Regeneration of condyle with a functional appliance.

Condylar regeneration with the use of functional appliances after condylectomy has been validated. However, the process during treatment remains unclear. In this study, we evaluated the condylar regeneration process and then examined mandibular growth and masticatory muscle activity after regeneration in growing rats. Seventy-five male Wistar rats aged 4 weeks were equally divided into 3 groups: unilateral condylectomy group, unilateral condylectomy + appliance group, or control group. The use of a functional appliance following condylectomy promoted mandibular growth and regeneration of the condyle 1 week after condylectomy. Condyle regeneration showing normal morphology was finally achieved 8 weeks after condylectomy. Asymmetrical masticatory muscle activity was observed after condylectomy. However, the use of a functional appliance produced symmetrical masticatory muscle activity. These results indicate a favorable regeneration process in the condylectomized area due to the use of a functional appliance. In addition, due to condylar regeneration, symmetrical masticatory muscle activity was achieved.

Cryopreservation of osteoblasts by use of a programmed freezer with a magnetic field.

In order to determine a suitable condition for osteoblasts cryopreservation, murine osteoblasts were freezed by programmed freezer with a magnetic field (CAS freezer). After 7 days cryopreservation at -150°, the number of survival cells immediately after thawing and the growth rate of cultured cells for 48 hours were examined. Gene and protein expression of alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) were compared between cryopreserved and non-cryopreserved groups. As a result, a plunging temperature of -30°, a hold-time at -5° for 15 minutes and a 0.1 mT of magnetic field led to the largest survival and growth rate. Moreover, there was no significant difference in ALP, OPN and BSP mRNA and protein expression between cryopreserved and control groups. From these results, it was suggested that the CAS freezer is available for osteoblast cryopreservation and bone tissue banking can be established in the future.

Mandibular and femoral growth alteration after sex hormone disruption in growing mice.

OBJECTIVES: To investigate how mandibular and femoral growth is affected when sex hormone- specific receptor antagonist is administered in growing mice. MATERIALS AND METHODS: Forty C57BL/6J mice were used in this experiment. At 5 days of age, the mice received daily injection of estrogen receptor alpha (ERα), beta (ERß), or androgen receptor (AR) antagonists, and their body weight was assessed every 4 days. One, four and eight weeks after the initial injection, radiographs of the mandible and femur were taken and measured. Analyses of variance and pairwise comparisons (Fisher) were performed to examine the differences in values measured among the groups. RESULTS: Mandibular growth was affected by ERß antagonist injection in male mice at 4 and 8 weeks. In female mice, the growth was affected during all the experimental period, when ERß was administered. Moreover, at 8 weeks, mandibular growth was also affected in male and female mice injected with ERα antagonist and in male mice injected with AR antagonist. Femoral growth was affected during all the experimental period in male and female mice injected with ERß antagonist. Moreover, at 8 weeks, the growth was affected in male and female mice injected with ERα antagonist and in male mice injected with AR antagonist. CONCLUSIONS: Growth of the mandible and femur in mice, in part, is induced in response to the stimulation of ERß in chondrocytes before and during early puberty. In late and after puberty, the growth is induced by the stimulation of ERα in male and female mice and that of AR in male mice.

Effects of cryopreservation with a newly-developed magnetic field programmed freezer on periodontal ligament cells and pulp tissues.

The purpose of this study was to evaluate the effects of long-term cryopreservation on the isolated human periodontal ligament cells (PDL) and pulp tissues. In the first part of study, 10 freshly extracted teeth were selected and divided into two groups. In the cryopreserved group, the teeth were frozen for 5 years using a programmed freezer combined with a magnetic field, known as Cells Alive System "CAS". As for the control group, freshly extracted teeth were used. In each group, extracted PDL tissues were cultured and gene expression and protein concentration of collagen type I, alkaline-phosphatase (ALP) and vascular endothelial growth factor (VEGF) was compared between the two groups. In the second part, pulp tissues were obtained from 10 mature and immature third molars which were freshly extracted or cryopreserved for three months. Expression of VEGF and nerve growth factor (NGF) mRNAs and the protein concentration in the supernatant were investigated. Results indicated that long-term cryopreservation with the use of CAS freezer cannot affect the growth rate and characteristics of PDL cells. There was no significant difference in VEGF expression and VEGF and NGF protein concentration of pulp cells derived from cryopreserved teeth with immature apex and control group with mature root formation. Finally, proper PDL regeneration and appropriate apexogenesis after transplanting magnetically cryopreserved immature tooth was clinically confirmed. These findings demonstrate that teeth banking with the use of magnetic field programmed freezer can be available for future autotransplantation as a treatment modality for replacing missing teeth.

Sex hormones receptors play a crucial role in the control of femoral and mandibular growth in newborn mice.

