Comparing the Effects of Krebs Plus Verapamil Solution on Endothelial Function of Harvested Human Greater Saphenous Vein with Heparinized Blood, an Invitro Study.

INTRODUCTION: Integrity of the great saphenous vein (GSV) endothelium is the most important key element for long-term patency rate of grafts in coronary artery bypass graft (CABG). Storage solutions play an important role in maintaining viability of vein endothelium. Diminished nitric oxide (NO) because of endothelial dysfunction may facilitate vascular inflammation and formation of atherosclerotic plaque. AIM: So, we decided to find a reasonable alternative preservative solution instead of heparinized blood (HB) by measuring NO concentration with Griess assay. MATERIAL AND METHOD: SVG samples were obtained from 54 patients undergoing elective CABG. 3 mm rings were stored in solutions: heparinized blood (HB), Krebs (K), Krebs + Propranolol (K+P) 6.66 g/l, Krebs + Adrenaline (K+A) 200 µl/l, and Krebs + Verapamil (K+V) 200 µl/l for 30, 45, 60 and 90 min. Nitrite concentration was measured by Griess assay at 540 nm. H&E staining was performed for histologic test. Statistical analysis was performed using SPSS (V16). Results were expressed as (Means ± SE) followed by One-Way ANOVA for finding best preservative solution. Repeated measurement test was used to investigate best time. In all analysis, (P<0.05) was considered significant. RESULTS: Average concentration of NO in (K+V) compare with HB (1st control), K (2nd control), (K+A) and (K+P) showed higher rate in all times from 30 to 90 min (16.55±1.85:) and in (K+A, K+P) compare with (HB) and (K) there was no statistically significant difference in the same times. Comparing the average concentration of (NO) between (HB) and (K) showed no significant difference (K+V>HB=K=K+A=K+P). Also, our investigations showed that NO concentration in (K+V) has the highest rate in time 90 min (10.07±0.56, p=0.002):. More than 50 percent of endothelial cells stay normal in (K+V) compare with other solutions. CONCLUSION: It seems that (K+V) is the best solution for the maintenance of normal physiology of SVGs endothelial cells. The most appropriate SVGs endothelial function is within 90 minutes after harvesting.

In vitro and in vivo evaluations of three-dimensional hydroxyapatite/silk fibroin nanocomposite scaffolds.

In this study, three-dimensional hydroxyapatite/silk fibroin (HAp/SF) nanocomposite scaffolds were successfully prepared through layer solvent casting combined with the freeze-drying technique for tissue engineering applications. Various SF aqueous concentrations, ranging from 2.5% to 10%, were used to control the physicochemical properties of the prepared scaffolds. Biologic responses of the rat bone marrow stromal cells (rBMSCs) to the HAp/SF scaffolds were examined by culturing the cells within them. In addition, biodegradation and biocompatibility of the scaffolds were evaluated in vitro and in vivo, respectively. Among the prepared scaffolds, HAp/SF-2.5% was the most brittle sample and showed porous structure with lowest mechanical properties. The average pore diameters were 350 ± 67 and 112 ± 89 µm and decreased with the increase in the SF concentration from 5% to 10%, respectively. The pores formed in the scaffolds, made up of the 5% SF, were more uniform and regular than those of the scaffolds made up of 5% and 10% SF. The HAp/SF scaffolds did not change the rBMSCs viability and were not cytotoxic compared with the control sample. The scanning electron microscopy micrographs showed that the cells migrated into the pores and well attached to the scaffolds and their cytoplasm was extended in all directions, indicating a promising cell adhesion, high biocompatibility, and no cytotoxicity of the HAp/SF-5% nanocomposite scaffolds. Subcutaneous implantation of the HAp/SF-5% scaffolds in rat models suggested an excellent biocompatibility. All data obtained from this study suggest the potential use of the HAp/SF-5% for hard tissue engineering.

The Protective Effects of L-Carnitine and Zinc Oxide Nanoparticles Against Diabetic Injury on Sex Steroid Hormones Levels, Oxidative Stress, and Ovarian Histopathological Changes in Rat.

