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Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-β1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-β1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-β1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-β1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-β1 added in culture media or those without TGF-β1. However, constructs with TGF-β1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-β1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-β1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy., (© 2024. The Author(s).)

In this study, the effect of alumina nanowire on the physical and biological properties of polyhydroxybutyrate-keratin (PHB-K) electrospun scaffold was investigated. First, PHB-K/alumina nanowire nanocomposite scaffolds were made with an optimal concentration of 3 wt% alumina nanowire by using the electrospinning method. The samples were examined in terms of morphology, porosity, tensile strength, contact angle, biodegradability, bioactivity, cell viability, ALP activity, mineralization ability, and gene expression. The nanocomposite scaffold provided a porosity of >80 % and a tensile strength of about 6.72 MPa, which were noticeable for an electrospun scaffold. AFM images showed an increase in surface roughness with the presence of alumina nanowires. This led to an improvement in the degradation rate and bioactivity of PHB-K/alumina nanowire scaffolds. The viability of mesenchymal cells, alkaline phosphatase secretion, and mineralization significantly increased with the presence of alumina nanowire compared to PHB and PHB-K scaffolds. In addition, the expression level of collagen I, osteocalcin, and RUNX2 genes in nanocomposite scaffolds increased significantly compared to other groups. In general, this nanocomposite scaffold could be a novel and interesting construct for osteogenic induction in bone tissue engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Because spermatogonia transmit genetic information across generations, their DNA must be protected from environmental damages, including exposure to zinc oxide nanoparticles (ZnO NPs), which are frequently used in modern technology. Here, we used an in vitro system enriched for spermatogonia and exposed them to 10 and 20 μg/ml ZnO NPs for one/seven days. We did not detect any significant cell death, chromosomal instability, or DNA fragmentation in the spermatogonia treated with the ZnO NPs following one-day treatment with 10 or 20 μg/ml ZnO NPs. However, ZnO NPs (both 10 and 20 μg/ml) induced chromosomal instability in the spermatogonia after seven days of treatment. Moreover, one-day exposure to these NPs induced reactive oxygen species (ROS) generation and upregulation of apoptotic pathway-related genes p53, Caspase3 and Il6, as an inflammatory factor. Taken together, our study provides preliminary evidence for possible damages induced by low concentrations of ZnO NPs in spermatogonia. We should pay increased attention when using these NPs because of the silent damages in spermatogonia that can be transmitted to the next generation and cause severe effects. However, more data and validation of these results are required to determine the extent of this concern., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Germ cell production from stem cells allows for studying the mechanisms involved in gamete development with the aim of helping infertile couples with the generation of healthy gametes. In this context, improving the protocols for in-vitro germ cell induction from stem cells is very important. Recently, SB4 small molecule has been introduced as a potent agonist for bone morphogenic protein 4 (BMP4). Herein, we investigated whether BMP4, is replaceable by SB4 for having affordable protocol for in vitro germ cell differentiation. We demonstrated that SB4 can induce Blimp1 (as the first gene induced germ line differentiation) expression significantly but at a lower level compared to BMP4. However, Tfap2c (a putative downstream target of Blimp1 during germ cell differentiation) expression level in SB4-induced aggregates was significantly higher than in BMP4-induced aggregates. Moreover, co-presence of both BMP4 and SB4 could increase the expression level of Prdm14, Nnose3 and Stella (Dppa3), and thereby improve establishment of the germ cell fate during in-vitro differentiation of embryonic stem cells. In summary, our data suggest that SB4 could improve germ line gene expression pattern induced by BMP4 during embryonic stem cells in-vitro differentiation., (Copyright © 2021. Published by Elsevier B.V.)

Breast cancer is a heterogeneous disease, which is the consequence of several genetic and environmental factors. Also, it is one of the most common causes of cancer death and second leading cancer among women all around the world. Therefore, it is necessary to develop novel therapeutic approaches useful for the successful treatment of breast cancer. As conventional treatments had limited success, alternative approaches for the treatment of breast cancer have been applied in recent years. Hence, the molecular basis of breast cancer has provided the opportunity of using genetic materials for therapeutic uses. In this regard, gene therapy as one of the potentially efficient and beneficial treatments among various techniques became a popular treatment for different cancers, especially breast cancer. Accordingly, there are plenty of targets available for gene therapy of breast cancer. Gene therapy strategies have the potential to correct molecular defects that contributed to the cancer progression. These techniques should selectively target tumor cells without affecting normal cells. Moreover, data of clinical trials in gene therapy for breast cancer indicated that this approach has little toxicity compared to other therapeutic approaches. In this study, different aspects of breast neoplasm, gene therapy techniques, challenges, and recent developments will be mentioned.

Psychiatric disorders such as schizophrenia can generate distress and disability along with heavy costs on individuals and health care systems. Different genetic and environmental factors play a pivotal role in the appearance of the mentioned disorders. Since the conventional treatment options for psychiatric disorders are suboptimal, investigators are trying to find novel strategies. Herein, stem cell therapies have been recommended as novel choices. In this context, the preclinical examination of stem cell-based therapies specifically using appropriate models can facilitate passing strong filters and serious examination to ensure proper quality and safety of them as a novel treatment approach. Animal models cannot be adequately helpful to follow pathophysiological features. Nowadays, stem cell-based models, particularly induced pluripotent stem cells reflected as suitable alternative models in this field. Accordingly, the importance of stem cell-based models, especially to experiment with the regenerative medicine outcomes for schizophrenia as one of the severe typing of psychiatric disorders, is addressed here.

