[Biological dental implant: myth or reality?]. / L'implant dentaire biologique : mythe ou réalité ?

The currently available options for tooth-loss are prostheses, implants, or surgery (auto-transplantation). They all have their limitations. The emergence of tissue engineering, 15 years ago, was made possible by a better knowledge of the various stages of dental development, and the mastery of stem cell differentiation. It opened a new alternative approach for tooth regeneration. Even if animal experiments have demonstrated that it was possible to obtain a biological tooth from stem cells, two major issues remain to be discussed. Is it possible to use induced pluripotent stem cells instead of embryonic stem cells, which raise an ethical problem? Is it possible to reproduce a dental crown with an adapted shape and colour? Or should we consider the simpler creation of a biological root secondarily covered by a ceramic prosthesis? Our study mentions the main landmarks and the key cells involved in the embryological development of the tooth, establishes a mapping and a list of the various types of stem cells. It details the various methods used to create a biological implant.

HUGO (FNDC3A): a new gene overexpressed in human odontoblasts.

Previously, we established a subtractive cDNA library enriched in odontoblast-specific genes and hypothesized that new, previously unidentified, markers would be present, associated with the odontoblast phenotype. In this paper, we report the first characterization of a new gene we have named HUGO, and its associated deduced protein sequence. This gene expression is under the control of two alternative promoters, resulting in the synthesis of two proteins, one of which, HUGO2, is included in the other, HUGO1. HUGO proteins are mainly composed of a proline-rich region at the N-terminus, 8 type III-fibronectin modules, and a transmembranous helix at the C-terminus. In odontoblasts, the proteins are located in Golgi vesicles. However, they display a broader expression pattern, since they are also expressed by nerve fibers in the dental pulp and other tissues (e.g., trachea, brain, kidney), as demonstrated by immunohistochemistry and qPCR, respectively. Their location in odontoblasts suggests a role in collagen and glycosaminoglycan synthesis.

Cloning, sequencing, and expression of the amelogenin gene in two scincid lizards.

Our knowledge of the gene coding for amelogenin, the major enamel protein, is mainly based on mammalian sequences. Only two sequences are available in reptiles. To know whether the snake sequence is representative of the amelogenin condition in squamates, we have studied amelogenin in two scincid lizards. Lizard amelogenin possesses numerous conserved residues in the N- and C-terminal regions, but its central region is highly variable, even when compared with the snake sequence. This rapid evolution rate indicates that a single squamate sequence was not representative, and that comparative studies of reptilian amelogenins might be useful to detect the residues which are really important for amelogenin structure and function. Reptilian and mammalian enamel structure is roughly similar, but no data support amelogenin being similarly expressed during amelogenesis. By performing in situ hybridization using a specific probe, we showed that lizard ameloblasts express amelogenin as described during mammalian amelogenesis. However, we have not found amelogenin transcripts in odontoblasts. This indicates that full-length amelogenin is specific to enamel matrix, at least in this lizard.

A substractive PCR-based cDNA library from human odontoblast cells: identification of novel genes expressed in tooth forming cells.

Odontoblasts are highly specialized cells aligned at the edge of the dental pulp. As a step towards understanding the complex mechanisms underlying their terminal differentiation, the gene expression pattern was examined in human cultured odontoblast cells. Suppression substractive hybridization (SSH) was used to establish a substracted cDNA library specific for human odontoblasts. For this purpose, cDNAs from human cultured fibroblastic pulp cells were substracted to cDNA from human cultured odontoblasts. The nucleotide sequence of 154 substracted cDNA clones was determined. We identified 130 preferentially expressed gene fragments in odontoblasts as compared with the fibroblastic pulp cells. Ten of them were already identified in odontoblasts such as DSPP, BSP, enamelysin and Col1A1. We confirmed their overexpression by RT-PCR on the cultured cells and in vivo by in situ hybridization on human molars. Another 64 clones corresponded to known genes. Among them, two clones were of particular interest: reelin, which was first detected in the brain and osteoadherin, which was first located in bone. Fifty-six clones were unknown genes even though 82% matched expressed sequence tags or genomic clones. A reverse Northern dot blot showed that 96% of them were overexpressed at different rates in cultured odontoblasts. These latest results indicate that there are still unknown genes that are associated with the control of the odontoblast phenotype. Thus, cloning of odontoblast differentiation-associated genes not only opens up new methods of elucidating the normal development but also the recruitment of odontoblasts when required to initiate repair of dentin.