Diabetes mellitus is a common chronic metabolic disorder. This study aimed to investigate the effects of co-treatment with L-carnitine (LC) and zinc oxide nanoparticles (ZnONPs) on serum levels of sex hormones, oxidative stress, and ovarian histopathology in streptozotocin (STZ)-induced diabetic rats. Female Wistar rats (n = 56, 180-220 g) received a single intraperitoneal (IP) injection of STZ (65 mg/kg). They were randomly assigned into the following groups: diabetic group (Dia), Dia+Met group (100 mg metformin/kg/day), Dia+LC group (200 mg/kg/day), Dia+ZnONPs group (10 mg/kg/day), and Dia+LC+ZnONPs group (200 mg LC/kg/day and 10 mg ZnONPs/kg/day). Control group (Ctl) received the same volume of STZ solvent. After 21 days of treatment, blood serum was centrifuged for sex hormone assays. The right ovary was used for biochemical analysis, and the left ovary was fixed in 10% neutral buffered formalin for histological assessment. The levels of estradiol, progesterone, FSH, and LH significantly increased in the Dia+ZnONPs+LC group (P < 0.001) compared with the Dia group. Co-treatment with LC and ZnONPs reduced malondialdehyde and carbonyl protein and increased glutathione, catalase, and superoxide dismutase activities in ovarian tissue compared with the Dia group (P < 0.05). Moreover, the number of all ovarian follicles significantly increased in this group compared with the Dia group (P < 0.05). The results of this study indicated that co-treatment with LC and ZnONPs could preserve ovarian function by increasing sex hormones levels and antioxidant activity and decreasing lipid peroxidation in diabetic rats. Therefore, this compound supplementation may improve ovulation and fertility in people with diabetes mellitus.

Differentiation of bone marrow stromal stem cells seeded on silk scaffold to mature oligodendrocyte using cerebrospinal fluid.

The differentiation of cultured Bone marrow stromal cells (BMSC) on silk scaffold into mature oligodendrocyte was done in the presence of cerebrospinal fluid (CSF). BMSC were isolated from Sprague-Dawley rats and were seeded on silk scaffold. The seeded cells were cultured in DMEM/F12 medium supplemented with CFS, basic fibroblast growth factor (bFGF), Retinoic acid (RA) and Epidermal growth factor (EGF). The glial differentiation was investigated using Real time-PCR and immunofluorescence techniques for specific glial markers: Oligo 2, NG2, PLP and MBP. Our dates showed that the differentiated cells expressed specific glial markers: Oligo 2, NG2, PLP and MBP. The specific mature oligodendrocyte genes were up regulated in cultured cells on silk scaffold in the presence of CSF. It is concluded that CSF leads to improve glial differentiation of seeded BMSC on silk scaffold using preparation of appropriate niche. This culture condition may be served as an efficient differentiation induction protocol for glial phenotype, with the perspective of therapeutic application in neuroregenerative medicine.

Antimicrobial peptides-loaded smart chitosan hydrogel: Release behavior and antibacterial potential against antibiotic resistant clinical isolates.

In this study, we synthesized thermo-responsive chitosan (TCTS) hydrogels, and loaded with different concentrations of antimicrobial peptide (AMP) (0, 4, 8 and 16 µg·ml-1) to fabricate an antibacterial wound dressing against resistant clinical isolates. Physico-chemical properties, release behavior, cytobiocompatibility and antibacterial activity of the AMP-TCTS hydrogels against standard strain and resistant Acinetobacter baumannii were fully determined in vitro. The TCTS-40% ß-glycerolphosphate hydrogels showed a gelation time of 15 min at 37 °C. 80% weight loss at day 35 with no changes in pH value was observed. AMP-TCTS hydrogels showed a burst release of AMP (around 40%) at day 1, and a controlled release up to day 7. A dramatic water uptake was observed at first 4 h, and then continued for 10 h in a steady manner. All the AMP-TCTS hydrogels showed excellent cytobiocompatibility for human fibroblasts. The TCTS showed no antibacterial activity against both standard strain and clinical isolates. All the AMP-TCTS hydrogels had strong antibacterial activity against standard strains, but only 16 µg·ml-1 showed antibacterial behavior against resistant A. baumannii. Our results strongly suggest the 16 µg·ml-1 AMP-TCTS hydrogel as an excellent antibacterial wound dressing against resistant A. baumannii, and now promises to proceed with pre-clinical investigations.

Evaluation of Differential Gene Expression during Transdifferentiation of Bone Marrow Stromal Cells to Glial Phenotype in the Presence of Cerebrospinal Fluid.

