Abstract
Introduction
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 1 to 8 (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.
Material and Methods
Caudal IVDs from the tails of 20 to 24-month old steers were isolated with adjacent vertebral bone. After 7 days of preconditioning in culture, degeneration was induced in IVDs by a single injection of 50 µg trypsin into the NP. After 7 days after induced-degeneration, the trypsin-treated discs were injected with either sLink N or Link N (100 µg/disc, n = 6 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 nondegeneration controls. At 2, 4, and 8 weeks posttreatment, 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 the 1,9-dimethylmethylene blue dye-binding assay, and Western blotting was performed to determine the expression of aggrecan and type II collagen in the tissue.
Results
Without intervention, at all-time points, the GAG content in degenerate discs dropped to approximately 50% of that in nondegenerate controls. In contrast, sLink N or Link N, significantly increased the GAG content of the discs compared with the GAG content in degeneration control discs. However, sLink N was more potent at inducing proteoglycan and type II content than Link N.
Conclusion
The results revealed that sLink N or Link N have the ability to restore tissue content and that sLink N is more potent than Link N in an early state of the disease. These results have implications in relation to using either sLink N or Link N when regenerating a functional NP in the degenerated IVD or in repairing cartilage.