Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo.

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF( E6-EGFP ) cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF( E6-EGFP ) cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF( E6-EGFP ) cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF( E6-EGFP ) cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF( E6-EGFP ) cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies.

Clinical and radiographic evaluation of root-canal obturation with obtura II.

This study evaluated clinical and radiographic healing of 236 root-canal treatments in 131 cases obturated with the Obtura II system. One operator performed all canal preparation and obturation with sealer. A standardized apical-coronal preparation technique instrumented all canals. Clinical symptoms, periodontal condition, and radiographic findings were evaluated at 3, 6, and 12 months. Radiographs taken immediately postobturation were compared to recall radiographs. The level of the final root filling was classified as short (more than 2 mm short of the apex), flush (within 2 mm), or over (beyond) in 12.7%, 81.4%, and 5.9% of cases, respectively. More than 96% of cases were treated successfully by the Obtura II system. Where roots were filled flush, over, or short, lesions healed in 97%, 93%, and 93% of cases, respectively, with no significant differences (p < 0.05). Root filling excess had no impact on the healing process.

Cementum matrix formation in vivo by cultured dental follicle cells.

Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.

Enhanced hydrolytic stability of dental composites by use of fluoroalkyltrimethoxysilanes.

The hydrolytic stability of a group of experimental composite materials was evaluated. Seven distinct composites were formed by the mixing of a resin monomer mixture with silica filler that had been pre-treated with one of 7 different ethanol solutions. In one case, the filler was treated with an ethanol solution that contained only 3-methacryloyloxypropyltrimethoxysilane. In 5 cases, it was treated with solution containing a mixture of 3-methacryloyloxypropyltrimethoxysilane and one of the following hydrophobic fluoroalkyltrimethoxysilanes: trifluoropropyl-, nonafluorohexyl-, tridecafluorooctyl-, heptadecafluorodecyl-, and henicosafluorododecyl-trimethoxysilane. The tensile strength, after being immersed in water for 1800 days, of 2 of the experimental composites, whose pre-treatment regimen had included a fluoroalkyltrimethoxysilane, was significantly higher than that of the composite whose pre-treatment regimen had not included a fluoroalkyltrimethoxysilane. Moreover, there was no significant difference between the tensile strength of fresh samples of these 2 composites and the tensile strength of identically produced samples that had remained under water for 1800 days or that had been subjected to 30,000 cycles of thermal stress.

The role of LFA-1 in osteoclast development induced by co-cultures of mouse bone marrow cells and MC3T3-G2/PA6 cells.

Interactions between leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) influence the development of osteoclasts. However, little is known about how these adhesion molecules are involved in the process of osteoclast development. This study evaluated the role of LFA-1 and its ligands in osteoclast development and bone resorption. Co-cultures of bone marrow cells from LFA-1-deficient mice and MC3T3-G2/PA6 (PA6) cells were cultured in the presence of 1alpha,25(OH)2D3 and dexamethasone for 7 days. The number of TRAP-positive cells that were generated by bone marrow cells from LFA-1-deficient mice was smaller than that generated by bone marrow cells from wild-type mice. In addition, the bone-resorbing activity of osteoclast-like cells that were generated from LFA-1-deficient mice was lower than that generated by osteoclast-like cells from wild-type mice. Immunofluorescence flow cytometry showed that osteoclast stromal PA6 cells expressed the cell adhesion molecules, ICAM-1 and VCAM-1. When monoclonal antibodies to mice VCAM-1, CD11b or CD18 were added separately to the co-culture system, the number of TRAP-positive cells that were generated from LFA-1-deficient mice was 20-30% smaller than that generated from wild-type mice. The formation of TRAP-positive cells from both LFA-1 deficient and wild-type mice was especially inhibited by anti-CD18 antibody, in comparison to the addition of normal IgG serum. These results suggest that LFA-1 adhesion molecules play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. CD18 appears to be a key adhesion molecule in cell-to-cell contacts during the early stage of osteoclast development.

Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis.

Cementum-derived attachment protein (CAP) is a 56 kDa collagenous protein that promotes attachment of mesenchymal cells. Previous studies have shown that the presence of CAP is restricted to cementum in adult human tissues. In this study, we report generation of a monoclonal antibody against CAP and its use for the investigation of CAP in developing bovine tooth germs. Mice were immunized with CAP purified from bovine cementum, and a monoclonal antibody, 3G9, was produced. Immunohistochemical staining of bovine tooth germ at root forming stage using 3G9 antibody showed that the tissue distribution of CAP expression was limited to cementum matrix and cementoblasts during cementogenesis. Alveolar bone did not stain with the 3G9 antibody, whereas anti-type I collagen stained positively. CAP was purified from bovine tooth germs with immunoaffinity purification using the 3G9 antibody. Examination of the immunoaffinity-purified fraction showed that CAP existed in tooth germ as a 65 kDa protein. The protein was susceptible to bacterial collagenase. To investigate the possible biological function of CAP during cementogenesis, we isolated dental follicle cells from the bovine tooth germ, and showed that they adhered to surfaces containing CAP. These data demonstrate that CAP is expressed by bovine cementoblasts as a 65 kDa protein and that the CAP may have a function in cementogenesis.

