Photobiomodulation, alone or combined with adipose-derived stem cells, reduces inflammation by modulation of microRNA-146a and interleukin-1ß in a delayed-healing infected wound in diabetic rats.

Diabetic wounds are categorized by chronic inflammation, leading to the development of diabetic foot ulcers, which cause amputation and death. Herewith, we examined the effect of photobiomodulation (PBM) plus allogeneic diabetic adipose tissue-derived stem cells (ad-ADS) on stereological parameters and expression levels of interleukin (IL)-1ß and microRNA (miRNA)-146a in the inflammatory (day 4) and proliferation (day 8) stages of wound healing in an ischemic infected (with 2×107 colony-forming units of methicillin-resistant Staphylococcus aureus) delayed healing wound model (IIDHWM) in type I diabetic (TIDM) rats. There were five groups of rats: group 1 control (C); group 2 (CELL) in which rat wounds received 1×106 ad-ADS; group 3 (CL) in which rat wounds received the ad-ADS and were subsequently exposed to PBM(890 nm, 80 Hz, 3.5 J/cm2, in vivo); group 4 (CP) in which the ad-ADS preconditioned by the PBM(630 nm + 810 nm, 0.05 W, 1.2 J/cm2, 3 times) were implanted into rat wounds; group 5 (CLP) in which the PBM preconditioned ad-ADS were implanted into rat wounds, which were then exposed to PBM. On both days, significantly better histological results were seen in all experimental groups except control. Significantly better histological results were observed in the ad-ADS plus PBM treatment correlated to the ad-ADS alone group (p<0.05). Overall, PBM preconditioned ad-ADS followed by PBM of the wound showed the most significant improvement in histological measures correlated to the other experimental groups (p<0.05). On days 4 and 8, IL-1 ß levels of all experimental groups were lower than the control group; however, on day 8, only the CLP group was different (p<0.01). On day 4, miR-146a expression levels were substantially greater in the CLP and CELL groups correlated to the other groups, on day 8 miR-146a in all treatment groups was upper than C (p<0.01). ad-ADS plus PBM, ad-ADS, and PBM all improved the inflammatory phase of wound healing in an IIDHWM in TIDM1 rats by reducing inflammatory cells (neutrophils, macrophages) and IL-1ß, and increasing miRNA-146a. The ad-ADS+PBM combination was better than either ad-ADS or PBM alone, because of the higher proliferative and anti-inflammatory effects of the PBM+ad-ADS regimen.

Preconditioning adipose-derived stem cells with photobiomodulation significantly increased bone healing in a critical size femoral defect in rats.

We assessed the combined impacts of human demineralized bone matrix (hDBM) scaffold, adipose-derived stem cells (hADS), and photobiomodulation (PBM) on bone repair of a critical size femoral defect (CSFD) in 72 rats. The rats were divided into six groups: control (group 1); ADS (group 2 - ADS transplanted into hDBM); PBM (group 3 - PBM-treated CSFDs); ADS + PBM in vivo (group 4 - ADS transplanted into hDBM and the CSFDs were treated with PBM in vivo); ADS + PBM in vitro (group 5 - ADS were treated with PBM in vitro, then seeded into hDBM); and ADS + PBM in vitro+in vivo (group 6 - PBM-treated ADS were seeded into hDBM, and the CSFDs were treated with PBM in vivo. At the anabolic phase (2 weeks after surgery), bone strength parameters of the groups 5, 6, and 4 were statistically greater than the control, ADS, and PBM in vivo groups (all, p = 0.000). Computed tomography (CT) scans during the catabolic phase (6 weeks after surgery) of bone healing revealed that the Hounsfield unit (HU) of CSFD in the groups 2 (p = 0.000) and 5 (p = 0.019) groups were statistically greater than the control group. The groups 5, 4, and 6 had significantly increased bone strength parameters compared with the PBM in vivo, control, and ADS groups (all, p = 0.000). The group 5 was statistically better than the groups 4, and 6 (both, p = 0.000). In vitro preconditioned of hADS with PBM significantly increased bone repair in a rat model of CSFD in vivo.

