In situ atomic force microscopy of partially demineralized human dentin collagen fibrils
Introduction
Dentin is the calcified tissue that forms the internal bulk of the tooth, lying between the enamel and the pulp chamber. It is a hydrated biological composite consisting of tubules, which were the pathways of the formative odontoblastic cells, the peritubular dentin, a highly mineralized zone surrounding the tubules, and the intertubular dentin (Ten Cate, 1994). The latter has an estimated composition of approximately 50 wt% mineral phase, 40 wt% organic phase, and 10 wt% aqueous fluids and is similar to the composition of bone (Weiner and Wagner, 1998). Ninety weight percent of the organic phase in dentin is collagen, which is almost exclusively type I (Goldberg and Takagi, 1993; Linde and Robins, 1988). Type I collagen forms a fibrous three-dimensional network which builds up the dentin matrix. Compared to bone, the collagen matrix in dentin is more interwoven with numerous crossings of fibrils (Kramer, 1951). Noncollagenous proteins, mostly glycoproteins and proteoglycans, cover the collagen fibrils and are associated with the inorganic phase. In particular, phosphoproteins are believed to be critical for inducing mineral nucleation and for binding to calcium phosphates (Begue-Kirn et al., 1998; Dahl et al., 1998; MacDougall et al., 1992; Saito et al., 1998). The mineral in dentin is a carbonated apatite, which is similar to the mineral in bone and calcified tendon and is located either in the gaps between collagen molecules (intrafibrillar) or attached to the collagen fibrils (extrafibrillar) (Lees et al., 1997; Wassen et al., 2000). Plate-like and cylindrical morphologies have been reported for the apatite crystals in dentin with dimensions between 20 and 5 nm (Arsenault, 1989; Kinney et al., 2001; LeGeros, 1991).
The structure–property relationship of collagen fibrils is critical for understanding many aspects of this tissue: alteration with disease, aging, or restorative treatments such as dentin bonding. Type I collagen molecules are composed of three supercoiled polypeptide chains of about 290 nm in length, held together by water bridges and hydrophobic cross-links. During dentinogenesis, collagen molecules are synthesized at the rough endoplasmic reticulum of the odontoblasts and extruded as a triple helix (tropocollagen) into the extracellular space. In a current model, five tropocollagen molecules stagger longitudinally, overlapping by about one quarter of their length, to form a microfibril about 4 nm in diameter (Nimni and Harkness, 1988). The microfibrils aggregate with their long axes in parallel to form collagen fibrils. In vitro observations of the fibrillogenesis showed an increase of the fibril diameter by increments of approximately 8 nm, attributed to units of microfibrils, which are the building blocks of the collagen fibril (Parry and Craig, 1988; Prockop and Fertala, 1998). Depending on the tissue type, age, and genetic defects, collagen fibrils vary in diameter from 30 to 500 nm, as determined by electron microscopy and X-ray diffraction (XRD) (Nimni and Harkness, 1988). Shrinkage due to dehydration by 10–40% has been reported (Brodsky et al., 1988). Human dentin collagen fibrils have been imaged at high resolution by electron microscopy. However, the fibril diameters reported were not consistent among studies. Fibril diameters around 100 nm have frequently been determined (Garberoglio and Brånnström, 1976; Pashley, 1991; Perdigao et al., 1996), but values as low as 30–60 nm were also found in the literature (Avery, 1988; Lin et al., 1993). Lin et al. (1993) reported fibrils of increased thickness at the dentino–enamel junction,with diameters between 80 and 120 nm.
The hierarchical synthesis of collagen fibrils leads to a high degree of organization and yields a strongly crystalline character, which is even more pronounced when the fibril is hydrated (Prockop and Fertala, 1998). The staggered arrangement, combined with gaps between the ends of successive collagen molecules, results in periodically alternating gaps and overlap zones. Their periodicity, or D-distance, depends on the state of hydration of the fibril and decreases from 67 nm for the hydrated fibril, to around 64 nm in air-dried samples, and down to 60 nm after dehydrothermal treatments at 120 °C (Baer et al., 1988; Bella et al., 1995; Wess and Orgel, 2000).