BACKGROUND: The present study assessed the alteration of gene expression during transdifferentiation of Bone Marrow Stromal Cells (BMSCs) into oligodendrocyte in the presence of Cerebrospinal Fluid (CSF). METHODS: BMSCs were collected from female Sprague-Dawley rats and were cultured in DMEM/F12 medium supplemented with Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), and Epidermal Growth Factor (EGF). CSF was added daily to the culture media. The oligoprogenitor and oligodendrocyte generation was assessed by immunocytochemistry for Oligo 2, A2B5, CNP and MBP markers. RESULTS: The mean percentages of immunopositive cells for Olig2 and A2B5 were 52.1±1.74 and 56.34±2.55%, respectively. The number of immunopositive cells for glial markers CNP and MBP were 48.8±3.12 and 40.5±8.92%, respectively. Alteration of gene expression of Oct4, Olig 2, PDGFR-α and PLP were examined by real time PCR during transdifferentiation of BMSC to oligodendrocyte. Immunocytochemical results indicate that oligoprogenitor cells were immunopositive for Oligo2 and A2B5 markers. Also, oligodendrocytes expressed the mature glial markers of CNP and MBP indicating successful differentiation. CONCLUSION: In conclusion, CSF promotes the transdifferentiation of BMSC into mature oligodendrocyte via providing an appropriate niche for glial maturation.

Effects of L-Carnitine on the sperm parameters disorders, apoptosis of spermatogenic cells and testis histopathology in diabetic Rats.

BACKGROUND: Diabetes mellitus affects male reproductive system that is known to cause male infertility. OBJECTIVE: The aim of the present study was to assess the effects of L-carnitine (LC) on sperm parameters, apoptosis of spermatogenic cells and testis histopathology in Streptozotocin-induced diabetic Rats. MATERIALS AND METHODS: The study was carried out on 36 male Wistar adult rats (220 ± 30 gr) randomly divided into six groups (n = 6/each). 1 (Control); 2 (LC 100 mg/kg); 3 (Diabetic); 4, 5, and 6 (Diabetic + LC 50 or 100 or 200 mg/kg, respectively). Daily injections were administered intraperitoneally for 48 days. Then, rats were sacrificed, left testis and epididymis were harvested for sperm analysis and histopathology, morphometric and spermatogenesis assessments, and Tunnel assay. RESULTS: L-carnitine in group 6 significantly decreased blood glucose level (p < 0.01) in comparison with group 3. L-carnitine in groups 5 and 6 significantly (p < 0.001) and dose-dependently increased the count, motility, viability, maturity, and chromatin quality of sperm and decreased the abnormal morphology of sperm in comparison with group 3. In groups 4, 5, and particularly 6, in comparison with group 3, there has been a significant difference in the increase of seminiferous tubule diameter, germinal epithelium height (p < 0.001), maturity quality of the seminiferous tubules (p < 0.001), decrease apoptosis of spermatogenic cells (p < 0.001), and testis tissue histopathological complications. CONCLUSION: The data obtained from the present study suggest that in the diabetic rats, LC decreases serum glucose level, improves the diameter and thickness of the epithelium of spermatogenic cells, reduces germ cells' apoptosis, and improves epididymal sperm parameters. Therefore, it seems that LC plays an effective role in diabetes-induced infertility.

Trans-Differentiation of Human Dental Pulp Stem Cells Into Cholinergic-Like Neurons Via Nerve Growth Factor.

INTRODUCTION: Cell therapy has been widely considered as a therapeutic approach for neurodegenerative diseases and nervous system damage. Cholinergic neurons as one of the most important neurons that play a significant role in controlling emotions, mobility, and autonomic systems. In this study, Human Dental Pulp Stem Cells (hDPSCs) were differentiated into the cholinergic neurons by ß-mercaptoethanol in the preinduction phase and also by the nerve growth factor (NGF) in the induction phase. METHODS: The hDPSCs were evaluated for CD73, CD31, CD34, and Oct-4. Concentration-time relationships for NGF were assessed by evaluating the viability rate of cells and the immune response to nestin, neurofilament 160, microtubule-associated protein-2, and choline acetyltransferase. RESULTS: The hDPSCs had a negative response to CD34 and CD31. The optimal dose for the NGF was 50 ng/mL seven days after the induction when the highest percentage of expressing markers for the Cholinergic neurons (ChAT) was detected. CONCLUSION: The results of this study provided a method for producing cholinergic neurons by hDPSCs, which can be used in cytotherapy for degenerative diseases of the nervous system and also spinal cord injury.

Effects of l-Carnitine on the Follicle-Stimulating Hormone, Luteinizing Hormone, Testosterone, and Testicular Tissue Oxidative Stress Levels in Streptozotocin-Induced Diabetic Rats.

