Chitin contributes to the formation of a feeding structure in a predatory nematode.

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P. pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicates a key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

Caffeic Acid Phenethyl Ester (CAPE) Induces VEGF Expression and Production in Rat Odontoblastic Cells.

Caffeic acid phenethyl ester (CAPE), the main component of propolis, has various biological activities including anti-inflammatory effect and wound healing promotion. Odontoblasts located in the outermost layer of dental pulp play crucial roles such as production of growth factors and formation of hard tissue termed reparative dentin in host defense against dental caries. In this study, we investigated the effects of CAPE on the upregulation of vascular endothelial growth factor (VEGF) and calcification activities of odontoblasts, leading to development of novel therapy for dental pulp inflammation caused by dental caries. CAPE significantly induced mRNA expression and production of VEGF in rat clonal odontoblast-like KN-3 cells cultured in normal medium or osteogenic induction medium. CAPE treatment enhanced nuclear factor-kappa B (NF-κB) transcription factor activation, and furthermore, the specific inhibitor of NF-κB significantly reduced VEGF production. The expression of VEGF receptor- (VEGFR-) 2, not VEGFR-1, was up regulated in KN-3 cells treated with CAPE. In addition, VEGF significantly increased mineralization activity in KN-3 cells. These findings suggest that CAPE might be useful as a novel biological material for the dental pulp conservative therapy.

Scaling of cytoskeletal organization with cell size in Drosophila.

Spatially organized macromolecular complexes are essential for cell and tissue function, but the mechanisms that organize micron-scale structures within cells are not well understood. Microtubule-based structures such as mitotic spindles scale with cell size, but less is known about the scaling of actin structures within cells. Actin-rich denticle precursors cover the ventral surface of the Drosophila embryo and larva and provide templates for cuticular structures involved in larval locomotion. Using quantitative imaging and statistical modeling, we demonstrate that denticle number and spacing scale with cell length over a wide range of cell sizes in embryos and larvae. Denticle number and spacing are reduced under space-limited conditions, and both features robustly scale over a 10-fold increase in cell length during larval growth. We show that the relationship between cell length and denticle spacing can be recapitulated by specific mathematical equations in embryos and larvae and that accurate denticle spacing requires an intact microtubule network and the microtubule minus end-binding protein, Patronin. These results identify a novel mechanism of micro-tubule-dependent actin scaling that maintains precise patterns of actin organization during tissue growth.

A biochemical and histopathological evaluation of generalized pulp calcification in young permanent teeth.

Pulp calcifications are a frequent finding on bitewing and periapical radiographs in older age-groups but their occurrence in the entire dentition in young subjects is unusual. We report such an unusual occurrence of generalized pulp calcification in a 13-year-old Indian female. Radiographic examination of the dentition revealed pulp calcifications in all permanent teeth, located mostly in the pulp chamber but with some in the root canals. The patient's dental, medical, and family history was noncontributory. Biochemical analysis of the removed pulp calcification from one of the teeth during endodontic treatment showed large amounts of calcium, phosphorus, and carbonate. However, metabolic evaluation of patient through liver and kidney function tests and other blood investigations did not reveal any metabolic disorder. The patient was also evaluated for any systemic, syndromic, or genetic involvement but this was also noncontributory. Therefore, we propose that this unusual case of generalized pulp calcification is of idiopathic origin. In this work, histopathological and biochemical evaluations of the pulp calcification was done to try and understand the initiation and progress of calcifications in pulpal tissue.

Adaptive calcified matrix response of dental pulp to bacterial invasion is associated with establishment of a network of glial fibrillary acidic protein+/glutamine synthetase+ cells.

We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein(-) cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP(+)/GS(+) cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP(+)/GS(+) interodontoblastic cells. A regulatory role for the networks of GFAP(+)/GS(+) cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response.

Immunohistochemical study of hard tissue formation in the rat pulp cavity after tooth replantation.

