Development of secretome-based strategies to improve cell culture protocols in tissue engineering.

Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration.

Is the bone tissue of the femoral neck demineralised in patients with hip fracture?

The aim of this study is to establish the falsifiability of the "osteoporotic hypothesis" for hip fracture, according to which the bone density and mineral composition of bone tissue in patients with hip fracture is poorer than when no such fracture is present, and that this circumstance is relevant to the occurrence of a fracture. The study population consisted of forty patients treated with arthroplasty. Twenty patients with femoral neck fracture and another twenty with hip osteoarthritis received the same diagnostic protocol and the same antibiotic, anaesthetic, surgical and antithrombotic prophylaxis. Levels of calcium (Ca), phosphorus (P) and vitamin D in blood, amongst other values, were determined, and five samples of bone tissue from the proximal femoral metaphysis were obtained and characterised by optical microscopy and microanalytical analysis. No statistically significant differences were observed between the two groups with respect to the trabecular number, area or thickness, or inter-trabecular distance. However, there were differences in the length of the trabeculae, which was greater in the patients with hip osteoarthritis (p = 0.002), but not when the groups were compared by gender. When compared by age, a greater inter-trabecular distance was observed in the patients aged over 75 years (p = 0.036) but there were no differences in the remaining parameters. Serum levels of Ca (p = 0.03), P (p < 0.01) and vitamin D (p < 0.01) were lower in the fracture group. In the quantitative microanalytical analysis, no significant differences were observed in bone levels of Ca or P or in the Ca/P index, nor was there any correlation between serum and levels of bone Ca or P (Ca-0.197:p = 0.314;P-0.274:p = 0.158).Multiple linear regression revealed no correlation between the diagnoses, vitamin D and bone levels of Ca or P. Despite the reduced serum levels of Ca and P in the patients with hip fracture, no correlation was observed with bone levels of Ca and P,which were similar in both groups. There were differences in the organic bone structure, in terms of length and inter-trabecular distance. For patients with osteoporosis, treatment should be aimed at increasing the synthesis of bone trabeculae to reinforce their structure. Nevertheless, no such treatment can prevent falls, and therefore no reduction in hip fractures amongst this population can be assured.

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)

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

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.

Effect of normobaric hypoxia on the testis in a murine model.

High-altitude hypoxia generates spermiogram impairment due to germinal epithelium, Leydig cells, sperm and seminal plasma alterations, but precise mechanisms involved are unknown. The objective of this work was to analyse the effect of normobaric hypoxia on the morphology of testicular interstitium and some associated molecular and hormonal factors. Twenty-four mice were exposed to normobaric hypoxia (8.1% inspired oxygen fraction) during 20 days. The effects on body weight, testicular weight, vascularisation, testosterone, HIF1-α and VEGF were analysed at different periods of exposure and compared to controls. Hypoxic mice had lower body weight than mice kept in normoxia. Testicular weight raised significantly the 1st day, but remained normal during the rest of experiment. Number of blood vessels per field and mean diameter of vessels were higher in hypoxic mice. Plasmatic and testicular testosterone raised during first 24 h of hypoxia, but decreased on the 5th day. Vascular/interstitial ratio (proportion of interstice occupied by blood vessels) duplicated at the end of the experiment. Most substantial early effects of hypoxia were testicular oedema, increase in number and diameter of blood vessels and elevation of plasmatic and testicular testosterone. Normobaric hypoxia generates similar effects to those induced by hypobaric hypoxia.

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering.

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

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.

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis.

Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P<0.001) and Cl (P<0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P<0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na+/K+ ATPase. This later phase was also characterized by a significant increase (P<0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.

The crystalline pattern of calcium in different topographical regions of the otoconial membrane. An electron probe and spectroscopic diffraction study.

Quantitative electron probe microanalysis and electron spectroscopic diffraction analysis was used to determine the gradient of distribution of calcium and its crystalline pattern at different levels (lower gelatinous membrane, upper gelatinous membrane and otoliths) in the otoconial membrane of adult OF1 mice. Our quantitative electron probe microanalytical data, obtained with scanning-transmission electron microscopy, indicated that there was a gradient in calcium concentration which increased from the vestibular surface towards the otoliths. Differences between the three regions of the otoconial membrane were statistically significant in both the utricle and saccule. Our results with electron spectroscopic diffraction revealed an increasing crystalline development from the lower gelatinous membrane towards the otoliths. Our findings with both techniques suggest that the gelatinous membrane is involved in the maturation and crystallization of the otoliths.

