Effect of teriparatide on the healing of mandibular condylar osteochondral defects: a preclinical study.

This study was performed to evaluate the effect of teriparatide therapy on the healing of osteochondral defects of the mandibular condylar. Ninety-six rats underwent surgery to create a defect in the condylar head on one side of the mandible, and were divided into two groups. One group received subcutaneous injection of 2 µg/kg/day teriparatide, and the other group received normal saline until sacrifice. On postoperative days 20, 40, and 60, 16 animals from each group were sacrificed, and bone and cartilage healing was histologically evaluated and semiquantitatively scored (1-5). The mean difference in healing score of the cartilaginous and subchondral parts of the defect between the teriparatide and control groups at days 20, 40 and 60 were 0.438 and 0.438, 0.813 and 0.750, and 1.125 and 0.813, respectively. The healing scores of the osteochondral defects in the teriparatide group were significantly (p< 0.05) higher than that in the control group at days 40 and 60. This study has shown beneficial effects of teriparatide on the healing of condylar osteochondral defects in rats. Clinical trials are required to extrapolate these findings to humans.

Anatomical characteristics of mandibular bone in skeletal class I, II and III patients by using cone beam computed tomography images in an iranian population / Características anatômicas do osso mandibular em pacientes Classe I, II e III esquelética usando imagens de Tomografia Computadorizada de Feixe Cônico: em uma População Iraniana

Objetive: This study aimed to compare the anatomical characteristics of the mandible in patients with skeletal class I, II and class III disorders using cone beam computed tomography (CBCT). Material and Methods: CBCT scans of patients between 17 to 40 years taken with NewTom 3G CBCT system with 12-inch field of view (FOV) were selected from the archive. Lateral cephalograms were obtained from CBCT scans of patients, and type of skeletal malocclusion was determined (Class I, II or III). All CBCT scans were evaluated in the sagittal, coronal and axial planes using the N.N.T viewer software. Results: The ramus height and distance from the mandibular foramen to the sigmoid notch in class II patients were significantly different from those in skeletal class I (P < 0.005). Distance from the mandibular canal to the anterior border of ramus in class III individuals was significantly different from that in skeletal class I individuals (P < .005). Conclusion: Length of the body of mandible in skeletal class I was significantly different from that in skeletal class II and III patients. Also, ramus height in skeletal class I was significantly different from that in skeletal class II patients. CBCT had high efficacy for accurate identification of anatomical landmarks. (AU)

Objetivo: Este estudo teve como objetivo comparar as características anatômicas da mandíbula em pacientes com desordem esquelética Classe I, II e III usando imagens de tomografia computadorizada de feixe cônico (TCFC). Material e Métodos: Foram selecionadas de arquivo, imagens de TCFC (Sistema NewTon 3G) com FOV (campo de visão) 12 polegadas e incluindo pacientes entre 17 a 40 anos. Cefalometrias laterais foram obtidas a partir das imagens de TCFC e o tipo de maloclusão esquelética foi determinada (Classe I, II ou III). Todas as imagens de TCFC foram avaliadas nos planos sagital, coronal e axial usando o software de visualização N.N.T. Resultados: A altura do ramo e distância do forame mandibular para a incisura da mandíbula em pacientes Classe II foi significativamente diferente daqueles Classe I esquelética (p< 0.005). A distância do canal mandibular até a borda anterior do ramo em indivíduos Classe III foi significativamente diferente daqueles indivíduos Classe I esquelética (p<0.005). Conclusão: O comprimento do corpo da mandíbula na Classe I esquelética foi diferente significativamente daqueles pacientes em Classe II e III esquelética. Além disso, a altura do ramo na Classe I esquelética foi significativamente diferente daqueles pacientes Classe II esquelética. A TCFC apresentou alta eficácia para a identificação precisa de marcos anatômicos. (AU)

Histological assessment of the effects of teriparatide therapy on mandibular fracture healing: A preclinical study.

