Microarray analysis identification of key pathways and interaction network of differential gene expressions during osteogenic differentiation.

BACKGROUND: Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that can differentiate into three lineages. They are suitable sources for cell-based therapy and regenerative medicine applications. This study aims to evaluate the hub genes and key pathways of differentially expressed genes (DEGs) related to osteogenesis by bioinformatics analysis in three different days. The DEGs were derived from the three different days compared with day 0. RESULTS: Gene expression profiles of GSE37558 were obtained from the Gene Expression Omnibus (GEO) database. A total of 4076 DEGs were acquired on days 8, 12, and 25. Gene ontology (GO) enrichment analysis showed that the non-canonical Wnt signaling pathway and lipopolysaccharide (LPS)-mediated signaling pathway were commonly upregulated DEGs for all 3 days. KEGG pathway analysis indicated that the PI3K-Akt and focal adhesion were also commonly upregulated DEGs for all 3 days. Ten hub genes were identified by CytoHubba on days 8, 12, and 25. Then, we focused on the association of these hub genes with the Wnt pathways that had been enriched from the protein-protein interaction (PPI) by the Cytoscape plugin MCODE. CONCLUSIONS: These findings suggested further insights into the roles of the PI3K/AKT and Wnt pathways and their association with osteogenesis. In addition, the stem cell microenvironment via growth factors, extracellular matrix (ECM), IGF1, IGF2, LPS, and Wnt most likely affect osteogenesis by PI3K/AKT.

Cartilage Repair by Mesenchymal Stem Cell-Derived Exosomes: Preclinical and Clinical Trial Update and Perspectives.

Osteoarthritis (OA) and other degenerative joint diseases are characterized by articular cartilage destruction, synovial inflammation, sclerosis of subchondral bone, and loss of extracellular matrix (ECM). Worldwide, these diseases are major causes of disability. Cell therapies have been considered to be the best therapeutic strategies for long-term treatment of articular cartilage diseases. It has been suggested that the mechanism of stem cell-based therapy is related to paracrine secretion of extracellular vesicles (EVs), which are recognized as the main secretion factors of stem cells. EVs, and in particular the subclass exosomes (Exos), are novel therapeutic approaches for treatment of cartilage lesions and OA. The results of recent studies have shown that EVs isolated from mesenchymal stem cells (MSCs) could inhibit OA progression. EVs isolated from various stem cell sources, such as MSCs, may contribute to tissue regeneration of the limbs, skin, heart, and other tissues. Here, we summarize recent findings of preclinical and clinical studies on different MSC-derived EVs and their effectiveness as a treatment for damaged cartilage. The Exos isolation techniques in OA treatment are also highlighted.

The Robust Potential of Mesenchymal Stem Cell-Loaded Constructs for Hard Tissue Regeneration After Cancer Removal.

Malignant bone tumors, although quite rare, are one of the causes of death in children and adolescents. Surgery as a common and primary treatment for removal of virtually bone cancer cause large bone defects. Thus, restoration of hard tissues like bone and cartilage after surgical tumor resection needs efficient therapeutic approaches. Tissue engineering (TE) is a powerful approach which has provided hope for restoration, maintenance, or improvement of damaged tissues. This strategy generally supplies a three-dimensional scaffold as an active substrate to support cell recruitment, infiltration, and proliferation for neo-tissues. The scaffold mimics the natural extracellular matrix (ECM) of tissue which needs to be regenerated. The use of potent cell sources such as mesenchymal stem cells (MSCs) has also led to remarkable progresses in hard tissue regeneration. Combination of living cells and various biomaterials have continuously evolved over the past decades to improve the process of regeneration. This chapter describes various strategies used in TE and highlights recent advances in cell-loaded constructs. We herein focus on cell-based scaffold approach utilized in hard tissue engineering and parameters determining a clinically efficient outcome. Also, we attempt to identify the potential as well as shortcomings of pre-loaded scaffolds for future therapeutic applications.

Correction to: Photobiomodulation with single and combination laser wavelengths on bone marrow mesenchymal stem cells: proliferation and differentiation to bone or cartilage.

In the originally published article, the name of the 3rd and 4th authors were labeled incorrectly. The correct names are Mohammadreza Baghaban Eslaminejad and Leila Taghiyar. Also, affiliation 4 has been corrected.

Evaluation of toll-like receptor 4 expression in human bone marrow mesenchymal stem cells by lipopolysaccharides from Shigella.

