Thermosensitive behavior in cell culture media and cytocompatibility of a novel copolymer: poly(N-isopropylacrylamide-co-butylacrylate).

Cell sheet technology is a promising step forward in tissue engineering. Cell sheets are usually generated using Poly(N-isopropylacrylamide) hydrogels due to their swelling change around the lower critical solution temperature (LCST). Nevertheless, LCST can be affected by cell culture medium components and therefore it is necessary to ensure that the polymer preserves its thermosensitivity under these conditions. We propose a novel thermosensitive crosslinked-copolymer: Poly(N-isopropylacrylamide-co-butylacrylate). This copolymer is shown to be cytocompatible and thermosensitive under cell culture medium conditions, and besides, it can be synthesized inexpensively. Thermosensitivity was investigated by determining the LCST with differential scanning calorimetry and swelling/ratio measurements. Cytocompatibility and capacity to deliver cell sheets were studied employing 3T3 and human oral epithelial cells. In conclusion, we obtained a thermosensitive copolymer that allows cell sheet formation/detachment by using a simple and low-cost polymerization method. Furthermore, crosslinking allows easy manipulation of cell sheets growing on the copolymer for potential in situ applications.

Viscoelastic properties of plasma-agarose hydrogels dictate favorable fibroblast responses for skin tissue engineering applications.

Dermal wound healing relies on the properties of the extracellular matrix (ECM). Thus, hydrogels that replicate skin ECM have reached clinical application. After a dermal injury, a transient, biodegradable fibrin clot is instrumental in wound healing. Human plasma, and its main constituent, fibrin would make a suitable biomaterial for improving wound healing and processed as hydrogels albeit with limited mechanical strength. To overcome this, plasma-agarose (PA) composite hydrogels have been developed and used to prepare diverse bioengineered tissues. To date, little is known about the influence of variable agarose concentrations on the viscoelastic properties of PA hydrogels and their correlation to cell biology. This study reports the characterization of the viscoelastic properties of different concentrations of agarose in PA hydrogels: 0 %, 0.5 %, 1 %, 1.5 %, and 2 % (w/v), and their influence on the cell number and mitochondrial activity of human dermal fibroblasts. Results show that agarose addition increased the stiffness, relaxation time constants 1 (τ1) and 2 (τ2), and fiber diameter, whereas the porosity decreased. Changes in cell metabolism occurred at the early stages of culturing and correlated to the displacement of fast (τ1) and intermediate (τ2) Maxwell elements. Fibroblasts seeded in low PA concentrations spread faster during 14 d than cells cultured in higher agarose concentrations. Collectively, these results confirm that PA viscoelasticity and hydrogel architecture strongly influenced cell behavior. Therefore, viscoelasticity is a key parameter in the design of PA-based implants.

Improving Fibrin Hydrogels' Mechanical Properties, through Addition of Silica or Chitosan-Silica Materials, for Potential Application as Wound Dressings.

Fibrin is a protein-based hydrogel formed during blood coagulation. It can also be produced in vitro from human blood plasma, and it is capable of resisting high deformations. However, after each deformation process, it loses high amounts of water, which subsequently makes it mechanically unstable and, finally, difficult to manipulate. The objective of this work was to overcome the in vitro fibrin mechanical instability. The strategy consists of adding silica or chitosan-silica materials and comparing how the different materials electrokinetic-surface properties affect the achieved improvement. The siliceous materials electrostatic and steric stabilization mechanisms, together with plasma protein adsorption on their surfaces, were corroborated by DLS and ζ-potential measurements before fibrin gelling. These properties avoid phase separation, favoring homogeneous incorporation of the solid into the forming fibrin network. Young's modulus of modified fibrin hydrogels was evaluated by AFM to quantitatively measure stiffness. It increased 2.5 times with the addition of 4 mg/mL silica. A similar improvement was achieved with only 0.7 mg/mL chitosan-silica, which highlighted the contribution of hydrophilic chitosan chains to fibrinogen crosslinking. Moreover, these chains avoided the fibroblast growth inhibition onto modified fibrin hydrogels 3D culture observed with silica. In conclusion, 0.7 mg/mL chitosan-silica improved the mechanical stability of fibrin hydrogels with low risks of cytotoxicity. This easy-to-manipulate modified fibrin hydrogel makes it suitable as a wound dressing biomaterial.

Seroprevalence of hepatitis B and factors potentially associated in a population-based study in Medellin, Colombia.

