Sperm and oocyte as carriers for bovine viral diarrhoea virus biotypes during in vitro fertilization.

Bovine viral diarrhoea virus (BVDV) is an important viral agent causing the reproductive failure in cattle. The objectives of the study were to assess the role of male and female gametes, as carriers of cytopathic (CP) and non-cytopathic (NCP) BVDV to embryonic cells during in vitro fertilization. In this respect, sperm and oocytes were separately exposed to concentrations of 104.5 or 105.5 TCID50 /mL CP and NCP BVDV, for 2 h before fertilization. After washing, the intact gametes with the infected gametes were inseminated. Seven days post-fertilization, the virus-exposed embryos were examined for presence of the viral genome by RT-PCR. One-way anova with post-hoc Tukey's HSD test and an independent samples t-test were used to compare within and between groups, respectively. The results presented a significant decrease in the blastocyst rates for CP-infected groups than NCP-infected groups (p ≤ .01). Compared to the controls and the infected oocyte groups, the cleavage rates of the infected sperm groups (NCP and CP BVDV) were significantly reduced both in low (104.5 TCID50 /mL) and high (105.5 TCID50 /mL) titres of the virus (p ≤ .01). The proportion of embryos which was developed to blastocyst stages was significantly lower for CP and NCP-infected groups than the control groups (p ≤ .001). According to the molecular results, all samples of the retarded/degenerated embryos (at least one blastocyst within each one) in CP and NCP groups, one sample (at least one blastocyst in that) within a CP-infected group, and six samples (at least one blastocyst in each one of those) of NCP-infected groups contained the viral nucleic acid. Likewise, the results of viral enrichment showed all reactions in which RT-PCR were positive induced CPEs in MDBK monolayers. In conclusion, it is clear that CP and NCP BVDV were able to traverse zona pellucida during fertilization, and they had also negative effects on embryo development.

In Vitro Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Diacerein.

Background: Tissue engineering is the application system that tries to restore damaged tissues by different approaches, such as cellular therapy, application of cell differential factors, and various materials. One of the important goals in tissue engineering is to guide stem cells directly to the desired tissue, and researchers tried to utilize different molecules as effective factors to improve this technique. Objectives: This study aims to demonstrate the effects of diacerein, a slow-acting drug for the treatment of osteoarthritis, on mesenchymal stem cell proliferation and evaluate its potential in the chondrogenesis process. Methods: Stem cells were isolated from adipose tissue, characterized by flow cytometry, and cells were treated with 10-5M diacerein for three weeks. Chondrogenic gene expression of SOX9, COL2A1, ACAN, and TGFB1 were analyzed by qRT-PCR and immunocytochemistry techniques. Results: Our results showed that diacerein increased the expression of the following genes involved in chondrogenesis: SOX9 (2.9-fold, P < 0.00), COL2A1 (2.2-fold, P < 0.00), ACAN (2.7-fold, P < 0.00), and TGFB1 (2.6-fold, P < 0.00). Immunocytochemistry results also showed increased production of collagen type II as the main protein marker for chondrocytes. Conclusions: We observed that diacerein alone could initiate and enhance chondrogenesis, and it can be used as a differentiation factor for stem cells to chondrocyte besides its ability to inhibit IL-1ß. Knowing the actual function of diacerein, it could be a good candidate for the treatment of osteoarthritis.

In-vitro cellular and in-vivo investigation of ascorbic acid and ß-glycerophosphate loaded gelatin/sodium alginate injectable hydrogels for urinary incontinence treatment.

