Calcified Tissue International

, Volume 105, Issue 6, pp 660–669 | Cite as

Substrate Strain Mitigates Effects of β-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking

  • Silvia P. Canelón
  • Joseph M. WallaceEmail author
Original Research


Enzymatic crosslinks stabilize type I collagen and are catalyzed by lysyl oxidase (LOX), a step interrupted through β-aminopropionitrile (BAPN) exposure. This study evaluated dose-dependent effects of BAPN on osteoblast gene expression of type I collagen, LOX, and genes associated with crosslink formation. The second objective was to characterize collagen produced in vitro after exposure to BAPN, and to explore changes to collagen properties under continuous cyclical substrate strain. To evaluate dose-dependent effects, osteoblasts were exposed to a range of BAPN dosages (0–10 mM) for gene expression analysis and cell proliferation. Results showed significant upregulation of BMP-1, POST, and COL1A1 and change in cell proliferation. Results also showed that while the gene encoding LOX was unaffected by BAPN treatment, other genes related to LOX activation and matrix production were upregulated. For the loading study, the combined effects of BAPN and mechanical loading were assessed. Gene expression was quantified, atomic force microscopy was used to extract elastic properties of the collagen matrix, and Fourier Transform infrared spectroscopy was used to assess collagen secondary structure for enzymatic crosslinking analysis. BAPN upregulated BMP-1 in static samples and BAPN combined with mechanical loading downregulated LOX when compared to control-static samples. Results showed a higher indentation modulus in BAPN-loaded samples compared to control-loaded samples. Loading increased the mature-to-immature crosslink ratios in control samples, and BAPN increased the height ratio in static samples. In summary, effects of BAPN (upregulation of genes involved in crosslinking, mature/immature crosslinking ratios, upward trend in collagen elasticity) were mitigated by mechanical loading.


Osteoblast Collagen Indentation BAPN FTIR 



The authors are grateful to the IU School of Medicine Department of Anatomy & Cell Biology, particularly Dr. William Thopmson, for providing access to the Flexcell system and laboratory space, as well as Donna Roskowski in the IUPUI Department of Chemistry and Chemical Biology for providing access to the Nicolet iN 10 infrared microscope.


This work was supported by funding from the National Institutes of Health (AR072609, AR067221).

Compliance with Ethical Standards

Conflict of interest

Silvia P. Canelón, Joseph M. Wallace have stated that they have no conflicts of interest.

Human and Animal Rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

This article does not contain any studies with human participants performed by any of the authors.