Multipotent human bone marrow-derived mesenchymal stem cells (hMSCs) are promising candidates for bone and cartilage regeneration. Toll-like receptor 4 (TLR4) is expressed by hMSCs and is a receptor for both exogenous and endogenous danger signals. TLRs have been shown to possess functional differences based on the species (human or mouse) they are isolated from therefore, the effects of knockdown of TLR4 were evaluated in humans during the differentiation of MSCs into bone, fat and chondrocyte cells in vitro. We investigated the expression profile of TLR4 during the differentiation of hMSCs into three different lineages on days 7, 14 and 21 and assessed the differentiation potential of the cells in the presence of lipopolysaccharide (LPS, as an exogenous agonist) and fibronectin fragment III-1c (FnIII-1c, as an endogenous agonist). TLR4 expression increased following the induction of hMSC differentiation into all three lineages. Alkaline phosphatase activity revealed that FnIII-1c accelerated calcium deposition on day 7, whereas LPS increased calcium deposition on day 14. Chondrogenesis increased in the presence of LPS; however, FnIII-1c acted as a reducer in the late stage. TLR4 silencing led to decreased osteogenesis and increased adipogenesis. Furthermore, Wnt5a expression was inversely related to chondrogenesis during the late stage of differentiation. We suggest that understanding the functionality of TLR4 (in the presence of pathogen or stress signal) during the differentiation of hMSCs into three lineages would be useful for MSC-based treatments., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Introduction: Cholinergic-associated diseases currently constitute a significant cause of neurological and neurodegenerative disabilities. As the drugs are not efficient in improving the suffered tissues, stem cell treatment is considered an effective strategy for substituting the lost cells., Methods: In the current study, we set out to investigate the differentiation properties of human Adipose-Derived Mesenchymal Stem Cells (AD-MSCs) into cholinergic-like cells by two morphogens of Retinoic Acid (RA) and Sonic Hedgehog (Shh) using a three-step in vitro procedure. The results were evaluated using real-time PCR, flow cytometry, and immunocytochemistry for two weeks., Results: Our data showed that the cells could express cholinergic specific markers, including Islet-1, Acetylcholinesterase (AChE), SMI-32, and Nestin, at mRNA and protein levels. We could also quantitatively evaluate the expression of Islet-1, AChE, and Nestin at 14 days post-induction using flow cytometry., Conclusion: Human AD-MSCs are potent cells to differentiate into cholinergic-like cells in the presence of RA and Shh through a three-step protocol. Thus, they could be a suitable cell candidate for the regeneration of cholinergic-associated diseases. However, more functional and electrophysiological analyses are needed in this regard., Competing Interests: Conflict of interest The authors declared no conflict of interest., (Copyright© 2021 Iranian Neuroscience Society.)

Rheumatoid arthritis as a common autoimmune inflammatory disorder with unknown etiology can affect 0.5-1% of adults in developed countries. It involves more than just the patient's joints and can be accompanied by several comorbidities and affect cardiovascular, pulmonary, and some other systems of the human body. Although cytokine-mediated pathways are mentioned to have a central role in RA pathogenesis, adaptive and innate immune systems and intracellular signaling pathways all have important roles in this process. Non-steroidal anti-inflammatory drugs, glucocorticoids, conventional disease-modifying anti-rheumatic drugs, and biological agents are some mentioned medications used for RA. They are accompanied by some adverse effects and treatment failures which elucidates the needing for novel and more powerful therapeutic approaches. Stem cell-based therapies and their beneficial effects on therapeutic processes of different diseases have been founded so far. They can be an alternative and promising therapeutic approach for RA, too; due to their effects on immune responses of the disease. This review, besides some explanations about RA characteristics, addresses the outcome of the stem cell-based therapies including mesenchymal stem cell transplantation and hematopoietic stem cell transplantation for RA and explains their effects on the disease improvement., (© 2020. Springer Nature Switzerland AG.)

Systemic sclerosis is a rare chronic autoimmune disease with extensive microvascular injury, damage of endothelial cells, activation of immune responses, and progression of tissue fibrosis in the skin and various internal organs. According to epidemiological data, women's populations are more susceptible to systemic sclerosis than men. Until now, various therapeutic options are employed to manage the symptoms of the disease. Since stem cell-based treatments have developed as a novel approach to rescue from several autoimmune diseases, it seems that stem cells, especially mesenchymal stem cells as a powerful regenerative tool can also be advantageous for systemic sclerosis treatment via their remarkable properties including immunomodulatory and anti-fibrotic effects. Accordingly, we discuss the contemporary status and future perspectives of mesenchymal stem cell transplantation for systemic sclerosis.

