The shaping effects of three nickel-titanium rotary instruments in simulated S-shaped canals.

The purpose of this study was to compare the shaping effects of three nickel-titanium rotary instruments, ProTaper, K3, and RaCe, with emphasis on canal transportation. Simulated canals with an S-shaped curvature in clear resin blocks were prepared with a torque-control, low-speed engine. Canals were prepared using the crown-down technique to the size of #30. Canal aberrations were assessed by comparing the pre- and postinstrumentation images under a stereomicroscope. ProTaper instruments caused greater widening of canals compared to K3 or RaCe. Furthermore, ProTaper files showed a tendency to ledge or zip formation at the end-point of preparation. These canal aberrations may be caused by ProTaper finishing files, which appear to be less flexible than other files of the same tip-size, because of their greater taper-size. These results suggest that nickel-titanium file systems including less tapered, more flexible instruments, like K3 and RaCe should be used in the apical preparation of canals with a complicated curvature.

In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue.

OBJECTIVES: This study examined the in situ expression of receptor activator of nuclear factor-kappaB ligand (RANKL), receptor activator of nuclear factor-kappaB (RANK), osteoprotegerin, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in the osteoclasts of rat periodontal tissue. BACKGROUND: In periodontal disease, osteoclasts cause resorption of the alveolar bone. The function of osteoclasts is regulated by interaction with periodontal ligament cells (PDLs). Furthermore, various kinds of molecules such as RANKL, RANK, osteoprotegerin, IL-1beta and TNFalpha are known to be related to the osteoclasts differentiation and function. It is therefore important to observe the expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts and PDLs. METHODS: Four-week-old Wistar rats were used. Tooth movement was performed by the Waldo method, and the pathological bone resorption was induced. The demineralized maxillae and mandiblae were embedded with paraffin. In situ hybridization was performed to detect RANKL, RANK, osteoprotegerin, IL-1beta, and TNFalpha mRNAs in osteoclasts and other cells using the specific RNA probes, respectively. RESULTS: Both RANKL and RANK were concomitantly expressed in some osteoclasts. RANKL was also positive in osteoblasts and PDLs. No IL-1beta- and TNFalpha-positive osteoclast was noted. The positive signals of osteoprotegerin were detected in almost all osteoblasts, PDLs and odontoblasts. No osteoprotegerin-positive osteoclasts were observed. The number and the distribution pattern of RANKL- and RANK-expressing osteoclasts changed when orthodontic excessive force was applied to periodontal tissue. In addition, IL-1beta and TNFalpha were shown to be expressed in osteoclasts under pathological status. CONCLUSION: These findings suggest that an autocrine mechanism of RANKL-RANK exists in osteoclast, which is heightened in the pathological conditions. Furthermore, the autocrine mechanism of IL-1beta and TNFalpha is also provided in osteoclast under pathological condition. These autocrine mechanisms therefore seem to regulate the osteoclast function in both physiological and pathological conditions.

In vitro evaluation of the cytocompatibility of a glass-lonomer cement sealer.

The purpose of this study was to evaluate the cytocompatibility of two different types of root canal sealers in cell culture. Human periodontal ligament cells were cultured with set materials from an experimental glass-ionomer cement sealer (KT-308) and a commercially available zinc oxide-eugenol-based sealer (Canals) for 1, 3, and 7 days. Cytotoxic effects were evaluated from the morphological changes under a light microscope. Canals induced severe degenerative alteration of human periodontal ligament cells. In contrast, human periodontal ligament cells adjacent to KT-308 showed normal morphology and growth during the culture period. These results suggest that the glass-ionomer cement sealer, KT-308, is cytocompatible and has good potential as a root canal sealer.

Identification of dexamethasone-dependent osteoprogenitors in cell populations derived from adult human female bone.

