Modification of tooth development by heat shock protein 60.

Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2-deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

TLR signalling can modify the mineralization of tooth germ.

OBJECTIVE: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. MATERIALS AND METHODS: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. RESULTS: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. CONCLUSION: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.