Composite Zonal Scaffolds of Collagen I/II for Meniscus Regeneration.

The meniscus is divided into three zones according to its vascularity: an external vascularized red-red zone mainly comprising collagen I, a red-white interphase zone mainly comprising collagens I and II, and an internal white-white zone rich in collagen II. Known scaffolds used to treat meniscal injuries do not reflect the chemical composition of the vascular areas of the meniscus. Therefore, in this study, four composite zonal scaffolds (named A, B, C, and D) were developed and characterized; the developed scaffolds exhibited the main chemical components of the external (collagen I), interphase (collagens I/II), and internal (collagen II) zones of the meniscus. Noncomposite scaffolds were also produced (named E), which had the same shape as the composite scaffolds but were entirely made of collagen I. The composite zonal scaffolds were prepared using different concentrations of collagen I and the same concentration of collagen II and were either cross-linked with genipin or not cross-linked. Porous, biodegradable, and hydrophilic scaffolds with an expected chemical composition were obtained. Their pore size was smaller than the size reported for the meniscus substitutes; however, all scaffolds allowed the adhesion and proliferation of human adipose-derived stem cells (hADSCs) and were not cytotoxic. Data from enzymatic degradation and hADSC proliferation assays were considered for choosing the cross-linked composite scaffolds along with the collagen I scaffold and to test if composite zonal scaffolds seeded with hADSC and cultured with differentiation medium produced fibrocartilage-like tissue different from that formed in noncomposite scaffolds. After 21 days of culture, hADSCs seeded on composite scaffolds afforded an extracellular matrix with aggrecan, whereas hADSCs seeded on noncomposite collagen I scaffolds formed a matrix-like fibrocartilage without aggrecan.

In vitro and in vivo assessment of oral autologous artificial connective tissue characteristics that influence its performance as a graft.

Several studies have evaluated proteins secreted by fibroblasts comprising skin substitutes, finding that they are secreted in combinations and concentrations that promote wound healing. However, assessment of proteins secreted by oral fibroblasts forming a part of oral substitutes is scarce. In our previous work, collagen type-I scaffolds (CSs) and autologous artificial connective tissue (AACT) were produced and implanted in rabbit oral lesions, evidencing that AACT outperforms CS. The present work determined the secreted factor profile of AACT in the time of grafting as well as that of the AACT embedded in the clot. It also evaluated the proliferation and viability of AACT fibroblasts to establish the dwell time of these cells in the grafted area. Finally, it assessed whether CS, AACT, and clot-embedded AACT increase fibroblast recruitment induced by a fibrin clot, because the cell migratory response has been associated with the wound-healing outcome. We found that some of the factors secreted by AACT fibroblasts are significantly different from those secreted by clot-embedded AACT fibroblasts. Also, that the profile of proteins secreted by AACT fibroblasts and clot-embedded AACT fibroblasts is different from already reported protein secretion profiles of other engineered tissues used in treating oral mucosa wounds. It was also found that AACT fibroblasts are viable when grafted and remain in the treated area for almost 2 weeks, and that the migratory response of fibroblasts to tissue-substitute stimulus is significantly less than the migratory response induced by the clot alone. Overall, data suggest that AACT secretion of proteins is modulated by three-dimensionality and environment factors. This bioactivity and the fact that AACT does not increase fibroblast migration can be held accountable for AACT's good performance as a graft.

Determining the pharmacological activity of Physalis peruviana fruit juice on rabbit eyes and fibroblast primary cultures.

PURPOSE: The pharmacologic activity of compounds isolated from Physalis peruviana has been demonstrated. The use of this fruit juice for treating pterygium has been reported in Colombian traditional medicine. However, studies demonstrating the fruit juice's pharmacologic activity when used in this disease have not been published to date. In the present study the anti-inflammatory and cytostatic activities of P. peruviana fruit juice in a rabbit eye inflammatory model were investigated. METHODS: A novel rabbit eye inflammation model was developed for studying the juice's anti-inflammatory activity (based on an adaptation of the Draize test). Cytostatic activity was evaluated by measuring and comparing growth rates of cultured fibroblasts exposed and not exposed to various fruit juice concentrations. RESULTS: P. peruviana fruit juice exhibited a mild anti-inflammatory activity compared with methylprednisolone, a known anti-inflammatory drug. An interesting dose-dependent cytostatic effect on cultured fibroblasts was also established. CONCLUSIONS: The data found suggest that the P. peruviana fruit juice anti-pterygium effect described in traditional medicine may be related to its inhibiting fibroblast growth. The present study contributes to the pharmacologic knowledge regarding a remedy commonly used in Colombian traditional medicine.