Lipopolysaccharides (LPS) from gram negative bacteria stimulate toll-like receptor 4 (TLR4) expression in immune cells. Recent reports state that bone marrow-derived cells such as mesenchymal stem cells (MSCs) also express TLR proteins. Numerous researches have studied the effect of a number of LPSs on TLR4 expression, but no data exists on the effect of LPSs from different strains of one bacterial genus on TLR4 expression. In this study, we investigate the effects of various concentrations of LPS from different Shigella strains on TLR4 expression in human bone marrow (hBM)-MSCs. At the mRNA level, we have found that untreated hBM-MSCs (control) did not express TLR4 compared to the experimental groups. Cells treated with LPS from Shigella flexneri had the highest expression of TLR4, whereas cells treated with LPS from Shigella sonnei had the lowest expression. We observed that LPSs had a dose-dependent effect on TLR4 expression in all of the treatment groups. ELISA findings for interleukin-6 secretion have confirmed mRNA expression results for all treatment groups. Hence, LPS from S. flexneri can be considered as an optimum LPS to stimulate the immune system for vaccine production against shigellosis. Also, TLR activation in hBM-MSCs can modulate their function such as homing.

Effects of Maternal Isocaloric Diet Containing Different Amounts of Soy Oil and Extra Virgin Olive Oil on Weight, Serum Glucose, and Lipid Profile of Female Mice Offspring.

BACKGROUND: Health status of offspring is programmed by maternal diet throughout gestation and lactation. The present study investigates the lasting effects of maternal supplementation with different amounts of soy oil or extra virgin olive oil (EVOO) on weight and biochemical parameters during gestation and lactation of female mice offspring. METHODS: Eight weeks old female C57BL/6 mice (n=40) were assigned through simple randomization into four isocaloric dietary groups (16% of calories as soy oil (LSO) or EVOO (LOO) and 45% of calories as soy oil (HSO) or EVOO (HOO)) during three weeks of gestation and lactation. After weaning (at 3 weeks), all offspring received a diet containing 16% of calories as soy oil and were sacrificed at 6 weeks. Two-way ANOVA was used to adjust for confounding variables and repeated measures test for weight gain trend. Statistical analyses were performed with the IBM SPSS package. RESULTS: At birth and adolescence, the weight of offspring was significantly higher in the soy oil than the olive oil groups (P<0.001 and P<0.001, respectively). Adolescence weight was significantly higher in the offspring born to mothers fed with 16% oil than those with 45% oil (P=0.001). Serum glucose, triglyceride and total cholesterol were significantly higher in the LSO than LOO (P<0.001, P<0.001 and P<0.001), LSO than HSO (P<0.001, P=0.03 and P<0.001), and LOO than HOO (P<0.001, P<0.001 and P<0.001) dietary groups, respectively. Serum triglyceride and total cholesterol were significantly higher in the offspring of HSO than HOO fed mothers (P<0.001 and P<0.001, respectively). CONCLUSION: A maternal diet containing EVOO has better effects on birth weight, as well as weight and serum biochemical parameters in offspring at adolescence.

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

Comparative analysis of neural differentiation potential in human mesenchymal stem cells derived from chorion and adult bone marrow.

The finding of a reliable and abundant source of stem cells for the replacement of missing neurons in nervous system diseases requires extensive characterization of neural-differentiation-associated markers in stem cells from various sources. Chorion-derived stem cells from the human placenta have recently been described as an abundant, ethically acceptable, and easily accessible source of cells that are not limited in the same way as bone marrow (BM) mesenchymal stem cells (MSCs). We have isolated and cultured chorion MSCs (C-MSCs) and compared their proliferative capacity, multipotency, and neural differentiation ability with BM-MSCs. C-MSCs showed a higher proliferative capacity compared with BM-MSCs. The expression and histone modification of Nestin, as a marker for neural stem/progenitor cells, was evaluated quantitatively between the two groups. The Nestin expression level in C-MSCs was significantly higher than that in BM-MSCs. Notably, modifications of lys9, lys4, and lys27 of histone H3 agreed with the remarkable higher expression of Nestin in C-MSCs than in BM-MSCs. Furthermore, after neural differentiation of MSCs upon retinoic acid induction, both immunocytochemical and flow cytometry analyses demonstrated that the expression of neural marker genes was significantly higher in neural-induced C-MSCs compared with BM-MSCs. Mature neuron marker genes were also expressed at a significantly higher level in C-MSCs than in BM-MSCs. Thus, C-MSCs have a greater potential than BM-MSCs for differentiation to neural cell lineages and can be regarded as a promising source of stem cells for the cell therapy of neurological disorders.

Stable overexpression of DNA fragmentation factor in T-47D cells: sensitization of breast cancer cells to apoptosis in response to acetazolamide and sulfabenzamide.

Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells treated by GSK-3 inhibitors.

A study of the cartilage differentiation of mesenchymal stem cells (MSCs) would be of particular interest since one strategy for cell-based treatment of cartilage defects emphasizes the use of cells that are in a differentiated state. The present study has attempted to evaluate the effects of two well-known glycogen synthase kinase-3 inhibitors, including lithium chloride (LiCl) and SB216763 on a human marrow-derived MSC (hMSC) chondrogenic culture. Passaged-3 MSCs were condensed into small pellets and cultivated in the following groups based on the supplementation of chondrogenic medium: transforming growth factor (TGF)-ß1, TGF-ß1 + LiCl, TGF-ß1 + SB216763, TGF-ß3, TGF-ß3 + LiCl, and TGF-ß3 + SB216763. The cultures were maintained for 21 days and then analyzed for expression of Sox9, aggrecan, collagen II, ß-catenin, and axin genes. Deposition of glycosaminoglycan (GAG) in the cartilage matrix was also measured for certain cultures. The presence of both LiCl and SB216763 along with TGF-ß in the MSC chondrogenic culture led to the up-regulation of cartilage-specific genes. TGF-ß3 appeared much better than TGF-ß1. Based on our findings, SB216763 was more effective in up-regulation of cartilage-specific genes. These chondrogenic effects appeared to be mediated through the Wnt signaling pathway since ß-catenin and axin tended to be up-regulated and down-regulated, respectively. In the culture with SB216763 + TGF-ß3, significantly more GAG was deposited (P < 0.05). In conclusion, addition of either SB216763 or LiCl to hMSC chondrogenic culture up-regulates cartilage-specific gene expression and enhances GAG deposition in the culture.

High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds.

Transplantation of stem cells using biodegradable and biocompatible nanofibrous scaffolds is a promising therapeutic approach for treating inherited retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. In this study, conjunctiva mesenchymal stem cells (CJMSCs) were seeded onto poly-L-lactic acid (PLLA) nanofibrous scaffolds and were induced to differentiate toward photoreceptor cell lineages. Furthermore, the effects of orientation of scaffold on photoreceptor differentiation were examined. Scanning electron microscopy (SEM) imaging, quantitative real time RT-PCR (qPCR) and immunocytochemistry were used to analyze differentiated cells and their expression of photoreceptor-specific genes. Our observations demonstrated the differentiation of CJMSCs to photoreceptor cells on nanofibrous scaffolds and suggested their potential application in retinal regeneration. SEM imaging showed that CJMSCs were spindle shaped and well oriented on the aligned nanofiber scaffolds. The expression of rod photoreceptor-specific genes was significantly higher in CJMSCs differentiated on randomly-oriented nanofibers compared to those on aligned nanofibers. According to our results we may conclude that the nanofibrous PLLA scaffold reported herein could be used as a potential cell carrier for retinal tissue engineering and a combination of electrospun nanofiber scaffolds and MSC-derived conjunctiva stromal cells may have potential application in retinal regenerative therapy.

Reconstruction of human mandibular continuity defects with allogenic scaffold and autologous marrow mesenchymal stem cells.

Mandibular continuity defects occur after tumor resection, maxillofacial injury, or osteomyelitis. In this clinical pilot study, we report a novel method for reconstruction of mandibular continuity defect by in vivo tissue engineering. In 3 patients with critical-size mandibular bone defects, the allogenic mandibular bone scaffold was customized, loaded by ex vivo expanded mesenchymal stem cells, and transplanted into the surgical defect site. According to the bone scintigraphy, vascularized bone was identified in 2 cases. In spiral computed tomography, normal bone healing without significant bone resorption was seen at the 2 viable grafts, but at the failed construction, there was a lack of osteointegration to the adjacent host bone and a higher density in the medullary bone. According to the serial panoramic imaging, the patients with viable bone grafts had normal bone healing, whereas the other patient had progressive overall bone resorption. Our results demonstrate the feasibility of allogenic bone scaffold loaded by mesenchymal stem cells in the reconstruction of mandibular continuity defects. Although long-term results are not yet available, it may be a novel method of reconstruction and a basis for further studies.

Vertical bone augmentation with simultaneous implant placement using particulate mineralized bone and mesenchymal stem cells: a preliminary study in rabbit.

