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Measuring Differences in Compositional Properties of Bone Tissue by Confocal Raman Spectroscopy

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Abstract

The full range of fracture risk determinants arise from each hierarchical level comprising the organization of bone. Raman spectroscopy is one tool capable of characterizing the collagen and mineral phases at a near submicron-length scale, but the ability of Raman spectra to distinguish compositional differences of bone is not well defined. Therefore, we analyzed multiple Raman peak intensities and peak ratios to characterize their ability to distinguish between the typically less mineralized osteonal tissue and the more mineralized interstitial tissue in intracortical human bone. To further assess origins of variance, we collected Raman spectra from embedded specimens and for two orientations of cut. Per specimen, Raman peak intensities or ratios were averaged among multiple sites within five osteons and five neighboring interstitial tissue. The peak ratios of ν1 phosphate (PO4) to proline or amide III detected the highest increases of 15.4 or 12.5%, respectively, in composition from osteonal to interstitial tissue. The coefficient of variance was less than 5% for each as opposed to a value of ~8% for the traditional ν1PO4/amide I, a peak ratio that varied the most between transverse and longitudinal cuts for each tissue type. Although embedding affected Raman peaks, it did not obscure differences in most peak ratios related to mineralization between the two tissue types. In studies with limited sample size but sufficient number of Raman spectra per specimen for spatial averaging, ν1PO4/amide III or ν1PO4/proline is the Raman property that is most likely to detect a compositional difference between experimental groups.

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References

  1. Kanis JA, Johnell O, Oden A, Dawson A, De Laet C, Jonsson B (2001) Ten year probabilities of osteoporotic fractures according to BMD and diagnostic thresholds. Osteoporos Int 12:989–995

    Article  PubMed  CAS  Google Scholar 

  2. Schnitzler CM (1993) Bone quality: a determinant for certain risk factors for bone fragility. Calcif Tissue Int 53(suppl 1):S27–S31

    Article  PubMed  Google Scholar 

  3. Turner CH (2002) Biomechanics of bone: determinants of skeletal fragility and bone quality. Osteoporos Int 13:97–104

    Article  PubMed  CAS  Google Scholar 

  4. Rho JY, Kuhn-Spearing L, Zioupos P (1998) Mechanical properties and the hierarchical structure of bone. Med Eng Phys 20:92–102

    Article  PubMed  CAS  Google Scholar 

  5. Opsahl W, Zeronian H, Ellison M, Lewis D, Rucker RB, Riggins RS (1982) Role of copper in collagen cross-linking and its influence on selected mechanical properties of chick bone and tendon. J Nutr 112:708–716

    PubMed  CAS  Google Scholar 

  6. Jonas J, Burns J, Abel EW, Cresswell MJ, Strain JJ, Paterson CR (1993) Impaired mechanical strength of bone in experimental copper deficiency. Ann Nutr Metab 37:245–252

    Article  PubMed  CAS  Google Scholar 

  7. Oxlund H, Barckman M, Ortoft G, Andreassen TT (1995) Reduced concentrations of collagen cross-links are associated with reduced strength of bone. Bone 17:365S–371S

    PubMed  CAS  Google Scholar 

  8. Akkus O, Polyakova-Akkus A, Adar F, Schaffler MB (2003) Aging of microstructural compartments in human compact bone. J Bone Miner Res 18:1012–1019

    Article  PubMed  CAS  Google Scholar 

  9. Lane NE, Yao W, Balooch M, Nalla RK, Balooch G, Habelitz S, Kinney JH, Bonewald LF (2006) Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-deficient mice. J Bone Miner Res 21:466–476

    Article  PubMed  CAS  Google Scholar 

  10. Carden A, Morris MD (2000) Application of vibrational spectroscopy to the study of mineralized tissues (review). J Biomed Opt 5:259–268

    Article  PubMed  CAS  Google Scholar 

  11. Bi X, Patil CA, Lynch CC, Pharr GM, Mahadevan-Jansen A, Nyman JS (2011) Raman and mechanical properties correlate at whole bone- and tissue-levels in a genetic mouse model. J Biomech 44:297–303

    Article  PubMed  Google Scholar 

  12. Timlin JA, Carden A, Morris MD, Rajachar RM, Kohn DH (2000) Raman spectroscopic imaging markers for fatigue-related microdamage in bovine bone. Anal Chem 72:2229–2236

    Article  PubMed  CAS  Google Scholar 

  13. Schulmerich MV, Dooley KA, Morris MD, Vanasse TM, Goldstein SA (2006) Transcutaneous fiber optic Raman spectroscopy of bone using annular illumination and a circular array of collection fibers. J Biomed Opt 11:060502

    Article  PubMed  Google Scholar 

  14. Yeni YN, Yerramshetty J, Akkus O, Pechey C, Les CM (2006) Effect of fixation and embedding on Raman spectroscopic analysis of bone tissue. Calcif Tissue Int 78:363–371

