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Mechanism of Proteoglycan Synthesis by Bovine and Human Link N
Introduction Link N peptide is the N-terminal region of link protein which stabilizes the interaction between aggrecan and hyaluronan in proteoglycan aggregates. In vivo, during tissue turnover, link protein is subject to proteolytic degradation by stromelysin and gelatinase A and B generating Link...
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| Published in: | Global Spine Journal 2014-05, Vol.4 (1_suppl), p.s-0034-1376653-s-0034-1376653 |
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| Main Authors: | , , , , , |
| Format: | Article |
| Language: | English |
| Online Access: | Request full text |
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| Summary: | Introduction
Link N peptide is the N-terminal region of link protein which stabilizes the interaction between aggrecan and hyaluronan in proteoglycan aggregates. In vivo, during tissue turnover, link protein is subject to proteolytic degradation by stromelysin and gelatinase A and B generating Link N peptide. It has been shown that human Link N (DHLSDNYTLDHDRAIH) can act as a growth factor and stimulate the synthesis of proteoglycans and collagen by IVD cells in vitro and improve disc height and proteoglycan levels in vivo in a rabbit model of IVD degeneration.1-3 To date, there have been no reports on the effect of bovine Link N (DHHSDNYTVDHDRVIH) on disc cells. The specific aim of this study is to compare the effects of bovine Link N (BLN) and human Link N (HLN) on bovine IVD cells to determine whether substitution of residues, as occurs in the BLN sequence, can alter Link N function.
Materials and Methods
Coccygeal IVDs from healthy 20 to 24 months old steers were obtained from a local abattoir at 2 to 3 hours after slaughter. The IVDs were separated from their adjacent vertebral bodies, and the NP and AF cells were isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) regions by sequential digestion with 0.2% pronase followed by 0.125% collagenase digestion as previously described.4 After isolation, the NP and AF cells were either immediately embedded in 1.2% alginate beads (350,000 cell/bead) for proteoglycan synthesis or were plated in six well plates for protein extraction.
Proteoglycan synthesis After 7 days of stabilization in complete DMEM high glucose medium, the alginate beads were placed in 24 well plates at a density of 9 bead/well and were incubated for 18 days in media supplemented with 1 µg/mL of either HLN or BLN (CanPeptide, Montreal). Beads cultured in media alone for the same period of time were used as the control (CTL). The culture media with or without Link N was changed every three days for 18 days and the sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of the media was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay.
Canonical SMAD-Mediated Signaling AF and NP cells were expanded in culture medium (Dulbecco's Modified Eagle Medium high glucose supplemented with 10% fetal bovine serum) into six well plates (7.5 × 105 cells/well) until reaching 80 to 90% confluence. The cells were preincubated overnight in serum-free medium, then were incubated in 1μg/mL HLN or BLN for different ti |
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| ISSN: | 2192-5682 2192-5690 |
| DOI: | 10.1055/s-0034-1376653 |