Extensive application of zinc oxide (ZnO) nanoparticles (NPs) in everyday life results in increased exposure to these NPs. Spermatogonial stem cells (SSCs) guarantee sperm production throughout the male reproductive life by providing a balance between self-renewal and differentiation. We used an in vitro platform to investigate the ZnO NPs effects on SSCs. We successfully synthesized ZnO NPs. In order to investigate these NPs, we isolated SSCs from mouse testes and cultured them in vitro. Our results confirmed the uptake of ZnO NPs by the cultured SSCs. We observed a dose- and time-dependent decrease in SSC viability. Both spherical and nanosheet ZnO NPs had the same cytotoxic effects on the SSCs, irrespective of their shapes. Moreover, we have shown that short time (one day) exposure of SSCs to a low concentration of ZnO NPs (10 μg/mL) promoted expressions of specific genes (Plzf, Gfr α1 and Bcl6b) for SSC self-renewal and differentiation genes (Vasa, Dazl, C-kit and Sycp3) expressed by spermatogonia during spermatogenesis. Our study provides the first insight into ZnO NPs function in SSCs and suggests a new function for ZnO NPs in the male reproductive system. We demonstrated that ZnO NPs might promote spermatogenesis via upregulation of gene expression related to SSC self-renewal and differentiation at low concentrations. Additional research should clarify the possible effect of ZnO NPs on the SSC genome and its effects on human SSCs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acute respiratory infections as one of the most common problems of healthcare systems also can be considered as an important reason for worldwide morbidity and mortality from infectious diseases. Coronaviruses are a group of well-known respiratory viruses that can cause acute respiratory infections. At the current state, the 2019 novel coronavirus is cited as the most worldwide problematic agent for the respiratory system. According to investigations, people with old age and underlying diseases are at higher risk of 2019 novel coronavirus infection. Indeed, they may show a severe form of the disease (with severe acute respiratory infections). Based on the promising role of cell therapy and regenerative medicine approaches in the treatment of several life-threatening diseases, it seems that applying cell-based approaches can also be a hopeful strategy for improving subjects with severe acute respiratory infections caused by the 2019 novel coronavirus. Herein, due to the amazing effects of mesenchymal stem cells in the treatment of various diseases, this review focuses on the auxiliary role of mesenchymal stem cells to reduce inflammatory processes of acute respiratory infections caused by the 2019 novel coronavirus.

Background: Regeneration of articular cartilage poses a tremendous challenge due to its limited self-repair capability and inflammation at the damaged site. To generate the desired structures that mimic the structure of native tissue, microtissues with repeated functional units such as cell aggregates have been developed. Multicellular aggregates of mesenchymal stem cells (MSCs) can be used as microscale building blocks of cartilage due to their potential for cell-cell contact, cell proliferation, and differentiation., Methods: Chondrogenic microtissues were developed through incorporation of kartogenin-releasing poly (lactic-co-glycolic acid) (PLGA) microparticles (KGN-MP) within the MSC aggregates. The chondrogenic potential of KGN-MP treated MSC aggregates was proven in vitro by studying the chondrogenic markers at the RNA level and histological analysis. In order to address the inflammatory responses at the defect site, the microtissues were delivered in vivo via an injectable, anti-inflammatory hydrogel that contained gelatin methacryloyl (GelMA) loaded with curcumin (Cur)., Results: The KGN-MPs were fabricated to support MSCs during cartilage differentiation. According to real-time RT-PCR analysis, the presence of KGN in the aggregates led to the expression of cartilage markers by the MSCs. Both toluidine blue (TB) and safranin O (SO) staining demonstrated homogeneous glycosaminoglycan production throughout the KGN-MP incorporated MSC aggregates. The curcumin treatment efficiently reduced the expressions of hypertrophy markers by MSCs in vitro. The in vivo results showed that implantation of chondrogenic microtissues (KGN-MP incorporated MSC aggregates) using the curcumin loaded GelMA hydrogel resulted in cartilage tissue regeneration that had characteristic features close to the natural hyaline cartilage according to observational and histological results., Conclusions: The use of this novel construct that contained chondrogenic cell blocks and curcumin is highly desired for cartilage regeneration.

Cartilage tissue engineering is the interdisciplinary science that will help to improve cartilage afflictions, such as arthrosis, arthritis, or following joints traumatic injuries. In the present work, we developed an injectable hydrogel which derived from decellularized extracellular matrix of sheep cartilage. Successful decellularization was evaluated by measuring the DNA, glycosaminoglycans (GAG), collagen contents, and histological analyses. There was a minor difference in GAG and collagen contents among natural cartilage and decellularized tissue as well as ultimate hydrogel. Rheological analysis showed that the temperature and gelation time of prepared hydrogel were 37°C and between 5 and 7 min, respectively. Mechanical properties evaluation indicated a storage modulus of 20 kPa. The results show that prepared hydrogel possessed cell-friendly microenvironment as confirmed via calcein staining and MTT assay. Also, cells were able to proliferate which observed by H&E and alcian blue staining. Cell attachment and proliferation at the surface of the decellularized hydrogel was apparent by Scanning Electron Microscope (SEM) images and microphotographs. Furthermore, the cells embedded within the hydrogel were able to differentiate into chondrocyte with limited evidence of hypertrophy and osteogenesis in utilized cells which proved by SOX9, CoL2, ACAN, and also CoL1 and CoL10 gene expression levels. In summary, the results suggest that developed novel injectable hydrogel from decellularized cartilage could be utilized as a promising substrate for cartilage tissue engineering applications., (© 2020 Wiley Periodicals, Inc.)

