RANKL and OPG expression: Jiggling force affects root resorption in rats.

OBJECTIVE: To immunohistochemically investigate the longitudinal changes in root resorption by jiggling force in experimental animal models. MATERIALS AND METHODS: Fifty-six 12-week-old male Wistar rats were used. The maxillary first molars were alternately moved in the buccal and lingual direction in 28 rats (experimental group) using an experimental appliance to produce jiggling forces of 10 g. In another 28 rats (control group), the maxillary first molars were moved in only the lingual direction with a force of 10 g. After 1, 3, 7, 10, 14, 17, and 21 days, the maxillae were resected and subjected to immunohistochemical analysis. The resorption area was quantified histomorphometrically and the number of odontoclasts on the root surface was counted. Expression of RANKL and OPG was also examined by immunohistochemical staining. RESULTS: The root resorption area and the number of odontoclasts were significantly greater in the experimental group than in controls. Odontoclasts were detected in the resorption lacunae and PDL in the experimental group, whereas osteoclasts were located only along the alveolar bone in controls. OPG was detected on the alveolar bone in the experimental group and on the root surfaces of the controls. CONCLUSIONS: Jiggling force is a critical factor in severe root resorption, affecting RANKL and OPG expression, which accelerates and inhibits odontoclastic induction, respectively.

A case of tooth autotransplantation after long-term cryopreservation using a programmed freezer with a magnetic field.

This case report describes the treatment of a skeletal Class III malocclusion with autotransplantation of a cryopreserved tooth. To gain an esthetic facial profile and good occlusion, extraction of bimaxillary premolars and surgical therapy were chosen. The patient had chronic apical periodontitis on the lower left first molar. Although she did not feel any pain in that region, the tooth was considered to have a poor prognosis. Therefore, we cryopreserved the extracted premolars to prepare for autotransplantation in the lower first molar area because the tooth would probably need to be removed in the future. The teeth were frozen by a programmed freezer with a magnetic field (CAS freezer) that was developed for tissue cryopreservation and were cryopreserved in -150°C deep freezer. After 1.5 years of presurgical orthodontic treatment, bilateral sagittal split ramus osteotomy was performed for mandible setback. Improvement of the facial profile and the occlusion were achieved in the retention phase. Six years after the initial visit, the patient had pain on the lower left first molar, and discharge of pus was observed, so we extracted the lower left first molar and autotransplanted the cryopreserved premolar. Three years later, healthy periodontium was observed at the autotransplanted tooth. This case report suggests that long-term cryopreservation of teeth by a CAS freezer is useful for later autotransplantation, and this can be a viable technique to replace missing teeth.

Effects of pulpectomy on the amount of root resorption during orthodontic tooth movement.

INTRODUCTION: Previous studies have revealed that orthodontic force affects dental pulp via the rupture of blood vessels and vacuolization of pulp tissues. We hypothesized that pulp tissues express inflammatory cytokines and regulators of odontoclast differentiation after excess orthodontic force. The purpose of this study was to investigate the effects of tensile force in human pulp cells and to measure inflammatory root resorption during tooth movement in pulpless rat teeth. METHODS: After cyclic tensile force application in human pulp cells, gene expression and protein concentration of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand, interleukin-1 beta, and tumor necrosis factor alpha were determined by real-time polymerase chain reaction and enzyme-linked immunoassay. Moreover, the role of the stretch-activated channel was evaluated by gadolinium (Gd(3+)) treatment. The upper right first molars of 7-week Wistar rats were subjected to pulpectomy and root canal filling followed by mesial movement for 6 months. RESULTS: The expression of cytokine messenger RNAs and proteins in the experimental group peaked with loading at 10-kPa tensile force after 48 hours (P < .01). Gd(3+) reduced the expression of these cytokine messenger RNAs and protein concentrations (P < .01). The amount of inflammatory root resorption was significantly larger in the control teeth than the pulpectomized teeth (P < .05). CONCLUSIONS: This study shows that tensile forces in the pulp cells enhance the expression of various cytokines via the S-A channel, which may lead to inflammatory root resorption during tooth movement. It also suggests that root canal treatment is effective for progressive severe inflammatory root resorption during tooth movement.

Cranial bone regeneration after cranioplasty using cryopreserved autogenous bone by a programmed freezer with a magnetic field in rats.