Sequential expression of matrix protein genes in developing rat teeth.

Tooth organogenesis is dependent on reciprocal and sequential epithelial-mesenchymal interactions and is marked by the appearance of phenotypic matrix macromolecules in both dentin and enamel. The organic matrix of enamel is composed of amelogenins, ameloblastin/amelin, enamelins and tuftelin. Dentin is mainly composed of type I collagen, but its specificity arises from the nature of the non-collagenous proteins (NCPs) involved in mineralization, phosphophoryn (DPP), dentin sialoprotein (DSP), osteocalcin, bone sialoprotein and dentin matrix protein-1 (Dmp1). In this paper, we studied the pattern of expression of four mineralizing protein genes (type I collagen, amelogenin, DSPP and osteocalcin) during the development of rat teeth by in situ hybridization on serial sections. For this purpose, we used an easy and rapid procedure to prepare highly-specific labeled single-stranded DNA probes using asymmetric polymerase chain reaction (PCR). Our results show that type I collagen is primarily expressed in polarizing odontoblasts, followed by the osteocalcin gene expression in the same polarized cells. Concomitantly, polarized ameloblasts start to accumulate amelogenin mRNAs and transiently express the DSPP gene. This latter expression switches over to odontoblasts whereas mineralization occurs. At the same time, osteocalcin gene expression decreases in secretory odontoblasts. Osteocalcin may thus act as an inhibitor of mineralization whereas DSP/DPP would be involved in more advanced steps of mineralization. Amelogenin and type I collagen gene expression increases during dentin mineralization. Their expression is spatially and temporally controlled, in relation with the biological role of their cognate proteins in epithelial-mesenchymal interactions and mineralization.

Expression of the small leucine-rich proteoglycan osteoadherin/osteomodulin in human dental pulp and developing rat teeth.

Because the extracellular matrices of dentin and bone are composed mainly of type I collagen, their characteristics are determined by the nature of noncollagenous proteins (NCPs). Among these NCPs, some proteoglycans (PGs) belong to the small leucine-rich proteoglycans (SLRPs). Recently, osteoadherin (OSAD) has been described as a new member of this family, that is expressed by mature bovine osteoblasts. Here, we report the expression of OSAD messenger RNA (mRNA) in human dental tissues and during the development of rat molars, using in situ hybridization. For this purpose, we constructed a probe for OSAD mRNA transcripts from human odontoblast cells cultured in vitro. Our results indicate that the mature human odontoblasts overexpress the OSAD gene as compared with cells present in the pulp core. In rat developing molars, mRNA transcripts were first detected in alveolar bone in 19-day-old embryos. At the same age, no signal was detected in any cell of the first molar. In more mature teeth (newborn and 2-day-old rats), OSAD expression starts in the polarized odontoblasts and increases in the secretory and mature odontoblasts, respectively. Interestingly, a similar pattern of expression was observed in the ameloblast layer responsible for the deposition of enamel mineralized matrix. Together, these results lead us to speculate that OSAD may be implicated in biomineralization processes.

Evaluation of the biocompatibility of a Ni-Cr-Mo dental alloy with human gingival explant culture in vitro: morphological study, immunodetection of fibronectin, and collagen production.