Cardiovascular diseases (CVD) are a major cause of mortality worldwide. Accessibility to heart tissue is limited due to sampling issues and lack of appropriate culture conditions. In addition, animal models are not an ideal choice for physiological, pharmacological, and fundamental evaluations in the cardiovascular field due to interspecies differences. Hence, there is an inevitable need for functional in vitro cardiac models. In this study, we have synthesized a novel electroconductive scaffold comprised of cardiac extracellular matrix (ECM) derived pre-cardiogel (pCG) blended with polypyrrole (Ppy). Our data revealed that 2.5% (w/v) pyrrole (Py) had the highest possible Py ratio that provided pCG-Ppy gel formation. The prepared mixture was fabricated into a scaffold by using the freeze-dried method. The scaffolds had open interconnected pores that ranged from 55 ± 24 μm for the cardiogel (CG)-Ppy to 74 ± 26 μm for the CG scaffolds, with no alterations in vital ECM components of collagen, polysaccharides, and glycosaminoglycans (GAGs). Incorporation of Ppy increased the CG stiffness with a final complex modulus from 80 pa to 140 pa. The CG-Ppy group had significantly greater electrical conductivity than the CG group. Scaffolds supported neonatal mouse cardiomyocyte (NMCM) adhesion, viability, cardiac-specific gene expression, and spontaneous beating up to 14 days after seeding. Among the fabricated hydrogels, the CG-Ppy group resulted in the synchronous beating of cardiomyocyte clusters and upregulation of cardiac genes involved in cardiac muscle contraction (cardiac troponin T [cTNT]) and cardiomyocyte electrical coupling (connexin 43 [Cx43]). Thus, this ECM-based electro-conductive scaffold might provide a promising substrate for constructing in vitro cardiac models for drug testing, disease modeling, developmental studies, and cardiac regenerative approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and β-tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen type 1 (Col 1) 30-fold, osteopontin (OPN) 10-fold, and osteocalcin (OCN) 3-fold) after 14 days of culture with the incorporated CMPs, while no significant upregulation was observed with the incorporation of gelatin microparticles. The porous structure of the CMPs was exploited for loading and sustained release of an angiogenic small molecule. Dimethyloxaloylglycine (DMOG) was loaded efficiently onto the CMPs (loading efficiency: 65.32 ± 6%) and showed a sustained release profile over 12 days. Upon incorporation of the DMOG-loaded CMPs (DCMPs) within the mesenspheres, a similar osteogenic differentiation and an upregulation in angiogenic genes (VEGF 5-fold and kinase insert domain (KDR) 2-fold) were observed after 14 days of culture. These trends were also observed in immunostaining analysis. To evaluate scalable production of the osteogenic micro-tissues, the incorporation of microparticles was performed during cell aggregation in a spinner flask. The DCMPs were efficiently incorporated and directed the mesenspheres toward osteogenesis and angiogenesis. Finally, the DCMP mesenspheres were loaded within a three-dimensional printed cell trapper and transplanted into a critical-sized defect in a rat model. Computed tomography and histological analysis showed significant bone formation with blood vessel reconstruction after 8 weeks in this group. Taken together, we provide a scalable and cost-effective approach for fabrication of osteogenic micro-tissues, as building blocks of macro-tissues, that can address the large amounts of cells required for cell-based therapies.

Interest in the role of male factor in infertility continues to mount with defects related to sperm movement considered as one of the more severe forms of subfertility. The peroxisome proliferator-activated receptor gamma (PPARγ) primarily regulates the expression of target genes involved in energy control as well as lipid and glucose metabolism. Although the pivotal roles of these receptors on female fertility have been reported, there are limited studies addressing PPARs role(s) in the male. This study was designed to determine and compare PPARα, PPARβ and PPARγ mRNA expression in sperm cells of normozoospermic and asthenozoospermic men. In addition, flow cytometric analyses, immunofluorescence and western blot were used to evaluate PPARγ protein levels in spermatozoa. We have compared the sperm PPARs mRNA relative expression in 27 normozoospermic and 28 asthenozoospermic samples and monitored sperm PPARγ protein levels in 39 normozoospermic and 40 asthenozoospermic samples using flow cytometry. We have also assessed in a sub-group of seven normozoospermic and eight asthenozoospermic samples, PPARγ protein levels by western blotting. Relative expression of PPARγ mRNA in normozoospermic men was found to be significantly higher (P = 0.004) than in asthenozoospermic men while PPARα and PPARβ relative expression was similar in the two groups. Likewise, PPARγ showed a positive correlation with motility (r = 0.34; P < 0.05), sperm concentration (r = 0.33) and the percentage of progressive motile spermatozoa (r = 0.31). In agreement with the mRNA behavior, sperm PPARγ protein levels as measured by flow cytometry (P = 0.066) and western blot (P = 0.089) showed a tendency to be higher in normozoospermic than asthenozoospermic men. The present study proposes a link between PPARγ gene expression level and motility in human sperm. Abbreviations: PPARs: Peroxisome Proliferator-Activated Receptors; CASA: Computer Assisted Semen Analysis; TFA: Trans Fatty Acids; HTF: Human Tubal Fluid; PBS: Phosphate-Buffered Saline; PPP: Pentose Phosphate Pathway; PI3K: Phosphoinositide 3-Kinase; G6PDH: Glucose 6-Phosphate Dehydrogenase.