OBJECTIVE: This study was performed to evaluate the effects of teriparatide therapy on mandibular fracture healing in a rat model. SUBJECTS AND METHODS: A unilateral mandibular fracture, 5 mm posterior to the last molar tooth, was surgically created in 120 rats. Half of the animals received a daily subcutaneous injection of 2 µg/kg teriparatide while the control rats received normal saline, starting from the day of surgery until sacrifice. Twenty rats from each group were sacrificed on postoperative days 10, 20, and 30. The healing process was evaluated histologically and scored using a grading system (ranging from 1 to 10). RESULTS: On day 10 the fracture gaps of the control and teriparatide groups were mainly filled with fibrous tissue and new trabecular bone, respectively. On day 20 a large amount of new trabecular bone and some areas of fibrocartilaginous tissue were seen in the fracture gaps of the control rats. In the teriparatide group the fracture area was entirely filled with trabecular bone, which in some areas had been replaced by mature bone. On day 30 the fracture gaps of the control group were entirely bridged by new trabecular bone, while in the teriparatide group they was predominantly filled with mature bone. At all three time-points the mean healing scores for the teriparatide group (6.20 ± 0.70, 8.50 ± 0.69, and 9.85 ± 0.37, respectively) were significantly higher (p < 0.001) than for the control group (4.90 ± 0.55, 7.15 ± 0.59, and 8.90 ± 0.64, respectively). CONCLUSION: Based on the results of this study, teriparatide should be tested in humans in order to establish whether comparable results can be achieved.

Histological evaluation of the healing process of autografted mandibular bone defects in rats under treatment with zoledronate.

OBJECTIVE: This study aims to evaluate the healing process of autografted mandibular bone defects in rats treated with zoledronate (ZOL). SUBJECTS AND METHODS: A total of 180 Wistar rats were divided into four groups: group L received intravenous infusion of two doses of 0.06 mg/kg ZOL, nine weeks apart; group H received 0.06 mg/kg ZOL, while groups C and NC received normal saline at three-week intervals for nine weeks. Three weeks following the last infusion, a unilateral mandibular bone defect (5 mm) was created. Except in the NC group, all defects were repaired with autologous iliac bone graft. Fifteen animals from each group were sacrificed on postoperative Day 20, Day 40, and Day 60. Graft healing was scored using a histological grading system (ranging from 1 to 6). RESULTS: Histological evaluations performed on postoperative Day 60 showed that the mandibular defects were mainly repaired with fibrous tissue in the NC and H groups (93.00% ± 7.51% and 82.67% ± 13.08%, respectively) and with bone in the C and L groups (75.33% ± 14.20% and 92.67% ± 8.84%, respectively). The percentage of fibrous tissue and bone as well as the healing score of the NC and H groups were significantly different (P = 0.001) from those of the C and L groups. However, these were not different between neither the NC and H groups nor the C and L groups. CONCLUSION: Based on the results of the present study the hypothesis can be established that there also might be a dose-dependent effect of ZOL on the healing of bone grafts in humans. This hypothesis has to be verified or rejected in clinical trials.

Evaluation of the effect of teriparatide therapy on mandibular fracture healing in rats with medication-related osteonecrosis of the jaw.

OBJECTIVE: The aim of this study was to evaluate the effect of teriparatide therapy on mandibular fracture healing in rats with medication-related osteonecrosis of the jaw (MRONJ). MATERIALS AND METHODS: To induce MRONJ, a total of 120 rats received intravenous zoledronate 0.06 mg/kg once a week for 6 weeks and their right mandibular first molar was extracted. Eighty of 94 rats with MRONJ were randomly selected and underwent unilateral mandibular osteotomy to replicate a fracture. After surgery, the rats were randomly assigned to T (teriparatide-treated) and C (control) groups. Group T (n = 40) received subcutaneous injection of 2 µg/kg/day teriparatide and group C (n = 40) received the same volume of normal saline until sacrifice. Four and 8 weeks after surgery, 20 rats in each group were sacrificed. Fracture healing was scored using a histological grading system (1 to 10). RESULTS: In group C, at 4 weeks and 8 weeks post-fracture, fibrous and cartilaginous tissues and scant bone formation at the fracture site and lacunae without osteocyte in adjacent mandibular bone were seen. In group T, substantial amounts of new trabecular bone rimmed by osteoblasts and some areas of remodeled mature bone were seen. After 8 weeks, extensive replacement of trabecular bone with mature bone occurred. Except between C4 and C8 groups, the healing score was significantly different between all subgroups. CONCLUSION: Teriparatide therapy successfully improved mandibular fracture healing in rats with MRONJ. However, this study was limited by the use of an animal model whose anatomy, physiology, and drug metabolism might be different from humans. CLINICAL RELEVANCE: The present study showed that teriparatide therapy may be used adjunctive to surgery in the treatment of mandibular fractures in MRONJ patients.

