Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice

Abstract

Background: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium.

Methods: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging.

Results: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and β-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance.

Conclusions: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Keywords: Antioxidants; Cartilage; Methylene blue; Mitoquinone; Osteoarthritis; Sub-chondral bone; UM-HET3.

Fig. 1

OARSI scoring of stifles from UMHET3 mouse model. A UM-HET3 is a genetically diverse mouse model, generated via 4way cross of (BALB/cByJ × C57BL/6J) F1 mothers and (C3H/HeJ × DBA/2J) F1 fathers. Each of the offspring is genetically unique but shares 50% of its genetic material with every other UM-HET3 mouse. B Shown are representative knee joint samples that were processed for safranin-O-red, or (C) H&E staining of the synovial membrane that were categorized based on the OARSI scoring system [–17]

Fig. 2

Prevalence and severity of OA in UM-HET3 mice during aging. The frequency of cAC scores at the medial (A, B) and lateral (C, D) sides of the knee joints from control (CTL males n=47, CTL females n=45), MB- (males n=22, females n=24), and MitoQ-treated (males n=22, females n=22) mice. The odd ratio (OR) and the coefficient interval 95% upper (U) and lower (L) of the main effects for treatment and sex, as well as the interaction between the two, were estimated using ordered logistic regression, are indicated in the tables

Fig. 3

Correlations between cumulative AC (cAC) scores and synovitis, osteophytosis, or ectopic chondrogenesis (Ec.Chond). Correlations between cAC scores and synovitis in male (A) and female (B) mice. Correlations between cAC scores and osteophyte formation in male (C) and female (D) mice. Correlations between cAC scores and ectopic chondrogenesis in in male (E) and female (F) mice. A summary table of the Z score differences between each correlation along with the p values (G). CTL males n=47, CTL females n=45, MB males n=22, MB females n=24, MitoQ males n=22, MitoQ females n=22

Fig 4

Correlations between cumulative AC scores and markers of inflammation. Correlations between cumulative AC scores at the medial (A) and lateral (B) side of the joint with the levels of MMP13 (males n=21, females n=15), iNOS (males n=19, females n=19), and NLRP3 (males n=17, females n=15) in AC chondrocytes. (C) Sections of the medial synovial membrane in control mice immunostained with iNOS (males n=19, females n=19), and NLRP3 (males n=17, females n=15)

Fig. 5

Subchondral bone (SCB) and subchondral plate (SCP) morphology analyzed by micro-CT. A 2D image of a micro-CT scan indicating the knee joint regions of interest that were analyzed including the SCB of the distal femur, SCB of the proximal tibia, and the SCB plate of the proximal tibia. Parameters assessed by micro-CT included femur (B) bone volume/total volume (BV/TV), (C) trabecular thickness (Tb.Th), and (D) SCB mineral density (BMD). Likewise, tibia BV/TV (E), Tb.Th of the tibia SCB (F), and tibia SCB mineral density (G). SCB plate (SCBp) thickness (H), and SCBp bone mineral density (I), were determined at the proximal tibia. Data presented as mean ± SEM, p values are indicated. CTL males n=44, CTL females n=44, MB males n=22, MB females n=24, MitoQ males n=22, MitoQ females n=22. SCB phenotypic measurements from PCA of both male (J) and female (K) mice. Positively correlated variables point to the same side of the plot; negatively correlated variables point to opposite sides of the plot. Length and color of arrows indicate contribution of the variable to the overall variance of the data. Arrows that are close together are strongly positively correlated, arrows that are about 90 degrees (orthogonal) to each other are uncorrelated, and those far away (such as 180 degrees) are strongly negatively correlated