Mineralogy of Hypermineralized Bone
Date of Award
Doctor of Philosophy (PhD)
Bone is composed mainly of mineral and collagen. The mineral, bioapatite, is a form of carbonated hydroxylapatite, which makes up over 50 wt.% of bone. Mineralogical features of bioapatite, e.g., chemistry, mechanical properties, degree of crystallinity, and morphology, thereby absorb mineralogists' interest. However, normal bone contains much collagen, which is interlaced with mineral crystallites at the nanometer and micrometer scale, making the study of bioapatite very difficult. In addition, removal of the collagen by chemicals usually changes some chemical and physical features of bone mineral. Hypermineralized bone with extremely high mineral contents (>80 wt.%) fortunately allows the application of many analytical techniques to investigate the mineralogy of bioapatite with very little interference from collagen. Therefore, such hypermineralized bones appear to be the ideal materials in which to study bioapatite in bone. In the present study, a rostrum bone from a Mesoplodon densirostris whale (~96 wt.% mineral) and bullae (ear bones) from Tursiops truncates dolphins (~85 wt.%) are analyzed by several techniques, e.g., Raman spectroscopy, electron microprobe, and scanning electron microscopy, to investigate the mineralogy, to compare hypermineralized bone to enamel, and to distinguish age-related changes of the bioapatite in bone.
Firstly, it is necessary to confirm that these hypermineralized materials are true bone, rather than hypermineralized enamel or other dense bio-tissues. The Raman spectra of the rostrum and bullae show similar peak occurrence as in other normal bones, except for the low intensity of peaks for organics. Raman spectra also confirm that the mineral in the rostrum and bulla is carbonated hydroxylapatite, just as in other normal bones. A set of bone features, e.g., collagen fibrils, lacunae, osteons, vascular holes, and blood vessels, are identified in the rostrum. The dolphin's bulla has long been recognized as true bone because it is actually the ear bone. However, these two types of hypermineralized bone show distinct processes of mineralization. The high density of the bulla results from complete filling of the inter-trabecular spaces with hypermineralized tissue, whereas the rostrum undergoes hypermineralization during extensive remodeling and development of secondary osteons.
Secondly, the hypermineralized rostrum, as a bone exemplar, was used to investigate the chemistry of bioapatite. Relatively few chemical analyses of major and minor elements of bone mineral have been reported. The existing analyses are usually based on ashed bone and therefore do not exclude effects from the abundant collagen. Electron microprobe analyses of the rostrum's bioapatite show an average carbonate content of ~8 wt% and an average Ca/P atomic ratio of 1.7. Electron microprobe and Raman analyses show a homogenous distribution of the mineral content, except around a few vascular holes and vessels. Hydroxyl depletion in the bioapatite is coupled with carbonate substitution and Ca can be substituted by Na and Mg in mineralization. Bulk analysis by X-ray fluorescence shows that the bioapatite in the rostrum has an average composition of (Ca8.40Mg0.20Na0.54)[(PO4)4.87(CO3)1.13](OH)0.87. In addition, the rostrum has some minor elements (K and Cl) and extremely low-concentration trace elements (Al, Si, Fe, Ti and Sr), as in typical bone materials.
Thirdly, the rostrum was compared to the tooth enamel. These two hypermineralized bio-tissues have similar mineral contents, but are different in mechanical properties, texture of their component mineral prisms, chemistry, crystal size and degree of crystallinity. The mineral prisms in enamel show extreme homogeneity of orientation, i.e., c axis of the crystals parallel the longitudinal direction, whereas there is some amount of variation in orientation in the crystals of the rostrum. Such heterogeneity, compared to enamel, also occurs in the chemistry and mechanical properties of the rostrum. For example, in biomedical tests, the bioapatite in the rostrum shows higher deviations in indentation depth than the enamel. In addition, substitutions of Na and Mg for Ca, as well as CO32- for PO43-, are more prominent in the rostrum than in enamel. At the micrometer scale, the lengths and widths of mineral prisms in enamel are four times larger than those in the rostrum. However, platelets that constitute the prisms are wider in the rostrum (~200 nm) than those in enamel (~70 nm). The bioapatite crystals in the rostrum also show a lower degree of crystallinity compared to those in enamel based on the widths of the ν1 P-O stretch at about 960 δcm-1 in Raman spectra. The hypermineralized bone therefore has a greater heterogeneity in almost every aspect of its mineralogy compared to enamel. The tooth enamel is much closer to the standard hydroxylapatite in its chemistry.
Finally, the mineralogical changes in bioapatite were investigated using dolphins' bullae. The bullae from dolphins at ages of < 3 months, 2.5 years, and 20 years were studied. Transverse sections show that the bullae have (previously undescribed) organic-enriched edge areas and mineral-enriched central areas. Additionally, the central areas have a ~2 wt.% more carbonate in their bioapatite than the edge areas. During aging, the abundant pores in the edge areas become filled with densely mineralized tissue whereas organic matter is reduced. These changes yield greater homogeneity in mineral content throughout the adult bullae. Ca/P atomic ratios and the concentrations of Mg, S, and other minor/trace elements, otherwise, are almost constant in the central areas over time. Enhancement of the coupled substitutions of CO32- for PO43- and Na for Ca during aging yield a carbonate content up to ~10 wt.% in the adult bulla, making its carbonate content at the high end of all bones. Remarkably, the degree of crystallinity of the bioapatite remains approximately constant with age despite the increase in carbonate contents.
Chair and Committee
JIll Dill Pasteris
Jill Dill Pasteris, Jeffrey G. Catalano, Robert F. Dymek, Alian Wang, Stavros Thomopoulous, Daniel Giammar
Li, Zhen, "Mineralogy of Hypermineralized Bone" (2013). Arts & Sciences Electronic Theses and Dissertations. 179.