Urinary incontinence is one of the most common disorders especially in adult women. In this study, cellular and in-vivo analyses were performed on (3-glycidyloxypropyl) trimethoxysilane (GPTMS) and CaCl2 cross-linked alginate and gelatin hydrogels containing ß-glycerophosphate and ascorbic acid to evaluate the regenerative potential as injectable compression agents for the treatment of urinary incontinence. The hydrogels were prepared with different percentages of components and were named as GA1 (7.2% w/v gelatin, 6% w/v sodium alginate, 0.5:1w/w GPTMS, CaCl2 1% (wt) sodium alginate, 50 µg/mL ascorbic acid, 1.5 mg/mL ß-glycerophosphate), GA2 (10% w/v gelatin, 8.5% w/v sodium alginate, 0.5:1 w/w GPTMS, CaCl2 1% (wt) sodium alginate, 50 µg/mL ascorbic acid, 1.5 mg/mL ß-glycerophosphate), and GA3 (10% (w/v) gelatin, 8.5% w/v sodium alginate, 1:1 w/w GPTMS, CaCl2 1% (wt) sodium alginate, 50 µg/mL ascorbic acid, 1.5 mg/mL ß-glycerophosphate) hydrogels. The results of cell studies showed that although all three samples supported cell adhesion and survival, the cellular behavior of the GA2 sample was better than the other samples. Animal tests were performed on the optimal GA2 sample, which showed that this hydrogel repaired the misfunction tissue in a rat model within 4 weeks and the molecular layer thickness was reached the normal tissue after this duration. It seems that these hydrogels, especially GA2 sample containing 10% (w/v) gelatin, 8.5% (w/v) sodium alginate, 0.5:1 (w/w) GPTMS, CaCl2 1% (wt) sodium alginate, 50 µg/mL ascorbic acid, and 1.5 mg/mL ß-glycerophosphate, can act as an injetable hydrogel for urinary incontinence treatment without the need for repeating the injection.

Design of a decellularized fish skin as a biological scaffold for skin tissue regeneration.

The use of decellularized natural skin as an extracellular matrix (ECM) may be a great candidate to regenerate damaged tissues. In this study, decellularized scaffolds from fish skin were designed by different techniques (physical, chemical, and enzymatic methods) and investigated by analyses such as Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), Tensile strength, Degradability, Histological studies, Toxicity test, and Determination of DNA content. Results showed that the best sample is related to the decellularized skin by hypertonic & hypotonic technique and Triton X100 solutions. Structural and mechanical results were demonstrated that samples have similar properties to human skin to regenerate it. The cytotoxicity results showed that decellularized skin by hypertonic & hypotonic method and Triton solution is non-toxic with minimal amount of genetic materials. Cellular results with epithelial cells indicated good adhesion on decellularized matrix, so it can be a suitable candidate for skin tissue regeneration.

Graphene Oxide Negatively Regulates Cell Cycle in Embryonic Fibroblast Cells.

BACKGROUND: Unique properties of graphene and its derivatives make them attractive in the field of nanomedicine. However, the mass application of graphene might lead to side effects, which has not been properly addressed in previous studies, especially with regard to its effect on the cell cycle. METHODS: The effect of two concentrations (100 and 200 µg/mL) of nano- and microsized graphene oxide (nGO and mGO) on apoptosis, cell cycle, and ROS generation was studied. The effect of both sizes on viability and genotoxicity of the embryonic fibroblast cell cycle was evaluated. MTT and flow cytometry were applied to evaluate the effects of graphene oxide (GO) nanosheets on viability of cells. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The results of this study showed that GO disturbed the cell cycle and nGO impaired cell viability by inducing cell apoptosis. Interestingly, both nGO and mGO blocked the cell cycle in the S phase, which is a critical phase of the cell cycle. Upregulation of TP53-gene transcripts was also detected in both nGO- and mGO-treated cells compared to the control, especially at 200 µg/mL. DNA content of the treated cells increased; however, because of DNA degradation, its quality was decreased. CONCLUSION: In conclusion, graphene oxide at both nano- and micro-scale damages cell physiology and increases cell population in the S phase of the cell cycle.

Bioactive and biostable hyaluronic acid-pullulan dermal hydrogels incorporated with biomimetic hydroxyapatite spheres.

