Towards refining Raman spectroscopy-based assessment of bone composition

www.nature.com/scientificreports OPEN Towards refining Raman spectroscopy‑based assessment of bone composition Furqan A. Shah Various compositional parameters are derived using intensity ratios and integral area ratios of different spectral peaks and bands in the Raman spectrum of bone. The ν1-, ν2-,ν3-, ν4 PO43−, and ν1 CO32− bands represent the inorganic phase while amide I, amide III, Proline, Hydroxyproline, Phenylalanine, δ(CH3), δ(CH2), and ν(C–H) represent the organic phase. Here, using high-resolution Raman spectroscopy, it is demonstrated that all PO43− bands of bone either partially overlap with or are positioned close to spectral contributions from the organic component. Assigned to the organic component, a shoulder at 393 cm−1 compromises accurate estimation of ν2 PO43− integral area, i.e., phosphate/apatite content, with implications for apatite-to-collagen and carbonate-to-phosphate ratios. Another feature at 621 cm−1 may be inaccurately interpreted as ν4 PO43− band broadening. In the 1020–1080 cm−1 range, the ~ 1047 cm−1 ν3 PO43− sub-component is obscured by the 1033 cm−1 Phenylalanine peak, while the ~ 1076 cm−1 ν3 PO43− sub-component is masked by the ν1 CO32− band. With ν1 PO43− peak broadening, ν2 PO43− integral area increases exponentially and individual peaks comprising the ν4 PO43− band merge together. Therefore, ν2 PO43− and ν4 PO43− band profiles are sensitive to changes in mineral crystallinity. Raman spectroscopy is a highly versatile and non-destructive tool for bone composition analysis. Using intensity ratios1 and integral area r atios2 of different spectral peaks and bands, a variety of Raman metrics are derived in order to describe various compositional parameters of bone. Spectral features assigned as amide I, amide III, Proline (Pro), Hydroxyproline (Hyp), Phenylalanine (Phe), δ(CH3), δ(CH2), and ν(C–H) are taken as markers of the organic component, i.e., collagen3–5, while ν1 PO43−, ν2 PO43−, ν3 PO43−, ν4 PO43−, and ν1 CO32− are the main bands associated with the inorganic component, i.e., apatite6. Of the various compositional parameters commonly considered, crystallinity of bone mineral is almost invariably estimated as the reciprocal of the full-width at halfmaximum (FWHM) of the ν1 PO43− peak, centred at 957–962 cm−1, while estimation of the apatite-to-collagen ratio and the carbonate-to-phosphate ratio remain arbitrary. Tissue-level mechanical properties of bone are interpreted from the apatite-to-collagen ratio, while tissue dynamics (i.e., maturation and turnover/remodelling) are interpreted from the carbonate-to-phosphate r atio1,7, for example in compromised systemic c onditions8–10 and at the bone-implant interface4,11. The ν2 PO43− and ν4 PO43− bands are observed in the 350–650 cm−1 spectral range of bone and synthetic hydroxyapatite12. However, two discrete bands attributable to type-I collagen in the rat tail tendon are also observed in the same spectral r ange13. CO32− substitution for P O43− influences physical properties including 14 crystallite size , thereby restricting mineral crystallinity to below that of carbonate-free apatites. At ≥ 6.5 wt% CO32−, which approximates to one CO32− per unit cell, mineral crystallinity is significantly affected15. In a typical Raman spectrum of B-type carbonated apatites, the ν1 CO32− mode overlaps the ν3 PO43− band16. In synthetic B-type carbonated apatites, the ν3 PO43− band may be visible up to ~ 3 wt% C O32− but is completely enveloped 17 2− 2− by the ν1 CO3 band in bone , where the CO3 content is much higher (~ 7–9 wt%)18. However, superimposition of spectral features associated with the inorganic and the organic components remains largely unreported. Using high-resolution Raman spectroscopy, this work examines the overlap between spectral contributions of the organic and inorganic components of the extracellular matrix in bovine and human bone. Furthermore, a comparative analysis of the ν1-, ν2-, ν3-, and ν4 PO43− regions of synthetic hydroxyapatite (HAp) and bone is reported. Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. email: furqan. Scientific Reports | (2020) 10:16662 | https://doi.org/10.1038/s41598-020-73559-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Comparison of whole bone, deproteinised bone, and demineralised bone (averaged Raman spectra, n = 6; 1800 g mm−1 grating). (a) 325–1225 cm−1 range. Inset in (a): 1200–1800 cm−1 range. (b) 2800–3100 cm−1 range. (c) Demineralisation removes the inorganic phase. Inset in (c): ν1 PO43− position (mean values ± standard deviations). (d–f) Spectral contributions of the organic (demineralised bone) and the inorganic (deproteinised bone) components tend to overlap each other. Results Spectral overlap between organic and inorganic components of bovine bone. Whole bone (Ca/P: 1.45 ± 0.01, N/Ca: 0.6 ± 0.1; in at.%) shows typical spectral features associated with the inorganic and organic components of the extracellular matrix. The organic and inorganic components are isolated by demineralisation using ethylenediaminetetraacetic acid (EDTA) and deproteinisation using sodium hypochlorite (NaOCl), respectively (Supplementary Figure S1). Demineralisation removes the inorganic phase (Ca and P: < 0.01, C: ~ 55, N: ~ 24, and O: ~ 21; in at.%). The ν1 PO43− band, typically the most prominent spectral feature of calcium phosphates and a patite19, is no longer visible (Fig. 1). Deproteinisation removes most of the organic component (Ca/P: 1.51 ± 0.01, N/Ca: 0.08 ± 0.05; in at.%), however, minor traces remain detectable. In the 350–650 cm−1 range, spectral contributions of the organic component are evident at 390–410 cm−1 as a well-defined shoulder on the lower wavenumber side of the ν2 PO43− band (380–410 cm−1) and at 520–545 cm−1. There is considerable overlap between the organic and inorganic components, particularly in the ν2 PO43− and ν4 PO43− regions. Demineralised bone comprises integral areas equivalent to 14.5% (at 410–460 cm−1) and 31.2% (at 570–620 cm−1), on average, those of whole bone. Peaks at 920 cm−1 and 940 cm−1 are attributable to ν(C–C) modes of Pro and Hyp. The 940 cm−1 peak is overlapped by the ν1 PO43− resulting in a minor broadening of the ν1 PO43− band and a small shift towards lower wavenumbers. Distinct features at 1004 cm−1 and 1033 cm−1 in whole bone and demineralised bone are attributable to Phe. FWHM ν1 PO43− correlates with CO32− content. Compared to synthetic HAp (fibres and powder), human and bovine bone generate markedly higher background fluorescence, which is particularly strong for demineralised bone (Fig. 2). HAp fibres (Ca/P: 1.60 ± 0.10 at.%) show a very intense and narrow ν1 PO43− peak (FWHM: 3.3 cm−1), while HAp powder (Ca/P: 1.56 ± 0.12 at.%) shows broadening of the ν1 PO43− peak (FWHM: 6.8 cm−1), indicating differences in mineral crystallinity (Fig. 3). In bone, the ν1 PO43− peak is significantly broader (FWHM: 13.25– (...truncated)