Use of a Fluorogenic Septapeptide Matrix Metalloproteinase Assay to Assess Responses to Periodontal Treatment

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Abstract
Quantification of gingival crevicular fluid matrix metalloproteinase activity may provide improved assessment of periodontal disease status and response to treatment. A fluorogenic matrix metalloproteinase substrate assay (FSA) has been developed using a methoxycoumarin-containing septapeptide analog of the alpha2(I) collagen cleavage site. This substrate exhibits increased fluorescence following cleavage by many matrix metalloproteinases, and the enzyme activity can be readily estimated with a fluorimeter. Here we compared this assay with classical methods of periodontal assessment including bleeding on probing, crevicular fluid flow, and probing depth to assess its utility as an indicator of changes in periodontal status and treatment response. Complete measurements of probing depth were obtained for Ramfjord teeth on subjects who had been previously treated for periodontitis. Subjects were subsequently divided into groups based on existing periodontal disease severity: gingivitis (n = 21), stable periodontitis (n = 41), and severe periodontitis (n = 50). Crevicular fluid volume, bleeding on probing, and FSA were measured at each Ramfjord tooth or substitute. After baseline measurements, subjects received subgingival scaling and prophylaxis; 3 months later, they were reassessed. FSA measurements were positively associated with severity of disease at baseline. After treatment there were substantial reductions of FSA in gingivitis (approximately 51%; P 0.2). All groups showed a positive association between FSA measurements and higher bleeding scores at individual sites. FSA measurements were also positively associated with crevicular fluid flow at baseline, but after treatment there was a approximately 67% decrease (P <0.01) in the highest crevicular fluid flow class. There were significant reductions of FSA at follow-up for sites with probing depths between 0 to 3 mm (23%; P <0.05) and 4 to 6 mm (31%; P <0.05). However, the largest reduction was for sites with probing depth between 7 to 9 mm (49%; P <0.001). These results indicate that monitoring patients by measurement of matrix metalloproteinase levels in gingival crevicular fluid with the quenched fluorescent substrate assay provides estimates of inflammatory status, periodontal destruction, and response to treatment, especially in more severe periodontitis lesions.

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