While mineralized tissue is formed in the pulp cavity after tooth replantation or transplantation, little is known of this hard tissue formation. Therefore, we conducted histological and immunohistochemical evaluations of hard tissue formed in the pulp of rat maxillary molars after tooth replantation. At 5 days after replantation, degenerated odontoblasts were lining the pulp cavity. At 14 days, dentin- or bone-like tissue was present in the pulp cavity. Immunoreactivity for osteopontin (OPN) and bone sialoprotein (BSP) was strong in the bone-like tissue, but weak in the dentin-like tissue. Conversely, dentin sialoprotein (DSP) was localized in the dentin-like tissue, but not in the bone-like tissue. Cells positive for BMP4, Smad4, Runx2, and Osterix were found around the blood vessels of the root apex at 5 days. At 14 days, these cells were also localized around the bone-like tissue. Cells expressing alpha-smooth muscle actin (SMA) were seen around the newly formed bone-like tissue, whereas no such cells were found around the newly formed dentin-like tissue. In an experiment involving the transplantation of a green fluorescent protein (GFP)-transgenic rat tooth into a wild-type rat tooth socket, GFP-positive cells were detected on the surface of the bone-like tissue and over all dentin-like tissue. These results indicate that the original pulp cells had the ability to differentiate into osteoblast-like cells as well as into odontoblast-like cells.

Hard tissue formation in subcutaneously transplanted rat dental pulp.

While dental pulp appears to be able to form mineralized matrices that do not always resemble dentin, the precise characteristics of the hard tissue and the mechanism of its induction remain unknown. Therefore, we evaluated hard tissue induced by transplantation of pulp into subcutaneous tissue. Seven days after transplantation, initial hard tissue was formed at the inner periphery of the pulp. After 14 days, this hard tissue expanded inwardly. Mineralized matrix was immunopositive for osteocalcin, osteopontin, and bone sialoprotein, but negative for dentin sialoprotein. Transplantation of GFP-labeled pulp into wild-type rats showed these formative cells to have been derived from the transplant. TEM observation revealed apatite crystals within necrotic cells and matrix vesicles at the initial stage of calcification. These results indicate that pulp cells possess the ability to form a bone- or cementum-like matrix. Calcification of the matrix may occur in necrotic cells and matrix vesicles, followed by collagenous calcification.

Stimulatory effects of hydroxyl radical generation by Ga-Al-As laser irradiation on mineralization ability of human dental pulp cells.

The present study was conducted to investigate the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells. HDP cells in vitro were irradiated once with a Ga-AL-As laser at 0.5 W for 500 s and at 1.0 W for 500 s in order to investigate free radicals as one mechanism for transmission of laser photochemical energy to cells. Production of the hydroxyl radical (*OH) was measured using the ESR spin-trapping method and was found to be increased by laser irradiation. The DMPO-OH was not detected in the presence of dimethyl sulfoxide (DMSO), a *OH scavenger. The formation of calcification nodule was also investigated by von Kossa staining. The number of calcified nodules was increased by 1.0 W-laser irradiation. Alkaline phosphatase (ALP) activity was higher in the 1.0 W-laser irradiation group. Expression of mRNAs for heat shock protein 27, bone morphogenetic proteins (BMPs) and ALP were greater in the 1.0 W-laser irradiation group. Expression of BMPs in the conditioned medium was also higher in the 1.0 W-laser irradiation group. In particular, DMSO decreased the number of calcified nodule produced by 1.0 W-laser irradiation. These results supposed that the mineralization of HDP cells is stimulated by laser irradiation, and that *OH generated by laser irradiation is a trigger for promotion of HDP cell mineralization.

Isolated rat dental pulp cell culture and transplantation with an alginate scaffold.