This study was undertaken to evaluate the biocompatibility of a Ni-Cr-Mo dental casting alloy by an in vitro explant culture method. Through ultrastructural features, the immunolabelling of fibronectin, and the immunodetection of type I and III collagen production, we investigated the behaviour of gingival cells (both fibroblasts and epithelial cells) in contact with the alloy in comparison with control cultures. Our results indicate that cultured gingival cells present a well preserved ultrastructure and synthesized fibronectin (the main glycoprotein involved in adhesion to substrates). The alteration of collagen production concerned only type III collagen which decreased significantly in the cultures on the dental alloy.

An image analysis method for the study of cell adhesion to biomaterials.

This fluorescence image analysis method for the quantitative determination of cell adhesion on biomaterials allows bone cells labelled with propidium iodide to be counted automatically, directly on their support. The reliability of the estimation by fluorescence image analysis was validated by comparison with visual counting and with results obtained by an electronic particle counter. In this way it was possible to demonstrate that the adhesive properties of bone cells are dependent on the type of substrate--enstatite (MgO, SiO2, CaO-P2O5-Al2O3), Thermanox (modified polyethyleneterephthalate), or glass. In contrast, the spread of the cell cytoplasm, labelled with fluorescein isothiocyanate and measured by image analysis, does not vary significantly according to the substrate. The characterisation by SKIZ tessellation of the spatial cell arrangement shows that the bone cells have a random organisation on Thermanox and glass, whereas they form aggregates on enstatite.

Expression and localization of TREK-1 K+ channels in human odontoblasts.

During tooth development, odontoblasts are the cells that form dentin and possibly mediate early stages of sensory processing in teeth. It is suggested that ion channels assist in these events. Indeed, mechanosensitive potassium currents, transducing mechanical stimuli into electrical cell signals, have been previously recorded in the human odontoblast cell membrane. Here, we show by RT-PCR that the mechanosensitive potassium channel TREK-1 (a member of the two-pore-domain potassium channel family) is overexpressed in these cultured cells compared with pulp cells in vitro. In situ hybridization showed that transcripts are detected in the odontoblast layer in vivo. The use of antibodies shows that TREK-1 is strongly expressed in the membrane of coronal odontoblasts and absent in the root. This distribution is related to the spatial distribution of nerve endings identified by labeling of the low-affinity nerve growth factor (NGF) receptor (p75(NTR)). These results demonstrate the expression of TREK-1 in human odontoblasts in vitro and in vivo.

Effects of TGFbeta1 on dental pulp cells in cultured human tooth slices.

Transforming growth factor-beta1 (TGF beta1) is a potent modulator of tissue repair in various tissues. To analyze its role during human dental repair, we used thick-sliced teeth cultured as described previously (Magloire et al., 1996). The supply of TGF beta1 to the pulp tissue was accomplished by means of a small tube glued onto the dentin. We show that this device allowed the growth factor to diffuse locally through dentinal tubules and to bind to the cells present in the coronal pulp opposite the TGF beta1-delivery tube. The tube was filled with 20 ng/mL TGF beta1, and slices were cultured for 4 days. Results show a preferential accumulation of cells in the odontoblastic and subodontoblastic layers in the vicinity of the tube. Cell proliferation increased in the subodontoblastic layer and in the underlying pulp, and BrdU-positive cells were abundant around the blood vessels. TGF beta1 induced type I collagen production by the odontoblastic/subodontoblastic/pulp cells in the stimulated zone, as demonstrated by in situ hybridization. These results suggest that TGF beta1 could be directly involved in the regulation of cell proliferation, migration, and extracellular matrix production in the human dental pulp and eventually in the repair process occurring after tooth injury.

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Expression of fibronectin and type I collagen by human dental pulp cells and gingiva fibroblasts grown on fibronectin substrate.

Specific antibodies and indirect immunoperoxidase labelling were used to study the intracellular production of collagen and fibronectin by cells grown on fibronectin-coated glass; the same cell populations seeded on uncoated glass were used as controls. Strong intracellular staining for type I collagen was seen in all cases, but immunostaining for fibronectin was very faint or negative in both gingival and pulp cells grown on the fibronectin substrate, in contrast to control cells. Thus, fibronectin substrate inhibited fibronectin synthesis by the cultured cells, but did not seem to influence type I collagen synthesis.

