title: Fibrotic-like changes in degenerate human intervertebral discs revealed by quantitative proteomic analysis.
abstract: OBJECTIVE
  Intervertebral disc degeneration (IDD) can lead to symptomatic conditions including sciatica and back pain. The purpose of this study is to understand the extracellular matrix (ECM) changes in disc biology through comparative proteomic analysis of degenerated and non-degenerated human intervertebral disc (IVD) tissues of different ages.
  
  
  METHODS
  Seven non-degenerated (11-46 years of age) and seven degenerated (16-53 years of age) annulus fibrosus (AF) and nucleus pulposus (NP) samples were used. Proteins were extracted using guanidine hydrochloride, separated from large proteoglycans (PGs) by caesium chloride (CsCl) density gradient ultracentrifugation, and identified using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). For quantitative comparison, proteins were labeled with iTRAQ reagents. Collagen fibrils in the NP were assessed using scanning electron microscopy (SEM).
  
  
  RESULTS
  In the AF, quantitative analysis revealed increased levels of HTRA1, COMP and CILP in degeneration when compared with samples from older individuals. Fibronectin showed increment with age and degeneration. In the NP, more CILP and CILP2 were present in degenerated samples of younger individuals. Reduced protein solubility was observed in degenerated and older non-degenerated samples correlated with an accumulation of type I collagen in the insoluble fibers. Characterization of collagen fibrils in the NP revealed smaller mean fibril diameters and decreased porosity in the degenerated samples.
  
  
  CONCLUSIONS
  Our study identified distinct matrix changes associated with aging and degeneration in the intervertebral discs (IVDs). The nature of the ECM changes, together with observed decreased in solubility and changes in fibril diameter is consistent with a fibrotic-like environment.
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: A
authors.initials: A
authors.lastname: Yee
authors.affiliation: Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: M P Y
authors.initials: MP
authors.lastname: Lam
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: V
authors.initials: V
authors.lastname: Tam
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: W C W
authors.initials: WC
authors.lastname: Chan
authors.affiliation: Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: I K
authors.initials: IK
authors.lastname: Chu
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: K S E
authors.initials: KS
authors.lastname: Cheah
authors.affiliation: Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong, China. Electronic address: cheungmc@hku.hk.
authors.forename: K M C
authors.initials: KM
authors.lastname: Cheung
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. Electronic address: chand@hku.hk.
authors.forename: D
authors.initials: D
authors.lastname: Chan
mesh_terms: D000293:Adolescent; D000328:Adult; D000375:Aging / Q000378:metabolism; D002648:Child; D003094:Collagen / Q000378:metabolism; D005355:Fibrosis; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D055959:Intervertebral Disc Degeneration / Q000378:metabolism* / Q000473:pathology; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008875:Middle Aged; D000070614:Nucleus Pulposus / Q000378:metabolism / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012995:Solubility; D055815:Young Adult
chemical_list: D011506:Proteins; D003094:Collagen
keywords: Disc degeneration; Fibrosis; Intervertebral disc; Mass spectrometry; Proteome; Quantitative method