Many studies have been conducted on tissue stem cells in the field of regenerative medicine, and cultured dental pulp mesenchymal cells have been reported to secrete dentin matrix. In the present study we used alginate as a scaffold to transplant subcultured rat dental-pulp-derived cells subcutaneously into the back of nude mice. We found that when beta-glycerophosphate was added to the culture medium, the mRNA of the dentin sialophosphoprotein (DSPP) gene coding dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) was expressed, and an increase in alkaline phosphatase, an early marker of odontoblast differentiation, was also demonstrated. Six weeks after implantation, subcutaneous formation of radiopaque calcified bodies was observed in situ. Immunohistochemical and fine structure studies identified expression of type I collagen, type III collagen, and DSP in the mineralizing transplants, and isolated odontoblast-like cells began to form dentin-like hard tissue formation. Scattered autolyzing apoptotic cells were also observed in the transplants. The study showed that subcultured rat dental-pulp-derived cells actively differentiate into odontoblast-like cells and induce calcification in an alginate scaffold.

Gene expression of bone matrix proteins in a calcified tissue appeared in subcutaneously transplanted rat dental pulp.

Dental pulp self-mineralizes or induces calcified tissue formation, when it is subcutaneously transplanted. This study aims to clarify the nature and process of this tissue calcification by using histochemical techniques and in situ hybridization methods. The dental pulps of incisors from Sprague-Dawley strain rats were homogeneously transplanted into the subcutaneous tissue of the head of another rat. On the 1st day of transplantation, the mRNAs of type I collagen (COL I) and bone sialoprotein (BSP) were expressed in the transplant. On the 5th to 7th days after transplantation, von Kossa-positive, calcified tissues appeared in the transplanted tissues. The mRNAs of COL I, BSP and osteopontin (OPN) were expressed with the same distributions as those of the von Kossa-positive areas, but those of type II collagen (COL II) and bone morphogenic protein 2 (BMP-2) were not expressed in the transplant. When the cellular incorporation of BrdU was examined after its subcutaneous injection into the animals, positive cells were present in the von Kossa-positive calcified foci as well as in their surrounding areas. These results indicate that the transplanted dental pulp possesses the ability to induce cell proliferation resulting in the synthesis of several bone matrix proteins and the formation of calcified tissue. This may give us an insight into the nature and processes of pulp obliteration, which occurs in a human tooth following an episode of trauma.

MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells.

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells.

Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts.

The purpose of the present study was to investigate the effect of laser irradiation on calcified nodule formation in human dental pulp (HDP) cells. HDP cells were irradiated once with a Ga-Al-As laser for 5 and 10 min, and calcified nodule formation was determined by von Kossa staining. The laser irradiation increased the number of calcified nodules in a time-dependent manner. The activity of alkaline phosphatase and production of collagen and osteocalcin in conditioned medium were measured. Both were higher in the irradiated group than in the nonirradiated group. These results suggested that formation of calcified nodules in HDP cells, as well as in alkaline phosphatase activity, the production of collagen and osteocalcin were enhanced by laser irradiation.

Dental manifestations of osteogenesis imperfecta and abnormalities of collagen I metabolism.

The in vitro protein-chemical features and the molecular background of osteogenesis imperfecta (OI), a heritable disorder of collagen I metabolism, have been elucidated in recent years. The aim of our study was to find the prevalence of dentinogenesis imperfecta (DI) and other dental anomalies in 88 patients with OI, to compare clinical with radiologic abnormalities, and to correlate these clinical/radiologic findings with the results of gel electrophoresis and molecular studies of collagen I. Twenty-eight percent of OI patients had DI. Most patients with DI had radiologic abnormalities, but some patients had radiologic signs compatible with DI, but no clinical signs of DI. OI type I patients with DI were more severely affected by OI than those without DI. In OI type III and IV, in contrast, there was no difference in overall severity between patients with and without DI. DI was not associated with any particular molecular aberration in any OI type. If defining DI from the presence of both clinical and radiologic signs, collagen I produced by cultured fibroblasts was qualitatively abnormal from all OI patients with DI. Some OI patients had dental abnormalities not resembling DI. A qualitative collagen abnormality could not be found in any of these patients. Denticles, i.e., calcifications within the pulpal cavity, were found more frequently in OI patients than in control subjects.

Characterization of a system of mineralized-tissue formation by rat dental pulp cells in culture.

