ON THE PROGENITOR CELL MIGRATION VELOCITY

An attempt is presented to extract cell kinetic information from histomorphological features. It is applicable to rapidly proliferating tissues like the intestinal epithelium. Each replicating tissue has an origin where cells are formed and a periphery toward which cells migrate. The migration path along which they move is denominated as tissue radius on which all cell positions are mapped. Cell migration on the radius is associated with cell proliferation at tissue origin. Each mitosis there is associated with the displacement of all cells distal to it by one cell position. The more mitoses positioned between a cell and tissue origin, the greater its migration velocity. It is possible therefore to derive the cell migration velocity v(x) from the cumulative mitotic distribution on the radius, N(x). v(x) = N(x)/t_m (t_m= mitotic time). In this form v(x) represents also cell production at any point on the radius and may serve for the computation of other cell kinetic parameters like generation time. These arguments are illustrated on the rat incisor tooth inner enamel epithelium which has been studied in the normal and rapidly erupting tooth.     

The role of preameloblast-conditioned medium in dental pulp regeneration

Pulp regeneration using human dental pulp stem cells (hDPSCs) maintains tooth vitality compared with conventional root canal therapy. Our previous study demonstrated that preameloblast-conditioned medium (PA-CM) from murine apical bud cells induces the odontogenic differentiation of hDPSCs and promoted dentin formation in mouse subcutaneous tissue. The purpose of the present study is to evaluate the effects of PA-CM with human whole pulp cells on pulp regeneration in an empty root canal space. Human pulp cells were seeded in the pulp cavities of 5 mm-thick human tooth segments with or without PA-CM treatment, and then transplanted subcutaneously into immunocompromised mice. In the pulp cell-only group, skeletal muscle with pulp-like tissue was generated in the pulp cavity. A reparative dentin-like structure with entrapped cells lined the existing dentin wall. However, in the PA-CM-treated group, only pulp-like tissue was regenerated without muscle or a reparative dentin-like structure. Moreover, human odontoblast-like cells exhibited palisade arrangement around the pulp, and typical odontoblast processes elongated into dentinal tubules. The results suggest that PA-CM can induce pulp regeneration of human pulp cells with physiological structures in an empty root canal space.     

Effect of glypican‐1 gene on the pulp cells during the reparative dentine process

GPC‐1 (glypican‐1) is a cell surface heparan sulfate proteoglycan that acts as a co‐receptor for heparin‐binding growth factors and members of the TGF‐β (transforming growth factor beta‐1) family. The function of cell‐surface proteoglycans in the reparative dentine process has been under investigation. Gpc‐1 was detected with similar frequency as tgf‐β1 in the cDNA library using mRNA from the odontoblast‐like cell‐enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc‐1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc‐1 on odontoblast‐like cell differentiation using siRNA (small interfering RNA) to down‐regulate gpc‐1 expression. Immunohistological examination showed that GPC‐1 was expressed in pulp cells entrapped by fibrodentine and odontoblast‐like cells as well as TGF‐β1. The mRNAs for gpc‐1, ‐3 and ‐4, except for gpc‐2, were expressed during odontoblast‐like cell differentiation in pulp cells. The relative levels of gpc‐1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down‐regulation of gpc‐1 expression resulted in a 3.9‐fold increase in tgf‐β1 expression in pulp cells and a 0.3‐fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc‐1 and tgfβ‐1 expression are necessary for the onset of differentiation, but should be down‐regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc‐1 expression in odontoblast‐like cells is associated with the early differentiation but not with the formation of reparative dentine.     

Responses of BrdU label-retaining dental pulp cells to allogenic tooth transplantation into mouse maxilla

Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5–7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation.     

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)_x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)₁₀ oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)_x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)_x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.     

CELL WALL CHEMISTRY AND FINE STRUCTURE IN LEPTOIDS OF DENDROLIGOTRICHUM (BRYOPHYTA): THE END WALL

Leptoids (sieve elements) of Dendroligotrichum are characterized by a highly oblique end wall which is composed of cellulose (birefringent; IKI-H₂SO₄-positive), polyuronides (toluidine blue-positive), pectins (hydroxylamine-positive) and natural aldehydes (silver hexamine and silver proteinate-positive). Cytochemically the end wall appears identical to the unevenly thickened lateral wall. Electron cytochemical localization of aldehydes with silver proteinate reveals two distinct wall layers in comparison to the 3-layered lateral wall. Plasmodesmata are present in the end wall with a frequency of 15-20 per μm². A characteristic feature of end wall plasmodesmata is an expanded median cavity which is 0.12-0.15 μm in diameter. Frequently an electron-dense substance, whose chemical nature and origin are unknown, occludes the plasmodesmata.     

