Fibrin-based scaffolds for dental pulp regeneration: from biology to nanotherapeutics.

Tissue engineering-based endodontic therapies, designed to regenerate the dental pulp (DP) in the devitalised endodontic space, have been proposed to improve tooth longevity compared to conventional root-filling therapies. Their aim is to restore tooth vitality and major DP functions necessary to maintain tooth health such as immunosurveillance, sensitivity and healing/repair/regenerative capacities. Several formulations based on the use of fibrin, the main component of the blood clot matrix, recently gave valuable results in the regeneration of the human DP. This review describes recent fibrin-based scaffolds designed for that purpose. After having presented the various strategies for DP regeneration, the main fibrin-based scaffolds reported so far for clinical use in endodontics were reviewed. Particular emphasis was given to hydrogel devices that may be improved by incorporation of bioactive molecules that stimulate vascularisation and tissue neoformation or provide antibacterial properties. Data indicate that fibrin-based scaffolds constitute a highly favourable environment for mesenchymal stem cells, which is maintained upon functionalisation. Additional knowledge is needed to understand how fibrin and functionalising agents affect adhesion, survival, proliferation, migration and differentiation of cells incorporated in the scaffold or which will colonise it from neighbouring host tissues. This knowledge is needed to adapt the hydrogel formulation for various clinical conditions.

Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering.

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 µg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 µg mL-1. MBIC50 and MBEC50 were 4 and 16 µg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

Dental pulp inflammatory/immune response to a chitosan-enriched fibrin hydrogel in the pulpotomised rat incisor.

Current pulpotomy is limited in its ability to induce regeneration of the dental-pulp (DP) complex. Hydrogels are reported to be well-suited for tissue engineering and are unlikely to induce an inflammatory response that might damage the remaining tissue. The present study investigated the molecular and cellular actors in the early inflammatory/immune response and deciphered M1/M2 macrophage polarisation to a chitosan-enriched fibrin hydrogel in pulpotomised rat incisors. Both fibrin and fibrin-chitosan hydrogels induced a strong increase in interleukin-6 (IL-6) transcript in the DP when compared to the DP of untreated teeth. Gene expression of other inflammatory mediators was not significantly modified after 3 h. In the viable DP cell population, the percentage of leukocytes assessed by flow cytometry was similar to fibrin and fibrin-chitosan hydrogels after 1 d. In this leukocyte population, the proportion of granulocytes increased beneath both hydrogels whereas the antigen-presenting cell, myeloid dendritic cells, T cells and B cells decreased. The natural killer (NK) cell population was significantly decreased only in DPs from teeth treated with fibrin-chitosan hydrogel. Immunolabeling analysis of the DP/hydrogel interface showed accumulation of neutrophil granulocytes in contact with both hydrogels 1 d after treatment. The DP close to this granulocyte area contained M2 but no M1 macrophages. These data collectively demonstrated that fibrin-chitosan hydrogels induced an inflammatory/immune response similar to that of the fibrin hydrogel. The results confirmed the potential clinical use of fibrin-chitosan hydrogel as a new scaffold for vital-pulp therapies.

Immune Cells and Molecular Networks in Experimentally Induced Pulpitis.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

Pattern-recognition receptors in pulp defense.

Initial sensing of infection is mediated by germline-encoded pattern-recognition receptors (PRRs), the activation of which leads to the expression of inflammatory mediators responsible for the elimination of pathogens and infected cells. PRRs act as immune sensors that provide immediate cell responses to pathogen invasion or tissue injury. Here, we review the expression of PRRs in human dental pulp cells, namely, receptors from the Toll-like (TLR) and Nod-like NLR families, by which cells recognize bacteria. Particular attention is given to odontoblasts, which are the first cells encountered by pathogens and represent, in the tooth, the first line of defense for the host. Understanding cellular and molecular mechanisms associated with the recognition of bacterial pathogens by odontoblasts is critical for the development of therapeutic strategies that aim at preventing excessive pulp inflammation and related deleterious effects.

[Dental ankylosis diagnosed by CT with tridimensional reconstructions]. / L'ankylose dentaire: diagnostic par tomodensitométrie et reconstruction tridimensionnelle.