Study Question: Could small molecules (SM) which target (or modify) signaling pathways lead to increased proliferation of undifferentiated spermatogonia following chemotherapy?, Summary Answer: Inhibition of transforming growth factor-beta (TGFb) signaling by SM can enhance the proliferation of undifferentiated spermatogonia and spermatogenesis recovery following chemotherapy., What Is Known Already: Spermatogonial stem cells (SSCs) hold great promise for fertility preservation in prepubertal boys diagnosed with cancer. However, the low number of SSCs limits their clinical applications. SM are chemically synthesized molecules that diffuse across the cell membrane to specifically target proteins involved in signaling pathways, and studies have reported their ability to increase the proliferation or differentiation of germ cells., Study Design, Size, Duration: In our experimental study, spermatogonia were collected from four brain-dead individuals and used for SM screening in vitro. For in vivo assessments, busulfan-treated mice were treated with the selected SM (or vehicle, the control) and assayed after 2 (three mice per group) and 5 weeks (two mice per group)., Participants/materials, Setting, Methods: We investigated the effect of six SM on the proliferation of human undifferentiated spermatogonia in vitro using a top-bottom approach for screening. We used histological, hormonal and gene-expression analyses to assess the effect of selected SM on mouse spermatogenesis. All experiments were performed at least in triplicate and were statistically evaluated by Student's t-test and/or one-way ANOVA followed by Scheffe's or Tukey's post-hoc., Main Results and the Role of Chance: We found that administration of SB431542, as a specific inhibitor of the TGFb1 receptor (TGFbR1), leads to a two-fold increase in mouse and human undifferentiated spermatogonia proliferation. Furthermore, injection of SB to busulfan-treated mice accelerated spermatogenesis recovery as revealed by increased testicular size, weight and serum level of inhibin B. Moreover, SB administration accelerated both the onset and completion of spermatogenesis. We demonstrated that SB promotes proliferation in testicular tissue by regulating the cyclin-dependent kinase (CDK) inhibitors 4Ebp1 and P57 (proliferation inhibitor genes) and up-regulating Cdc25a and Cdk4 (cell cycle promoting genes)., Limitations, Reasons for Caution: The availability of human testis was the main limitation in this study., Wider Implications of the Findings: This is the first study to report acceleration of spermatogenesis recovery following chemotherapy by administration of a single SM. Our findings suggest that SB is a promising SM and should be assessed in future clinical trials for preservation of fertility in men diagnosed with cancer or in certain infertility cases (e.g. oligospermia)., Study Funding/competing Interest(s): This study was supported by Royan Institute and National Institute for Medical Research Development (NIMAD, grant no 963337) granted to H.B. The authors have no conflict of interest to report., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Large bone defects treatment is one of the challenges in current bone tissue engineering approaches. Various strategies have been proposed to address this issue, among which, prevascularization by coculturing of angiogenic and osteogenic cells on the scaffolds can alleviate this problem. In the present study, modified fibrous scaffolds were prepared by electrospinning and subsequent ultrasonication of polycaprolactone (PCL) containing nano-hydroxyapatite (n-HA), with/without nano-zinc oxide (n-ZnO), and polyethylene oxide [PEO] as a sacrificial agent. The physical, mechanical, and chemical characteristics of the scaffolds were evaluated. The results showed the presence of n-ZnO, which in turn increased Young's module of the scaffolds from 5.5 ± 0.67 to 6.7 ± 1.77 MPa. Moreover, MTT, SEM, alkaline phosphatase (ALP) activity, chicken embryo chorioallantoic membrane (CAM) assay, and real-time RT-PCR were utilized to investigate the biocompatibility, cell adhesion and infiltration, osteoconductivity, angiogenic properties, and expression of osteogenic and angiogenic related genes. ALP assay showed that the highest enzyme activity was noted when the modified scaffolds containing n-ZnO were seeded with HUVEC:hBMSC at the cell ratio of 1:5. CAM assay showed induction of angiogenesis for the scaffolds containing n-ZnO. Real-time RT-PCR results showed significant upregulation of angiogenic related genes. Thus, the scaffolds containing n-ZnO may have great potential for osteogenesis and angiogenesis in tissue engineering applications., (© 2019 Wiley Periodicals, Inc.)

This study aimed at investigating the expression of osteoblast and chondrocyte-related genes in mesenchymal stem cells (MSCs), derived from rabbit adipose tissue, under mechanical vibration. The cells were placed securely on a vibrator's platform and subjected to 300 Hz of sinusoidal vibration, with a maximum amplitude of 10 μm, for 45 min per day, and for 14 consequent days, in the absence of biochemical reagents. The negative control group was placed in the conventional culture medium with no mechanical loading. The expression of osteoblast and chondrocyte-related genes was investigated using real-time polymerase chain reaction (real-time PCR). In addition, F-actin fiber structure and alignment with the help of actin filament fluorescence staining were evaluated, and the level of metabolic activity of MSCs was determined by the methyl thiazolyl tetrazolium assay. The real-time PCR study showed a significant increase of bone gene expression in differentiated cells, compared with MSCs (P < 0.05). On the other hand, the level of chondrocyte gene expression was not remarkable. Applying mechanical vibration enhanced F-actin fiber structure and made them aligned in a specific direction. It was also found that during the differentiation process, the metabolic activity of the cells increased (P < 0.05). The results of this work are in agreement with the well-accepted fact that the MSCs, in the absence of growth factors, are sensitive to low-amplitude, high-frequency vibration. Outcomes of this work can be applied in cell therapy and tissue engineering, when regulation of stem cells is required.

Critical-sized bone defects constitute a major health issue in orthopedics and usually cause mal-unions due to an inadequate number of migrated progenitor cells into the defect site or their incomplete differentiation into osteogenic precursor cells. The current study aimed to develop an optimized osteoinductive and angiogenic scaffold by incorporation of strontium (Sr) and bioglass (BG) into gelatin/nano-hydroxyapatite (G/nHAp) seeded with bone marrow mesenchymal stem cells to enhance bone regeneration. The scaffolds were fabricated by a freeze-drying technique and characterized in terms of morphology, structure, porosity and degradation rate. The effect of fabricated scaffolds on cell viability, attachment and differentiation into osteoblastic lineages was evaluated under in vitro condition. Micro computed tomography scan, histological and histomorphometric analysis were performed after implantation of scaffolds into the radial bone defects in rat. RT-PCR analysis showed that G/nHAp/BG/Sr scaffold significantly increased the expression level of osteogenic and angiogenic markers in comparison to other groups (P < 0.05). Moreover, the defects treated with the BMSCs-seeded scaffolds showed superior bone formation and mechanical properties compared to the cell-free scaffolds 4 and 12 weeks post-implantation. Finally, the BMSCs-seeded G/nHAp/BG/Sr scaffold showed the greatest bone regenerative capacity which was more similar to autograft. It is concluded that combination of Sr, BG, and nHAp can synergistically enhance the bone regeneration process. In addition, our results demonstrated that the BMSCs have the potential to considerably increase the bone regeneration ability of osteoinductive scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 50-64, 2019., (© 2018 Wiley Periodicals, Inc.)