Topical Application of Lithium Chloride on the Pulp Induces Dentin Regeneration

We herein describe a novel procedure for dentin regeneration that mimics the biological processes of tooth development in nature. The canonical Wnt signaling pathway is an important regulator of the Dentin sialophosphoprotein (Dspp) expression. Our approach mimics the biological processes underlying tooth development in nature and focuses on the activation of canonical Wnt signaling to trigger the natural process of dentinogenesis. The coronal portion of the dentin and the underlying pulp was removed from the first molars. We applied lithium chloride (LiCl), an activator of canonical Wnt signaling, on the amputated pulp surface to achieve transdifferentiation toward odontoblasts from the surrounding pulpal cells. MicroCT and microscopic analyses demonstrated that the topical application of LiCl induced dentin repair, including the formation of a complete dentin bridge. LiCl-induced dentin is a tubular dentin in which the pulp cells are not embedded within the matrix, as in primary dentin. In contrast, a dentin bridge was not induced in the control group treated with pulp capping with material carriers alone, although osteodentin without tubular formation was induced at a comparatively deeper position from the pulp exposure site. We also evaluated the influence of LiCl on differentiation toward odontoblasts in vitro. In the mDP odontoblast cell line, LiCl activated the mRNA expression of Dspp, Axin2 and Kallikrein 4 (Klk4) and downregulated the Osteopontin (Osp) expression. These results provide a scientific basis for the biomimetic regeneration of dentin using LiCl as a new capping material to activate dentine regeneration.     

Capacity of Dental Pulp Differentiation in Mouse Molars as Demonstrated by Allogenic Tooth Transplantation

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5–7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase–positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue. (J Histochem Cytochem 56:1075–1086, 2008)     

Microscopic and histochemical study of odontoclasts in physiologic resorption of teeth of the polyphyodont lizard, Liolaemus gravenhorsti

Using tartrate-resistant acid phosphatase (TRAP), we examined the cytodifferentiation of odontoclast cells in resorbing areas of dental tissues during the replacement of teeth in a polyphyodont lizard, Liolaemus gravenhorsti. We also report, by means of Lectin-HRP histochemistry, the distribution pattern of some specific sugar residues of TRAPase-positive cells. For detection of TRAPase activity, the azo dye-coupling technique was used. Lectin binding sites were demonstrated by means of specific HRP-lectins. The process of tooth resorption was divided into four stages: 1) preresorption-the wall of the dental pulp is covered with an odontoblast layer, and no TRAP-positive cells are in the dental pulp; 2) early resorption-TRAP-positive multinucleate odontoclasts are present on the dental wall, but the rest of the pulp surface is still covered with an odontoblast layer; 3) later resorption-the entire surface of the pulp chamber is lined with multinucleate odontoclasts; and 4) final resorption-the tooth has been totally resorbed. Odontoclasts are usually detached from the resorbed surface, and show signs of degeneration. Of the six lectins used, PNA, ECA, and UEA-1 bind to multinucleated but not mononuclear cells. All the remaining lectins, BS-1, RCA(120), and LTA showed no binding to any cells of the teeth. The significance of saccharidic moieties such as acetyl-galactosamine, acetyl-glucosamine, and fucose sugar residues is difficult to ascertain. Perhaps these oligosaccharides might be borne on molecules associated with odontoclastic resorption or associated with multinucleation of odontoclasts after attachment to the dentine surface.     

THE RODENT INCISOR TOOTH PROLIFERON

The rodent incisor tooth is the site of five cell populations proliferating in harmony: amelocytes, odontocytes, pulp cells, endothelial cells and the periodontal ligament. Their proliferating regions are located in the apex tip, where the various cells originate. Cells displaced from the tooth origin at the apex toward the periphery, mature to perform their specified function. The proliferative events in the tooth are summarized in a conceptual model of the incisor proliferon. The proliferon is an oriented structure with an origin and periphery. It consists of four basic elements: parenchyma, connective tissue, blood vessels and nerve fibres, all interacting continuously. All four are indispensable in the definition of the proliferon.     