Effects of pentoxifylline and alendronate on fracture healing in ovariectomy-induced osteoporosis in rats.

Osteoporosis is determined by decreased bone strength that increases the threat of fractures. The aim of this study was to evaluate the effects of pentoxifylline (PTX) and alendronate (ALN), on the stereological parameters, and gene expression in callus of fracture in an experimental rat model of ovariectomy-induced osteoporosis (OVX). The OVX was induced in 90 female rats. Fourteen weeks later, a complete fracture on the right femur was made. Rats were divided into five groups: 1) control: no treatment; 2) sham: received daily distilled water; 3) daily 3.00 mg kg-1 ALN subcutaneously (SC); 4) daily 200 mg kg-1 PTX (SC) and 5) daily PTX (SC) + ALN (same doses). The osteoclast count was significantly lower in all treatment groups, at 21 and 56 days post-surgery, compared to the control and sham groups. The PTX significantly increased total callus volume at 21 and 56 days post-surgery, compared to the other groups. The PTX+ALN treatment significantly increased both cortical bone volume on day 21, and osteocyte and osteoblast numbers on day 56, compared to the control and sham groups. It can be concluded that PTX and ALN have antiresorptive effects, in OVX rats. Also, PTX has increased the extracellular matrix on both 21 and 56 days after surgery, compared to the other groups. PTX+ALN elevated cortical bone volume on day 21, and osteocyte and osteoblast numbers compared to the control and sham groups on day 56.

Effect of Artificial Oocyte Activation on Intra-Cytoplasmic Sperm Injection Outcomes in Patients with Lower Percentage of Sperm Containing Phospholipase Cζ: A Randomized Clinical Trial.

BACKGROUND: Artificial oocyte activation (AOA) is a specialized method in assisted reproductive technique (ART). According to increasing concern about using AOA, it is necessary to evaluate sperm-borne oocyte activating factors (SOAFs) including phospholipase C zeta (PLCζ). In this study, PLCζ before AOA was evaluated first and then the impact of AOA on pre-implantation embryo development was investigated. METHODS: This prospective clinical trial enrolled couples subjected to ICSI. By evaluating PLCζ, semen samples were categorized into two groups; I (Control) and II (PLCζ deficient). Retrieved oocytes from partners were put into three categories: control group (Injected with sperm from group I, n=113), group without AOA (Injected with sperm from group II and no exposure to AOA, n=106), and group AOA (Injected with sperm from group II and exposure to AOA, n=114). Finally, fertilization results were compared via Kruskal-Wallis followed by Dunn's multiple comparison test. The p<0.05 was considered statistically significant. RESULTS: Fertilization rate was significantly lower in the group without AOA compared to control group (41.9±6.3 vs. 78.1±4.7, p<0.001). AOA improved fertilization rate in group AOA compared to the group without AOA (69.5±3.9 vs. 41.9±6.3, p<0.01); however, cleavage (91.7±2.8, 90.9±4.6, and 95.2±3.4, respectively) and embryo quality (2.5±0.1, 2.3±0.2, and 2.4±0.2, respectively) scores were not substantially different between groups of control, with and without AOA. CONCLUSION: We showed that PLCζ can be considered as a good biomarker in evaluation of oocyte activation capability. Further studies are required to establish the best use of PLCζ as a biomarker in clinics.

Anacardic Acid Reduces Acetylation of H4K12 in Mouse Oocytes during Vitrification.

