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BACKGROUND@#Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). @*METHODS@#The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. @*RESULTS@#The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. @*CONCLUSION@#Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.
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BACKGROUND@#Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). @*METHODS@#The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. @*RESULTS@#The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. @*CONCLUSION@#Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.
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Objective: Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells [MSCs] to improve in vitro maintenance conditions for hepatocytes
Materials and Methods: In this experimental study, following serum deprivation, human adipose tissue-derived MSCs [hAT-MSCs] were cultured for 24 hours under normoxic [N] and hypoxic [H] conditions. Their conditioned media [CM] were subsequently collected and labeled as N-CM [normoxia] and H-CM [hypoxia]. Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal [William's] medium supplemented with 4% N-CM or H-CM. Untreated William's and hepatocyte-specific media [HepZYM] were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells
Results: We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS [3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]- 2H-tetrazolium] assay. Indocyanine green [ICG] uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin [Alb] and urea secretion compared to the other groups [P<0.0001]. However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor [VEGF] in the H-CM group compared to the N-CM group [P=0.063]
Conclusion: The enrichment of William's basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture
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Introduction: Memory and cognitive impairments are some of devastating outcomes of Multiple Sclerosis [MS] plaques in hippocampus, the gray matter part of the brain. The present study aimed to evaluate the intrahippocampal injection of Ethidium Bromide [EB] as a simple and focal model to assess cognition and gray matter demyelination
Methods: Thirty Wistar rats were divided into three groups: control group, which received saline, as solvent of EB, into the hippocampus; and two experimental groups, which received 3 microL of EB into the hippocampus, and then, were evaluated 7 and 28 days after EB injection [n=10 in each group], using a 5-day protocol of Morris Water Maze [MWM] task as well as Transmission Electron Microscopy [TEM] assay
Results: Seven days after EB injection, the behavioral study revealed a significance increase in travelled distance for platform finding in the experimental group compared to the control group. In addition, the nucleus of oligodendrocyte showed the typical clumped chromatin, probably attributed to apoptosis, and the myelin sheaths of some axons were unwrapped and disintegrated. Twenty-eight days after EB injection, the traveled distance and the time spent in target quadrant significantly decreased and increased, respectively in experimental groups compared to the control group. Also, TEM micrographs revealed a thin layer of remyelination around the axons in 28 days lesion group
Discussion: While intracerebral or intraventricular injection of EB is disseminated in different parts of the brain and can affect the other motor and sensory systems, this model is confined locally and facilitates behavioral study. Also, this project could show improvement of memory function subsequent to the physiological repair of the gray matter of the hippocampus
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Animales de Laboratorio , Hipocampo , Ratas Wistar , Trastornos del Conocimiento , Inyecciones , Lesiones EncefálicasRESUMEN
The effect of pentoxifylline (PTX) administration on histomorphological parameters of streptozotocin (STZ)-induced type 1 diabetes mellitus (DM) in male rat testes were evaluated. We randomly divided 40 male rats into the following four groups: group 1: control or normal glycemic (NG) rats; group 2 or NG rats that received only normal saline (NS), (NG+NS); group 3 or diabetic rats which were not treated by PTX (DM+vehicle solution (NS)); and group 4 which comprised diabetic rats treated with 50 mg/kg of PTX (DM+PTX). Type 1 DM was induced by intraperitoneal injection of STZ (55 mg/kg). Rats were held for 30 days after which the experimental group received PTX twice daily (25 mg/kg) or NS. After 14 days of treatment by PTX or NS, the left testes from all rats were extracted and prpared for histological study. Apoptotic cells, blood vessel density, and spermatogenesis were evaluated. Data were analyzed by ANOVA test. PTX-treated-diabetic rats showed a significant decrease in number of apoptotic cells and decrease in blood vessel density compared to the DM+NS rats. A significant increase in spermatogenesis was observed in the PTX-treated diabetic group, compared to the DM+NS groups. It was concluded that PTX administration to STZ-induced type 1 DM rats affected apoptotic cell number positively. Moreover, blood vessel density significantly decreased and improvements were observed in spermatogenesis.
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Animales , Humanos , Masculino , Ratas , Células Sanguíneas , Vasos Sanguíneos , Recuento de Células , Diabetes Mellitus , Diabetes Mellitus Tipo 1 , Inyecciones Intraperitoneales , Pentoxifilina , Espermatogénesis , Estreptozocina , TestículoRESUMEN
Wound healing represents a dynamic physiological process influenced by many factors. The aim of the present study was to evaluate the effects of hydro-alcoholic extracts of aerial components of Teucrium polium on the tensile strength in rat. Twelve Wistar rats were randomly divided into two equal [n= 6] treatment and control groups, pressure ulcer were made over the dorsal thoracic region according to the shyn model. Animals were treated with topical hydro-alcoholic extracts of aerial components of Teucrium polium twice a day post surgery until complete healing was achieved. Tensiometry were then studied. No significant difference in biomechanical parameters was observed among the ointment containing of extract 1%, vehicle and control groups. This study shows that topical application of Teucrium polium. Aerial components of Teucrium polium did not have any effect on healing of pressure ulcer in an animal model. Further studies are requiring for assessment of other extracts by different solvents of this plant on pressure ulcer wound healing
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Animales de Laboratorio , Fitoterapia , Extractos Vegetales , Ratas Wistar , Úlcera por Presión/terapia , Cicatrización de Heridas , Resistencia a la TracciónRESUMEN
Tubal ectopic pregnancy [tEP] is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide [NO] is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase [eNOS] enzyme in the regulation of many reproductive events that occur in the fallopian tube [FT]. The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women [n=10]. Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase [p=0.00]. Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups [p=0.78]. This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP.
