Optimizing Protein Crosslinking Formulations for Degenerating Spinal Disks

Degenerative disc disease is a debilitating chronic condition with a US economic cost estimated at $100 billion. Spinal disc cells, through a complex but defined process, experience a decline in the tissue’s ability to repair the mechanical damage caused by daily physiological loading and unloading. Over time the tissue’s ability to support loads lessens, leading to fissure formation, stress intensification and loss of disc height. Abnormal bulging of the weakened disc can impinge upon nerve roots, leading to the generation of pain and, in extreme cases, disc herniation. Non-surgical exogenous crosslinking therapy is a potential new, non-invasive technology for the treatment of degenerative disc disease. The technology is based upon the injection of protein crosslinking reagents into the pathological disc to restore its mechanical properties and to facilitate the exchange of waste products and nutrients. Mechanical tests using a TestResources biomedical test machine supported this research effort to improve treatment for degenerative disk disease. Tests involved circumferential specimens of annulus cut from bovine lumbar discs, further cut to narrow a central region. The smallest cross-section in the necked-down region of each sample was measured using a rotating laser micrometer. The specimen was positioned in custom grips and a tensile test run at a constant displacement rate of 0.3 mm/sec. Peak modulus, ultimate tensile strength, yield stress (using a 0.1% offset method), yield strain, and resilience (total energy absorbed at the yield point) were calculated from the stress-strain data. Data were expressed as the percentage change in the parameter in a treated specimen compared to the mean of the untreated samples. A Mann-Whitney non-parametric test was used to determine significance of mean differences between groups (α≤0.05). After treatment and incubation, specimens were clamped into place on a TestResources 100R materials test system such that the load axis was parallel to the specimen’s anatomical superior-inferior axis and centered on the disc’s center of mass. Cyclic compressive load from 0N to 400N was applied at 0.05 mm/sec for 40 cycles to condition the specimen. A rotating Keyence laser micrometer measured the anterior surface of the disc at three heights (midline and 2 mm above and below) at static loads of 50 and 400N. Disc surface position was analyzed for each applied load and at each height using custom Octave code. Bulge was calculated as the difference in disc surface position at each height between the 50 and 400N loads. One specimen from the treatment group was 2 standard deviations away from the mean of the remaining samples and was discarded as an outlier. Statistical significance was determined using the Mann-Whitney-test. After analyzing results, the group found that formulations lacking amines and containing phosphate ions appear to be promising candidates for clinical use of the crosslinkers genipin and methylglyoxal. Learn more: Optimization of Protein Crosslinking Formulations Research for the Treatment of Degenerative Disc Disease

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