Matrix metalloproteinase-2 in dentin matrix mineralization.

In the serum-free culture medium of bovine odontoblasts we detected active gelatinolytic metalloproteinases, matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinases A and B). The activity of MMP-2, in particular, appeared suddenly around day 21 in the culture, coinciding with the development of odontoblastic cell processes and the loss of alkaline phosphatase. Reverse transcriptase-polymerase chain reaction analysis of these odontoblasts demonstrated that messages of MMP-2 but not MMP-9 increased significantly between day 15 and day 21. The in vitro observation indicates that medium conditioned by these odontoblasts and containing significant amounts of MMP-2 degrades not only the collagenous substrates but also purified dentin phosphophoryn as well. We have also observed that dephosphorylated dentin phosphoprotein becomes a better substrate for casein kinase II after limited proteolysis with MMP-2. These results support our working hypothesis that MMP-2-mediated proteolytic processing is an important step in accelerating the process of dentin matrix maturation, which includes phosphorylation and subsequent mineralization. As has been suggested previously, extracellular phosphorylation of matrix proteins is an important step in biomineralization both in bone and in dentin (Mikuni-Takagaki et al., J Bone Miner Res 1995;10:231-42; Zhu et al., Biochem J 1997; 323:637-43). Our present histochemical analysis in MMP-2 knockout mice confirms the concept with the delayed formation of mineralized tissues, dentin, and bone.

Autocrine regulation of osteoclast formation and bone resorption by IL-1 alpha and TNF alpha.

Bone resorption is regulated by the cytokines within marrow cells that mediate osteoclast formation and activation. IL-1 and TNF induce bone resorption by stimulating the production of osteoclast-like multinucleated cells and by increasing the bone-resorbing activity of formed osteoclasts. This study was designed to detect IL-1 and TNF in osteoclasts in vitro and to determine whether these cytokines up-regulate osteoclast differentiation and bone resorption. The production of IL-1 alpha, -beta, and TNF alpha, beta in osteoclasts was examined immunohistochemically and by in situ hybridization. In the co-culture of C57BL/6N mouse bone marrow and MC3T3-G2/PA6 cells, a colony of osteoclasts formed, and IL-1 alpha and TNF alpha were detected. However, IL-1 beta and TNF beta were not detected. To investigate the role of IL-1 alpha and TNF alpha from osteoclasts, we enumerated TRAP-positive cells and measured the resorption pit areas in the presence of antibodies against IL-1 alpha and TNF alpha. The addition of antibodies against IL-1 alpha and TNF alpha to the co-culture system decreased the number of TRAP-positive colonies at seven days after incubation (anti-IL-1 alpha, 25.0 +/- 2.3%; anti-TNF alpha, 41.7 +/- 3.7%; anti-IL-1 alpha + anti-TNF alpha, 40.5 +/- 1.3%; and control, 100%), and the ratio of mononuclear to multinuclear cells had changed (anti-IL-1 alpha, 90:10; anti-TNF alpha, 75:25; anti-IL-1 alpha+ anti-TNF alpha, 88:12; and control, 60:40). The total pit areas per dentin slice also decreased with the addition of antibodies (anti-IL-1 alpha, 28,828; anti-TNF alpha, 49,249; anti-IL-1 alpha + anti-TNF alpha, 30,685; and control, 303,139 mm2). These results suggest that local production of IL-1 alpha and TNF alpha by osteoclasts is an important mechanism for regulating the osteoclast differentiation and bone resorptive process.

Dentinal tubule occlusion with lanthanum fluoride and powdered apatite glass ceramics in vitro.

To simulate hypersensitive dentin, the smear layer and dentinal plugs of bovine root dentin specimens were removed by immersion in 10% phosphoric acid, polishing with hydroxyapatite particles, and ultrasonic cleansing. The fluoride-tannic acid-lanthanum-apatite (FTLA) group was treated with acidulated phosphate fluoride (APF) containing tannic acid followed by rubbing with a paste of lanthanum chloride (LaCl3) and powdered apatite glass ceramics. The treated specimens were immersed in a remineralizing solution that mimics saliva for 6 weeks. The SEM observations revealed that the treated surfaces of the FTLA group were completely covered with fine spherical compounds and the dentinal tubules were occluded with plugs to a depth of about 3 microns. Fluoride and lanthanum were detected to a depth of over 20 microns by EPMA observation. After the remineralization, the surface of FTLA-treated specimen did not have any opened tubules and showed a remarkable increase in the number of fine spherical deposits in the dentinal tubules. These results suggest that the reaction products produced by sequential treatment with acidic fluoride and LaCl3 and powdered apatite glass ceramics are able to effectively occlude dentinal tubules.