The seroprevalence of hepatitis B (HB) and of potentially associated factors in Medellin, Colombia, were investigated 17years after the start of universal vaccination. Biological and sociodemographic data from a population survey with a multistage random sampling were analyzed in 6-64year old individuals. HB surface antigen, total HB core antibodies and HB surface antibodies, and in some cases IgM antibodies to HB core antigen, were tested in 2077 samples. Factors potentially associated with and natural, and vaccine immunity relative to susceptibility (absence of any marker) were analyzed using a multinomial logistic regression. The prevalence of serological patterns was: chronic infection 0.20% (95% CI 0.11-0.71), vaccine immunity 25.10% (95% CI 21.72-28.83) and natural immunity 2.60% (95% CI 1.80-3.74). No markers were detected in 71.30% (95% CI 67.70-74.83) of the individuals and evidence of recent infection was not detected. Relative to the absence of markers, natural immunity was potentially associated with age (6-17years and 41-64years) and sleeping less than 6 hours, while vaccine immunity was associated with age (6-17years), reporting vaccination against HB, belonging to high socioeconomic strata, home ownership and being obese, after adjusting for other variables. These results may be a population effect of mass vaccination. It is recommended to complete the vaccination schedule and to study in detail, persistence of antibodies and the role of obesity and socioeconomic strata in the vaccine immunity.

Electrospinning of gelatin and SMPU with carbon nanotubes for tissue engineering scaffolds.

The nanofibres created by electrospinning technique are currently used for a variety of applications in tissue engineering; and Gelatin and Polyurethane Shape-Memory (SMPU) have important results in biomedicine. Similarly, carbon nanotubes combined with other biomaterials change important properties, opening new opportunities for biomedical applications. In this work, we constructed scaffold using electrospinning technique based in bovine-hide gelatin, SMPU and both materials hybrid with carbon nanotube. Morphology and cytotoxicity were evaluated and mechanical properties for two materials were obtained in scaffold building. Morphological, mechanical and citotoxic properties of the electrospun fibers were found to be dependent of alteration in materials concentration, electrospinning conditions and MWCNT concentration. According to morphological, cytotoxic and mechanical analysis, SMPU more MWCNT were the best material, with nanofibers of 451 nm, tensile strength of 1.912 MPa, and a high ratio surface volume.

Seroprevalence of mumps in an epidemic period in Medellín, Colombia.

OBJECTIVE: We related seroprevalence and outbreaks data in order to identify factors that could explain the occurrence of outbreaks despite high vaccination coverage in Medellín Colombia. METHODS: Samples from a population seroprevalence data obtained in 2009 in a random survey were analyzed. IgG levels were determined for mumps using 2 commercial tests of 2119 individuals aged 6-64 years. A comparative analysis was undertaken using age-specific mumps seroprevalence data and information of 98 epidemiological investigations of mumps outbreaks reported in 2009. RESULTS: Overall, seroprevalence was 91.6% (95% CI=89.3-93.5%). The age-specific seronegativity was 20.3% and 20.6% in age groups 11-15 years and 16-20 years respectively. Individuals aged 6-20 years were the most affected during outbreaks. In individuals born in 2003, a year after the change in the booster schedule from 10 to 5 years, the proportion of unvaccinated individuals (14%) and those who received only one dose of MMR (45%) increased substantially. On average, 23.5 days elapsed between the onset of symptoms in secondary cases and the outbreak investigation. CONCLUSION: Potential contributing factors for the occurrence of outbreaks of mumps were the relatively high prevalence of seronegativity among individuals aged 11-20 years, delays in investigation and control of outbreaks, and incomplete vaccination schedules.

Tratamiento de las úlceras crónicas en los miembros inferiores con un equivalente cutáneo autólogo y desbridación con larvas de Lucilia sp. (Diptera: Calliphoridae). Reporte de un caso / Chronic lower limb ulcers treatment with autologous skin equivalent and larvae debriding (Diptera Calliphoridae)

