LINK N CANNOT STIMULATE MATRIX PRODUCTION BUT INCREASES CELL DIVISION OF YOUNG BOVINE DISC CELLS

Abstract

Recent evidence indicates that link N can stimulate synthesis of proteoglycans and collagen by adult (2–4 years old) bovine disc tails. Here we sought to determine the effect of link N on the accumulation of disc matrix proteins from young (eight to twenty month old) bovine tails. We show that degradation products of link protein generated by matrix metalloproteinases cannot “feed-back” and stimulate matrix assembly of the disc matrix from young bovine tails but may have a regulatory role in cell proliferation. Link N may have value only in stimulating the growth and regeneration of the old damaged intervertebral disc.

To date, there have been no reports on the effect of the amino terminal peptide of link protein (DHLSD-NYTLDHDRAIH) (link N) on disc cells from young (eight to twenty month old) bovine coccygeal discs. Link N is produced when removed by proteolysis from the N-terminus of the link protein of cartilage proteoglycan aggregates. We recently showed that link N can act directly on disc cells from adult (two to four years old) bovine discs to stimulate matrix production (J Cell Biochem, 2003; 88:1202–13).

To examine whether link N can play a role in maintaining the matrix integrity of young bovine disc cells.

Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from fresh grade I discs from young steers, and cultured in pellets at 1 million cell per tube in 1 ml of DMEM-high glucose supplemented with 1% 100X Pen-Strep, 1% ITS, 1 mg/ml BSA, and 50 μg/ml ascorbic acid. Cell pellets were digested and then analysed for sulfated glycosaminoglycan, type II collagen, percent denatured type II collagen, type IX collagen, and DNA content, using specific assays.

A concentration of 100 ng/ml link N significantly increased the DNA content of AF cells. However, link N had no significant effect on proteoglycan, type II and type IX collagen accumulation.

This study demonstrates that link N at a concentration of 10 ng/ml and 100 ng/ml cannot stimulate matrix production but may increase cell division in young bovine disc tails.