Intrafibrillar demineralization study of single human dentin collagen fibrils by AFM
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Intrafibrillar demineralization study of single human dentin collagen fibrils by AFM M. Balooch, G. Balooch, S. Habelitz, S. J. Marshall and G. W. Marshall Department of Preventive and Restorative Dental Sciences Division of Biomaterials and Bioengineering University of California, San Francisco 707 Parnassus Ave. San Francisco, Ca 94143 ABSTRACT In situ atomic force microscopy (AFM) was used to investigate the kinetics of demineralization of human dentin collagen fibrils. Individual dentin collagen fibrils containing intrafibrillar mineral were isolated, transferred onto a glass slide and exposed to water for a day prior to demineralization studies. The fibrils then were exposed to trypsin for removal of non-collagenous proteins and subsequently demineralized in 10 vol % citric acid. Topographic images showed a gradual increase in gap-overlap depth of the fibril. The gap-overlap depth varied linearly with the square root of time before saturation at 7 nm in approximately sixty minutes, suggesting a diffusion process for demineralization of intrafibrillar mineral. Micro Raman studies of partially demineralized dentin revealed the existence of a phosphate peak at wave number close to 960 cm-1. The peak gradually disappeared in 60 minutes as the samples were exposed to 10% citric acid, supporting the notion that AFM topography may be correlated to the degree of intrafibrillar mineralization.
INTRODUCTION Dentin is a calcified tissue that forms the internal bulk of the tooth, lying between the enamel and pulp chamber. It has a composition of approximately 50 wt% mineral phase, 40 wt% organic phase, and 10 wt% aqueous fluid. More than ninety percent of the organic phase in dentin is collagen type I (1, 2), formed in a three-dimensional fibril network which builds the dentin matrix. In dentin and related calcified tissues such as bone and cementum, collagen type I fibrils are mineralized to provide the fundamental building blocks of the calcified tissue. It is believed that collagen and non-collagenous proteins are required for proper biomineralization of dentin. The major non-collagenous proteins of dentin are dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) (3), which are encoded by the dentin sialophosphoprotein (DSPP) gene (4). Non-collagenous proteins (NCP’s) are believed to associate at specific sites on the collagen molecule to promote the nucleation and growth of apatite crystals (5). Collagen fibrils are formed by self assembly of triple helical collagen molecules that have a distinctive arrangement in which the adjacent molecules are arranged with a regular staggered off-set of ¼ the length of the triple helical molecule (6). This leads to the formation of typical banding or repeat patterns along the length of collagen fibrils that have a normal 67 nm repeat distance. Each repeat is further subdivided into overlap (27 nm.) and gap zones (40 nm.)
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based on the periodic staggering of the molecules (7). The gap zones are thought to be the key site for the initiation of biomineralization.
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