Ultrastructural investigation of matrix-mediated biomineralization in echinoids (Echinodermata,Echinoida)

Summary The formation of echinoderm endoskeletons is studied using echinoid teeth as an example. Echinoid teeth grow rapidly. They consist of many calcareous elements each produced by syncytial odontoblasts. The calcification process in echinoderms needs (1) syncytial sclerocytes or odontoblasts, (2) a spacious vacuolar cavity within this syncytium, (3) an organic matrix coat in the cavity. As long as calcite is deposited, the matrix does not touch the interior face of the syncytium. The cooperation between syncytium, interior cavity and matrix coat during the mineralization process is discussed. The proposed hypothesis applies to the formation of larval skeletons, echinoderm ossicles and echinoid teeth.When calcite deposition ceases the syncytium largely splits up into filiform processes, and the skeleton is partly exposed to the extracellular space. However, the syncytium is able to reform a continuous sheath and an equivalent of the cavity and may renew calcite deposition.The tooth odontoblasts come from an apical cluster of proliferative cells, each possessing a cilium. The cilium is lost when the cell becomes a true odontoblast. This suggests that cilia are primitive features of echinoderm cells. The second step in calcification involves the odontoblasts giving rise to calcareous discs which unite the hitherto single tooth elements. During this process the odontoblasts immure themselves. The structures necessary for calcification are maintained until the end of the process.The mineralizing matrix is EDTA-soluble. The applied method preserves the matrix coating the calcite but more is probably incorporated into the mineral phase and dissolved with the calcite.Abbreviations A adhesive point (LNC) - B adaxial bag - bb basal body (ci) - CA calcareous deposits - cb cytoplasmic bladder (cp) - ce centriole - ci cilium - cp cable-like cell process - cv condensing vacuole - dp distal processes (sh) - E epithelium of the tooth - ex extracellular space - f extracellular fibrils - ga gasket (sh) - ic interior cavity - L lamellae (LNC) - LNC lamellae needle complex - m mitochondrium - mc matrix coat - MF main fold (P) - MI mitosis - mt microtubules - N nucleus - O odontoblast - P primary plate - Ph phagocyte - PR proliferative cell - pr prism - rb reserve body - RER rough endopl. reticulum - rl rootlet (ci) - RY relatively youngest plate - s satellite (bb, ce) - sh synplasmic sheath (O) - SP secondary plate - sv smooth-walled vesicle - TF transversal fold (P) - U umbo (P) - v Golgi vesicle - Y youngest tooth element     

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.     

The Quiescin Q6 Gene (QSCN6) Is a Fusion of Two Ancient Gene Families: Thioredoxin and ERV1

Cell and tissue growth is a dynamic process determined by the fraction of cells in the proliferative cycle, the fraction of cells in quiescence, and the rate of cell death. Genes whose expression is induced at the beginning of the transition from the proliferative cell cycle to quiescence may play an important role in this process. We have identified a gene, Quiescin Q6 (QSCN6), whose expression is induced just as fibroblasts begin to leave the proliferative cycle and enter quiescence. QSCN6 is located on human chromosome 1q24, near the putative hereditary prostate cancer locus (HPC1). A triplet repeat (CTG)_nencodes a putative signal sequence. The gene encodes a 582-amino-acid open reading frame that has domains that are members of two ancient gene families. These domains apparently underwent a gene fusion event during metazoan evolution to create QSCN6. QSCN6 is most closely related to three genes of unknown function fromCaenorhabditis elegansas well as a gene from guinea pig. Analysis of this relationship showed nine Quiescin homology zones (QHZ). QHZ 0 is the putative signal sequence, QHZ 1 is homologous to a thioredoxin domain, and QHZ 2, 3, 4, and 8 are homologous only to themselves, while QHZ 5, 6, and 7 are homologous to the ERV1 gene ofSaccharomyces cerevisiae.In both thioredoxin and ERV1 gene superfamilies, QSCN6 sequences appear to be on distinct branches of their respective phylogenetic trees, consistent with an ancient origin of the QSCN6 gene. We present a model of the origin of QSCN6 and discuss its potential role in growth regulation.     

Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

TGF-β subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-β signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-β signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2^fl/fl;Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-β2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-β signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.     

FIBROBLAST CELL KINETICS IN THE PERIODONTAL LIGAMENT OF THE MOUSE

Twelve male mice were injected intraperitoneally with tritiated thymidine. Six were sacrificed after 1 hr and six after 7 days. The right manibular incisors were dissected, cut sagittally and dipped in liquid emulsion. In the exposed and stained slides, observation was restricted to the lingual side of the periodontal ligament. Cells were evaluated sagittally from the basal tooth end up to the distance of 5 mm and up to the depth of 100 μm in the direction of socket wall. Cell and grain count was evaluated separately in 100 × 10 μm rectangles, creating a two-dimensional array onto which the periodontal ligament was mapped. The progenitor compartment extends up to the distance of 2400 μm from origin. Fibroblasts leaving this compartment migrate at different velocities, creating a velocity profile across the ligament. Adjacent to the socket wall cell movement is sluggish, whereas the fastest cell movement is exhibited by cells located 20–30 μm from the tooth. The existence of such a profile indicates a continuous renewal of intercellular bonds, consistent with a process of actively pulling the incisor from its socket by the migrating fibrocytes.     