This study aimed to assess vertical bone augmentation with simultaneous implant placement in rabbit tibiae using particulate mineralized bone/fibrin glue/mesenchymal stem cell. Bone marrow was aspirated from tibiae of five 10-week-old New Zealand White male rabbits. Right and left tibiae of each rabbit were prepared, and a 3-mm protruding implant from tibial bone was placed in each side. Particulate allogenic bone/fibrin glue/mesenchymal stem cell combination was placed around test implants and particulate bone graft/fibrin glue around controls. Two months postoperatively, the animals were euthanized, and sections were prepared for histological analysis. The mean amount of vertical bone length was higher in the experimental group than the control group (2.09 mm vs 1.03 mm; P < .05). New supracrestal trabecular bone formation was also significantly higher in the test group (28.5 ± 4.5% vs 4.3 ± 1.8%; P < .05). Mesenchymal stem cell/particulate allograft/fibrin glue appears to be a promising combination for vertical bone augmentation around simultaneously inserted implants in rabbit tibia.

Autologous dental pulp stem cells in regeneration of defect created in canine periodontal tissue.

This study aimed to investigate effects of dental pulp stem cells (DPSCs) on regeneration of a defect experimentally created in the periodontium of a canine model. Surgically created mesial 3-walled periodontal defects with ligature-induced periodontitis were produced bilaterally in the first lower premolar teeth of 10 mongrel dogs. Simultaneously, DPSCs were derived from the maxillary premolar teeth of the same dogs. Four weeks after creation of the periodontitis model, autologous passaged-3 DPSCs combined with Bio-Oss were implanted on one side as the test group. On the other side, only Bio-Oss was implanted as a control. Eight weeks after surgery, regeneration of the periodontal defects was evaluated histologically and histomorphometrically in terms of bone, periodontal ligament (PDL), and cement formation. Histologically, in all test specimens (10 defects), regeneration of cementum, bone, and PDL was observed. In the control groups, although we observed the regeneration of bone in all defects, the formation of cementum was seen in 9 defects and PDL was seen in 8 defects. Histomorphometric analyses showed that the amount of regenerated cementum and PDL in the test groups (3.83 ± 1.32 mm and 3.30 ± 1.12 mm, respectively) was significantly higher than that of the control groups (2.42 ± 1.40 mm and 1.77 ± 1.27 mm, respectively; P < .05). A biocomplex consisting of DPSCs and Bio-Oss would be promising in regeneration of periodontal tissues.

Advances in mechanically robust and biomimetic polysaccharide-based constructs for cartilage tissue engineering.

The purpose of cartilage tissue engineering is to provide artificial constructs with biological functions and mechanical features that resemble native tissue to improve tissue regeneration. Biochemical characteristics of the cartilage extracellular matrix (ECM) microenvironment provide a platform for researchers to develop biomimetic materials for optimal tissue repair. Due to the structural similarity of polysaccharides into physicochemical characteristics of cartilage ECM, these natural polymers capture special attention for developing biomimetic materials. The mechanical properties of constructs play a crucial influence in load-bearing cartilage tissues. Moreover, the addition of appropriate bioactive molecules to these constructs can promote chondrogenesis. Here, we discuss polysaccharide-based constructs that can be used to create substitutes for cartilage regeneration. We intend to focus on newly developed bioinspired materials, fine-tuning the mechanical properties of constructs, the design of carriers loaded by chondroinductive agents, and development of appropriate bioinks as a bioprinting approach for cartilage regeneration.

Preparation of gum tragacanth/poly (vinyl alcohol)/halloysite hydrogel using electron beam irradiation with potential for bone tissue engineering.

Nanocomposite hydrogels based on tyramine conjugated gum tragacanth, poly (vinyl alcohol) (PVA), and halloysite nanotubes (HNTs) were prepared by electron beam irradiation and characterized. The FTIR, 1H NMR, and TGA results confirmed the chemical incorporation of HNTs into gum tragacanth. Gel content and swelling of hydrogels decreased with HNTs loading up to 20 % wt. The mechanical strength of hydrogels increased by increasing HNTs content up to 10 % with 371 kPa fracture stress at 0.95 fracture strain, compared to 312 kPa stress at 0.79 strain for gum tragacanth/PVA hydrogel. Hydrogel's biocompatibility and osteogenic activity were tested by seeding rabbit bone marrow mesenchymal stem cells. The cell viability was >85 % after 7 days of culture. In vitro secretion of ALP and calcium deposition on hydrogels in alizarin red assay after 21 days of culture indicated hydrogel potential for bone tissue engineering.

Development of osteon-like scaffold-cell construct by quadruple coaxial extrusion-based 3D bioprinting of nanocomposite hydrogel.