OBJECTIVE: Over the last years, vitrification has been widely used for oocyte cryopreservation, in animals and humans; however, it frequently causes minor and major epigenetic modifications. The effect of oocyte vitrification on levels of acetylation of histone H4 at lysine 12 (AcH4K12), and histone acetyltransferase (Hat) expression, was previously assessed; however, little is known about the inhibition of Hat expression during oocyte vitrification. This study evaluated the effect of anacardic acid (AA) as a Hat inhibitor on vitrified mouse oocytes. MATERIALS AND METHODS: In this experimental study, 248 mouse oocytes at metaphase II (MII) stage were divided in three experimental groups namely, fresh control oocytes (which were not affected by vitrification), frozen/thawed oocytes (vitrified) and frozen/thawed oocytes pre-treated with AA (treatment). Out of 248 oocytes, 173 oocytes were selected and from them, 84 oocytes were vitrified without AA (vitrified group) and 89 oocytes were pretreated with AA, and then vitrified (treatment group). Fresh MII mouse oocytes were used as control group. Hat expression and AcH4K12 levels were assessed by using real-time quantitative polymerase chain reaction (PCR) and immunofluoresce staining, respectively. In addition, survival rate was determined in vitrified and treatment oocytes. RESULTS: Hat expression and AcH4K12 modification significantly increased [4.17 ± 1.27 (P≤0.001) and 97.57 ± 6.30 (P<0.001), respectively] in oocytes that were vitrified, compared to the fresh oocytes. After treatment with AA, the Hat mRNA expression and subsequently H4K12 acetylation levels were significantly reduced [0.12 ± 0.03 (P≤0.001) and 89.79 ± 3.20 (P≤0.05), respectively] in comparison to the vitrified group. However, the survival rate was not significantly different between the vitrified (90.47%) and treatment (91.01%) groups (P>0.05). CONCLUSION: The present study suggests that AA reduces vitrification risks caused by epigenetic modifications, but does not affect the quality of vitrification. In fact, AA as a Hat inhibitor was effective in reducing the acetylation levels of H4K12.

Evaluation of the Effects of Photobiomodulation on Partial Osteotomy in Streptozotocin-Induced Diabetes in Rats.

OBJECTIVE: We examined the effects of photobiomodulation (PBM) on stereological parameters, and gene expression of Runt-related transcription factor 2 (RUNX2), osteocalcin, and receptor activator of nuclear factor kappa-B ligand (RANKL) in repairing tissue of tibial bone defect in streptozotocin (STZ)-induced type 1 diabetes mellitus (TIDM) in rats during catabolic response of fracture healing. BACKGROUND DATA: There were conflicting results regarding the efficacy of PBM on bone healing process in healthy and diabetic animals. MATERIALS AND METHODS: Forty-eight rats have been distributed into four groups: group 1 (healthy control, no TIDM and no PBM), group 2 (healthy test, no TIDM and PBM), group 3 (diabetic control, TIDM and no PBM), and group 4 (diabetic test, no TIDM and PBM). TIDM was induced in the groups 3 and 4. A partial bone defect in tibia was made in all groups. The bone defects of groups second and fourth were irradiated by a laser (890 nm, 80 Hz, 1.5 J/cm2). Thirty days after the surgery, all bone defects were extracted and were submitted to stereological examination and real-time polymerase chain reaction (RT-PCR). RESULTS: PBM significantly increased volumes of total callus, total bone, bone marrow, trabecular bone, and cortical bone, and the numbers of osteocytes and osteoblasts of callus in TIDM rats compared to those of callus in diabetic control. In addition, TIDM increased RUNX2, and osteocalcin in callus of tibial bone defect compared to healthy group. PBM significantly decreased osteocalcin gene expression in TIDM rats. CONCLUSIONS: PBM significantly increased many stereological parameters of bone repair in an STZ-induced TIDM during catabolic response of fracture healing. Further RT-PCR test demonstrated that bone repair was modulated in diabetic rats during catabolic response of fracture healing by significant increase in mRNA expression of RUNX2, and osteocalcin compared to healthy control rats. PBM also decreased osteocalcin mRNA expression in TIDM rats.

Evaluation of PAWP and PLC? Expression in Infertile Men with Previous ICSI Fertilization Failure.