Evaluation of teriparatide effect on healing of autografted mandibular defects in rats.

OBJECTIVE: To evaluate the effects of short-term teriparatide administration on healing of autologous bone graft in mandibular critical-size defects. SUBJECTS AND METHODS: A 5-mm mandibular bone defect was created and iliac bone graft was harvested in 135 rats. Rats were randomly divided into 3 groups of negative control (NC), control (C), and study (S). In groups S and C, iliac graft was placed in defect and 2 µg/kg/day teriparatide or saline, respectively, was administered for 20 days. In group NC, iliac graft was not transferred to the defect and saline was injected for 20 days. Twenty, 40, and 60 days after surgery, 15 rats in each group were euthanized and the healing process was histologically evaluated and scored using a grading system (1-6). RESULTS: In group NC, defects did not heal or were predominantly filled with fibrous tissue. At day 20, bone defects in both C and S groups contained a large area of graft particles, numerous collagen fibers and some areas of new trabeculae. At the day 40, defects in group S showed a larger bone graft area, more new bone formation, smaller connective tissue area, and a higher healing score compared to group C (P < 0.05). At day 60, most of the defect in group S was filled with graft particles and mature bone while in group C, new trabecular bone formation was still underway (P < 0.05). CONCLUSION: Teriparatide therapy improves healing of bone defects reconstructed with autograft by reducing bone graft resorption and enhancing new bone formation and maturation.

Effects of grape seed extract, quercetin and vitamin C on ovine oocyte maturation and subsequent embryonic development.

The present study aimed to evaluate the effects of grape seed extract (GSE) versus quercetin and vitamin C on in vitro oocyte maturation and embryo development in sheep. The free radical scavenging activity of different concentrations of each product was measured by 1, 1- diphenyl-2-picryl hydrazyl (DPPH). Oocytes were collected from ovaries of slaughtered ewes and matured in TCM-199 medium containing fetal calf serum, follicle stimulating hormone (FSH), estradiol-17 ß, sodium pyruvate, and gentamicin sulfate. The in vitro fertilization and culture were performed using Bracket and Oliphant's (BO) medium and modified Charles Rosenkrans medium with amino acids (mCR2aa), respectively. The results showed that the hydroalcoholic extract of grape seed had free radical scavenging activity. IC50 value for GSE, vitamin C, and quercetin was found to be 585 µg/mL, 53 µg/mL, and 43 µg/mL, respectively. The concentrations, which showed beneficial effects on oocyte maturation and early development based on the mean number of cleavage, morula and blastocyst rates, were 25-200 µg/mL, 5 or 15 µg/mL, and 800 µg/mL, respectively, for vitamin C, quercetin and GSE. However, there were no significant differences between different concentrations of GSE and control. Findings also highlight the great effect on blastocyst rate while adding GSE at 800 µg/mL. However, the best rate of blastocyst production was obtained in presence of quercetin. Findings suggested the need for further studies on special molecules derived from GSE.

Effect of different doses and durations of teriparatide therapy on resolution of medication-related osteonecrosis of the jaw: A randomized, controlled preclinical study in rats.

OBJECTIVE: To evaluate the effects of different doses and durations of teriparatide therapy on MRONJ resolution in rats. SUBJECTS AND METHODS: A total of 120 rats that had been affected with MRONJ (after six weekly zoledronate injections and tooth extraction) were randomly divided into eight subgroups: 2, 10, and 20 µg/kg/day teriparatide were administered to L4, M4, and H4 for 4 weeks, and to L8, M8, and H8 for 8 weeks, respectively. C4 and C8 received saline for 4 and 8 weeks, respectively. One week after the final injection, rats were sacrificed and assessed clinically (bone exposure/fistula) and histologically (number of osteocytes in extraction socket and empty lacunae in alveolar bone). RESULTS: MRONJ was clinically improved in 72.2%, 61.5%, and 40% of stage I, II, and III experimental rats, respectively. In the control rats, the results were 20.8% for stage I and no improvement for stages II and III. Aside from L4 and L8, the experimental subgroups had a significantly higher rate of clinical and histological improvement compared with their corresponding controls. There was a significantly higher number of osteocytes and lower number of empty lacunae in M4 and H4 compared with C4, in H4 compared with L4, in M8 and H8 compared with C8, and in H8 compared with L8. CONCLUSION: Teriparatide therapy improved clinical and histological features of MRONJ in a dose-dependent manner, but clinically relevant doses of teriparatide might not be sufficient for MRONJ resolution in rats. Extending the duration of teriparatide therapy from 4 to 8 weeks did not affect treatment outcomes.