Despite advances in bone tissue engineering, fabricating a scaffold which can be used as an implant for large bone defects remains challenge. One of the great importance in fabricating a biomimetic bone implant is considering the possibility of the integration of the structure and function of implants with hierarchical structure of bone. Herein, we propose a method to mimic the structural unit of compact bone, osteon, with spatial pattern of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) in the adjacent layers that mimic Haversian canal and lamella, respectively. To this end, coaxial extrusion-based bioprinting technique via a customized quadruple-layer core-shell nozzle was employed. 3D implant scaffold-cell construct was fabricated by using polyethylene glycol as a hollowing agent in the first layer, gelatin methacryloyl (GelMA) and alginate blended hydrogel encapsulating HUVEC cells with vascular endothelial growth factor nanoparticles in the second layer (vasculogenic layer) to mimic vascular vessel, and GelMA and alginate blended hydrogel containing hMSCs cells in the outer osteogenic layer to imitate lamella. Two types of bone minerals, whitlockite and hydroxyapatite, were incorporated in osteogenic layer to induce osteoblastic differentiation and enhance mechanical properties (the young's modules of nanocomposite increased from 35 kPa to 80 kPa). In-vitro evaluations demonstrated high cell viability (94 % within 10 days) and proliferation. Furthermore, ALP enzyme activity increased considerably within 2 weeks and mineralized extra cellular matrix considerably produced within 3 weeks. Also, a significant increase in osteogenic markers was observed indicating the presence of differentiated osteoblast cells. Therefore, the work indicates the potential of single step 3D bioprinting process to fabricate biomimetic osteons to use as bone grafts for regeneration.

Rheumatoid arthritis: the old issue, the new therapeutic approach.

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease of unknown etiology. The most common form of this disease is chronic inflammatory arthritis, which begins with inflammation of the synovial membrane of the affected joints and eventually leads to disability of the affected limb. Despite significant advances in RA pharmaceutical therapies and the availability of a variety of medicines on the market, none of the available medicinal therapies has been able to completely cure the disease. In addition, a significant percentage (30-40%) of patients do not respond appropriately to any of the available medicines. Recently, mesenchymal stromal cells (MSCs) have shown promising results in controlling inflammatory and autoimmune diseases, including RA. Experimental studies and clinical trials have demonstrated the high power of MSCs in modulating the immune system. In this article, we first examine the mechanism of RA disease, the role of cytokines and existing medicinal therapies. We then discuss the immunomodulatory function of MSCs from different perspectives. Our understanding of how MSCs work in suppressing the immune system will lead to better utilization of these cells as a promising tool in the treatment of autoimmune diseases.

An efficient method for cell sheet bioengineering from rBMSCs on thermo-responsive PCL-PEG-PCL copolymer.

Utilizing both medium enrichment and a thermos-responsive substrate to maintain the cell-to-cell junctions and extracellular matrix (ECM) intact, cell sheet technology has emerged as a ground-breaking approach. Investigating the possibility of using sodium selenite (as medium supplementation) and PCL-PEG-PCL (as vessel coating substrate) in the formation of the sheets from rat bone marrow-derived mesenchymal stem cells (rBMSCs) was the main goal of the present study. To this end, first, Polycaprolactone-co-Poly (ethylene glycol)-co-Polycaprolactone triblock copolymer (PCEC) was prepared by ring-opening copolymerization method and characterized by FTIR, 1 H NMR, and GPC. The sol-gel-sol phase transition temperature of the PCEC aqueous solutions with various concentrations was either measured. Next, rBMSCs were cultured on the PCEC, and let be expanded in five different media containing vitamin C (50 µg/ml), sodium selenite (0.1 µM), vitamin C and sodium selenite (50 µg/ml + 0.1 µM), Trolox, and routine medium. The proliferation of the cells exposed to each material was evaluated. Produced cell sheets were harvested from the polymer surface by temperature reduction and phenotypically analyzed via an inverted microscope, hematoxylin and eosin (H&E) staining, and field emission scanning electron microscopy (FESEM). Through the molecular level, the expression of the stemness-related genes (Sox2, Oct-4, Nanog), selenium-dependent enzymes (TRX, GPX-1), and aging regulator gene (Sirt1) were measured by q RT-PCR. Senescence in cell sheets was checked by beta-galactosidase assay. The results declared the improved ability of the rBMSCs for osteogenesis and adipogenesis in the presence of antioxidants vitamin C, sodium selenite, and Trolox in growth media. The data indicated that in the presence of vitamin C and sodium selenite, the quality of the cell sheet was risen by reducing the number of senescent cells and high transcription of the stemness genes. Monolayers produced by sodium selenite was in higher-quality than the ones produced by vitamin C.