Saburo Kurihara

Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.

BACKGROUND AND OBJECTIVE A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. MATERIAL AND METHODS Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. RESULTS Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. CONCLUSION These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.

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 1α, 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.

Orthodontic Treatment of an Impacted Dilacerated Maxillary Central Incisor Combined with Surgical Exposure and Apicoectomy

The impacted incisor was moved into its proper position with surgical exposure and orthodontic traction. Although apicoectomy was performed during the orthodontic treatment, the incisor showed good stability after the long retention period.

Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors.

Prostaglandin E2 (PGE2) has been proposed to be a potent stimulator of bone resorption. However, PGE2 itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE2 in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE2 receptors EP1, EP2, EP3β, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-κB ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE2 as well as calcitonin caused intracellular Ca2+ influx in osteoclasts. However, PGE2 and 17-phenyltrinol-PGE2 (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE2 further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE2 on bone resorption.

Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices — A simple method for identifying polarized osteoclasts☆

Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayers hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayers hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts.

Immunohistochemical expression of heat shock proteins in the mouse periodontal tissues due to orthodontic mechanical stress

The histopathology of periodontal ligament of the mouse subjected to mechanical stress was studied. Immunohistochemical expressions of HSP27 and pHSP27 were examined. Experimental animals using the maxillary molars of ddY mouse by Waldo method were used in the study. A separator was inserted to induce mechanical stress. After 10 minutes, 20 minutes, 1 hour, 3 hours, 9 hours and 24 hours, the regional tissues were extracted, fixed in 4% paraformaldehyde and 0.05 M phosphate-buffered fixative solution. Paraffin sections were made for immunohistochemistry using HSP27 and p-HSP27. In the control group, the periodontal ligament fibroblasts expressed low HSP27 and p-HSP27. However, in the experimental group, periodontal ligament fibroblasts expressed HSP27 10 minutes after mechanical load application in the tension side. The strongest expression was detected 9 hours after inducing mechanical load. p-HSP27 was also expressed in a time-dependent manner though weaker than HSP27. The findings suggest that HSP27 and p-HSP27 were expressed for the maintenance of homeostasis of periodontal ligament by the activation of periodontal ligament fibroblasts on the tension side. It also suggests that these proteins act as molecular chaperones for osteoblast activation and maintenance of homeostasis.

Histopathological Evaluation of Subcutaneous Tissue Reaction in Mice to a Calcium Hydroxide Paste Developed for Root Canal Fillings

We aimed to evaluate the subcutaneous tissue reaction to a new calcium hydroxide paste for root canal fillings developed by Neo Dental Chemical Products Co., Tokyo, Japan. We injected calcium hydroxide paste and a water-based control subcutaneously into the dorsal area of 6-week-old anaesthetized male ddY mice. The tissue surrounding the injection sites was removed from mice in each group, 2 days, 1 week, 3 weeks and 12 weeks after the injection, and histopathologically examined. We found necrotic and degenerative changes, as well as foreign body reaction or proliferation of granulation tissue in specimens from both groups until 3 weeks after the injection. Some lymphocyte infiltration was observed in the 12-week specimens of the control group only. We conclude that calcium hydroxide paste is safe to use as a root canal filling material.

Histopathological Safety Evaluation of Polyethylene Glycol Applied Subcutaneously in Mice

Polyethylene glycol is used as a nonaqueous solvent for a variety of medical agents. We believe it could also be used as a base component in root canal filling material and aimed to test the histological safety of subcutaneously injected polyethylene glycol. We injected polyethylene glycol subcutaneously into the dorsal area of 30 6-week-old anaesthetized male ddY mice. An equal number of mice were injected with propylene glycol as controls. One week, 3 weeks and 9 weeks after injection, the tissue surrounding the injection site was removed from 10 mice in each group and examined. We found no histopathological changes, degeneration, necrosis, foreign body reactions or granulation tissue proliferation in either group. Some inflammatory cell infiltration was observed in both groups. Based on these results, we believe that polyethylene glycol could be used safely as a base component of biomaterials for internal medical use, such as in root canal filling material.

LPS-stimulated Apert Syndrome Gingival Keratinocytes Show Markedly Suppressed Inflammatory Cytokine Production

Apert syndrome (AS) is an autosomal dominant disorder characterized by craniosynostosis and bony syndactyly from a gain-of-function mutation of the fibroblast growth factor receptor 2 gene (FGFR2) (Ser252Trp and Pro253Arg mutations). However, the effects of FGFR2 mutation on oral conditions are not fully understood. To elucidate the effects of AS-type FGFR2 mutation on periodontal disease, we examined the production of matrix metalloproteinases (MMPs) and inflammatory cytokines. Gingival keratinocytes were obtained from a normal subject and an AS patient with Ser252Trp mutation. Cells were treated with lipopolysaccharide (LPS), and the production of MMPs and inflammatory cytokines was examined. No difference in MMP-2 production and MMP-2 activation was observed between normal and AS keratinocytes treated with LPS. LPS-treated normal keratinocytes produced interleukin (IL)-6 and IL-8 in a dose-dependent manner. However, LPS-treated AS keratinocytes produced only very low levels. LPS-induced cytokine production was not significantly decreased in normal keratinocytes when cells were treated with LPS and FGF-7. A mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059, had no effect on LPS-induced cytokine production in AS keratinocytes. These results suggest that AS-type FGFR2 mutation causes the hypoproduction of inflammatory cytokines independent of the MEK/ERK pathway, and that AS-type FGFR2 mutation is an inhibitory factor for periodontal disease.

Bone Mineral Density in Hemifacial Microsomia

Abstract We aimed to assess whether patients with hemifacial microsomia can be quantitatively identified using bone mineral density information. Mandibular bone mineral density was studied using computer assisted analysis between the nonaffected (r) and the affected (1) sides with an orthopantomograph in a patient with hemifacial microsomia with median mandibular cleft, four patients who suffered from hemifacial microsomia in the left side. Fifty controls without bone diseases were randomly selected. Bone mineral density r/l ratios in the controls ranged from 0.479 to 2.064, those in two patients that were associated with and without median mandibular cleft were higher than those in the controls, with a maximum of 8.622 in a particular male with median mandibular cleft after bone graft, whereas the r/l ratios in the other three cases were similar to the controls. Our findings indicate that the quantitative character in the case with median mandibular cleft reveals a large discrepancy of bone mineral density between the nonaffected and the affected sides. This may suggest a compensatory mechanism for bone hypertrophy from regulated bone mineral density with underdevelopment in hemifacial microsomia.