Hypoxia-inducible factor-1α (HIF-1α), a well-studied angiogenesis pathway, plays an essential role in angiogenesis-osteogenesis coupling. Targeting the HIF-1a pathway frequently leads to successful reconstruction of large-sized bone defects through promotion of angiogenesis. Dimethyloxalylglycine (DMOG) small molecule regulates the stability of HIF-1α at normal oxygen tension by mimicking hypoxia, which subsequently accelerates angiogenesis. The current study aims to develop a novel construct by seeding adipose derived mesenchymal stem cells (ADMSCs) onto a scaffold that contains DMOG to induce angiogenesis and regeneration of a critical size calvarial defect in a rat model. The spongy scaffolds have been synthesized in the presence and absence of DMOG and analyzed in terms of morphology, porosity, pore size, mechanical properties and DMOG release profile. The effect of DMOG delivery on cellular behaviors of adhesion, viability, osteogenic differentiation, and angiogenesis were subsequently evaluated under in vitro conditions. Histological analysis of cell-scaffold constructs were also performed following transplantation into the calvarial defect. Physical characteristics of fabricated scaffolds confirmed higher mechanical strength and surface roughness of DMOG-loaded scaffolds. Scanning electron microscopy (SEM) images and MTT assay demonstrated the attachment and viability of ADMSCs in the presence of DMOG, respectively. Osteogenic activity of ADMSCs that included alkaline phosphatase (ALP) activity and calcium deposition significantly increased in the DMOG-loaded scaffold. Computed tomography (CT) imaging combined with histomorphometry and immunohistochemistry analysis showed enhanced bone formation and angiogenesis in the DMOG-loaded scaffolds. Therefore, spongy scaffolds that contained DMOG and had angiogenesis ability could be utilized to enhance bone regeneration of large-sized bone defects.

Bioengineering of whole hearts using human embryonic stem cells (hESCs)-derived cardiovascular progenitor cells (CPCs) and natural matrices is a promising approach to overcome organ donor shortage threatening millions of patients awaiting for heart transplantation. Here, we developed a novel strategy for generation of heart constructs by repopulating engineered decellularized rat hearts using hESCs-derived CPCs. Careful expansion of CPCs in a scalable stirred-suspension bioreactor combined with step-wise seeding (60 million cells in 3 steps of 20 million per 1.5 h) onto decellularized hearts containing immobilized basic fibroblast growth factor (bFGF) resulted in improved retention of CPCs and differentiation to cardiomyocytes, smooth muscle cells and endothelial cells as evaluated by immunohistochemistry and qRT-PCR. We observed spontaneous and synchronous contractions of humanized hearts after 12 days of perfusion as well as advanced alignment of myofilaments. Our study provides a robust platform for generation of artificial human hearts and resolves major bottlenecks hindering further development of this technology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Musculodegenerative diseases threaten the life of many patients in the world. Since drug administration is not efficient in regeneration of damaged tissues, stem cell therapy is considered as a good strategy to restore the lost cells. Since the efficiency of myogenic differentiation potential of human Chorion- derived Mesenchymal Stem Cells (C-MSCs) has not been addressed so far; we set out to evaluate myogenic differentiation property of these cells in comparison with Umbilical Cord Blood- derived Mesenchymal Stem Cells (UCB-MSCs) in the presence of 5-azacytidine., Materials & Methods: To do that, neonate placenta Umbilical Cord Blood were transferred to the lab. After characterization of the isolated cells using flowcytometry and multilineage differentiation capacity, the obtained Mesenchymal Stem Cells were cultured in DMEM/F12 supplemented with 2% FBS and 10μM of 5-azacytidine to induce myogenic differentiation. Real-time PCR and immunocytochemistry were used to assess the myogenic properties of the cells., Results: Our data showed that C-MSCs and UCB-MSCs were spindle shape in morphology. They were positive for CD90, CD73 and CD44 antigens, and negative for hematopoietic markers. They also differentiated into osteoblast and adipoblast lineages. Real-time PCR results showed that the cells could express MyoD, desmin and α-MHC at the end of the first week (P<0.05). No significant upregulation was detected in the expression of GATA-4 in both groups. Immunocytochemical staining revealed the expression of Desmin, cTnT and α-MHC., Conclusions: Results showed that these cells are potent to differentiate into myoblast- like cells. An upregulation in the expression of some myogenic markers (desmin, α- MHC) was observed in C-MSCs in comparison with UCB-MSCs., (Copyright © 2017. Published by Elsevier Ltd.)

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox ( Msx ) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx -regulated genes ( Bmp4 , Fgf8 , and keratin 14 ( K14 )) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx -overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8 , and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx -transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition., Competing Interests: Author Disclosure Statement No competing financial interests exist.