Pulp tissue was obtained from maxillary incisors of young adult male Wistar rats, minced and digested with 0.5% trypsin and 0.02% EGTA at 37 degrees C for 30 min. Dissociated cells were cultured with or without 10 nM dexamethasone using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. Confluent cells were subcultured at 7 days and the medium further supplemented with beta-glycerophosphate (beta-GP). Dexamethasone in primary culture and/or secondary culture enhanced the formation of mineralized tissue while > 5 mM beta-GP was necessary for mineralization to occur. Biochemical analysis of the radiolabelled medium revealed that these cells produced type I, type I trimer and type III collagens. Analysis of [32PO4]-labelled medium, using DEAE-Sephacel ion-exchange chromatography and sodium dodecylsulphate-polyacrylamide gel electrophoresis, showed that these cells produced phosphophoryn-like protein. These results indicate that some of the rat dental pulp cells in culture express an odontoblast-like phenotype.

Pathological mineralization in a serially passaged cell line from rat pulp.

The ultrastructure of crystal formation in association with dental pulp cells isolated from rat incisor was studied in vitro. A clone, RPC-K, was obtained and incubated with Na-beta-glycerophosphate (BGP). Growing pulp cells showed low alkaline phosphatase (ALP) activity, which began to increase with cell proliferation. Pulp cells formed cell multilayers after day 14 of culture. Mineralized tissues were observed within cell multilayers on day 28 of culture. Vesicular structures were found around degenerate and necrotic cells. Some of these vesicles contained needle-like crystals. Organic structures appeared at the periphery of mineralized tissues with a post-embedding demineralization and staining method. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite (HAP). These findings suggest that the RPC-K cell line might be useful for a model system to investigate pathological mineralization.

Electron probe micro-analysis of human dental pulp stones.

The mineral composition of ten human dental pulp stones presenting various morphological aspects has been studied by electron probe micro-analysis. The denticles were composed of two major chemical elements: Ca and P with mean concentrations 32.12% and 14.69% respectively giving a Ca/P weight ratio of 2.19 which is very close to the weight ratio of pure stoichiometric hydroxyapatite (2.15). The concentration of some other elements was much lower (0.88% for F; 0.75% for Na; 0.51% for Mg). The other analysed constituents (K, Cl, Mn, Zn, Fe) were present at trace concentrations. The mineral composition of sound human dentine from one tooth containing a pulpal calcification was also analysed for comparative purpose.

Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.

Pulp fibroblasts were isolated from human deciduous and supernumerary teeth and cultured in vitro. With continued culture in normal tissue-culture medium, six pulp fibroblast strains formed cell nodules after 10-15 days. By electron microscopy the nodules had matrix vesicles, and needle-shaped crystals associated with a dense network of collagen fibrils. The crystalline material exhibited a pattern consistent with hydroxyapatite when nodules were examined by X-ray diffractometry. Furthermore, the cells showed high levels of alkaline phosphatase activity, which could be increased more than seven-fold by the addition of 1,25(OH)2D3 (5 x 10(-9)-5 x 10(-8) M). In addition to the production of type I collagen, these cells also synthesized fibronectin and osteonectin. The formation of mineralized tissue nodules by pulp cells in vitro provides a useful system for study of the pathological calcification of pulp tissues.

A study of the mineral phase of pulp calcification.

Physico-chemical properties of pulp calcification were studied by means of X-ray microbeam diffraction, electron spin resonance (ESR), X-ray energy-dispersive analysis, and chemical analysis. The material was obtained from the second molar of the right mandible of a 25-year-old woman. X-ray diffraction and ESR analyses showed that calcium salts of the calcification are deposited in the form of apatitie, possibly carbonate-containing apatite. Furthermore, the mineral phase of the calcification was found to be similar to that of bone rather than that of dentin with regard to crystallinity and inorganic content. An unexpected finding was that high concentrations of iron were detected in some areas near the surface of the calcification. The question remains open as to what role, if any, iron may play in the formation of pulp calcification.