BACKGROUND: The purpose of this study was to develop a bone tissue bank using a programmed freezer with a magnetic field. Parietal bones were removed from rats and used for organ culture examination (non-cryopreserved, cryopreserved with a magnetic field (CAS) and cryopreserved without a magnetic field group). Next, other parietal bones were used for histological examination. The cryopreserved bones by a CAS freezer and dried bones were transplanted respectively. Control bones were replanted without cryopreservation. Animals were sacrificed at 4, 8, 12 and 24 weeks after surgery. After organ culture, the isolated osteoblasts from parietal bones which were cryopreserved by a CAS freezer can survive and proliferate as much as non-cryopreserved group. Histological examinations showed new bone formation in control and CAS group. These results suggest that bone tissue cryopreservation by CAS freezer can be successfully used for bone grafting which may be a novel option for regeneration medicine.

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field.

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of -150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.

Open bite as a risk factor for orthodontic root resorption.

The purpose of the present study was to clarify the prevalence and degree of root resorption induced by orthodontic treatment in patients with and without open bite. One hundred and eleven patients treated with multibracket appliances were retrospectively selected from the patients and divided into non-open bite (NOB) and open bite (OB) groups. The severity of root resorption and the root shape were classified into five groups on periapical radiographs before and after treatment. Moreover, only in the OB group, all teeth were sub-divided into functional and hypofunctional ones that are occluding and non-occluding. As the results of multiple linear regression analysis of patient characteristics and clinical variables with the number of overall root resorption, the independent variables that were found to contribute significantly to root resorption were bite and abnormal root shape. The prevalences of root resorption evaluated in the number of patients were significantly higher in OB group than in NOB group, and those in the number of teeth were significantly higher in OB group than in NOB group, in particular anterior and premolar teeth. The prevalence of resorbed teeth with abnormal root shapes was also significantly higher in OB group than in NOB group. On the other hand, in OB group, the prevalences of root resorption and teeth with abnormal root shape were significantly greater in hypofunctional teeth than in normal functional teeth. There are more teeth with root resorption and abnormal root shape in open bite cases than in normal bite cases, and more teeth with abnormal root shapes and root resorption in hypofunctional teeth than in functional teeth.

Effects of vascular endothelial growth factor-C and -D on osteoclast differentiation and function in human peripheral blood mononuclear cells.

OBJECTIVE: The purpose of this study was to clarify the interaction of vascular endothelial growth factors (VEGFs)-C and -D with cell surface foetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-4 (Flt-4) receptors in the induction and activity of osteoclasts in cultured human peripheral blood mononuclear cells (PBMCs). DESIGN: PBMCs were cultured on chamber slides or on ivory discs for 2 or 3 weeks in the presence of macrophage-colony stimulating factor (M-CSF), VEGF-A, -C or -D, or placental growth factor (PlGF) with or without receptor activator of nuclear factor kappa-B ligand (RANKL). The number of osteoclasts in each group was counted and the area of ivory resorption was measured. In addition, osteoclast differentiation was further analysed under the same conditions, but with the addition of specific neutralizing antibodies against Flk-1 and Flt-4. RESULTS: RANKL was essential for the induction of osteoclasts in PBMCs. However, significant differences were found in the number of osteoclasts induced by VEGF-A, -C, -D or M-CSF with RANKL compared with control groups lacking or containing RANKL. Blocking of either Flk-1 or Flt-4 resulted in a reduction in the enhancement of osteoclast differentiation in PBMCs by VEGF-C or -D with RANKL. The osteoclasts induced by VEGF-A, -C, -D or M-CSF with RANKL formed significantly larger resorption lacunae than those formed by osteoclasts induced by RANKL alone. CONCLUSIONS: This study showed that VEGF-C and -D play a role in the induction of osteoclast differentiation through both Flk-1 and Flt-4 receptors and influence the area of the ivory resorption in PBMCs.

Effects of long-term occlusal hypofunction and its recovery on the morphogenesis of molar roots and the periodontium in rats.