Objectives: Maternal high-fat diet has been shown to have deleterious effects on the offspring bones. However, there is no study to assess the effects of type and amount of maternal dietary oil in an isocaloric diet, with focus on extra virgin olive oil (EVOO). The objective of the current study was to test the hypothesis that type of maternal dietary oil has more effects than its amount in an isocaloric diet during gestation and lactation on bone genes expression in offspring in adolescence., Materials and Methods: Virgin female C57BL/6 mice were impregnated and fed either the AIN 93G diet (received 16% of calories as soybean oil, as a control diet, or EVOO) or a high fat AIN 93G diet (received 45% of calories as soybean oil or EVOO) from the time of vaginal plug confirmation until offspring's weaning., Results: After adjusting for the amount of oils, osteoprotegerin/receptor activator of nuclear factor NF-κB ligand (OPG/RANK-L) and OPG expressions were 6.1- and 2.8-folds higher in offspring born to EVOO compared with soybean oil-fed mothers. OPG, beta-catenin, and OPG/RANK-L expression were 88%, 94%, and 70% lower in offspring born to the 45% oil-fed mothers compared with the 16% group. In contrast, peroxisome proliferator-activated receptor gamma-2 (PPARγ2) gene expression was higher in the 45% oil group, adjusted for the types of oil., Conclusion: Maternal EVOO consumption, but not soybean oil increased osteoblastic gene expression, and high amounts of both oils decreased osteoblastic and increased adipogenic genes expression in adolescent offspring.

Objective: In this study, we investigate the effects of preconception endurance training with or without voluntary exercise during pregnancy on indices of bone formation and resorption in female offspring bone., Methods: Twenty-four C57BL/6 female mice were randomly divided into four groups: trained in preconception period and exercised during pregnancy (TE); trained in preconception periods but unexercised during pregnancy (TC); untrained in preconception periods but exercised during pregnancy (CE); untrained and unexercised (CC). Trained dams were subjected to a protocol of moderate exercise training over a period of 4 weeks before pregnancy. Analyses were performed on the adult female offspring that did not have access to running wheels in any portion of their lives., Results: The OPG, Runx2, COLI, ALP, and OPN mRNA expression was significantly up-regulated in offspring born to dams that was trained in preconception period. However, there was no significant difference in OPG, COLI, Runx2, and ALP expression in TE and TC offspring (p > 0.05). RANKL and osteocalcin expression were significantly down-regulated in TE offspring group (p < 0.001)., Conclusions: Improved physical fitness in preconception period results in significant changes in bone gene expressions of female offspring, in particular towards osteogenic responses with improved RANKL/OPG ratio.

Many people worldwide suffer from motor neuron-related disorders such as amyotrophic lateral sclerosis and spinal cord injuries. Recently, several attempts have been made to recruit stem cells to modulate disease progression in ALS and also regenerate spinal cord injuries. Chorion-derived mesenchymal stem cells (C-MSCs), used to be discarded as postpartum medically waste product, currently represent a class of cells with self renewal property and immunomodulatory capacity. These cells are able to differentiate into mesodermal and nonmesodermal lineages such as neural cells. On the other hand, gelatin, as a simply denatured collagen, is a suitable substrate for cell adhesion and differentiation. It has been shown that electrospinning of scaffolds into fibrous structure better resembles the physiological microenvironment in comparison with two-dimensional (2D) culture system. Since there is no report on potential of human chorion-derived MSCs to differentiate into motor neuron cells in two- and three-dimensional (3D) culture systems, we set out to determine the effect of retinoic acid (RA) and sonic hedgehog (Shh) on differentiation of human C-MSCs into motor neuron-like cells cultured on tissue culture plates (2D) and electrospun nanofibrous gelatin scaffold (3D).

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.

Background: Previous studies have suggested mesenchymal stem cells (MSCs) as a suitable source for cell replacement therapy in diabetes. MSCs have successfully isolated from different adult and fetal tissues, including the pancreas. In vitro studies have shown that human fetal pancreatic stem cells could be extensively expanded and differentiated into islet-like structures. Here, we introduce a simple and cost-effective method for the generation of MSCs from the human fetal pancreas (FPMSCs)., Methods: To isolate FPMSCs, pancreata from four aborted fetuses (second trimester) were processed with short collagenase digestion. The resulting tissue fragments were transferred to a basic media (DMEM+15%FBS) without adding any growth factor., Results: After 10 to14 days, fibroblast-like cells were harvested and passaged six times for further evaluations. Flow cytometry analysis and three-lineage differentiation capacity have demonstrated that these cells have MSC-like properties. We also continuously passaged samples of FPMSCs and found no evidence for chromosomal instability and morphological changes until 10th subculture. Moreover, our cell culture protocol can be easily modified and translated into a GMP-compliant one., Conclusion: The results of current study demonstrated that our simple and inexpensive method could yield a pure population of FPMSCs that might be suitable for transplantation.

