Effects of insulin-like growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice.

It is well accepted that mechanical loading inhibits bone resorption and increases in vivo bone formation. It is also known that cyclic mechanical loading, in particular, can enhance bone formation significantly. These findings suggest a significant role for mechanical stimuli in bone remodelling mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Earlier studies showed that the nasal bone length and premaxillary bone width were significantly greater in mice fed a solid diet rather than a granulated diet, and that these dimensions increased significantly in a solid-diet group treated with IGF-I. The present study sought to examine the effect of IGF-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture subjected to different masticatory loadings. For the solid-diet groups, the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells and osteoblasts were significantly greater in the group injected with IGF-I than in the animals injected with physiological saline. In the groups fed a granulated diet, no significant differences in the numbers of TRAP-positive osteoclastic cells and osteoblasts were found over the entire experimental period between mice injected with either IGF-I or physiological saline. It is shown that IGF-I significantly induces the expression of osteoclasts and osteoblasts and the subsequent bone remodelling, and that the effect may be additive as compared to that of mechanical masticatory loading, which seems to be more important in bone remodelling in terms of the numbers of osteoclasts and osteoblasts.

Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement.

Vascular endothelial growth factor (VEGF) has an ability to induce functional osteoclasts as well as neovascularization. We recently reported that the number of osteoclasts was enhanced by the injection of recombinant human VEGF (rhVEGF) with the application of mechanical force for experimental tooth movement. In this study, the expression of VEGF was detected in osteoblasts on the tension side of the alveolar bone. Moreover, the rate of tooth movement was significantly increased in the rhVEGF injection groups compared with the controls. These results suggested that VEGF, highly expressed by mechanical stimuli, enhances the number of osteoclasts as a paracrine factor, and that the amount of tooth movement is accelerated by both endogenous VEGF and injected rhVEGF.

Effects of vascular endothelial growth factor on osteoclast induction during tooth movement in mice.

UNLABELLED: For orthodontic tooth movement, remodeling of the alveolar bone is maintained by a repeated process of bone resorption and new bone formation, controlled, respectively, by osteoclasts and osteoblasts. Recently, we have found that recombinant human vascular endothelial growth factor (rhVEGF) acts as a macrophage colony-stimulating factor in osteoclast induction in osteopetrotic (op/op) mice. The purpose of this study was to investigate whether rhVEGF stimulates osteoclast differentiation during experimental tooth movement. Purified rhVEGF was injected once into the buccal gingival groove around the incisors. An experimental appliance with a helical loop was bonded onto the upper incisors, and an initial force of 1.0 g was applied for three days. The number of osteoclasts appearing in the periodontal ligament space on the pressure side of the alveolar bone was increased markedly. These results suggest that local administration of rhVEGF enhances the number of osteoclasts, and may increase the rate of orthodontic tooth movement. ABBREVIATIONS: recombinant human vascular endothelial growth factor (rhVEGF), macrophage colony-stimulating factor (M-CSF), osteopetrotic mice (op/op mice),fms-like tyrosine kinase (Flt-1), periodontal ligament (PDL), phosphate-buffered saline (PBS), tartrate-resistant acid phosphatase (TRAP), and analysis of variance (ANOVA).

Transplantation of new autologous biomaterials into jaw cleft.

This study compared bone-marrow grafting using chondroid or fibula bone grafts transplanted into simulated alveolar bone defects in mice. The osteogenic procedure was also investigated. As an experimental model of the maxillary alveolar bone cleft, suitable for testing bone-inductive materials, a surgical trephine with a low-speed dental engine was used to form critical-sized defects in the pre-maxillary bones of male mice. Distraction osteogenesis was performed using an external fixation device. The osteotomy site was surrounded by an external callus, consisting of hyaline cartilage, that contained a large quantity of chondroid bone. Transplanted bone within chondroid bone was characterized by bone formation and remodelling 30 days post-transplantation, and bone adhesion following chondroid bone grafting was better than adhesion following fibula grafting. The present findings are the first to demonstrate the potential of chondroid bone transplantation as a new therapeutic system of bone grafting, suitable for bone substitution in craniofacial bone defects.

Breadth of the mandibular condyle affected by disturbances of the sex hormones in ovariectomized and orchiectomized mice.

