pp 1–8 | Cite as

Structural and biomechanical changes to dentin extracellular matrix following chemical removal of proteoglycans

  • Ana Paula Farina
  • Cristina M. P. Vidal
  • Doglas Cecchin
  • Thaiane R. Aguiar
  • Ana K. Bedran-RussoEmail author
Original Article


Proteoglycans are biomacromolecules with significant biomineralization and structural roles in the dentin extracellular matrix. This study comprehensively assessed the mechanical properties and morphology of the dentin extracellular matrix following chemical removal of proteoglycans to elucidate the structural roles of proteoglycans in dentin. Dentin extracellular matrix was prepared from extracted teeth after complete tissue demineralization. Chemical removal of proteoglycans was carried-out using guanidine hydrochloride for up to 10 days. The removal of proteoglycans was determined by dimethylmethylene blue colorimetric assay and histological staining analyses using transmission electron microscopy and optical microscopy. The modulus of elasticity of dentin matrix was determined by a 3-point bending test method. Partial removal of proteoglycans induced significant modifications to the dentin matrix, particularly to type I collagen. Removal of proteoglycans significantly decreased the modulus of elasticity of dentin extracellular matrix (p < 0.0001). In conclusion, the subtle disruption of proteoglycans induces pronounced changes to the collagen network packing and the bulk modulus of elasticity of dentin matrix.


