Advances in Exosome-Based Therapies for the Repair of Peripheral Nerve Injuries.

Peripheral nerve injuries (PNIs) are the term used to describe injuries that occur to the nerve fibers of the peripheral nervous system (PNS). Such injuries may be caused by trauma, infection, or aberrant immunological response. Although the peripheral nervous system has a limited capacity for self-repair, in cases of severe damage, this process is either interrupted entirely or is only partially completed. The evaluation of variables that promote the repair of peripheral nerves has consistently been a focal point. Exosomes are a subtype of extracellular vesicles that originate from cellular sources and possess abundant proteins, lipids, and nucleic acids, play a critical role in facilitating intercellular communication. Due to their modifiable composition, they possess exceptional capabilities as carriers for therapeutic compounds, including but not limited to mRNAs or microRNAs. Exosome-based therapies have gained significant attention in the treatment of several nervous system diseases due to their advantageous properties, such as low toxicity, high stability, and limited immune system activation. The objective of this review article is to provide an overview of exosome-based treatments that have been developed in recent years for a range of PNIs, including nerve trauma, diabetic neuropathy, amyotrophic lateral sclerosis (ALS), glaucoma, and Guillain-Barre syndrome (GBS). It was concluded that exosomes could provide favorable results in the improvement of peripheral PNIs by facilitating the transfer of regenerative factors. The development of bioengineered exosome therapy for PNIs should be given more attention to enhance the efficacy of exosome treatment for PNIs.

Fabrication and application of salicin-polycaprolactone 3D-printed scaffold in the healing of femur bone defects.

Polycaprolactone (PCL) is a suitable material for bone repair due to good biocompatibility and mechanical properties. However, low bioactivity and hydrophobicity pose major challenges for its biomedical applications. To overcome these limitations, PCL-based scaffolds loaded with bioactive agents have been developed. Salicin (Sal) is an anti-inflammatory and analgesic herbal glycoside with osteogenic potential. In the present study, we aimed to produce a Sal-laden PCL (PCL-Sal) scaffold for bone healing applications. Three-dimensional scaffolds were produced and their biocompatibility, and physical-chemical characteristics were determined. The osteogenic potential of the PCL (PCL) and PCL-Sal scaffolds was evaluated using bone marrow mesenchymal stem cells (BMSCs). Scaffolds were implanted into a 5 mm bone defect created in the femur of adult rats, and the new bone fraction was determined using micro-computed tomography scanning at one-month follow-up. PCL-Sal scaffold had a structure, porosity, and fiber diameter suitable for bone construction. It also possessed a higher rate of hydrophilicity and bioactivity compared to the PCL, providing a suitable surface for the proliferation and bone differentiation of BMSCs. Furthermore, PCL-Sal scaffolds showed a higher capacity to scavenge free radicals compared to PCL. The improved bone healing potential of the PCL-Sal scaffold was also confirmed according toin vivoimplantation results. Our findings revealed that the Sal-laden implant could be considered for bone repair due to desirable characteristics of Sal such as hydrophilicity, surface modification for cell attachment, and antioxidant properties.

Selenium enhances the expression of miR-9, miR-124 and miR-29a during neural differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: Selenium (Se) is a trace element that plays important role in antioxidant defense in the brain. Sodium selenite (Na2SeO3) is an inorganic salt of Se which has an antioxidant function. In the present study, we investigated the effect of Sodium selenite on the expression of important neuronal microRNAs during neural differentiation of bone marrow-derived stem cells (BMSCs). METHODS: Mesenchymal stem cells were collected from rat bone marrow and cultured in the Dulbecco's Modified Eagle Medium (DMEM) medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was conducted to determine the toxicity of Na2SeO3. For neural induction, BMSCs were divided into control, Na2SeO3 containing (10 ng/mL) and Na2SeO3 free groups and cultured in DMEM medium supplemented with Isobutyl-l-methylxanthine (IBMX), Fibroblast growth factor 2 (FGF2), B27, Retinoic acid, and brain derived neurotrophic factor (BDNF) for 14 days. At the end of the differentiation, immunostaining against Microtubule associated protein 2 (Map-2) and Choline acetyltransferase (ChAT) proteins was performed. Also, the total RNA is extracted from control and neural differentiated cells using a special kit, and the expression of miR-9, miR-124, and miR-29a was analyzed using real-time polymerase chain reaction (RT-PCR). RESULTS: Increasing Na2SeO3 concentrations had increasing toxicity; therefore, the concentration of 10 ng/mL was used as a supplement during neural differentiation. Examination of the expression of Map-2 and ChAT proteins showed that Na2SeO3 increased the expression of them and consequently the neuronal differentiation of BMSCs. Na2SeO3 also significantly increased the expression of miR-9, miR-124, and miR-29a in BMSCs undergoing neuronal differentiation. CONCLUSIONS: Our results suggest that the protective effect of selenium on neural differentiation of stem cells may be mediated through neuron specific microRNAs. This result further highlights the importance of selenium supplementation in preventing neuronal diseases.

