A study in situ of the effect of metallo- and serine proteinase inhibitors on the birefringence of the secretory stage enamel organic extracellular matrix

Autor(es)

do Espirito Santo, A. R. ^{[1, 2]} ; Marques, M. R. ^[1] ; Line, S. R. P. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Campinas UNICAMP, Piracicaba Dent Sch, Dept Morphol, Sao Paulo - Brazil

^[2] Fed Univ Bahia UFBA, Dept Biomorphol, Inst Hlth Sci, Salvador, BA - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOTECHNIC & HISTOCHEMISTRY; v. 86, n. 2, p. 108-114, APR 2011.
• Citações Web of Science: 0

Resumo

Dental enamel formation occurs extracellularly and establishment of an ordered enamel organic extracellular matrix (ECM) seems to be crucial for proper construction of the enamel mineral phase. Polarizing microscopy shows that the ordered supramolecular structure of the secretory stage enamel organic ECM exhibits strong birefringence. We reported earlier that this birefringence is lost in unfixed specimens, probably due to extensive proteolytic cleavage of enamel proteins. Therefore, we investigated the association between enamel proteinase activities by analyzing the effects of metallo-and serine proteinase inhibitors in situ on the birefringence of the secretory stage enamel organic ECM. Male rats were used in the present study. After sacrifice, distal 10 mm fragments of upper incisors were removed and immersed for 15 h under continuous shaking at 37 degrees C in one of the following solutions: 1) 10 mM Tris, pH 8.0; 150 mM NaCl (negative control, n = 8); 2) 2% paraformaldehyde and 0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2 (positive control, n = 5); 3) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline (n = 9); 4) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM phenylmethyl-sulfonyl fluoride (PMSF) (n = 8); 5) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline; 2 mM PMSF (n = 9). Samples then were immersed in fixative solution for 24 h and processed to obtain 5 mu m thick longitudinal sections of the secretory stage enamel organic ECM. The sections were immersed in 80% glycerin for 30 min and analyzed by transmitted polarizing light microscopy. 1,10-Phenanthroline (inhibitor of metalloproteinases) and 1,10-phenanthroline + PMSF (inhibitor of serine proteinases) clearly prevented a decrease in the optical retardation of birefringence brightness from the tissue. PMSF alone promoted a slight preservation of the birefringence exhibited by the secretory stage enamel organic ECM. Rapid loss of birefringence in secretory stage enamel organic ECM that is not fixed immediately is caused by enamel proteinases and the activity of metalloproteinases seems to lead to preliminary degradation of the enamel organic ECM, which in turn facilitates subsequent serine proteinase activity. (AU)

• Processo FAPESP: 04/13255-8 - Efeito de alterações genéticas e ambientais sobre a birrefringência da matriz orgânica do esmalte dentário
• Beneficiário: Alexandre Ribeiro do Espírito Santo
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 04/10994-4 - Defeitos na formação do órgão dental
• Beneficiário: Sergio Roberto Peres Line
• Modalidade de apoio: Auxílio à Pesquisa - Temático