Morphological and immunocytochemical characterization of cultured rat incisor cervical epithelial cells.

Epithelial cells from the cervical loop of the rat incisor were isolated by co-culture of apical explants with growth-arrested 3T3 fibroblasts. The epithelial phenotype of the expanding outgrowths was confirmed 10 days after the seeding of the explants by phase-contrast microscopy and immunocytochemical identification of cytokeratins. After 3 weeks in culture, the epithelial cells covered the entire surface of the coverslips and were then passaged. Subcultures gave rise to a confluent sheet within 10-12 days. Light and electron microscopy showed that confluent cervical epithelial cells generally reconstituted a bi-layered structure similar to Hertwig's epithelial sheath. Epithelial cells from the rat palate, cultured and subcultured according to the same procedure, organized themselves in 5-6 cell layers, the upper cells having generally a squamous morphology. Synthesis of extracellular matrix molecules by rat incisor cervical epithelial cells was studied with specific antibodies. These cells failed to produce type I collagen, but synthesized all the major basement membrane components (type IV collagen, laminin, heparan sulphate proteoglycan and fibronectin). These observations suggest that the culture conditions allowed the reconstitution of a typical Hertwig's epithelial sheath by rat incisor cervical epithelial cells.

Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium hydroxide-containing cement.

The cement produced microcrystals of calcite by reaction with culture medium supplemented with calf serum. Human dental pulp cells seeded on such a substrate preferentially adhered and aggregated around the microcrystals. Immunofluorescence and immunogold labelling revealed a high affinity of serum fibronectin molecules for the calcite crystals. At 4 weeks in culture, the cells had various features of differentiated odontoblasts, notably nuclear polarization, typical appearance of the Golgi apparatus, synthesis of type I collagen and absence of type III, and apical accumulation of actin and vimentin. These cells also elaborated a collagenous extracellular matrix which did not mineralize.

Ultrastructural immunohistochemical study of interstitial collagenous components of the healthy human keratinized mucosa surrounding implants.

The aim of this study was to investigate the ultrastructural and immunohistochemical organization of the main collagenous components of healthy human keratinized mucosa surrounding endosseous implants. Eight patients with completely edentulous mandibles were selected. Four endosseous implants were placed in the mandible of each patient, connected with a bar to support a complete overdenture, and loaded 4 months later. Two years after placement, biopsies of surrounding soft tissue, including the sulcular and junctional epithelium with the underlying and supracrestal connective tissue, were routinely prepared for standard electron microscopy and for ultrastructural immunolabeling of Types I, III, and IV collagen. The connective tissue located under the junctional epithelium comprised Types I and III collagen, whereas the supracrestal connective tissue was composed mainly of Type I collagen. Type IV collagen was located exclusively in the basement membrane of the junctional epithelium.

The biological effects of fluoride on tooth development: possible use of cell culture systems.

The action of fluoride on tooth development and the observed side-effects of fluorosis are not yet fully understood. The authors briefly review the literature relating to hypotheses on dental fluorosis and describe in vitro cell culture techniques which could help to clarify the biological effect of fluoride on dental pulpal cells.

Cell differentiation and matrix organization in engineered teeth.

Embryonic dental cells were used to check a series of criteria to be achieved for tooth engineering. Implantation of cultured cell-cell re-associations led to crown morphogenesis, epithelial histogenesis, organ vascularization, and root and periodontium development. The present work aimed to investigate the organization of predentin/dentin, enamel, and cementum which formed and mineralized after implantation. These implants were processed for histology, transmission electron microscopy, x-ray microanalysis, and electron diffraction. After two weeks of implantation, the re-associations showed gradients of differentiating odontoblasts. There were ciliated, polarized, and extended cell processes in predentin/dentin. Ameloblasts became functional. Enamel crystals showed a typical oriented arrangement in the inner and outer enamel. In the developing root, odontoblasts differentiated, cementogenesis occurred, and periodontal ligament fibroblasts interacted with the root surface and newly formed bone. The implantation of cultured dental cell re-associations allows for reproduction of complete functional differentiation at the cell, matrix, and mineral levels.

Primary cilia of odontoblasts: possible role in molar morphogenesis.

A primary cilium, a sensory organelle present in almost every vertebrate cell, is regularly described in odontoblasts, projecting from the surfaces of the cells. Based on the hypothesis that the primary cilium is crucial both for dentin formation and possibly in tooth pain transmission, we have investigated the expression and localization of the main cilium components and involvement of the OFD1 gene in tooth morphogenesis. Odontoblasts in vitro express tubulin, inversin, rootletin, OFD1, BBS4, BBS6, ALMS1, KIF3A, PC1, and PC2. In vivo, cilia are aligned parallel to the dentin walls, with the top part oriented toward the pulp core. Close relationships between cilium and nerve fibers are evidenced. Calcium channels are concentrated in the vicinity of the basal body. Analysis of these data suggests a putative role of cilia in sensing the microenvironment, probably related to dentin secretion. This hypothesis is enhanced by the huge defects observed on molars from Ofd1 knockout mice, showing undifferentiated dentin-forming cells.

Expression and localisation of alphav integrins in human odontoblasts.

Integrin alphabeta heterodimers mediate adhesion to the extracellular matrix and at cell-cell contacts and initiate intracellular signalling cascades in response to a variety of inductive factors. Apart from the expression of alphavbeta3 that we have previously reported, little is known about the expression of integrins in odontoblasts. Here, we investigated the expression of alphav-binding beta integrin subunits in healthy human dental pulp in vivo and in odontoblasts differentiated in vitro. Reverse transcription/polymerase chain reaction analysis revealed the expression of alphav, beta1, beta5 and beta8 integrin mRNA, but not beta6, in whole pulp cells. Flow cytometry showed that the alphav and beta1 subunits were the most intensely expressed. Immunohistochemistry demonstrated that the beta1 subunit was localised in newly differentiated odontoblasts in the root and in mature odontoblasts in the crown, including their intradentinal cell processes. The alphav chain was predominantly expressed by mature odontoblasts and alphavbeta5 was only observed in mature odontoblasts. In vitro differentiated odontoblasts expressed genes for alphav, beta1 and beta5, but not for beta6 and beta8. A comparison of integrin profiles between cultured pulp cells and in vitro differentiated odontoblasts revealed that odontoblast maturation was characterised by a significant increase in the expression of alphav and beta1 subunits and alphavbeta5 integrin. The beta8 subunit was detected in nerve cells only. Histological analysis of teeth from alphav knockout mice showed no obvious structural modification in the odontoblast layer. Thus, human mature odontoblasts express alphavbeta3, alphavbeta5 and perhaps alphavbeta1 integrins, with the possible presence of alpha-beta1 pairs. The roles that these molecules play in the exchange of information throughout the odontoblast layer remain to be determined.

Immunohistochemical study of types I, III and IV collagen in diseased human gingiva of patients with rapidly progressive periodontitis: a light and electron microscopic study.

The distribution of type I, III and IV collagens and their ultrastructural organization have been studied in diseased gingival connective tissue of patients with rapidly progressive periodontitis. This disease is characterized by acute destruction of the gingival collagenous components. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and III collagens but that type III collagen was less resistant to acute inflammation. Ultrastructural immunolabelling, using the peroxidase procedure has shown that the large, dense bundles of type I collagen of PI, the main pattern of organization of the gingival connective tissue offered a better resistance to acute destruction than PII, a loose pattern of organization mainly composed of type III collagen. Type IV collagen was exclusively located in degraded lamina densa of basement membrane.