In this study, hyaluronic acid-pullulan injectable hydrogels were incorporated with biomimetic hydroxyapatite spheres and were modified using silane coupling agents in order to improve the physicochemical, mechanical, and biological performance of hydrogels. So the biomimetic hydroxyapatite spheres were synthesized through immersion of gelatin-siloxane microspheres in the simulated body fluid. The results of field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction spectra confirmed the formation of hydroxyapatite on the surface of hybrid spheres. The morphology observation of the prepared hydrogels confirmed the uniform distribution of hydroxyapatite spheres in gel structure. The chemical characterization proved the possible interactions between the polymers and the created complex with a silane coupling agent to provide more durability. Improvement of storage modulus and viscosity indicated the positive role of hydroxyapatite spheres on the stability and long-lasting durability of hydrogels. A slight reduction was observed in the absorption capacity and water retention in hybrid hydrogels; even though, the great resistance to enzymatic biodegradation led to higher durability of hydroxyapatite-contained gels. Improvement in L-929 fibroblast cell adhesion and spreading especially around the biomimetic hydroxyapatite spheres along with higher cell viability demonstrated the initial potential of hydrogels for further pre-clinical and clinical studies in order to recommend the gels for dermal rejuvenation applications.

Effects of Different Concentrations of Reversine on Plasticity of Mesenchymal Stem Cells.

Dedifferentiation can be induced by small molecules. One of these small molecules used in this study in order to increase the plasticity of differentiation of stem cells was reversine. The objective of present study was to investigate the effect of different concentrations of reversine on the plasticity of ovine fetal bone-marrow mesenchymal stem cells (BM-MSCs). BM-MSCs were extracted from ovine fetal and cultured. Passaged-3 cells were evaluated for their differentiation potential into osteocytes and adipocytes cells. In the present study, BM-MSCs were culture plated in the presence of 0, 300, 600, 900 and 1200 nM of reversine. The number of viable cells was determined by MTT test after addition of different concentrations of reversine. Furthermore, expression of the nanog gene was evaluated. The culture without reversine was taken as the control group. Expression of nanog was analysed by immunocytochemistry. Multi-lineage differentiation showed that the BM-MSCs could be differentiated into adipose cells and osteocytes. Our results indicated that the addition of 1200 nM of reversine to medium significantly decreased overall proliferation compared to the other treatment groups (p > 0.05). Real-time PCR analysis showed that after addition of 600 nM of reversine significantly increased nanog expression compared to other treatments. All treatments received reversine were seen to be relative expression of nanog. Our findings confirm that low concentrations reversine increases the plasticity of ovine BM-MSCs.

Exposing Mouse Oocytes to MitoQ During In Vitro Maturation Improves Maturation and Developmental Competence.

BACKGROUND AND PURPOSE: Mitochondrion is the main indicator of oocyte quality and one of the components of oocyte, which is sensitive to oxidative damage during the maturation process. Mitoquinone mesylate (MitoQ) is a strong antioxidant targeting mitochondria as well as anti-apoptotic agent. However, the effect of MitoQ on the quality of oocytes during in vitro maturation (IVM) is still unknown. OBJECTIVES: This study investigated the possible effects of MitoQ on maturation and developmental competency in mice oocytes. MATERIALS AND METHODS: The oocytes were collected at germinal vesicle stage from 6-8-week old female NMRI mice and then cultured in TCM-199 medium supplemented with 0, 0.01, 0.02 and 0.04 µM MitoQ. The sham group was treated with DMSO (0.01% v.v). Then intracellular Glutathione (GSH), reactive oxygen species (ROS) levels, mitochondria membrane potential (ΔΨm), as well as in vitro fertilization (IVF) rate in the 18-20 h matured oocytes and metaphase II (MII) oocytes (in vivo-control), were assessed. RESULTS: The results showed that between three dose of MitoQ, the 0.02 µM significantly increased nuclear maturation rate, GSH level, fertilization rate and blastulation (92.6, 231.7, 90.19 and 81.66%, respectively) than the in vitro-control (71.14, 152, 78.84 and 73.50%, respectively) and more comparable to that of the in vivo matured oocytes (100, 243.5, 92.10 and 83%, respectively). Also, the mitochondria membrane potential in the 0.02 µM MitoQ was significantly higher compared with those in the other groups (4.4). However, the intracellular ROS level in 0.02 µM MitoQ was significantly decreased (38.72%) compared to in vitro-control (82.2%) and was similar to the in vivo-control (33.5%). CONCLUSION: The results indicated that supplementation of IVM medium with MitoQ (specially 0.02 µM) enhance maturation and fertilization rate. In conclusion, MitoQ might be considered as a novel component that could be added to IVM media.