Proteoglycans Collagen Dentin Modulus of elasticity Histology 



The study was supported by the National Institute of Health—NIH/NIDCR (#DE021040) and CAPES Foundation-Brazil (#BEX 17764/12-2). The authors would like to thank Ariene Leme-Kraus for the support with the statistical analysis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Bertassoni LE, Swain MV. The contribution of proteoglycans to the mechanical behavior of mineralized tissues. J Mech Behav Biomed Mater. 2014;38:91–104.CrossRefGoogle Scholar
  2. 2.
    Imbeni V, Kruzicm JJ, Marshall GW, Marshall SJ, Ritchie RO. The dentin-enamel junction and the fracture of human teeth. Nat Mater. 2005;4:229–32.CrossRefGoogle Scholar
  3. 3.
    Bertassoni LE, Orgel JP, Antipova O, Swain MV. The dentin organic matrix—limitations of restorative dentistry hidden on the nanometer scale. Acta Biomater. 2012;8:2419–33.CrossRefGoogle Scholar
  4. 4.
    Dechichi P, Biffi JC, Moura CC, de Ameida AW. A model of the early mineralization process of mantle dentin. Micron. 2007;38:486–91.CrossRefGoogle Scholar
  5. 5.
    Goldberg M, Ono M, Septier D, Bonnefoix M, Kilts TM, Bi Y, et al. Fibromodulin-deficient mice reveal dual functions for fibromodulin in regulating dental tissue and alveolar bone formation. Cells Tissues Organs. 2009;189:198–202.CrossRefGoogle Scholar
  6. 6.
    Waddington RJ, Hall RC, Embery G, Lloyd DM. Changing profiles of proteoglycans in the transition of predentine to dentine. Matrix Biol. 2003;22:153–61.CrossRefGoogle Scholar
  7. 7.
    Ho SP, Sulyanto RM, Marshall SJ, Marshall GW. The cementum-dentin junction also contains glycosaminoglycans and collagen fibrils. J Struct Biol. 2005;151:69–78.CrossRefGoogle Scholar
  8. 8.
    Bourdon MA, Oldberg A, Pierschbacher M, Ruoslahti E. Molecular cloning and sequence analysis of a chondroitin sulfate proteoglycan cDNA. Proc Natl Acad Sci. 1985;82:1321–5.CrossRefGoogle Scholar
  9. 9.
    Scott JE. Alcian blue. Now you see it, now you don’t. Eur J Oral Sci. 1996;104:2–9.CrossRefGoogle Scholar
  10. 10.
    Vogel KG, Paulsson M, Heinegård D. Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J. 1984;223:587–97.CrossRefGoogle Scholar
  11. 11.
    Hedbom E, Heinegård D. Binding of fibromodulin and decorin to separate sites on fibrillar collagens. J Biol Chem. 1993;268:27307–12.Google Scholar
  12. 12.
    Kobe B, Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci. 1994;19:415–21.CrossRefGoogle Scholar
  13. 13.
    Schönherr E, Witsch-Prehm P, Harrach B, Robenek H, Rauterberg J, Kresse H. Interaction of biglycan with type I collagen. J Biol Chem. 1995;270:2776–83.CrossRefGoogle Scholar
  14. 14.
    Septier D, Hall RC, Lloyd D, Embery G, Goldberg M. Quantitative immunohistochemical evidence of a functional gradient of chondroitin 4-sulphate/dermatan sulphate, developmentally regulated in the predentine of rat incisor. Histochem J. 1998;30:275–84.CrossRefGoogle Scholar
  15. 15.
    Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem. 1998;67:609–52.CrossRefGoogle Scholar
  16. 16.
    Goldberg M, Septier D, Oldberg A, Young MF, Ameye LG. Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation. J Histochem Cytochem. 2006;54:525–37.CrossRefGoogle Scholar
  17. 17.
    Bedran-Russo AK, Pashley DH, Agee K, Drummond JL, Miescke KJ. Changes in stiffness of demineralized dentin following application of collagen crosslinkers. J Biomed Mater Res B Appl Biomater. 2008;86:330–34.CrossRefGoogle Scholar
  18. 18.
    Mazzoni A, Pashley DH, Ruggeri A Jr, Vita F, Falconi M, Di Lenarda R, Breschi L. Adhesion to chondroitinase ABC treated dentin. J Biomed Mater Res B Appl Biomater. 2008;86:228–36.CrossRefGoogle Scholar
  19. 19.
    Chandrasekhar S, Esterman MA, Hoffman HA. Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal Biochem. 1987;161:103–08.CrossRefGoogle Scholar
  20. 20.
    Barbosa I, Garcia S, Barbier-Chassefière V, Caruelle JP, Martelly I, Papy-García D. Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies. Glycobiology. 2003;13:647–53.CrossRefGoogle Scholar
  21. 21.
    Yang Y, Rupani A, Bagnaninchi P, Wimpenny I, Weightman A. Study of optical properties and proteoglycan content of tendons by polarization sensitive optical coherence tomography. J Biomed Opt. 2012;17:081417.CrossRefGoogle Scholar
  22. 22.
    Bedran-Russo AK, Castellan CS, Shinohara MS, Hassan L, Antunes A. Characterization of biomodified dentin matrices for potential preventive and reparative therapies. Acta Biomater. 2011;7:1735–41.CrossRefGoogle Scholar
  23. 23.