[Morphological and functional correlations of the protective action of coenzyme A in cisplatin ototoxicity (II)]. / Correlaciones morfofuncionales de la acción protectora del coenzima A en la ototoxia por cisplatino (II).

Earlier studies have shown that coenzyme A (CoA) protects against cisplatin (CP) induced deafness in guinea pigs. We studied the correlation between the latency of the N1 wave in electrocochleography and total loss of cilia in hair cells by scanning electron microscopy.

Pantothenic acid and coenzyme A in experimental cisplatin-induced ototoxia.

We have observed that pantothenic acid (PA) prevents deafness induced by cisplatin (CP) in the guinea pig if both drugs are administered jointly. When deafness was previously produced, recovery was sometimes obtained after the administration of PA; so, we studied the effects of PA on cisplatian-induced ototoxia in guinea pigs, both as a prophylactic agent in healthy animals, and as a therapeutic agent in animals previously made deaf by the drug. To elucidate why PA protects the ear from the toxic effects of CP, we used coenzyme A (CoA) instead of PA-since PA is a component of CoA-to test the hypothesis that the action of PA is due to CoA. The results were practically the same in both experiences, the compound action potential of the auditory nerve (CAP) was tested and cochleas were examined by scanning electron microscopy (SEM). Our results suggest that the protective effect of PA takes place through CoA. Both substances had the same effect on CP ototoxicity, but CoA appears to be much more active, since the dose tested here was much lower than that of PA.

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.

Gentamicin ototoxicity in otoconia: quantitative electron probe X-ray microanalysis.

Chronic gentamicin ototoxicity was evaluated in the otolithic membrane of adult OF1 mice at the otoconial layer of the saccule and utricle by quantitative electron probe X-ray microanalysis of Ca and K. The otolithic membranes were plunge-frozen and freeze-dried. The analysis was carried out with an energy dispersive detector using the peak-to-back-ground ratio method and different inorganic salts of Ca and K as standards to calibrate the microprobe. Ca and K in the otoconia are related via a linear function in both the saccule and the utricle. This association is not maintained after exposure to gentamicin, which suggests that this aminoglycoside antibiotic interferes with the Ca-K equilibrium in the otoconia. A dose of 200 mg/kg gentamicin twice a day for 5 days did not affect Ca in the mineral phase of the otoconia, but did increase K in both saccular (p < 0.05) and utricular (p < 0.01) otoconia. These increases in K may reflect a modification in the composition of the endolymph, resulting from cellular damage at the plasma membrane.

Early morphofunctional alterations induced by cisplatin in the cochlea. A quantitative and electrocochleographic study.

The early morphological and functional alterations of the cochlea were studied in guinea pigs treated with cisplatin, administered to two groups of animals as a single dose of 4 or 10 mg/kg body weight. Morphological changes were observed by scanning electron microscopy, and functional alterations were investigated by electrocochleography to record compound action potentials. Statistically significant alterations in neurosensory stereocilia, consisting of partial or total denudation, were observed only after the experimental administration of the higher dose of cisplatin; these changes were consistently associated with distortions in the compound action potential.

Anatomical and clinical correlation with histological and histometric patterns in cryptorchism.

One feature rarely included in studies on cryptorchism is an adequate correlation between anatomical and clinical data and histological patterns of the testis. The present study correlates anatomical and clinical data from 50 patients (age range 2 months-14 years) using 4 different histological and histometric patterns established previously. Three different patterns of progressively severe histological changes corresponding to increasingly marked dysgenetic alterations in the anatomical and clinical patterns were detected. Less severe changes were correlated with mature anatomical and clinical features, whereas testes showing severe histological-histometric changes were correlated with immature anatomical and clinical features. Non-abdominally located testes with Sertoli cell hyperplasia were not significantly correlated with any of the anatomical and clinical features. The histometric patterns described should facilitate the diagnosis of the stage of testicular development on the basis of anatomical and clinical data.

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: calibration curves and electron beam sensitivity.

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K K alpha P/B or Y = (P/B).Z2/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10-25 kV for Ca standards. K standards were also independent when P/B was corrected for Z2/A. Background counts in the otoconia (Bo) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. Bo was not dependent on the spectra acquisition time, with the exception of Bo under K alpha K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z2/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.

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.