PURPOSE: The aim of this study was to evaluate postacrosomal sheet WW domain binding protein (PAWP) and phospholipase C ? (PLC?) protein expression in patients with fertilization failure. MATERIALS AND METHODS: Semen samples were collected from 15 fertile men (control group) and 15 patients with previous fertilization failure following ICSI (FF group) and were analyzed according to World Health Organization (WHO) criteria. The mean percentages of PAWP and PLC? positive sperm and the total level of PAWP and PLC? proteins were assessed using immunofluorescence staining. RESULTS: A significantly lower level and lower percentage of PAWP positive sperm in patients with fertilization failure was found compared to the control group (P = 0.01 and P = 0.03, respectively). The mean percentage ofPLC? positive sperm and level of PLC? protein were significantly lower in FF group compared to the control group (P = 0.0003 and P = 0.04, respectively). Significant positive correlations was observed between PAWP and PLC? positive sperms (r = 0.4, P = 0.008) and also total level of expression of PLC? and PAWP proteins (r = 0.4, P = 0.02) in all participants in the study. CONCLUSION: This is the first study that evaluates two main candidates for sperm-borne oocyte activating factors (SOAFs) simultaneously in patients with fertilization failure. Considering lower expression of PAWP and PLC? proteins in such patients, it seems like both factors might have the potential to be considered as SOAFs and diagnostic markers for the oocyte activation ability.

The Effects of In Vitro Maturation Technique on The Expression of Genes Involved in Embryonic Genome Activation of Human Embryos.

OBJECTIVES: In vitro maturation technique (IVM) is shown to have an effect on full maturation of immature oocytes and the subsequent embryo development. Embryonic genome activation (EGA) is considered as a crucial and the first process after fertilization. EGA failure leads to embryo arrest and possible implantation failure. This study aimed to determine the role of IVM in EGA-related genes expression in human embryo originated from immature oocytes and recovered from women receiving gonadotrophin treatment for assisted reproduction. MATERIALS AND METHODS: In this experimental study, germinal vesicle (GV) oocytes were cultured in vitro. After intracytoplasmic sperm injection of the oocytes, fertilization, cleavage and embryo quality score were assessed in vitro and in vivo. After 3-4 days, a single blastomere was biopsied from the embryos and then frozen. Afterwards, the expression of EGA-related genes in embryos was assayed using quantitative reverse transcriptase-polymerase chain reaction (PCR). RESULTS: The in vitro study showed reduced quality of embryos. No significant difference was found between embryo quality scores for the two groups (P=0.754). The in vitro group exhibited a relatively reduced expression of the EGArelated genes, when compared to the in vivo group (all of them showed P=0.0001). CONCLUSIONS: Although displaying the normal morphology, the IVM process appeared to have a negative influence on developmental gene expression levels of human preimplanted embryos. Based on our results, the embryo normal morphology cannot be considered as an ideal scale for the successful growth of embryo at implantation and downstream processes.

Oligoasthenoteratozoospermic (OAT) men display altered phospholipase C ζ (PLCζ) localization and a lower percentage of sperm cells expressing PLCζ and post-acrosomal sheath WW domain-binding protein (PAWP).

