Purpose: Quantitative MRI is currently being tested as an early and non-invasive diagnostic tool of disc problems prior to the appearance of symptoms. The aim of the present study was to determine the effects of cyclic loading and enzymatic digestion on quantitative MRI, biochemical composition, and mechanical properties of intervertebral disc tissue.

Methods: Bovine tail segments consisting of three discs were subjected to 16h of cyclic compression loading (50N–300N–50N at 1Hz) or left unloaded for 16h while in saline solution at 37°C. Prior to loading, the nucleus pulposus were injected with either a trypsin or buffer solution. MR examinations were carried out in a 1.5T Siemens` Avanto system to measure T1 and T2 relaxation times, magnetization transfer ratio (MTR), and trace of the apparent diffusion coefficient (TrD). The nucleus pulposus and annulus fibrosus were dissected and analyzed for contents of water, glycosaminoglycan, total collagen, and denatured collagen. Cylindrical nucleus pulposus and annulus fibrosus tissue plugs were harvested, prepared, and tested under confined compression to measure compressive modulus (HA) and hydraulic permeability (k). ANOVA and linear regression analyses were performed (p< 0.05).

Results: Loading decreased the T1, T2, and TrD of NP while it increased MTR. Only water content in the nucleus pulposus was significantly influenced by loading. T1, water content, and k of the annulus fibrosus tissue were significantly reduced with loading.|Enzymatic treatment of the nucleus pulposus had no effect on its MR properties, but increased the percent of denatured collagen and thus decreased HA. None of the biochemical, mechanical, and MR parameters of the annulus fibrosus changed with trypsin treatment.

Conclusions: Dynamic loading of the disc segments for 16h decreased the permeability of both disc tissues. This was consistent with the measured drop in tissue hydration and was observed as a decrease in T1. Targeted trypsin digestion of the nucleus pulposus was confirmed with no detectable changes in the biochemical, biomechanical, or MR properties of the annulus fibrosus. Future studies will address additional quantitative MR parameters such as T1-rho, a higher strength magnet, and different enzymatic treatments. Funding: Other Education Grant Funding Parties: Canadian Institutes of Health Research, McGill William Dawson Scholar Award, and Whitaker Foundation

Recent evidence indicates that link N can stimulate synthesis of proteoglycans and collagen by adult (2–4 years old) bovine disc tails. Here we sought to determine the effect of link N on the accumulation of disc matrix proteins from young (eight to twenty month old) bovine tails. We show that degradation products of link protein generated by matrix metalloproteinases cannot “feed-back” and stimulate matrix assembly of the disc matrix from young bovine tails but may have a regulatory role in cell proliferation. Link N may have value only in stimulating the growth and regeneration of the old damaged intervertebral disc.

To date, there have been no reports on the effect of the amino terminal peptide of link protein (DHLSD-NYTLDHDRAIH) (link N) on disc cells from young (eight to twenty month old) bovine coccygeal discs. Link N is produced when removed by proteolysis from the N-terminus of the link protein of cartilage proteoglycan aggregates. We recently showed that link N can act directly on disc cells from adult (two to four years old) bovine discs to stimulate matrix production (J Cell Biochem, 2003; 88:1202–13).

To examine whether link N can play a role in maintaining the matrix integrity of young bovine disc cells.

Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from fresh grade I discs from young steers, and cultured in pellets at 1 million cell per tube in 1 ml of DMEM-high glucose supplemented with 1% 100X Pen-Strep, 1% ITS, 1 mg/ml BSA, and 50 μg/ml ascorbic acid. Cell pellets were digested and then analysed for sulfated glycosaminoglycan, type II collagen, percent denatured type II collagen, type IX collagen, and DNA content, using specific assays.

A concentration of 100 ng/ml link N significantly increased the DNA content of AF cells. However, link N had no significant effect on proteoglycan, type II and type IX collagen accumulation.

This study demonstrates that link N at a concentration of 10 ng/ml and 100 ng/ml cannot stimulate matrix production but may increase cell division in young bovine disc tails.