Development of a simvastatin loaded injectable porous scaffold in situ formed by phase inversion method for bone tissue regeneration.

INTRODUCTION:: The use of injectable scaffolds as a minimally invasive method is a good choice in tissue engineering applications. A critical parameter for the tissue engineering scaffolds is a suitable morphology with interconnected pores. We present the development of a simvastatin loaded scaffold that forms in situ and provides the porous structure with interconnected pores. METHODS:: The formulation of these scaffolds includes a polymeric solution of poly lactic-co-glycolic acid (25 wt%) in N-methyl-2-pyrrolidone containing 6 wt% deionized water and porogen (mannitol, four times the weight of the polymer). We have grafted simvastatin to poly lactic-co-glycolic acid by the esterification reactions. Simvastatin or simvastatin-grafted poly lactic-co-glycolic acid in different levels was added to polymer solution and finally the solution was injected into phosphate buffered saline. The simvastatin-grafted poly lactic-co-glycolic acid was characterized by attenuated total reflection Fourier-transform infra-red and 1H-nuclear magnetic resonance spectroscopy. The morphology, porosity, and biocompatibility of the scaffolds were evaluated. The in vitro simvastatin release from the various formulations was studied. Osteogenic differentiation of the adipose-derived stem cells was investigated using alkaline phosphatase activity assay and cell mineralization was evaluated using Alizarin red staining. RESULTS:: The morphology results showed the resultant scaffold was porous with the interconnected pores. The scaffolds presented 91% porosity. Non-toxic doses of simvastatin in the scaffolds were determined by methyl-thiazolyl diphenyl-tetrazolium bromide assay. The released simvastatin from the scaffolds continues over 80 days. Alkaline phosphatase activity and Alizarin red results indicated that cell osteogenic differentiation is promoted. CONCLUSION:: The results demonstrated that release of simvastatin from the injectable scaffolds can have positive effects on osteogenic differentiation of the adipose-derived stem cells.

Low oxygen tension promotes invasive ability and embryo implantation rate.

Low oxygen concentrations during in vitro embryo development not only improving the embryo quality but also can lead to successful implantation. Yet, there is no investigation at the molecular level to indicate the association between increased implantation rate and invasive ability of blastocyst and its inner cell mass quality with in vitro culture under a hypoxic condition. Therefore, the present study was designed to investigate blastocyst formation, total cell number, hatching and implantation rates. In addition we assessed the transcription levels of invasion-(Mmp-9 and uPA) and pluripotency-related genes (Pou5f1, Nanog) in mouse blastocyst under hypoxic condition. In vivo two-cell embryos were randomly divided into two groups; 5% O2 and 20% O2. Embryos were then cultured to the blastocyst stage and evaluated in terms of cellular parameters. The expression levels of selected genes were also analyzed both in experimental group and in vivo blastocysts recovered from uteri as control group. Results indicated the blastocyst formation, hatching and implantation rates were improved when the embryos were cultured in hypoxic condition. Furthermore, the expression levels of Mmp-9, Nanog and Pou5f1 showed an increase in 5% O2 in comparison with 20% O2 group. In conclusion, it seems that hypoxic condition by increasing the quality and invasion ability of the blastocyst can improve implantation rate.

