The molecular basis of bone structure and strength is mineralized collagen fibrils at the submicron scale (∼500 nm). Recent advances in optical photothermal infrared (O-PTIR) spectroscopy allow the investigation of bone composition with unprecedented submicron spatial resolution, which may provide new insights into factors contributing to underlying bone function. Here, we investigated (i) whether O-PTIR-derived spectral parameters correlated to standard attenuated total reflection (ATR) Fourier transform infrared spectroscopy spectral data and (ii) whether O-PTIR-derived spectral parameters, including heterogeneity of tissue, contribute to the prediction of proximal femoral bone stiffness. Analysis of serially demineralized bone powders showed a significant correlation (r = 0.96) between mineral content quantified using ATR and O-PTIR spectroscopy, indicating the validity of this technique in assessing bone mineralization. Using femoral neck sections, the principal component analysis showed that differences between O-PTIR and ATR spectra were primarily attributable to the phosphate ion (PO4) absorbance band, which was typically shifter toward higher wavenumbers in O-PTIR spectra. Additionally, significant correlations were found between hydrogen phosphate (HPO4) content (r = 0.75) and carbonate (CO3) content (r = 0.66) quantified using ATR and O-PTIR spectroscopy, strengthening the validity of this method to assess bone mineral composition. O-PTIR imaging of individual trabeculae at 500 nm pixel resolution illustrated differences in submicron composition in the femoral neck from bones with different stiffness. O-PTIR analysis showed a significant negative correlation (r = –0.71) between bone stiffness and mineral maturity, reflective of newly formed bone being an important contributor to bone function. Finally, partial least squares regression analysis showed that combining multiple O-PTIR parameters (HPO4 content and heterogeneity, collagen integrity, and CO3 content) could significantly predict proximal femoral stiffness (R2 = 0.74, error = 9.7%) more accurately than using ATR parameters. Additionally, we describe new findings in the effects of bone tissue orientation in the O-PTIR spectra. Overall, this study highlights a new application of O-PTIR spectroscopy that may provide new insights into molecular-level factors underlying bone mechanical competence.
ReznikovN.ShaharR.WeinerS.. “Bone Hierarchical Structure in Three Dimensions”. Acta Biomater. 2014. 10(9): 3815–3826.
2.
ArunK.N.GautieriA.ChangS.W.BuehlerM.J.. “Molecular Mechanics of Mineralized Collagen Fibrils in Bone”. Nat. Commun. 2013. 4: 1724. 10.1038/ncomms2720
3.
HassenkamT.FantnerG.E.CutroniJ.A.WeaverJ.C., et al. “High-Resolution AFM Imaging of Intact and Fractured Trabecular Bone”. Bone. 2004. 35(1): 4–10.
4.
HimadriS.G.SetoJ.WagermaierW.ZaslanskyP., et al. “Cooperative Deformation of Mineral and Collagen in Bone at the Nanoscale”. Prof. Natl. Acad. Sci. U.S.A. 2006. 103(47): 17741–17746. 10.1073/pnas.0604237103
5.
OttS.M.. “Cortical or Trabecular Bone: What's the Difference?”. Am. J. Nephrol. 2018. 47(6): 373–375.
6.
OftadehR.Perez-ViloriaM.Villa-CamachoJ.C.VaziriA.NazarianA.. “Biomechanics and Mechanobiology of Trabecular Bone: A Review”. J. Biomech. Eng. 2015. 137: 0108021–5. 10.1115/1.4029176
7.
KreiderJ.M.GoldsteinS.A.. “Trabecular Bone Mechanical Properties in Patients with Fragility Fractures”. Clin. Orthop. Relat. Res. 2009. 467(8): 1955–1963.
8.
BayraktarH.H.MorganE.F.NieburG.L.MorrisG.E., et al. “Comparison of the Elastic and Yield Properties of Human Femoral Trabecular and Cortical Bone Tissue”. J. Biomech. 2004. 37(1): 27–35.
