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Development of an ex vivo model for the study of microbial infection in human teeth
Author(s) -
Patel B.,
Pratten J.,
Mordan N.,
Gulabivala K.
Publication year - 2007
Publication title -
international endodontic journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.988
H-Index - 119
eISSN - 1365-2591
pISSN - 0143-2885
DOI - 10.1111/j.1365-2591.2007.01277_4.x
Subject(s) - root canal , biology , ex vivo , amplicon , in vivo , 16s ribosomal rna , ribosomal rna , polymerase chain reaction , microbiology and biotechnology , bacteria , gene , dentistry , genetics , medicine
Aims (1) To infect human teeth artificially to mimic root canal and dentine infection, using the Constant Depth Film Fermenter (CDFF); (2) To verify the similarity of the infections to those found, in vivo , using culture and microscopy (SEM, LM and TEM). Methodology Human teeth [ n = 38 and n = 28, for phases I (preliminary) and II (definitive), respectively] were infected within the CDFF for a period of 28 days and at pre‐selected time points were removed, externally decontaminated using validated protocols and subjected to either culture‐dependent or microscopy protocols. The condition of the teeth was varied in phase I to establish the feasibility of the approach and identify optimal conditions. This informed the selection of optimal conditions for definitive test in phase II. For culture‐dependent analysis in this phase, a dentine filing sample was obtained from the apical 5 mm of the root canal and cultured anaerobically to allow isolation of individual strains. Bacterial DNA was extracted from purified isolates, the 16S rRNA genes amplified by PCR and the amplicons sequenced for identity using sequence databases. Teeth assigned for microscopy were post‐fixed in 3% gluteraldehyde after removal from the CDFF and then subjected to appropriate protocols prior to microscopic evaluation of the infection. Results All three microscopy techniques and culture‐dependent analysis confirmed infection of the human teeth using the CDFF, with root canal infections visually resembling closely those seen in vivo. Furthermore, partial 16S rRNA gene sequencing of DNA from cultured isolates confirmed a selective number of 7–9 genera/species in the apical portion of two teeth each at 7 and 28 days; these taxa are also commonly recovered from teeth with apical periodontitis, in vivo . There were no objective measures other than speciation and topographical evaluation to compare the artificial and real ( in vivo ) infections. Conclusions The proposed ex vivo model has the potential for development into an investigative tool for studying the dynamics of bacterial ecology in infected root canals, both before and after treatment. Its advantage is the ability to control both the abiotic and biotic factors. There is a need for the development of objective measures to compare artificial and real bacterial biofilms.