    Article  PubMed  CAS  Google Scholar 

  15. Kazanci M, Roschger P, Paschalis EP, Klaushofer K, Fratzl P (2006) Bone osteonal tissues by Raman spectral mapping: orientation–composition. J Struct Biol 156:489–496

    Article  PubMed  CAS  Google Scholar 

  16. Kazanci M, Wagner HD, Manjubala NI, Gupta HS, Paschalis E, Roschger P, Fratzl P (2007) Raman imaging of two orthogonal planes within cortical bone. Bone 41:456–461

    Article  PubMed  CAS  Google Scholar 

  17. Bromage TG, Goldman HM, McFarlin SC, Warshaw J, Boyde A, Riggs CM (2003) Circularly polarized light standards for investigations of collagen fiber orientation in bone. Anat Rec B New Anat 274:157–168

    Article  PubMed  Google Scholar 

  18. Morris MD (2010) Raman spectroscopy of bone and cartilage. In: Matousek P, Morris MD (eds) Emerging Raman applications and techniques in biomedical and pharmaceutical fields. Springer, New York, pp 347–364

    Chapter  Google Scholar 

  19. Boivin G, Bala Y, Doublier A, Farlay D, Ste-Marie LG, Meunier PJ, Delmas PD (2008) The role of mineralization and organic matrix in the microhardness of bone tissue from controls and osteoporotic patients. Bone 43:532–538

    Article  PubMed  CAS  Google Scholar 

  20. Bergot C, Wu Y, Jolivet E, Zhou LQ, Laredo JD, Bousson V (2009) The degree and distribution of cortical bone mineralization in the human femoral shaft change with age and sex in a microradiographic study. Bone 45:435–442

    Article  PubMed  CAS  Google Scholar 

  21. Rho JY, Roy ME II, Tsui TY, Pharr GM (1999) Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. J Biomed Mater Res 45:48–54

    Article  PubMed  CAS  Google Scholar 

  22. Lieber CA, Mahadevan-Jansen A (2003) Automated method for subtraction of fluorescence from biological Raman spectra. Appl Spectrosc 57:1363–1367

    Article  PubMed  CAS  Google Scholar 

  23. Hofmann T, Heyroth F, Meinhard H, Franzel W, Raum K (2006) Assessment of composition and anisotropic elastic properties of secondary osteon lamellae. J Biomech 39:2282–2294

    Article  PubMed  Google Scholar 

  24. Nie S, Bergbauer KL, Ho JT, Kuck JF Jr, Yu NT (1990) Applications of near-infrared Fourier transform Raman spectroscopy in biology and medicine. Spectroscopy 5:24–32

    Google Scholar 

  25. Giraud-Guille MM (1988) Twisted plywood architecture of collagen fibrils in human compact bone osteons. Calcif Tissue Int 42:167–180

    Article  PubMed  CAS  Google Scholar 

  26. Bandekar J (1992) Amide modes and protein conformation. Biochim Biophys Acta 1120:123–143

    Article  PubMed  CAS  Google Scholar 

  27. Gourion-Arsiquaud S, Burket J, Havill L, Dicarlo E, Doty S, Mendelsohn R, van der Meulen M, Boskey A (2009) Spatial variation in osteonal bone properties relative to tissue and animal age. J Bone Miner Res 24:1271–1281

    Article  PubMed  Google Scholar 

  28. Akkus O, Adar F, Schaffler MB (2004) Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone. Bone 34:443–453

    Article  PubMed  CAS  Google Scholar 

  29. Donnelly E, Boskey AL, Baker SP, van der Meulen MC (2010) Effects of tissue age on bone tissue material composition and nanomechanical properties in the rat cortex. J Biomed Mater Res A 92:1048–1056

    PubMed  Google Scholar 

  30. Yerramshetty JS, Lind C, Akkus O (2006) The compositional and physicochemical homogeneity of male femoral cortex increases after the sixth decade. Bone 39:1236–1243

    Article  PubMed  CAS  Google Scholar 

  31. Yerramshetty JS, Akkus O (2008) The associations between mineral crystallinity and the mechanical properties of human cortical bone. Bone 42:476–482

    Article  PubMed  CAS  Google Scholar 

  32. de Carmejane O, Morris MD, Davis MK, Stixrude L, Tecklenburg M, Rajachar RM, Kohan DH (2005) Bone chemical structure response to mechanical stress studied by high pressure Raman spectroscopy. Calcif Tissue Int 76:207–213

    Article  PubMed  Google Scholar 

  33. Ramasamy JG, Akkus O (2007) Local variations in the micromechanical properties of mouse femur: the involvement of collagen fiber orientation and mineralization. J Biomech 40:910–918

