Abstract
Purpose
Although the disc has limited endogenous repair activity, induced repair of disc tissue may be possible by the intradiscal injection of growth factors to stimulate the production of disc matrix. We previously demonstrated that Link N (DHLSDNYTLDHDRAIH), a naturally occurring peptide generated by the N-terminal proteolytic fragmentation of link protein during tissue turnover, can act as a growth factor in the disc. It can stimulate matrix production in vitro, in vivo and in intact ex vivo human intervertebral discs (IVDs). We have recently discovered that AF cells have the ability to proteolytically process Link N resulting in a fragment spanning amino acid residues one-eight (US Patent # 61870394) – short Link N (sLink N). Our in vitro data indicates that the biologically active sequence is preserved within this fragment and, thus, sLink N could represent a potential stable growth factor able to stimulate disc repair. Separately, we developed a long-term organ culture model with vertebral bone. The purpose of the present study was to evaluate the effect of sLink N and compare its efficacy to Link N in this novel organ culture model of early disc degeneration.
Method
Caudal IVDs from the tails of 20–24 months old steers were isolated with adjacent vertebral bone. After seven days of preconditioning in culture, degeneration was induced in IVDs by a single injection of 50 μg trypsin into the NP. Seven days after induced-degeneration, the trypsin-treated discs were injected with either sLink N or Link N (100 μg/disc, n = six discs/group). Four of the trypsin-treated degenerate discs were injected with PBS alone to serve as a control for degeneration while four discs served as non-degeneration controls. At two, four and eight weeks post treatment, two discs from each treatment and control groups were processed for biochemical analyses. Proteoglycan (predominantly aggrecan) synthesis in the NP was monitored as sulfated glycosaminoglycan using dimethylmethylene blue dye-binding assay, and Western blotting was performed to determine the expression of aggrecan and type two collagen in the tissue.
Results
Without intervention, at all time points, the GAG content in degenerate discs dropped to ~50% of that in non-degenerate controls. In contrast, sLink N, significantly increased the GAG and collagen content of the discs compared with that in degeneration control discs.
Conclusion
The results revealed that sLink N has the ability to restore tissue content in an early state of disc degeneration. These results have implications in relation to using either sLink N in regenerating a functional NP in the degenerated IVD or in retarding disc degeneration. Separately, we have developed and validated a novel long-term IVD organ culture model that retains vertebral bone and easy to prepare. Our model is ideal for testing potential drugs and alternate-based therapies, in addition to investigating the long-term effects of loading paradigms on disc degeneration and repair.
Disclosures: None