Hypertrophic differentiation and calcification during intervertebral disc degeneration.
BACKGROUND
In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications.


OBJECTIVE
The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration.


METHODS
IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining.


RESULTS
Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs.


CONCLUSIONS
This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.
Rutges
J P H J
JP
Department of Orthopaedics, University Medical Center Utrecht, The Netherlands. j.rutges@umcutrecht.nl
Duit
R A
RA
Kummer
J A
JA
Oner
F C
FC
van Rijen
M H
MH
Verbout
A J
AJ
Castelein
R M
RM
Dhert
W J A
WJ
Creemers
L B
LB
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002114:Calcinosis / Q000503:physiopathology*; D002454:Cell Differentiation / Q000502:physiology; D002648:Child; D002675:Child, Preschool; D024121:Collagen Type X / Q000032:analysis; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006984:Hypertrophy / Q000503:physiopathology*; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000378:metabolism / Q000503:physiopathology*; D055959:Intervertebral Disc Degeneration / Q000503:physiopathology*; D008297:Male; D008875:Middle Aged; D053244:Osteoprotegerin / Q000032:analysis; D055114:X-Ray Microtomography; D055815:Young Adult
D024121:Collagen Type X; D050659:Core Binding Factor Alpha 1 Subunit; D053244:Osteoprotegerin; C493709:RUNX2 protein, human