Reproduction and differentiation of cells in the tissues of growing rat teeth in response to parathyroid hormone and hydrocortisone

Influence of parathyroid hormone (PTH) and hydrocortisone (HC) on growth and development of teeth during prefunctional period has been studied, employing radioactive tracers. Biosynthetic and reproductive activity of cells is estimated in the molar and incisor tissues. It has been established that PTH accelerates the process of local resorption of the bone along the alveolar crest, but does not influence upon the teeth growth, functional and reproductive activity of the pulp cells. HC inhibits protein and glycoprotein synthesis, decreases amount of proliferating cells, accelerates their differentiation and, thus, delays formation of the root and tooth growth.     

Regional ultrastructural and cytochemical comparisons of the epithelial-mesenchymal interface during rat incisor development

A major theme in understanding epithelial-mesenchymal interactions during development focuses upon regional mesenchyme specification of epithelial differentiation. One particularly useful epidermal organ system for studying this issue is the rodent continuously growing and erupting incisor tooth organ. One advantage of this particular system resides in the regional features of the rodent incisor tooth organ. Along the labial surface, inner dental epithelial cells differentiate into ameloblasts that produce enamel extracellular matrix, whereas the epithelia along the lingual surface do not become ameloblasts and do not produce enamel matrix. This study has been designed to compare ultrastructural features of labial versus lingual surfaces, with particular emphasis upon mesenchymal cell shape, the orientation of extracellular matrix collagen, the basal lamina, and the distribution of sulfated glycoconjugates. Critical analyses of the data indicated that different microenvironments exist between epithelia and mesenchyme in the labial versus the lingual surfaces of the developing rodent incisor tooth organ.     

The functions of mechanosensitive ion channels in tooth and bone tissues

Tooth and bone are independent tissues with a close relationship. Both are composed of a highly calcified outer structure and soft inner tissue, and both are constantly under mechanical stress. In particular, the alveolar bone and tooth constitute an occlusion system and suffer from masticatory and occlusal force. Thus, mechanotransduction is a key process in many developmental, physiological and pathological processes in tooth and bone. Mechanosensitive ion channels such as Piezo1 and Piezo2 are important participants in mechanotransduction, but their functions in tooth and bone are poorly understood. This review summarizes our current understanding of mechanosensitive ion channels and their roles in tooth and bone tissues. Research in these areas may shed new light on the regulation of tooth and bone tissues and potential treatments for diseases affecting these tissues.     

A tracer study with systemically and locally administered dinitrophenylated osteopontin.

Osteopontin (OPN), a major non-collagenous matrix protein of bone, is also found in tissue fluids and in the circulation. It is still not clear whether circulating OPN contributes to bone formation. To elucidate this question, rat OPN was tagged with dinitrophenol groups and administered to rats either intravenously or by infusion with an osmotic minipump through a "surgical window" in the bone of the hemimandible. Dinitrophenylated rat albumin (ALB) was used as a control. The presence and distribution of tagged proteins were revealed by immunogold labeling on sections of tibia and alveolar bone. Tagged molecules of OPN were found in mineralization foci, surfaces and interfaces, and matrix accumulations among calcified collagen fibrils. Even though dinitrophenylated ALB was administered at several-fold higher concentrations, it did not accumulate in these sites. These results show that circulating OPN can be incorporated into specific compartments of forming bone and suggest that such molecules may play a more important role than previously suspected.     

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, α-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth’s MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300 μg per ml), hydrocortisone (1 μg per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeably promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporation of collagen-promoting factors to the in vitro environment particularly with regard to the controlling role implicated for collagen in a variety of biological processes. Some of the work reported here was undertaken while A. H. Melcher was a member of the Department of Dental Science, Royal College of Surgeons of England, London, England.     

