientes de plântulas já estabelecidas in vitro com aproximadamente 0,5 cm foram inoculados em meio MS, suplementado com diferentes associações de BAP e KIN, em adição de 30 g L-1 de sacarose. As concentrações de citocininas utilizadas foram 0,0; 1,0 e 2,0 mg L-1 de BAP e 0,0; 0,5 e 1,0 mg L-1 de KIN. O delineamento experimental foi o inteiramente casualizado, com nove tratamentos consistindo de cinco frascos cada um, sendo que cada frasco continha quatro explantes. A utilização de 2,0 mg L-1 de BAP promove a multiplicação in vitro de hortelã-pimenta, porém, diminui o índice de sobrevivência. O uso de citocininas aumenta a massa fresca e seca dos explantes e a ausência destes reguladores propicia o alongamento nesta espécie.

Peppermint is used for medical treatments of nausea, gastrointestinal cramps, flatulence, gallstones, jaundice, anxiety, sputum and expulsion of intestinal worms. In micropropagation, BAP (6-benzylaminopurine) and KIN (kinetin) have been the most widely used sources of cytokinins. The aim of this study was to evaluate the effect of plant growth regulators in multiplication of peppermint. Explants consisting of nodal segments from seedlings already established in vitro with approximately 0,5 cm were inoculated on MS medium supplemented with different combinations of BAP and KIN and 30 g L-1 of sucrose. The concentrations of cytokinins used were 0,0; 1,0 and 2,0 mg L-1 of BAP and 0,0; 0,5 and 1,0 mg L-1 of KIN. The experiment was set up in a completely randomized design with nine treatments consisting of five bottles each, and each vial contained four explants. The use of 2,0 mg L-1 of BAP promotes the in vitro multiplication of peppermint, but decreases the survival rate. Cytokinins increase fresh and dry weight of explants and the absence of these regulators provides the elongation in this specie.

Multiplicação in vitro e aclimatação de Vernonia condensata Baker / In vitro multiplication and acclimation of Vernonia condensata Baker

A espécie medicinal Vernonia condensata, vulgarmente conhecida por alumã, pertencente à família Asteraceae, possui propriedades analgésicas e de proteção gástrica. A crescente utilização dessa planta no Nordeste, pelas propriedades terapêuticas, justifica a necessidade de medidas que minimizem o impacto de sua exploração nas reservas naturais. O objetivo desse trabalho foi multiplicar in vitro plantas de alumã sob diferentes concentrações de BAP e aclimatá-las. Gemas axilares foram desinfestadas em solução de álcool etílico 70 por cento, durante 2 minutos e em solução de hipoclorito de sódio (2 por cento de cloro ativo) na concentração de 3:1, durante 15 minutos, seguido de três lavagens em água destilada estéril. Os tratamentos para multiplicação consistiram em doses de BAP (0,0; 1,0; 2,0; 3,0; 4,0 e 5,0 mg L-1) em meio MS semi-sólido. O delineamento experimental foi o inteiramente casualizado, com 5 repetições, contendo 10 gemas por repetição. Após 30 dias de cultivo observou-se maior taxa de explantes responsivos, 84 por cento na concentração de 1,0 mg L-1 de BAP, com produção de 4,0 brotos/explante. Nos tratamentos 3,0; 4,0 e 5,0 mg L-1 ocorreu hiperhidricidade nas folhas. As microplantas de alumã provenientes da metodologia utilizada neste trabalho alcançaram 100 por cento de sobrevivência na aclimatação.