Evaluation of mandibular fracture healing in rats under zoledronate therapy: A histologic study.

INTRODUCTION: To evaluate fracture healing in mandible of rats under zoledronate therapy. METHODS: A total of 135 Wistar rats were randomly allocated into 3 groups. Group L received two intravenous infusion of 0.06 mg/kg zoledronate 6 weeks apart. Group H received the same dose of zoledronate as group L once a week for 6 weeks and group C were treated with normal saline. Seven days after the last infusion, rats underwent unilateral mandibular osteotomy to replicate a fracture. Fifteen rats from each group were sacrificed 2, 4, and 6 weeks after surgery. Fracture calluses were examined and scored using a histological grading system (1 to 10). RESULTS: After 2 weeks, substantial woven bone and some lamellar bone were seen in control and L groups. In group H, healing was delayed and consisted of fibrous and cartilaginous tissue and some woven bone. After 4 weeks, most of woven bone in control group was replaced with lamellar bone but in group L, comparatively less bone remodeling occurred. In group H, healing process was nearly the same as that at 2 weeks. After 6 weeks, complete bone remodeling was seen in control group. In group L, bone remodeling was under way and in group H, histological findings were nearly the same as those at 2 and 4 weeks. Except for L and control groups at 2 weeks, healing score was significantly different between all corresponding groups. CONCLUSION: Zoledronate therapy delayed healing process of mandibular fracture in rats in a dose-dependent manner.

Short-term perioperative teriparatide therapy for the prevention of medication-related osteonecrosis of the jaw: A randomized, controlled preclinical study in rats.

OBJECTIVE: Dentoalveolar procedures in patients receiving bisphosphonates and other antiresorptive agents are associated with an increased risk of medication-related osteonecrosis of the jaw (MRONJ). The aim of present study was to evaluate the effects of perioperative teriparatide (TPD) therapy in prevention of MRONJ. SUBJECTS AND METHODS: Two protocols of TPD therapy were studied. For protocol A, 25 TPD-treated (AT) and 25 control (AC) rats received 5 weekly injection of 0.06 mg/kg zoledronate. At the end of week 5, extraction of bilateral mandibular first molars was performed for all rats, and 4-week TPD (20 µg/kg/day) and saline therapy was started for AT and AC rats, respectively. For protocol B, 25 TPD-treated (BT) and 25 control (BC) rats received 5 weekly injection of 0.06 mg/kg zoledronate. One week later, 4-week TPD and saline therapy was started for BT and BC rats, respectively. Both groups underwent tooth extraction at the end of week 7 of the experiment. All rats were sacrificed 8 weeks after tooth extraction and assessed clinically for bone exposure/fistula, and histologically for density of osteocytes in newly formed bone and empty osteocyte lacunae in alveolar bone. RESULTS: Incidence of bone exposure/fistula and mean numbers of osteocytes and empty lacunae per 25 mm2 (at 400× magnification) were 20%, 15.36, and 2.63 in AT group; 78%, 5.78, and 6.81 in AC group; 14%, 16.94, and 2.08 in BT group; and 78%, 7.54, and 5.95 in BC group; respectively. The differences between AT and AC and between BT and BC were statistically significant (P < 0.001). However, no statistically significant difference between AT and BT and between AC and BC was found. CONCLUSION: Four weeks of TPD therapy, beginning at the same day or 2 weeks before tooth extraction, had a potential role in prevention of ONJ.

The starting point for bisphosphonate-related osteonecrosis of the jaw: Alveolar bone or oral mucosa? A randomized, controlled experimental study.

