Generación in vitro de un sustituto de pared vesical humana obtenido mediante ingeniería tisular / In vitro generation of a human bladder wall substitute by tissue engineering

Objetivos. Numerosas patologías que afectan a la vejiga, de origen congénito (extrofia) o adquirido (traumatismos, tumores), requieren la reconstrucción de la pared vesical utilizando intestino delgado, sigma o estómago, los cuales no están exentos de complicaciones. Por ese motivo, en el presente trabajo pretendemos desarrollar un nuevo modelo de pared vesical humana mediante ingeniería tisular que pudiese tener una utilidad clínica. Material y métodos. En primer lugar, se procedió a generar cultivos primarios de células epiteliales y estromales de la mucosa vesical a partir de pequeñas biopsias de la pared vesical humana, utilizando para ello técnicas de digestión enzimática mediante tripsina-EDTA y colagenasa. Posteriormente, se generó un sustituto tridimensional de la mucosa vesical utilizando como soporte biomateriales de fibrina-agarosa. El análisis de las muestras se realizó a los 14 días mediante examen histológico de muestras teñidas con hematoxilina-eosina. Resultados. La aplicación de los métodos de digestión enzimática permitió generar eficientemente cultivos primarios de células epiteliales y estromales de la mucosa vesical humana, comprobándose que la tasa de proliferación de las células estromales era superior a la de las células epiteliales. Una vez generados los sustitutos de la pared vesical, se comprobó el adecuado nivel de biocompatibilidad del biomaterial y las células estromales y epiteliales. La estructura histológica de los sustitutos de pared vesical presentaba una gran analogía con la mucosa vesical humana nativa. Conclusiones. El tejido vesical generado por ingeniería tisular muestra importantes similitudes estructurales e histológicas con el tejido vesical nativo. Estos resultados sugieren que los tejidos generados mediante ingeniería tisular podrían tener utilidad terapéutica en el futuro (AU)

Introduction. Certain urological congenital conditions, such as bladder exstrophy and acquired conditions such as trauma and tumors may require the use of different tissues like small bowel, sigmoid colon or stomach for bladder reconstruction. However, these tissues are often associated to important complications. The aim of this study is to develop a novel substitute of the human bladder wall by tissue engineering. Material and methods. We first generated primary cell cultures of epithelial and stromal bladder mucosa cells from small tissue biopsies of human bladder by using enzymatic methods based on trypsin-EDTA and collagenase I. Then, a three-dimensional substitute of the bladder mucosa was generated using fibrin-agarose biomaterials. The analysis of the tissue substitutes was carried out at day 14th of development by histological examination of samples stained with hematoxylin-eosin. Results. The use of enzymatic digestion methods allowed us to efficiently generate primary cell cultures of the human bladder epithelial and stromal cells. The proliferation rate was higher in stromal cells as compared to epithelial cells. Once the bladder mucosa substitutes were generated, a good biocompatibility of the stromal and epithelial cells into the biomaterial was found. The histological structure of the bladder wall substitutes was analogue to that of the native human bladder mucosa. Conclusions. The bladder mucosa substitute generated by tissue engineering showed structural and histological similarities with the native human bladder tissues and open the door to the future therapeutic use of these bioengineered tissues (AU)

Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis.

Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X-ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering.

Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells.

Decreases in the intracellular concentrations of both K(+) and Cl(-) have been implicated in playing a major role in the progression of apoptosis, but little is known about the temporal relationship between decreases in electrolyte concentration and the key events in apoptosis, and there is no information about how such decreases affect different intracellular compartments. Electron probe X-ray microanalysis was used to determine changes in element concentrations (Na, P, Cl, and K) in nucleus, cytoplasm, and mitochondria in U937 cells undergoing UV-induced apoptosis. In all compartments, the initial stages of apoptosis were characterized by decreases in [K] and [Cl]. The largest decreases in these elements were in the mitochondria and occurred before the release of cytochrome c. Initial decreases in [K] and [Cl] also preceded apoptotic changes in the nucleus. In the later stages of apoptosis, the [K] continued to decrease, whereas that of Cl began to increase toward control levels and was accompanied by an increase in [Na]. In the nucleus, these increases coincided with poly(ADP-ribose) polymerase cleavage, chromatin condensation, and DNA laddering. The cytoplasm was the compartment least affected and the pattern of change of Cl was similar to those in other compartments, but the decrease in [K] was not significant until after active caspase-3 was detected. Our results support the concept that normotonic cell shrinkage occurs early in apoptosis, and demonstrate that changes in the intracellular concentrations of K and Cl precede apoptotic changes in the cell compartments studied.