Synthesis of silane coupling agents containing fluorocarbon chain and applications to dentistry: plaque-controlling surface modifiers.

Silane coupling agents containing a fluorocarbon chain were prepared in high yields. It was found that silanes can be useful modifiers of the surfaces of glass, metals, and resin composites for dental use. The silane coupling agent CF3(CF2)9CH2CH2Si(OCH3)3 was the best modifier of these surfaces in terms of water and oil repellency. Colorants and experimental bacterial plaque detached much more easily from, and adhered less well to, surfaces modified with this silane coupling agent compared with unmodified surfaces. The surfaces of four teeth of a denture were modified with this silane coupling agent by spreading the agent on the surfaces with a small brush followed by brief drying with a hair drier. The modified tooth surfaces of the denture, which was worn for four months in a heavy smoker's oral cavity, were more stain-resistant than the unmodified tooth surfaces. It is expected that silane coupling agents containing a fluorocarbon chain will be surface modifiers for enhancement of oral health.

Extracellular processing of bone and dentin proteins in matrix mineralization.

There are two steps in the process of matrix-mediated bone and dentin mineralization. First, as in other soft tissues, osteoblasts/odontoblasts synthesize collagenous matrices and second, mineral deposits in these matrices at a location distant from the cells that synthesized the matrices. We suggest a sequence of events that lead the matrix to mineralization: the phosphoproteins of bone and dentin are posttranslationally processed by limited proteolysis, then they are extracellularly processed into a more phosphorylated species that, we believe, facilitates mineralization. Our in situ phosphorylation experiments done with [gamma-32P] GTP suggest the existence of extracellular phosphorylation by a casein kinase II (CKII)-like enzyme, the enzyme known to phosphorylate most of the phosphate residues in dentin phosphophoryn and bone sialoproteins (osteopontin and BSP II).

Extracellular processing of dentin matrix protein in the mineralizing odontoblast culture.

Odontoblasts that we prepared from bovine incisors produced a dentin-specific protein, phosphophoryn, and accumulated it in mineralized nodules. The time course of mineralization was detected by measuring osteocalcin and mineral in the nodules. The sequence of developmental expression of proteins in this mineralizing dentin cell culture is very similar to that in bone cells, suggesting a common mechanism for matrix mineralization in bone and dentin. Casein kinase II, which phosphorylates bone phosphoproteins and dentin phosphorylates bone phosphoproteins and dentin phosphophoryn, also emerges coinciding with the initiation of mineralization. Furthermore, we have detected extracellular phosphorylation by casein kinase II of a dentin protein of M(r) 60,000, which we recovered from the phosphophoryn fraction in CaCl2 precipitate.

The fracture toughness characteristics of three dental composite resins.

A fracture mechanics approach has been adopted in the present study to investigate the fracture toughness behavior of three commercial composite resins for dental use named Clearfil photo posterior, P-50 and Occlusin. The resins have been cast in to ring-shaped specimens for the determination of individual fracture toughness values. The experimentally determined values have been compared with fracture toughness values obtained from the indentation micro-fracture method, which is the currently more popular test method for determining fracture toughness characteristics of composite resins. Occlusin was found to exhibit higher fracture toughness values than any other resin, employing the ring specimen test procedure. However, this was not the case when using the indentation method which gave comparable fracture toughness values for all three resins. It was concluded that the ring specimen provides a simple, accurate and reproducible method for providing a measure of the materials fracture toughness.

Protein content and amino-acid content of consolidated carious lesions in human enamel and of experimental lesions in bovine enamel exposed to the human mouth.

Consolidation is a natural defence reaction that results in arrest of enamel caries. Experimental consolidated lesions (ECL) were compared with naturally-consolidated lesions (NCL): ECL were obtained by exposing pre-softened, bovine-enamel slabs to the oral environment in 3 subjects for 2 h, 24 h or 7 days, and NCL were sampled from extracted human teeth with white or yellow spots of arrested caries. Protein content of ECL from 2 subjects were similar to each other and to that of NCL throughout the experimental period. The ECL of the other subject showed a gradual increase in protein content with significantly higher values at day 7. The predominant amino acids in ECL were glutamic acid, proline and alanine, and in NCL, glutamic acid, glycine and alanine. The amino-acid composition of the 7-day ECL was closer to that of NCL than were that of the 2 and 24 h ECL. Intra-oral ageing caused significant reductions in proline and glycine and pronounced increases in aspartic acid, threonine, alanine and leucine. Thus the adsorbed or incorporated organic material in the ECL changed from having components dissimilar to NCL to ones similar to NCL. This intra-oral model might be useful for studies of the organic material incorporated into enamel during the process of consolidation.