Effects of Endothelial and Mesenchymal Stem Cells on Improving Myocardial Function in a Sheep Animal Model.

Background: Myocardial infarction is the main cause of death worldwide. Angiogenesis, a promising new therapy for the treatment of diffuse coronary artery disease, shows a poor response to conventional revascularization techniques. This study focused on improving myocardial function using endothelial cells (ECs) and mesenchymal stem cells (MSCs) in a sheep animal model. Methods: Acute myocardial infarction was induced in 18 sheep (12 treated cases and 6 controls). Autologous MSCs and ECs were injected in the infarcted area and the border zone. Two months after transplantation, echocardiography, electron microscopy, and immunohistochemistry were performed. Results: Echocardiography in both MSC and EC groups revealed a significant improvement in the ejection fraction compared with the control group (p value < 0.05). Vascular density, estimated by antibodies against the von Willebrand factor and smooth muscle actin, increased in both study groups. The pattern of vascularity in the MSC and EC groups was diffused. The electron microscopic evaluation of the infracted areas revealed cardiomyocytes in variable stages of development in the border zone in both EC and MSC groups. Conclusion: Both ECs and MSCs were able to promote angiogenesis and improve cardiac function. Presumably, MSCs differentiate into ECs and cause angiogenesis as it occurs for ECs.

Expressional and Bioinformatic Analysis of Bovine Filia/Ecat1/Khdc3l Gene: A Comparison with Ovine Species.

Maternal effect genes have highly impressive effects on pre-implantation development. Filia/Ecat1/Khdc3l is a maternal effect gene found in mouse oocytes and embryos, loss of which causes a 50% decrease in fertility. In the present study, we investigated Filia mRNA expression in bovine oviduct, 30- to 40-day fetus, liver, heart, lung, and oocytes (as a positive control), by RT-PCR and detected it only in oocytes. A 443 bp fragment was amplified only in oocytes and was sequenced as a part of bovine predicted Filia mRNA. We analyzed bovine and ovine Filia N-terminal peptide sequence in PHYRE2, and a KH domain was predicted. Protein alignment using ClustalW indicated a highly identical N-terminal extention between the 2 species. Immunohistochemical analysis using anti-bovine Filia antibody showed the expression of Filia protein in the zone surrounding the nuclear membrane, and in the subcortex of ovine oocytes of primary and antral follicles. However, in the bovine, Filia has been found through the oocyte cytoplasm of antral follicles, and here it is further confirmed in the primary follicles. Our data suggests a difference in Filia expression pattern between cow and sheep, although the sequence is highly conserved.

Ovine fetal mesenchymal stem cell differentiation to cardiomyocytes, effects of co-culture, role of small molecules; reversine and 5-azacytidine.