The purpose of this investigation was to establish whether or not dexamethasone (Dex)-dependent osteoprogenitors with sufficient proliferative capacity to form a colony of bone-forming osteoblasts could be identified in cell populations isolated from adult human bone. This question is relevant because of the ongoing controversy regarding the effects of dexamethasone on bone formation in humans, the clearly different effects of dexamethasone on osteoprogenitor differentiation in mouse vs. rat bone cell populations, and the related question of whether observations in either rat or mouse systems are applicable to human systems. To answer the question, we isolated cell populations from distal femoral cancellous bone of 8 female patients with osteoarthritis and quantitated the number of Dex-dependent osteoprogenitors in these populations by counting the number of osteoblastic colonies forming bone (bone nodules) or unmineralized bone matrix (osteoid nodules). Dex increased alkaline phosphatase (AP) content in all populations, induced bone nodule formation in 2 of the 8 populations, and induced formation of AP-positive clusters of cells with osteoblastic morphology in one. Treatment with 1,25-dihydroxyvitamin D3 increased osteocalcin (OC) production in the nodule forming populations, but not in the non-nodule-forming populations. Our results thus establish that Dex-dependent osteoprogenitors with sufficient proliferative capacity to form bone or osteoid nodules are present in cell populations derived from adult human bone. They also show that frozen primary human bone cell populations that have been characterized previously in terms of the number of Dex-dependent osteoprogenitors present can be used to further study the characteristics of such progenitors.

[An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2000].

An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho in 2000. The results obtained were as follows. 1) 63 patients out of 69 patients with Yusho, who were measured periodontal pocket depth according to Ramfjord' methods, had at least one tooth with periodontal pocket deeper than 3 mm. Similarly, 188 teeth out of a total 285 examined teeth showed periodontal pocket with more than 3 mm depth. 2) In this examination, intraoral sinus tracts stoma were observed in 9 patients out of 70 patients. Radiographic examination and probing examination of pocket depth indicated that periapical lesions were involved in these intraoral sinus tract formation. 3) Oral pigmentation was observed in 46 out of 76 patients with Yusho. In this study, gingival pigmentation was most predominant among oral pigmentation. These results indicated that PCBs had yet affected the mechanism of oral pigmentation and metabolism of alveolar bone.

Extensive subdural empyema treated with drainage and barbiturate therapy under intracranial pressure monitoring: case report.

In subdural empyema (SDE), if the mass effect and vasogenic edema are not controlled, the brain can be fatally damaged. Massive SDE over the skull base often requires repeated surgical drainage for removal of accumulated pus. Intracranial pressure (ICP) management until obliteration of the empyema is important to the improvement of clinical outcome. An 18-year-old man was admitted to our center in a nearly comatose state and with a mild fever. CT scan showed massive SDE extending to the skull base and parafalx. ICP was measured with a pressure transducer through an intraventricle tube. Repeated surgical drainage was performed while ICP was controlled with barbiturate therapy. He was discharged with no neurological deficits. In patients with an extensive SDE over the cerebral hemisphere, ICP control with barbiturate therapy may enhance the therapeutic effect of surgical drainage.

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts.

Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts.

Increased expression of cathepsins E and D in neurons of the aged rat brain and their colocalization with lipofuscin and carboxy-terminal fragments of Alzheimer amyloid precursor protein.

Age-related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Nonlysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age. Western blot analyses revealed that CE existed predominantly as the mature enzyme at 2 and 17 months of age, whereas it was present as not only the mature enzyme but also the proenzyme at 30 months of age. On the other hand, CD was mainly present in the mature form throughout development, although its level in these tissues was also significantly increased with aging. The CE-positive cortical and brainstem neurons of the aged rat corresponded well with cells emitting autofluorescence for lipopigments. By the double-staining technique, most of the CE-positive cortical and brainstem neurons of the aged rat were also positive for antibody to the carboxyl-terminal fragments of amyloid precursor protein (APP634-695), intracellular accumulation of which is thought to be associated with age-related changes in the endosome/lysosome system. It is important that electron microscopy revealed that CE in brainstem neurons of the aged rat colocalized with CD in the lipofuscin-containing lysosomes. These results indicate that aging results in the increased expression and lysosomal localization of CE in cortical and brainstem neurons and changes in the endosomal/lysosomal proteolytic system, which may be related to lipofuscinogenesis and altered intracellular APP metabolism.

In vitro interaction between tetracalcium phosphate-based cement and calvarial osteogenic cells.