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Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury [SCI]. However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Mesenchymal stem cells were cultured from adult rats' bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups [n = 10 each]: laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment
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Masculino , Animales de Laboratorio , Células de Schwann , Traumatismos de la Médula Espinal , Ratas Wistar , Médula Ósea , Diferenciación Celular , Inmunohistoquímica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To study the impact of ultraweb nano-fibrillar substrates on in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells into hepatocyte-like cells. For a duration of up to 36 days, mouse bone marrow mesenchymal stem cells [MSCs] were cultured on an artificial basement membrane containing ultraweb nanofiber [nano+] and plates [nano] coated with Matrigel and used growth and differentiating factors for hepatogenic differentiation. Then, to study the impact of nanofibers on hepatocyte differentiation, mRNA expressions for liver-specific genes [CK7, AFP, FOXa2, ALB, CYP1a1, HNF4a] were analyzed using real-time PCR, and ultrastructures of differentiated cells were evaluated using scanning and transmission electron microscope. Both experimental groups expressed mRNA of liver-specific genes in a time dependent manner. However, mRNA expression trends of liver-specific genes, especially those of FOXa2 and HNF4a, were better in the nano+ culture as compared to the nanoculture [p<0.05]. Furthermore, the ultrastructures of differentiated cells in the 36 day-old nano+ culture were more mature as compared to those of the nanogroup. The results suggest that topographical properties of the extracellular matrices produced by ultraweb nanofibers are effective for the in vitro reproduction of more differentiated MSC-derived hepatocyte-like cells
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Animales de Laboratorio , Médula Ósea , Hepatocitos , Ratones , Nanofibras , ARN Mensajero , Reacción en Cadena de la Polimerasa , Microscopía Electrónica de Transmisión de RastreoRESUMEN
The aim of this study was to differentiate human mesenchymal stem cells [hMSCs] into cartilage in a micromass culture system and study of their structure by light and electron microscopy. Human bone marrow cells obtained from volunteer patients were plated in 75-cm2 flasks and their MSCs were expanded through several sub-cultures. The passage 4 cells were used to establish micromass culture system for chondrogenic differentiation. For this purpose, 200,000 fibroblastic cells were placed in centrifuge tubes and pelleted at 250 g for 5 minutes. About 0.5 ml chondrogenic induction medium was then added to the pellet and the culture incubated in 5% CO2 at 37°C for 21 days. Then, some pellets were utilized to evaluate chondrogenic differentiation by either RT-PCR analysis of some cartilage marker molecules or specific staining for detecting cartilage matrix, and other pellets were used for light and electron microscopic study of differentiated tissue. Primary culture of the bone marrow cells were initially composed of the spindle- and round shaped cells, from which the spindle cells remained and expanded through several passages. At the end of differentiation period, RT-PCR analysis showed high production of collagen II and X and aggrecan mRNA inside the differentiated cells, and toluidine blue staining indicated intermediate accumulation of the metachromatic matrix among the inddced cells. In general, light micrograph indicated a rather cellular state of the differentiated tissue in which the peripheral part had more metachromatic matrix than central zone. More detailed study of the sections revealed that induced aggregates of the cells were composed externally of very thin layer of elongated cells reminiscent of perichondrium and internally a mass of oval cells comprising the main part of the pellet. Ultra-thin sections showed that the cells in perichondrium-like layer were very similar to fibroblastic cells and those located centrally had a set of well-developed organelles, characteristic of highly active cells. Some fat cells were seen in central zone. Conclusion: Cartilage tissue differentiated from MSCs in micromass culture system seemed to be structurally very similar to developing cartilage not to adult mature cartilage
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Introduction: Laser pointers are devices that produce a weak laser beam of 630-680 nmwavelength and 1-5 mW power [Class II or III A laser]. These devices generally emit a red beam that is used by lecturers and teachers for presentations. Some children use pointers as toys and sometimes direct the beam to their own or others' eyes
Material and Methods: Following irradiation by a laser pointer beam for 8 seconds the eyes of Chinchilla rabbits were examined by opthalmoscope, and fluorescein angiography was performed 5, 10 and 15 min after the exposure. The rabbits were killed immediately or 24h after exposure, the eyes were enucleated, and the histological features of sections from fundus, retina and choroid were observed by transmission electron microscopy
Results: A fluorescein block was found in the irradiated area immediately after irradiation and it increased in size with increasing time after exposure. The ultrastructural study showed acute oedema shortly after exposure, and thick collagenic bundles after 24h
Conclusion: Laser pointers with labelled power of less than 1mW are capable of producing visible and ultrastructual lesions in pigmented rabbit eyes