Mineral Trioxide Aggregate (MTA) Upregulates the Expression of DMP1 in Direct Pulp Capping in the Rat Molar.

Mineral trioxide aggregate (MTA) is an alternative endodontic material that predicts conductive or inductive calcified tissue formation from immature pulp mesenchymal stem cells (IPMSCs). The purpose of this study was to investigate whether MTA could promote reparative odontoblast differentiation via IPMSCs in the early phase of regeneration and compare with calcium hydroxide (CH). Direct pulp capping using calcium hydroxide (CH), MTA, and MTA with platelet-rich plasma (MTA + PRP) was performed on maxillary first molars of 8-week-old male Wistar rats (n = 36). After 3, 7, or 14 days, the teeth were analyzed for mineral density (MD) and volume of MD (VMD) via micro-focusing computed tomography (µCT), nestin, dentin matrix acidic phosphoprotein 1 (DMP1) immunohistochemistry, and real-time PCR for DMP1 mRNA expression. MTA stimulated the early phase differentiation of the IPMSCs into odontoblasts, with positive results for nestin and DMP1 compared with CH. Moreover, MTA + PRP stimulated calcified granule and dentin bridge formation through calcium mineral deposition, following the induction of DMP1 mRNA expression in IPMSCs. Our results suggested that the combination of MTA and PRP is an effective and clinically applicable method for activating endogenous dental pulp stem cells into odontoblasts in the early stages of pulp regeneration.

Horseradish peroxidase interacts with the cell wall peptidoglycans on oral bacteria.

Salivary peroxidase and myeloperoxidase are known to display antibacterial activity against oral microbes, and previous indications have pointed to the possibility that horseradish peroxidase (HRP) adsorbs onto the membrane of the major oral streptococci, Streptococcus mutans and Streptococcus sanguinis (S. sanguinis). However, the mechanism of interaction between HRP and the bacterial cell wall component is unclear. Dental plaques containing salivary glycoproteins and extracellular microbial products are visualized with 'dental plaque disclosing agent', and are controlled within dental therapy. However, current 'dental plaque disclosing agents' are difficult to evaluate with just dental plaques, since they stain and disclose not only dental plaques but also pellicle formed with salivary glycoproteins on a tooth surface. In this present study, we have demonstrated that HRP interacted with the cell wall component of the major gram-positive bacterial peptidoglycan, but not the major cell wall component of gram-negative bacteria lipopolysaccharide. Furthermore, we observed that the adsorbed HRP labeled with fluorescence was detected on the major oral gram-positive strains S. sanguinis and Streptococcus salivarius (S. salivarius), but not on a gram-negative strain, Escherichia coli (E. coli). Furthermore, we have demonstrated that the combination of HRP and chromogenic substrate clearly disclosed the dental plaques and the biofilm developed by S. sanguinis, S. salivarius and the major gram-postive bacteria Lactobacillus casei on tooth surfaces, and slightly disclosed the biofilm by E. coli. The combination of HRP and chromogenic substrate did not stain either the dental pellicle with the salivary glycoprotein mucin, or naked tooth surfaces. These results have suggested the possibility that the adsorption activity of HRP not only contributes to the evaluation of dental plaque, but that enzymatic activity of HRP may also contribute to improve dental hygiene.

Immunohistochemical and ultrastructural evaluation of matrix components in mandibular condylar cartilage in comparison with growth plate cartilage in cartilage calcification insufficient rats.

Matrix components of growth plate cartilage and mandibular condylar cartilage were immunohistochemically analyzed in cartilage calcification insufficient (CCI) rats, a model for dwarf rats. Reduction in total tibial length, elongation of growth plate, and appearance of noncartilaginous regions in the growth plate were observed in CCI rats. Immunoreactivity for type I collagen and hyaluronic acid (HA) staining were observed in the noncartilaginous region. However, weak immunoreactivity was observed for aggrecan, collagen types II and X, and decorin in this region. Transmission electron microscopy indicated that the noncartilaginous region showed a loose network of thin collagen fibrils, indicating that HA is predominantly involved in capturing space of the noncartilaginous region in the growth plate. Meanwhile, the mandibular condylar cartilage in CCI rats also showed elongation of the cartilaginous region and had a noncartilaginous region, predominantly comprising thick collagen fibrils. The structural difference between the two types of cartilages in CCI rats may be due to the presence of the fibrous cell zone and the fibrocartilaginous nature of the normal condylar cartilage. Additionally, the reduction in mandibular length was relatively less than the reduction in tibial length. The outline of the condylar process showed only slight abnormality. These results suggest that the condylar cartilage compensated its growth by supplying the characteristic noncartilaginous region effectively and may adapt to severe structural changes observed in CCI rats.

Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats.

Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

Pthlh, a promising cancer modifier gene in rat tongue carcinogenesis.

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA x WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.

Regulation of CCN2 gene expression and possible roles in developing tooth germs.

CCN proteins are extracellular and cell-associated molecules involved in several developmental processes, but their expression patterns and regulation in tooth development remain unclear. Here we first determined the expression patterns of CCN genes in mouse tooth germs. We found that at early stages CCN2 was detected in dental lamina, dental mesenchyme, and primary enamel knot, while other CCN family members were expressed broadly. By the bell stage, all members were expressed in differentiating odontoblasts and ameloblasts, but CCN1 and CCN2 transcripts were conspicuous in differentiating osteoblasts in dental follicle. Next, we asked what signalling molecules regulate CCN2 expression and what roles CCN2 may have. We found that upon surgical removal of dental epithelium CCN2 was not longer expressed in dental mesenchyme in cultured bud stage germs. Implantation of beads pre-coated with BMPs and FGFs onto E12-13 mandibular explants induced CCN2 expression in dental mesenchyme. There was a dose-dependent effect of BMP-4 on CCN2 induction; a concentration of 100 ng/µl was able to induce strong CCN2 expression while a minimum concentration of 25 ng/µl was needed to elicit appreciable expression. Importantly, Noggin treatment inhibited endogenous and BMP-induced CCN2 expression, verifying that CCN2 expression in developing tooth germs requires BMP signalling. Lastly, we found that rCCN2 stimulated proliferation in dental mesenchyme in a dose-dependent manner. Together, the data indicate that expression of CCN genes is spatio-temporally regulated in developing tooth germs. CCN2 expression appears to depend on epithelial and mesenchymal-derived signalling factors, and CCN2 can elicit strong proliferation in dental mesenchyme.

Immunohistochemical analysis of osteoconductivity of beta-tricalcium phosphate and carbonate apatite applied in femoral and parietal bone defects in rats.

The feature of osteoconductivity, and expression of inductive BMP and transcription factors (Runx2 and Osterix) for osteoblast differentiation, which was related to conductive bone formation, were observed in experimentally created defects in rat femoral and parietal bones filled with beta-tricalcium phosphate (beta-TCP) or carbonate apatite (CAP). Femoral cortical bone defects were repaired by conductive bone formed by osteoblasts differentiated around beta-TCP and CAP, and immunohistochemical observation revealed that the osteoblasts expressed BMPs, Runx2, and Osterix. However, the repair in parietal bone defects was incomplete despite the beta-TCP and CAP filling. Only cells, which differentiated around beta-TCP or CAP, and formed conductive bone expressed BMPs, Runx2, and Osterix. These findings revealed that the osteoconductivity of calcium phosphate materials required the expression of BMPs as the prerequisite for Runx2 and Osterix expression. Therefore, it is suggested that when calcium phosphate ceramics are used as bone substitute materials, BMPs are essential for osteoconductivity.

Garlic allyl derivatives interact with membrane lipids to modify the membrane fluidity.

As a novel approach to the mode of medicinal action of garlic, its constituents were comparatively studied with respect to their interactions with membrane lipids to modify the membrane fluidity. Allyl derivatives rigidified tumor cell and platelet model membranes consisting of unsaturated phospholipids and cholesterol at 20-500 muM with the potency being diallyl trisulfide (DATS) > diallyl disulfide (DADS) by preferentially acting on the hydrocarbon cores of lipid bilayers. They were also effective in rigidifying candida cell model membranes prepared with ergosterol and phospholipids at 100-500 microM with the potency being DADS > DATS > diallyl sulfide (DAS), but not bacteria cell model membranes without ergosterol. Alliin, a precursor of these DASs, was not active on any membranes at 500 microM. Both relative intensity and selectivity in membrane effects correlated with those in antiproliferative, antiplatelet and antimicrobial effects. In cell culture experiments, membrane-active DASs inhibited the growth of tumor cells cultured for 24 and 48 h at 20-500 muM to show the potency being DATS > DADS, together with rigidifying cell membranes by acting on their deeper regions more intensively. However, membrane-inactive allyl derivatives were not growth-inhibitory on tumor cells. The membrane lipid interactions of DASs appear to be one of possible mechanisms underlying different effects of garlic.