9.
FajarJ.K.TaufanT.SyarifM.AzharuddinA.. “Hip Geometry and Femoral Neck Fractures: A Meta-Analysis”. J. Orthop. Translat. 2018. 13: 1–6.
10.
ClynesM.A.HarveyN.C.CurtisE.M.FuggleN.R., et al. “The Epidemiology of Osteoporosis”. Br. Med. Bull. 2020. 133(1): 105–117.
11.
SözenT.ÖzışıkL.BaşaranN.. “An Overview and Management of Osteoporosis”. Eur. J. Rheumatol. 2017. 4(1): 46–56.
12.
ParfittA.M.. “Skeletal Heterogeneity and the Purposes of Bone Remodeling: Implications for the Understanding of Osteoporosis”. Osteoporosis. 2013. 4: 855–872. 10.1016/B978-0-12-415853-5.00036-4
13.
HuiskesR.RuimermanR.van LentheG.H.JanssenJ.D.. “Effects of Mechanical Forces on Maintenance and Adaptation of Form in Trabecular Bone”. Nature. 2022. 405(6787): 704–706. 10.1038/35015116
14.
XiaoW.WangY.PaciosS.LiS.GravesD.T.. “Cellular and Molecular Aspects of Bone Remodeling”. Front. Oral. Biol. 2016. 18: 9–16. 10.1159/000351895
15.
EriksenE.F.. “Cellular Mechanisms of Bone Remodeling”. Rev. Endocr. Metab. Disord. 2010. 11(4): 219–227.
16.
RoblingA.G.CastilloA.B.TurnerC.H.. “Biomechanical and Molecular Regulation of Bone Remodeling”. Annu. Rev. Biomed. Eng. 2006. 8(1): 455–498.
17.
TaiK.QiH.J.OrtizC.. “Effect of Mineral Content on the Nanoindentation Properties and Nanoscale Deformation Mechanisms of Bovine Tibial Cortical Bone”. J. Mater. Sci. Mater. Med. 2005. 15: 947–959. 10.1007/s10856-005-4429-9
18.
RitchieR.O.. “The Conflicts Between Strength and Toughness”. Nat. Mater. 2011. 10(11): 817–822.
19.
KanisJ.A.HarveyN.C.JohanssonH.LiuE., et al. “A Decade of FRAX: How Has It Changed the Management of Osteoporosis?”. Aging Clin. Exp. Res. 2020. 32(2): 187–196.
20.
BlakeG.M.FogelmanI.. “The Role of DXA Bone Density Scans in the Diagnosis and Treatment of Osteoporosis”. Postgrad Med. J. 2007. 83(982): 509–517.
21.
JonesB.C.JiaS.LeeH.FengA., et al. “MRI-Derived Porosity Index is Associated with Whole-Bone Stiffness and Mineral Density in Human Cadaveric Femora”. Bone. 2021. 143: 115774.
22.
AbrahamA.C.AgarwallaA.YadavalliA.McAndrewC., et al. “Multiscale Predictors of Femoral Neck In Situ Strength in Aging Women: Contributions of BMD, Cortical Porosity, Reference Point Indentation, and Nonenzymatic Glycation”. J. Bone Miner. Res. 2015. 30(12): 2207–2214.
23.
JohannesdottirF.ThrallE.MullerJ.KeavenyT.M., et al. “Comparison of Non-Invasive Assessments of Strength of the Proximal Femur”. Bone. 2017. 105: 93–102.
24.
OttS.M.. “Bone Strength: More Than Just Bone Density”. Kidney Int. 2016. 89(1): 16–19.
25.
FriedmanA.W.. “Important Determinants of Bone Strength: Beyond Bone Mineral Density”. J. Clin. Rheumatol. 2006. 12(2): 70–77.
26.
Gourion-ArsiquaudS.FaibishD.MyersE.SpevakL., et al. “Use of FTIR Spectroscopic Imaging to Identify Parameters Associated with Fragility Fracture”. J. Bone Miner. Res. 2009. 24(9): 1565–1571.
