Hyperspectral Raman imaging of bone growth and regrowth chemistry

ABSTRACT Hyperspectral Raman microscopic imaging of carbonated hydroxyapatite (HAP) is used to follow the chemistry of bonegrowth and regrowth. Deep red (785 am) excitation is employed to minimize protein fluorescence interference. A passive linegenerator based on Powell lens optics and a motorized translation stage provide the imaging capabilities. Raman imagecontrast is generated from several lines of the HAP Raman spectrum, primarily the PO stretch at ca. 960cm1. Factor analysis is used to minimize the integration time needed for acceptable conirast and to explore the chemical species withinthe bone. Bone age is visualized as variations in image intensity. High definition, high resolution images of newly formedbone and mature bone are compared qualitatively. The technique is currently under evaluation for study of experimentaltherapies for fracture repair.Keywords: Raman imaging, hydroxyapatite, factor analysis, bone growth 1. INTRODUCTION Bone consists of an organic matrix onto which an inorganic mineral phase is deposited. In mature bone the inorganic phase isa complex non-stoichiometric hydroxyapatite species. The composition is nominally Ca10(P04)6(OH)2. The most commonsubstitution is replacement of hydroxide or phosphate in the crystal structure by carbonate ion. Small amounts of fluoride,chloride and hydrogen phosphate are also found in the lattice. Several other insoluble calcium phosphates, such as amorphouscalcium phosphate, dicalcium phosphate, and octacalcium phosphate, precede the formation of hydroxyapatite. As boneadvances from the initial stages of growth to full maturity the degree of mineralization and substitution vary.In an effort to completely characterize bone growth, studies are underway to understand both the chemistry involved in bonegrowth and the physical properties that correlate to changes in the chemical composition of bone. Progress in these areaswould impact many diverse arenas from clinical orthopedics to bioengineering. A more complete understanding of bonechemistry could aid in the design and implementation of rational bone growth therapies and thus speed fracture healing,improve implant technology, and stimulate the design of novel synthetic bone graft materials.Bone growth is dynamic process occurring throughout a volume. Spectroscopic imaging can reveal information that pointspectroscopy alone can not. The chemical information, micron-scale spatial resolution, and high definition available withRaman imaging are important benefits when examining biological samples. The mineral phase within bone is very similar tothe mineral in teeth. The success of Raman imaging in the dental materials arena, coupled with the successful use of infraredand Raman spectroscopy to analyze bone, makes Raman imaging a logical next step in studying bone growth chemistry. Thedevelopment of stable high power near-infrared diode lasers and red-optimized CCD detectors make near-infrared excitationpractical for Rainan imaging. With the excitation wavelength in the near-infrared, most of the interference from fluorescenceis eliminated.While bone composition and growth have been studied by a variety of methods, vibrational spectroscopy is particularlyuseful to study the chemical nature of biological hydroxyapatite. Paschalis and Boskey have developed novel methods for