    Bedran-Russo AK, Pereira PN, Duarte WR, Okuyama K, Yamauchi M. Removal of dentin matrix proteoglycans by trypsin digestion and its effect on dentin bonding. J Biomed Mater Res B Appl Biomater. 2008;85:261–6.CrossRefGoogle Scholar
  24. 24.
    Castellan CS, Pereira PN, Viana G, Chen SN, Pauli GF, Bedran-Russo AK. Solubility study of phytochemical cross-linking agents on dentin stiffness. J Dent. 2010;38:431–6.CrossRefGoogle Scholar
  25. 25.
    Reddy GK, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem. 1996;29:225–9.CrossRefGoogle Scholar
  26. 26.
    Vogel KG, Peters JA. Histochemistry defines a proteoglycan-rich layer in bovine flexor tendon subjected to bending. J Musculoskelet Neuronal Interact. 2005;5:64–9.Google Scholar
  27. 27.
    Mazzocca AD, McCarthy MB, Ledgard FA, Chowaniec DM, McKinnon WJ Jr, Delaronde S, et al. Histomorphologic changes of the long head of the biceps tendon in common shoulder pathologies. Arthroscopy. 2013;29:972–81.CrossRefGoogle Scholar
  28. 28.
    Fisher MC, Li Y, Seghatoleslami MR, Dealy CN, Kosher RA. Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation. Matrix Biol. 2006;25:27–39.CrossRefGoogle Scholar
  29. 29.
    Scott JE. Proteoglycan histochemistry a valuable tool for connective tissue biochemists. Coll Relat Res. 1985;5:541–75.CrossRefGoogle Scholar
  30. 30.
    Rabau MY, Dayan D. Polarization microscopy of picrosirius red stained sections: a useful method for qualitative evaluation of intestinal wall collagen. Histol Histopathol. 1994;9:525–8.Google Scholar
  31. 31.
    Yamauchi N, Nagaoka H, Yamauchi S, Teixeira FB, Miguez P, Yamauchi M. Immunohistological characterization of newly formed tissues after regenerative procedure in immature dog teeth. J Endod. 2011;37:1636–41.CrossRefGoogle Scholar
  32. 32.
    Ribeiro JF, dos Anjos EH, Mello ML, de Campos Vidal B. Skin collagen fiber molecular order: a pattern of distributional fiber orientation as assessed by optical anisotropy and image analysis. PLoS One. 2013;8:e54724.CrossRefGoogle Scholar
  33. 33.
    Dolber PC, Spach MS. Picrosirius red staining of cardiac muscle following phosphomolybdic acid treatment. Stain Technol. 1987;62:23–6.CrossRefGoogle Scholar
  34. 34.
    Diaz Encarnacion MM, Griffin MD, Slezak JM, Bergstralh EJ, Stegall MD, Velosa JA, et al. Correlation of quantitative digital image analysis with the glomerular filtration rate in chronic allograft nephropathy. Am J Transplant. 2004;4:248–56.CrossRefGoogle Scholar
  35. 35.
    Lattouf R, Younes R, Lutomski D, Naaman N, Godeau G, Senni K, et al. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem. 2014;62:751–8.CrossRefGoogle Scholar
  36. 36.
    Provenzano PP, Vanderby R Jr. Collagen fibril morphology and organization: implications for force transmission in ligament and tendon. Matrix Biol. 2006;25:71–84.CrossRefGoogle Scholar
  37. 37.
    Zhang G, Ezura Y, Chervoneva I, Robinson PS, Beason DP, Carine ET, et al. Decorin regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development. J Cell Biochem. 2006;98:1436–49.CrossRefGoogle Scholar
  38. 38.
    Bertassoni LE, Stankoska K, Swain MV. Insights into the structure and composition of the peritubular dentin organic matrix and the lamina limitans. Micron. 2012;43:229–36.CrossRefGoogle Scholar
  39. 39.
    Scott JE. Proteoglycan–fibrillar collagen interactions. Biochem J. 1988;252:313–23.CrossRefGoogle Scholar
  40. 40.
    Breschi L, Gobbi P, Lopes M, Prati C, Falconi M, Teti G, Mazzotti G. Immunocytochemical analysis of dentin: a double-labeling technique. J Biomed Mater Res A. 2003;67:11–7.CrossRefGoogle Scholar
  41. 41.
    Hsueh MF, Khabut A, Kjellström S, Önnerfjord P, Kraus VB. Elucidating the molecular composition of cartilage by proteomics. J Proteome Res. 2016;15:374–88.CrossRefGoogle Scholar
  42. 42.
    Goldberg M, Takagi M. Dentine proteoglycans: composition, ultrastructure and functions. Histochem J. 1993;25:781–806.CrossRefGoogle Scholar

Copyright information

© The Society of The Nippon Dental University 2019

Authors and Affiliations

  • Ana Paula Farina
    • 1
    • 2
  • Cristina M. P. Vidal
    • 1
    • 3
  • Doglas Cecchin
    • 1
    • 2
  • Thaiane R. Aguiar
    • 1
    • 4
  • Ana K. Bedran-Russo
    • 1
    Email author
  1. 1.Department of Restorative Dentistry, College of DentistryUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of Restorative Dentistry, Passo Fundo Dental SchoolUniversity of Passo Fundo, UPFPasso FundoBrazil
  3. 3.Department of Operative Dentistry, College of DentistryUniversity of IowaIowa CityUSA
  4. 4.Department of Clinical Dentistry, School of DentistryFederal University of BahiaSalvadorBrazil

Personalised recommendations