  1. 1.
    Burr DB, Allen MR (2013) Basic and applied bone biology, 1st edn. Elsevier, San DiegoGoogle Scholar
  2. 2.
    Boskey AL, Wright T, Blank R (1999) Collagen and bone strength. J Bone Miner Res 14:330–335CrossRefGoogle Scholar
  3. 3.
    Burr DB (2002) The contribution of the organic matrix to bone’s material properties. Bone 31:8–11. CrossRefPubMedGoogle Scholar
  4. 4.
    Viguet-Carrin S, Garnero P, Delmas PD (2006) The role of collagen in bone strength. Osteoporos Int 17:319–336. CrossRefPubMedGoogle Scholar
  5. 5.
    Avery NC, Sims TJ, Bailey AJ (2009) Quantitative determination of collagen cross-links. Methods Mol Niol 522:103–121. CrossRefGoogle Scholar
  6. 6.
    Eyre DR, Paz M, Gallop P (1984) Cross-linking in collagen and elastin. Annu Rev Biochem 53:717–748. CrossRefPubMedGoogle Scholar
  7. 7.
    Eyre DR, Wu J-J (2005) Collagen Cross-Links. Top Curr Chem 247:207–229. CrossRefGoogle Scholar
  8. 8.
    Saito M, Marumo K (2010) Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus. Osteoporos Int 21:195–214. CrossRefPubMedGoogle Scholar
  9. 9.
    Dasler W (1954) Isolation of toxic crystals from sweet peas (Lathyrus odoratus). Science 120:307–308CrossRefGoogle Scholar
  10. 10.
    Nimni ME (1977) Mechanism of inhibition of collagen crosslinking by penicillamine. Proc R Soc Med 70(Suppl 3):65–72PubMedPubMedCentralGoogle Scholar
  11. 11.
    Peng J, Jiang Z, Qin G, Huang Q, Li Y, Jiao Z, Zhang F, Li Z, Zhang J, Lu Y, Liu X, Liu J (2007) Impact of activity space on the reproductive behaviour of giant panda (Ailuropoda melanoleuca) in captivity. Appl Anim Behav Sci 104:151–161. CrossRefGoogle Scholar
  12. 12.
    Norris RA, Damon B, Mironov V, Kasyanov V, Ramamurthi A, Moreno-Rodriguez R, Trusk T, Potts JD, Goodwin RL, Davis J, Hoffman S, Wen X, Sugi Y, Kern CB, Mjaatvedt CH, Turner DK, Oka T, Conway SJ, Molkentin JD, Forgacs G, Markwald RR (2007) Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues. J Cell Biochem 101:695–711. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Maruhashi T, Kii I, Saito M, Kudo A (2010) Interaction between Periostin and BMP-1 promotes proteolytic activation of lysyl oxidase. J Biol Chem 285:13294–13303. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fogelgren B, Polgár N, Szauter KM, Újfaludi Z, Laczkó R, Fong KS, Csiszar K (2005) Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation. J Biol Chem 280:24690–24697. CrossRefPubMedGoogle Scholar
  15. 15.
    Turner CH, Pavalko FM (1998) Mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. J Orthop Sci 3:346–355. CrossRefPubMedGoogle Scholar
  16. 16.
    Ward DF, Salasznyk RM, Klees RF, Backiel J, Agius P, Bennett K, Boskey A, Plopper GE (2007) Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway. Stem Cells Dev 16:467–480. CrossRefPubMedGoogle Scholar
  17. 17.
    Warden SJ, Galley MR, Hurd AL, Wallace JM, Gallant MA, Richard JS, George LA (2013) Elevated mechanical loading when young provides lifelong benefits to cortical bone properties in female rats independent of a surgically induced menopause. Endocrinology 154:3178–3187. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    McNerny EM, Gong B, Morris MD, Kohn DH (2015) Bone fracture toughness and strength correlate with collagen cross-link maturity in a dose-controlled lathyrism mouse model. J Bone Miner Res 30:455–464. CrossRefPubMedGoogle Scholar
  19. 19.
    Hammond MA, Wallace JM (2015) Exercise prevents β-aminopropionitrile-induced morphological changes to type I collagen in murine bone. Bonekey Rep 4:645. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Canelón SP, Wallace JM (2016) β-Aminopropionitrile-induced reduction in enzymatic crosslinking causes in vitro changes in collagen morphology and molecular composition. PLoS ONE 11:1–13. CrossRefGoogle Scholar
  21. 21.
    Fernandes H (2009) The role of collagen crosslinking in differentiation of human mesenchymal stem cells and MC3T3-E1 cells. Tissue Eng A 15:3857–3867CrossRefGoogle Scholar
  22. 22.
    Thaler R, Spitzer S, Rumpler M, Fratzl-Zelman N, Klaushofer K, Paschalis E, Varga F (2010) Differential effects of homocysteine and beta aminopropionitrile on preosteoblastic MC3T3-E1 cells. Bone 46:703–709. CrossRefPubMedGoogle Scholar
  23. 23.
    Turecek C, Fratzl-Zelman N, Rumpler M, Buchinger B, Spitzer S, Zoehrer R, Durchschlag E, Klaushofer K, Paschalis E, Varga F (2008) Collagen cross-linking influences osteoblastic differentiation. Calcif Tissue Int 82:392–400. CrossRefPubMedGoogle Scholar
  24. 24.
    Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46:69–81. CrossRefPubMedGoogle Scholar
  25. 25.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:45e-45. CrossRefGoogle Scholar
  26. 26.
    Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST(C)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:36e-36. CrossRefGoogle Scholar
  27. 27.
    Vande Geest JP, Di Martino ES, Vorp DA (2004) An analysis of the complete strain field within Flexercell(TM) membranes. J Biomech 37:1923–1928. CrossRefPubMedGoogle Scholar
  28. 28.
    Yang H, Yang S, Kong J, Dong A, Yu S (2015) Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy. Nat Protoc 10:382–396. CrossRefPubMedGoogle Scholar
  29. 29.
    Dong A, Huang P, Caughey WS (1990) Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry 29:3303–3308. CrossRefPubMedGoogle Scholar
  30. 30.
    Paschalis E, Verdelis K, Doty SB, Boskey AL, Mendelsohn R, Yamauchi M (2001) Spectroscopic characterization of collagen cross-links in bone. J Bone Miner Res 16:1821–1828. CrossRefPubMedGoogle Scholar
  31. 31.
    Paschalis E, Gamsjaeger S, Tatakis D, Hassler N, Robins S, Klaushofer K (2014) Fourier transform infrared spectroscopic characterization of mineralizing type I collagen enzymatic trivalent cross-links. Calcif Tissue Int 96:18–29. CrossRefPubMedGoogle Scholar
  32. 32.
    Kemp AD, Harding CC, Cabral WA, Marini JC, Wallace JM (2012) Effects of tissue hydration on nanoscale structural morphology and mechanics of individual Type I collagen fibrils in the Brtl mouse model of Osteogenesis Imperfecta. J Struct Biol 180:428–438. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ (1992) Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res 7:683–692. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Weldon School of Biomedical EngineeringPurdue UniversityWest LafayetteUSA
  2. 2.Department of Biomedical EngineeringIndiana University-Purdue University at IndianapolisIndianapolisUSA
  3. 3.Department of Orthopaedic SurgeryIndiana University School of MedicineIndianapolisUSA

Personalised recommendations