OBJECTIVES: To investigate the effects of long-term, artificially created, hypofunctional occlusion and its recovery on the morphology of rat molar roots. MATERIAL AND METHODS: Eighteen 5-week-old Wistar-strain male rats were randomly divided according to their periodontal conditions into normal, hypofunctional, and recovery groups (n = 6 in each). In the experimental hypofunctional and recovery groups, a bite-raising appliance was set to produce hypofunction at the molar region. All groups were analyzed at 16 weeks of age using three-dimensional micro-computed tomography. Root length, width, and area as well as the thickness and the area of the periodontal ligament (PDL) space of the maxillary first molar were calculated. RESULTS: Roots were longer and narrower in the hypofunctional group than in the control group. The mesial root in particular showed a dramatic change. Root area also decreased significantly in the hypofunctional group compared to the other groups. Moreover, the PDL thickness and area decreased significantly in the hypofunctional group compared to the control group, but increased in the recovery group compared to the hypofunctional group. CONCLUSIONS: These findings suggest that root size and PDL structure may be reduced due to disuse atrophy resulting from a defect in occlusal function, but may be recovered following a gain of occlusal stimuli.

Gummy smile and facial profile correction using miniscrew anchorage.

This case report describes the treatment of a case involving a skeletal Class II facial profile with a gummy smile. While treating a facial profile and a gummy smile, the outcome may not always be successful with orthodontic therapy alone. For this reason, surgical therapy is often chosen to gain an esthetic facial profile and a good smile. However, sometimes the patients reject surgical treatment and an alternative method must be considered. Skeletal anchorage systems such as miniscrews are now frequently used for correcting severe malocclusion that should be treated by surgical therapy. In this case report, we treated a skeletal Class II malocclusion with a convex profile and a gummy smile using miniscrews, which were placed in the upper posterior and anterior areas. The active treatment period was 3.5 years, and the patient's teeth continued to be stable after a retention period of 36 months.

Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in osteopetrotic (op/op) mice.

OBJECTIVE: Expression of osteoclasts in osteopetrotic (op/op) mice is substantially reduced by the absence of functional macrophage colony-stimulating factor (M-CSF). However, it has been reported that osteoclasts do gradually appear in the bones of op/op mice and spontaneously correct the osteopetrosis. DESIGN: Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in op/op mice were examined. RESULTS: The number of femoral osteoclasts, and the serum levels of VEGF, both gradually increased in op/op mice after birth and reached a peak in 120- and 60-day-old mice, respectively. However, the serum levels of RANKL showed an inverse relationship to osteoclast number. CONCLUSIONS: These findings suggest that the appearance of osteoclasts may be influenced by the serum levels of VEGF and that the serum levels of RANKL may be influenced by the appearance of osteoclasts.

Association between root resorption incident to orthodontic treatment and treatment factors.

The purpose of this study was to clarify the prevalence and degree of root resorption induced by orthodontic treatment in association with treatment factors. The files of 243 patients (72 males and 171 females) aged 9-51 years were randomly selected from subjects treated with multi-bracket appliances. The severity of root resorption was classified into five categories on radiographs taken before and after treatment. The subjects were divided into extraction (n = 113 patients, 2805 teeth) and non-extraction (n = 130 patients, 3616 teeth) groups and surgical (n = 56 patients, 1503 teeth) and non-surgical treatment (n = 187 patients, 4918 teeth) groups. These subjects were also divided into two or three groups based on the duration of multiloop edgewise archwire (MEAW) treatment, elastic use, and total treatment time: 0 month (T1; n = 184 patients, 4831 teeth), range 1-6 months (T2; n = 37 patients, 994 teeth), more than 6 months (T3; n = 22 patients, 596 teeth); range 0-6 months (n = 114 patients, 3016 teeth) more than 6 months (n = 129 patients, 3405 teeth); range 1-30 months (n = 148 patients, 3913 teeth) and more than 30 months (n = 95 patients, 2508 teeth). The prevalence of overall and severe root resorption evaluated by the number of subjects and teeth was compared with a chi-square test. A Student's t-test for unpaired data was used to determine any statistically significant differences. The prevalence of severe root resorption based on the number of teeth was significantly higher in the group with extractions (P < 0.01). Longer use of a MEAW appliance and elastics also produced a significantly higher prevalence of root resorption (P < 0.05). On the other hand, the prevalence of severe root resorption was not significantly different between the subjects treated with or without surgery, but there was a significant increase when treatment time was prolonged (P < 0.05). A significant difference was found in the amount of root movement of the upper central incisors and the distance from their root apices to the cortical bone surface (P < 0.05). These are regarded as essential factors in the onset of root resorption. These results indicate that orthodontic treatment with extractions, long-term use of a MEAW appliance and elastics, treatment time, and distance of tooth movement are risk factors for severe root resorption.