[Specific expression of inflammatory genes in macrophage subpopulations]. / Expresión específica de los genes de la respuesta inflamatoria en subpoblaciones de macrófagos.

Macrophages serve as an effective component of innate immunity in their ability to recognize, engulf and kill potential pathogens. They also coordinate additional host responses by synthesizing a range of inflammatory mediators that can activate the adaptive immune response and establish protective immunity. Although they are a key component of mammalian defense system, macrophage activity is not always beneficial to the host. The centrality of macrophages in disease processes makes macrophage regulation a major target in the prevention, control and cure of inflammatory processes. Consequently, macrophage-restricted genes may be crucial targets for therapeutic intervention. A review is presented of the use of large-scale cDNA microarrays to compare macrophage inflammatory genes differentially expressed in two distinct macrophages populations--bone marrow derived macrophages (bmm) and inflammatory thioglycolate-elicited peritoneal macrophages (tepm)--to non-macrophage cell populations consisting of primary embryonic fibroblast and spleen non-adherent cells. Expression profiles indicate that macrophage inflammatory genes are associated with expected functional categories, such as lysosomal degradation, phagocytosis, host defense and homeostasis.

Expresión específica de los genes de la respuesta inflamatoria en subpoblaciones de macrófagos / Specific expression of inflammatory genes in macrophage subpopulations

Como brazo efectivo de la respuesta inmune, los macrófagos reconocen, fagocitan y destruyen patógenos potenciales. También, coordinan respuestas adicionales del hospedero mediante la síntesis de un amplio rango de mediadores de la inflamación que, en últimas, activan la respuesta inmune adaptativa y establecen la inmunidad protectora. Aunque son componentes claves del sistema de defensa de los mamíferos, el resultado de su actividad no siempre es beneficioso para el hospedero. El papel central jugado en la enfermedad hace que su regulación se convierta en un blanco importante en la prevención, el control y la curación de los procesos inflamatorios. De hecho, los genes de expresión restringida en macrófagos pueden ser puntos cruciales de la intervención terapéutica. Este trabajo revisa el uso de los microarreglos de cADN para la comparación de los genes de la inflamación que se expresan de diferente forma en dos poblaciones de macrófagos, derivados de la médula ósea y macrófagos peritoneales obtenidos después de inducir una inflamación con tioglicolato, con genes expresados en fibroblastos primarios aislados de embrión y células esplénicas no adherentes. La comparación de los perfiles de expresión indica que los genes de la inflamación de los macrófagos están asociados con categorías funcionales esperadas como la degradación lisosómica, la fagocitosis, la defensa del hospedero y de la homeostasis. La información revisada por este estudio contribuye a entender la biología de los macrófagos

Cultivos primarios de células endoteliales aisladas de cordón umbilical humano: un modelo biológico para el estudio de los mecanismos de infección por enterococos / Primary cultures of human umbilical chord vein endothelial cells: a biological model for studying enterococcal infection mechanisms

Aunque las especies de enterococos son flora normal del tracto gastrointestinal humano, se encuentran entre los tres agentes patógenos microbianos que más se asocian con infecciones intrahospitalarias. Tradicionalmente, los enterococos se han considerado bacterias extracelulares. Sin embargo, es creciente la información que reporta su ingreso a través de líneas celulares epiteliales o macrófagos. A pesar de que estos microorganismos constituyen el tercer grupo de aislamientos obtenido de pacientes con bacteriemia o endocarditis, su interacción con la célula endotelial no se ha descrito completamente. En el presente trabajo evaluamos si el aislamiento intrahospitalario de Enterococcus faecalis (Ef2880) podía penetrar en células endoteliales de la vena de cordón umbilical humano (HUVEC) cultivadas in vitro. Nuestros resultados indican que los cultivos primarios HUVEC después de ser inoculados con Ef2890, incubados y tratados con antibióticos bactericidas para las bacterias extracelulares y adheridas a la cara externa de la membrana celular, exhiben bacterias intracelulares que pueden ser recuperadas vivas cuatro horas después de la inoculación. El modelo biológico desarrollado se puede constituir en una herramienta útil para el estudio de las interacciones que establecen los enterococos con el endotelio

[Primary cultures of human umbilical chord vein endothelial cells: a biological model for studying enterococcal infection mechanisms]. / Cultivos primarios de células endoteliales aisladas de cordón umbilical humano: un modelo biológico para el estudio de los mecanismos de infección por enterococos.