The present study was designed to investigate the antioxidant property of l-carnitine (LC) on serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (TH) and testis oxidative stress in streptozotocin (STZ)-induced diabetic rats. The rats were divided into the following groups: group I, control; group II, LC 100 mg/kg/d; group III, diabetic; and groups IV to VI, diabetic rats treated with 50, 100, and 200 mg/kg/d of LC, respectively. Daily injections were given intraperitoneally for 7 weeks. At the end of experimental period, after sacrificing the rats, FSH, LH, TH, total antioxidant capacity (TAC), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), mitochondrial function (MTT), protein carbonyl (PC), and reactive oxygen species (ROS) levels were measured. STZ caused an elevation of MDA, ROS, and PC ( P < .001) with reduction of GSH, CAT, TAC, and MTT ( P < .001) in the serum levels. Group VI had significantly increased FSH, LH, and TH levels versus the untreated diabetic group ( P < .001). Although groups V and VI significantly decreased MDA ( P < .001), PC ( P < .01), and ROS ( P < .01) compared with the untreated diabetic group; only in group VI, the activity of GSH ( P < .001), CAT ( P < .01), TAC ( P < .001), and MTT ( P < .001) significantly increased. The results of the present study suggest that LC decreased diabetes-induced oxidative stress complications and also improved serum level of FSH, LH, and TH by reducing levels of lipid peroxidation and increasing antioxidant enzymes.

Repair of Critical-Sized Rat Calvarial Defects With Three-Dimensional Hydroxyapatite-Gelatin Scaffolds and Bone Marrow Stromal Stem Cells.

INTRODUCTION: The repair of critical-sized defects (CSDs) are one of the most challenging orthopedic problems and the attempts for development of an ideal scaffold for treatment of large bone defect are ongoing. AIM: The aim of this study was the effectiveness of hydroxyapatite-gelatin seeded with bone marrow stromal cells construct for healing of critical-sized bone defect in vivo. MATERIAL AND METHODS: In this experimental study, the bone marrow stromal cells (BMSCs) were isolated by flushing method. For in vitro study, the cells were seeded on the scaffold and the cell viability as well as cytotoxicity were tested by MTT and LDH specific activity. The scaffold-cell construct was implanted into the critical-sized bone defect created in calvaria of Wistar male rats.15 rats were randomly divided into 3 groups (n=5), group 1 (control group): Injury without transplantation, group 2: implanted with hydroxyapatite-gelatin scaffold, group 3: hydroxyapatite-gelatin scaffold seeded with BMSCs. At different days post-implantation, the implanted site was collected and the bone healing was evaluated through H&E and Masson's Trichrome staining. ANOVA and paired t-test were used for data comparison and P<0.05 was considered significant. RESULTS: The results of MTT showed that the scaffold has no toxic effects on stromal cells. The first signs of ossification in hydroxyapatite-gelatin with BMSCs cells group appeared in the first week. However, in the fourth week, ossification was completed and the scaffold remaining was found as embedded islands in the spongy bone tissue. The greatest number of lymphocytes in the experimental group was observed after one week of planting scaffold. CONCLUSION: Hydroxyapatite-gelatin scaffold coated with BMSCs cells has a potential role in the healing process of bone and would be a possible new therapeutic strategy to repair extensive bone lesions.

Combined protective effect of zinc oxide nanoparticles and melatonin on cyclophosphamide-induced toxicity in testicular histology and sperm parameters in adult Wistar rats.

BACKGROUND: Cyclophosphamide (CP) has been known as an anticancer drug with several side effects on various organs such as a male reproductive system that can cause infertility. OBJECTIVE: To evaluate the possible combined effects of zinc oxide nanoparticles (nZno) and melatonin (Mel) on sperm parameters and histopathological changes of the testis in CP-treated rats. MATERIALS AND METHODS: 42 adult male Wistar rats were divided into six groups. GI: control, GII: 60 mg/kg/wk CP, GIII and GIV, 10 mg/kg/wk Mel and 5mg/kg/wk nZno and GV: 5 mg/kg/wk nZno and 10 mg/kg/wk Mel were given 2 hr prior to CP injection, respectively,GVI: 5mg/kg/wk nZno and 10 mg/kg/wk Mel simultaneously. After 8 wk of treatment, rats were sacrificed and testis and epididymis were harvested for further evaluation. RESULTS: The CP-treated group showed significant decreases in the body, testes and epididymis weights and sperm parameters (sperm count, viability, motility) with an increase abnormal sperms when compared with the control (p<0.001), as well as many histological alterations included decreased diameters of seminiferous tubules and Johnsen's Testicular Score (with degeneration, desquamation, multi-nucleated giant cell formation), whereas combined treatment (GV), showed more protective effects on CP-induced reproductive system damage compared with groups III or IV (p<0.001). CONCLUSION: These results suggest simultaneous administration of Mel and nZno have more effectively protections against CP-induced reproductive damage than Mel or nZno alone.