Because both bone and teeth are essentially the only substrates used for the various examinations and analyses presented, it is necessary to provide a description of the individual structures pertinent to this study. According to White & Folkens (1991), human skeletal elements display a great variety of shapes and sizes, yet can be categorized into several basic forms that often share certain features amongst one another. Long bones, which are cylindrically shaped with flaring ends, belong to the elements comprising the upper and lower extremities. Flat, tabular shaped bones can be seen in the regions of the skull, shoulder, pelvis and rib cage. The carpal, tarsal and vertebral bones are block-like and exhibit numerous different forms. Irregardless of shape or size, bone tissue is basically the same at the macroscopic and molecular level. Bone tissue is composed of two major components: an inorganic mineral fraction (bone ash), which composes 70% of bone’s dry weight in adult humans, and an organic matrix, which composes approximately 30% of bone's dry weight. Collagen composes 90% of the organic matrix, also called osteoid, in dry, fat free bone (Price 1989). A number of other components are also contained in bone to a lesser extent, yet play important roles in its makeup and function and include water, ground substance proteoglycans, non-collagenous proteins, which are thought to be involved in regulation of bone mineralization, and also fats, vascular elements and cells (Wheater et al. 1987). Collagen is the most abundant protein in vertebrates and comprises the primary fibrous component of supporting tissues. Nineteen different types have been identified (I-XIX), and are distinguished based upon morphology, amino acid composition and physical properties (Martin et al. 1998). In bone, Type-I collagen, which belongs to a group known as fibrillar collagens, functions to provide tensile strength and resilience, two important attributes that make bone such a dynamic tissue during life and so durable after death. Type-I collagen is also found in the skin, tendons and ligaments and its structural arrangement differs in these tissues according to the mechanical support required. According to Burkitt et al. (1993), collagen is secreted into the extracellular matrix in the form of tropocollagen or propeptide, which consists of three polypeptide chains (primarily two α1 chains and one α2 chain) bound together to form a triple helical structure 300nm long and 1.5nm in diameter. The amino acid sequence of the individual polypeptide chain is Gly-X-Y repeats, a peptide arrangement that results in a coiled, left-handed helix (Currey 2002). The Gly residues are at the center of the triple helix and the X and Y residues at the surface of the helix. In one-third of the cases X is a proline and Y is hydroxyproline; the presence of hydroxyproline is essential to stabilize the triple helix and is a unique characteristic of collagen molecules. When three of the helically
coiled polypeptide chains assemble, they form a right-handed triple helix (Rossert & de Crombrugghe 1996). These molecules subsequently polymerize to form collagen fibrils. Parallel collagen fibrils further aggregate in a process of systematic “quarter staggered” overlapping to form strong bundles 2-10µm in diameter. It is precisely this tough, fibrous quality of Type-I collagen combined with the rigid, hardness of inorganic hydroxyapatite crystals interspersed within the gaps between the collagen molecules that produces the unique structural integrity characterizing both human bone tissue and the skeletal framework as a whole (Lowenstam & Weiner 1989).
In order to facilitate the analysis of this organic component, collagen must first be isolated from the other bone constituents. The process of collagen extraction from bone involves its separation from the inorganic components of bone resulting in the formation of gelatin (see methods, section 8). The complex triple helix structure of this protein is destabilized when the intermolecular bonds holding them together are broken following a heating step during the last phase of the extraction procedure, which causes the otherwise tough and insoluble polypeptide chains to disband. Gelatin is created when these unwound chains randomly tangle and fold back upon themselves during cooling. In order to isolate the mineral fraction, the same structural characteristics that make collagen and non-collagenous proteins extractable are utilized to facilitate its denaturing and removal.
According to McCarthy and Frassica (1998), the mineral component of the human skeleton including the teeth, is comprised primarily of a crystalline calcium-phosphate composite (Ca10[PO4]6[OH]2), a molecular complex that is analogous to the geological molecule
hydroxyapatite (Grupe et al. 2005). In comparison to this form of hydroxyapatite, bone mineral itself possesses deficiencies with respect to calcium and hydroxyl ions, and is also characterized by numerous substitutions, mainly through carbonate. The majority of this carbonate is type-B, which substitutes for phosphate ions, and is different from type-A in that this form substitutes for OH groups (Boskey 1999). The quantity and type of substitution have a direct influence on the solubility of the bone mineral. During adulthood the fraction of carbonate increases, in contrast to the phosphate fraction which decreases, however, the total ion sum of carbonate and phosphate always remains constant. Therefore the actual mineral fraction of the human skeleton can be illustrated with the molecular combination Ca8.3(PO4)4.3(CO3)x(HPO4)y(OH)0.3, with x + y = 1.7 (const.) The mineral’s apatite crystal
structure is small relative to tooth apatite or the geological analog, and are hexagonal, symmetric, and average 5 x 5 x 40nm in size (Martin et al. 1998).
Bone continually undergoes both a modeling and remodeling process (Boskey 1999). Modeling simply involves the formation of bone in places where it has not been before and remodeling refers to the formation of bone in places where it has already been. Bone is actively remodeled for two primary reasons. The main functions are to maintain mechanical strength by replacing fatigued bone whose structural integrity is compromised and to facilitate mineral homeostasis. Within the span of one year, approximately 2-3% of cortical bone is turned over consistent with the maintenance of mechanical properties (Dempster 1999). Cancellous bone experiences a greater amount of turnover, primarily because it is more heavily involved in mineral homeostasis.