Rat Alveolar Bone Research Articles

Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0–200 ng ml−1 of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P. gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.

Oral Administration of Vitamin C Prevents Alveolar Bone Resorption Induced by High Dietary Cholesterol in Rats

A high-cholesterol diet stimulates alveolar bone resorption, which may be induced via tissue oxidative damage. Vitamin C reduces tissue oxidative damage by neutralizing free radicals and scavenging hydroxyl radicals, and its antioxidant effect may offer the clinical benefit of preventing alveolar bone resorption in cases of hyperlipidemia. We examined whether vitamin C could suppress alveolar bone resorption in rats fed a high-cholesterol diet. In this 12-week study, rats were divided into four groups: a control group (fed a regular diet) and three experimental groups (fed a high-cholesterol diet supplemented with 0, 1, or 2 g/l vitamin C). Vitamin C was provided by adding it to the drinking water. The bone mineral density of the alveolar bone was analyzed by microcomputerized tomography. As an index of tissue oxidative damage, the 8-hydroxydeoxyguanosine level in the periodontal tissue was determined using a competitive enzyme-linked immunosorbent assay. Hyperlipidemia, induced by a high-cholesterol diet, decreased rat alveolar bone density and increased the number of tartrate-resistant acid phosphatase-positive osteoclasts. The expression of 8-hydroxydeoxyguanosine was upregulated in the periodontal tissues. Intake of vitamin C reduced the effect of a high-cholesterol diet on alveolar bone density and osteoclast differentiation and decreased periodontal 8-hydroxydeoxyguanosine expression. In the rat model, vitamin C suppressed alveolar bone resorption, induced by high dietary cholesterol, by decreasing the oxidative damage of periodontal tissue.

Inorganic Polyphosphate: a Possible Stimulant of Bone Formation

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

The quantitative analysis of rat alveolar bone regeneration and the regenerative activities of Emdogain(R)

The quantitative analysis of rat alveolar bone regeneration and the regenerative activities of Emdogain^(R)

エムドゲインゲル(R)による歯槽骨再生誘導活性の検討

エムドゲインゲル(R)による歯槽骨再生誘導活性の検討

The Effect of Cyclosporin A on Alveolar Bone in Rats Subjected to Experimental Periodontal Disease

Cyclosporine A (CsA), broadly used in organ transplantation, may contribute to pathogenesis of osteoporosis. The aim of this study was to investigate the effects of CsA on alveolar bone in rats subjected or not to experimental periodontal disease using biochemical, radiographic, and histometric analysis. Forty Wistar rats were divided into 4 equal groups: Group I (Control), Group II (CsA was injected subcutaneously in a daily dose of 10 mg/kg), Group III (Ligature was placed around the mandibular molars), Group IV (Ligature+CsA). After 60 days, rats were decapitated, serum alkaline phosphatase and calcium levels were measured. Radiographic-alveolar bone loss (ABL), histometric-ABL, and percentage of new alveolar bone formation (NABF%) were determined on mandibular molars. Significant increase in serum alkaline phosphatase levels (p < 0.001), no significant difference in calcium levels were observed (p > 0.05) in Group IV compared to Group III. Radiographic and histometric-ABL were significantly less (p < 0.001), NABF% was significantly greater (p < 0.05) in Group IV than in Group III. No significant difference in any of the parameters between Group II and Group I was found. It can be concluded that in the presence of periodontal disease, CsA treatment may bring out an imbalance in the alveolar bone homeostasis by decreasing resorption and stimulating formation of alveolar bone in rats.