OBJECTIVE: Although over a decade has passed since first introduction of BRONJ, the exact pathophysiology of this disease is still unclear. The present experimental study aimed to determine whether the oral mucosa or alveolar bone serves as the starting point for BRONJ development. SUBJECTS AND METHODS: Sixty male Wistar rats were randomly assigned into study and control groups (each, n = 30), and received intraperitoneal injection of 0.06 mg/kg zoledronate and saline, respectively, once a week for 12 weeks. At the end of the week 4 of the experiment, all 60 rats underwent unilateral mandibular first molar extraction. A 4 mm defect was made in the contralateral canine alveolar mucosa. At the end of the experiment, rats were sacrificed, and the three areas of interest including extraction, soft tissue defect, and the non-intervention (canine area on the same side of extraction) sites were assessed clinically for presence of bone exposure/fistula, and histologically for status of bone remodeling (only at extraction site) and osteonecrosis. RESULTS: In the study group, the frequency of bone exposure/fistula was 80%, 0%, and 0%; and the rate of histological bone necrosis was 83.3%, 20%, and 0%; at the extraction, soft tissue defect, and non-intervention sites, respectively. No clinical and histological sign of bone necrosis was found in the control group. Normal bone remodeling was observed in 0% and 100% of the extraction sockets in the study and control groups, respectively. CONCLUSION: Injury to alveolar bone was a stronger trigger for BRONJ development compared to oral mucosal damages.

The necessity of pterygomaxillary disjunction in surgically assisted rapid maxillary expansion: A short-term, double-blind, historical controlled clinical trial.

PURPOSE: To evaluate and compare the short-term skeletal and dental changes following surgically assisted rapid maxillary expansion (SARME) with or without pterygomaxillary disjunction (PTMD) using cone beam computed tomography (CBCT). MATERIAL AND METHODS: In present study, a total of 15 consecutive patients with transverse maxillary deficiency underwent SARME without PTMD. This prospective group was compared to one arm of a randomized controlled clinical trial the authors of present study published in 2014, in which 15 patients underwent SARME with PTMD. The age range of patients, inclusion and exclusion criteria, surgical procedure (except PTMD), operating surgeon, the appliance and protocol of expansion, and the method of outcomes evaluation were the same in both prospective (-PTMD) and historical control (+PTMD) groups. Before treatment and immediately after consolidation period, CBCT was obtained and the nasal floor width, interdental root distance, palatal bone width and interdental cusp distance were measured at first premolar and first molar regions of maxilla. RESULTS: In both -PTMD and +PTMD groups, the greatest expansion occurred at the dental arch, followed by palatal bone and nasal floor level (V-shaped expansion in the coronal plane), and the amount of expansion achieved at the first premolar and molar areas was comparable (uniform widening of maxilla posteroanteriorly). There was no statistically significant difference in the amount and pattern of maxillary expansion between two groups. CONCLUSION: Because of favorable surgical outcomes and decreased risk of possible perioperative complications, SARME without PTMD was recommended for the treatment of transverse maxillary deficiencies.

Introducing a protocol to create bisphosphonate-related osteonecrosis of the jaw in rat animal model.

OBJECTIVE: Previously published animal investigations on bisphosphonate-related osteonecrosis of the jaws (BRONJ) showed a variety of methods for BRONJ induction and inconsistent findings. The aim of present study was to develop a reliable protocol for BRONJ induction in rat animal model. SUBJECTS AND METHODS: In a pilot study, 64 rats were randomly divided into 4 groups and 16 subgroups (each containing 2 experimental and 2 control rats) based on the timing of tooth extraction and euthanasia. The experimental and control rats received intraperitoneal injection of 0.06 mg/kg zoledronate and saline, respectively, once a week until sacrificed, and evaluated for presence of bone exposure clinically, and osteonecrosis and new bone formation histologically. The protocol that successfully produced BRONJ in pilot study was tested in a randomized controlled experimental investigation using 45 rats. RESULTS: In pilot investigation, the highest rate of BRONJ was obtained after four weekly zoledronate injections, at least 4 weeks after tooth extraction. The randomized controlled experimental study verified this finding with a success rate of 83%, and also showed that more prolongation of zoledronate therapy did not increase the BRONJ rate. CONCLUSION: The protocol developed in the present study could be used reliably for future BRONJ investigations on rats.

