Calcified Tissue International

, Volume 79, Issue 3, pp 160–168 | Cite as

Degree of Mineralization-related Collagen Crosslinking in the Femoral Neck Cancellous Bone in Cases of Hip Fracture and Controls

  • Mitsuru Saito
  • Katsuyuki Fujii
  • Keishi Marumo


Based on the present definition of osteoporosis, both bone density and quality are important factors in the determination of bone strength. Collagen crosslinking is a determinant of bone quality. Cross-links can form enzymatically by the action of lysyl oxidase or non-enzymatically, resulting in advanced glycation end products. Collagen crosslinking is affected by tissue maturation as well as the degree of mineralization. Homocysteine and vitamin B6 (pyridoxal) are also regulatory factors of collagen crosslinking. We elucidate the relationship between the degree of mineralization and collagen cross-links in cancellous bone from hip fracture cases. We also determined plasma levels of homocysteine and pyridoxal. Twenty-five female intracapsular hip fracture cases (78 ± 6 years) and 25 age-matched postmortem controls (77 ± 6 years) were included in this study. Collagen crosslinking was analyzed after each bone specimen was fractionated into low (1.7–2.0 g/ml) and high (>2.0 g/ml) density fractions. The content of enzymatic (immature reducible and mature nonreducible cross-links) and nonenzymatic cross-link (pentosidine) were determined. In the controls, there was no difference in total enzymatic cross-links between low and high density bone, while pentosidine content was significantly higher in high density bone. In the fracture cases, not only reduced enzymatic cross-links in high density bone and increased pentosidine in both low and high density bone, but also higher plasma homocysteine and lower pyridoxal levels were evident compared with the controls. These results indicate that detrimental crosslinking in both low and high mineralized bone result in impaired bone quality in osteoporotic patients.


Cross-links Degree of mineralization Homocysteine Vitamin B6 Trabecular bone 



The authors are grateful to Takaaki Tanaka Ph.D (NHO, National Utsunomiya Hospital, Tochigi, Japan), Ryuichi Fujisawa Ph.D (Hokkaido University, Japan) and Kazumi Hirakawa (Jikei University School of Medicine) for technical support.