Introducción: las úlceras en los miembros inferiores constituyen una causa importante de hospitalización y deterioro en la calidad de vida de los pacientes, al interferir con sus actividades laborales y sociales. Estas lesiones obedecen a diferentes enfermedades, la más frecuente de las cuales en Medellín es la insuficiencia venosa; una vez diagnosticadas, existen muchas alternativas de tratamiento, que buscan crear un lecho apropiado para el cierre de la herida. El uso de larvas para el desbridamiento es una alternativa rápida y de bajo costo, con la cual se logra un tejido de granulación adecuado para la aplicación de equivalentes cutáneos autólogos, que constituyen una opción para pacientes refractarios a otros tratamientos. Materiales y métodos: se seleccionó y evaluó a una paciente de acuerdo con los criterios de inclusión establecidos para el estudio. Para desbridar la úlcera se recurrió a la aplicación de larvas de Lucilia sp. (Diptera: Calliphoridae), btenidas a partir de huevos previamente desinfectados con hipoclorito de sodio y sembrados en un medio de cultivo con antibióticos. Se aplicaron las larvas directamente en la lesión y posteriormente se procedió a tapar esta con muselina, permitiendo la obtención de oxígeno y facilitando el drenaje del tejido necrótico. Las larvas se dejaron por 48 horas, al cabo de las cuales se retiraron con una pinza estéril y se descartaron en alcohol al 70%; luego se procedió a evaluar la limpieza de la lesión. Este procedimiento se llevó a cabo en el Laboratorio de Entomología (GIEM) de la Universidad Antioquia. Se procesó una biopsia de piel de la paciente, obtenida siguiendo un protocolo previamente establecido, para obtener queratinocitos y fibroblastos. Para el cultivo primario de queratinocitos el 90% de las células obtenidas de la biopsia se sembró en cajas de cultivo de 75 cm2, en presencia de 7 x 106 células de la línea 3T3-Swiss tratadas con mitomicina C (10 μg/mL) como capa alimentadora; para el cultivo primario de fibroblastos, se sembró el 10% de las células en cajas de cultivo de 75 cm2 con el medio DMEM suplementado. En la producción del equivalente cutáneo se utilizó un gel obtenido con una mezcla de plasma humano de sangre AB de banco de sangre, 6-7,5 x 104 fibroblastos, CaCl2 y ácido tranexámico. Sobre dicho gel se sembraron luego los queratinocitos provenientes del cultivo primario y se hizo seguimiento del cultivo en microscopio invertido. Cuando se obtuvo una confluencia celular cercana al 100%, se desprendió el equivalente cutáneo con pinzas estériles para aplicarlo inmediatamente sobre la úlcera. Se reportan los resultados del tratamiento de una úlcera en el miembro inferior izquierdo de una paciente de 66 años con la terapia combinada de desbridamiento con larvas y cultivo de equivalentes cutáneos autólogos. Después de 15 meses de iniciado el tratamiento, la úlcera continúa cerrada y la paciente no tiene dolor ni impedimentos funcionales en la extremidad.

Transferencia de genes. Bases y aplicaciones

En la última década el enorme desarrollo de la biología molecular ha posibilitado que la tecnología del DNA recombinante se aplique a la transferencia de genes con fines terapéuticos, pasando del sueño a la realidad. Los sistemas de vectorización de los diferentes genes incluyen, a los retrovirus, adenovirus, herpesvirus, lipososmas, etc., cada uno con ventajas y desventajas, de acuerdo con la enfermedad a ser tratada y con el tejido objeto de la corrección genética. Hasta el momento aproximadamente 80 protocolos clínicos están siendo aplicados en el mundo y su número auemnta día a día. El control y regulación de la aplicación de estos protocolos implica la evaluación rigurosa de cada uno de ellos, tanto en modelos in vitro como en animales, antes de su aplicación final en el hombre. El país debe prepararse tanto científica y tecnológicamente como desde el punto de vista ético y legal, para enfrentar los retos de este nuevo enfoque terapéutico.

Determinación de anticuerpos y aloanticuerpos linfocitoxicos en pacientes candidatos a trasplante renal

Las pruebas cruzadas para detectar la presencia de anticuerpos linfocitotóxicos en el suero de candidatos a trasplante renal, son decisivas en el estudio pretrasplante. En el presente estudio se reportan los hallazgos en 97 pacientes con insificiencia renal crónica, candidatos a trasplantes y 127 posibles donantes vivos relacionados. Las pruebas incluyerón detección de anticuerpos antilifocitos T y B separadamente a 4,20 y 37 grados contra linfocitos del posible donante y contra las células autólogas. Se incluye además el tratamiento con ditiotreitol (DTT) para diferenciar entre los isotopos IgM e IgG. Los resultados muestran que 40.2 por ciento de los pacientes y 16.5 por ciento (p<=0.04) de los donantes clínicamente sanos tiene anticuerpos que pueden reaccionar con células alogénicas. Los aloanticuerpos se dectectaron en 38 por ciento de los pacientes. La mayoria de los anticuerpos estaban dirigidos contra los linfocitos B (71.7 por ciento) y correspondieron al isotipo IgM (66.7 por ciento), aunque tambien se detectaron tanto auto como aloanticuerpos IgG.No se detectó un efecto significativo de las trasfuciones o embarazos previos en el desarrollo de anticuerpos ni de aloanticuerpos. De otro lado se observó una frecuencia significativamente mayor (p=0.0009) de donantes intrafamiliares con autoanticuerpos positivos, en pacientes tambien positivos para autoanticuerpos que de receptores negativos para autoanticuerpos positivos que fueron trasplantados con riñones provenientes de sus donantes intrafamiliares o de cadáver, tienen sus injertos funcionantes hasta un año después. Los pacientes sin autoanticuerpos presentaron una menor sobrevida de sus injertos especialmente en el caso de los injertos de cadáver, de los cuales soló sobrevivió la tercera parte después de un año. Los resultados hacen énfasis en la necesidad de realizar las pruebas cruzadas previas al trasplante en condiciones que permitan obtener la mayor información posible sobre los anticuerpos linfocitotóxicos presentes en el suero de los pacientes candidatos a trasplante renal.