The etiology of unerupted teeth often is difficult to establish. It may relate to abnormal orientation of the tooth bud, an anatomical obstacle, pathology of the dental sac or ankylosis. The ankylosis process and diagnosis are analyzed in this clinical series. Ankylosis and its underlying physiological process are abundantly described in the literature. Nonetheless, diagnosis remains difficult to achieve. Clinical evaluation and complementary radiographic techniques such as dental radiographs and orthopantomogram are considered by some authors as poorly reliable. These clinical examinations based on a series of subjective criteria cannot be performed on unerupted teeth. In a series of patients with unerupted or retained teeth of undetermined etiology or with traction failure, 15 were diagnosed with ankylosis. The diagnosis of ankylosis could not be achieved based on clinical or radiographic evaluation and required CT imaging. Source images along with tridimensional reconstructed images were evaluated using the MVS software (Hospices Civils of Lyon). The radicular volume involved by ankylosis could be measured and its exact location on the tooth could be demonstrated. This imaging technique with tridimensional reconstruction provided positive diagnosis of ankylosis.

Different roles of odontoblasts and fibroblasts in immunity.

Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.

Localization of STRO-1, BMP-2/-3/-7, BMP receptors and phosphorylated Smad-1 during the formation of mouse periodontium.

Bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) are known to regulate the development of calcified tissues by directing mesenchymal precursor cells differentiation. However, their role in the formation of tooth-supporting tissues remains unclear. We investigated the distribution pattern of STRO-1, a marker of mesenchymal progenitor cells and several members of the BMP pathway during the development of mouse molar periodontium, from the post-natal days 6 to 23 (D6 to D23). STRO-1 was mainly localized in the dental follicle (DF) at D6 and 13 then in the periodontal ligament (PDL) at D23. BMP-2 and -7 were detected in Hertwig's epithelial root sheath (HERS) and in DF, then later in differentiated periodontal cells. BMP-3 was detected after D13 of the periodontal development. BMPRs-Ib, -II, the activin receptor-1 (ActR-1) and the phosphorylated Smad1 were detected in DF and HERS at D6 and later more diffusely in the periodontium. BMPR-Ia detection was restricted to alveolar bone. These findings were in agreement with others data obtained with mouse immortalized DF cells. These results suggest that STRO-1 positive DF cells may be target of BMPs secreted by HERS. BMP-3 might be involved in the arrest of this process by inhibiting the signaling provided by cementogenic and osteogenic BMPs.

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.

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.

TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer.

TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents is suspected to influence dental pulp response, including the modulation of cell migration. To determine the consequences of TGF-beta1 action on pulp immune cells, we analyzed, by immunohistochemistry, the effect of transdentinally diffusing TGF-beta1 on their localization in a human tooth slice culture model. TGF-beta1 induced an accumulation of HLA-DR-positive cells in both odontoblast and subodontoblast layers of the stimulated zone. Together with HLA-DR, these cells co-expressed Factor XIIIa and CD68, two features of immature antigen-presenting dendritic cells (DC), as well as the TGF-beta1 specific receptor TbetaRII. In contrast, no effect could be detected on the localization of either mature DC-LAMP-positive DC or of T- and B-lymphocytes. Analysis of these data suggests that TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents could be involved in the immune response of the dental pulp resulting from tooth injury.

New genes involved in odontoblast differentiation.

The odontoblast phenotype has been mainly approached by the biochemical characterization of dentin matrix proteins and by extrapolation of the knowledge of bone cell biology, since dentin and bone share many similarities. In fact, direct investigations of the odontoblast phenotype have been hindered by the limited number of cells within the dental pulp and the difficulty in microdissection and isolation of a pure population of these cells. To overcome these obstacles, we previously developed a cell-culture system that promotes differentiation of human dental pulp cells into odontoblasts. This material now permits the study of odontoblasts through molecular biology techniques. Therefore, we constructed a cDNA library enriched for odontoblast-specific genes using the suppression subtractive hybridization technique (SSH). This library led us to identify new genes expressed by odontoblasts. In this paper, we will focus on some genes implied in various functions associated with odontoblast differentiation, such as cell polarization (MAP1B), dentin mineralization (PHEX, osteoadherin), and relationships between odontoblasts and nerve cells (reelin). Another important fact is that about 40% of the cDNA were unknown genes. Therefore, one can speculate that some of them will be odontoblast-specific, since, until now, only one gene (DSPP) presents this characteristic.