Oligoasthenoteratozoospermia (OAT) is demonstrated to be one of the most common causes of male subfertility. Phospholipase C ζ (PLCζ), a sperm-specific protein, is considered to be one of the sperm-borne oocyte activating factors (SOAFs), which play a vital role in fertilization. The post-acrosomal sheath WW domain-binding protein (PAWP) is another candidate for SOAF. The aim of this study was to compare the PLCζ localization patterns and percentage of PLCζ- and PAWP-positive sperm cells in patients with OAT and fertile men with normozoospermia. A total of 40 men included in this study were classified into two groups: OAT (n = 25) and control group (n = 15). Semen samples were collected and analyzed using conventional semen analysis according to the World Health Organization guidelines. The percentage of PLCζ- and PAWP-positive sperm cells and localization patterns of PLCζ were evaluated using immunofluorescence staining. The mean percentage of sperm cells expressing PAWP and PLCζ was significantly lower in OAT compared to control group (52.8 ± 4.2 vs. 76.8 ± 5 and 63.4 ± 3.5 vs. 86.7 ± 2.1, respectively). In addition, statistically significant differences were found with regard to the PLCζ localization patterns, including equatorial, acrosomal + equatorial, and equatorial + post-acrosomal pattern, between the two groups (p < 0.01). The present study showed a lower percentage of sperm cells expressing PLCζ and PAWP, as well as altered localization patterns of PLCζ in men with OAT. Given the role of PLCζ and PAWP in fertilization, as two major candidates for SOAFs, our findings indicate that PLCζ and PAWP impairments may be one of the possible etiologies of decreased fertility in OAT.

Phospholipase C zeta parameters in sperm from polymorphic teratozoospermic men.

Teratozoospermia is a disorder associated with high abnormal sperm morphology which affects fertility in males. In recent years, it has been described that biomarker-based sperm quality evaluation can alleviate male infertility treatment. Phospholipase C zeta (PLCζ) is a sperm-specific factor which appears to be a predicting biomarker for fertilization potential of males. Following fertilization, PLCζ enters into oocyte cytoplasm and induces oocyte activation, a fundamental stage in initiation of embryo development. Currently, PLCζ parameters, including localization patterns, the proportion of PLCζ-expressing sperm and the expression level, are not defined in polymorphic teratozoospermic men. This study aimed to evaluate PLCζ parameters in polymorphic teratozoospermic men, and compare these parameters with fertile normozoospermic men. Semen samples from thirteen normozoospermic fertile men and twenty-three polymorphic teratozoospermic men were included in this study and evaluated using western blotting and immunofluorescence analyses. Our data indicated significantly lower expression of PLCζ in polymorphic teratozoospermic men, as compared with control men; however, there was no significant difference in localization patterns and the proportion of PLCζ-expressing sperm between polymorphic teratozoospermic patients and control men. Collectively, findings from the present study demonstrated that polymorphic teratozoospermic men did not show abnormal localization patterns or the absence of PLCζ, as compared to the control men; nonetheless, lower expression of PLCζ, considering its role in oocyte activation, might be one of the possible causes of infertility in these patients.

Recognition of a rare intrathoracic rib with computed tomography: a case report.

One of the uncommon congenital variations is intrathoracic rib which a normal, a bifid, or an accessory rib lies within the thoracic cavity that is founded accidentally. Clinically, in most cases they are without symptoms; however, it may cause intrathoracic problems therefore it is important for radiologists and physicians to identify to prevent of excessive intervention and treatment during imaging diagnostic techniques of thoracic problems. In this report, we provide the case of a rare presentation of an intrathoracic rib in a 3-year-old boy arising from the inferior portion of a second rib based on findings from computed tomography. To our knowledge, this is only the second reported case of this type of intrathoracic rib that demonstrated with computed tomography.

Supplementation of L-carnitine during in vitro maturation of mouse oocytes affects expression of genes involved in oocyte and embryo competence: An experimental study.