Histological and histomorphometrical changes in rat alveolar bone following antagonistic tooth extraction and/or ovariectomy

To examine changes appearing in the alveolar bone following the removal of the mechanical stress of occlusal loading, as well as the added influence of estrogen deficiency on such changes. The right mandibular molars of female rats were extracted. After 8 weeks, 12 animals were ovariectomized (OVX), and the other 12 were subjected to sham surgery (sham). Four weeks after surgery, all rats were sacrificed. The left-half and right-half maxillas of the sham group (the sham-occluded side and the sham-extruded side, respectively) and right-half maxilla of OVX group (the OVX-extruded side) were examined by histological observation and bone histomorphometry. The vertical height of alveolar bone in the sham-extruded and the OVX-extruded sides increased as compared with that of the sham-occluded side. In both extruded sides, active bone formation occurred on the surface of the alveolar bone facing the periodontal ligament, but the bone marrow was expanded and the bone volume had decreased in the internal area of the alveolar bone. In the OVX-extruded side, the bone marrow expanded more remarkably than that of the sham-extruded side, and the highest percentage of osteoclast surface was detected. Around the extruded teeth, there were regional differences in bone dynamics between the internal area of the alveolar bone and the bone surface facing the periodontal ligament, and estrogen deficiency seems to have caused further loss of bone volume in the interior of the alveolar bone supporting the extruded tooth.

Evaluation of the Tissue Response to MTA and MBPC: Microscopic Analysis of Implants in Alveolar Bone of Rats

The aim of this study was to evaluate and compare the quantitative and qualitative inflammatory responses and bone formation potential after implantation of polyethylene tubes filled with a new calcium hydroxide containing sealer (MBPc) and ProRoot mineral trioxide aggregate (MTA). There were 48 Wistar rats divided in three groups: Group I (control group) empty polyethylene tubes were implanted in the extraction site; group II and III, polyethylene tubes were implanted filled with ProRoot mineral trioxide aggregate (MTA) and MBPc, respectively. At 7, 15, and 30 days after tube implantation, the animals were killed, the hemi-maxillas were removed and prepared to light microscopic analyses. The scores obtained were submitted to Kruskal-Wallis statistical test (p < 0.05). Significant differences between the materials were not observed. The results showed that both materials had similar biological response.

The Effect of Fluoridation and Its Discontinuation on Fluoride Profiles in the Alveolar Bone of Rat

We investigated the effect of fluoridation and its discontinuation on fluoride content in the alveolar portion of the mandible in rats. Drinking water with three different fluoride contents (0, 50, 100 ppmF) was given to rats for three different periods (4, 13 and 25 weeks). Fluoride concentrations were measured in the crest, the middle, and the apical parts of the alveolar bone and in the body of the mandible. Furthermore, after fluoridated drinking water was given to rats for 4 or 13 weeks, distilled water was given to them for 21 or 12 weeks respectively; and the effect of the discontinuation on fluoride profiles was investigated. Layer samples were analyzed by abrasive microsampling. Fluoride and phosphorus concentrations were determined by ion-specific electrode and colorimetric procedures, respectively. There was an increase in fluoride concentrations in the mandible in proportion to the fluoride content in the drinking water and the duration of fluoridation. After fluoridation was discontinued, fluoride concentrations in the surface layers of the mandible presented a decrease. Among the four different parts of the mandible, the upper part of the alveolar bone and the alveolar crest part presented the highest rates of reduction. The relative reduction rate of fluoride concentration was closely related to the duration of discontinuation. The alveolar crest was affected most by the discontinuation of fluoridation, presenting the greatest reduction.

Influence of Occlusal Stimuli on the Remodelling of Alveolar Bone in a Rat Hypofunction-Recovery Model