Electron probe microanalysis of gentamicin-induced changes on ionic composition of the vestibular gelatinous membrane.

Gentamicin-induced changes in ionic composition in the otolithic membrane of adult OF1 mice were evaluated in the gelatinous layers of the saccule and utricle by quantitative electron probe X-ray microanalysis. The otolithic membranes were plunge-frozen and freeze-dried to prevent the redistribution of elements. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background (P/B) ratio method and different salts dissolved in dextran as standards to calibrate the P/B ratio against the concentration of the elements P, S and K in the microprobe. Gentamicin selectively decreased the concentrations of P (P < 0.001) and S (P < 0.01) in the gelatinous membrane of the saccule, and had no effect in the utricle. The concentration of K also increased in the utricular gelatinous membrane (P < 0.05). The mechanism of ototoxicity in the gelatinous membrane is unknown, but the ability of aminoglycosides to block calcium channels may induce disturbances in the ionic equilibrium of the endolymphatic fluid, and thus affect the biochemical composition of the gelatinous membrane. This technique can be useful to evaluate the distribution of ions in the process of drug-induced ototoxicity.

X-ray microanalytical determination of P, S and K concentrations in the gelatinous membrane of the utricle.

Electron probe X-ray microanalysis was used to determine the concentrations of P, S and K (Cp, CS, CK) in the gelatinous membrane of the mouse utricle. The otolithic membranes were plunge-frozen in liquid N2, freeze-dried and carbon-coated. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 and K2SO4 salts dissolved in dextran solutions to calibrate the microprobe. P, S and K were measured and their concentrations plotted as bar graphs to study the frequency distributions. Regression analysis revealed a dependence between the concentrations of P and K (CK = 1454.10 - 2.83 CP, r = -0.68745, p < 0.05), and P and S (CS = 43.18 + 0.23 CP, r = 0.66949, p < 0.05); however, no correlation was found between CK and CP (r = -0.25424). The findings obtained in the present study show an inverse relationship between P and K ions, and direct relationship between P and S in the gelatinous membrane of the utricle.

Quantitative histochemistry of phosphorus in the vestibular gelatinous membrane: an electron probe X-ray microanalytical study.

Electron probe X-ray microanalysis was used to study the phosphorus concentration in the otolithic gelatinous membrane of the saccule and the utricle with scanning electron microscopy. The otolithic membranes were plunge-frozen in liquid N2 and freeze-dried. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 salts dissolved in dextran solutions. The otolithic gelatinous membrane consists of a 25-30 microns-thick layer overlying the cilia of the hair cells. Elements detected in the gelatinous membrane are: Na, P, S, Cl, K and Ca. Although Student's t-test did not show significant differences between saccular and utricular concentrations of phosphorus, the distribution of this element in the two organs was different. Regression analysis established that the concentrations of phosphorus in the saccular and utricular gelatinous membrane were dependent. The regression equation was: y = 18.02x2 + 133.9 (r = 0.83, P < 0.05) where y is the concentration of phosphorus in the utricle, and x2 the concentration of phosphorus in the saccule. The findings obtained in the present study could be related to structural differences in organic phosphate residues of the phosphoproteins associated to collagen, or to different polyphosphoinositide turnover rates in the cell membrane.

X-ray microanalytical histochemistry of human circumpulpar and mantle dentine.

An X-ray microanalytical histochemistry study was carried out, on thick sections observed under scanning electron microscopy, of five freshly extracted human premolars and five molars. In particular the circumpulpar and mantle dentine were examined to determine levels of calcium, phosphorus and sulphur, the basic elements involved in the process of biomineralization. Semiquantitative analysis was subsequently performed after the appropriate ZAF (Z, atomic number; A, absorption; F, secondary fluorescence) correction. Ca/P mass ratio was found to be similar for both regions in molars and slightly higher in circumpulpar dentine in premolars implying that this parameter is independent of the processes of biomineralization in the two dentinal regions. In contrast, statistically significant differences were recorded in sulphur content upon comparing mantle and circumpulpar dentine between premolars and molars (P less than 0.02). If sulphur, which has been associated with sulphated glycosaminoglycans (GAGs) decreases as mineralization progresses it would therefore seem reasonable to posit sulphur-rich areas in both topographical regions of the matrix--more significant in premolars than molars--able to undergo subsequent mineralization if required. The possible application of these findings in remineralization therapies is suggested.