Although enterococcus bacteria are normal human intestinal flora, they rank as the third most common pathogen involved in hospital acquired infections. Generally, these bacteria are considered extracellular pathogens; however, an increasing number of reports indicate invasiveness to epithelial cell lines and macrophages. Despite their importance as nosocomial infection agents in patients suffering bacteremias and endocarditis, their interaction with endothelial cells has not been fully described. Herein, the nosocomial Enterococcus faecalis isolate Ef2890 from a hospitalized patient was exposed to cultured human venous endothelial cells from the umbilical chord. When the primary cell cultures were inoculated with Ef2890 and treated with bactericidal antibiotics to kill extracellular and adhered bacteria, intracellular bacteria were recovered and plated 4 h post-infection. These observations indicate that cell cultures provide a valuable biological model to study interactions between endothelium and enterococci.

Aislamiento y caracterización de macrófagos- células espusas a partir de aorta de un modelo de ateroesclerosis en conejo hipercolesterolémico / Isolation and characterization of macrophages-foam cells from aorta of hypercholesterolemic rabbit

Actualmente existe suficiente evidencia que sustenta que la ateroesclerosis es una patología en la cual están involucrados no sólo procesos de desequilibrio y aumento de lípidos, sino también procesos inflamatorios mediados por macrófagos-células espumosas. Estos hallazgos han sido encontrados en estudios llevados a cabo con animales de experimentación. Con el propósito de racionalizar la utilización de animales y de proponer un modelo biológico alterno en el que se puedan estudiar los mecanismos de patogenicidad que involucren tipos celulares relacionados con la ateroesclerosis, en el presente trabajo se estandarizó una técnica de aislamiento y cultivo de macrófagos-células espumosas, así como, los procedimientos para caracterizar los cultivos establecidos mediante la detección de esterasas no específicas. Para el análisis de la expresión de estas enzimas, se utilizó una técnica histoquímica y electroforesis en gel de poliacrilamida en condiciones no denaturantes. En la literatura revisada, este último método no ha sido empleado para evidenciar expresión de esterasas no específicas en leucocitos. El modelo biológico aportado por este trabajo puede ser usado para estudiar respuestas de los macrófagos activados y células espumosas relacionadas con la ateroesclerosis.

[Isolation and characterization of macrophages-foam cells from aorta of hypercholesterolemic rabbit]. / Aislamiento y caracterización de macrófagos-células espumosas a partir de aorta de un modelo deateroesclerosis en conejo hipercolesterolémico.

Evidence has accumulatd to support the hypothesis that atherosclerosis involves lipid imbalance as well as inflammatory responses mediated by macrophage and foam cells. These findings have been based on animal models. To rationalize animal use and to propose an alternative biological model, a technique was standardized for macrophage-foam cell isolation and culture. The cultures were characterized by non-denaturing polyacrylamide gel electrophoresis (PAGE) of nonspecific esterases and histochemical staining. This method has not been applied previously for the characterization of the non specific esterases from leucocytes. The biological model presented here can be used to study macrophage-foam cell responses related to atherosclerosis.

Sustitutos de mucosa oral creados mediante ingeniería tisular: una alternativa para la reconstrucción de defectos de mucosa oral / Oral mucosa substitutes developed by Tissue Engineering: an alternative for the reconstruction of mucosal defects

Varias lesiones que tienen como resultado pérdida de mucosa oral (malformaciones, enfermedad periodontal, trauma y tumores entre otras) pueden afectar la integridad de la cavidad oral humana. La vida de quienes la sufren, se altera clínica-psicosocial y económicamente debido a la estética y funcionamiento defectuosos, mayor riesgo de infecciones, dolor y molestia severa. Reemplazar la mucosa oral puede llegar a ser difícil, debido a la cantidad limitada de tejido disponible para el autoinjerto y a la morbilidad en el sitio donante. Cuando la mucosa oral es escasa, se emplean como injertos piel antóloga o mucosa gástrica. Infortunadamente, estas aproximaciones no siempre dan buenos resultados debido a la aparición de secreciones, crecimiento de anexos de la piel y patrones diferentes de queratinización. Por lo anterior, se hace necesario buscar nuevas fuentes de tejido oral. En otros países se adelantan investigaciones para desarrollar, con células vivas unidas a un substituto de matriz, un tejido de mucosa oral funcional. De hecho, cultivos de epitelio gingival y láminas de mucosa artificiales han sido probados con algún éxito como injertos en ensayos preclínicos y clínicos. Este artículo presenta una revisión de los modelos de tejido mucoso oral desarrollados in Vitro y las valoraciones de esos productos efectuadas hasta el momento.