Remodelling of alveolar bone is a crucial factor determining tooth movement against orthodontic forces. Occlusion is the most influential stimulus causing physiological remodelling of alveolar bone. A removable metal appliance was devised, which can sequentially induce hypofunctional occlusion at molar teeth and reestablish normal occlusion. Using this appliance, remodelling of the alveolar bone was examined by histological morphometric analyses, with particular attention to localized regions of bone resorption by identifying the receptor activator of NF-κB ligand (RANKL), a growth factor inducing osteoclastogenesis, and osteoclasts. In the occlusal hypofunction period, the number of TRAP-positive cells increased at the margin of marrow spaces, and the marrow space enlarged. RANKL-positive osteoblasts also increased, especially around the blood vessels. In bone morphometric parameters, BV/TV, Tb. Th and Tb. N decreased, and Tb. Sp and Oc. N increased. These results are consistent with active bone remodelling consisting of active resorption at the inner alveolar bone and bone formation on the outer alveolar bone surface. In the 3 days recovery group, many TRAP-positive cells were observed at the inner alveolar bone and around the bone surface. RANKL-positive osteoblasts were observed at the margin of the marrow space in alveolar bone. However, BV/TV, Tb. Th, and Tb. Sp returned to the control level in the 7 days recovery group, with decreased TRAP-positive cells and RANKL-positive osteoblasts. These findings suggest that occlusal stimuli play important roles in the maintenance of functional alveolar structure and the regulation of alveolar bone in rats.

Immunohistochemical and Electron Microscopic Study of the Biodegradation Processes of Chitin and Chitosan Implanted in Rat Alveolar Bone

Immunohistochemical and Electron Microscopic Study of the Biodegradation Processes of Chitin and Chitosan Implanted in Rat Alveolar Bone

Identification of genes differentially regulated in rat alveolar bone wound healing by subtractive hybridization.

Periodontal healing requires the participation of regulatory molecules, cells, and scaffold or matrix. Here, we hypothesized that a certain set of genes is expressed in alveolar bone wound healing. Reciprocal subtraction gave 400 clones from the injured alveolar bone of Wistar rats. Identification of 34 genes and analysis of their expression in injured tissue revealed several clusters of unique gene regulation patterns, including the up-regulation at 1 wk of cytochrome c oxidase regulating electron transfer and energy metabolism, presumably occurring at the site of inflammation; up-regulation at 2.5 wks of pro-alpha-2 type I collagen involving the formation of a connective tissue structure; and up-regulation at 1 and 2 wks and down-regulation at 2.5 and 4 wks of ubiquitin carboxyl-terminal hydrolase l3 involving cell cycle, DNA repair, and stress response. The differential expression of genes may be associated with the processes of inflammation, wound contraction, and formation of a connective tissue structure.

Influence of age on combined effects of cyclosporin and nifedipine on rat alveolar bone.

There is some evidence showing that cyclosporin A (CsA) and nifedipine (NIF) affect bone metabolism. The purpose of this work was to study the effects of CsA and NIF, given alone or concurrently, on alveolar bone of rats of different ages. Rats 15, 30, 60, and 90 days old were treated daily with 10 mg/kg body weight of CsA subcutaneously injected and/or 50 mg/kg body weight of NIF/day given orally for 60 days. Alveolar bone of the first lower molars was morphologically and stereologically evaluated in serial 5 microm bucco-lingual paraffin sections, stained with hematoxylin and eosin. Serum calcium and alkaline phosphatase levels were measured in all animals at the end of the experimental period. Rats treated with CsA or NIF alone or CsA and NIF concurrently showed decreased alveolar bone density. CsA was more effective than NIF. A significant decrease in serum calcium was found only in animals treated with CsA or CsA/NIF. The results were similar regardless of age. These results indicate that the decrease in the alveolar bone volume in rats caused by CsA and NIF alone or concurrently is not age dependent. Furthermore, NIF (50 mg/kg) did not further increase the loss of alveolar bone volume induced by CsA (10 mg/kg).

Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction

Objective: To understand bone regeneration process after tooth extraction could be a clue to develop a new strategy for alveolar bone reconstruction. Recently, accumulated evidences support that connective tissue growth factor (CTGF) is implicated in tissue repair of many tissues. In this study, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets. Design: Five weeks old wild type male rats (weighing 120 g) were used for this experiment. Expression of CTGF was determined by immunohistochemistry and in situ hybridization in the rat upper molar tooth extraction sockets at 2, 4, 7, 10 and 14 days after tooth extraction. Results: CTGF was expressed strongly in the endothelial cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. During the reparative process, no apparent chondrocyte-like cell appeared in the sockets, while osteoblast-like cells proliferated in the sockets with low CTGF expression at 7, 10, 14 days after extraction. As expected, no staining was observed with the preimmune rabbit IgG and CTGF sense probe. CTGF may play an important role in angiogenesis and granulation tissue formation specifically at early healing stage after tooth extraction to initiate alveolar bone repair. Conclusion: CTGF was expressed at early healing stage of the rat tooth extraction wound.

Effects of ovariectomy on trabecular structures of rat alveolar bone

To clarify the relationship between estrogen deficiency and tooth loss, we analyzed the trabecular structural changes of mandibular alveolar bone in ovariectomized rats. Two months after ovariectomy, the bone resorptive activity remarkably accelerated and caused high bone turnover. On the other hand, one year after ovariectomy, bone loss and trabecular fragmentation occurred in the alveolar bone, although the accelerated degree of bone resorptive activity was moderate. If elderly women could experience similar changes on their alveolar bone, we must pay more attention to the bone resorption presently after menopause and the trabecular fragility long after menopause.

Long-term changes in trabecular structure of aged rat alveolar bone after ovariectomy.

Trabecular structural changes in the jaw after long-term estrogen deficiency are not well understood. Therefore, we sought to observe the changes in rat alveolar bone for 1 year. Six-month-old female rats were ovariectomized (OVX) or underwent a sham operation. After 1 year, bone histomorphometry and a node-strut analysis were performed on the interradicular septum of the mandibular first molar by using micro computed tomography and confocal laser scanning microscopy. Statistical analysis was carried out by using analysis of variance. The alveolar trabeculae of rats in the sham group had network structures, whereas the trabeculae of rats in the OVX group became fragmented. The trabecular bone volume, number, and thickness in the OVX group were significantly lower than those found in the sham group, and the trabecular separation was 4-fold higher in the OVX group than in the sham group. Bone resorptive and formative activity appeared to be moderately higher in the OVX group than in the sham group, but only the difference in bone formation was of statistical significance. By 1 year after ovariectomy, bone loss and trabecular fragmentation had occurred in the rat mandibular alveolar bone.

Effects of ovariectomy on trabecular structures of rat alveolar bone.

An association between postmenopausal osteoporosis and tooth loss has been proposed. However, histomorphometrical changes in alveolar bone following estrogen deficiency are rarely reported with data on microtrabecular structural changes. To clarify the relationship between estrogen deficiency and tooth loss, we histomorphometrically analyzed the trabecular structural changes of mandibular alveolar bone in ovariectomized rats. Twenty-four adult female Fischer rats were used. Eight rats were sacrificed on day 0 (baseline). The remaining 16 rats were divided into two groups. One group was ovariectomized bilaterally (OVX) and the other group was subjected to sham surgery (Sham). After administration of tetracycline and calcein, the animals were sacrificed 60 days after surgery. Bone histomorphometry, node-strut analysis and measurement of thickness of alveolar bone proper were performed on the interradicular septum of the first molar on the sagittal surface. The trabecular bone volume and trabecular number of the OVX group were significantly lower than those of the baseline and Sham groups. All of the bone resorptive and formative parameters of the OVX group were significantly higher (about one-and-a-half times) than those of the Sham group. Several osteoclasts were seen lining the irregular, eroded surface facing the bone marrow in the OVX group. Furthermore, the OVX group tended to have low microtrabecular stiffness and showed significantly thinner distal alveolar bone proper than in the baseline and Sham groups. In summary, estrogen deficiency caused osteoporotic changes and thin alveolar bone proper in the interradicular septum of rat first molar. This phenomenon might accelerate destruction of alveolar bone and tooth loss, especially in elderly women affected by periodontal disease.

Cell death of osteocytes occurs in rat alveolar bone during experimental tooth movement.