Sex hormones are important for bone growth. However, the mechanism by which sex hormone receptors influence bone growth remains unclear. In orthodontic treatment, there is a need to develop an indicator of bone maturity to accurately predict the beginning and end of growth. This indicator might be developed from the screening of sex hormones. The purpose of this study was to investigate the role of each sex hormone receptor on bone growth in newborn mice. Five-day-old C57BL/6J mice were used in this experiment. Forty mice underwent an orchiectomy (ORX), ovariectomy (OVX), or sham surgery. One week after surgery, the femur and the mandible were resected for immunohistochemical staining. Alternatively, 80 mice were daily injected with antagonist against receptors oestrogen alpha (ERα), beta (ERß), or androgen receptor (AR). One week after the first injection, radiographs of the femur and mandible were taken and then measured. Analysis of variance and pairwise comparisons (Fisher) were performed to examine the differences in values measured among the groups In the sham-operated male and female mice, ERß was found to be more prominent than ERα and AR during all experimental periods. In the ORX and OVX groups, the expressions of all receptors were significantly reduced in comparison with the sham-operated control group throughout the experiment. Moreover, femur and mandibular growth were significantly affected in the group injected with ERß antagonist. The deficiency of any sex hormone leads to reduced bone growth. In particular, a disturbance in ERß produces a greater aberrance in both male and female mice immediately after birth.

Cryopreservation of periodontal ligament cells with magnetic field for tooth banking.

The purpose of this study was to establish a long-term tooth cryopreservation method that can be used for tooth autotransplantation. Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide (Me(2)SO) using a programmed freezer with a magnetic field. Cells were cryopreserved for 7 days at -150 degrees C. Immediately after thawing, the number of surviving cells was counted and the cells were cultured; cultured cells were examined after 48 h. Results indicated that a 0.01 mT of a magnetic field, a 15-min hold-time, and a plunging temperature of -30 degrees C led to the greatest survival rate of PDL cells. Based on these findings, whole teeth were cryopreserved under the same conditions for 1 year. The organ culture revealed that the PDL cells of cryopreserved tooth with a magnetic field could proliferate as much as a fresh tooth, although the cells did not appear in the cryopreserved tooth without a magnetic field. Histological examination and the transmission electron microscopic image of cryopreserved tooth with a magnetic field did not show any destruction of cryopreserved cells. In contrast, severe cell damage was seen in cells frozen without a magnetic field. These results indicated that a magnetic field programmed freezer is available for tooth cryopreservation.

Effects of mandibular advancement on growth after condylectomy.

Previous studies have indicated that an injured condyle during adolescence is a causative factor for reduced mandibular growth and resulting asymmetry of the mandible. The aim of this study was to examine the nature of mandibular growth after unilateral condylectomy and to elucidate the effects of mandibular advancement. Sixty growing mice were subjected to unilateral condylectomy, and then one-half of them underwent treatment with a functional appliance. After 4 wks, a unilateral condylectomy produced reduced growth of the mandible and a subsequent lateral shift to the affected side. However, reduced growth and a lateral shift of the mandible were eliminated by a functional appliance, and prominent regeneration of the condyle was also demonstrated. It was shown that mandibular advancement provides for the regeneration of cartilaginous tissues on injured condyles and recovery of reduced mandibular growth, leading to correction of the lateral shift of the mandible.

Correction of severe open bite using miniscrew anchorage.

This report describes the treatment of a case of severe open bite with posterior crossbite. While treating open bite, the outcome may not always be successful with orthodontic therapy alone. In such cases, surgical therapy is often chosen to gain a stable occlusion. Skeletal anchorage systems such as miniscrews are now frequently used for correcting severe malocclusion. In this report, we treated an open bite by intruding the molars with miniscrews placed bilaterally in the interdental space between both the upper and lower posterior teeth. The active treatment period was 36 months and the patient's teeth continued to be stable after a retention period of 36 months.

Influence of sex hormone disturbances on the internal structure of the mandible in newborn mice.

It has not yet been clarified how sex hormones affect craniofacial bone development immediately after birth. The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial bone development immediately after birth, in terms of the internal structure of the mandible in newborn mice with orchiectomy (ORX) and ovariectomy (OVX). ORX, OVX and a sham-operation were performed on 40 five-day-old C57BL/6J mice. Eight weeks after surgery, each mandible was subjected to histomorphometric analysis of trabecular (Tr) and cortical (Ct) bone mineral density (BMD) by peripheral quantitative computed tomography (pQCT). In the experimental groups, a significant reduction in BMD was found in comparison with the control groups. In histomorphometric analysis, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the condyle and the thickness of the condylar cartilage layer was significantly greater in the experimental mice than in the controls. Trabecular bone volume of the condyle measured on azocarmine-aniline blue (AZAN) sections was significantly less in the experimental mice than in the controls. These results indicate that mandibular growth is inhibited by sex hormone disturbances and the relevant internal structures changed. The findings show that sex hormones are one of the key determinants of mandibular growth and development immediately after birth.

Amyloid beta protein deposition in osteopetrotic (op/op) mice is reduced by injections of macrophage colony stimulating factor.