It is well known that the size of the condyle is different between males and females in humans. It has also been documented that ovariectomy (OVX) and orchiectomy (ORX) influence the remodeling of the condylar head in experimental animals. However, influences of sex hormones on the condyle in terms of its breadth have not been examined. With these considerations, this study was designed to examine the breadth of the mandibular condyle in OVX and ORX mice by histomorphometry 8 weeks after surgery. In the OVX mice, the condylar breadth was significantly larger than in the controls, while no significant differences in the breadth were found between the ORX mice and the controls. The thickness of the condylar cartilage layers exhibited similar findings to the breadth of the condyle. On the contrary, the trabecular bone volume was significantly smaller in the OVX and ORX mice than in the corresponding controls, and the difference was more substantial in female mice than in the males. These findings emphasize that a reduction of sex hormone levels, induced by OVX and ORX, significantly influences condylar morphogenesis.

Analysis of histochemically and morphometrically in the anterior belly digastric muscle of osteopetrotic (op/op) mice.

The fibers of the anterior belly digastric muscle of mice, fed a granulated diet for various periods, have been studied histochemically and morphometrically. The diameters of the anterior belly digastric fibers in normal mice fed only a granulated diet were smaller than those in mice fed a solid diet. Differences in the succinate dehydrogenase (SDH) activity of muscle fibers between op/op and normal mice gradually appeared in the anterior belly digastric muscle and, by the age of 90 days, under-development of muscle fibers was observed in the mild-belly region of the anterior belly digastric muscle of op/op mice fed a granulated diet. These results indicate mechanical stress in mastication plays an important role in the development of the anterior belly digastric muscle structures.

Influence of oestrogen and androgen on modelling of the mandibular condylar bone in ovariectomized and orchiectomized growing mice.

Oestrogen and androgen exert a substantial influence on bone metabolism, but any differences in their influence on modelling of the condyle, a mandibular growth site, have not been fully clarified. The purpose here was to examine histological and histochemical differences in the condyle of ovariectomized (OVX) or orchiectomized (ORX) mice given injections of oestrogen (E(2), 17 beta-oestradiol) or non-aromatizable androgen (DHT, 5 alpha-dihydrotestosterone). Eight-week-old C57BL/6J mice (n=170) were used: they were divided equally into six experimental groups (OVX, ORX, OVX+E(2), ORX+E(2), OVX+DHT, ORX+DHT), and non-treatment male and female control groups. In each experimental group, five mice were killed 2,4,8 and 12 weeks after OVX and ORX. Oestrogen or androgen were given daily after the surgery by subcutaneous injection of E(2) or DHT. Increases in the number of tartrate-resistant acid phosphatase-positive cells induced in the OVX and ORX mice from 4 to 12 weeks after surgery were obviously suppressed by E(2) and DHT. The trabecular bone volume in the OVX and ORX mice treated with DHT had only increased at 12 weeks after surgery, whereas the E(2) injected mice exhibited a substantial increase from 4 to 12 weeks after surgery. E(2) injected into the OVX and ORX mice increased the trabecular bone volume earlier than did DHT, and both E(2) and DHT suppressed osteoclast differentiation similarly during the same period. These results suggest that metabolic responses of osteoclasts and osteoblasts to E(2) and DHT may be different, producing somewhat different patterns of bone modelling in males and females.

Influences of osteoclast deficiency on craniofacial growth in osteopetrotic (op/op) mice.

It is well known that the defect in bone resorption in osteopetrotic (op/op) mice brings about deformation of the cranium and failure of tooth eruption. However, the influences on longitudinal growth of the craniofacial skeleton have not been elucidated. This study was thus conducted to examine craniofacial morphology and longitudinal changes in the op/op mice by means of morphometric analysis with lateral cephalograms. Lateral cephalograms, taken every 10 days from 10- to 90-day-old mice, were analyzed on a personal computer for 11 measurement items. For the nasal bone region, the most prominent differences were found between the op/op and normal mice. The anterior cranial base and occipital bone height presented almost equivalent growth changes in both the op/op and normal mice. The size of mandible, meanwhile, was significantly smaller in the op/op mice than in the normal controls. The gonial angle was also significantly larger in the op/op mice than in the normal mice throughout the experimental period. Thus, substantial differences in craniofacial growth were demonstrated in various areas of the craniofacial complex, which are assumed essentially due to the lack of osteoclastic bone resorption during growing period. Since the difference became more prominent in the anatomic regions relevant to the masticatory functions, it would be a reasonable assumption that reduced masticatory function is also a key determinant for the less-developed craniofacial skeleton in the op/op mouse.

Effects of insulin-like growth factor-I on nasopremaxillary growth under different masticatory loadings in growing mice.