Activation and Inhibition of The Wnt3A Signaling Pathway in Buffalo (Bubalus bubalis) Embryonic Stem Cells: Effects of WNT3A, Bio and Dkk1.

BACKGROUND: This research studies the effects of activation and inhibition of Wnt3A signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells. MATERIALS AND METHODS: To carry on this experimental study, the effects of activation and inhibition of Wnt3A signaling in buffalo ES cell-like cells were examined using Bio (0.5 mM) combined with WNT3A (200 ng/ml), as an activator, and Dickkopf-1 (Dkk1, 250 ng/ml), as an inhibitor, of the pathway. ES cells were cultured up to three weeks in ES cell medium without fibroblast growth factor-2 (FGF-2) and leukemia inhibitory factor (LIF), but in the presence of Bio, WNT3A, Bio+WNT3A and Dkk1. The effects of these supplements were measured on the mean area of ES cell colonies and on the expression levels of a number of important genes related to pluripotency (Oct4, Nanog, Sox2 and c-Myc) and the Wnt pathway (ß-catenin). ES cell colonies cultured in ES cell medium that contained optimized quantities of LIF and FGF-2 were used as the control. Data were collected for week-1 and week-3 treated cultures. In addition, WNT3A-transfected ES cells were compared with the respective mock-transfected colonies, either alone or in combination with Dkk1 for expression of ß-catenin and the pluripotency-related genes. Data were analyzed by ANOVA, and statistical significance was accepted at P<0.05. RESULTS: Among various examined concentrations of Bio (0.5-5 mM), the optimum effect was observed at the 0.5 mM dose as indicated by colony area and expressions of pluripotency-related genes at both weeks-1 and -3 culture periods. At this concentration,the expressions of Nanog, Oct3/4, Sox2, c-Myc and ß-catenin genes were nonsignificantly higher compared to the controls. Expressions of these genes were highest in the Bio+WNT3A treated group, followed by the WNT3A and Bio-supplemented groups, and lowest in the Dkk1-treated group. The WNT-transfected colonies showed higher expressions compared to both mock and Dkk1-treated mock transfected colonies. CONCLUSION: WNT3A functions to maintain the pluripotency of ES cell-like cells both as an exogenous growth factor as well as an endogenously expressed gene. It complements the absence of FGF-2 and LIF, otherwise propounded essential for buffalo ES cell culture. WNT3A antagonizes the inhibitory effects of Dkk1 and acts in combination with its activator, Bio, to activate the Wnt signaling pathway.

Perioperative discontinuation of intravenous bisphosphonate therapy reduces the incidence and severity of bisphosphonate-related osteonecrosis of the jaw: A randomized, controlled, prospective experimental study in rats.

OBJECTIVE: To evaluate the effects of intravenous bisphosphonate discontinuation on incidence and severity of bisphosphonate-related osteonecrosis of the jaw (BRONJ). MATERIAL AND METHODS: Seventy rats were randomly divided into 7 groups. In control and S0 groups, weekly injection of saline and 0.06 mg/kg zoledronate (respectively) for 4 weeks, tooth extraction, continuation of injections for 2 months and euthanasia were performed. In group S1, zolendronate injection for 4 weeks, tooth extraction, zolendronate discontinuation for 2 months, and euthanasia were done. For groups S2, S3, S4, and S5, zolendronate injections for 4 weeks, drug holiday for 1-4 months (respectively) before and 2 months after tooth extraction, and euthanasia were performed. Presence of bone exposure, osteonecrosis, and new bone formation were clinically and histologically evaluated. RESULTS: The rate of BRONJ in control, S0, S1, S2, S3, S4, and S5 groups was 0%, 85%, 80%, 65%, 60%, 50%, and 40%, respectively. In control group, epithelial healing, bone formation, and absence of osteonecrosis; and in S0 group, unhealed epithelium, osteonecrosis, and impaired bone formation were histologically observed. In study groups, prolongation of drug holiday caused diminished osteonecrosis, and improved bone and epithelial healing. CONCLUSION: Zolendronate discontinuation significantly decreased the incidence and severity of BRONJ in rats.

Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System.

OBJECTIVE: In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. MATERIALS AND METHODS: In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. RESULTS: The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. CONCLUSION: We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro-Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos.

