Dentine caries: acid tolerant microorganisms and aspects on collagen degradation

Sammanfattning: Dental caries is a common disease all over the world, despite the fact that it can be both effectively prevented as well as treated. It is driven by acids produced by oral microorganisms as a consequence of their metabolism of dietary carbohydrates. Given enough acid challenge, eventually the enamel barrier will be broken down, and the carious lesion will extend into underlying hard tissue, forming a macroscopic cavity in the dentine. In comparison to plaque biofilm on enamel, this dentine carious lesion will form a vastly different environment for the residing microorganisms. The environment will influence the types and numbers of microorganisms that will be able to colonize the dentine caries lesion. The overall aims for this thesis is to enumerate and further study microorganisms found in established dentine caries lesions, and also to illuminate how host-derived proteolytic enzymes might contribute to this degradation, in order to better understand the caries process in dentine, but also to find incitements for new methods to influence the natural progression of caries lesions. In Paper I, the number of remaining viable microorganisms after completed excavation using two different excavation methods were investigated. Samples of carious dentine tissue was collected before and after excavation, and cultivated on different agar media in different atmospheres. Analysis was performed by counting the number of colony forming units (cfu). Key findings: Numbers of remaining microorganisms after excavation was low for both methods, but some microorganisms always remained in the cavity floors even when the cavities were judged as ”caries free” using normal clinical criteria. In Paper II, the acid tolerant microbiota in established dentine caries lesions were investigated. Samples were taken as in Paper I, but on three levels (superficial, center of lesion, cavity floor). The samples were cultivated in anaerobic conditions on solid pH-selective agar media of different acidity. Key findings: Each investigated lesion harbored a unique microbiota, both in terms of species composition and numbers of microorganisms. This indicate that various combinations of aciduric microorganisms can colonize, survive in and probably also propagate dentine carious lesions. We also found that solid pH-selective agars successfully can be used to select acid tolerant microorganisms in caries lesions. This would preserve their phenotypic traits for further study. In Paper III, the relation between salivary levels of matrix metalloproteinase-8 (MMP-8), salivary levels of tissue inhibitor of MMP (TIMP-1) and presence of manifest caries lesions in a large number of subjects was investigated. Saliva samples were collected and analyzed for concentrations of MMP-8, TIMP-1 and total protein using immunofluorometric assay, enzyme linked immunosorbent assay and Bradford assay, respectively. Key findings: Subjects with manifest caries lesions had significantly elevated levels of salivary MMP-8 compared to subjects without caries lesions. TIMP-1 was not significant in any case. In Paper IV, a new method for generating bioactive demineralized dentine matrix substrate (DDM) was developed, using a dialysis system and two different demineralization approaches (acetic acid (AA) or EDTA). The generated DDM was subsequently analyzed for presence of type 1 collagen, active MMP-8 and hydroxyproline (HYP) levels using SDS-PAGE, ELISA or immunofluorescence assay. Key findings: Both demineralization methods produced a substrate rich in collagen and with preserved MMP-8 activity. The report presents new knowledge on the composition of the acid tolerant dentine caries microbiota from three levels in dentine carious lesions and on the efficacy of operative caries removal on the numbers of viable microorganisms in the caries free cavity using two different operative methods. Moreover, the basic mechanisms behind collagen degradation in the dentine caries process are studied from both a clinical and laboratory perspective. The report provides a reference for further studies on dentine caries microbiology and dentine caries collagen degradation mechanisms, both of which are only known in part.