27.
FelsenbergD.BoonenS.. “The Bone Quality Framework: Determinants of Bone Strength and their Interrelationships, and Implications for Osteoporosis Management”. Clin. Ther. 2005. 27(1): 1–11.
28.
ChavassieuxP.SeemanE.DelmasP.D.. “Insights into Material and Structural Basis of Bone Fragility from Diseases Associated with Fractures: How Determinants of the Biomechanical Properties of Bone are Compromised by Disease”. Endocr Rev. 2007. 28(2): 151–164.
29.
DonnellyE.ChenD.X.BoskeyA.L.BakerS.P.van der MeulenM.C.. “Contribution of Mineral to Bone Structural Behavior and Tissue Mechanical Properties”. Calcif. Tissue Int. 2010. 87(5): 450–460.
30.
HuntH.B.DonnellyE.. “Bone Quality Assessment Techniques: Geometric, Compositional, and Mechanical Characterization from Macroscale to Nanoscale”. Clin. Rev. Bone Miner. Metab. 2016. 14(3): 133–149.
31.
DonnellyE.. “Methods for Assessing Bone Quality: A Review”. Clin. Orthop. Relat. Res. 2011. 469(8): 2128–2138.
32.
CampbellG.M.SophocleousA.. “Quantitative Analysis of Bone and Soft Tissue by Micro-Computed Tomography: Applications to Ex Vivo and In Vivo Studies”. BoneKEy Rep. 2014. 3: 564. 10.1038/bonekey.2014.59
33.
QueridoW.AilavajhalaR.PadalkarM.PleshkoN.. “Validated Approaches for Quantification of Bone Mineral Crystallinity Using Transmission Fourier Transform Infrared (FT-IR), Attenuated Total Reflection (ATR) FT-IR, and Raman Spectroscopy”. Appl Spectrosc. 2018. 72(11): 1581–1593.
34.
BoskeyA.Pleshko CamachoN.. “FT-IR Imaging of Native and Tissue-Engineered Bone and Cartilage”. Biomaterials. 2007. 28(15): 2465–2478.
35.
QueridoW.KandelS.PleshkoN.. “Applications of Vibrational Spectroscopy for Analysis of Connective Tissues”. Molecules. 2021. 26(4): 922.
36.
TaylorE.A.DonnellyE.. “Raman and Fourier Transform Infrared Imaging for Characterization of Bone Material Properties”. Bone. 2020. 139: 115490.
ZhangD.LiC.ZhangC.SlipchenkoM.N., et al. “Depth-Resolved Mid-Infrared Photothermal Imaging of Living Cells and Organisms with Submicrometer Spatial Resolution”. Sci. Adv. 2016. 2(9): e1600521.
39.
AilavajhalaR.QueridoW.RajapakseC.S.PleshkoN.. “Near Infrared Spectroscopic Assessment of Loosely and Tightly Bound Cortical Bone Water”. Analyst. 2020. 145(10): 3713–3724.
40.
RajapakseC.S.FaridA.R.KargilisD.C.JonesB.C., et al. “MRI-Based Assessment of Proximal Femur Strength Compared to Mechanical Testing”. Bone. 2020. 133: 115227.
41.
RajapakseC.S.HotcaA.NewmanB.T.RammeA., et al. “Patient-Specific Hip Fracture Strength Assessment with Microstructural MR Imaging-Based Finite Element Modeling”. Radiology. 2017. 283(3): 854–861.
42.
KeyakJ.H.RossiS.A.JonesK.A.LesC.M.SkinnerH.B.. “Prediction of Fracture Location in the Proximal Femur Using Finite Element Models”. Med. Eng. Phys. 2001. 23(9): 657–664.
43.
QueridoW.FalconJ.M.KandelS.PleshkoN.. “Vibrational Spectroscopy and Imaging: Applications for Tissue Engineering”. Analyst. 2017. 142(21): 4005–4017.
