Label-free highly multimodal nonlinear endoscope.

We demonstrate a 2 mm diameter highly multimodal nonlinear micro-endoscope allowing label-free imaging of biological tissues. The endoscope performs multiphoton fluorescence (3-photon, 2-photon), harmonic generation (second-SHG and third-THG) and coherent anti-Stokes Raman scattering (CARS) imaging over a field of view of 200 µm. The micro-endoscope is based on a double-clad antiresonant hollow core fiber featuring a high transmission window (850 nm to 1800 nm) that is functionalized with a short piece of graded-index (GRIN) fiber. When combined with a GRIN micro-objective, the micro-endoscope achieves a 1.1 µm point spread function (PSF). We demonstrate 3-photon, 2-photon, THG, SHG, and CARS high resolution images of unlabelled biological tissues.

Double clad tubular anti-resonant hollow core fiber for nonlinear microendoscopy.

We report the fabrication and characterization of the first double clad tubular anti-resonant hollow core fiber. It allows to deliver ultrashort pulses without temporal nor spectral distortions in the 700-1000 nm wavelength range and to efficiently collect scattered light in a high numerical aperture double clad. The output fiber mode is shaped with a silica microsphere generating a photonic nanojet, making it well suitable for nonlinear microendoscopy application. Additionally, we provide an open access software allowing to find optimal drawing parameters for the fabrication of tubular hollow core fibers.

Dentonin, a MEPE fragment, initiates pulp-healing response to injury.

Phosphorylated extracellular matrix proteins, including matrix extracellular phosphoprotein (MEPE), are involved in the formation and mineralization of dental tissues. In this study, we evaluated the potential of Dentonin, a synthetic peptide derived from MEPE, to promote the formation of reparative dentin. Agarose beads, either soaked with Dentonin or unloaded, were implanted into the pulps of rat molars, and examined 8, 15, and 30 days after treatment. At day 8, Dentonin promoted the proliferation of pulp cells, as visualized by PCNA-labeling. RP59-positive osteoblast progenitors were located around the Dentonin-soaked beads. PCNA- and RP59-labeling were decreased at day 15, while osteopontin, weakly labeled at day 8, was increased at 15 days, but dentin sialoprotein was undetectable at any time. At 8 days, precocious reparative dentin formation occurred in pulps containing Dentonin-soaked beads, with formation slowing after 15 days. These results suggest that Dentonin affects primarily the initial cascade of events leading to pulp healing.

Amelogenin gene splice products A+4 and A-4 implanted in soft tissue determine the reorientation of CD45-positive cells to an osteo-chondrogenic lineage.

Several molecules such as bone morphogenetic protein-7, bone sialoprotein (BSP), or amelogenin gene splice products (A+4 or A-4) have been shown to induce reparative dentin formation in a rat model. However, at the moment, the origin and the mechanism of differentiation of the pulp cells stimulated by the bioactive molecules remain poorly understood. The present investigation was undertaken to validate an ectopic oral mucosal mouse model to evaluate the effects of amelogenin gene splice product implantation in a non-mineralizing tissue. Agarose beads, alone or coated with amelogenin gene splice products, were implanted in the mucosa of the cheeks in mouse. An immunohistochemical characterization of the recruited cells was undertaken for 3 days, 8 days, and 30 days after the implantation. The results showed that the implantation of agarose beads in mucosa induced the recruitment of inflammatory CD45 positive cells. When the beads were coated with amelogenin gene splice products (A+4 or A-4), the expression of osteo-chondrogenic markers (RP59, Sox9, or BSP) was also observed. However, no mineralization nodule was observed, even after 30 days of implantation. The present investigation suggests that amelognin gene splice products have the capacity of recruiting among inflammatory cell mesenchymal progenitors that eventually differentiate into osteo-chondrogenic cells. Altogether, the results obtained in the pulp model and the present data suggest the existence of different pathways of cell recruitment and differentiation in different cellular environments.

Is the lingual forming part of the incisor a structural entity? Evidences from the fragilitas ossium (fro/fro) mouse mutation and the TGFbeta1 overexpressing transgenic strain.

