A needle micro-osmometer for determination of glycosaminoglycan concentration in excised nucleus pulposus tissue
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Aggrecan is one of the major macromolecular components of the intervertebral disc (IVD) and its loss is an early sign of degeneration. Restoration of aggrecan, and hence of biomechanical properties, is a major objective of biological therapies. At present, assessment of aggrecan concentration via its glycosaminoglycan (GAG) content is accomplished using biochemical and histological methods which require sacrifice of tissue. A minimally invasive method for assessing GAG, and hence aggrecan, which can avoid destruction of tissue, would be of benefit.
We have developed a needle micro-osmometer that is capable of measuring flux of saline into excised human nucleus pulposus (NP) tissue. Using the isotropic osmotic stress technique to assess the swelling pressure of the excised NP tissue and assuming negligible collagen tensile stress, we were able to relate the flux to the tissue fixed charge density (FCD). GAG concentration is evaluated from its FCD via the radioactive tracer technique. Samples representing different ages (28–59 years) and degeneration grades (1–4) were analyzed.
The flux is controlled by both the osmotic pressure difference across the probe’s semi-permeable membrane and by the tissue permeability. A linear correlation was found between flux and the tissue FCD. The equation describing the linear fit is FCD/(total tissue hydration) = 1.97 × 10−4 + 8283 × flux (R = 0.836, p < 10−4). Thus, by measuring saline flux, the concentration of GAG can be determined.
Micro-osmometry provides a reliable and minimally invasive tool for assessing GAG content in excised NP tissue. This method may be usefully applied in tissue engineering applications. It may also be useful for in vivo measurements if the question of the degenerative effect of needle puncture can be overcome.
KeywordsSwelling pressure Microdialysis Intervertebral disc (IVD) Fixed charge density (FCD) Micro-osmometry
The authors would like to thank Prof. Peter-Paul Varga from the National Center for Spinal Disorders (Budapest, Hungary) and Prof. Marco Brayda-Bruno from the Galeazzi Orthopaedic Institute (Milan, Italy) for providing MRI-graded IVD samples. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013) under Grant agreement no. HEALTH-F2-2008-201626. The partial financial support from the FP7-People-2007-2-2-ERG (Grant agreement 224834) is gratefully acknowledged (S.S). We acknowledge the support from the Charles W. McCutchen Foundation (A.M. and Y.M.).
Conflict of interest
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