The aim of the present study was to investigate the effect of small molecules: Reversine and 5-azacytidine (5-AC), in an indirect co-culture condition with the cardiac fibroblasts as well as non co-culture condition, in order to explore the effect of such molecules in the process of differentiation of the ovine bone-marrow mesenchymal stem cells (BM-MSCs) towards cardiomyocytes. Surface antigens of the isolated cells were analysed using flow-cytometry. In addition, following to three passages cells were examined for their differentiation capacity into osteocytes and adipose cells, in order to ensure the mesenchymal origin of the stem cells. Six types of treatments were carried out in the present investigation, such that, in the first treatment BM-MSCs were cultured for 28 days as control group; the second treatment was composed of culturing ovine fetal cardiac fibroblasts on inserts, aiming to use these inserts for culturing plates which were seeded with BM-MSCs (Chamber group). As the third treatment, BM-MSCs were supplemented with 10-µM 5-AC and incubated for 48 h. The fourth treatment was composed of supplementing BM-MSCs with the 600-nM reversine, incubated for 48 h, and subsequently the incubation was further extended for another 48 h in the presence of 5-AC. The fifth treatment was composed of supplementing the chamber group with 10-µM 5-AC and incubation for 48 h, and the last or the sixth treatment was such that chamber group was supplemented with 600-nM reversine and an incubation period of 48 h. Following to the incubation, medium was replaced with 10-µM 5-AC and further incubated for another round of 48 h. In all treatments, following to addition of the small molecules incubations were carried out for 28 days; same as controls. Expression of cardiac alpha-actinin was analysed by immunocytochemistry. BM-MSCs have shown to express CD44 and CD166 along with a weak expression of the CD90, CD34, in addition to CD45. Multilineage differentiation has indicated that BM-MSCs could differentiate into adipose and osteocytes cells as well. In the treatment 4 it was observed that FGF signalling involved genes and all cardiac-related genes (ANP, MYH6 and Troponin I) were significantly expressed, except connexin 43 compared to other treatments. All treatments received small molecules, either alone or as a co-culture were seen to express sarcomeric alpha-actinin. This finding was partially supported by immunocytochemistry. These results validate that reversine and 5-AC have an effect on ovine BM-MSC differentiation into cardiomyocytes. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro maturation of ovine oocyte in a modified granulosa cells co-culture system and alpha-tocopherol supplementation: effects on nuclear maturation and cleavage.

This study was designed to investigate the effects of α-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of α-tocopherol (0, 5, 10, 15 µg/ml), oocytes were incubated at 39 °C with 5 % CO2 for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that α-tocopherol had no effect on GVBD and MII as compared to control group, but when α-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

Preparation and characterization of (PCL-crosslinked-PEG)/hydroxyapatite as bone tissue engineering scaffolds.

In this study, interconnected porous bioactive scaffolds were synthesized for bone tissue engineering. At the first step, poly( ɛ-caprolactone) (PCL) diols were diacrylated with acryloyl chloride. Then, the scaffolds were synthesized by radical crosslinking reaction of PCL and poly(ethyleneglycol) (PEG) diacrylates in the presence of hydroxyapatite (HA) particles. Morphological, swelling, thermal, and mechanical characteristics as well as degradability of the scaffolds were investigated. Results showed that increasing the ratio of PEG to PCL led to significant increase of swelling ratio and degradation rate, and decrease of crystallinity and compressive modulus of the networks, respectively. It was found that the incorporation of HA particles with the polymer matrices resulted in an augmented crystallinity, a decreased swelling ratio, and also a significantly increased compressive modulus of the networks. Cytocompatability and osteoconductivity of the scaffolds were assessed by MTT and alkaline phosphatase (ALP) assays, respectively. The results confirmed the cytocompatible nature of PCL/PEG/HA scaffolds with no toxicity. MG-63 cells attached and spread on the pore walls offered by the scaffolds. PCL/PEG/HA scaffolds compared with PCL/PEG ones showed higher ALP activity. Thus, the results indicated that the PCL/PEG/HA scaffolds have the potential of being used as promising substrates in bone tissue engineering.

A Comparative Study of MTA Solubility in Various Media.

INTRODUCTION: Solubility of root filling materials is heavily influenced by the environment they are in contact with. This study compared the solubility of ProRoot MTA in deionized water and synthetic tissue fluid. MATERIALS AND METHODS: Forty specimens of prepared MTA were immersed in deionized water and synthetic tissue fluid (20 samples each). The solubility was assessed after 7 and 28 days. Scanning electron microscope observation was also performed. The mean weight loss was evaluated using a digital scale. Data were analyzed using one-way ANOVA. Tukey test was performed for multiple comparisons. RESULTS: MTA solubility in synthetic tissue fluid was significantly lower than deionized water after 7 and 28 days (P<0.05). Secondary electron detectors revealed the presence of lumps and platelets on the surfaces of both specimens. Also, more voids were observed in specimen stored in deionized water. CONCLUSION: MTA dissolved faster in deionized water than synthetic tissue fluid. Despite this, the solubility of this material in both media was acceptable.