It is well accepted that reduced masticatory function induced by a diet with soft physical consistency causes alterations in the craniofacial morphology in growing animals. It is assumed that these alterations are associated with reduced proliferative activity of osteoblasts on the bone surface, indicating a significant role for mechanical stimuli mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Here, the effects of IGF-I on the linear growth of nasal and premaxillary bones subjected to different masticatory loadings were examined. The length of the nasal bone and the width of the premaxilla were measured. These dimensions were significantly greater in mice fed a solid diet than in mice fed a granulated diet. In animals treated with IGF-I, the nasal bone length and premaxillary width increased significantly in a subgroup receiving a solid diet, but these changes were not found in a similar group fed a granulated diet. No statistically significant differences in these dimensions were found between solid-diet mice injected with saline and granulated-diet group injected with IGF-I. It is concluded that IGF-I induces nasal and premaxillary growth, and that its effect is enhanced or accelerated by increased mechanical masticatory loading.

Long-term granular diet may lead to declined resistance to trauma of the temporomandibular joint.

This study was carried out to determine the effect of food texture on the response to trauma of the temporomandibular joint in mice. Animals were fed either a solid or granular diet after weaning. At 8 weeks old, condylar hypermobility was induced by forcing the mouth wide open ten times under ether anaesthesia. This procedure was repeated 10 times once daily for 10 consecutive days. The temporomandibular joint tissues were observed microscopically on day 0 and then 2 days, 5 days, 2 weeks, 3 weeks and 5 weeks after the last forced opening. Eighteen male mice were used in total, three for each timepoint. A proliferation in synovial surface cells and an increased number of capillaries were seen at day 2 in the mice fed a granular diet, but not in those fed a solid diet. Multiple cell layers of the synovial membrane, dilated capillaries and fibrin deposits were expanded further between day 2 and week 5 in the mice fed a granular diet, but not until day 5 to week 5 in the mice that had been fed a solid diet. At week 3, the mice fed a granular diet showed erosion of the anterior articular disc. This was observed in both dietary groups at week 5. This study suggests that, in advanced countries, people who habitually eat easily chewable food should be advised that such practice may lead to a decline in the resistance to trauma of the masticatory system.

New biomaterials and methods for craniofacial bone defect: chondroid bone grafts in maxillary alveolar clefts.

The purpose of this study was to evaluate autogenous osteogenic marrow within chondroid bone grafts in simulated alveolar defects of mice in order to determine the ability of the graft material to effectively close the cleft from an osseous standpoint and to observe the effect of the grafting procedure. Critical-sized defects were made in the premaxillary bones of male mice using a surgical trephine and a low-speed dental engine as a model of the maxillary alveolar cleft for testing bone-inductive agents. Premaxillary trephine defects were not repaired by fibrous tissue or bone formation 30 days after operation. This nonhealing bony wound of the premaxilla in mice may be useful as a model for studying the effect of bone-inductive agents on the healing of alveolar clefts. Distraction osteogenesis is a recently advanced principle of bone lengthening in which a long bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. The osteotomy site was surrounded by an external callus consisting of hyaline cartilage. The callus contained a lot of chondroid bone. The transplant bone within chondroid bone was characterized by bone formation and remodeling 30 days after transplantation. Throughout the experiment, our findings demonstrated, for the first time, that the transplant bone that contains chondroid bone may be used clinically in relation to craniofacial bone defects to improve the treatment of bone grafts.

Mechanism in inhibitory effects of vitamin K2 on osteoclastic bone resorption: in vivo study in osteopetrotic (op/op) mice.

Osteoclast deficiency in op/op mice was cured by a single injection of 5 micrograms recombinant human macrophage colony-stimulating factor (M-CSF). On d 5, the osteoclast number reached a maximum value. By d 15, the osteoclast number had decreased to about 70% of the maximum level. Moreover, by d 20, the osteoclast number had decreased to about 30% of its maximum level. On d 5, the osteoclast number of vitamin K2 12 h previously had decreased to about 30% of the M-CSF-only injected mice. Moreover, on d 5, the osteoclast number of the mice receiving a single injection of vitamin K2 24 h previously had decreased to about 15% that of mice injected only with M-CSF. These results indicate that vitamin K2 inhibits in vivo osteoclast formation. On d 20, the osteoclast number of the mice injected with a single dose of vitamin K2 12 or 24 h previously had decreased to 0% compared with those receiving only M-CSF. The present results suggest that the vitamin K2 "causes cell death" to mature osteoclasts and inhibits in vivo osteoclast formation.

Remodeling of the sagittal suture in osteopetrotic (op/op) mice associated with cranial flat bone growth.