BACKGROUND: Oocyte developmental competence is one of the key factors for determining the success rate of assisted reproductive technique. OBJECTIVE: The aim of the current study was to investigate the effect of L-carnitine (LC) supplementation during in vitro maturation (IVM), on preimplantation embryo development and expression of genes involved in embryo competence derived from oocytes selected with brilliant cresyl blue (BCB) test. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) were obtained from NMRI mice ovaries. COCs were stained with BCB and then BCB+ (colored cytoplasm) oocytes cultured in IVM medium supplemented with 0.3 or 0.6 mg/ml LC. COCs untreated with LC were used as control. Fertilization rate and blastocyst development rate were determined after in vitro fertilization. In addition, quantitative reverse transcriptase polymerase chain reaction was used to measure relative genes expression related with development (Ccnb1, Mos, Ces5, and Dppa2) and apoptosis (Bax and Bcl-xL) in oocytes and embryos. RESULTS: Oocytes treated with both LC concentrations showed higher blastocyst development rate compared with untreated oocytes (p<0.01). Moreover, fertilization rate was increased in oocytes treated with 0.6 mg/ml LC (p<0.01). Treatment of oocytes with both LC concentrations increased (p<0.01) the level of Ccnb1 mRNA in MII oocytes. The two-cell stage embryos and blastocysts derived from LC-treated oocytes (0.6 mg/ml) showed increased the expression levels of Dppa2 and Bcl-xl mRNA, respectively (p<0.01). CONCLUSION: The results of the present study show that adding of LC to the IVM medium of BCB+ oocytes can ameliorate reproductive success following in vitro fertilization.

Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ≤30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p < 0.05). The rate of fertilization, cleavage, and embryo quality score were not significantly different between the two groups (all p > 0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p > 0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte.

Evaluation of the effects of LLLT on biomechanical properties of tibial diaphysis in two rat models of experimental osteoporosis by a three point bending test.

Osteoporosis (OP) is a disease which causes bone loss and fractures, leading to severe pain and deformity. This study has aimed to assess the effects of pulsed wave low-level laser therapy (PW LLLT) on cortical bone in two experimental models of OP in rats. There were four ovariectomized (OVX-d) groups and four dexamethasone-treated groups. The healthy group were considered for baseline evaluations. At 14 weeks following ovariectomy, the OVX-d rats were further subdivided into the following: control rats with OP, OVX-d rats that received alendronate (1 mg/kg), OVX-d rats treated with LLLT, and OVX-d rats treated with alendronate and LLLT. The remaining rats received dexamethasone for 5 weeks and were divided into four groups: control, alendronate-treated rats (1 mg/kg), laser-treated rats, and laser-treated rats with concomitant administration of alendronate. The rats received alendronate for 30 days. LLLT (890 nm, 80 Hz, 0.972 J/cm(2)) was performed on the tibias three times per week for 8 weeks. After 8 weeks, tibias were extracted and submitted to a three-point bending test. PW LLLT did not increase the biomechanical parameters of osteoporotic bones compared to controls and healthy rats. PW LLLT associated with alendronate treatment significantly increased stress high load in OVX-d rats compared to the healthy group. PW LLLT at the current study parameters failed to cause beneficial biomechanical effects in the examined osteoporotic cortical bones. PW LLLT associated with alendronate treatment produced a more remarkable effect on bone strength in the ovariectomized induced OP rat model.

Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining.

PURPOSE: The present study was designed to investigate the effect of L-carnitine treatment during IVM on nuclear and cytoplasmic maturation of immature oocytes selected by Brilliant Cresyle Blue (BCB) staining, and their subsequent developmental competence. MATERIALS & METHODS: Compact cumulus-oocyte complexes (COCs) were collected from NMRI mice ovaries and stained with BCB staining. BCB+ (colored cytoplasm) oocytes were then cultured in tissue culture medium (TCM) 199 with 0.0, 0.3 and 0.6 mg/ml L-carnitine. RESULTS: The both L-carnitine concentrations significantly increased the intracellular glutathione (P<0.001), nuclear maturation (P<0.01) and expression levels of cyclin-dependent kinase1 (CDK1) (P<0.05). Moreover, treated oocytes with 0.6 mg/ml L-carnitine showed increased (P < 0.05) expression of mitogen-activated protein kinase1 (MAPK1) mRNA. Also, adding L-carnitine (0.6 mg/ml) to IVM medium significantly increased the cleavage rate (P<0.05). The blastocyst development rate (BDR) in the both L-carnitine treated groups was significantly higher (P<0.001) than the control group. L-carnitine had no significant effect on total blastocyst cell numbers. CONCLUSIONS: These data indicated that L-carnitine supplementation during IVM of immature BCB+ oocytes improved preimplantation developmental competence of oocytes after IVF, probably by accelerating cytoplasmic and nuclear maturation of oocytes. It may provide a novel approach to improving ART outcomes in infertile couples.

