A Novel Therapeutic Agent for Rescuing the Adverse Effects of Advanced Glycation End-products in the Intervertebral Disk

Abstract

IntroductionLong-lived structural proteins such as collagen and aggrecan, particularly in tissues with low turnover, such as the intervertebral disk, are susceptible to accumulation of advanced glycation end-products (AGEs).1 The increase in AGEs, along with other changes in the matrix, has been implicated in the degeneration of the IVD.1,2 The changes induced by increased AGEs include specific deteriorations in the elastic and viscoelastic mechanical behaviors of the IVD tissues.3 We aim to mechanistically investigate the relationship between AGEs and the IVD tissue material behavior. We hypothesize that it may be possible to rescue the deleterious effects of AGEs using a novel AGEs-breaker4 and restore the tissue mechanical function. To investigate this hypothesis, we sought out to (1) investigate the effects of tissue mechanical behavior by inducing the formation of AGEs in vitro; and (2) reduce the amount of AGEs and restore the tissue mechanical behavior from both in vitro inductions and in vivo accumulations of the AGEs using a novel AGEs breaker.Materials and MethodsA total of 11 fresh frozen lumbar spines (T12/L1 - L5/S1) were collected (5M/6F) with a mean age of 82.5 years?±?10.2. The cross-sections of the lumbar disk were visually categorized into Thompson grades 1 to 55 and corroborated with fluoroscopic imaging. The grade 1 (?Control?) and grade 5 (?Degenerate?) disks were used for this study (total 27 disks). The disks were separated into annulus fibrosus (AF) and nucleus pulposus (NP) tissues. The Control disks were tested mechanically and then divided to undergo 0, 2, 4, 8, and 10 days of ribosylation in 0.6 M ribose at 37°C.6 The treated control disks, along with the degenerated disks, were then tested mechanically. The resulting force-deformation curves, accounting for the probe geometry, were used to compute elastic modulus and tan δ.Following mechanical measurements, the 0-day and 4-day treated control NP tissues were incubated in 0.15 M of a thiazolium salt known to cleave AGEs products4 for 4 days. The degenerate NP tissues were also incubated for 4 days in the thiazolium salt solution. Following the second incubation, all tissues were mechanically assessed again, and then subjected to acid hydrolysis by 6N hydrochloric acid (16 hours, 110C). The collagen cross-linking by AGEs, a sugar-derived posttranslational modification of amino acids, was quantified by measuring the autofluorescence of the acid hydrolysates at 370 nm emission and 440 nm excitation and divided by collagen content. The collagen content was measured from the acid-hydrolysates using a chloramine T absorbance assay that measured the hydroxyproline, an amino acid on the collagen protein that constitutes approximately 14% of collagen by mass7.ResultsThe in vitro ribosylation of the Control AF and NP tissues resulted in a significant increase in the advanced glycation end-products (AGEs). Correspondingly, the changes in AGEs resulted in changes in the elastic (p?