Molecular regulation of odontoblast activity under dentin injury.

Pulp tissue responds to dentin damage by laying down a tertiary dentin matrix (reactionary or reparative) beneath the site of injury. Reactionary dentin is secreted by surviving odontoblasts in response to environmental stimuli, leading to an increase in metabolic activities of the cells. The inductive molecules that determine the success of the pulp healing may be released from the damaged dentin as well as from the pulp tissue subjacent to the injury. This paper will schematically consider two major growth factors probably implicated in the control of odontoblast activity: TGF beta-1 released from demineralized dentin and NGF from pulp. To analyze their role with an in vitro system that mimics the in vivo situation, we have used thick-sliced teeth cultured as described previously. The supply of factors was accomplished by means of a small tube glued onto the dentin. The tube was filled with TGF beta-1 (20 ng/mL) or NGF (50 ng/mL), and slices were cultured for 4 or 7 days. Results showed that TGF beta-1 binding sites are strongly detected on odontoblasts in the factor-rich zone. A strong expression of alpha 1(I) collagen transcripts was also detected. In the NGF-rich environment, p75NTR was re-expressed on odontoblasts and the transcription factor NF-kappa B activated. Modifications in the odontoblast morphology were observed with an atypical extension of the cell processes filled with actin filaments. These results suggest that odontoblasts respond to influences from both dentin and pulp tissue during pulp repair.

Expression of TGF-beta receptors I and II in the human dental pulp by in situ hybridization.

Members of the TGF-beta family of growth factors are important in modulation of odontoblast secretory activity during dental tissue repair. Odontoblast expression of TGF-beta isoforms during development leads to their sequestration within the dentin matrix, from where they may be released during carious injury and participate in reparative processes. Two receptors, implicated in TGF-beta-mediated cell signaling, have been identified immunohistochemically in both odontoblasts and pulpal cells of healthy and carious human molar teeth. This study aimed to characterize the expression of the TGF-beta receptors I and II in sound and carious teeth by means of in situ hybridization, to help our understanding of the response of these cells to TGF-beta stimulation. Sound and carious human third molar teeth were routinely processed immediately following extraction, and 10-microns paraffin-embedded sections prepared. These sections were hybridized with 32P-labeled probes to TGF-beta receptors I and II, and the subsequent signal was detected by autoradiography. mRNA for both receptors I and II was mainly detected within the odontoblasts and nerve-associated cells of healthy tissues, with expression at lower levels seen within the subodontoblast and pulp core cells. The expression in odontoblasts was higher for TGF-beta receptor I than for receptor II. Expression of both receptors was more homogenous in all pulp cells within carious teeth, because of an increase of signal within the underlying pulp cell population, including blood-vessel-associated cells. We conclude that the TGF-beta receptors I and II were expressed in odontoblasts and pulp cells, and that subtle variations in the levels of their expression could be involved in the tissue response to injury.

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.

Odontoblast differentiation of human dental pulp cells in explant cultures.

In order to elucidate the mechanisms involved in human dentin formation, we developed a cell culture system to promote differentiation of dental pulp cells into odontoblasts. Explants from human teeth were cultured in Eagle's basal medium supplemented with 10% or 15% fetal calf serum, with or without beta-glycerophosphate (beta GP). Addition of beta GP to the culture medium induced odontoblast features in the cultured pulp cells. Cells polarized and some of them exhibited a typical cellular extension. In some cases, cells aligned with their processes oriented in the same direction and developed junctional complexes similar to the terminal web linking odontoblasts in vivo. Fine structural analyses showed the presence of typical intracellular organelles of the odontoblast body, whereas the process contained only cytoskeleton elements and secretory vesicles. Polarized cells deposited onto the plastic dishes an abundant and organized type I collagen-rich matrix with areas of mineralization appearing thereafter. X-ray microanalysis showed the presence of calcium and phosphorus and the electron diffraction pattern confirmed the apatitic crystal structure of the mineral. High expression of alpha 1 (1) collagen mRNAs was detected in all polarized cells whereas dentin sialoprotein gene was mainly expressed in mineralizing areas. This cell culture system allowed for the differentiation of pulp cells into odontoblasts, at both the morphological and functional level. Moreover, these cells presented a spatial organization similar to the odontoblastic layer.