It is well known that cranial flat bone experiences growth and development at the sutural interface, which is regarded as a neutral zone to control mechanical stimuli. In osteopetrotic (op/op) mice, meanwhile, cranial deformation is produced by the deficiency of osteoclasts and the subsequent defect of bone resorption. It would be a reasonable assumption that such disturbance in bone remodeling affects sutural modification and the relevant cranial flat bone development. The present study was thus conducted to examine histological features of the sagittal sutures in op/op mice, with special reference to the relevant bone remodeling. The sagittal sutures in 10-, 15-, 30-, and 60-day-old normal and op/op mice were observed microscopically. Furthermore, osteoclastic activity was evaluated on the sections stained with tartrate-resistant acid phosphatase (TRAP). The sutures of 15-day-old op/op mice showed stenosis and synostosis, and less-developed collagen fibers associated with an irregular arrangement of fibroblasts, whereas these changes were rarely found in normal mice. Osteoclasts were hardly detected in the parietal bones around the sutures of op/op mice, although the number was numerous in normal mice. These results emphasize that congenital deficiency in osteoclast produces unbalanced bone remodeling at the sutural interface and on the surfaces of the cranial bones, which is assumed to be closely related to cranial bone deformity in op/op mice.

Lack of the bone remodeling in osteopetrotic (op/op) mice associated with microdontia.

Osteopetrosis is an inherited metabolic disease characterized by an excessive accumulation of bone. This is associated with an osteoclast deficiency. Osteopetrosis is always accompanied by the failure and/or delay of tooth eruption. The present study was conducted to examine in detail the morphological and histological changes of growth of the third molars in the osteopetrosis (op/op) mouse. At the age of 10 days, normal and op/op mice showed no detectable difference in the shape of the third molar follicles. However, the third molars in the op/op mouse became obscured by the proliferation of neighboring bone trabeculae. Moreover, no tartrate-resistant acid phosphatase-positive cells were detected on the bone surfaces of 10-day-old op/op mice. Ankylosis between the root dentin and proliferating bone trabeculae was a common feature in the 20- and 30-day-old op/op mice. The third molars erupted into the oral cavity before the age of 30 days in normal mice, and the crowns, roots, and periodontal ligaments appeared well developed. Throughout the experiment, it seemed that the primary cause of the microdontia and ankylosis of the developing root in the mutant mouse was a deficiency of osteoclasts, with attendant lack of bone remodeling.

Morphological change of the nasopremaxillary suture in growing "toothless" osteopetrotic (op/op) mice.

Osteopetrotic (op/op) mice are known to commonly show a failure of tooth eruption. It is also well understood that masticatory function is highly associated with the craniofacial morphology of the growing mouse; however, the effects on sutural growth have not been studied. The present study was conducted to examine, in detail, the morphological and histological changes of the nasopremaxillary suture in these mutant mice and to assess a role of mechanical stress from mastication in the sutural growth. The width of the nasopremaxillary suture was measured on the section for the superior (P1), middle (P2), and inferior (P3) levels. The width of the nasopremaxillary suture for the P1 level in the normal mice fed a solid diet was significantly smaller in 30-day-old mice than in 15-day-old mice, whereas the width for the level P3 was significantly greater in the 30-day-old mice than in the 15-day-old mice. These changes in the sutural space were more prominent in the normal mice fed a solid diet than in the normal mice fed a granular diet. The sutural widths for all the levels became smaller in the 30-day-old op/op mice than in the 10-day-old op/op mice. The endocranial area of the nasopremaxillary suture showed synostosis in 30-day-old op/op mice. In both the normal and op/op mice, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells was greatest at the age of 15 days. Moreover, the TRAP-positive cell number was smaller in the op/op mice than in the normal mice for all the experimental stages. Since, in general, mastication begins in mice after tooth eruption, i.e. from 15 to 30 days after birth, the present findings suggest that failure of tooth eruption and the reduced masticatory function restrict sutural modification.

Recruitment of osteoclasts in the mandibular condyle of growing osteopetrotic (op/op) mice after a single injection of macrophage colony-stimulating factor.

The purpose was to elucidate histological changes in the mandibular condyle and ramus in growing osteopetrotic (op/op) mice after a single injection of macrophage colony-stimulating factor (M-CSF). M-CSF (5 microg) was injected into 6-, 11-, 26-, 56- and 86-day-old op/op mice, and the mice were killed 4 days after the injection. In normal mice, the condyle was substantially wider than the ramus beneath it, and enlargement and ossification of the condyle occurred after weaning. These changes were not found in the uninjected and injected op/op mice, the condyles of which were occupied by hypertrophic cartilage cells, and the hypertrophic cell layer was thicker and more irregular in the arrangement of epiphyseal cell columns. In spite of the lack of bone resorption in uninjected and injected op/ op mice, ossification of the mandibular ramus occurred, but later than that of normal mouse. The number of tartrate-resistant acid phosphatase-positive cells in the injected op/op and normal mice approached a maximum at 30 days and then gradually decreased up to 90 days of age, although the numbers were substantially different for all ages. The uninjected op/op mice had no visible osteoclasts until 15 days and their number then increased significantly from 60 to 90 days of age. These results were considered due to the difference in biological responses of bony structures to M-CSF injection in the op/op mice. The influences of mechanical stimuli from masticatory functions, which are deficient in op/op mice, might also be responsible for the differences in bony architecture between the op/op and normal mice.

