Cell Journal (Yakhteh)
Year:2009 | Volume:11 | Issue:3 (43)|Papers Count:11

Regeneration of large bone defects is considered a challenging task by facio-mandibular and orthopedic surgeons. In these circumstances, either bone grafts or metal implants are currently being used. The inherent limitations associated with these methods have directed the attention of investigators to new technologies such as bone tissue engineering, a multidisciplinary field in which life science as well as engineering is involved to manufacture an appropriate bone construct. The objectives of scientists involved in this field are to design and manufacture scaffolds with appropriate chemical and physical features, to direct cell differentiation within the scaffold using appropriate culture conditions and finally to render the engineered construct applicable for clinical use. In this article, the main components involved in the bone tissue engineering process have been reviewed. These include cells (with an emphasis on mesenchymal stem cells), scaffolds, growth factors and bioreactors, and tissue engineering approaches to tissue regeneration.

Objective: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Antibiotic therapies do not protect from potential re-infection and have a risk for development of drug resistance. Therefore, prophylactic vaccine mediated protection against H. pylori is an attractive clinical interest. H. pylori adhesin A (HpaA) is a conserved surface lipoprotein and plays important roles in the pathogenesis of infection. In this study the recombinant protein (rHpaA) was over-expressed in E.coli.Materials and Methods: The hpaA gene was amplified by PCR. Prokaryote expression vector pET28a-hpaA was constructed, and used to transform E.coli BL21DE3. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of rHpaA by a rabbit polyclonal antibodies against whole cell of H. pylori.Results: The hpaA gene nucleotide sequence in the recombinant plasmid vector of pET-28a- hpaA was consistent with that of H.pylori hpaA as published in the GenBank. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced rHpaA at the 1.5mmol/L of IPTG. HpaA fusion protein was able to react with the rabbit polyclonal antibody against whole cells of H. pylori.Conclusion: A prokaryotic expression system pET-28a-hpaA-BL21 with high efficiency of H. pylori hpaA gene was successfully established and the HpaA fusion protein showed satisfactory immunoreactivity. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

Objective: In vertebrates, bone morphogenetic proteins (BMPs) and activin signals play multiple roles in dorso-ventral patterning and development of the spinal cord. Here the inductions of BMP 4 and activin A on embryonic stem cells (ESCs) into dorsal interneurons have been studied.Materials and Methods: Four different treatments have been used for mESC derived neural precursors; they include BMP4 (1ng/ml and 10ng/ml), activin A (100ng/ml), and activin A+BMP4 (100ng/ml, 10ng/ml). Induction’s effect on expression of specific dorsal interneuron markers in mature neurons have been evaluated by the use of immunocytochemistry and RT-PCR.Results: Treatment of ESC-derived neural precursors with BMP4, activin A, or both showed an increased generation of both dI1 and dI3 interneurons (Lhx2 and Isl-1-positive cells) compared to the control group. However, the synergistic effect in generation of dI3 was not observed when both factors were used. Moreover, RT-PCR analysis of differentiated cells showed the expression of Lhx9, Lhx1, and Isl1, the transcription factors that are markers of dI1, dI2, and dI3 interneurons respectively.Conclusion: Our results showed that specific combination of developmental signaling molecules can direct the differentiation of ESCs into dorsal interneurons. Furthermore, qualitative and quantitative differences in signaling by different members of the TGF-β superfamily may play a role in the specification of different types of dorsal interneurons.

Objective: Recently, we reported the presence of metabotropic glutamate receptor type 5 (mGluR-5) dependent long-term potentiation (LTP) at excitatory synapses on fast-spiking GABAergic (FS-GABA) cells in the visual cortex. In this study, we report a Ca2+ signaling pathway involved in this LTP type.Materials and Methods: Brain slices from GAD67-GFP knock-in mice were used. Using whole-cell patch-clamp recording followed by immunohistochemical staining on parvalbumin- positive (PV+) FS-GABA cells, we studied the Ca2+ signaling pathway involved in excitatory LTP of certain visual cortical interneuron subtypes.Results: U-73122- a phospolipas c (PLC) inhibitor (10mM), inositol triphosphate (IP3) inhibitors such as 2-APB (3mM) and heparin (10IU/ml), and CPA- the internally stored Ca2+ release inhibitor- (5mM) blocked the mGluR5 signaling pathway to induce LTP at excitatory synapses on PV+ fast-spiking cells in the visual cortex. However, application of the vehicles alone had no effect.Conclusion: Our results indicate that mGluR-5 at FS-GABA neurons activate PLC and IP3 production. This leads to Ca2+ release, promotes LTP induction, and its maintenance is supported by internal Ca2+ stores. Considering the key role of PV+ FS inhibitory neurons in the visual cortex circuits, we suggest that the metabotropic glutamate receptor-dependent LTP of excitatory synapses to FS cells plays a crucial role in the visual cortex plasticity.

