Regulated gene expression dictates enamel structure and tooth function.

Enamel is a complex bioceramic tissue. In its final form, enamel is a reflection of the unique molecular and cellular activities occurring during organogenesis. From the ectodermal origins of ameloblasts, their gene activity and protein expression profiles exist for the sole purpose of producing a mineralized shell, almost entirely devoid of protein, deposited over the 'bone-like' dentine. The interface between enamel and dentine is referred to as the dentine enamel junction and it is also unique in its biology. This review article is narrow in its scope. We restrict our review to selected advances in our understanding of the genetic, molecular and structural aspects of enamel biology. We present a model of enamel formation that relates gene expression to the assembly of an extracellular protein matrix that in turn controls the structural hierarchy and mechanical aspects of enamel and the tooth organ.     

Osteonectin is a minor component of mineralized connective tissues in rat

Osteonectin is a major glycoprotein of porcine and bovine bones and teeth that is found associated with hydroxylapatite crystal surfaces. From the ability of osteonectin to bind calcium ions, it has been proposed as a possible nucleator of hydroxylapatite crystal formation. Analysis of hydroxylapatite-bound proteins of rat bone and dentine, however, has revealed that osteonectin represents only 2.5 +/- 1.5% of the hydroxylapatite-bound protein in long bones, 0.9 +/- 0.5% in calvariae, and less than 0.1% in incisor dentine of animals of different ages. Further, in vivo pulse-chase studies carried out in young adult rats have shown osteonectin to be synthesized at low levels in these tissues. Similarly, low levels of osteonectin were synthesized by rat calvarial cells in vitro. In contrast, fibroblastic cells from periodontal ligament and gingiva synthesized significantly greater amounts of osteonectin. These studies indicate that the low quantities of osteonectin in rat mineralized tissues are a consequence of low rates of formation rather than being due to rapid turnover. The virtual absence of osteonectin in incisor dentine correlates with the lack of peritubular dentine in rat, whereas the low osteonectin content of rat bones may reflect differences in their structure and biophysical properties compared with bones of larger mammals.     

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.     

Visualization of glycosaminoglycans in rat incisor extracellular matrix using a hyaluronidase-gold complex

Summary The enzyme-gold technique was used on dental tissues. Hyaluronidase was complexed with gold, and ultrathin sections of rat incisors were incubated with the hyaluronidase-gold complex to localize chondroitin-sulphate and hyaluronic acid at the ultrastructural level. The hyaluronidase-gold complex was, found in predentine and dentine, especially at the mineralization front, in interglobular spaces and around dentinal canaficuli. The very young enamel was labelled, but not the later stages of formation. This method allowed a very precise localization of hyaluronic acid and/or chondroitin sulphate in rat incisors extracellular matrices. These findings support the important role of glycosaminoglycans in dentine mineralization.     

Production and characterization of antibodies against murine dentine phosphoprotein.

Experiments were designed to produce and characterize a polyclonal antibody directed against mouse dentine phosphoprotein, the major non-collagenous protein of the dentine extracellular matrix. Dental extracellular matrix proteins from 2-day-postnatal Swiss-Webster-mouse tooth organs were extracted with 0.5 M-acetic acid, followed by 4 M-guanidinium chloride/0.5 M-EDTA. Mouse dentine phosphoprotein yields were further increased by precipitation with 1 M-CaCl2. Final purification was achieved by excising and eluting dentine phosphoprotein polypeptide bands from preparative sodium dodecyl sulphate/urea/polyacrylamide gels. Mouse dentine phosphoprotein is a single component of approx. 72 kDa and has a characteristic amino acid composition of 33% aspartic acid and 55% serine/phosphoserine. A polyclonal antibody was raised in rabbits against purified mouse dentine phosphoprotein and was shown to be monospecific by enzyme-linked immunoabsorbent, dot-immunobinding and 'Western transfer' assays. This antibody was used to detect the expression and localization of dentine phosphoprotein in 1-day-postnatal mouse tooth organs. This antigen was localized intracellularly within the monolayer of odontoblasts, which line the perimeter of the dental papilla mesenchyme, and within the odontoblastic cell processes, which traverse the predentine matrix. Newly forming mineralized dentine matrix was also cross-reactive with the dentine phosphoprotein specific antibody. The non-mineralized predentine matrix did not contain any detectable cross-reactive antigens.     