Effect of Vitex agnus-castus ethanolic extract on hypothalamic KISS-1 gene expression in a rat model of polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is an endocrine system disruption that affects 6-10% of women. Some studies have reported the effect of Vitex agnus-castus (Vitagnus) on the hypothalamic-pituitary-gonad axis (HPG). This study was conducted to investigate Vitagnus effect on the expression of kisspeptin gene in a rat model of PCOS. MATERIALS AND METHODS: Thirty-two female rats were distributed into: control, Vitagnus-treatment (365 mg/kg for 30 days), PCOS (Letrozole for 28 days) and PCOS animals treated with Vitagnus (30 days of Vitagnus after PCOS induction). At the end of the treatments, serum and ovaries were collected for analysis. Expression level of KISS-1 gene in the hypothalamus was investigated, using Real-Time-PCR. RESULTS: In the PCOS group compared to control, FSH, progesterone and estradiol levels were decreased, whereas testosterone and LH levels were significantly increased. No significant changes were observed in the Vitagnus-treated animals in compare to control. However, Vitagnus treatment in the PCOS group, resulted in a raise in progesterone, estrogen and FSH levels and a reduction in the levels of testosterone and LH. Quantitative gene expression analysis showed that PCOS induction resulted in over-expression of KISS-1 gene, however, Vitagnus treatment reduced this up-regulated expression to normal level. CONCLUSION: In conclusion, our results indicated that Vitagnus extract inhibited downregulation of KISS-1 gene in the hypothalamus of PCOS rats. Because of the master role of kisspeptin in adjusting the HPG axis, Vitagnus is likely to show beneficial effects in the treatment of PCOS via regulation of kisspeptin expression. This finding indicates a new aspect of Vitagnus effect and may be considered in its clinical applications.

Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats.

Paternal metabolic status is an important factor in the health status of offspring. Cholestasis, as a metabolic disorder, significantly disrupts spermatogenesis. Spermatogonial stem cells (SSCs) are considered the dividing germ cells, which maintain spermatogenesis throughout the lifespan. Here, we investigated the in vivo and in vitro effect(s) of cholestasis on SSCs. Cholestasis was induced in rats by bile duct ligation. Four weeks after the cholestasis induction, testicular tissues were analysed using histopathological examinations. The expression of SSC markers, including Plzf and Thy-1, was determined using the immunofluorescent technique. Also, SSCs were isolated from animals, and their proliferation was examined in vitro. The histological examinations revealed that cholestasis caused irregularities in the structure of seminal tubes. Immunostaining showed that the total number of Thy-1- and Plzf-expressing cells was declined in the cholestasis group compared with the control group. In vitro culture of SSCs indicated that the number of SSC colonies and those expressing Plzf were significantly reduced in the culture medium of the cholestasis group. Our results indicated that cholestasis affects the functionality of SSCs and reduces the number and proliferation of them. This finding may be of interest to the effect of metabolic diseases such as cholestasis on spermatogenesis.

Comparison the Effect of Metformin and Clomiphene Citrate on Sirtuin3 gene Expression in the Oocytes of Mice with Polycystic Ovary Syndrome.

Oxidative stress (OS) is a common biological event in polycystic ovarian syndrome (PCOS), causing oocytes to undergo OS-induced changes. Sirtuin3 (Sirt3) has a critical role in oocyte maturation through the modulation of OS. In the current study, we compared the effects of metformin and clomiphene citrate on the expression of the Sirt3 gene in oocytes obtained from the mice, induced by PCOS. The induction of PCOS was performed by the single injection of estradiol valerate. The animals were divided into control, PCOS, metformin (500 mg/Kg), and clomiphene (18 mg/kg) groups. At the end of the experiment, the levels of LH and FSH were determined using the ELISA method. The ovarian tissues were evaluated histologically, and the expression of the Sirt3 gene was analyzed by the Real-time PCR. The induction of PCOS led to an increase in the ratio of LH/FSH elevation, the number of follicle atresia, as well as the presence of hydrated cysts. The results showed that both treatment regimens returned the altered parameters to the baseline values. The gene of Sirt3 was significantly (P < 0.001) reduced in the PCOS group compared to the control. Also, no significant difference was found in the expression of Sirt3 between clomiphene and PCOS group, whereas, in the metformin group, Sirt3 expression had the higher rate of expression in comparison with the PCOS group (P < 0.05). The administration of metformin and clomiphene showed that metformin is capable of preventing the downregulation of the Sirt3 gene in oocytes, collected from PCOS mice.

The synergistic cytotoxic effects of doxorubicin and Viola odorata extract on human breast cancer cell line T47-D.