  1. 1.
    Burr BB (2002) Bone material properties and mineral matrix contributions to fracture risk or age in women and men. J Musculoskelet Neuronal Interact 2:201–204PubMedGoogle Scholar
  2. 2.
    Gearnero P, Delmas PD (2004) Contribution of bone mineral density and bone turnover markers to the estimation of risk of Osteoporotic fracture in postmenopausal wemen. J Musculoskelet Neuronal Interact 4:50–63Google Scholar
  3. 3.
    Paschalis EP, Shne E, Lyritis D, Skaeantavos G, Mandelsohn R, Boskey AL (2004) Bone fragility and collagen cross-links. J Bone Miner Res 19:2000–2004PubMedCrossRefGoogle Scholar
  4. 4.
    Boivin GY, Meunier PJ (2003) The mineralization of bone tissue: a forgotten dimension in osteoporosis research. Osteopor Int 14 supple 3:S19–S24Google Scholar
  5. 5.
    Mashiba T, Mori S, Burr DB, Komatsubara S, Cao Y, Manabe T, Norimatsu H (2005) The effects of suppressed bone remodeling by bisphosphonates on microdamage accumulation and degree of mineralization in the cortical bone of dog rib. J Bone Miner Metab 23 Supple:36–42Google Scholar
  6. 6.
    Mori S,Harruff R, Ammrosius W, Burr DB (1997) Trabecular bone volume and microdamage accumulation on the femoral heads of women with and without femoral neck fractures. Bone 21:521–526PubMedCrossRefGoogle Scholar
  7. 7.
    Wang X, Shen X, Li X, Agrawal CM (2002) Age-related changes in the collagen network and toughness of bone. Bone 31:1–7PubMedCrossRefGoogle Scholar
  8. 8.
    Banse X, Sims TJ, Bailey AJ (2002) Mechanical properties of adult vertebral cancellous bone: correlation with collagen intermolecular cross-links. J Bone Miner Res 17:1621–1628PubMedCrossRefGoogle Scholar
  9. 9.
    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–371SPubMedGoogle Scholar
  10. 10.
    Oxlund H, Mosekilde L, Ortoft G (1996) Reduced concentration of collagen reducible crosslinks in human trabecular bone with respect to age and osteoporosis. Bone 19:479–484PubMedCrossRefGoogle Scholar
  11. 11.
    Zioupos P, Currey JD, Hamer AJ (1999) The role of collagen in the declining mechanical properties of aging human cortical bone. J Biomed Mater Res 45:108–116PubMedCrossRefGoogle Scholar
  12. 12.
    Kuboki Y, Kudo A, Mizuno M, Kawamura M (1992) Time-dependent changes of collagen cross-links and their precursors in the culture of osteogenic cells. Calcif Tissue Int 50:473–480PubMedCrossRefGoogle Scholar
  13. 13.
    Saito M, Fujii K, Tanaka T, Soshi S (2004) Effect of low- and high-intensity pulsed ultrasound on collagen post-translational modifications in MC3T3-E1 osteoblasts Calcif Tissue Int 75:384–385PubMedCrossRefGoogle Scholar
  14. 14.
    Saito M, Soshi S, Tanaka T, Fujii K (2004) Intensity-related differences in collagen post-traslational modification in MC3T3-E1 osteoblasts after exposure to low and high intensity pulsed ultrasound. Bone 35:644–655PubMedCrossRefGoogle Scholar
  15. 15.
    Saito M, Soshi S, Fujii K (2003) Effect of hyper- and microgravity on collagen post-translatonal controls of MC3T3-E1 osteoblasts. J Bone Miner Res 18:1695–1705PubMedCrossRefGoogle Scholar
  16. 16.
    Uzawa K, Grzesik WJ, Nishiura T, Kuznetsov SA, Robey PG, Brenner DA, Yamauchi M (1999) Differential expression of human lysyl hydroxylase genes, lysyl hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro. J Bone Miner Res 14:1272–1280PubMedCrossRefGoogle Scholar
  17. 17.
    Yamauchi M, Katz EP (1993) The post-translational chemistry and molecular packing of mineralizing tendon collagens. Connect Tissue Res 29:81–98PubMedGoogle Scholar
  18. 18.
    Eyre DR, Dickson IR, Ness KV (1988) Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochem J 252:495–500PubMedGoogle Scholar
  19. 19.
    Marumo K, Saito M, Yamagishi M, Fujii K (2005) The “ligamentization” process in human anterior cruciate ligament reconstruction with autogenous patellar and hamstring tendons. Am J Sports Med 33:1166–1173PubMedCrossRefGoogle Scholar
  20. 20.
    Saito M, Marumo K, Fujii K, Ishioka N (1997), Single column high – performance liquid chromatographic - fluorescence detection of immature, mature and senescent cross-links of collagen. Anal Biochem 253:26–32PubMedCrossRefGoogle Scholar
  21. 21.
    Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J, Fyhrie DP (2001) Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone 28:195–201PubMedCrossRefGoogle Scholar