The deposition of amyloid beta (Abeta) protein is a neuropathological change that characterizes Alzheimer's disease. Animals with the osteopetrosis (op/op) mutation suffer from a general skeletal sclerosis, a significantly reduced number of macrophages and osteoclasts in various tissues, and have no systemic macrophage colony stimulating factor (M-CSF). This study examined the effect that M-CSF injections had on Abeta deposition and microglial cell distribution in the brains of normal and op/op mice. Abeta-positive plaques were detected in the cerebral cortex of op/op mice, but not in normal mice. M-CSF reduced the numbers of Abeta-positive plaques in op/op mice. The microglial cell population was reduced in op/op mice compared with normal mice, and M-CSF increased the numbers to 65.8% of that observed in normal mice. Our results suggest that a clearer understanding of the role that microglial cells play in Abeta deposition may help determine the mechanisms involved in the pathogenesis of Alzheimer's disease.

Effects of cyclic tensile forces on the expression of vascular endothelial growth factor (VEGF) and macrophage-colony-stimulating factor (M-CSF) in murine osteoblastic MC3T3-E1 cells.

It has been reported that vascular endothelial growth factor (VEGF), expressed by osteoblasts, can induce osteoclast recruitment and thus affects bone remodeling. The purpose of this study was to investigate the effects of cyclic tensile forces on the expression of VEGF and macrophage-colony-stimulating factor (M-CSF) in osteoblastic MC3T3-E1 cells. VEGF and M-CSF gene expression and protein concentration were determined by real-time PCR and enzyme-linked immunoassay. The expression of VEGF and M-CSF mRNA in the experimental group was higher than in the control group. The increase in the concentration of VEGF and M-CSF protein in the experimental group was time-dependent. Moreover, gadolinium (an S-A channel inhibitor), but not nifedipine (L-Type Ca2+ channel blocker), treatment reduced the concentration of VEGF and M-CSF mRNA and protein in the experimental groups. These findings suggest that cyclic tensile forces increase the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells via a stretch-activated channel (S-A channel).

Effects of sex hormone disturbances on craniofacial growth in newborn mice.

It is well-known that sex hormones influence bone metabolism. However, it remains unclear as to how sex hormones affect bone growth in newborn mice. In this study, we performed orchiectomy (ORX) and ovariectomy (OVX) on newborn mice, and examined the effects on craniofacial growth morphometrically. ORX and OVX were performed on five-day-old C57BL/6J mice. Four weeks after surgery, lateral cephalograms were taken of all of the mice, with the use of a rat and mouse cephalometer. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer. Inhibition of craniofacial growth was found in the experimental groups but not in the sham-operated groups. In the nasomaxillary bone and mandible, the amount of growth was significantly reduced. These results suggest that craniofacial growth is inhibited by sex hormone disturbances not only in puberty but also immediately after birth.

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.

The Progression of Cavitation in Earlywood Vessels of Fraxinus mandshurica var japonica during Freezing and Thawing.

For an examination of the progression of cavitation in large-diameter earlywood vessels of a deciduous ring-porous tree, potted saplings of Fraxinus mandshurica var japonica Maxim. were frozen and then thawed. The changes in the amount and distribution of water in the lumina of the current year's earlywood vessels during the course of the freezing and thawing were visualized by cryo-scanning electron microscopy. When samples were frozen, most of the current year's earlywood vessels were filled with water. After the subsequent thawing, the percentage of cavitated current-year earlywood vessels gradually increased with time. All of the current year's earlywood vessels were cavitated within 24 h, and only limited amounts of water remained in the lumina of earlywood vessels. Similar cavitation of earlywood vessels was observed after thawing of frozen, excised stem pieces. In contrast, many vessels of the current year's latewood retained water in the lumina during freezing and thawing. These observations indicate that the cavitation of the current year's earlywood vessels is not produced during freezing but progresses during rewarming after freezing in F. mandshurica var japonica.

Seasonal changes in the freezing behavior of xylem ray parenchyma cells in four boreal hardwood species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. Copyright 1999 Academic Press.

Visualization of cavitated vessels in winter and refilled vessels in spring in diffuse-porous trees by cryo-scanning electron microscopy

Xylem cavitation in winter and recovery from cavitation in the spring were visualized in two species of diffuse-porous trees, Betula platyphylla var. japonica Hara and Salix sachalinensis Fr. Schm., by cryo-scanning electron microscopy after freeze-fixation of living twigs. Water in the vessel lumina of the outer three annual rings of twigs of B. platyphylla var. japonica and of S. sachalinensis gradually disappeared during the period from January to March, an indication that cavitation occurs gradually in these species during the winter. In April, when no leaves had yet expanded, the lumina of most of the vessels of both species were filled with water. Many vessel lumina in twigs of both species were filled with water during the period from the subsequent growth season to the beginning of the next winter. These observations indicate that recovery in spring occurs before the onset of transpiration and that water transport through twigs occurs during the subsequent growing season. We found, moreover, that vessels repeat an annual cycle of winter cavitation and spring recovery from cavitation for several years until irreversible cavitation occurs.