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis.

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Comparison of carbonate apatite and beta-tricalcium phosphate (resorbable calcium phosphates) implanted subcutaneously into the back of rats.

Bioresorption and biocompatibility of carbonate apatites, both sintered and non-sintered (S-CAP and N-CAP), and of sintered beta-tricalcium phosphate (beta-TCP) were compared by implanting particles of these materials into the back of adult rats. Bioresorption--when evaluated non-destructively with non-decalcified tissues using microfocus X-ray tomography--was essentially the same for N-CAP and beta-TCP, while S-CAP exhibited statistically lower bioresorption at 2, 4, and 12 weeks postoperatively. Biocompatibility--when evaluated by ED1 immunostaining--was in the order of beta-TCP > N-CAP > S-CAP. The intensity of ED1 immunostaining decreased with time, but persisted longer in beta-TCP than in S-CAP and N-CAP, indicating that beta-TCP produced the strongest and most enduring stimulation of macrophages. Although no statistical differences were found in tartrate-resistant acid phosphatase (TRAP) staining among the materials at each implantation period, the degree of TRAP staining for S-CAP was statistically greater at 12 weeks than at 2 and 4 weeks, indicating that osteoclast-like cells were in part responsible for the resorption of the carbonate apatite.

Influence of secondary colonizers and human plasma on the adherence of Porphyromonas gingivalis in vitro.

The influence of secondary colonizers (Fusobacterium nucleatum and Actinomyces naeslundii) and the effect of human plasma on the adherence of Porphyromonas gingivalis were investigated. Hydroxyapatite (HAP) discs coated with Streptococcus sanguis were immersed in a 3H-labeled bacterial cell suspension of F. nucleatum or A. naeslundii and then in a 14C-labeled P. gingivalis cell suspension. Bacterial cells on the discs were pyrolysed to quantify the radioisotopes released. The cell numbers of secondary colonizers on the discs increased with immersion time and this, in turn, resulted in significantly elevated adherence of P. gingivalis. These two secondary colonizers had very similar positive effects on the adherence of P. gingivalis. Human plasma significantly inhibited the adherence of P. gingivalis and secondary colonizers to S. sanguis-coated HAP discs. Adherence of P. gingivalis and A. naeslundii was strongly inhibited by plasma, while that of F. nucleatum was affected the least. Treatment with plasma, after immersion of streptococcal-coated discs in individual cell suspension of secondary colonizers, also reduced subsequent adherence of P. gingivalis. The rate of decrease was much smaller in F. nucleatum. These results indicate that both F. nucleatum and A. naeslundii enhance the adherence of P. gingivalis, and that the former may play a more important role in the establishment of P. gingivalis in dental plaque where plasma-derived components are present.

Membrane-rigidifying effects of anti-cancer dietary factors.

Since several anti-cancer drugs interact with cell membrane lipids, the effects of anti-cancer dietary factors on liposomal membranes with different lipid composition were comparatively studied by measuring fluorescence polarization. Fluidity was imparted on both hydrophobic and hydrophilic regions of lipid bilayers by decreasing cholesterol and increasing unsaturated phosphatidylcholine in membranes. At 0.625-10 microM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-cancer drug, doxorubicin, rigidified the tumor cell model membranes consisting of 20 mol% cholesterol and 80 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 1.0, but not daidzein. They were more effective on the membrane core than the membrane surface. Quercetin showed a biphasic effect on the hydrophobic regions of membrane lipid bilayers to rigidify above 5 microM and fluidize below 2.5 microM. In contrast, anti-cancer dietary factors and doxorubicin were not or much less effective in rigidifying the normal cell model membranes consisting of 40 mol% cholesterol and 60 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 0.5. The membrane-rigidifying effects were greater depending on a decrease of the cholesterol/phosphatidylcholine ratio and an increase of the phosphatidylcholine unsaturation degree. Membrane-active dietary factors and doxorubicin inhibited the growth of mouse myeloma cells at 10-100 microM, while the growth inhibition by membrane-inactive daidzein was relatively weak. Anti-cancer dietary factors appear to act on more fluid membranes like tumor cells as well as doxorubicin to induce rigidification, especially in the hydrocarbon core of membrane lipids, which is determined by the composition of cholesterol and unsaturated phospholipids.