  22. 22.
    McCarthy AD, Etcheverry SB, Bruzzone L, Lettieri G, Barrio DA, Cortizo AM (2001) Non-enzymatic glycation of a type I collagen matrix: effect on osteoblastic development and oxidative stress. BMC Cell Biol 2:16PubMedCrossRefGoogle Scholar
  23. 23.
    Paul RG, Bailey AJ (1996) Glycation of collagen: the basis of its central role in the late complications of ageing and diabetes. Int. J Biochem Cell Biol 28:1297–1310PubMedCrossRefGoogle Scholar
  24. 24.
    Sell DR, Monnier VM (1989) Structure elucidation of a senescence cross-link from human extracellular matrix, Implication of pentoses in the aging process. J Biol Chem 264:21597–21602PubMedGoogle Scholar
  25. 25.
    McLean RR, Jacques PF, Selhub J, Tucker KL, Samelson EJ, Broe KE, Hannan MT, Cupples LA, Kiel DP (2004) Homocysteine as a predictive factor for hip fracture in older persons. N Engl J Med 350:2042–2049PubMedCrossRefGoogle Scholar
  26. 26.
    van Meurs JB, Dhonukshe-Rutten RA, Pluijm SM, van der Klift M, de Jonge R, Lindemans J, de Groot LC, Hofman A, Witteman JC, van Leeuwen JP, Breteler MM, Lips P, Pols HA, Uitterlinden AG (2004) Uitterlinden, Homocysteine levels and the risk of osteoporotic fracture. N Engl J Med 35:2033–2041CrossRefGoogle Scholar
  27. 27.
    Reynolds TM, Marshall PD, Brain AM (1992) Hip fracture patients may be vitamin B6 deficient. Controlled study of serum pyridoxal 5 phosphate. Acta Orthop Scand 63:635–638PubMedGoogle Scholar
  28. 28.
    Khan M, Yamauchi M, Srisawasdi S, Stiner D, Doty S, Paschalis EP, Boskey AL (2001) Homocysteine decrease chondrocyte-mediated matrix mineralization in differentiating limb-bud mesenchymal cell micro-mass cultures. Bone 28:387–398PubMedCrossRefGoogle Scholar
  29. 29.
    Lubec B, Fang-Kircher S, Lubec T, Blom HJ, Boers GHJ (1996) Evidence for McKusick’s hypothesis of deficient collagen cross-linking in patients with homocystinuria. Biochim Biophys Acta 13:159–162Google Scholar
  30. 30.
    Bird TA, Levene CI (1982) Lysyl oxidase: evidence that pyridoxal phosphate is a co-factor. Biochem Biophys Res Commun 108:1172–1180PubMedCrossRefGoogle Scholar
  31. 31.
    Grynpas MD (1993) Age and disease-related changes in the mineral of bone. Calcif Tissue Int 53 supple1:S57–S64CrossRefGoogle Scholar
  32. 32.
    Turner CH (2002) Biomechanics of bone: determinants of skeletal fragility and bone quality. Osteoporos Int 13 (2002) 97–104PubMedCrossRefGoogle Scholar
  33. 33.
    Jowsey J (1964) Variation in bone mineralization with age and disease. In: Bone Biodynamics, Little Brown, Boston, pp. 461–479Google Scholar
  34. 34.
    Meunier PJ, Boivin G (1997) Bone mineral density reflects bone mass but also the degree of mineralization of bone: Therapeutic implications. Bone 21:373–377PubMedCrossRefGoogle Scholar
  35. 35.
    Muthusami S, Ramachandran I, Muthusamy B, Vasudevan G, Prabhu V, Subramaniam V, Jagadeesan A, Narasimhan S (2005) Ovariectomy induces oxidative stress and impairs bone antioxidant system in adult rats. Clin Chim Acta 360:81–86PubMedCrossRefGoogle Scholar
  36. 36.
    Russell JE, Avioli LA (1972) Effect of experimental chronic renal insufficiency on bone mineral and collagen maturation. J Clin Invest 51:3072–3079PubMedGoogle Scholar
  37. 37.
    Grynpas MD, Hunter GK (1988) Bone mineral and glycosaminoglycans in newborn and mature rabbits. J Bone Miner Res 3:159–164PubMedGoogle Scholar
  38. 38.
    Saito M (1999) Age-related changes in biochemical characteristics of collagen from human weight-bearing and non-weight-bearing bone. Tokyo Jikeikai Medical Journal 114:327–337 (in Japanese)Google Scholar
  39. 39.
    Grynpas MD, Marie PJ (1990) Effects of low doses of strontium on bone quality and quantity in rats. Bone 11:313–319PubMedCrossRefGoogle Scholar
  40. 40.
    Sodek KL, Tupy LH, Sodek J, Grynpas MD (2000) Relationship between bone protein and mineral developing porcine long bone and calvaria. Bone 26:189–198PubMedCrossRefGoogle Scholar
  41. 41.
    Termine JD, Eanes ED, Greenfield DJ, Nylen MU (1973) Hydrazine deproteinated bone mineral. Calcif Tissue Res 12:73–90PubMedCrossRefGoogle Scholar
  42. 42.