Effect of Pentoxifylline Administration on an Experimental Rat Model of Femur Fracture Healing With Intramedullary Fixation.

BACKGROUND: Globally, musculoskeletal injuries comprise a major public health problem that contributes to a large burden of disability and suffering. Pentoxifylline (PTX) has been originally used as a hemorheologic drug to treat intermittent claudication. Previous test tube and in vivo studies reported the beneficial effects of PTX on bony tissue. OBJECTIVES: This study aims to evaluate the effects of different dosages of PTX on biomechanical properties that occur during the late phase of the fracture healing process following a complete femoral osteotomy in a rat model. We applied intramedullary pin fixation as the treatment of choice. MATERIALS AND METHODS: This experimental study was conducted at the Shahid Beheshti University of Medical Sciences, Tehran, Iran. We used the simple random technique to divide 35 female rats into five groups. Group 1 received intraperitoneal (i.p.) PTX (50 mg/kg, once daily) injections, starting 15 days before surgery, and group 2, group 3, and group 4 received 50 mg/kg, 100 mg/kg, and 200 mg/kg i.p. PTX injections, respectively, once daily after surgery. All animals across groups received treatment for six weeks (until sacrificed). Complete surgical transverse osteotomy was performed in the right femur of all rats. At six weeks after surgery, the femurs were subjected to a three-point bending test. RESULTS: Daily administration of 50 mg/kg PTX (groups 1 and 2) decreased the high stress load in repairing osteotomized femurs when compared with the control group. The highest dose of PTX (200 mg/kg) significantly increased the high stress load when compared with the control group (P = 0.030), group 1 (P = 0.023), group 2 (P = 0.008), and group 3 (P = 0.010), per the LSD findings. CONCLUSIONS: Treatment with 200 mg/kg PTX accelerated fracture healing when compared with the control group.

Effect of Pulsed Wave Low-Level Laser Therapy on Tibial Complete Osteotomy Model of Fracture Healing With an Intramedullary Fixation.

BACKGROUND: Fractures pose a major worldwide challenge to public health, causing tremendous disability for the society and families. According to recent studies, many in vivo and in vitro experiments have shown the positive effects of PW LLLT on osseous tissue. OBJECTIVES: The aim of this study was to evaluate the outcome of infrared pulsed wave low-level laser therapy (PW LLLT) on the fracture healing process in a complete tibial osteotomy in a rat model, which was stabilized by an intramedullary pin. MATERIALS AND METHODS: This experimental study was conducted at Shahid Beheshti University of Medical Sciences in Tehran, Iran. We performed complete tibial osteotomies in the right tibias for the population of 15 female rats. The rats were divided randomly into three different groups: I) Control rats with untreated bone defects; II) Rats irradiated by a 0.972 J/cm(2) PW LLLT; and III) Rats irradiated by a 1.5 J/cm(2) PW LLLT. The right tibias were collected six weeks following the surgery and a three-point bending test was performed to gather results. Immediately after biomechanical examination, the fractured bones were prepared for histological examinations. Slides were examined using stereological method. RESULTS: PW LLLT significantly caused an increase in maximum force (N) of biomechanical repair properties for osteotomized tibias in the first and second laser groups (30.0 ± 15.9 and 32.4 ± 13.8 respectively) compared to the control group (8.6 ± 4.5) LSD test, P = 0.019, P = 0.011 respectively). There was a significant increase in the osteoblast count of the first and second laser groups (0.53 ± 0.06, 0.41 ± 0.06 respectively) compared to control group (0.31 ± 0.04) (LSD test, P = 0001, P = 0.007 respectively). CONCLUSIONS: This study confirmed the efficacy of PW LLLT on biomechanical strength, trabecular bone volume, callus volume, and osteoblast number of repairing callus in a complete tibial osteotomy animal model at a relatively late stage of the bone healing process.