In situ observation of details of dentin collagen structure has largely been achieved by electron microscopy (Arsenault, 1989; Lin et al., 1993; Perdigão et al., 1999). Usually transmission electron microscopy (TEM) has been used to obtain the high resolution necessary to observe the structural features of the fibrils. TEM studies, however, require intensive sample preparation and often staining to resolve gap and overlap zones of collagen fibrils. Furthermore, the vacuum in electron microscopy does not facilitate imaging of the tissue in its natural hydrated state and raises concerns of introducing artifacts due to desiccation (El Feninat et al., 2001; Lee, 1984; Reedy et al., 1983). TEM studies frequently showed an axial repeat distance around 64 nm, attributed to the dehydrated fibril. Beniash et al. (2000) avoided fibril contraction during TEM observation by using vitrified ice sections and determined a D-periodicity of 67 nm, as documented for the hydrated fibril. They also reported D-distances as low as 23.5 nm for forming fibrils in rat predentin, with diameters of around 30 nm.
Scanning probe microscopes and, in particular, the atomic force microscope have facilitated the imaging and analysis of biological surfaces with little or no sample preparation. Atomic force microscopy (AFM) can operate in air or in liquid, and the imaging of macromolecules, like proteins or DNA, has been reported by several authors (Chen and Hansma, 2000; Hansma, 2001; Scheuring et al., 2001). Assembled proteins, e.g., collagen fibrils, also have been resolved successfully. Several authors (Chernoff and Chernoff, 1992; Raspanti et al., 1997; Revenko et al., 1994) revealed the D-periodicity of reconstituted collagen fibrils on a mica or glass substrate using AFM. Subfibrillar structures of 4–5 nm of dehydrated native rat tail and reconstituted bovine dermal collagen were resolved by Baselt et al. (1993). Reconstitution of collagen fibrils, however, involves a series of chemical treatments to first dissolve and then reassemble the fibrils. Thus, the fibril is no longer imaged in its native environment and original structural features could be lost or altered during reassembly.
The composite nature of dentin and bone, where fibrils are covered by noncollagenous matrix proteins and apatite minerals, increases the difficulty of imaging of the fibrils by topographic techniques like AFM. Recent in situ studies of sequential demineralization and deproteinization of dentin have shown that, under certain circumstances, high-resolution AFM imaging of collagen fibrils is possible (Marshall et al., 2001). This suggested that dissolution of most of the mineral phase, followed by gradual removal of extracellular matrix proteins, would expose collagen fibrils, which could be revealed by AFM. In the present study, this method is applied to dentin to obtain information on collagen fibril organization and the effects of hydration and dehydration on fibril diameter and axial repeat distance.
Section snippets
Materials and methods
Human third molars with documented history were extracted from patients of ages of between 22 and 34 years according to protocols approved by the University of California, San Francisco Committee on Human Research. Teeth were sterilized by γ-radiation and stored in deionized water at 4 °C until prepared (White et al., 1994). Sagittal midcoronal sections of six teeth (thickness of 2 mm) were prepared by polishing through a series of SiO2 papers and with water-based diamond paste to
Results
Figs. 1a–d show a series of 10×10-μm contact mode images of hydrated human intertubular dentin after different chemical treatments. The polished and untreated tooth specimen of Fig. 1a shows its characteristic structure (Marshall et al., 1997). A layer of higher mineralization, the peritubular dentin, lines tubules of about in diameter. The matrix phase between the tubules is intertubular dentin, consisting of extracellular matrix proteins and carbonated apatite mineral. Fig. 1b shows the
Discussion
In this study, a chemical process was used to expose type I collagen fibrils of midcoronal dentin. Polished dentin sections prepared from documented human third molars were partially demineralized by citric acid. Acid etching is a common procedure to dissolve the calcium-phosphate mineral phases in dentin in order to form a porous collageneous surface layer, which allows the penetration of a monomer that forms a layer of collagen and polymer, resulting in a superior interface for the bonding of
Conclusions
Etching and controlled deproteinization enabled the in situ imaging of the fibrous network of partially demineralized dentin collagen by AFM. Fully hydrated and dehydrated type I collagen fibrils were analyzed at high resolution and information on the organization of the fibrillar network of collagen was obtained. Dehydration induced significant structural changes to the fibrils. While three major groups of fibril diameters were observed when the substrate was hydrated, fibril diameter spread
Acknowledgements
AFM-images of Figs. 1c and d are courtesy of Dr. Nil Yücel, Department of Preventive and Restorative Dental Sciences, University of California, San Francisco. This research was supported by NIH/NIDCR, Grant PO1 DE09859.
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