Kelp versus coralline: cellular basis for mechanical strength in the wave‐swept seaweed Calliarthron (Corallinaceae,Rhodophyta)1

Previous biomechanical studies of wave‐swept macroalgae have revealed a trade‐off in growth strategies to resist breakage in the intertidal zone: growing in girth versus growing strong tissues. Brown macroalgae, such as kelps, grow thick stipes but have weak tissues, while red macroalgae grow slender thalli but have much stronger tissues. For example, genicular tissue in the articulated coralline Calliarthron cheilosporioides Manza is more than an order of magnitude stronger than some kelp tissues, but genicula rarely exceed 1 mm in diameter. The great tissue strength of Calliarthron genicula results, at least in part, from a lifelong strengthening process. Here, a histological analysis is presented to explore the cellular basis for mechanical strengthening in Calliarthron genicula. Genicula are composed of thousands of fiber‐like cells, whose cell walls thicken over time. Thickening of constitutive cell walls likely explains why older genicula have stronger tissues: a mature geniculum may be >50% cell wall. However, the material strength of genicular cell wall is similar to the strength of cell wall from a freshwater green alga, suggesting that it may be the quantity—not the quality—of cell wall material that gives genicular tissue its strength. Apparent differences in tissue strength across algal taxa may be a consequence of tissue construction rather than material composition.     

COMPARATIVE BUNDLE SHEATH AND MESOPHYLL DIFFERENTIATION IN THE LEAVES OF THE C4 GRASSES PANICUM EFFUSUM AND P. BULBOSUM

The two C₄ Panicum species examined differ in C₄ acid decarboxylation type and in developmental origin of bundle sheaths in major veins of their leaf blades. In Panicum effusum R.Br. (NAD-malic enzyme type) both mesophyll (PCA) and chlorenchymatous bundle sheath (PCR) cells are derived from ground meristem. In contrast, in Panicum bulbosum H.B.K. (NADP-malic enzyme type), bundle sheath cells are derived from procambium, while mesophyll develops from ground meristem. To test the hypothesis that the developmental divergence of bundle sheath and mesophyll cells would occur earlier when these two tissues had different ontogenetic origins (in P. bulbosum) than when these tissues had the same origin (in P. effusum), the development of major veins in each species was investigated. We measured cell length and cross sectional area, plastid and mitochondrial number, plastid area, vacuole area fraction, wall thickness and fraction adjacent to intercellular space using direct and digitizer measurements of transmission electron micrographs of leaf cross sections of successive developmental stages. Many of the statistically significant changes in the structural parameters measured occurred late in development of both species. The magnitude of developmental change in P. effusum PCR cells was sometimes more dramatic, viz. changes in cell and PCR plastid area, and in mitochondrion number per cell. However, earlier divergence of PCR and PCA length and volume, and wall fraction adjacent to intercellular space in P. bulbosum than in P. effusum indicates that ontogenetic origin of PCR cells from procambium could determine the timing of at least some developmental events.     

Apparent and true digestibility of the Weende components, cell content and cell wall of ryegrass

For 14 samples of ventilated hay and 6 of fresh grass, digestibility was determined with young wethers. The contribution to the digestibility was calculated for the Weende components (crude protein, crude fibre, nitrogen-free extractives + fat) and for cell content and cell wall (Van Soest) components. For the cell content and crude protein, a hyperbolic relationship, y' = a − b/x between the digestibility coefficient of the components (y') and the percentage component in dry matter (DM, x) can be accepted with a sufficient degree of certainty. This means that the apparent digestibility of the cell content and crude protein increase with increasing concentration. The latter formula can be transformed into a linear regression y = ax − b where y = percentage digestible component in DM, a being an estimate of the true digestibility of the component and b an estimate of the endogenous excretion of the component. From these relationships some important practical conclusions can be drawn. For other dry matter components, such as crude fibre, nitrogen free extractives + fat and cell wall, the relationship between the digestibility coefficient and the percentage of the component in DM cannot be expressed by a simple regression line. The advantages of splitting the digestibility into a contribution from cell content and cell wall components versus splitting into contributions from the Weende components are discussed.By determination of the cell wall and its digestibility in vitro, a very good prediction for the dry matter digestibility of grass can be given, which is of great importance for plant breeding and grassland research.