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Quantitative second harmonic generation imaging of cartilage damage

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Abstract

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples. SHG imaging of the frozen cryopreserved specimens showed a dramatic loss of mean gray value intensities when compared to both fresh and vitrified tissues (P < 0.05), indicating structural changes of the extracellular matrix, in particular the deformation and destruction of the collagen fibers in the analyzed articular cartilage. A 0.9974 correlation coefficient was observed between the metabolic cell activity assessed by the alamarBlue technique, and retention of collagen structure between the three experimental groups. These studies suggest that multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling may prove to be useful tools for the investigation of extracellular matrix changes in preserved cartilage, giving insights on the structural quality prior to implantation.

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Abbreviations

3D:

Three-dimensional

DIC:

Transmitted light-differential interference-contrast

DMEM:

Dulbecco’s modified Eagle medium

DMSO:

Dimethyl sulfoxide

ECM:

Extracellular matrix

FT-IRIS:

Fourier transform infrared imaging spectroscopy

GAG:

Glycosaminoglycan

GVI:

Gray value intensities

MRI:

Magnetic resonance imaging

μCT:

Contrast-enhanced microcomputed tomography

SEM:

Standard error of the mean

SHG:

Second harmonic generation

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Acknowledgements

We would like to thank Mrs. Elizabeth Greene and Dr. Simona Baicu for their assistance in review and preparation of this manuscript. This work was financially supported by the NIH (5T32HL007895-10) (K.S-L.); EY-11386 (S.H-A.); and AR47273 (K.G.M.B.).

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Authors and Affiliations

  1. Cell & Tissue Systems, Inc., 2231 Technical Parkway, Suite A, North Charleston, SC, 29406, USA

    Kelvin G. M. Brockbank & Zhen Zhen Chen

  2. The Georgia Tech/Emory Center for The Engineering of Living Tissues, Georgia Institute of Technology, Atlanta, GA, USA

    Kelvin G. M. Brockbank

  3. Department of Medicine/Cardiology, Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, 675 Charles E. Young Drive South, MRL-3579, Los Angeles, CA, 90095-1760, USA

    W. Robb MacLellan & Katja Schenke-Layland

  4. Department of Pharmaceutical Sciences, School of Pharmacy, USC, Los Angeles, CA, USA

    Jiansong Xie & Sarah F. Hamm-Alvarez

Authors
  1. Kelvin G. M. Brockbank
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  2. W. Robb MacLellan
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  3. Jiansong Xie
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  4. Sarah F. Hamm-Alvarez
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  5. Zhen Zhen Chen
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  6. Katja Schenke-Layland
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Corresponding authors

Correspondence to Kelvin G. M. Brockbank or Katja Schenke-Layland.

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Brockbank, K.G.M., MacLellan, W.R., Xie, J. et al. Quantitative second harmonic generation imaging of cartilage damage. Cell Tissue Bank 9, 299–307 (2008). https://doi.org/10.1007/s10561-008-9070-7

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  • DOI: https://doi.org/10.1007/s10561-008-9070-7

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