The medicinal species Vernonia condensata, commonly known as "alumã", belongs to the family Asteraceae and has analgesic and gastric protective properties. The increasing use of this plant in the Northeast of Brazil due to its therapeutic properties justifies the need of measures to minimize the impact of its exploitation in natural reserves. The aim of this study was to multiply, in vitro, "alumã" plants under different BAP levels, acclimating them. Axillary buds were sterilized in 70 percent (v/v) alcohol solution for 2 minutes and in 75 percent sodium hypochlorite solution (2 percent active chlorine) at 3:1 concentration for 15 minutes, followed by three washings in sterile distilled water. Multiplication treatments consisted of different BAP levels (0.0; 1.0; 2.0; 3.0; 4.0 and 5.0 mg L-1) in semi-solid MS medium. The experimental design was completely randomized, with 5 replicates and 10 buds per replicate. After 30 days of cultivation, the highest rate of responsive explants was obtained: 84 percent at 1.0 mg L-1 BAP, producing 4.0 sprouts/explant. In the treatments 3.0, 4.0 and 5.0 mg L-1, there were vitrified leaves. The "alumã" microplants used in this study had 100 percent survival in acclimation.

Evaluation of polyvinyl alcohol for fatty acid supplementation in adipose tissue explant culture

Cultures of adipose tissue explants are a valuable tool for studying the intracellular mechanisms involving hormones and nutrients. However, testing how fatty acids affect cells requires a carrier molecule; bovine serum albumin (BSA) has been used for this purpose. However, contaminants can alter the cellular response. Our objectives were to: 1) test BSA as a fatty acid carrier and 2) evaluate polyvinyl alcohol (PVA) as a replacement for BSA. Adipose tissue explants from nine pigs were cultured in medium 199 for 4, 12, 24, and 48 h, with the following treatments: control, PVA (100 mM PVA added) and PVA + pGH (100 mM PVA plus 0.1 mg/mL porcine growth hormone). After each culture period, explants were collected and assayed for lipogenesis. After 48 h in culture, explants were assayed for lipolysis. A preliminary study with different commercial sources and high concentrations showed that BSA affected lipogenic rates. On the other hand, there were no effects of PVA on lipid synthesis, while pGH (positive control) reduced glucose incorporation into lipids (P < 0.01) when compared to both control and PVA (P < 0.05). There was no difference between control and PVA for lipolysis rates. However, pGH increased lipolysis when compared to control (P < 0.01) and PVA (P < 0.05). We demonstrated that BSA can alter lipogenesis, which precludes its use as a carrier molecule. On the other hand, addition of PVA had no effect on lipolysis or lipogenesis. We suggest the use of PVA instead of BSA for adding bioactive fatty acids to cultures of adipose tissue

Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation.

BACKGROUND: The technique of transplantation of cultivated limbal epithelium rather than direct limbal tissue isa novel method of "cell therapy" involved in reconstructing the ocular surface in severe limbal stem cell deficiency [LSCD], caused by chemical burns. AIM: To describe a simple feeder-cell free technique of cultivating limbal epithelium on human amniotic membrane[HAM]. MATERIALS AND METHODS: The limbal tissues (2 mm) were harvested from patients with LSCD. These tissues were proliferated in vitro on HAM supplemented by human corneal epithelial cell medium and autologous serum. Cultures covering more > or = 50% area of 2.5 x 5 cm HAM were considered adequate for clinical use. The cultured epithelium was characterized by histopathology and immunophenotyping. RESULTS: A total of 542 cultures out of 250 limbal tissues were cultivated in the laboratory from January 2001 through July 2005. The culture explants showed that clusters of cells emerging from the edge of the explants in one-three days formed a complete monolayer within 10-14 days. In 86% of cultures (464 of 542), the growth was observed within one-two days. Successful explant cultures were observed in 98.5% (534 of 542 cultures) with 91% explant cultures showing an area of > or = 6.25 cm2 (6.25 - 12.5 cm2 range). The cultivated epithelium was terminated between 10-14 days for clinical transplantation. The problems encountered were inadequate growth (2 of 542) and contamination (2 of 542). CONCLUSIONS: We demonstrate a simple technique of generating a sheet of corneal epithelium from a limbal biopsy. This new technique could pave the way for a novel form of cell therapy.