In-vitro and in-vivo study of periodontal ligament cryopreserved with a magnetic field.

INTRODUCTION: The purpose of this study was to examine the effect of a new cryopreservation method with a magnetic field on periodontal regeneration in vitro and in vivo. METHODS: Human periodontal ligament cells were frozen in 10% dimethyl sulfoxide by using a programmed freezer with a magnetic field. Cells were cryopreserved for 3 days at -150°C. Immediately after thawing, collagen type I and alkaline phosphatase gene expression were determined by real-time polymerase chain reaction. Incisors were extracted from 15-week-old Wistar rats and cryopreserved or dried for 3 days. Then the incisors were replanted into the same sockets. Ninety days after transplantation, they were observed under light microscopy. RESULTS: There was no difference in the messenger RNA expression of collagen type I between the cryopreserved and the control groups. The expression of alkaline phosphatase messenger RNA in the cryopreserved group was slightly decreased compared with the control group. There was no progressive root resorption in the teeth that were replanted immediately (control group) or cryopreserved. However, there was widespread root resorption and ankylosis in the dried teeth. CONCLUSIONS: These results show that a magnetic field programmed freezer can be successfully used for cryopreservation of teeth.

Treatment of a case of skeletal class II malocclusion with temporomandibular joint disorder using miniscrew anchorage.

At the present time, there are no reports in the literature on the treatment of temporomandibular joint disorder (TMD) by intrusion of molars using mini-screws. This case report describes the treatment for a female patient, aged 19 years seven months, with a TMD and an excessive lower anterior facial height. Overjet and overbite were +5.0 mm and +0.5 mm, respectively. The patient had a history of orthodontic treatment in which her first premolars were all extracted. During the first orthodontic treatment, a clockwise mandibular rotation was observed as a result of the increase of posterior dentoalveolar height. She had temporomandibular joint (TMJ) pain during mouth opening and complained of difficulty in eating due to masticatory dysfunction. The pretreatment Schuller views of both TMJ showed a posterior condyle position. In order to correct the overjet, molar relationship and the mandibular condyle position, a miniscrew was inserted into the palatal region of the upper first molar to intrude the upper posterior teeth. As the upper molars were intruded, the overjet was decreased, and a class I molar relationship was achieved by a counterclockwise mandibular rotation. After one year of treatment, an acceptable occlusion was achieved, and the condyle moved into centric position in the glenoid fossa. The patient's teeth continued to be stable, and she had no pain in TMJ after a retention period of three years. The result of this treatment showed that molar intrusion using miniscrew anchorage is effective for treatment of a TMD patient with a posterior condyle position.

Changes in cortical bone mineralization in the mouse mandible with regenerated condyle.

The objective of this study was to evaluate the effects of condylectomy and of a functional appliance on the mineral content, density, and area of the mandibular cortical bone in a growing mouse model. In the condylectomy group, a unilateral condylectomy was performed on the right side of the mouse mandible. In the condylectomy + appliance group, a functional appliance was used to reposition the mandible after the unilateral condylectomy. All mice were killed 4 wk after surgery. Each mandible was then subjected to analyses of cortical bone mineral content (CRT_CNT), cortical bone density (CRT_DEN), and cortical bone area (CRT_A) by peripheral quantitative computed tomography (pQCT). The CRT_CNT, CRT_DEN, and CRT_A values were significantly lower in the condylectomized mandible (right side) than in the non-condylectomized mandible (left side). However, in the condylectomized animals in which a functional appliance was used, the CRT_CNT, CRT_DEN, and CRT_A values became higher than those in the mice treated with condylectomy alone. No significant differences were found in the CRT_CNT, CRT_DEN, and CRT_A between non-condylectomized (left side) mandibles, mandibles treated with condylectomy + functional appliance (right side), and control mandibles. It was thus shown that a functional appliance used to reposition the condylectomized mandible forward in a symmetric position induced improvement of the cortical bone in a mouse model in terms of cortical bone mineral content, density, and area.