Objective: Receptor activator for nuclear factor kappa B ligand (RANKL), which is also called osteoclast differentiation factor, is an important regulatory factor in osteoclast maturation. Knowledge of bone and cementum similarities and RANKL role in bone resorption suggests the possibility of a role for this protein in root resorption induced by orthodontic tooth movement. The aim of this study is to examine the expression of RANKL mRNA during root resorption induced by orthodontic tooth movement in rats.Materials and Methods: In order to move maxillary right first molars mesially fixed Ni-Ti closed coil springs (Dentaurum®-Germany) were tightened to the teeth. Sample consisted of 20 male seven week old Wistar rats. For each animal, the contralateral tooth was used as, an internal control. At day 21 the rats were sacrificed. Tissues from 10 rats were embedded in paraffin for histologic examination. Scratched material from resorptive lacunae on mesial sides of the roots of the other ten rats was used for extracting mRNA by RTPCR.Results: The histologic sections, analyzed histomorphometrically, showed a significant increase in root resorption in the case group as compared to the control (p<0.001). Densitometric studies of RANKL mRNA expression band on gel electrophoresis showed significantly increased RANKL expression in the resorptive lacunae of the case group (p<0.001).Conclusion: This observation indicates increased RANKL expression is associated with orthodontic tooth movement induced root resorption.

Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation, fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV) oocytes were obtained from 26 ovaries of 3-4 week old ICR female mice 48 hours after injection of 5 IU pregnant mare serum gonadotropin (PMSG). Collected oocytes were divided into two groups; group I: GV oocytes without cumulus cells (denuded oocyte), group II: GV oocytes with cumulus cells (cumulus-oocyte complex). The oocytes in both groups were cultured in TCM-199 medium supplemented with 10% fetal bovine serum (FBS) for 22-24 hours in a humidified atmosphere of 5% CO2 in air at 37oC. Oocyte maturation was scored under inverted microscope. To do in vitro fertilization, matured oocytes from each group were placed in T6 medium and capacitated spermatozoa were added. Then the fertilized oocytes were cultured and assessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hours after fertilization. Data was analyzed by chi-square test and differences in the values were considerable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rates were: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complex were significantly higher than those of denuded oocytes (p<0.05).Conclusion: The present study indicates that cumulus cells have important role during maturation, fertilization and subsequent embryo development to the blastocyst stage.

Objective: Newcastle disease virus (NDV) or avian paramyxovirus type1 possesses several unique properties that make it an excellent anticancer agent. The hemagglutini neuraminidase (HN) protein of NDV plays an important role in viral infection. The purpose of the present study is to investigate the dissemination of Newcastle disease virus (NDV) AF2240 strain in the liver during intratumoral injection in 4T1 breast cancer in female BALB/c mice.Materials and Methods: A total of 200 female BALB/c mice were divided randomly into 10 cancerous groups consisting of 20 mice per group. The mice were initially induced with 104 4T1 cells, NDV-AF2240 and tamoxifen co-culture. Cancerous groups were divided into: cancer control (CC), cancer treated with 0.5mg/ml tamoxifen citrate (CT), cancer treated with 8, 16, 32 and 64HA units of NDV-AF2240 (respectively named C/NDV8, C/NDV16, C/NDV32, C/NDV64), cancer treated with 8, 16, 32 and 64 HA units of NDV-AF2240 and tamoxifen (respectively as CT/NDV8, CT/NDV16, CT/ NDV32 and CT/NDV64 daily for four weeks). In situ reverse transcription polymerase chain reaction (In situ RT-PCR), negative staining electron microscopy (NSEM), polyclonal chicken antibody and goat anti-chicken antibody conjugated with fluorescein isothiocynate (FITC) using confocal laser scanning microscopy (CLSM) were used to detect the virus in the tumor and liver.Results: In situ RT-PCR, NSEM and CLSM successfully detected NDV-AF2240 in tumor cells and liver.Conclusion: The findings showed NDV-AF2240 disseminated into liver during intratumoral injection.