Three-dimensional printing biotechnology for the regeneration of the tooth and tooth-supporting tissues

The tooth and its supporting tissues are organized with complex three-dimensional (3D) architecture, including the dental pulp with a blood supply and nerve tissues, complex multilayer periodontium, and highly aligned periodontal ligament (PDL). Mimicking such 3D complexity and the multicellular interactions naturally existing in dental structures represents great challenges in dental regeneration. Attempts to construct the complex system of the tooth and tooth-supporting apparatus (i.e., the PDL, alveolar bone, and cementum) have made certain progress owing to 3D printing biotechnology. Recent advances have enabled the 3D printing of biocompatible materials, seed cells, and supporting components into complex 3D functional living tissue. Furthermore, 3D bioprinting is driving major innovations in regenerative medicine, giving the field of regenerative dentistry a boost. The fabrication of scaffolds via 3D printing is already being performed extensively at the laboratory bench and in clinical trials; however, printing living cells and matrix materials together to produce tissue constructs by 3D bioprinting remains limited to the regeneration of dental pulp and the tooth germ. This review summarizes the application of scaffolds for cell seeding and biofabricated tissues via 3D printing and bioprinting, respectively, in the tooth and its supporting tissues. Additionally, the key advantages and prospects of 3D bioprinting in regenerative dentistry are highlighted, providing new ideas for dental regeneration.     

The cryomicrotomy of rat dental tissues: a technique for histological and immunohistochemical analysis

Summary We have applied a method developed for the cryomicrotomy of non-decalcified bone to the histological preparation of the tooth and related dental tissues. Cryosections of rat mandible have been cut on a heavy-duty freezing microtome. Both cellular and extracellular structure was well preserved and the sections of tooth and bone appeared to be suitable for optical and scanning electron microscopy and for immunohistochemical analysis. However, there was an overall strong non-specific binding of immunohistochemical reagents to enamel which was not evident in the other mineralized tissues of the mandible. This may relate to important differences in the nature of this tissue.     

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.     

Immunolocalization of connexin 43 in the tooth germ of the neonatal rat

Summary Rabbit polyclonal antibodies to amino acids 346–360 of connexin 43, the ‘heart’ gap junction protein, were employed to immunolocalize connexin 43 gap junctions in the neonatal rat molar tooth germ. Connexin 43 appears early in the differentiation of both ectodermally derived and ectomesenchymally derived cells of the developing tooth. Connexin 43 immunoreactivity is present in the epithelial components of the enamel organ, including the area of the proximal and distal junctional complexes of the ameloblast layer, and the stratum intermedium, stellate reticulum and outer enamel epithelium. Secretory odontoblasts and developing alveolar bone also display a pattern of connexin 43 immunostaining. Both the epithelial and ectomesenchymally-derived components of the developing tooth acquire connexin 43 channels in a manner that correlates with cell differentiation. In addition, three regions can be defined by connexin 43 immunostaining: the epithelia of the enamel organ that are derived from the oral epithelium, the odontoblast layer derived from the ectomesenchyme, and the alveolar bone. The results suggest that connexin 43 may provide the mechanism for functional compartmentalization of the tissues associated with tooth formation. Compartmentalization suggested by connexin 43 expression could play important roles in the development and functions of these tissues.     

Immunohistochemical evidence for the intracellular formation of basement membrane collagen (type IV) in developing tissues

Antibodies to type IV collagen obtained from the basement membrane of the mouse EHS tumor were incubated with sections of rat incisor teeth and other tissues for immunostaining by direct or indirect methods. In all locations, the immunostaining was pronounced in basement membranes in which it was restricted to the "basal lamina" layer, from which "bridges" often extended to nearby basal laminae. Usually no immunostaining was detectable in the cells associated with the basement membranes. However, examination of the capillaries at the posterior extremity of the rat incisor tooth, where tissues are at an early stage of development, showed immunostaining not only of the basement membrane, but also of the endothelial cells. The staining was localized in rough endoplasmic reticulum cisternae, some Golgi saccules and their peripheral distensions, and structures believed to be secretory granules. These findings suggest that the synthesis of type IV collagen proceeds along the classical secretory pathways through rough endoplasmic reticulum and Golgi apparatus. At the same time, immunostaining was usually lacking in the cells of the capillaries that had migrated about 2 mm away from the posterior end of the incisor tooth and also in the cells of most other tissues examined, even though the associated basal laminae were reactive. It is, therefore, presumed that the production of type IV collagen may be high in cells at an early stage of development and that any later production and turnover of basement membrane collagen can only be minimal.     