44.
ShanasN.QueridoW.OswaldJ.JepsenK., et al. “Infrared Spectroscopy-Determined Bone Compositional Changes Associated with Anti-Resorptive Treatment of the Oim/Oim Mouse Model of Osteogenesis Imperfecta”. Appl Spectrosc. 2022. 76(4): 416–427.
MieczkowskaA.MansurS.A.IrwinN.FlattP.R., et al. “Alteration of the Bone Tissue Material Properties in Type 1 Diabetes Mellitus: A Fourier Transform Infrared Microspectroscopy Study”. Bone. 2015. 76: 31–39.
47.
Locci-LopezD.ZhangR.OyemA.CastagnaJ.. “The Multiscale Fourier Transform”. Poster presented at: Society of Economic Geologists (SEG) 2018: Metals, Minerals, and Society. Keystone, Colorado; 22–24 September 2018. Pp. 4176–4180. 10.1190/segam2018-2994723.1
48.
JolliffeI.T.CadimaJ.. “Principal Component Analysis: A Review and Recent Developments”. Philos. Trans. R. Soc., A. 2016. 374(2065): 20150202.
49.
RosipalR.KrämerN.. “Overview and Recent Advances in Partial Least Squares”. In: SaundersC.GrobelnikM.GunnS.Shawe-TaylorJ., editors. Subspace, Latent Structure, and Feature Selection. Vol. 3940. Berlin, Heidelberg: Springer, 2006. Pp. 34–51.
50.
WoldS.SjöströmM.ErikssonL.. “PLS-Regression: A Basic Tool of Chemometrics”. Chemom. Intell. Lab. Syst. 2001. 58(2): 109–130.
51.
BakirG.GirouardB.E.WiensR.MastelS., et al. “Orientation Matters: Polarization Dependent IR Spectroscopy of Collagen from Intact Tendon Down to the Single Fibril Level”. Molecules. 2020. 25(18): 4295.
52.
BiX.LiG.DotyS.B.CamachoN.P.. “A Novel Method for Determination of Collagen Orientation in Cartilage by Fourier Transform Infrared Imaging Spectroscopy (FT-IRIS)”. Osteoarthritis Cartilage. 2005. 13(12): 1050–1058.
53.
Arteaga-SolisE.Sui-ArteagaL.KimM.SchafflerM.B., et al. “Material and Mechanical Properties of Bones Deficient for Fibrillin-1 or Fibrillin-2 Microfibrils”. Matrix Biol. 2011. 30(3): 188–194.
54.
CoatsA.M.HukinsD.W.ImrieC.T.AspdenR.M.. “Polarization Artefacts of An FTIR Microscope and the Consequences for Intensity Measurements on Anisotropic Materials”. J. Microsc. 2003. 211(Pt 1): 63–66.
55.
TaiK.DaoM.SureshS.PalazogluA.OrtizC.. “Nanoscale Heterogeneity Promotes Energy Dissipation in Bone”. Nat Mater. 2007. 6(6): 454–462.
56.
ImbertL.Gourion-ArsiquaudS.Villarreal-RamirezE.SpevakL., et al. “Dynamic Structure and Composition of Bone Investigated by Nanoscale Infrared Spectroscopy”. PLoS One. 2018. 13(9): e0202833.
57.
ChenX.HughesR.MullinN.HawkinsR.J., et al. “Mechanical Heterogeneity in the Bone Microenvironment as Characterized by Atomic Force Microscopy”. Biophys. J. 2020. 119(3): 502–513.
58.
AhnT.JueckstockM.MandairG.S.HendersonJ., et al. “Matrix/Mineral Ratio and Domain Size Variation with Bone Tissue Age: A Photothermal Infrared Study”. J. Struct. Biol. 2022. 214(3): 107878.
59.
