Facet joint degeneration in adolescent idiopathic scoliosis

Abstract

Adolescent idiopathic scoliosis (AIS) is a poorly understood deformity of the thoracolumbar spine which affects the intervertebral discs (IVDs) and the articular facet joints. The knowledge concerning facet joints in this context is very limited, although facet joint degeneration is a known contributor of back pain. In this study, a comprehensive investigation was performed to characterize the facet joint chondrocytes and extracellular matrix within the scoliotic spine. Surgically removed articular facet joint tissues were collected from patients undergoing spinal corrective surgery for AIS deformities, while non-scoliotic articular facet joint tissues were obtained from cadaveric organ donors. Alterations in cartilage tissue structure were evaluated histologically with safranin-O fast green and a modified OARSI grading scale. Pro-inflammatory cytokines, matrix-degrading proteases, and fragmented matrix molecules associated with cartilage degradation were analyzed by immunohistochemistry and western blotting. Safranin-O fast green staining revealed that young scoliotic facet joints show clear signs of degeneration with substantial proteoglycan loss, similar to osteoarthritis (OA). The proteoglycan levels were significantly lower than in healthy asymptomatic non-scoliotic control individuals. In comparison to controls, scoliotic articular facets showed increased cell density, increased expression of the proliferation marker Ki-67, and higher expression of MMP-3, MMP-13, and IL-1β. Expression and fragmentation of the small leucine-rich proteins (SLRPs) chondroadherin, decorin, biglycan, lumican, and fibromodulin were analyzed with western blot. Chondroadherin and decorin were fragmented in cartilage from patients with a curve greater than 70°, whereas biglycan and fibromodulin did not show curve-related fragmentation. AIS facet joint cartilage shows hallmarks of OA including proteoglycan loss, overexpression of pro-inflammatory mediators, increased synthesis of matrix-degrading proteases and fragmentation of SLRPs. As with patients with age-related OA, the premature joint degeneration seen in scoliotic patients is likely to contribute to the pain perceived in some individuals.

Keywords: cartilage; cell proliferation; cytokines; extracellular matrix; facet joint; metalloproteases; osteoarthritis; proteoglycans; scoliosis.

Figure 1

Safranin‐O fast green staining of bilateral facet joint cartilage from A, Scoliotic and B, Non‐scoliotic donors with red staining quantification using MATlab. C, Overall safranin‐O intensity and OARSI grading comparison between Scoliotic and non‐Scoliotic donors. D, Schematic representing a facet joint pair from the same vertebrae. E, Osteoarthritic markers found in scoliotic cartilage and highlighted by arrow (fissure) and thinning (erosion). Error bars shown are 95% CI. Unpaired parametric student's t test (**** = P < .0001, * = P < .05)

Figure 2

Safranin‐O fast green staining of articular cartilage from 3 facet joint pairs (left and right side for 3 spinal levels for 6 samples overall) for each of 6 Lenke types

Figure 3

Cell density and proliferative marker Ki‐67 IHC within facet joint cartilage from A, AIS and B, Non‐Scoliotic groups revealed by hematoxylin and DAB staining respectively. C, Cell density quantification between scoliotic and non‐scoliotic groups as well as paired comparison in high cellularity and low cellularity subgroups within the same donors. D, Ki‐67 immunopositivity quantification and comparison between scoliotic groups and the same cellularity subgroups as C). E, High magnification Ki‐67 IHC to show positive cells (green arrow) and negative cells (red arrow) and negative control (secondary antibody only) F, Image of scoliotic facet joint cartilage showing cell cluster, as revealed by hematoxylin. Error bars shown are 95% CI. Unpaired t test for overall comparisons and paired t test for paired comparisons (**** = P < .0001, * = P < .05)

Figure 4

Scoliotic and non‐scoliotic cartilage immunohistochemistry of proteases MMP‐3, MMP‐13, and pro‐inflammatory cytokines IL‐1β and IL‐6 with cell‐positivity quantification. Positive cells are shown by green arrows and negative cells by red arrows. Error bars shown are 95% CI. Unpaired t test (*** = P < .001, ** = P < .01, * = P < .05)

Figure 5

Western blot analysis of SLRPs on a 10% gel. Gels were loaded based on ascending grade of curvatures. The curves ranged from 50° to 100°; the first lane is occupied by non‐scoliotic, non‐degenerated age‐matched control sample. A, Chondroadherin core protein at 38 Kda & its fragments below the core protein are indicated by the arrow. B, Decorin core protein at 45 kDa with fragments. Note fragments for Decorin & Chondroadherin appear as the spinal curves magnitudes reach 70° of cobb angle. C, Doublet bands of Biglycan at 45 kDa along with its fragmented species. D, Double and single bands of fibromodulin appearing around 65 kDa. E, Non‐fragmented Lumican appears as single band at 55 kDa