Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Improving the growth rate of human adipose-derived mesenchymal stem cells in alginate/gelatin versus alginate hydrogels

Background: Expansion and differentiation of stem cells relies on the soluble materials as well as the physical conditions of their microenvironment. Several methods have been studied in attempt to enhance the growth and differentiation rates of different adult stem cells extracted from different sources

Objectives: The purpose was to improve the three-dimensional [3D] culture condition of the semi-permeable polymeric beads for encapsulation of the human adipose-derived mesenchymal stem cells [hADSCs] by modifying the ratio of the alginate-gelatin composition

Materials and Methods: Following isolation and characterization of hADSCs by flow cytometry and their functional differentiation, encapsulation in the alginate and alginate/gelatin compositions were performed. Moreover, the stability, swelling, size frequency, growth kinetics, and cytotoxicity of the beads were measured to meet proper condition in the designed experimental and control culture conditions. Finally, the growth rates of the cells in different experimental groups and control were measured and analyzed statistically

Results: Viability decreased in 2 and 3 percent alginate once compared to 1% alginate in beads [p

Conclusions: These findings suggested for the first time that the composite of alginate/gelatin beads with the ratio of 50:50 might provide a suitable culture condition for the encapsulation and in vitro expansion of the hADSCs

Expression of galectin-8 on human endometrium: molecular and cellular aspects

Background: the up-regulation of galectin-3, galectin-9, and galectin-15 expression in the luminal and glandular epithelium was reported in preparation of the endometrium for embryo implantation at the midlutheal phase. However, no data was available regarding the expression and the distribution pattern of galectin-8 in the human endometrium during a regular menstrual cycle

Objective: the current study designed to investigate the expression and the distribution pattern of galectin-8, a beta-galactoside-binding lectin in the human endometrium during both proliferative and luteal phases of a regular menstrual cycle

Materials and Methods: endometrial biopsies were obtained from the anterior wall of the uterine cavity of 16 women [proliferative phase: n=4, lutheal phase: n=12]. All female patients with mean age of 37.5 years were fertile [range 25-45]. Each biopsy was divided into three pieces; one piece was fixed in formaldehyde for light microscopy and immunohistochemistry. The second portion fixed in glutaraldehyde for scanning electron microscopy and the third portion was prepared for western blot analysis

Results: data of immunoblotting revealed a molecular weight of 34 KD band with high intensity in the lutheal phase samples. The immunohistochemistry staining demonstrated that galectin-8 expressed at a very low concentration during the proliferative phase, but showed a high expression throughout the lutheal phase. The expression of galectin-8 observed in luminal surface epithelium, glandular epithelium and stroma

Conclusion: the up-regulation of the expression of galectin-8 during lutheal phase may suggest galectin-8 as one of the potential molecular marker of the endometrial receptivity. These data propose that galectin-8 may play an important role during the initial events of human embryo implantation

novel in vitro model for cancer stem cell culture using ectopically expressed Piwil2 stable cell line

Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells [CSCs], and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. In this experimental study, mouse embryonic fibroblasts [MEFs] were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter [CMV]. Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. The results of reverse transcriptase-polymerase chain reaction [RT-PCR], western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells [ANOVA, p<0.05]. Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells [ANOVA, p<0.05]. The MEF-Piwil2 cells tolerated hypoxia mimetic conditions [CoCl[2]] with more than 95% viability. According to the molecular and functional studies, it has been realized that Piwil2 plays a key role[s] in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse

Co-expression of galectin-3 and alpha v beta 3 integrin at pinopodes of human endometrium

Pinopodes are suggested as biological markers of uterine receptivity, but their molecular components are unknown. Co-expression of galectin-3 and alpha v beta integrin at human pinopodes has been examined in this study to propose a role for them during adhesion phase of embryo implantation. Biopsies were obtained from early and mid luteal phase endometrium of 12 fertile women with regular menstrual periods [25-35 days] and the mean age of 37 years [range 25-45]. Then, they were examined under light and scanning electron microscopy for detection and dating of pinoposes. Using immunofluorescent staining and immunogold electron microscopy, the expression of galectin-3 and alpha v beta integrin in human endometrium and pinopodes was detected. Further, statistical analysis was performed using immunogold electron microscopy to investigate the expression and subcellular distribution of these, before and during the frame of implantation window. The results demonstrated that pinopodes of luminal epithelia cells exhibited immnoreactivity for both galectin-3 and alpha v beta integrin, which was increased statistically significant [p<0.05] at the time of implantation window. Furthermore, area-related distribution of these proteins was found higher in pinopodes compared to the neighboring apical membrane without pinopode. Temporal and spatial expression of galectin-3 and alpha v beta integrin at pinopodes proposes a role for pinopodes in the adhesion of embryo and the involvement of galecting-3 as a binding partner of integrins in the human utero-fetal complex

Endometrial receptivity to implantation in humans: biochemical and molecular aspects

The success rate of several advanced basic and clinical techniques in the field of mammalian biotechnology, including cloning, pre-implantation genetic diagnosis, and assisted reproductive techniques [ART] depends mainly on the success rate of pregnancy following in vitro fertilization-embryo transfer [IVF-ET]. The techniques used in ART have advanced considerably since the first in vitro fertilization birth in 1978. However, despite these advances, pregnancy rates are still relatively low and have not increased significantly in the last decade. Based on the facts that embryo implantation is considered as the last barrier in ART and that inadequate endometrial receptivity is responsible for approximately two-thirds of implantation failures, intensive research work has been performed to understand the physiology, regulation, and the clinical assessments of the endometrial receptivity to improve the success rate of IVF-ET. This and the ongoing reviews tend to cover the different aspects of the endometrial receptivity mainly in human model. The present part of this series primarily concerns with biochemical and molecular events in the endometrium coordinated within its receptivity period termed as the window of implantation. Successive sections will deal with its ultrastructural changes, biomarkers, clinical assessments and regulators of endometrium within the window of implantation