B o n e.

CONTRIBUTED BY: FIONA PETCHEY

Introduction

Bone is a connective tissue largely composed of an organic protein; collagen and the inorganic mineral hydroxyapatite, which combine to provide a mechanical and supportive role in the body (Smith et al. 1983:211). Depending on the orientation of collagen fibres, two types of bone can be distinguished: lamellar bone (cortical bone) and non lamellar (trabecular or cancellous bone), which is found in vertebrae, at the ends of long bones, the mammalian foetus, at fracture joints, and in many lower vertebrates (Pritchard 1974:5; Smith et al. 1983:447). Three types of cavities exit in bone; Haversian canals, marrow cavities, and the lacuna, which contain the bone cells (osteocytes) from which canalicuae (small tunnels) extend (Pritchard 1974:3-4; Hare 1980:208). A number of different bone extracts have been used to obtain 14C determinations. Initially analyses were carried out on whole bone, later the organic (collagen) or inorganic component of bone (hydroxyapatite) were separated and dated. A lack of success saw the dating of different fractions obtained during bone pretreatment, including the acid soluble (humics) as well as the "collagen" (acid insoluble component). More recently 14C determinations have been carried out on mixtures of amino acids (Ho et al. 1969; Stafford et al. 1987; Law and Hedges 1989; Long et al. 1989; Gurfinkel 1987); specific amino acids, for example hydroxyproline and proline (Wand 1981 in Taylor 1992; Stafford et al. 1982, 1988; Gillespie et al. 1984); a series of individual amino acids (Hare and Estep 1983 in Stafford et al. 1988; van Klinken and Mook 1990:155; Stafford et al. 1988); larger or smaller (peptide) parts of collagen (Brown et al. 1988; van Klinken and Hedges 1992); and non-collagenous proteins such as osteocalcin (Ajie et al. 1990).

Some extracted fractions of bone:

Collagen

Around 30% of bone is composed of organic compounds, of which 90 to 95% is collagen, the rest being non-collagenous proteins. Collagen is a fibrous protein which provides the bone with strength and flexibility, and is an important component of many other tissues, including skin and tendon. Individual collagen molecules contain three polypeptides of about 1000 amino acids per chain with a high glycine and hydroxyproline content. Bundles of these collagen molecules are arranged in fibrils with a molecular weight close to 97.1 Daltons. These fibrils are twisted into a right handed coil (fibre) with a total weight of between 95000 and 102 000 Daltons (Waterlow et al. 1978:512; Woodhead-Galloway 1980:1-3, 23; Garlick 1969:504; Smith et al. 1983:211-12).

For the collagen fibre to fully mature a number of chemical bonds must form. These include hydrogen bonds involving hydroxyproline, which stabilise the helix, and cross linkages involving hydroxylysine and lysine, which stabilise the fibrillar structure (Smith et al. 1983:447). These processes occur throughout the growth and maturity of an individual, consequently the density and stability of the bone tends to increase while the solubility decreases (Waterlow et al. 1978:512-14; Hare 1980:209; Smith et al. 1983:450). Once these bonds form only a small fraction of collagen can be extracted by neutral salt solutions and organic acids or acid-citrate buffers. The insoluble collagen which remains from such dissolutions, can however, be solubilised by heating above 58'C. At this temperature the triple helix denatures, but will partly reform into a gel when cooled (Waterlow et al. 1978:512; Woodhead-Galloway 1980:55; Smith et al. 1983:215).

Collagen in its unaltered state is also very resistant to proteolytic enzymes, however a group of enzymes exist which degrade native collagen fibrils under physiological conditions of temperature and pH; these are the collagenases (Waterlow 1978:516; Smith et al. 1983:223). An enzyme secreted by the gas gangrene bacteria (Clostridium perfringens and Cl. histolyticum) and Bacteroides melaninogenicus, a bacterium common in the gingival crevice of the tooth, will also cleave the triple helix (Woodhead-Galloway 1980:59). The peptide's produced in such cleavage are then open to proteolytic attack from the more conventional enzymes (Waterlow 1978:516).