Boulet-AudetM.BuffeteauT.BoudreaultS.DaugeyN.PézoletM.. “Quantitative Determination of Band Distortions in Diamond Attenuated Total Reflectance Infrared Spectra”. J. Phys. Chem. B. 2010. 114(24): 8255–8261.
60.
Gourion-ArsiquaudS.LukashovaL.PowerJ.LoveridgeN., et al. “Fourier Transform Infrared Imaging of Femoral Neck Bone: Reduced Heterogeneity of Mineral-to-Matrix and Carbonate-to-Phosphate and More Variable Crystallinity in Treatment-Naive Fracture Cases Compared with Fracture-Free Controls”. J. Bone Miner. Res. 2013. 28(1): 150–161.
61.
BoskeyA.L.DonnellyE.BoskeyE.SpevakL., et al. “Examining the Relationships Between Bone Tissue Composition, Compositional Heterogeneity, and Fragility Fracture: A Matched Case-Controlled FTIRI Study”. J. Bone Miner. Res. 2016. 31(5): 1070–1081.
62.
RuppelM.E.BurrD.B.MillerL.M.. “Chemical Makeup of Microdamaged Bone Differs from Undamaged Bone”. Bone. 2006. 39(2): 318–324.
63.
Gourion-ArsiquaudS.BurketJ.C.HavillL.M.DiCarloE., et al. “Spatial Variation in Osteonal Bone Properties Relative to Tissue and Animal Age”. J. Bone Miner. Res. 2009. 24(7): 1271–1281.
64.
DonnellyE.MeredithD.S.NguyenJ.T.GladnickB.P., et al. “Reduced Cortical Bone Compositional Heterogeneity with Bisphosphonate Treatment in Postmenopausal Women with Intertrochanteric and Subtrochanteric Fractures”. J. Bone Miner. Res. 2012. 27(3): 672–678.
65.
Gourion-ArsiquaudS.AllenM.R.BurrD.B.VashishthD., et al. “Bisphosphonate Treatment Modifies Canine Bone Mineral and Matrix Properties and their Heterogeneity”. Bone. 2010. 46(3): 666–672.
66.
NilssonB.E.WiklundP.E.. “Iliac Crest Biopsy in the Diagnosis of Metabolic Bone Disease. A Method Study”. Acta Med. Scand. 1983. 213(2): 151–155. 10.1111/j.0954-6820.1983.tb03707.x
67.
BoussonV.BergotC.WuY.JolivetE., et al. “Greater Tissue Mineralization Heterogeneity in Femoral Neck Cortex from Hip-Fractured Females than Controls: A Microradiographic Study”. Bone. 2011. 48(6): 1252–1259.
68.
BusseB.HahnM.SoltauM.ZustinJ., et al. “Increased Calcium Content and Inhomogeneity of Mineralization Render Bone Toughness in Osteoporosis: Mineralization, Morphology, and Biomechanics of Human Single Trabeculae”. Bone. 2009. 45(6): 1034–1043.
69.
DonnellyE.MeredithD.S.NguyenJ.T.BoskeyA.L.. “Bone Tissue Composition Varies Across Anatomic Sites in the Proximal Femur and the Iliac Crest”. J. Orthop. Res. 2012. 30(5): 700–706.
70.
ChenJ.AhnT.Colón-BernalI.D.KimJ.Banaszak HollM.M.. “The Relationship of Collagen Structural and Compositional Heterogeneity to Tissue Mechanical Properties: A Chemical Perspective”. ACS Nano. 2017. 11(11): 10665–10671.
71.
DemirtasA.CurranE.UralA.. “Assessment of the Effect of Reduced Compositional Heterogeneity on Fracture Resistance of Human Cortical Bone Using Finite Element Modeling”. Bone. 2016. 91: 92–101.
72.
WangZ.X.LloydA.A.BurketJ.C.Gourion-ArsiquaudS.DonnellyE.. “Altered Distributions of Bone Tissue Mineral and Collagen Properties in Women with Fragility Fractures”. Bone. 2016. 84: 237–244.
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