Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development.

Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA.

Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA.

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different soft and hard musculosk-eletal tissues. In the intervertebral disc (IvD) the major SLRPs involved in regulation of types I & II collagen fibril size are believed to be decorin, fibromodulin and lumican. Research into IvD degeneration and backpain is hampered by a lack of specific biomarkers to detect and monitor the disease process. We have discovered that two keratan sulphate (KS) substituted members of the SLRP family, Keratocan and Lumican (that are major KS-pro-teoglycans found in cornea) were unusually expressed in extracts from degenerative disc tissues.

Methods: Non-degenerate disc tissue (n=10) was obtained from 2 scoliosis patients and degenerate disc tissue from 11 patients undergoing surgery. The degenerate discs were graded using criteria described by Pfir-rman et al (Spine26: 1873; 2001). Tissue samples were extracted with 4M guanidine HCl and after dialysis subjected to SDS-PAGE and Western blot analyses using monoclonal antibodies that recognise epitopes on kera-tocan and lumican.

Results & Discussion: Keratocan was not found in the non-degenerate disc tissue but was present in all degenerate IvD tissues tested. Lumican showed and increased expression in extracts of degenative IvD tissues. Our working hypothesis is that the increased expression of these two SLRPs in degenerative disc tissue results from a reparative depostion of a type I collagen fibrillar ‘scar’. This unusual expression suggests their potential as biomarkers for detecting the onset of degenrative disc disease.

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different musculoskeletal tissues. In hyaline articular cartilage the major SLRPs involved in regulation of type II collagen fibrils are believed to be decorin and fibromodulin. These two SLRPs along with another family member, lumican, have also been identified in intervertebral disc tissues. In recent studies, we serendipitously discovered that, keratocan and lumican [two keratan sulphate (KS) substituted members of the SLRP family] were unusually expressed in extracts from degenerative joint and degenerative disc tissues. The object of this study has been to further investigate this finding with a view to examining the increased expression of keratocan and lumican using qualitative Western blot analysis and quantitative ELISA methods. Our working hypothesis is that the increased expression of these two SLRPs in degenerative joint and disc tissue results from a reparative deposition of a type I collagen fibrillar ¡®scar¡^-.

Methods: Monoclonal antibodies were produced to core protein epitopes in lumican and keratocan. Degenerate cartilage was obtained from patients undergoing routine joint replacement for either hip or knee joints, whilst normal articular cartilage tissue was obtained from surgical knee procedures. In addition, disc samples were obtained from patients undergoing a variety of spinal procedures and were Graded I-IV using a modified Thompson score. The tissue was diced and extracted in a 4M guanidine HCl buffer, pH6.8 containing an inhibitor cocktail for 48 h at 4¢ªC. Samples were then dialysed exhaustively against Milli Q water and assayed for glycosaminoglycan (GAG) content using the DMMB assay. Cartilage extracts containing equal amounts of GAG were then separated by SDS-PAGE and transferred to nitrocellulose for Western blotting using mMAbs to either keratocan or lumican. In addition, a competitive ELISA has been developed for quantifying keratocan and lumican.

Results: Western blot analysis of normal and degenerative articular cartilage revealed the presence of both keratocan and lumican. However, the presence of these SLRPs was substantially increased in the degenerate articular cartilge extracts. In addition, these proteins were also present in extracts of intervertabral disc with an increase being apparent in those disc samples with increased pathology. Preliminary data for the development of a quantitative ELISA for these two SLRPs shows promise.

Discussion: The unexpected increase in the detection of keratocan and lumican in degenerative articular cartilage and disc suggests their potential as biomarkers for the onset of degenerative joint and disc disease. However, this will involve the development of a quantitative assay and the investigation of the presence of these molecules in synovial fluid and serum.