Comparison of tryptophan-labeled constituents of developing rodent molar enamel matrix, non-enamel occlusal cusp, Hertwig's epithelial root sheath, and presumptive root furcation regions: light microscopic autoradiography

During the process of organogenesis involving the developing rodent molar and incisor tooth organs, novel gene products termed enamel proteins are expressed by ectodermally-derived enamel organ epithelia at precise times and positions within the course of morphogenesis. The present studies were designed to identify the relative distribution of tryptophan-labeled, non-collagenous, epithelial-derived proteins associated with rat maxillary first molar crown (M') and initial root formation. Our experimental strategy was to utilize semi-quantitative autoradiography methods to compare and contrast the distribution of silver grains resulting from tryptophan incorporation into developing postnatal pups associated with enamel matrix, non-enamel occlusal cusp, Hertwig's Epithelial Root Sheath (HERS), and presumptive root furcation regions of M'. Five-day-old Wistar rats were injected with 14C-labeled tryptophan. Four animals were sacrificed at 15 minutes and then at 1, 2, 4, and 24 hour intervals following the administration of this essential aromatic amino acid. Following fixation and subsequent processing for autoradiography, semiquantitative analyses were performed of the silver grain distribution localized within selected regions of the developing M' tooth organs. All enamel organ epithelia were found to incorporate tryptophan and silver grains were identified (above background) in the extracellular matrices (ECM) of the enamel matrix, non-enamel occlusal cusp adjacent to the inner enamel epithelia, and the ECM (2-4, micron) adjacent to presumptive root furcation and HERS regions. Tryptophan incorporation was not significant in the odontoblasts or dentine ECM of the crown or forming presumptive root regions. These results support the hypothesis that inner enamel epithelia associated with rat molar crown formation, as well as HERS, synthesize tryptophan-labeled, non-collagenous, ECM molecules. We speculate that HERS participates in root development by possibly producing non-collagenous proteins for intermediate cementum formation.     

Ribonuclease-RNAase inhibitor complex from rat testis. Purification of the RNAase inhibitor

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicle-associated nucleation.     

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.     

In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate–fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies. (J Histochem Cytochem 57:615–622, 2009)     

Amelogenesis in vitro: a model for studies of epithelial postsecretory processing during tissue-specific extracellular matrix biomineralization

The extracellular matrix (ECM) of developing mammalian enamel comprises a complex of unusual epithelial-derived proteins, which appear to function in concert to initiate and propagate tissue-specific biomineralization. Following enamel protein synthesis by ameloblast cells within the enamel organ, the subsequent steps of posttranslational modification, secretion, postsecretory processing and eventual removal of these proteins from forming enamel are largely unknown. To address this issue we have designed studies to investigate the hypothesis that enamel proteins are removed from enamel and translocated into the vasculature as relatively high-molecular-weight components. We examined enamel proteins recovered from serumless medium during prolonged organ culture of mouse capstage mandibular first molars. By 21 days in vitro the tooth crown formed and dentine and enamel biomineralization were apparent. At 31 days, explants retained metabolic activity and the enamel matrix showed extensive transformation. Immunologically identified enamel proteins of 26-18 k Da were produced by cultured tooth organs, translocated from tooth explants to the culture medium, recovered from the medium and then compared to control enamel protein from in vivo preparations. Comparable postsecretory processing of the 26-k Da amelogenin protein was observed in vitro and in vivo. We speculate that the pathway reported in the present studies is comparable to the processing of the enamel protein polypeptides of the maturing enamel which occurs in vivo. The in vitro organ culture model described in this report provides an approach with which to investigate the molecular events associated with epithelial-derived postsecretory processing of ECM molecules associated with tissue-specific biomineralization.     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Summary Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicleassociated nucleation.     

5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes.

A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.     

Scanning electron microscopy and dentinal permeability analysis of smear layer.

The aim of the present study was to evaluate the surface morphology and the permeability of dentine after different acid treatments: polyacrylic acid, maleic acid, phosphoric acid and saline solution as control. Dentine permeability was expressed as hydraulic conductance. All the acid treatments removed the smear layer and increased the dentine permeability.     