Our objective was to study the teeth of a mutant mice fro/fro that display severe forms of osteogenesis imperfecta. One day and 8 week-old fro/fro and +/fro heterozygote mice (wild type, WT) were processed for light and scanning electron microscopy. The genetic defect, shown to be located on chromosome 8, induced alveolar bone and teeth hypomineralisation. Due to defective cell proliferation in the fro/fro, the distal growth of the mandibular incisors was impaired. Immunolabelling revealed an increase of chondroitin/dermatan sulphate, whereas no difference was detected in dental tissues for decorin and biglycan. Amelogenin expression was decreased in the incisor and enhanced in the molar. Dentin sialoprotein was below the level of detection in the fro/fro, whereas osteonectin and osteopontin were unchanged. The main target of the mutation was seen in the lingual part of the incisor near the apex where dentine formation was delayed. In the molars, bulbous roots with obliteration of the pulp chamber were seen. In the TGFbeta1 overexpressing mice, the lingual root-analogue part of the incisor was missing. In the molar, short roots, circumpulpal dentine of the osteodentine type and pulp obliteration were seen. It may be noted that, although the mutant and transgenic strains mutations are two different genetic alterations not related to the same defective gene, in both cases the expression of the dentin sialoprotein is altered. Altogether, the present data suggest that the lingual forming part of the incisor seems to be an anatomical entity bearing its own biological specificities.

Dental mineralization.

Extracellular matrix components and cell-derived microstructures are implicated in mineralization processes which occur in dental tissues. The respective role(s) of collagenic and non-collagenic matrix components are reviewed: phosphorylated and non-phosphorylated proteins, proteoglycans and phosphpholipids. Space-filling amphiphilic molecules seem to play an important role in the preorganization and oriented deposition of calcium phosphate on structures serving more or less as passive support in dentine as well as in enamel.

Visualization of glycosaminoglycans in rat incisor predentin and dentin with cetylpyridinium chloride-glutaraldehyde as fixative.

Using cetylpyridinium chloride (CPC) in glutaraldehyde as fixative, we observed sinuous fiber-like structures 300-500 nm long and 7-14 nm thick in the spaces between the collagen fibers of rat incisor predentin. Small granules and fibrils were also detected. Electron-dense vesicles were seen inside the odontoblast processes. The plasma membrane was irregularly stained with material that adhered to its surface. In demineralized dentin, needle-like structures were seen at the periphery of globular structures which were not stained. Staining the sections with Alcian blue did not greatly improve the visualization of CPC-precipitated glycosaminoglycans. The specificity of staining was assessed on serial sections by selective dissociation of glycosaminoglycan aggregates with 2 M calcium chloride and their digestion by bovine testicular hyaluronidase. The glycosaminoglycans were probably combined with lipids, because treatment of the sections with a chloroform/methanol mixture removed the CPC-induced precipitates from both predentin and dentin.

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue.

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.

Inositol hexasulphate, a casein kinase inhibitor, alters enamel formation in cultured embryonic mouse tooth germs.

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

Ultrastructural study of the protein-polysaccharides distribution in enamel from rat incisors.

The distribution pattern of the protein-polysaccharides in the rat incisor enamel was similar whether stained for glycoproteins (phosphotungstic acid) or for proteoglycans (alcian blue, bismith nitrate). Except for the holes at the dentino-enamel junction, where these components seem to exist as amorphous gel, protein-polysaccharides are closely associated to the proteinic fibrous matrix in rods and interrods structures as in prism sheaths where they are particularly abundant.

Electron microscopic visualization of proteoglycans in rat incisor predentine and dentine with cuprolinic blue.

Cuprolinic blue in a MgCl2-critical electrolyte concentration solution allows a larger surface representation of proteoglycans than other methods. Glycoaminoglycans in predentine were ribbon-like structures 14 nm thick and 60 nm long, with radiating filaments (3-4 nm) located in spaces between collagen fibres. Glycoaminoglycans and collagen together displayed longitudinal and orthogonal relationships. The ground substance was slightly electron dense. In dentine, the precipitate was less in quantity and consisted of rounded granules (15 nm in diameter, and filaments/3 nm in width) closely associated with the surfaces of collagen fibres.

Visualization of proteoglycans and membrane-associated components in rat incisor predentine and dentine using ruthenium hexammine trichloride.

Ruthenium hexammine trichloride (RHT) used as a probe to visualize anion groups in the predentine and dentine of rat incisors, showed a complex distribution pattern including: intercellular proteoglycans (Pg), detected in the predentine as granules 10-15 nm in diameter and as filaments. Non-aggregating Pg was observed in the spaces between collagen fibres as an amorphous group substance. The dentine included smaller RHT-positive granules, observed after thin-section demineralization; pericellular aggregates, 30-50 nm in diameter, which were absent at the onset of the cell coat along the plasma-membrane of the odontoblast process and of the membrane itself. All these RHT-positive components might be Pg and/or sialoglyconjugates and glyco- or phospholipids present on the plasma membrane.

