ABSTRACT
Matrix vesicles (MV) having high alkaline phosphatase (ALP) activity act as initiators of biological mineralization. Although bacteria have similar membranous structures to MV, ALP mediated mineralization has not been studied in bacterial cells. Escherichia coli was transformed with a bacterial ALP gene in this study. Recombinant E. coli overproducing ALP induced mineralization through hydrolysis of calcium-glycerophosphate (Ca-GP). Fourier transform infrared spectroscopy and electron microscopy combined with electron diffraction revealed newly formed hydroxyapatite mineral deposits. These findings suggest that hydrolysis of Ca-GP through ALP induced high Ca and Pi concentrations within bacterial cells followed by complete bacterial mineralization.
Subject(s)
Alkaline Phosphatase/genetics , Calcification, Physiologic , Escherichia coli/genetics , Alkaline Phosphatase/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/ultrastructure , Kinetics , Transformation, BacterialABSTRACT
Transformation in the structure and composition of calcium-glycerophosphate (Ca-GP) was investigated using a scanning electron microscope fitted with an energy dispersive X-ray microanalysis (EDX) system. Ca-GP was packed inside roots that were subsequently implanted into the mandibles of rats to stimulate the formation of an apical barrier. Scanning electron microscopic observations of packed Ca-GP revealed three types of structures: globular aggregates, plate or flake-like aggregates, and granular aggregates. The average Ca/P molar ratios of EDX by area mode--postoperatively obtained at days 1, 3, 5, and 7--were 1.300, 1.533, 1.472, and 1.495 (analytic area: 0.01 to 0.03 mm2) and those of EDX by point mode were 1.220, 1.451, 1.487, and 1.467 (analytic point: 0.05 micron 2). The magnesium (Mg) weight percentage was found to be approximately 1.9 at days 3, 5, and 7, similar to Mg-substituted whitlockite. These findings suggest that Ca-GP are transformed, over a period of 3 days into Mg-substituted whitlockite as a result of being hydrolyzed by tissue fluid.
Subject(s)
Glycerophosphates/therapeutic use , Tooth Apex/drug effects , Analysis of Variance , Animals , Calcium/analysis , Calcium Phosphates/analysis , Chemical Precipitation , Confidence Intervals , Crystallography , Dental Pulp Cavity/chemistry , Dental Pulp Cavity/drug effects , Dental Pulp Cavity/pathology , Electron Probe Microanalysis , Glycerophosphates/metabolism , Hydrolysis , Magnesium/analysis , Male , Mandible/surgery , Microscopy, Electron , Microscopy, Electron, Scanning , Models, Animal , Phosphorus/analysis , Rats , Rats, Wistar , Tooth Apex/chemistry , Tooth Apex/pathology , Tooth Calcification/drug effects , X-Ray DiffractionABSTRACT
Tissue changes in rat incisors after direct pulp capping with hydroxyapatite (OH-AP) have been investigated using light and electron microscopy. Immediately after direct pulp capping, necrotic cells and cell debris were scattered only in the superficial zone of an exposed site. At day 1, both neutrophils having many vacuoles and macrophages phagocytizing numerous OH-AP granules appeared in the lower part of the OH-AP layer. At day 3, the cells surrounding the basal part of the OH-AP layer had large nuclei and well-developed rough-surfaced endoplasmic reticulum. At day 5, the newly formed mineralized tissue consisting of needle-like crystals was observed directly adjacent to the deepest zone of the OH-AP layer. However, fibroblast-like cells phagocytosing applied OH-AP granules were seen even at this period. These findings suggest that the applied OH-AP could directly substitute for osteodentin.
