Mapping Bone Surface Composition Using Real-Time Surface Tracked Micro-Raman Spectroscopy

Abstract

The surface of bone tells a story – one that is worth a thousand words – of how it is built and how it is repaired. Chemical (i.e., composition) and physical (i.e., morphology) characteristics of the bone surface are analogous to a historical record of osteogenesis and provide key insights into bone quality. Analysis of bone chemistry is of particular relevance to the advancement of human health, cell biology, anthropology/archaeology, and biomedical engineering. Although scanning electron microscopy remains a popular and versatile technique to image bone across multiple length scales, limited chemical information can be obtained. Micro-Raman spectroscopy is a valuable tool for nondestructive chemical/compositional analysis of bone. However, signal integrity losses occur frequently during wide-field mapping of non-planar surfaces. Samples for conventional Raman imaging are, therefore, rendered planar through polishing or sectioning to ensure uniform signal quality. Here, we demonstrate ν<sub>1</sub> PO<sub>4</sub><sup>3-</sup> and ν<sub>1</sub> CO<sub>3</sub><sup>2-</sup> peak intensity losses where the sample surface and the plane of focus are offset by over 1–2 μm when underfocused and 2–3 μm when overfocused at 0.5–1 s integration time (15 mW, 633 nm laser). A technique is described for mapping the composition of the inherently irregular/non-planar surface of bone. The challenge posed by the native topology characteristic of this unique biological system is circumvented via real-time focus-tracking based on laser focus optimization by continuous closed-loop feedback. At the surface of deproteinized and decellularized/defatted sheep tibial cortical bone, regions of interest up to 1 mm<sup>2</sup> were scanned at micrometer and submicrometer resolution. Despite surface height deviations exceeding 100 μm, it is possible to seamlessly probe local gradients in organic and inorganic constituents of the extracellular matrix as markers of bone metabolism and bone turnover, blood vessels and osteocyte lacunae, and the rope-like mineralized bundles that comprise the mineral phase at the bone surface.

The long‐term impact of alveolar ridge preservation with xenograft bone mineral on peri‐implant health after 5 years in function: A retrospective cohort study of 108 patients assessed clinically and radiologically

“…Nevertheless, histological studies of Bio‐Oss® reveal slow resorption (Orsini et al, 2007 ; Schlegel & Donath, 1998 ), which is related to the physicochemical characteristics of Bio‐Oss®. More specifically, the CO 3 2 − content (ν 1 CO 3 2 − /ν 1 PO 4 3 − intensity ratio) is ∼44%–52% lower than values typically reported for whole bone (Shah, Ruscsák, et al, 2020 ; Shah, Snis, et al, 2016 ), indicating significant decarboxylation, while the mineral crystallinity is ∼71.5% higher than whole bovine bone (Shah, 2020 ).…”

“…Bio‐Oss® granules, ∼250 µm to ∼1 mm in size, exhibit surface microstructure similar to deproteinized bone (Figure 4 ), where mineralized collagen fibrils assemble into a network of twisted rope‐like bundles (Shah, Ruscsák, et al, 2020 ; Shah, Zanghellini, et al, 2016 ). Fractured surfaces reveal a lamellar pattern.…”

Bone without borders – Monetite-based calcium phosphate guides bone formation beyond the skeletal envelope

“…1 H). Real-time surface tracked micro-Raman spectroscopy enabled mapping the phase composition of intact CaP without mechanically rendering the surface planar (e.g., by polishing) or excluding topological irregularities [ 31 ]. All the characteristic spectral features of monetite (at ∼900 and ∼986 cm −1 ), β -TCP (at ∼947 cm −1 and ∼970–972 cm −1 ), and Ca-PP (at ∼1043–1048 cm −1 ) are identified [ 33 ] ( Fig.…”

“…Micro-Raman spectroscopy was performed using a confocal Raman microscope (Renishaw inVia Qontor, Renishaw plc. Wotton under Edge, UK) equipped with a 633 nm laser and LiveTrack focus-tracking technology, which enables enhanced signal stability at non-planar and non-plane parallel surfaces [ 31 ]. The laser was focussed down on to the surface of intact CaP, resin embedded/polished intact CaP, resin embedded/polished CaP-bone (52 w CaP) blocks using a 100×/0.9 NA objective [ 32 ].…”

Physicochemical Understanding of Biomineralization by Molecular Vibrational Spectroscopy: From Mechanism to Nature

“…Anders et al solve the challenge brought by the inherent topology of this unique biological system by using the real-time focusing and tracking technology of continuous closed-loop feedback to optimize laser focusing. [218] In situ, analysis of organic and inorganic components of the biomineralization is possible despite surface height deviations of more than 100 μm in the femur. There are also some Raman spectroscopic studies focusing on bone diseases and the analysis of bone complications caused by other conditions, such as osteoporosis and type-II diabetes.…”