Newly-developed tetracalcium phosphate-based cement (4CP cement) and cells derived from neonatal rat calvaria were cocultured to study the in vitro reaction of osteoblastic cells to the biomaterial at light and electron microscopic levels. Three-dimensional nodular structures covered with active osteoblastic cells were formed in the periphery of the test material and they contained a mineralized tissue that exhibited features closely resembling bone formed in vivo. Ultrastructurally, the test material was circumscribed with an electron-dense structure, and was immediately adjacent to elongated cyoplasmic processes with intact morphology or collagen fibrils with periodic structures. Furthermore, the mineralization of the extracellular collagenous matrix occurred directly on the surface of the material. These in vitro findings suggest the ability of 4CP cement to bind directly with newly-formed hard tissues.

Effects of a combination of an antibacterial agent (ofloxacin) and a collagenase inhibitor (FN-439) on the healing of rat periapical lesions.

To investigate the effects of a combination of an antibacterial agent (ofloxacin) and a collagenase inhibitor (FN-439) in the root canal treatment of apical periodontitis, we studied the healing process of experimentally induced periapical lesions in rats by using immunohistochemical methods. With a topical application of a combination of ofloxacin and FN-439 following experimentally induced periapical lesions, both neutrophils and macrophages became significantly decreased in number, while active cementogenesis and extensive bone formation were seen in the periapical region. However, the use of ofloxacin alone also demonstrated a beneficial effect on periapical inflammation and healing. Therefore, it is suggested that ofloxacin is powerful against bacterial infection whether FN-439 is added. The only observed effect of a combination of ofloxacin and FN-439 is that it may more effectively inhibit osteoclastic bone resorption and activate the remodeling of the apical periodontal tissue if this combined medicament is used in a stage of active bone destruction characterized by high production of tissue collagenase.

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts.

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.

Histologic evaluation of tetracalcium phosphate-based cement as a direct pulp-capping agent.

Histologic healing processes were observed at 1, 3, 7, and 10 days after application with either tetracalcium phosphate cement or calcium hydroxide cement to the exposed pulp of the rat maxillary incisors. In teeth applied with calcium hydroxide cement, necrotic tissue was present beneath the cement before new hard tissue formed. In contrast, tetracalcium phosphate cement elicited a dentine bridge formation with no evidence of either intervening tissue necrosis or marked inflammation. Furthermore on ultrastructural examination the newly formed hard tissue was in direct contact with the material. This study suggests that 4CP cement possesses a biocompatible property, which indicates its potential for use as a direct pulp-capping agent.

Biocompatibility of tetracalcium phosphate cement when used as a bone substitute.

We evaluated the biocompatibility of tetracalcium phosphate (4CP) cement, made of 4CP powder and 40 wt% copolymer of polyacrylic acid/itaconic acid and 10 wt% citric acid solution. Light and electron microscopic characteristics were studied 3, 10 and 30 d after implantation. Neither inflammation nor foreign-body giant cell reaction was observed in the tissue adjacent to the implanted material. After 30 d, this material was surrounded with newly formed bone. Ultrastructural examination showed that osteogenesis occurred directly on the surface of the material. These findings suggest that this 4CP cement is biocompatible and possesses osteoconductive properties.

Bone-like nodules formed in vitro by rat periodontal ligament cells.

The periodontal ligament has been shown to possess the ability to regenerate both new cementum and alveolar bone as well as a self-regenerative capacity; however, the source of cementoblasts and osteoblasts is not still clear. We investigated the development of bone-like tissue in vitro by periodontal ligament cells, in order to determine whether the periodontal ligament contains osteoprogenitor cells. Periodontal ligament cells were obtained from periodontal ligament tissue attached to the maxillary incisors of 6-week-old WKA rats by means of the explant technique. Cells at passage #3 were cultured for long term in alpha-minimum essential medium containing 10% fetal bovine serum, antibiotics, and 50 micrograms/ml ascorbic acid, and were then examined using phase-contrast microscopy, histochemistry, transmission electron microscopy, X-ray microanalysis, and electron diffraction. Nodules were formed in the cultures, and when 10 mM Na-beta-glycerophosphate was added, these nodules became mineralized. The mineralized nodules were identified as bone-like elements in view of the presence of osteoblast-like and osteocyte-like cells, collagenous matrix, a mineral composed of hydroxyapatite, and intense alkaline phosphatase activity. The results show that the periodontal ligament contains osteoprogenitor cells, which differentiate into osteoblasts and produce bone-like tissue.