Alterations in the incisor development in the Tabby mouse

The X-linked tabby (Ta) syndrome in the mouse is homologous to the hypohidrotic ectodermal dysplasia (HED) in humans. As in humans with HED, Ta mice exhibit hypohidrosis, characteristic defects of hairs and tooth abnormalities. To analyze the effects of Ta mutation on lower incisor development, histology, morphometry and computer-aided 3D reconstructions were combined. We observed that Ta mutation had major consequences for incisor development leading to abnormal tooth size and shape, change in the balance between prospective crown- and root-analog tissues and retarded cytodifferentiations. The decrease in size of Ta incisor was observed at ED13.5 and mainly involved the width of the tooth bud. At ED14.5-15.5, the incisor appeared shorter and narrower in the Ta than in the wild type (WT). Growth alterations affected the diameter to a greater extent than the length of the Ta incisor. From ED14.5, changes in the shape interfered with the medio-lateral asymmetry and alterations in the posterior growth of the cervical loop led to a loss of the labio-lingual asymmetry until ED17.0. Although the enamel organ in Ta incisors was smaller than in the WT, a larger proportion of the dental papilla was covered by preameloblasts-ameloblasts. These changes apparently resulted from reduced development of the lingual part of the enamel organ and might be correlated with a possible heterogeneity in the development of the enamel organ, as demonstrated for upper incisors. Our observations suggest independent development of the labial and lingual parts of the cervical loop. Furthermore, it appeared that the consequences of Ta mutation could not be interpreted only as a delay in tooth development.     

Distribution of γ-carboxyglutamic acid in calcified tissues

γ-Carboxyglutamic acid, previously identified in the vertebrate mineralized tissues of bone and dentin, is not detectable in the calcified skeletons of six invertabrate species representing five phyla. Its absence in all analyzed invertebrate tissues (including calcitic, aragonitic, and apatitic mineral phases) indicates that matrix protein-bound γ-carboxyglutamic acid is not obligatory for the calcification process in the invertebrates. Further, these data raise the possibility that invertebrates as a group may lack the enzymatic capability for biosynthesizing γ-carboxyglutamic acid. In contrast, the distribution of γ-carboxyglutamic acid in the vertebrates has been further extended by this study to include an aptitic shark tooth and an aragonitic fish otolith. No γ-carnoxyglutamic acid was detected, however, in the organic matrix of the calcitic hen egg shell.     

High-resolution immunocytochemistry of noncollagenous matrix proteins in rat mandibles processed with microwave irradiation.

The mineral phase in calcified tissues represents an additional factor to be considered during their preservation for ultrastructural analyses. Microwave (MW) irradiation has been shown to facilitate fixative penetration and to improve structural preservation and immunolabeling in a variety of soft tissues. The aim of the present study was to determine whether MW processing could offer similar advantages for hard tissues. Rat hemimandibles were immersed in 4% formaldehyde + 0.1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2, and exposed to MWs for three periods of 5 min at temperatures not exceeding 37C. They were then decalcified in 4.13% EDTA, pH 7.2, for 15 hr, also under MW irradiation. Osmicated and non-osmicated samples were dehydrated in graded concentrations of ethanol and embedded in LR White resin. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against ameloblastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural preservation of tissues was in most cases comparable to that obtained by perfusion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreactivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating ameloblasts and labeling for osteopontin appeared over the Golgi apparatus of odontoblasts after initiation of dentine mineralization. We conclude that MW irradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention. (J Histochem Cytochem 49:1099-1109, 2001)     

The distribution of fibronectin in rat tooth and periodontal tissues: an immunofluorescence study using a monoclonal antibody

The distribution of fibronectin (FN) in longitudinal, buccolingual sections of decalcified adult rat periodontium and teeth was studied by indirect immunofluorescence using a monoclonal antibody. FN was present in virtually all regions of the periodontium, including the gingiva, periodontal ligament, many blood vessel walls, alveolar bone, incisor and molar predentine and dentine, and molar acellular and cellular cementum. The cementum of the incisor, ameloblasts, stratum intermedium and stellate reticulum, and the connective tissue of the pulp and the surface of ondontoblasts facing the pulp in the incisor and molar were not labeled for FN. FN distribution was not always uniform either within a given connective tissue or between different connective tissues of the same organ.     

E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor

Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor.     

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.