The purpose of this study was to examine the morphological changes in alveolar bone osteocytes on the pressure side during experimental tooth movement, using quantitative evaluation on hematoxylin and eosin-stained sections, the TUNEL method, confocal laser scanning microscopy (CLSM), and transmission electron microscopy. In 8-week-old Wistar rats, the left first molar was forced to move mesially with an average load of 10 g by a nickel-titanium superelastic wire. After 6 hours, nuclear condensation and fragmentation appeared in osteocytes adjacent to the hyalinized periodontal ligament (PDL). These cells showed TUNEL-positive reaction. The number of osteocytes with apoptosis progressively increased up to 1 day. At 1 and 2 days, cytoplasmic and nuclear destruction and distribution within the lacunae occurred and increased up to 4 days. The proportion of necrotic osteocytes and near empty lacunae peaked at 2 and 4 days, respectively. At 7 days, necrotic osteocyte and empty lacunae numbers returned to the level of control bone, probably due to resorption of the alveolar bone containing apoptotic and necrotic osteocytes. Ultrastructually, the osteocytes showed apoptotic morphology at 6 and 12 hours and 1 day; at 2 and 4 days, several osteocytes exhibited characteristics of necrosis and destructive images of the surrounding bone matrix, which resulted in enlargement of the lacunae. The present results demonstrate that osteocytes in alveolar bone adjacent to the hyalinized PDL underwent cell death via apoptosis and "secondary necrosis" during orthodontic tooth movement, which may be associated with the subsequent bone resorption.

Experimental renal failure and iron overload: A histomorphometric study in the alveolar bone of rats

Renal failure is a serious disease of a relatively high incidence, which among other lesions, causes bone alterations. These patients frequently suffer from anemia that is generally treated with iron. Given that iron overload inhibits bone formation, our aim was to evaluate the effect of iron on the interradicular bone of animals with experimental renal failure. Acute renal failure was induced in male rats by 5/6 nephrectomy. The animals were subjected to iron overload in the form of daily intraperitoneal injections of 88 mg/kg body weight of iron-dextran over a period of 16 days. Tetracyclines were injected intraperitoneally 14 days apart to evaluate dynamic parameters of bone. Serum urea and creatinine levels were evaluated immediately before the animals were killed on day 30. Static and dynamic histomorphometric measurements were performed. The data indicate that the iron overload modified the response of the renal failure animals which showed decreased interradicular bone volume and adynamic bone disease, characterized by reduced cell activity. These results should be taken into account when renal failure patients treated with iron must undergo dental treatments that depend on bone forming activity for their success.

Immunodetection of noncollagenous matrix proteins during periodontal tissue regeneration.

The interface between denuded dentin and regenerative periodontal tissue was investigated in a rat alveolar bone defect model using morphological and immunocytochemical approaches. The dentin surface was surgically exposed along the palatal roots of maxillary first molars. At 3 weeks post treatment, animals were perfused and treated regions from decalcified mandibles were embedded in Epon for ultrastructural studies or LR White for post-embedding immunogold labeling. Thin tissue sections were incubated with antibodies against noncollagenous matrix (osteopontin, bone sialoprotein, osteocalcin and fibronectin) and plasma (alpha2HS-glycoprotein and albumin) proteins. While in some cases, regenerative events took place directly on the denuded dentin surface, the interface between the denuded dentin and regenerating periodontal tissue was frequently characterized by the presence of an interfacial zone. This zone sometimes showed an electron-dense, cement line-like, planar accumulation of organic material immunoreactive for osteopontin and bone sialoprotein. Immunolabeling for osteocalcin and alpha2HS-glycoprotein was moderate and diffuse throughout the interfacial zone, whereas labeling with antibodies to albumin and fibronectin resulted in a weak reaction. It is concluded that accumulation of bone sialoprotein and osteopontin is a primary event during the formation of regenerative cementum onto denuded root surfaces.