Isolation and in vitro cultivation of human skin keratinocytes and preparation of epidermal sheet

Human keratinocytes have been cultivated in vitro using different methods. Using small size skin, large epidermal sheets have been produced. These epidermal sheets have been used for the treatment of injured epidermis, especially burn and other skin clinical disorders, and also for research purposes. The objective of this study was isolation and in vitro cultivation of human skin keratinocytes and preparation of epidermal sheet without using mouse 3T3 fibroblast feeder layer. Small size human skin with a relative thickness was divided to small pieces and the epidermis was separated from dermis using warm trypsin. Then keratinocytes were isolated from epidermis and cultured with high density in DMEM containing FBS and other supplements like cholera toxin and epidermal growth factor. The resulting epidermal sheet was separated using dispase grade II and transferred to vaseline gauze. Histological studies were also carried out on the epidermal sheet. Keratinocytes grew as multiple colonies and produced a thin confluent epidermal sheet consisted of a few layers of cells. With time, number of cell layers was increased and a thicker epidermal sheet was produced. Fibroblasts, which were present in the original cell suspension, grew in the culture when the growth of keratinocyte was slow or when a low density of keratinocytes was used. The histological examination of epidermal sheet showed the presence of germinative basal cells, suprabasal cells with relative differentiation and also melanocytes. The dimension of epidermal sheet was decreased when it was separated from tissue culture flask using dispase. Cultured epidermal autografts have several clinical and investigative indications. Even though the cultivation of keratinocytes with high density is easy, however, the surface area of resulting epidermal sheet is limited and its subculture is difficult. In this method there is the risk of growth of dermal fibroblasts. To produce thicker epidermal sheet with more cellular layers, it is better to separate it within two to three weeks after cultivation

Tissue factors in acute haematogenous osteomyelitis

80 successive cases of acute haematogenous osteomyelitis [AHO]. They were all treated and investigated in the Hadra Orthopaedic and Traumatolology University Hospital, November 1986- May 1988. The immune system was investigated by testing the skin reaction to purified protein derivative [PPD] and dinitrochlorobenzene [DNCB]. The percentage of T-lymphocytes was detected by the E-rosette test and their function was evaluated by the leucocyte migration inhibition test and the phytohaemagglutinin stimulation test. The phagocytic function of the neutrophils was assessed by the nitroblue tetrazolium dye reduction test. All patients were subjected to HLA typing. The percentage and function of T-lymphocytes were found subnormal in patients with AHO. The neutrophil phagocytic function and skin reactivity to PPD were also subnormal. HLAA-A9, B5, B7 and Bw73 were found more in patients with AHO than in normal controls. The HLA-B22, B42 and B15 [63] were more frequent in normal controls

Indirect immunoflourescent technique versus tissue culture method in diagnosis of poliomyelitis

Eighty stool samples were collected from paralytic children at Imbaba Poliomyelitis Institute. Isolation, identification and typing of polioviruses were done by both neutralization test [NT] and indirect immunofluorescent [IF] test. The indirect IF test gave negative results after 2 and 4 hours post-virus inoculation in Buffalo Green Monkey [BGM] cell line for all cases. After 6 hours post inoculation, it was found that the indirect IF test has a specificity rate of 100% in diagnosing and typing of polioviruses in comparison with the NT. The sensitivity of indirect IF test varies according to the type of polioviruses. It was high for type 1 [91%], the most prevalent one in Egypt, 75% for type 2, 88% for type 3 and 100% in cases of mixed infection with type 1 and 3. No cross reactions between the different types of polioviruses were detected by indirect IF technique, and its results were equivocal to that of the NT. The indirect IF test is a rapid test with 100% specificity and high sensitivity in diagnosing and typing polioviruses, and it is recommended to be used as a screening test, especially in endemic areas

Tissue culture for studying the effect of mechanical pressure on cartilage

Tissue culture for cartilagenous plates was done to study the effect of pressure from different directions unmodified by the presence of other tissues such as muscles, nerves and blood vessels. The chondrocytes lost their orientation and became parallel with the long axes of the pressure lines. The ground substance gradually disappeared and replaced by collagen fibrils that became parallel with the long axes of the packed and elongated cartilagenous cells