Amino Acids

Collagen molecules are composed of linear, unbranching sequences of approx 20 naturally occurring amino acids. The structure of the molecule is stabilised by hydrogen bonds; the most common being between the amino group (-NH2) of one residue and the carboxyl group (-COOH) of a second residue, resulting in both acidic and basic properties. Uncharged side-chains also interact with one another, but by excluding water from their mutual interfaces (i.e. hydrophobic reaction) (Woodhead-Galloway 1980:10, 23-24, 38). All amino acids, except glycine, exhibit optical activity, existing in the natural state as laevo-rotary compounds, a property apparently restricted to amino acids of a biological origin (Wyckoff 1972:53), a property which is exploited in amino acid racemisation dating (e.g. Masters 1987).

In general the composition of mammalian collagens shows little variability (see Hare 1980:209). Of special significance to recent AMS works is the amino acid hydroxyproline (i.e. Stafford et al. 1987, 1988, 1991). Hydroxyproline is found rarely in other proteins but comprises about 10% of all amino acids in collagen. However, from a practical point of view the use of hydroxyproline for 14C analysis is limited as it does not occur in large quantities in fossil bones, has been detected in natural waters (Long et al. 1989), is excreted in the urine, and can be found in some plants (Waterlow et al. 1978:510; Woodhead-Galloway 1980:12; Taylor 1982:468).

Apatite

Seventy percent of bone is made up of the inorganic mineral hydroxyapatite, which includes calcium phosphate, calcium carbonate, calcium fluoride, calcium hydroxide and citrate. This inorganic component ([Ca3(P)4)2]3.Ca(OH)2) is predominantly crystalline, though may be present in amorphous forms (Hedges and van Klinken 1992:284). The crystals are platelets or rods, about 8 to 15A thick, 20 to 40A wide and 200 to 400A long. The substitution mechanisms that occur in the hydroxyapatite of bone include intercrystalline exchange and a recrystallisation due to dissolution and reformation of crystals, with the addition of new ions into the crystal structure replacing Ca2+ or being adsorbed on the crystal surfaces (Smith et al. 1983:446).

Teeth

The tooth is constructed of three layers; the pulp cavity, containing blood vessels and nerves; this is covered with the dentine; where the tooth is exposed the dentine is covered by enamel; and the submerged roots are covered with cementum. The cementum closely resembles cortical bone in composition, except that dentine is hard and dense, being almost 75% mineral but with a higher collagen content than bone (30% compared to 15% in bone). The enamel is denser and harder and is almost 98% mineral with the hydroxyapatite crystals being much larger than those of dentine, cementum or bone and consequently more resistant. Fully formed enamel contains a small amount of low-molecular weight peptides that are almost devoid of proline and hydroxyproline (Smith et al. 1983:449, 452; Protsch 1991:282, 287).

Bone Weathering

Little attention has been given to the environmental conditions of bone preservation (Sobel and Berger 1994), however, the quantities and composition of surviving organic materials in a specimen are dependent on their burial environment (Garlick 1969:503). Environmental factors which have been suggested as influencing the rate at which collagen degrades include the composition, pH and hydrology of the matrix; oxygenation; temperature; and changes brought about by soil flora and fauna (Henderson 1987; Shiffer 1987).

In a generalised view of bone degradation the protein component undergoes relatively slow hydrolysis to peptides, which then break down into amino acids. At the same time there is spontaneous rearrangement of the inorganic crystalline matrix which weakens the protein-mineral bond and leaves the bone susceptible to dissolution by the action of internal and external agents (Henderson 1987:44). Alterations during diagenesis are believed to include random cross-linking, humification of parts of the molecule, attachment of exogenous humic materials, and hydrolysis with preferential loss of some amino acids (Hedges and van Klinken 1992:281-2).

Two major groups of contaminants exist; humic and non-humic substances. Humic substances are dark coloured acids moderately high-molecular-weight polymers of indefinite structure. They represent an extremely heterogeneous mixture of molecules, including amino acids which, in any given soil or sediment may range in molecular weight from 2,000 to over 3000000 and have a range of properties depending on size and attached functional group (Stevenson and Butler 1969:534). While humic substances are generally insoluble in acid and soluble in alkali, one component, the fulvic acids, are soluble in both mediums making them extremely difficult to separate from collagen (Stevenson and Butler 1969:535). Non-humic substances include all classes of organic compounds. The major contaminants are polyphenols, polysaccharides, lignins as well as degraded collagen and other broken down bone components (Morrison 1969:559-60).

Bone references

Bone Pretreatments