Iodopolatinate visualization of phospholipids in rat incisor predentine and dentine,compared with malachite green aldehyde

The iodoplatinate (IP) reaction, a selective method for visualization of phospholipids, was applied to the predentine and dentine of rat incisors and compared with malachite green aldehyde (MG) fixation/staining. Spot tests indicated (1) that IP specifically stains phospholipids, but not amino acids, displaying as do phospholipids, quaternary ammonium groups; and (2) phosphatidylserine and sphingomyelin were also stained by MGA. Although this reagent is known to interact with phosphorus, phosphoproteins remained unstained. In the rat incisor, an IP-positive network including granules and thin filaments was seen in predentine in the inter-collagen spaces, in many cases closely associated with collagen fibres and their periodic striations. In dentine, positively stained needle-like structures were located along individual collagen fibres, or at the surface of groups of collagen fibres. This staining pattern was unchanged on sections of material pretreated with acetone, whereas the staining was abolished or markedly reduced when the samples were treated either with chloroform/methanol or phospholipase C prior to the IP reaction. Pretreatment of the samples with hyaluronidase promoted subsequent diffusion of the staining. A very similar staining pattern was observed with MGA, in accordance with earlier reports. The present findings validate the histochemical results reported previously on the distribution and potential role(s) of phospholipids in dentine biomineralization.     

Cell-free translation of messenger RNAs of embryonic tooth organs: Synthesis of the major extracellular matrix proteins

In order to understand the regulation of embryonic mammalian enamel and dentine extracellular matrix protein synthesis, the biological activity of embryonic rabbit molar tooth organ messenger RNAs has been examined. Total RNA was extracted from 26-day embryonic tooth organs and fractionated by chromatography on oligo(dT)-cellulose. Replicate samples were fractionated on sucrose density gradients and the poly(A)-containing distribution determined using a poly(U) ${}^3$H assay. The poly(A)-containing fractions stimulated ${}^3$H-proline incorporation 10-fold in wheat germ cell-free extracts. Analysis of the labelled reaction products on sodium dodecyl sulphate-polyacrylamide gels revealed seven major peaks, one co-migrating with procollagen alpha chains (circa 145,000 daltons) and the others migrating slightly faster than the various extracellular matrix proteins which characterize amelogenesis and dentinogenesis. Purified collagenase digestion of the cell-free reaction products eliminated the 145,000 dalton procollagen-like polypeptide. This is the first demonstration of the isolation of embryonic tooth organ messenger RNAs and provides an experimental approach by which to study the regulation of extracellular matrix formation during tooth morphogenesis. We predict that the non-collagenous proteins synthesized $\text{in vitro}$ represent enamel proteins, alkaline phosphatase, dentine phosphoproteins and proteins associated with proteoglycans.     

Multiphoton imaging of the dentine‐enamel junction

Multiphoton microscopy has been used to reveal structural details of dentine and enamel at the dentin‐enamel junction (DEJ) based on their 2‐photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). In dentine tubule 2PEF intensity varies due to protein content variation. Intertubular dentin produces both SHG and 2PEF signals. Tubules are surrounded by a thin circular zone with a lower SHG signal than the bulk dentine and the presence of collagen fibers perpendicular to the tubule longitudinal axis is indicated by strong SHG responses. The DEJ appears as a low intensity line on the 2PEF images and this was never previously reported. The SHG signal is completely absent for enamel and aprismatic enamel shows a homogeneous low 2PEF signal contrary to prismatic enamel. The SHG intensity of mantle dentine is increasing from the dentine‐enamel junction in the first 12 μm indicating a progressive presence of fibrillar collagen and corresponding to the more external part of mantle dentine where matrix metallo‐proteases accumulate. The high information content of multiphoton images confirms the huge potential of this method to investigate tooth structures in physiological and pathological conditions. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Regulation of cellular retinol binding protein type II by 1,25-dihydroxyvitamin D3.

Previously we purified and sequenced an 18-kDa chick duodenal protein that was modulated by 1,25-dihydroxyvitamin D3. The N-terminus of this protein has striking sequence homology to cellular retinol binding protein type II (CRBP II). Furthermore, this purified chick protein binds retinol. Antibodies have now been generated to the chick protein and used for immunoblot analysis to demonstrate that the chick protein has molecular weight, tissue distribution, and subcellular localization similar to rat CRBP II. These antibodies also cross-reacted with rat CRBP II. Antibodies to rat CRBP II cross-react with the chick protein. Northern analysis using a cDNA probe for rat CRBP II showed a single 860 base pair mRNA in both chick and rat intestinal RNA preparations. These results demonstrate that the 1,25-dihydroxyvitamin D3 modulated protein in chick embryonic organ culture is chick CRBP II. Pulse-chase experiments in chick embryonic duodenal organ culture strongly suggest that 1,25-dihydroxyvitamin D3 markedly decreases the synthesis of CRBP II, while not changing the degradation rate. The concentration of 1,25-dihydroxyvitamin D3 required for the decrease in CRBP II synthesis is approximately that required to stimulate calcium uptake into embryonic chick duodenal organ cultures.     

Green fluorescent protein-transgenic rat: a tool for organ transplantation research

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.