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Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation

Nature Materials volume 8, pages 763–770 (2009)Cite this article

Abstract

An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the ‘bone nodules’ formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

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Figure 1: Mineralized nodules formed from three different cell sources analysed by standard microscopy techniques.
Figure 2: Raman spectroscopy, immunostaining and gene expression evidence for type-II collagen in differentiating cultures.
Figure 3: Raman spectra of mineralized nodules and native bone, and their properties as determined by univariate peak analysis.
Figure 4: Factor analyses of mature mineralized nodules after 28 days in culture.
Figure 5: Immunostaining, gene expression and alkaline-phosphatase (ALP) evidence of osteoblastic differentiation.
Figure 6: TEM micrographs of mineralized nodules formed after 28 days in culture.

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Acknowledgements

The authors would like to thank the EPSRC for funding, R. Carzaniga of the Electron Microscopy Centre, Imperial College London, for invaluable assistance with TEM and N. Walters for laboratory support. We are also grateful to R. Hill for help with the hydroxyapatite standard. R.J.S. gratefully acknowledges funding from the Rothermere Foundation, the National Science and Engineering Research Council Canada and the Canadian Centennial Scholarship Fund. N.D.E. was supported by a career development fellowship in stem cell research from the Medical Research Council, UK.

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Authors and Affiliations

  1. Department of Materials, Imperial College London, London SW7 2AZ, UK

    Eileen Gentleman, Robin J. Swain, Nicholas D. Evans, Suwimon Boonrungsiman, Gavin Jell, Michael D. Ball, Alexandra Porter & Molly M. Stevens

  2. Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, UK

    Eileen Gentleman, Nicholas D. Evans, Suwimon Boonrungsiman, Gavin Jell, Michael D. Ball & Molly M. Stevens

  3. Cambridge University Engineering Department, Cambridge CB2 1PZ, UK

    Tamaryn A. V. Shean & Michelle L. Oyen

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  1. Eileen Gentleman
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Contributions

E.G. carried out the cell culture work and the ALP assays and wrote the majority of the manuscript. R.J.S. carried out the Raman measurements and spectral processing and analysis and also contributed to manuscript writing and revision. N.D.E contributed some cell culture work, immunostaining results and the quantitative PCR data and helped with manuscript revision. G.J. and M.D.B carried out the SEM imaging and some TEM preparation. G.J. also contributed to manuscript revision. S.B. carried out the majority of the TEM imaging. T.A.V.S. and M.L.O. carried out the nanoindentation testing. A.P. helped with the TEM imaging and manuscript revision. M.M.S. was involved in study design, supervision of the work and manuscript revision.

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Correspondence to Molly M. Stevens.

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Gentleman, E., Swain, R., Evans, N. et al. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation. Nature Mater 8, 763–770 (2009). https://doi.org/10.1038/nmat2505

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