Mesenchymal stem cell-induced cranial suture-like gap in rats.

BACKGROUND: Mesenchymal stem cells are used in the reconstruction of many organs and tissues. However, there are no data regarding cranial suture regeneration using mesenchymal stem cells. Because cranial sutures are important for cranial bone growth, the authors examined whether mesenchymal stem cells could aid with suture reformation in adolescent rats. METHODS: Mesenchymal stem cells were isolated from bone marrow of rat femora. Twenty 4-week-old Fischer 344 male rats received sagittal suture and bone defects with a diameter of 6.0 mm, and mesenchymal stem cells were grafted with membranes. Twenty control rats underwent empty membrane transplantation. The mesenchymal stem cell and control groups were killed at 4, 8, 16, and 24 weeks after surgery. The parietal bones, including the sagittal suture, were observed under a light microscope. Bone structures were measured on digitized photomicrographs captured in a computer. For each sample, bone and suture regeneration were observed by dorsoventral cephalograms. RESULTS: In 4- and 8-week control and mesenchymal stem cell groups, a small volume of new bone formation was observed. In the 16- and 24-week mesenchymal stem cell groups, a large amount of new bone formation and a suture-like gap were identified. In contrast, only a small volume of new bone formation along with craniosynostosis was detected in the 24-week control group. CONCLUSION: These results suggest that mesenchymal stem cell grafting may be a novel option for cranioplasty, not only repairing bone tissue but also resulting in suture-like gap formation, which may advance cranial bone growth.

Expression of Sox 9 and type II and X collagens in regenerated condyle.

The present study was designed to examine the expression of Sox 9 and type II and X collagens in regenerated condyle resulting from the use of a functional appliance. Ninety, 3-week-old, mice were divided equally into the following groups: two experimental groups (condylectomy group and condylectomy with functional appliance group) and the corresponding control group. In the condylectomy group, a unilateral condylectomy was performed on the right side. In the condylectomy with appliance group, the mandible was repositioned in a forward direction using a functional appliance after unilateral condylectomy. The expression of Sox 9 and type II and X collagens in the condyle was determined immunohistochemically 4 weeks after surgery. In mice with a condylectomy, the expression was minimal. On the other hand, these factors were highly expressed in the condylectomized side with the appliance. It is thus speculated that cartilaginous regeneration is due to the expression of chondrogenic factors, such as Sox 9 and type II and X collagens. It is also suggested that condyle regeneration results from an optimal intra-articular environment with appropriate joint spaces achieved by condylar repositioning.

A newly developed snack effective for enhancing bone volume.

BACKGROUND: The incidence of primary osteoporosis is higher in Japan than in USA and European countries. Recently, the importance of preventive medicine has been gradually recognized in the field of orthopaedic surgery with a concept that peak bone mass should be increased in childhood as much as possible for the prevention of osteoporosis. Under such background, we have developed a new bean snack with an aim to improve bone volume loss. In this study, we examined the effects of a newly developed snack on bone volume and density in osteoporosis model mice. METHODS: Orchiectomy (ORX) and ovariectomy (OVX) were performed for C57BL/6J mice of twelve-week-old (Jackson Laboratory, Bar Harbar, ME, USA) were used in this experiment. We prepared and given three types of powder diet e.g.: normal calcium diet (NCD, Ca: 0.9%, Clea Japan Co., Tokyo, Japan), low calcium diet (LCD, Ca: 0.63%, Clea Japan Co.,) and special diet (SCD, Ca: 0.9%). Eighteen weeks after surgery, all the animals were sacrified and prepared for histomorphometric analysis to quantify bone density and bone mineral content. RESULTS: As a result of histomorphometric examination, SCD was revealed to enhance bone volume irrespective of age and sex. The bone density was increased significantly in osteoporosis model mice fed the newly developmental snack as compared with the control mice. The bone mineral content was also enhanced significantly. These phenomena were revealed in both sexes. CONCLUSION: It is shown that the newly developed bean snack is highly effective for the improvement of bone volume loss irrespective of sex. We demonstrated that newly developmental snack supplements may be a useful preventive measure for Japanese whose bone mineral density values are less than the ideal condition.