    Vukicevic S, Stavljenic A, Boll T, Vujicic G, Degenhardt C, Cervar M, Krempien B (1989) Effects of parathyroidectomy on tissue calcium, phosphorus, magnesium, and copper concentrations in aluminum-loaded uremic rats. Biol Trace Elem Res 22:45–53PubMedCrossRefGoogle Scholar
  43. 43.
    Eyre DR, Koob TJ, Van Ness KP (1984) Quantification of hydroxypyridinium crosslinks in collagen by High-Performance Liquid Chromatography. Anal Biochem 137:380–388PubMedCrossRefGoogle Scholar
  44. 44.
    Araki A, Sako Y (1987) Detremination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 422:43–52PubMedGoogle Scholar
  45. 45.
    Tani Y (1983) Assay methods of vitamin B6 1. Separation and determination of vitamin B6. Vitamins 57:263–271 (in Japanese)Google Scholar
  46. 46.
    Yoshida T, Yunoki N, Nakajima Y, Kaito T, Anmo T (1978) Simultaneous determination of vitamin B6 group in blood by high-performance liquid chromatography (HPLC). Yakuzai Zassi 98:1319–1326 (in Japanese)Google Scholar
  47. 47.
    Paschalis EP, ,Recker R, Dicarlo E, Doty SB, Atti E, Boskey AL (2003) Distribution of collagen cross-links in normal human trabecular bone. J Bone Miner Res 18:1942–1946PubMedCrossRefGoogle Scholar
  48. 48.
    Fujii K, Kajiwara T, Kurosu H (1979) Effect of vitamin B6 deficiency on the crosslink formation of collagen. FEBS Lett 97:193–195PubMedCrossRefGoogle Scholar
  49. 49.
    Masse PG, Rimnac CM, Yamauchi M, Coburn SP, Rucker RB, Howell DS, Boskey AL (1996) Pyridoxine deficiency affects biomechanical properties of chick tibial bone. Bone 18:567–574PubMedCrossRefGoogle Scholar
  50. 50.
    Grynpas M (1993) Age and disease-related changes in the mineral of bone. Calcif Tissue Int 53(supple 1):S57–S64CrossRefGoogle Scholar
  51. 51.
    Mbyu-Muamba JM, Dequeker J, Gevers G (1989) Collagen and non-collagenous proteins in different mineralization stages of human femur. Acta Anat 134:265–268Google Scholar
  52. 52.
    Simmons DJ, Russel JE, Geynpas MD (1986) Bone maturation and quality of bone material in rats flown on the space shuttle Spacelab-3Mission. Bone and Mineral 1:485–493PubMedCrossRefGoogle Scholar
  53. 53.
    Boskey AL, Marks SC (1985) Mineral and matrix alterations in the bones of incisors-absent (ia/ia) osteopetrotic rats. Calcif Tissue Int 37:287–292PubMedGoogle Scholar
  54. 54.
    Paschalis EP, V Paschalis EP, Verdelis K, Doty SB, Boskey AL, Mendelsohn R, Yamauchi M (2001) Spectroscopic characterization of collagen cross-links in bone. J Bone Miner Res 16:1821– 1828PubMedCrossRefGoogle Scholar
  55. 55.
    Yamauchi M, Katz EP (1993) The post-translational chemistry and molecular packing of mineralizing tendon collagens. Connect Tissue Res 29:81–98PubMedGoogle Scholar
  56. 56.
    Katayama Y, Akatsu T, Yamamoto M, Kugai N, Nagata N (1996) Role of nonenzymatic glycosylation of type I collagen in diabetic osteopenia. J Bone Miner Res 11:931–937PubMedGoogle Scholar
  57. 57.
    Fujii K, Kuboki Y, Sasaki S (1976) Aging of human bone and cartilage collagen: changes in the reducible cross-links and their precursors. Gerontology 22:363–370PubMedCrossRefGoogle Scholar
  58. 58.
    Baynes JW (1991) Role of oxidative stress in development of complications in diabetes. Diabetes 40:405–412PubMedGoogle Scholar
  59. 59.
    Starkebaum G, Harlan JM (1986) Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77:1370–1376PubMedGoogle Scholar
  60. 60.
    Eberhardt RT, Forgione MA, Cap A, Leopold JA, Rudd MA, Trolliet M, Heydrick S, Stark R, Klings ES, Moldovan NI, Yaghoubi M, Goldschmidt-Clermont PJ, Farber HW, Cohen R, Loscalzo J (2000) Endothelial dysfunction in a murine model of mild hyper homocyst(e)inemia. J Clin Invest 106:483–491PubMedGoogle Scholar
  61. 61.
    Bailey AJ, Wotton SF, Simes TJ, Thompson PW (1992) Post-translational modification in the collagen human osteoporotic femoral head. Biochem Biophys Res Commun 185:801–805PubMedCrossRefGoogle Scholar
  62. 62.
    Robins SP (1982) Analysis of the crosslinking components in the collagen and elastin. Methods Biochem Anal 28:329–379PubMedGoogle Scholar
  63. 63.
    Reiser KM (1991) Nonenzymatic glycation of collagen in aging and diabetes. Proc Soc Exp Biol Med 196:17–29PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  1. 1.Department of Orthopaedic SurgeryJikei University School of MedicineTokyoJapan

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