    Article  PubMed  CAS  Google Scholar 

  34. Silva MJ, Brodt MD, Wopenka B, Thomopoulos S, Williams D, Wassen MH, Ko M, Kusano N, Bank RA (2006) Decreased collagen organization and content are associated with reduced strength of demineralized and intact bone in the SAMP6 mouse. J Bone Miner Res 21:78–88

    Article  PubMed  Google Scholar 

  35. Freeman JJ, Wopenka B, Silva MJ, Pasteris JD (2001) Raman spectroscopic detection of changes in bioapatite in mouse femora as a function of age and in vitro fluoride treatment. Calcif Tissue Int 68:156–162

    Article  PubMed  CAS  Google Scholar 

  36. Kavukcuoglu NB, Arteaga-Solis E, Lee-Arteaga S, Ramirez F, Mann AB (2007) Nanomechanics and Raman spectroscopy of fibrillin 2 knock-out mouse bones. J Mater Sci 42:8788–8794

    Article  CAS  Google Scholar 

  37. Kavukcuoglu NB, Denhardt DT, Guzelsu N, Mann AB (2007) Osteopontin deficiency and aging on nanomechanics of mouse bone. J Biomed Mater Res A 83:136–144

    PubMed  Google Scholar 

  38. Kavukcuoglu NB, Patterson-Buckendahl P, Mann AB (2009) Effect of osteocalcin deficiency on the nanomechanics and chemistry of mouse bones. J Mech Behav Biomed 2:254–348

    Article  Google Scholar 

  39. Wallace JM, Golcuk K, Morris MD, Kohn DH (2009) Inbred strain-specific response to biglycan deficiency in the cortical bone of C57BL6/129 and C3H/He mice. J Bone Miner Res 24:1002–1012

    Article  PubMed  Google Scholar 

  40. Kohn DH, Sahar ND, Wallace JM, Golcuk K, Morris MD (2009) Exercise alters mineral and matrix composition in the absence of adding new bone. Cells Tissues Organs 189:33–37

    Article  PubMed  Google Scholar 

  41. Balooch G, Balooch M, Nalla RK, Schilling S, Filvaroff EH, Marshall GW, Marshall SJ, Ritchie RO, Derynck R, Alliston T (2005) TGF-beta regulates the mechanical properties and composition of bone matrix. Proc Natl Acad Sci USA 102:18813–18818

    Article  PubMed  CAS  Google Scholar 

  42. Ager JW, Nalla RK, Breeden KL, Ritchie RO (2005) Deep-ultraviolet Raman spectroscopy study of the effect of aging on human cortical bone. J Biomed Opt 10:034012

    Article  PubMed  CAS  Google Scholar 

  43. Shen J, Fan L, Yang J, Shen AG, Hu JM (2010) A longitudinal Raman microspectroscopic study of osteoporosis induced by spinal cord injury. Osteoporos Int 21:81–87

    Article  PubMed  CAS  Google Scholar 

  44. McCreadie BR, Morris MD, Chen TC, Sudhaker Rao D, Finney WF, Widjaja E, Goldstein SA (2006) Bone tissue compositional differences in women with and without osteoporotic fracture. Bone 39:1190–1195

    Article  PubMed  CAS  Google Scholar 

  45. Tarnowski CP, Ignelzi MA Jr, Morris MD (2002) Mineralization of developing mouse calvaria as revealed by Raman microspectroscopy. J Bone Miner Res 17:1118–1126

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

Supported in part by NIH grants NCI U54 CA-126505 and NIAMS R21 AG-029413.

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Authors and Affiliations

  1. Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA

    Jeffry S. Nyman & Alexander J. Makowski

  2. Department of Orthopaedics and Rehabilitation, Vanderbilt University Medical Center, Nashville, TN, USA

    Jeffry S. Nyman & Elizabeth C. O’Quinn

  3. Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN, USA

    Jeffry S. Nyman

  4. Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA

    Jeffry S. Nyman, Alexander J. Makowski, Chetan A. Patil, Xiaohong Bi & Anita Mahadevan-Jansen

  5. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA

    T. Philip Masui & Scott A. Guelcher

  6. Materials Engineering Department, Southwest Research Institute, San Antonio, TX, USA

    Daniel P. Nicollela

  7. Vanderbilt Orthopaedic Institute, Medical Center East, South Tower, Suite 4200, Nashville, TN, 37232, USA

    Jeffry S. Nyman

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  1. Jeffry S. Nyman
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Correspondence to Jeffry S. Nyman.

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Nyman, J.S., Makowski, A.J., Patil, C.A. et al. Measuring Differences in Compositional Properties of Bone Tissue by Confocal Raman Spectroscopy. Calcif Tissue Int 89, 111–122 (2011). https://doi.org/10.1007/s00223-011-9497-x

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