Comparison of vehicles to collect dentinal fluid for molecular analysis.

OBJECTIVES: To test the hypothesis that a material with higher water absorption than polyvinylidene fluoride (PVDF) could increase the yield of target molecules from exposed dentine. METHODS: In a series of standard tests, different cellulose membranes were compared to a PVDF counterpart for their ability to absorb water and release protein. In a subsequent randomized clinical trial, the cellulose material with the most favourable values was compared to PVDF regarding the levels of MMP-2 that could be collected from exposed dentine of healthy human teeth during filling replacement. MMP-2 levels were determined by enzyme-linked immunosorbent assay (ELISA). Data from the laboratory experiments were compared between materials using the appropriate parametric tests. The frequency of cases yielding quantifiable levels of MMP-2 was compared between materials by Fisher's exact test. The level of significance was set at 5%. RESULTS: The cellulose membrane with the largest pore size (12-15µm) absorbed significantly (P<0.05) more water than PVDF. It showed a protein release that was similar to that of PVDF, while the cellulose membranes with smaller pore size retained significantly more protein (P<0.05). Using the large-pore cellulose membrane, MMP-2 could be collected at a quantifiable level from the dentine of healthy teeth in 9 of 13 cases, compared to 1 of 13 with the PVDF membrane (P<0.05). CONCLUSIONS: Under the current conditions, a large-pore cellulose membrane yielded more of a molecule of diagnostic value compared to a standard PVDF membrane. CLINICAL SIGNIFICANCE: Molecular diagnostics of dentinal fluid are hampered by low yields. In the current study, it was shown that cellulose membranes are more useful to collect MMP-2 from dentinal fluid than PVDF membranes.

A first study on the usefulness of matrix metalloproteinase 9 from dentinal fluid to indicate pulp inflammation.

INTRODUCTION: Pulpal diagnostics might be improved by using molecular markers found in dentinal fluid. In the current pilot study, we tested whether matrix metallopeptidase 9 (MMP-9) levels in dentinal fluid were detectable and differed between pulps from symptomatic teeth diagnosed with irreversible pulpitis and healthy counterparts. METHODS: Thirty-one patients participated; 19 were diagnosed with irreversible pulpitis, and 12 were in need of replacement of a filling close to the pulp space in a clinically healthy tooth. Dentinal fluid was collected during a period of 2 minutes from dentin cavities by using folded polyvinylidene difluoride (PVDF) membranes, which were then transferred to microcentrifugation tubes containing physiologic saline solution. Total MMP-9 levels in these solutions were assessed by using a human MMP-9 fluorescent assay, detection limit 0.25 ng/mL. MMP-9 levels between groups were compared by using Mann-Whitney U test (alpha <0.05). RESULTS: Three specimens from the symptomatic teeth were not included because coronal pulps proved to be necrotic on access. Dentinal fluid samples from symptomatic teeth had significantly higher MMP-9 levels than those from clinically healthy counterparts (P < .05). However, merely 7 of the 16 pulpitis samples contained detectable levels of MMP-9. None of the clinically healthy control specimens contained any detectable amounts of MMP-9. CONCLUSIONS: With a sensitive assay, it was possible to detect an enzyme linked with pulp tissue destruction (MMP-9) in dentinal fluid. However, the collection method needs to be improved to provide predictable fluid yields. Longitudinal studies should be performed to assess the predictive value of molecular markers in dentinal fluid on pulpal pathosis.

The localization of matrix metalloproteinase-20 (MMP-20, enamelysin) in mature human teeth.

MMP-20 (enamelysin), the matrix metalloproteinase family member discovered in the enamel organ, has also been detected in odontoblasts during dentin formation. We studied the presence and localization of MMP-20 in mature human teeth in health and disease. In immunohistochemistry, MMP-20-positive staining was observed most intensively in the radicular odontoblastic layer and also in dilated dentinal tubuli of caries lesions. By Western blotting, MMP-20 was detected in odontoblasts and pulp tissue of both sound and carious teeth, in dentinal fluid and dentin of sound teeth, but not in soft carious dentin. We conclude that MMP-20 produced during primary dentinogenesis is incorporated into dentin and may be released during caries progression. The main cellular source of MMP-20 in the dentin-pulp complex is the odontoblasts, which secrete MMP-20 into the dentinal fluid.