BACKGROUND: Breast cancer accounts for one-third of cancer cases in women. Doxorubicin (Dox) is one of the chemotherapeutical compounds widely used to treat breast cancer. Chemical drugs have several side effects and their continuous administration leads to drug resistance in patients. To decrease such side effects in cancer treatment, combination therapy as well as application of natural and herbal compounds has been taken into consideration. The aim of this study was to investigate the cytotoxic effect of Viola odorata (Vo) extract on T47-D human breast cancer cells, alone and in combination with Dox. MATERIALS AND METHODS: The cytotoxic effects of V. odorata and Dox were studied by morphological examination and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flowcytometric analysis was performed to determine the type of cell death. Moreover, scratch healing assay was conducted to investigate antimigration effect of V. odorata. RESULTS: The results of MTT assay showed that V. odorata and Dox-induced cell death in T47-D cells in a dose- and time-dependent manner. Morphological analysis revealed that V. odorata and Dox-induced features of apoptotic cell death in T47-D cells. These results were confirmed by flow cytometry analysis. Scratch healing assay revealed that migration rate was reduced in the V. odorata- treated cells. CONCLUSIONS: Our findings suggest that components of V. odorata exert antitumor effects on human breast cancer and could be administered with lower doses of antitumor agent Dox, in combination therapy, to decrease its side effects.

Potential Use of Amniotic Membrane - Derived Scaffold for Cerebrospinal Fluid Applications.

Scaffolds derived from decellularized tissues provide a natural microenvironment for cell culture. Embryonic cerebrospinal fluid (e-CSF) contains factors which play vital roles in the development of the nervous system. This research was aimed to survey the effect of Wistar rat e-CSF on neural differentiation of bone marrow derived mesenchymal stem cells (BM-MSCs) cultured on the human amniotic membrane (AM). BM-MSCs were collected from femurs and tibias, and were cultured in Dulbecco's Modified Eagle's Medium. The placenta was harvested from healthy women during cesarean section and AM was acellularized using EDTA and physical scrubbing. e- CSF was harvested from rat fetuses at E17. Adequate numbers of BM-MSCs were cultured on acellularized membrane, and were treated with E17 CSF for 7 days. MTT (3-(4, 5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) assay confirmed the survival and proliferation of BM-MSCs cultured on AM derived scaffold. Hematoxylin/eosin staining and scanning electron microscopy showed the morphological and the structural changes of BM-MSCs throughout the culture and treatment with e-CSF. The results of immunocytochemistry showed that microtubule associated protein 2 and beta-III tubulin were expressed in BM-MSCs cultured on acellular amnion scaffold and treated with e-CSF. Our results showed for the first time that the combination of acellular AM as a natural scaffold and e-CSF as a source of neurological factors could effectively improve the BM-MSCs cultivation and differentiation.

Oleic acid promotes the expression of neural markers in differentiated human endometrial stem cells.

Variety of neurodegenerative diseases in humans are caused by loss of cells along with loss of function and disability. Cell replacement therapy is a potential strategy to cure neurodegenerative diseases. Mesenchymal stem cells are pluripotent non-hematopoietic cells that can be isolated from numerous tissues. Human endometrial-derived stem cell (hEnSC) are the abundant and easy available source with no immunological response, for cell replacement therapy. In the nervous system, where fatty acids are found in huge amounts, they participate in its development and maintenance throughout life. Oleic acid is a kind of the saturated fatty acids which plays crucial role in brain development. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. Human endometrial-derived stem cells in the third passage were divided into 3 groups including: control, sham (cultured in full differentiation medium without oleic acid) and experimental group (cultured in full differentiation medium with oleic acid) to differentiate over a 18-day period. Data from Real-Time PCR showed that mRNA levels of NF and ß-TUBULIN were increased significantly (p<0.05) in oleic acid treated cells in comparison to control and sham groups. Immunocytochemistry analysis of Chat and NF expression also showed the same results. The present study clearly demonstrates that oleic acid promotes neural differentiation of hEnSC through regulation of gene expression.

Unrestricted somatic stem cells as vehicle for nerve growth factor gene transfer.

OBJECTIVE: Nerve growth factor (NGF), a member of the neurotrophic factor family, plays a critical role in the maintenance and regeneration of different types of neurons. To overcome drastic challenges in the peripheral delivery of NGF, transplantation of NGF secreting stem cells to the target site of an injury may be an effective procedure. Unrestricted somatic stem cells (USSCs), a subtype of umbilical cord blood (UCB) stem cells, have shown promise for gene therapy purposes, and proper results have been observed from transplantation experiments in neurodegenerative disorders. Based on the considerable potential of USSCs for gene delivery applications, the goal of the current study was to establish a betaNGF gene containing USSCs, which is able to secrete functional recombinant betaNGF protein. METHODS: Unrestricted somatic stem cells were isolated from UCB and were cultured in a DMEM medium. The betaNGF gene was cloned in the EFalpha-promoting lentiviral vector, and virus production was performed as a third generation lentivirus packaging system. Enzyme-linked immunosorbent assay, real-time PCR, co-culture, MTT, and immunocytochemistry assays were performed to evaluate the genetically engineered USSCs. RESULTS: Overexpression of betaNGF gene in human USSCs created a USSC line that is able to secrete high amounts of functional betaNGF protein. betaNGF-secreting USSCs showed a high rate of viability along with acceptable immunological and morphological properties for transplantation into the nervous system. DISCUSSION: Long-term expression of functional betaNGF, high viability of betaNGF producing USSCs, and expression of primary neuronal markers suggest that USSCs may be useful to deliver betaNGF into targeted sites of the nervous system in neurodegenerative disorders.