The protective effects of TGF-β have been documented in various autoimmune diseases, mostly in organ-specific autoimmunity including type 1 diabetes mellitus (T1DM). However, TGF-β also plays a role as a pro-inflammatory mediator by induction of Th17 cytokine production. IL-23 also plays a key role in differentiation of Th17 cells, which are implicated in pathogenesis of autoimmune conditions including T1DM. The aim of this study was to investigate and compare the difference in the level of TGF-β1 and IL-23 gene expression in unstimulated peripheral blood mononuclear cells (PBMCs) of patients with different forms of diabetes compared with normal healthy controls subjects. Patients with T1DM were grouped as early-onset T1DM (N = 20) with age at diagnosis <18 years and late-onset T1DM (N = 20) with the age at onset >18 years. Patients with T2DM (N = 20) and normal healthy controls (N = 20) were recruited from the same area. TGF-β1 and IL-23 gene expression in fresh unstimulated PBMCs was determined in each group using quantitative real-time PCR. The results confirmed that a significant difference in TGF-β1 and IL-23 gene expression was observed in both forms of juvenile-onset T1DM and adult-onset T1DM compared to the controls and T2DM patients. There was no significant difference for TGF-β gene expression in patients with T2DM and controls. We therefore conclude that our results support the previous data on TGF-β gene down-regulation in T1DM. Also up-regulation of IL-23 has been observed in T1DM whilst it was down-regulated in T2DM. We also found no significant difference between juvenile-onset and adult-onset T1DM indicating same mechanism might be involved in the pathogenesis of both types. More studies on different cytokines in Th17 pathways are required to further confirm our finding.

Background: Activating mutations of potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11), which encodes Kir6.2 (beta-cell adenosine triphosphate-sensitive potassium [K(ATP)] channel subunit), have been associated with neonatal diabetes mellitus (NDM) in different studies. Treatment with oral sulfonylureas in place of exogenous insulin injections results in improved glycemic control in most patients carrying these mutations. Exploration of genetic causes of NDM occurring before the age of 6 months has been proposed as an important issue in identification of monogenic forms of diabetes, which might be critical in their therapeutic management, as a consequence., Methods: Mutation screening of the KCNJ11 gene was carried out using PCR amplification followed by direct sequencing in three family members: the proband, ND1, diagnosed at 40 days of age (current age 7 years); his sibling, ND2, diagnosed at 2 years of age (current age 14 years); and their father, ND3, diagnosed at 15 years of age (current age 35 years), who had been exclusively treated with insulin. The effect of the E227K mutation was also examined in a homology model of Kir6.2., Results: Our results revealed the presence of the heterozygous missense mutation c. 679 G/A (E227K) in all three patients, who were all able to successfully transfer from insulin injections to an oral sulfonylurea, with improved glycemic control., Conclusion: We found that three members of a family with highly variable age of onset of insulin-treated diabetes, diagnosed at 40 days, 2 years, and 15 years of age, all carried the E227K mutation in KCNJ11 and could switch to an oral sulfonylurea. This mutation has been previously reported in patients with permanent and transient NDM, as well as later-onset diabetes; this report adds to the variability in phenotypic presentation and further supports genetic testing in all diabetic members of any family affected by NDM.

Survivin expression is correlated with suppression of apoptosis in human solid tumors. A polymorphism at position -31 (G/C) (rs 9904341) has been associated with cancer risk in several studies. We evaluated the correlation of this polymorphism with the risk of papillary thyroid carcinoma (PTC) in an Irananian population. The cases consisted of patients with PTC (n=123) and normal controls, composed of non-related healthy people (n=131). The frequency of GC or CC genotype in patients with PTC was significantly higher than in the controls [GC+CC vs GG, p=0.02 OR; 1.7, 95%CI (1.05-3.04)]. There was a significant difference between patients with more aggressive clinical manifestations, including lymphatic involvement compared to the controls [GC+CC vs GG, p=0.0006, OR; 3.7, 95%CI (1.6-9.2)]. The presence of C allele was significantly associated with the presence of more profound manifestations, including lymph node involvement, vascular involvement and multifocality., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Objective: Survivin is an inhibitor of apoptosis protein, which is up-regulated in endometrial cancer (EC). A promoter region polymorphism (-31G/C) in the survivin gene has been reported as a modulator of gene expression. The aim of this study was to explore the frequency of survivin -31G/C polymorphism in tumor tissues from patients with EC in an Iranian population compared to that of healthy controls., Materials and Methods: Paraffin-embedded tissue sections from patients diagnosed with EC (n = 31) and healthy controls (n = 30) were examined. Genotyping for survivin -31G/C polymorphism was performed using polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP)., Results: The presence of allele C was found to be significantly increased in EC tissues compared to the healthy tissues (GG vs GC + CC, P = 0. 01; OR, 3.6; 95% CI, 1.1-11.9)., Conclusion: Our data are in keeping with a previous finding regarding the role of survivin gene polymorphism in malignancies. This finding highlights the role of survivin in pathogenesis of various carcinomas, which might have therapeutic implications.

Objective: Functional polymorphisms within vascular endothelial growth factor (VEGF) gene have shown association with various conditions including diabetic neuropathy and retinopathy. In this study we have performed a candidate gene association study in order to examine VEGF gene polymorphism association with diabetic foot ulcer (DFU)., Methods: The study group comprised of type 2 diabetes patients with (N=247) and without (N=241) DFU. Healthy control subjects (N=98) were also recruited from the same area. The ARMS-PCR technique was applied for genotyping of VEGF gene SNPs at positions -7*C/T and -2578*C/A., Results: The frequency of genotype AA was significantly decreased in patients with DFU compared with diabetic subjects without DFU (AA vs CA+CC, p=0.003, OR=0.44, CI=0.24-0.80). Also there was a significant decrease in frequency of A allele in patients with DFU compared to the controls (p=0.02, OR=0.68, CI=0.48-0.96)., Conclusion: It seems that lower frequency of A allele in patients with DFU is conferring a protective effect which might be as a result of increased angiogenesis in patients carrying this allele., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)