Journal of Materials Science: Materials in Medicine

, Volume 13, Issue 12, pp 1265–1269 | Cite as

Evaluating 3D bone tissue engineered constructs with different seeding densities using the alamarBlue™ assay and the effect on in vivo bone formation

  • C. E. Wilson
  • W. J. A. Dhert
  • C. A. van Blitterswijk
  • A. J. Verbout
  • J. D. de Bruijn


Bone tissue engineering using patient derived cells seeded onto porous scaffolds has gained much attention in recent years. Evaluating the viability of these 3D constructs is an essential step in optimizing the process. The alamarBlue™ (aB) assay was evaluated for its potential to follow in vitro cell proliferation on architecturally standardized hydroxyapatite scaffolds. The impact of the aB assayed and seeding density on subsequent in vivo bone formation was investigated. Twelve scaffolds were seeded with various densities from 250 to 2.5×106 cells/scaffold and assay by aB at 5 time points during the 7-day culture period. Twelve additional scaffolds were seeded with 2.5×105 cells/scaffold. Two control and 2 aB treated scaffolds were subcutaneously implanted into each of 6 nude mice for 6 weeks. Four observers ranked bone formation using a pair wise comparison of histological sections form each mouse. The aB assay successfully followed cell proliferation, however, the diffusion kinetics of the 3D constructs must be considered. The influence of in vitro aB treatment on subsequent in vivo bone formation cannot be ruled out but was not shown to be significant in the current study. The aB assay appears to be quite promising for evaluating a maximum or end-point viability of 3D tissue engineered constructs. Finally, higher seeding densities resulted in more observed bone formation.


Bone Formation Hydroxyapatite Bone Tissue Seeding Density Bone Tissue Engineering 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    J. D. De Bruijn, I. Van Den Brink, S. Mendes, R. Dekker, Y. P. Bovell and C. A. Van Blitterswijk, Adv. Dent. Res. 13 (1999) 74.Google Scholar
  2. 2.
    J. Goshima, V. M. Goldberg and A. I. Caplan, Clin. Orthop. 262 (1991) 298.Google Scholar
  3. 3.
    K. Anselme, B. Noel, B. Flautre, M. C. Blary, C. Delecourt, M. Descamps and P. Hardouin, Bone 25 (1999) 51S.Google Scholar
  4. 4.
    H. Ohgushi, M. Okumura, S. Tamai, E. C. Shors and A. I. Caplan, J. Biomed. Mater. Res. 24 (1990) 1563.Google Scholar
  5. 5.
    alamarBlue™ Product Booklet (BioSource International, Camarillo, USA.Google Scholar
  6. 6.
    M. Gilbert, W. J. Shaw, J. R. Long, K. Nelson, G. P. Drobny, C. M. Giachelli and P. S. Stayton, J. Biol. Chem. 275 (2000) 16213.Google Scholar
  7. 7.
    L. Gilbert, X. He, P. Farmer, S. Boden, M. Kozlowski, J. Rubin and M. S. Nanes, Endocrinology 141, (2000) 3956.Google Scholar
  8. 8.
    C. E. Wilson, J. D. De Bruijn, C. A. Van Blitterswijk, M. C. Kruyt, S. Van Gaalen, W. J. A. Dhert and A. J. Verbout, in Transactions of 27th Annual Meeting of the Society For Biomaterials, Saint Paul, April 2001.Google Scholar
  9. 9.
    S. A. Ahmed, R. M. Gogal and J. E. Walsh, J. Immunol. Methods 170 (1994) 211.Google Scholar
  10. 10.
    H. Yuan, M. Van Den Doel, C. A. Van Blitterswijk, K. De Groot and J. D. De Bruijn, in Transactions of 28th Annual Meeting of the Society For Biomaterials, Tampa, April 2002.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • C. E. Wilson
    • 1
  • W. J. A. Dhert
    • 1
  • C. A. van Blitterswijk
    • 2
    • 3
  • A. J. Verbout
    • 1
  • J. D. de Bruijn
    • 2
  1. 1.Department of OrthopaedicsUniversity Medical Center UtrechtThe Netherlands
  2. 2.IsoTis NV BilthovenThe Netherlands
  3. 3.Twente UniversityThe Netherlands

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