Tartrate-resistant acid phosphatase in mononuclear and multinuclear cells during the bone resorption of tooth eruption

Tartrate-resistant acid phosphatase (TRAP) has been used as a cytochemical marker for the cell mediators of bone resorption, osteoclasts and their mononuclear precursors. We have applied a cytochemical method for TRAP to study the dependence of the osteoclast-mediated bone resorption of tooth eruption on the dental follicle, a connective tissue investment of the developing tooth, by analyzing the TRAP activity of mononuclear cells in the dental follicle before and during pre-molar eruption in dogs. The percentage of TRAP-positive monocyte cells increases until mid-eruption, slightly preceding a previously demonstrated rise in numbers of osteoclasts on adjacent bone surfaces. These data suggest an ontogenetic relationship between follicular mononuclear cells and osteoclasts on adjacent alveolar bone surfaces during tooth eruption. However, because TRAP occurs in other tissues and is not an exclusive indicator of pre-osteoclasts, proof of their relationship will have to await application of more definitive techniques.     

Ultrastructure of alveolar bone during tooth eruption in the dog

Previous work from our laboratories has established that eruption of the permanent mandibular premolars in dogs is dependent upon the presence of the dental follicle and that it involves resorption of alveolar bone and the roots of the deciduous predecessor above and formation of alveolar bone below the developing crown. This study illustrates the topography of the bone surfaces of the crypt by scanning electron microscopy and the ultrastructure of the cells on alveolar bone surfaces during tooth eruption. Above the developing crown where the eruption pathway forms, the bone surface is a pitted sheet, the characteristic topographic feature of bone resorption; between the crown and the mandibular canal, the bone surface has numerous interconnecting trabeculae. Transmission electron microscopy of bone cells lining the eruption pathway area of the crypt showed numerous osteoclasts with adjacent mononuclear cells. Both cell types contained specific, membrane-bound cytoplasmic vesicles shown by the work of others to be characteristic features of osteoclasts and their precursors. Basal trabeculated bone in the crypt was covered by plump osteoblasts. These data show that the metabolic events in alveolar bone associated with tooth eruption have the appropriate cellular and bony surface correlates and that the suspected control of alveolar bone resorption by the dental follicle may be mediated by its recruiting and directing to adjacent bone surfaces the mononuclear precursors of osteoclasts.     

Structure of alveolar bone during tooth eruption in the dog: preliminary findings

The structure of the alveolar bone during the tooth eruption in the young dog mandibles was investigated by microradiographic and polarized light techniques. Around the first erupting molar root a trabecular network of primary alveolar bone, less mineralized than the surrounding cortical one, was found. Numerous calcified spicules parallel one to others radiate out the spongiosa near the periodontal ligament. The collagen fiber bundles of the alveolar, woven, bone are continuous with the periodontal ligament ones. This finding indicates that the alveolar bone increases by ossification of the periodontal ligament. Therefore the latter is the forming alveolar bone substratum. The trabeculae of the occlused premolar alveolar bone are ticker and more mineralized. This modification of the occlused tooth alveolar bone could be related to the occlusal stresses.     

The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog

Previous studies from our laboratories have shown that premolar eruption in dogs depends upon the presence of the dental follicle and is independent of root or crown growth or attachment to the oral epithelium. The present study is an analysis of the cellular composition of the dental follicle and the cellular investment of the adjacent walls of the bony crypt before and during eruption of the third and fourth mandibular permanent premolars in young beagle dogs. Four premolar follicles and their adjacent bony crypts were examined at 2-week intervals over 12 weeks before and during eruption of these teeth. Tissues were removed, fixed, processed, and oriented so that each follicle and the adjacent crypt wall could be reproducibly examined in vertical and horizontal planes. Mononuclear cells with abundant cytoplasm, euchromatic nuclei, and prominent nucleoli were present in juxtavascular location in the coronal part of the dental follicle; and these cells increased in number immediately preceding and during tooth eruption in parallel with an increase in osteoclasts on the adjacent crypt wall. These data are interpreted to mean that the coronal part of the dental follicle may coordinate the alveolar bone resorption required for tooth eruption by attracting and directing to the crypt wall a population of mononuclear cells, which either become osteoclasts and/or direct osteoclastic activity during tooth eruption.     