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.

Cytokeratin expression in human oral gingival epithelial cells: in vitro regulation by titanium-based implant materials.

To evaluate whether cytokeratin expression in human oral epithelial cells could be influenced by implant materials used in dental surgery, passaged human oral gingival epithelial cells were seeded on commercially pure titanium (CP-Ti) or on Ti6Al4V titanium alloy. Confluence was achieved after about 15 days on both substrates. Cells formed at that time, an organized layer of densely packed polygonal cells, and harbored a filamentous cytokeratin network typical of epithelial cells. Immunochemistry and immunoblot analysis were used to detect modifications of the amount of individual CK polypeptides (CK7, 8, 13, 18 and 19) in function of the culture substrate. Results showed that the level of CK8, CK18 and CK19 expression was not altered whatever the culture substrate used. The expression of CK13 was reduced in epithelial cells cultured on the titanium alloy, as compared with commercially pure titanium. Conversely, the level of CK7 was higher on the Ti6Al4V alloy than on commercially pure titanium. This study suggests that titanium-based implant materials could influence differently the phenotype of oral gingival epithelial cells.

Immunocytochemical localization of fibronectin and a 165-kDa membrane protein in the odontoblast layer under initial carious lesions in man.

The possible role of fibronectin in dental tissue repair was investigated by comparing its distribution and that of the 165-kDa fibronectin-binding membrane protein (165 kDa-FnBP) in odontoblasts underlying carious and sound dentine. By immunoperoxidase and light microscopy, fibronectin was localized in the dentine underlying the carious lesion, mainly on the surface of the tubule walls, whereas it could not be detected in neighbouring sound zones. The antibody to the 165 kDa-FnBP strongly reacted with the membrane of odontoblasts underlying the lesion, although those facing sound dentine did not express this antigen. Ultrastructurally the 165 kDa-FnBP was localized in the cell membrane at the apical portion of odontoblasts, including the process membrane, beneath the initial lesion; fibronectin was detected in the dentinal area close to the process, and also in contact with its external surface. By a high-resolution immunogold procedure, the proteins were colocalized at the external surface of odontoblast processes. These data suggest that fibronectin present in human carious dentine could modulate the behaviour of underlying odontoblasts by means of newly expressed 165 kDa-FnBP.

Ultrastructural characterization of mesenchymal and epithelial cells co-cultured from human dental root apical explants.

Previous studies have shown the role of cell-cell and cell-matrix interactions in the differentiation of the specific secretory cells of the tooth. In order to elucidate the mechanisms implicated in root dentin formation, we developed a co-culture system of human pulpal mesenchymal and epithelial root sheath cells. Root tips of premolars were cultured in Eagle's basal Medium supplemented with fetal calf serum, ascorbic acid, antibiotics and, for some of them, with sodium beta-glycerophosphate. After 60 days of culture, cells were prepared for light and electron microscopy. Three main cell types were observed: (1) polygonal mesenchymal cells showing a functional polarity and producing a dense network of tactoid collagenous fibers. The latter had a specific circular organization that delimited small lacunae around the cells and mineralized in the presence of beta-glycerophosphate; (2) spindle-shaped mesenchymal cells mainly localized inside epithelial-mesenchymal knots and synthesizing an abundant collagenous matrix; and (3) epithelial cells lying on the plastic culture dish, on the dense collagenous matrix, or on spindle-shaped cells. Epithelial cells deposited a structured basement membrane when they were lying on the plastic culture dish or on spindle-shaped cells. On the contrary, no basement membrane was found when epithelial cells were overlying the dense collagenous network. Immunoelectron microscopic analysis of type IV collagen and laminin indicated that these two specific basement membrane components were produced by all cell types. These results show that the co-culture system should be valuable for (1) studying the in vitro formation of human dental root hard tissues, (2) characterizing cell-cell and cell-matrix interactions implicated in dental basement membrane production, and (3) isolating populations of cells implicated in dental root formation.