Histological and biomechanical analysis of the effects of streptozotocin-induced type one diabetes mellitus on healing of tenotomised Achilles tendons in rats.

BACKGROUND: Tendon healing is impaired in patient with diabetes mellitus. The effects of streptozotocin-induced type 1 diabetes (STZ-D) on the healing of the transected Achilles tendon in rats was studied. METHODS: In the experimental group, type one diabetes was induced via administration of STZ. The right Achilles tendon of all the rats was transected 30 days after the STZ administration. The Achilles tendons were examined for biomechanical and histological examinations. RESULTS: The statistical analysis showed that Young's modulus of elasticity and stress tensile load of the control group were significantly higher than those of the experimental group, and inflammation in the experimental group was significantly higher than that in the control group. At the same time, fibrosis in the experimental group was significantly lower than that of the control group. CONCLUSION: Induction of type 1 diabetes by STZ significantly delayed the healing of the transected Achilles tendon in rats.

New single nucleotide variation in the promoter region of androgen receptor (AR) gene in hypospadic patients.

BACKGROUND: Hypospadias is one of the most common congenital abnormalities in the male which is characterized by altered development of urethra, foreskin and ventral surface of the penis. Androgen receptor gene plays a critical role in the development of the male genital system by mediating the androgens effects. OBJECTIVE: In present study, we looked for new variations in androgen receptor promoter and screened its exon 1 for five single nucleotide polymorphisms (SNP) in healthy and hypospadias Iranian men. MATERIALS AND METHODS: In our study, at first DNA was extracted from patients (n=100) and controls (n=100) blood samples. Desired fragments of promoter and exon 1 were amplified using polymerase chain reaction. The promoter region was sequenced for the new variation and exone 1 screened for five SNPs (rs139767835, rs78686797, rs62636528, rs62636529, rs145326748) using restriction fragment length polymorphism technique. RESULTS: The results showed a new single nucleotide variation (C→T) at -480 of two patients' promoter region (2%). None of the mentioned SNPs were detected in patients and controls groups (0%). CONCLUSION: This finding indicates that new single nucleotide polymorphism in androgen receptor promoter may have role in etiology of hypospadias and development of this anomaly. This article extracted from Ph.D. thesis. (Nasim Borhani).

The Effect of Melatonin on Maturation, Glutathione Level and Expression of H MGB1 Gene in Brilliant Cresyl Blue (BCB) Stained Immature Oocyte.

OBJECTIVE: Nutrients and antioxidants in the medium of immature oocyte have a profound effect on maturation, fertilization and development of resulting embryos. In this study the effects of melatonin as an antioxidant agent on maturation, glutathione level and expression of High mobility group box-1 (HMGB1) gene were evaluated in immature oocytes of mice stained with brilliant cresyl blue (BCB). MATERIALS AND METHODS: In this experimental study, immature oocytes were harvested from ovaries of Naval Medical Research Institute (NMRI) mice. Oocytes were stained with 26 µM BCB for 90 minutes and transferred to in vitro maturation medium containing varying doses of melatonin (10-12, 10-9, 10-6, 10-3 M) and without melatonin, for 22-24 hours. Maturation was monitored using an inverted microscope. Glutathione was assessed by monochlorobimane (MCB) staining and HMGB1 expression in mature oocyte was analyzed using real-time polymerase chain reaction (PCR). RESULTS: Melatonin in the concentration of 10-6 M had the most effect on maturation and HMGB1 expression of BCB+ oocytes (p<0.05). Meanwhile melatonin had no effects on glutathione levels. Additionally in immature BCB- oocytes, compared to the control group, melatonin did not affect cytoplasm maturation (p>0.05). CONCLUSION: In vitro treatment with melatonin increases the maturation and HMGB1 expression in BCB+ immature oocytes and has no significant effect on glutathione levels.