Changes in Biomechanical, Histological and Quantitative MRI Parameters in Lumbar Caprine Intervertebral Discs Subjected to Chondroïtinase-Induced Degeneration
Authors: Paul C. P. , Strijkers G. J. , de Graaf M. , Bisschop A. , Smit T. H. , Helder M. N. , van Royen B. J. , Mullender M. G. 
Published: June 2012

Introduction

Intervertebral disk (IVD) degeneration is one of the major causes of low back pain. In the onset of IVD degeneration, the nucleus pulposus of the disk loses proteoglycans, decreasing the discs' capability to retain water. This is considered to detrimentally affect its biomechanical properties, making the disk prone to further degradation. Quantitative magnetic resonance imaging (MRI) techniques, such as T2- and T1-rho mapping, provide valuable information on the matrix status (proteoglycan content and distribution) of healthy and degenerating IVDs. We have developed a model to culture and load large (lumbar) IVDs, in which we can simulate IVD degeneration by injection of chondroitinase ABC (cABC; an enzyme that cleaves proteoglycans). The purpose of the current study is to characterize the changes in biomechanical, histological, and quantitative MRI parameters of caprine lumbar IVDs subjected to intranuclear cABC-injection.

Materials and Methods

Lumbar IVDs (36 total) with cartilaginous endplates were dissected from the spines of mature goats under sterile conditions. IVDs were randomly assigned to receive either no injection or injection (29G needle) of P83 µL PBS, 0.25 U/mL cABC, or 0.5 U/mL cABC. All IVDs were cultured in a bioreactor for 21 days under simulated-physiological dynamic loading conditions. Before injection and directly after culture, IVDs were scanned with a high-field MRI scanner (9T). T1-, T2-, and T1-rho mapping sequences were used to quantify the relaxation time parameters. IVDs axial deformation behavior during loading was analyzed and IVD stiffness was calculated from the load and displacement data collected during culture in the bioreactor. Matrix integrity of IVDs was studied on transverse paraffin sections stained with Safranin-O to assess proteoglycan content and distribution and Masson's Trichrome to assess collagen composition and structure.

Results

All biomechanical properties of the IVDs injected with cABC showed significant changes over the culture period. During preloading of the IVDs, within the first 8 hours of culture, creep behavior differed significantly between groups. A cABC dose-dependent subsidence was observed. Over the total culture period, disk height was reduced in all groups. However, cABC-treated groups showed significantly more loss in height. These discs progressively lost height during the entire culture period and stiffness of IVDs increased gradually over time. When comparing the images and quantitative MRI data from day 0 and day 21, significant changes could be observed in T2 and T1-rho values in the nucleus region for both the 0.25U and 0.5U cABC group. In the annulus, the apparent diffusion coefficient (ADC) changed significantly in the anterior and lateral regions. On histological sections, we observed a clear loss in staining intensity in the nucleus on Safranin-O sections, especially in the 0.5U cABC injected IVDs, with blurring of the posterior demarcation zone between the nucleus and annulus. In Masson's Trichrome stained sections, a clearly defined lamellar structure in the anterior and lateral annulus could be observed in day 0 discs, which showed degradation in the cABC-injected IVDs at day 21.

Conclusion

cABC injection into the nucleus had a rapid and potent effect on the measured characteristics of caprine lumbar IVDs. Enzymatic cleavage of proteoglycans from the nucleus directly affected biomechanical properties of the discs and we observed dose-dependent degenerative changes. We also found comparative changes in matrix content and integrity as observed on histological section and quantified by MRI. This study shows the parallels between the functional and histological changes in a large lumbar IVD during the first stages of degeneration. We demonstrate that T1rho MRI measurements can be used to accurately quantify this early degenerative process. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared