Cranial suture-like gap and bone regeneration after transplantation of cryopreserved MSCs by use of a programmed freezer with magnetic field in rats.

Mesenchymal stem cells (MSCs) can be used for regeneration of various organs and tissues. A previous study revealed that cryopreserved MSCs, which were frozen by a programmed freezer with a magnetic field (Cells Alive System: CAS) and cryopreserved for 7 days in a -150°C deep freezer, can maintain high survival and proliferation rates while retaining both adipogenic and osteogenic differentiation abilities. The purpose of this study was to examine MSC viability and tissue regenerative ability after long-term cryopreservation using a CAS freezer. MSCs were isolated from rat femora bone marrow and cryopreserved in a -150°C deep freezer (CAS group) or directly cryopreserved in a deep freezer (Direct group). After 3 years, the cells were thawed and the number of viable cells was counted. Cell proliferation was also examined after 14 days in culture. For histological examination, forty 4-week-old Fischer 344 male rats received bone and sagittal suture defects with a diameter of 6.0mm, and MSCs (CAS or Direct group) cryopreserved for 1 year were grafted with membranes. Non-cryopreserved MSCs (Control group) were transplanted to an additional twenty rats. The rats were sacrificed at 4, 8, 16, and 24 weeks after surgery. The parietal bones, including the sagittal suture, were observed under a light microscope and the extent of bone regeneration was measured. Our results indicate that MSCs survival and proliferation rates were significantly higher in the CAS group than in the Direct group. In the Control and CAS groups, a large amount of new bone formation and a suture-like gap was identified 24 weeks after transplantation, whereas only a small amount of new bone formation was observed in the Direct group. These results suggest that the CAS freezer is amenable to long-term cryopreservation of MSCs, which can be applied to the regeneration of various tissues, including bone tissue with suture-like gap formation.

Effects of occlusal hypofunction and its recovery on PDL structure and expression of VEGF and bFGF in rats.

OBJECTIVES: The purpose of this study was to clarify whether occlusal hypofunction and its recovery affect the structure of the periodontal ligament (PDL) and expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rats. MATERIALS AND METHODS: Forty-eight Wistar rats aged 5 weeks were used and randomly divided into three groups: the hypofunctional group (HG), recovery group (RG), and control group (CG). In HG and RG, appliances were attached to the maxillary and mandibular incisors. In HG, appliances were set for 11 weeks. In RG, appliances were set for 7 weeks. Appliances were then removed at 0, 1, 3, 7, 14, and 28 days. Untreated rats served as CG. Histological sections were prepared and immunohistochemically stained for VEGF and bFGF. Three groups were evaluated for PDL area and the number of VEGF and bFGF immunopositive cells in PDL. RESULTS: The number of immunopositive cells and PDL area in CG and RG were significantly larger when compared with HG, and PDL area in RG was similar to that in CG. In the recovery process, PDL area and number of VEGF-positive cells in PDL increased from days 0 to 7 and decreased from days 7 to 28. Conversely, the number of bFGF-positive cells in PDL increased significantly after day 1 and peaked at 28 days. CONCLUSIONS: This study suggests that occlusal stimuli regulate PDL area through expression of VEGF and bFGF in rat PDL. CLINICAL RELEVANCE: Occlusal stimuli are able to regulate the expression of VEGF and bFGF in PDL cells, and these growth factors may lead to alveolar bone remodeling in PDL.

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.

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.

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.

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.

PR3-ANCA-positive crescentic necrotizing glomerulonephritis accompanied by isolated pulmonic valve infective endocarditis, with reference to previous reports of renal pathology.

Patients with infective endocarditis (IE) often have renal complications which may include infarcts, abscesses and glomerulonephritis (GN). Furthermore, it is generally accepted that there is an association between IE and anti-neutrophil cytoplasmic antibody (ANCA). Here, we report the case of a 24-year-old man who developed rapidly progressive GN in the course of IE due to infection with alpha-streptococcus. The initial clinical manifestation of the condition was severe sacroiliitis without fever. Sandwich ELISA showed that the patient was positive for PR3-ANCA at low titer, and the classical complement pathway was also activated. Renal biopsy demonstrated several lesions: focal embolic GN, GN with immune deposits and focal and segmental crescentic necrotizing GN. Treatment with antibiotics and steroids led to eradication of the infection, and resolution of the renal disease was accompanied by immediate disappearance of PR3-ANCA and hypocomplementemia. During a 4-year follow-up period, no recurrence was observed. There have only been 7 case reports of GN associated with IE and PR3-ANCA in which the renal pathology has been described, and the current report is the first to document renal pathology in a patient with isolated pulmonic valve IE and PR3-ANCA. Moreover, this report is the first to show a change in renal biopsy findings in response to treatment. A review of the 7 literature cases and that of our patient showed that none involved pauci-immune GN. Hence, further studies are needed to clarify the prevalence of pauci-immune GN in ANCA-positive IE patients.

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).

Immunotactoid glomerulopathy with microtubular deposits, with reference to the characteristics of Japanese cases.

We present the case of a 69-year-old man with nephrotic syndrome and renal insufficiency, who developed lobular glomerulonephritis. An electron microscopy examination of a renal biopsy showed microtubular structures of 24 nm in diameter in the subendothelial space and the paramesangial area. These deposits were PAS-positive and Congo red-negative, and revealed predominantly positive staining for kappa light chain. There was no evidence of diseases with highly organized glomerular deposits, such as amyloidosis, cryoglobulinemia, systemic lupus erythematosus or paraproteinemia. Therefore, the patient was diagnosed to have immunotactoid glomerulopathy (ITG). During a seven-year course he has not developed any disease known to be associated with organized glomerular immune deposits. Hence, we believe ITG occurred as a primary glomerular disease in this case. We also highlight cases of ITG with microtubular deposits that have been reported in Japan, compare these cases to previous reports, and show that the characteristics of the Japanese cases are male predominance; a high incidence of membranoproliferative glomerulonephritis (MPGN); a low incidence of monoclonal gammopathy and hematological malignancies and a higher incidence of hypocomplementemia.

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.

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 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.

Hypolipemia associated with the wasting condition of rabbits infected with Strongyloides papillosus.

Rabbits develop a wasting condition in the intestinal stage of Strongyloides papillosus infection. Serum inflammatory cytokine and lipid profiles were investigated in five rabbits infected with S. papillosus and five uninfected pair-fed controls to ascertain whether the disease is inflammatory cytokine-mediated cachexia. Tumor necrosis factor alpha (TNF alpha) was detected in one infected animal at Day 7 after infection. Interleukin (IL)-1 was detected in three infected, and one control, animals at Day 28. IL-6 remained unchanged in both the groups. Infected animals developed hypolipemia, including hypotriglyceridemia in the intestinal stage of infection. Control animals lost body weight in the same manner as the infected animals, but had elevated cholesterols and phospholipids with normal triglyceride concentrations. The results suggested that the wasting condition has no association with cachexia induced by TNF alpha. IL-1 or IL-6, and that hepatic function for lipid synthesis is affected during the intestinal stage of S. papillosus infection.

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.

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.