41. THE POSITIVE EFFECTS OF POLY-N-ACETYL GLUCOSAMINE ON HUMAN INTERVERTEBRAL DISC CELL METABOLISM IN VITRO

Abstract

Purpose: There is interest in biologic strategies that can potentially treat degenerative disc disease (DDD). A new deacetylated derivative of a marine diatomic glycosaminoglycan (DEAC) was developed and incorporated into two sulphated hydrogel formulations; Gel 1 and 2. These materials were proposed to have a reparative effect on damaged tissue. Biochemical studies were conducted using primary human disc cell (HDC) cultures.

Method: HDCs were isolated from surgical specimens by sequential enzymatic digestion (pronase and collagenase). Time-course in-vitro studies were conducted on cell cultures treated with DEAC, Gel 1 or Gel 2 (28 day period). Proteoglycan content (alcian blue), cellular viability/proliferation (MTT assay), and type collagen II, aggrecan expression (RT-PCR, immunohistochemistry) was assessed.

Results: When compared to controls, the DEAC, Gel 1 and 2 treated HDC groups showed significant increases in proteoglycan content as early as day 12. The greatest effect was observed with Gel 1 (78.4±1.9 fold greater optical density compared to control, p < 0.05). The amount of proteoglycan quantified on DEAC treated HDCs on day 28 was 27.7±0.09 times higher than control (p< 0.05). MTT results demonstrated that Gel 1 group showed the highest viability over the study period (mean optical density 0.13+.01 versus 0.039+0.01 in controls). There were no significant differences in cell proliferation of Gel 2, DEAC and untreated control groups. RT-PCR and immunohistochemistry demonstrated expression of type II collagen and aggrecan consistent with the disc phenotype.

Conclusion: The results of this study demonstrates that formulations derived from poly-N-acetyl glucosamine (pGLcNAc) have positive effects of disc cell metabolism as quantified by proteoglycan content, cellular viability and proliferation, and the expression of key extra-cellular matrix molecules. The sulphated formulation of deacetylated pGLcNAc (Gel 1) appeared to have the greatest in-vitro effect followed by DEAC and the short fiber construct of Gel 2. It is possible that the pGlcNAc fibers in Gel 2 were not as soluble to the extent of DEAC due to their inability to form strong hydrogen bonds. This study shows promise towards ongoing evaluation of novel biomaterials for the potential DDD treatment through tissue regenerative or reparative schemes.