We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro-fertilized, somatic cell nuclear-transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro-produced blastocysts. Most of the ICMs (45-55%) resulted in formation of primary colonies that were subcultured after 8-10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture-derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture-derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium.

Evaluation of third molar development and its relation to chronological age: a panoramic radiographic study.

INTRODUCTION: Third molar development, in comparison to other teeth in the dentition, has the greatest variation in morphology, anatomical position, and time of development and eruption, and its reliability for chronological age estimation is controversial. The aim of the present study was to evaluate third molar development and its relation to chronological age using panoramic radiography. MATERIAL AND METHODS: A total of 2536 digital panoramic radiographs of individuals aged between 5 and 26 years were selected. The developmental status of the third molars was assessed using eight-stage developmental scoring proposed by Demirjian et al., with one modification: a stage 0 was added. The collected data were entered into a checklist and subjected to statistical analyses. RESULTS: The mean ages of the first appearance of third molar bud, complete crown formation, and root apex closure were around 9, 14, and 22 years, respectively. In both jaws, third molar development occurred symmetrically, and sexual dimorphism was observed at some developmental stages. Finally, two formulas were presented to estimate age of the juveniles and adolescents based on their gender and developmental stages of the third molars, and validated on a second sample consisting of 523 individuals aged between 8 and 22. CONCLUSION: Assessment of third molar development was found to be a reliable method for age estimation of individuals between 11 and 22 years. Because of possible ethnic and geographic differences in third molar development, population specific researches were recommended.

Objectivity and reliability of panoramic radiographic signs of intimate relationship between impacted mandibular third molar and inferior alveolar nerve.

INTRODUCTION: The results of prior studies assessing the accuracy of panoramic radiographic signs of intimate relationship between inferior alveolar nerve (IAN) and impacted molars are controversial. This may be partly due to inadequate objectivity and reliability of these radiographic signs, which is evaluated in the present study. MATERIALS AND METHODS: Three hundred radiographs in which impacted third molar reached the superior border of the inferior alveolar canal or was superimposed by the canal were evaluated by three examiners independently, twice 3 months apart. Inter- and intra-examiner agreements were analyzed using kappa statistics. RESULTS: The inter-examiner agreement for all radiographic signs was poor (k < 0.2). The intra-examiner agreement for radiographic signs 2, 3, and 6 was poor with mean kappa values of 0.08, 0.00, and 0.09, respectively. Concerning the radiographic signs 4, 5, 7, and 8, the intra-examiner agreement was moderate with mean kappa values of 0.54, 0.49, 0.44, and 0.57, respectively. The mean kappa coefficient for the radiographic sign 1 yielded a good agreement (k = 0.65). CONCLUSIONS: In the present study, the examiners were unable to reliably assess radiographic signs of intimate relationship between IAN and third molar, indicating that panoramic images should not be relied upon for preoperative prediction of IAN injury.

Hypothalamic Expression of Melanocortin-4 Receptor and Agouti-related Peptide mRNAs During the Estrous Cycle of Rats.

Melanocortin- 4 receptor (MC4R) and agouti- related peptide (AgRP) are involved in energy homeostasis in rats. According to MC4R and AgRP effects on luteinizing hormone (LH) secretion, they may influence the estrous cycle of rats. Therefore, the aim of this study was to investigate the expression of MC4R and AgRP mRNAs at different stages of estrous cycle in the rat's hypothalamus. The estrous cycle stages (proestrus, estrus, metestrus and diestrus) were determined in 20 adult female rats using vaginal smears. The rats were divided into four equal groups (n=5). Four ovariectomized rats were selected as controls two weeks after surgery. Using real- time PCR, relative expressions (compared to controls) of MC4R and AgRP mRNAs in the hypothalamus of rats were compared in four different groups of estrous cycle. The relative expression of MC4R mRNA in the hypothalamus of female rats during proestrus stage was higher than those in other stages (P=0.001). Despite a lower mean of relative expression of AgRP mRNA at proestrus stage, the relative expression of AgRP mRNA of the four stages of estrous cycle did not differ (P>0.05). In conclusion, changes in the relative expression of MC4R and AgRP mRNAs in four stages of rat estrous cycle indicated a stimulatory role of MC4R in the proestrus and preovulatory stages and an inhibitory role of AgRP in gonadotropin releasing hormone (GnRH) and LH secretions.