ABSTRACT
Methods for rapid preclinical testing of antiplaque agents in vitro using hydroxyapatite (HT)-coated glass beads are described. The assays developed could reliably detect (i) prevention of growth in the culture fluid or on the HT surfaces, (ii) the effect of transient exposure of a bactericidal agent on the viability of cells in a preformed bacterial mat, (iii) reversible adsorption of a bactericidal agent on an HT surface, and (iv) the ability of an agent to inhibit adsorption of Streptococcus sanguis to an HT surface or to salivary proteins adsorbed to an HT surface.
Subject(s)
Cariostatic Agents/pharmacology , Dental Plaque/prevention & control , Drug Evaluation, Preclinical/methods , Adsorption , Drug Evaluation, Preclinical/instrumentation , Fluorides/pharmacology , Glass , Humans , Hydroxyapatites , Placebos , Salivary Proteins and Peptides/metabolism , Streptococcus mutans/drug effects , Streptococcus sanguis/drug effects , Veillonella/drug effectsABSTRACT
A model system for the study of bacterial colonization and growth on a hydroxyapatite (HT) surface is described. Hydroxyapatite was crystallized over the surface of porous glass beads. Chemical analysis of the product showed that the ratio of Ca2+/P042- was indistinguishable from that of commercial HT powder. X-ray diffraction analysis supported the conclusion that the product was HT. A system employing [14C]polyethylene glycol, which selectively adsorbs to the glass surface of the beads, was developed to determine the amount of glass surface covered by HT. Over 90% of the glass surface could be covered by our method. The product, HT beads, consisted of approximately 20% (dry weight) HT. The HT beads possess several properties which make them potentially useful for studying microbial adherence, growth, and interactions. These include: (i) chemical similarity to the tooth surface, (ii) large surface area, and (iii) high density. We also describe a method for direct measurement of the microbial mass of cells growing on beads. The method entails immobilizing a sample on a membrane filter (Millipore), staining it with amido black dye, and eluting the dye for spectrophotometric measurement. Streptococcus mutans served as the test organism. For free-growing bacteria the values measured with the filter assay were directly proportional to cell number, with a value of 1 mug of "protein" corresponding to about 1.5 X 10(6) colony-forming units, determined by viable count. For bacteria colonizing the beads, 1 mug of protein corresponded to about 2 X 10(7) colony-forming units on the beads during logarithmic growth. As the culture approached stationary phase, the efficiency of the assay decreased. These data indicate that multiple random samples, taken at a given time, are representative of the entire culture.