Objective: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Low activity phenotypes are correlated with several mutations in the TPMT gene and adverse drug reactions. The molecular basis for dissimilar enzymatic activity of TPMT has been established in Caucasians, African-Americans and Southwest Asians, but it remains to be elucidated in Iranian population. Until present, no study on Iranian population has been performed on the known alleles of TPMT. The aim of this study was to investigate the frequencies of four of the most common variants of this gene.Materials and Methods: This study was conducted during 2007 at the Department of Hematology, Tarbiat Modares University, Tehran, Iran. Using PCR-RFLP and allele specific PCR techniques, allelic variants of the TPMT gene TPMT*2 (G238C), TPMT*3B (G460A), TPMT*3C (A719G) and TPMT*3A (G460A and A719G) were genotyped in a normal population of 127 Iranians.Results: In this study TPMT*2 showed a prevalence of 7.08%. TPMT*3C and *3A were found in 2.47% and 2.18% of the samples, respectively. TPMT*3B variant was not detected in Iranian subjects. 112 out of 127 participants showed homozygote wild type allele.Conclusion: This study is the first to analyze TPMT allele frequencies in a sample of Iranian population and indicates that TPMT*2 is the most common allele (7.08%) in this population. These results can help to organize national pretreatment strategies in patients with acute lympho blastic leukemia (ALL) or other diseases requiring thiopurine medication in their standard therapy.

Objective: Mesenchymal stem cells (MSCs) are obtained from a variety of sources, mainly the bone marrow. These cells have a great potential for clinical research, however they cannot stay alive for long periods in culture. The aim of this study is to determine whether vitrification can be a useful freezing method for the storage of MSCs.Materials and Methods: Mesenchymal stem cells were isolated from rat bone marrow based on their capacity to adhere to plastic culture surfaces. MSCs were cryopreserved using both the vitrification method and open-pulled straw (OPS) vitrification and stored in liquid nitrogen with ethylene glycol ficoll (EFS) as a cryoprotectant for two months. The morphology and viability of thawed MSCs were evaluated by trypan blue staining. Furthermore, pre and post cryopreserved MSCs were induced to osteocyte and adipocyte with corresponding osteogenic and adipogenic medium.Results: After thawing, the viability rates were 81.33%±6.83 for the vitrification method and 80.83%±6.4 for OPS vitrification, while the values in the pre-vitrification control group were 88.16%±6.3 (Mean±SD, n=6). Post-cryopreserved cells from both the vitrification method and OPS vitrification also had a similar cellular morphology and colony-formation that was indistinguishable from non-vitrified fresh MSCs. In addition, the resuscitated cells cultured in induction medium showed osteogenesis. Mineral production and deposition was detectable by alizarine red S staining. Moreover, by applying an adipogenic differentiation condition, both pre and post cryopreserved cells differentiated into adipocyte and lipid vacuole accumulation that was stained by oil red O.Conclusion: Vitrification is a reliable and effective method for the cryopreservation of MSCs.

Objective: Carrot (Daucus carota L.) is known to possess antifertility properties in female. However, according to Iranian traditional medicine, it can increase the potency in men. The aim of this study was to investigate the influence of carrot seed extract (CSE) on spermatogenesis, number and motility of sperms in cauda epididyme in male rats.Materials and Methods: Forty adult male rats were randomly divided into 5 groups: control group, groups receiving low- and high doses of CSE, animals that received high-dose of CSE with gentamicin, and a gentamicin only group. After 4 weeks treatment, fasting serum samples were obtained for the sex hormone analysis. Under anesthesia, testis, cauda epididymides and sperm ducts were dissected and sperm count, motility and cauda epididymis sperm reserves (CESR) were determined. Histopathological changes of testis were also studied to assess spermatogenesis. Data analysis was performed using one-way ANOVA followed by Tukey HSD tests.Results: Administration of CSE caused a significant increase in CESR compared with the control (28.2±1.8 vs. 45.1±2.0×106). The extract could also protect testis from the gentamicin- induced necrosis. The CSE administration caused about 3.5-times increase in the LH levels even in spite of receiving 5 mg/kg/day gentamicin with no significant effect on FSH levels. The testosterone concentrations in the group received 400 mg/kg CSE were 30% and 83% higher than its levels in the control and the gentamicin treated group, respectively.Conclusion: CSE can overcome reproductive toxicity of gentamicin and induces spermatogenesis probably mainly through the elevation of testosterone levels. It appears that this extract has opposite effects on male and female reproductive systems.