A comparison between BMP4 and SB4 in inducing germ line gene expression pattern during embryonic stem cells differentiation.

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.

The Outcome of Stem Cell-Based Therapies on the Immune Responses in Rheumatoid Arthritis.

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.

Stem cell-based models and therapies: a key approach into schizophrenia treatment.

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.

Auxiliary role of mesenchymal stem cells as regenerative medicine soldiers to attenuate inflammatory processes of severe acute respiratory infections caused by COVID-19.

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.

Suppression of transforming growth factor-beta signaling enhances spermatogonial proliferation and spermatogenesis recovery following chemotherapy.

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.

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice.

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.

3D-porous ß-tricalcium phosphate-alginate-gelatin scaffold with DMOG delivery promotes angiogenesis and bone formation in rat calvarial defects.

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.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

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-ß1) 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-ß1-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-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/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-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Biological evaluation and osteogenic potential of polyhydroxybutyrate-keratin/Al2O3 electrospun nanocomposite scaffold: A novel bone regeneration construct.

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.

In vitro investigation of zinc oxide nanoparticle toxic effects in spermatogonial cells at the molecular level.

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.

Differentiation of Mesenchymal Stem Cells Derived From Human Adipose Tissue Into Cholinergic-like Cells: An in Vitro Study.

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.

Gene Therapy as an Emerging Therapeutic Approach to Breast Cancer: New Developments and Challenges.

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.

Mesenchymal stem cell as a novel approach to systemic sclerosis; current status and future perspectives.

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.

Design and characterization of an electroconductive scaffold for cardiomyocytes based biomedical assays.

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.

Production and evaluation of decellularized extracellular matrix hydrogel for cartilage regeneration derived from knee cartilage.

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.

Short time exposure to low concentration of zinc oxide nanoparticles up-regulates self-renewal and spermatogenesis-related gene expression.

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.

Synergistic effect of strontium, bioactive glass and nano-hydroxyapatite promotes bone regeneration of critical-sized radial bone defects.

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.

The effect of modified electrospun PCL-nHA-nZnO scaffolds on osteogenesis and angiogenesis.

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.

Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids.

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 ß-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.

Peroxisome Proliferator-Activated Receptors (PPARs) levels in spermatozoa of normozoospermic and asthenozoospermic men.

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, PPARß 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 PPARß 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.