RESUMEN
Collagen and elastin are the main structural proteins in mammal bodies. They provide mechanical support, strength, and elasticity to various organs and tissues, e.g. skin, tendons, arteries, and bones. They are readily available, biodegradable, biocompatible and they stimulate cell growth. The physicochemical properties of collagen and elastin-based materials can be modified by cross-linking. Glutaraldehyde is one of the most efficient cross-linking agents. However, the unreacted molecules can be released from the material and cause cytotoxic reactions. Thus, the aim of our work was to investigate the influence of a safer, macromolecular cross-linking agent--dialdehyde starch (DAS). The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5 and 10% of DAS and neutralized via dialysis against deionized water were tested. The homogenous, transparent, stiff hydrogels were obtained. The DAS addition causes the formation of intermolecular cross-linking bonds but does not affect the secondary structure of the proteins. As a result, the thermal stability, mechanical strength, and, surprisingly, swelling ability increased. At the same time, the surface properties test and in vitro study show that the materials are attractive for 3T3 cells. Moreover, the materials containing 10% of DAS are more resistant to enzymatic degradation.
Asunto(s)
Colágeno/química , Elastina/química , Almidón/análogos & derivados , Células 3T3 , Animales , Materiales Biocompatibles/química , Conformación de Carbohidratos , Movimiento Celular , Ratones , Microscopía Electrónica de Rastreo , Espectroscopía Infrarroja por Transformada de Fourier , Almidón/químicaRESUMEN
Hydrogels based on collagen and elastin are very valuable materials for medicine and tissue engineering. They are biocompatible; however their mechanical properties and resistance for enzymatic degradation need to be improved by cross-linking. Up to this point many reagents have been tested but more secure reactants are still sought. Squaric acid (SqAc), 3,4-dihydroxy 3-cyclobutene 1,2-dione, is a strong, cyclic acid, which reacts easily with amine groups. The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5%, 10% and 20% of SqAc and neutralized via dialysis against deionized water were tested. Cross-linked, 3-D, transparent hydrogels were created. The cross-linked materials are stiffer and more resistant to enzymatic degradation than those that are unmodified. The pore size, swelling ability and surface polarity are reduced due to 5% and 10% of SqAc addition. At the same time, the cellular response is not significantly affected by the cross-linking. Therefore, squaric acid would be regarded as a safe, effective cross-linking agent.
Asunto(s)
Materiales Biocompatibles/química , Colágeno/química , Ciclobutanos/química , Elastina/química , Hidrogeles/química , Reactivos de Enlaces Cruzados/químicaRESUMEN
The aim of this study is to present the comparison of four different methods for urothelial cell isolation and culture and compare them to methods cited in the literature. Four different techniques were examined for urothelium isolation from rat bladders. Isolation effectiveness was calculated using trypan blue assay. Confirmation of isolated cell phenotype and comparison with native bladder tissue was confirmed using immunohistochemical (IHC), immunocytochemical (ICC) and immunofluorescence (IF) analysis. The method with bladder inversion and collagenase P digestion resulted in the highest number of isolated cells. These cells showed positive expression of cytokeratin 7, 8, 18, α6-integrin and p63. Our results and the literature review showed that the best method for urothelium bladder isolation is dissection of the epithelium layer from other bladder parts and digestion of mechanically prepared tissue in a collagenase solution.
Asunto(s)
Separación Celular/métodos , Vejiga Urinaria/citología , Urotelio/citología , Animales , Células Cultivadas , Colagenasas/metabolismo , Integrina alfa6/metabolismo , Queratina-18/metabolismo , Queratina-7/metabolismo , Queratina-8/metabolismo , Masculino , Ratas , Ratas Wistar , Regeneración , Soluciones , Urotelio/metabolismo , Urotelio/fisiologíaRESUMEN
BACKGROUND: Numerous studies are ungoing to develop a substitute for the native urinary bladder wall. The principals of tissue engineering approaches to urinary bladder wall augmentation require a favorable environment for smooth muscle regeneration, which is crucial for bladder function. This study was performed to evaluate bone marrow mesenchymal stem cells (BMSC) seeded on to amniotic membranes fixed to Tachosil sponges as grafts for urinary bladder muscle layer augmentation in a syngenic rat model. MATERIALS AND METHODS: Amniotic membranes seeded with BMSC and covered by Tachosil sponges were implanted as multilayer grafts into nine rats to regenerate the urinary bladder wall. The control group consisted of 12 healthy rats. Urodynamic examinations included contraction, elasticity, compliance, and urinary bladder motor activity. Hematocylin and eosin and Masson's trichrome stains were used to evaluate muscle regeneration; histological data were digitally analyzed with the ImageJ tool. RESULTS: The area of muscle bundles ranged from 5% to 25% or 32% to 41% in control versus reconstructed bladders, respectively. Among nine animals with reconstructed urinary bladders, urodynamic evaluation revealed bladder motor hyperactivity with regular (n = 4) or irregular (n = 1) storage and voiding phases, as well as proper bladder motor activity with a large bladder capacity (n = 1). No bladder contractility was recorded in one case and large stones developed in two animals, which made functional studies impossible. CONCLUSIONS: Regenerated smooth muscle cells created an autonomic cell population that was poorly assimilated to the rest of the urinary bladder wall. The histological presence of a regenerated muscle layer did not guarantee proper urinary bladder function.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Contracción Muscular , Músculo Liso/cirugía , Regeneración , Medicina Regenerativa/métodos , Ingeniería de Tejidos , Vejiga Urinaria/cirugía , Urodinámica , Amnios/trasplante , Animales , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Adaptabilidad , Combinación de Medicamentos , Fibrinógeno/farmacología , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Músculo Liso/fisiopatología , Ratas , Ratas Wistar , Regeneración/efectos de los fármacos , Trombina/farmacología , Técnicas de Cultivo de Tejidos , Andamios del Tejido , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/patología , Vejiga Urinaria/fisiopatología , Urodinámica/efectos de los fármacosRESUMEN
The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells research as this work generates questions about undocumented sources of oviduct progenitor cells.
Asunto(s)
Técnicas de Cultivo de Célula/veterinaria , Pollos , Células Epiteliales/fisiología , Oviductos/citología , Células 3T3 , Animales , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Técnicas de Cocultivo/veterinaria , Medios de Cultivo , Femenino , Regulación de la Expresión Génica/fisiología , Ratones , Propiedades de Superficie , TemperaturaRESUMEN
More evidence indicate that prostate inflammation can lead to prostate cancer development. Prostate cancer affects elderly men. Prostate cancer prophylaxis is an important issue because life expectancy is very long now. Ciprofloxacin is an antibacterial agent used mainly in urinary tract infections and prostate inflammation. This drug acts also against cancer cells by the inhibition of topoisomerase II. These properties should allow it to inhibit the development of prostate cancer. Firstly, ciprofloxacin can stop the acute and chronic prostate inflammation which can lead to cancer development. Secondly, ciprofloxacin can potentially kill prostate cancer cells in their early stage of development. Ciprofloxacin accumulates mainly in the prostate after oral intake thus ciprofloxacin seems to be a perfect candidate as a prophylactic agent.