Facial skeletal growth in growing "toothless" osteopetrotic (op/op) mice: radiographic findings.

The defective bone resorption in the osteopetrotic (op/op) mouse brings about failure of tooth eruption. Furthermore, the op/op mouse has been studied as a "toothless" mouse in recent morphological and physiological investigations of the relationship between mastication and masseter muscle development. The present study was conducted to examine in detail the nasal bone and the premaxillary bone in this mutant mouse and to assess the roles of incisor growth and the mechanical stress of mastication in nasal bone and premaxillary bone growth. The forms of the nasal bone and the premaxillary bone were observed using roentgenography in both toothless op/op and normal (control) mice. In the op/op mouse, the nasal bone and the premaxillary bone show remarkable deformity. In contrast, the normal mouse appears well developed. This suggests that growth of the incisor root is important to normal upper jaw growth in the mouse. Furthermore, it is proposed that the upper facial phenotype seen in the op/op mice results from not only decreased bone resorption, but also from absence of the mechanical stress provided by normal mastication.

Influences of ovariectomy and orchiectomy on the remodeling of mandibular condyle in mice.

Ovariectomy (OVX) and orchiectomy (ORX) are well known to increase bone turnover; however, the influences on condylar remodeling are not fully understood. The purpose of this study was to examine histological changes of the mandibular condyles in OVX and ORX mice in comparison to the femora. Eighty 8-week-old mice were used in this study. In each of the experimental and control groups, five mice were sacrificed 2, 4, 8, and 12 weeks after surgery for OVX and ORX. Histological changes of the mandibular condyle were studied in comparison to the femur, which is liable to undergo the influences of OVX and ORX. The number of TRAP-positive cells and the thickness of articular cartilage layer were also quantified. TRAP-positive cells in the condylar head of OVX and ORX mice exhibited the most prominent increase 4 weeks after the surgery and approached the control levels at the later experimental stages. Trabecular bone volume in the condyle of the OVX and ORX mice also decreased, although the rate was less than in the femora. It is shown that influences of OVX on the remodeling of femora appear earlier than those of ORX, whereas OVX and ORX affect the remodeling of condyle during the same growth period. It is also demonstrated that the influences of OVX and ORX are less in the condyle than in the femora. These findings emphasize that the influences of OVX and ORX on bone remodeling are varied by sexual difference and the type of bones.

Suspension "hypokinesia/hypodynamia" may decrease bone mass by stimulating osteoclast production in ovariectomized mice.

This study was conducted to examine, in detail, the histological changes in the femurs of suspended ovariectomized (OVX) mice to assess the role of mechanical stress on bone remodeling. Suspended-OVX, suspended-sham-ope, nonsuspended-OVX, and nonsuspended-sham-operated mice underwent operations 8 weeks after birth. Immediately after operation, hypokinesia/hypodynamia was created by a suspension harness for one week. Five specimens in each group were sacrificed 9 weeks after birth. The trabecular bone of the femurs in the suspended-OVX mice was removed and replaced extensively by bone marrow. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was larger in the suspended-OVX mice than in the remaining three groups. No significant differences in the number of TRAP positive cells were found between the suspended-sham-ope, nonsuspended-OVX and nonsuspended-sham-ope mice. The femurs of the OVX mouse with suspension "hypokinesia/hypodynamia" thus exhibits extensive trabecular bone loss in association with an increase of osteoclasts.

Midpalatal suture of osteopetrotic (op/op) mice exhibits immature fusion.

The midpalatal suture was observed histologically in both toothless osteopetrotic (op/op) and normal (control) mice. The normal mice had a mature sutural structure, which consists of a well-developed cartilage cell zone and palatal bone. In contrast, the thickness of the cartilage cell zone was substantially greater in the op/op mice than that in the controls. Moreover, the cartilage cells in the op/op mice were frequently found in the palatal bone as well as in the sutural space, exhibiting an imperfect fusion. It seems that immature fusion at the sutural interface in the op/op mice is related to a decrease in biting or masticatory force accompanied by the failure of tooth eruption in addition to an essential defect in osteoclast differentiation, which is a congenital symptom in op/op mice.