Enzymatic isolation of viable human odontoblasts.

AIM: To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. METHODOLOGY: Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. RESULTS: MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. CONCLUSION: The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts.

Mature human odontoblasts express virus-recognizing toll-like receptors.

AIM: To study the expression of toll-like receptors (TLR) -3, -7, -8 and -9 as well as interferon receptors alpha and gamma (IFNAR1/IFNAR2 and IFNGR1/IFNGR2), which play important roles in the defence against viruses. METHODOLOGY: DNA microarray and quantitative PCR analyses of TLR3, -7, -8 and -9 as well as IFNAR1/IFNAR2 and IFNGR1/IFNGR2 genes in mature native human odontoblasts and pulp were performed. Immunohistochemistry was used to confirm TLR8 protein in odontoblasts of healthy and carious human teeth. RESULTS: TLR3, -7, -8 and -9 mRNAs were detected both in odontoblasts and in pulp, but TLR8 expression level was higher in the odontoblasts. IFNAR and IFNGR expression was observed in both tissues. Immunohistochemical analysis of healthy teeth revealed positive TLR8 staining in the pre-dentine and the dentine but varying staining patterns in the different portions of tooth. Lighter TLR8 staining was observed in dentine of mildly carious teeth. In teeth with carious lesions extending into the mid-dentine, only very weak staining was detected. CONCLUSIONS: The finding of these virus-recognition-related genes in odontoblasts strengthens the view that odontoblasts participate in the immune response of the dentine-pulp complex.

The expression and role of Lysyl oxidase (LOX) in dentinogenesis.

AIM: To establish whether eliminating Lysyl oxidase (LOX) gene would affect dentine formation. METHODOLOGY: Newborn wild-type (wt) and homo- and heterozygous LOX knock-out (Lox(-/-) and Lox(+/-) , respectively) mice were used to study developing tooth morphology and dentine formation. Collagen aggregation in the developing dentine was examined histochemically with picrosirius red (PSR) staining followed by polarized microscopy. Because Lox(-/-) die at birth, adult wt and Lox(+/-) mouse tooth morphologies were examined with FESEM. Human odontoblasts and pulp tissue were used to study the expression of LOX and its isoenzymes with Affymetrix cDNA microarray. RESULTS: No differences between Lox(-/-) , Lox(+/-) and wt mice developing tooth morphology were seen by light microscopy. Histochemically, however, teeth in wt mice demonstrated yellow-orange and orange-red polarization colours with PSR staining, indicating thick and more densely packed collagen fibres, whilst in Lox(-/-) and Lox(+/-) mice, most of the polarization colours were green to green-yellow, indicating thinner, less aggregated collagen fibres. Fully developed teeth did not show any differences between Lox(+/-) and wt mice with FESEM. Human odontoblasts expressed LOX and three of four of its isoenzymes. CONCLUSIONS: The data indicate that LOX is not essential in dentinogenesis, even though LOX deletion may affect dentine matrix collagen thickness and packing. The absence of functional LOX may be compensated by LOX isoenzymes.

Polarity of mature human odontoblasts.

Odontoblast polarization is based on histological appearance as columnar cells with asymmetric disposition of organelles and plasma membrane domains. However, little is known about the odontoblast plasma membrane organization. We investigated odontoblast membrane polarity using influenza virus hemagglutinin and vesicular stomatitis virus glycoprotein as model proteins in mature human odontoblast organ culture. We also examined the distribution patterns of aquaporin 4 and 5, which are basolateral and apical proteins in epithelial cells, respectively. Confocal microscopy immunofluorescence and electron microscopy demonstrated that the apical markers located at the surface toward pulp and basolateral markers located at the plasma membrane of odontoblast processes. Therefore, odontoblast plasma membrane polarity was different from that in epithelial cells. Also, certain lectins stained odontoblast processes while others stained the soma, reflecting the different natures of their membrane domains. Strong ZO-1 and weaker claudin expression suggest weak tight junctions in the odontoblasts. TGF-ß1 showed a tendency to reinstate the expression of selected TJ genes, indicating that TGF-ß1 may control odontoblast cell layer integrity by controlling tight junction protein expression.

Comparative gene expression profile analysis between native human odontoblasts and pulp tissue.

AIM: To undertake a large-scale analysis of the expression profiles of native human pulp tissue and odontoblasts, and search for genes expressed only in odontoblasts. METHODOLOGY: Microarray was performed to pooled pulp and odontoblasts of native human third molars and to pooled +/- TGF-beta1 cultured pulps and odontoblasts (137 teeth). The repeatability of microarray analysis was estimated by comparing the experimental pulp samples with expression profiles of two pulp samples downloaded from the GEO database. The genes expressed only in the experimental pulp samples or in odontoblasts were divided into categories, and the expression of selected odontoblast-specific genes of extracellular matrix (ECM) organization and biogenesis category was confirmed with RT-PCR and Western blot. RESULTS: A 85.3% repeatability was observed between pulp microarrays, demonstrating the high reliability of the technique. Overall 1595 probe sets were positive only in pulp and 904 only in odontoblasts. Sixteen expressed sequence tags (ESTs), which represent transcribed sequences encoding possibly unknown genes, were detected only in odontoblasts; two consistently expressed in all odontoblast samples. Matrilin 4 (MATN4) was the only ECM biogenesis and organization related gene detected in odontoblasts but not in pulp by microarray and RT-PCR. MATN4 protein expression only in odontoblasts was confirmed by Western blot. CONCLUSIONS: Pulp tissue and odontoblast gene expression profiling provides basic data for further, more detailed protein analysis. In addition, MATN4 and the two ESTs could serve as an odontoblast differentiation marker, e.g. in odontoblast stem cell research.