Subchondral bone microstructural damage by increased remodelling aggravates experimental osteoarthritis preceded by osteoporosis

Abstract

Introduction: Osteoporosis (OP) increases cartilage damage in a combined rabbit model of OP and osteoarthritis (OA). Accordingly, we assessed whether microstructure impairment at subchondral bone aggravates cartilage damage in this experimental model.

Methods: OP was induced in 20 female rabbits, by ovariectomy and intramuscular injections of methylprednisolone hemisuccinate for four weeks. Ten healthy animals were used as controls. At week 7, OA was surgically induced in left knees of all rabbits. At 22 weeks, after sacrifice, microstructure parameters were assessed by micro-computed tomography, and osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), alkaline phosphatase (ALP) and metalloproteinase 9 (MMP9) protein expressions were evaluated by Western Blot at subchondral bone. In addition, cartilage damage was estimated using the histopathological Mankin score. Mann-Whitney and Spearman statistical tests were performed as appropriate, using SPSS software v 11.0. Significant difference was established at P < 0.05.

Results: Subchondral bone area/tissue area, trabecular thickness and polar moment of inertia were diminished in OPOA knees compared with control or OA knees (P < 0.05). A decrease of plate thickness, ALP expression and OPG/RANKL ratio as well as an increased fractal dimension and MMP9 expression occurred at subchondral bone of OA, OP and OPOA knees vs. controls (P < 0.05). In addition, the severity of cartilage damage was increased in OPOA knees vs. controls (P < 0.05). Remarkably, good correlations were observed between structural and remodelling parameters at subchondral bone, and furthermore, between subchondral structural parameters and cartilage Mankin score.

Conclusions: Microstructure impairment at subchondral bone associated with an increased remodelling aggravated cartilage damage in OA rabbits with previous OP. Our results suggest that an increased subchondral bone resorption may account for the exacerbation of cartilage damage when early OA and OP coexist simultaneously in same individuals.

Figure 1

Experimental design. This study evaluated the effect of increased remodelling on subchondral bone microstructure and biomechanical-related genometrics in a combined rabbit model of osteoarthritis (OA) aggravated by previous osteoporosis (OP). (a)Experimental scheme. OVX: ovariectomy; MPH: methylprednisolone hemysuccinate. (b)Qualitative 2D models were reconstructed from images selected of a highly representative sample for each group (HEALTHY, OA, OP and OPOA), by using the CTAn software (Skyscan, Aartselaar, Belgium).

Figure 2

Microstructure parameters at subchondral bone. (a)Bone area fraction (B.Ar/T.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were assessed at subchondral bone. (b)Subchondral plate thickness. (c)Biomechanical-related structural parameters, fractal dimension (FD) and polar moment of inertia (Ip), at subchondral bone. HEALTHY (n = 7), OA (n = 7), OP (n = 8) and OPOA (n = 8) knees. OA, osteoarthritis; OP, osteoporosis. Values are expressed as mean ± SEM; *P < 0.05 vs. HEALTHY, #P < 0.05 vs. OA.

Figure 3

Remodelling parameters at subchondral bone and systemic level. (a)Alkaline hosphatise (ALP) and metalloproteinase 9 (MMP9) protein expressions at subchondral bone. At top: Densitometric analysis of ALP and MMP9 protein expressions at subchondral bone. At bottom: representative Western Blot images of ALP and MMP9 for 40 μg of total subchondral bone protein. The number of samples assayed for ALP expression were: HEALTHY n = 10; OA n = 8; OP n = 8 and OPOA n = 10, and for MMP9 were: n = 6 in each group. OA, osteoarthritis; OP, osteoporosis (b)Enzymatic activity for ALP and tartrato-resistant alkaline phosphatase (TRAP) in serum from OP animals (OP and OPOA groups; n = 8; white bars) and non-OP animals (healthy and OA groups; n = 7; black bars). OA, osteoarthritis; OP, osteoporosis. Bar represents the mean ± SEM. *P < 0.05 vs. HEALTHY, #P < 0.05 vs. OA.

Figure 4

Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB (RANKL) protein expression at subchondral bone. Densitometric analysis of OPG, RAKL and OPG/RANKL ratio, as well as, representative Western Blot images for OPG and RANKL are shown. To perform this blot, 40 μg of total protein from subchondral bone was used for HEALTHY, OA, OP and OPOA groups (n = 6, each group). OA: osteoarthritis; OP: osteoporosis. Data are displayed as arbitrary units ± SEM. *P < 0.05 vs. HEALTHY, #P < 0.05 vs. OA, &P < 0.05 vs. OP.

Figure 5

Total Mankin score of histological cartilage damage. The assessment was carried out at the weight bearing area of medial femoral condyle. HEALTHY (n = 7), OA (n = 7), OP (n = 8) and OPOA (n = 8) knees. OA: osteoarthritis; OP: osteoporosis. Results are expressed as mean ± SEM; *P < 0.05 vs. HEALTHY, #P < 0.05 vs. OA, &P < 0.05 vs. OP.