Effects of prostaglandin E2 on alveolar bone resorption during orthodontic tooth movement

Twenty Sprague-Dawley rats weighing 280-300 g were divided into two groups of ten animals each. They were treated by daily submucosal injections of 50 micrograms prostaglandin E2 (PGE2) per kilogram body weight into the region below the apex of the left first maxillary molar (experimental), or vehicle into the region below the apex of the right first molar (control), for a period of 5 days. The animals of the first group were sacrificed immediately following the treatment period, while those of the second group were sacrificed 5 days after the treatment period. Twenty-two hours prior to sacrifice, a piece of latex orthodontic elastic was secured to the adjacent area between the first and second maxillary molars of both sides of each rat by using two mosquito hemostats. The periodontal ligament (PDL) mesial to the mesiobuccal root of the first maxillary molar was assayed for changes in PDL cell factors. The results showed that immediately following the 5-day treatment period the left PDL had a significant decrease in the total number of fibroblasts and a significant increase in the total number of both osteoclasts and nuclei per osteoclast, while no significant changes in the osteoblasts when compared with those of the right control PDL. The left PDL of animals which were sacrificed 5 days after the treatment period revealed a significant decrease in the number of total fibroblasts and only a slight decrease in both numbers of total osteoclasts and total nuclei per osteoclast, but again no significant changes in osteoblasts when compared with those of the right control PDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

A morphometric study of bone and tooth volumes in the pipid frog Xenopus laevis (Daudin), with comments on the importance of tooth resorption during normal tooth replacement

Volumetric estimations of teeth and bone on serial sections using a semiautomatic image analyzer indicate that, in the polyphyodont dentition of the pipid anuran Xenopus laevis (Daudin), the mean volume of the dentine composing the teeth is about 23.5% of the volume of the supporting maxillae and premaxillae. During tooth replacement, osteoclasts resorb up to 98% of the dentine. Teeth may be resorbed rather than shed in order to conserve tooth constituents because, if shedding of complete teeth did occur, a quantity of calcified tissue equal to perhaps 45 times the volume of the bone of the upper jaw might be lost over an animal's projected life span of about 13-15 years.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are characteristics of the osteosclerotic mutation which suggest that it is a unique osteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

Structural and functional relations between the connective tissue and epithelium of enamel organ and their role during enamel maturation

Journal of Molecular Histology - The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel...     

Conservation and variation in enamel protein distribution during vertebrate tooth development

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.     

Structure of the periodontal ligament during tooth eruption in the dog: descriptive aspects

Serially stained uncalcified sections of young dog mandibles were examined to study the structure of the periodontal ligament of the erupting first right molar. The periodontal ligament around tooth crown presents three zones. The first, near the dental follicle, is a tick layer of parallel collagen bundles with numerous flattened fibroblasts. The second, intermediate, contains a blood vessels network, particularly veins and capillaries. The third, outer, is occupied by a continuous layer of osteoclasts and osteoblasts. Also the periodontal ligament around the tooth presents three layers, the outer and the intermediate rich of cells more than the inner. Particularly, the outer layer shows numerous osteoblasts surrounding the developing trabeculae of the alveolar bone and the collagen fiber bundles of the periodontal ligament. These penetrate into the trabeculae and appear similar to the osteoid layer. These results indicate that the alveolar bone increases by ossification of the connective tissue of the periodontal ligament.     

A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.     

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling.     

Acoustic microscope study of the elastic properties of fluorapatite and hydroxyapatite, tooth enamel and bone.

Measurements of Rayleigh velocity and attenuation were taken in single mineral crystals of hydroxyapatite and fluorapatite at angular intervals relative to their c axes, using an acoustic microscope. These results are compared with the values that were calculated using the elastic constants of apatite from Yoon and Newnham [(1969) Am. Miner. 54, 1193-1197.] and Katz and Ukraincik [(1971) J. Biomechanics 4, 221-227.]. The slowness curves of various wave modes are plotted and discussed in relation to cross-coupling effects that were found to cause instability in measurements of attenuation for the c axes direction. Velocity measurements were taken in specimens of tooth enamel and bone. Here comparisons are made with the values that were calculated by modelling the 'z' scan response of the microscope, using published data for the elastic and acoustic properties. Comparisons are also made with the measurements on single crystals, since apatite is a major component of enamel and bone.     

Effect of calcium ions on human calcitonin. Possible implications for bone resorption by osteoclasts

Calcium ions (Ca²⁺) are indispensable for life and are involved in important physiological actions, which makes maintaining a constant level of blood Ca²⁺ essential. Ca²⁺ is mainly stored in bones which serve as a reservoir and its homeostasis is modulated by various hormones. Human calcitonin (hCt) is a small peptide hormone that exerts its physiological effect on Ca²⁺ metabolism by means of osteoclast-mediated bone resorption inhibition. Most of these actions are mediated through peptide/receptor interaction that acts via a second messenger. However, in vitro studies have shown that hCt can interact with membrane lipids to form ion channels in membrane models. This ability is due to the peptide’s secondary structure and aggregation state, that can be modulated by different molecules. In our study, we evaluated the effect of Ca²⁺, at different concentrations, both on the hCt ion channel incorporated into a planar lipid membrane made up of phosphatidylcholine containing 15 % phosphatidylglycerol and on the secondary structure of hCt in an aqueous environment. Ca²⁺ is able to interact with the hCt peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both by increasing hCt channel frequency and in solution promoting α-helix formation, that counteracts the fibrillating process. These experimental observations, suggesting that hCt senses Ca²⁺ concentration variations, strengthen the hypothesis that channel formation represents an extra source of Ca²⁺ entry into osteoclasts in addition to the well-known interaction of the monomer with the specific receptor.