Improved lipid preservation by malachite green-glutaraldehyde fixation in rat incisor predentine and dentine.

A network of granules and filaments stained with malachite green-aldehyde (MGA) was observed in predentine in the spaces between the collagen fibres and many small granules were associated with these fibres in peripheral and circumpulpal-dentine. The granules were present throughout the entire thickness of the dentine, especially at the periphery of the calcospherites, although scarce or absent at their centre. This MGA-stainable material (i) resisted demineralization in acetic acid and EDTA solutions, and (ii) differed from non-collagenous material and crystal ghosts which stained on control sections after prior demineralization. In predentine, transport and diffusion of MGA-stainable material occurred in the direction of the dentine in which this material was associated with periodical striation of mineralized collagen fibres. This suggests that true matrix-associated lipids might play a role in dentine mineralization.

Effect of dissociative extraction with 1.5 M calcium chloride on proteoglycans in rat-incisor predentine visualized with cuprolinic blue.

Treatment of rat-incisor predentine for 48 h in a 1.5 M CaCl2 solution halved the number and size of the proteoglycan precipitates visualized using a critical MgCl2 concentration and cuprolinic blue staining. About 75 per cent of the aggregable proteoglycans were extracted; 25 per cent resisted extraction. Treatment with CaCl2 also completely abolished the electron density of the amorphous ground substance, an observation consistent with the possible existence of two classes of proteoglycans in predentine.

Ultrastructure of inter-odontoblastic fibres in the rat molar.

Radially directed inter-odontoblastic collagen fibres were observed in ultra-thin sections of the radicular pulp/predentine complex of rat molars. Bundles of fibres crossed the distal junctional complexes of the odontoblasts, went through the whole thickness of the predentine and were incorporated into the mineralized dentine. Scanning electron microscopy showed that bundles of radial fibres are not found between the coronal odontoblasts of the rat molar but only in the root. The radial fibres were inserted into the predentine along vertical long-axial crests. The intercrest interval was not tightly fixed and the pattern of fibre-bundle insertion did not show a regular periodicity.

Differences in the pattern of lanthanum diffusion into predentine and dentine in mouse incisors and molars.

Lanthanum nitrate was either perfused intravascularly or segments of mouse tooth were immersed in a fixative solution containing the tracer. The tracer deposits were examined in young (8-day-old) and older (8-week-old) mouse incisors and molars, demineralized or undemineralized. Lanthanum passed the distal junctional complex of odontoblasts and appeared in the predentine of incisors as large electron-dense stellate aggregates, 40-70 nm in diameter, and in molars as round, 20-40 nm dots. In dentine, tracer deposits were detected at three locations. Near the predentine dentine junction, the tracer densely stained a band 0.5-2.5 microm in width, also termed metadentine; in the inner circumpulpal dentine, the staining was weaker or lacking in an area extending 5-7 microm from the predentine-dentine junction; in outer circumpulpal dentine, lateral diffusion had occurred in porosities of intertubular dentine. Lanthanum impregnated the walls of dentine tubules and a peritubular-like dentine. In contrast, the mantle dentine was never stained. These differences in the pattern of diffusion prove that lanthanum staining is age-dependent and varies between mouse incisors and molars, independently of tissue processing. Architectural properties and driving flux are involved in the transport and localization of lanthanum in predentine and dentine.

Zinc deficiency-induced changes in the lipid composition and ultrastructure of rat incisor teeth.

The lipids and ultrastructure of the forming, maturing and erupted parts of incisors were compared in rats fed a zinc-deficient diet for 28 days, pair-fed rats and control rats. The lipid levels in the forming portions of zinc-deficient incisors were 30-50% below control levels and were associated with longer Tomes' processes in the secretory ameloblasts, porosities in the forming enamel and fewer malachite green-aldehyde-phospholipid aggregates in the predentine. No marked structural changes were seen in the erupted portions of the teeth, although variations in lipid composition were detected both in the maturing and erupted parts. No differences were found between teeth from the 3 groups of animals for Na, Mg, Cl, Ca and P signals with the electron microprobe.