Systemic iron overload exacerbates osteoarthritis in the strain 13 guinea pig

Abstract

Objective: Iron is emerging as a key player in aging-associated diseases due to its propensity for driving free radical formation. Studies examining the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. Our objective was to establish a direct relationship between excess iron and OA by administering iron dextran to a guinea pig strain with decreased propensity for developing this disease.

Design: Twenty, 12-week-old Strain 13 guinea pigs received either iron dextran or dextran control intraperitoneally once weekly for 4 weeks; termination occurred at 16 weeks of age. Iron levels were determined systemically (serum and liver) and within diarthrodial joints [femoral head articular cartilage and infrapatellar fat pads (IFPs) of knee joints]. One knee was collected to score structural changes associated with OA via microcomputed tomography (microCT) and histology using published grading schemes. Articular cartilage and IFPs were harvested from contralateral knees for gene expression analyses.

Results: Iron overload was confirmed systemically via increased serum iron and liver iron concentration. Articular cartilage and IFPs in the iron dextran group also had higher levels of iron. Excess iron worsened knee OA using both microCT and histologic scoring systems. Gene analyses revealed that exogenous iron altered the expression of iron trafficking proteins, select cytokines, and structural components of cartilage.

Conclusion: These results demonstrate that systemic iron overload caused cellular iron accumulation in the knee joint. This excess iron is associated with increased expression of local inflammatory mediators and early onset and progression of knee joint OA in Strain 13 animals.

Keywords: Aging; Guinea pig; Iron; Strain 13.

Figure 1.

Systemic iron quantification. Black lines on graphs indicate mean values. [A] Liver iron concentrations determined by iron AAS^×. Livers from animals in the iron overload group had a mean iron concentration of 10,448.00 ppm (n = 9). Livers from animals in the dextran control group had a mean iron concentration of 1,214.00 ppm (n = 10; 9,777.00 ppm difference between medians). One animal from the iron overload group was unable to be included in liver iron analysis due to lack of available tissue. [B] Serum iron concentration^×. Mean serum iron concentration was 939.10 μg/dl in iron overloaded animals (n = 7) and was 281.90 μg/dl in dextran control animals (n = 10; 689.50 μg/dl difference between medians). Serum was unable to be collected from 2 animals in the iron overload group and, as such, these animals were not included in this analysis. Additionally, 1 serum sample from an iron overload animal was identified for preanalytical sample preclusion due to hemolyzed serum. As serum iron measurement can be influenced by hemolysis during blood collection, the hemolyzed sample was excluded from serum iron analysis (n = 7). Data with non-Gaussian distribution were compared using non-parametric Mann-Whitney tests^×. No significant sex differences were present for liver iron concentration or serum iron concentration.

Figure 2.

Iron content of knee joint tissue. Black lines on graph represent mean values. [A-C] Enhanced iron stain of IFP^◊. Representative images of iron staining in the IFP of a control animal [A] and an iron overload animal [B]. [C] Mean surface area of iron staining was 54.70 square microns in the iron overload group (n = 10) and 37.22 square microns in the dextran control group (n = 9; 95% CI 12.01 – 22.94 square microns). One animal from the dextran control group was unable to be evaluated due to an appropriate tissue section being unavailable. [D] Iron concentration of femoral head articular cartilage by AAS^×. Mean concentration of iron within articular cartilage of iron overloaded animals was 2495.00 ppm (n = 10). Mean concentration of iron within articular cartilage of dextran control animals was 765.40 ppm (n = 10; 1,667 ppm difference between medians). Normally distributed data with significant differences in variance were compared using parametric t tests with Welch’s correction^◊. Data with non-Gaussian distribution were compared using non-parametric Mann-Whitney tests^×. No significant sex differences were present for surface area of iron staining in the IFP. Sex differences observed for cartilage iron concentration are presented and discussed in Supplemental Figure S4.

Figure 3.

Structural analysis of knee joints. Black lines on graphs represent mean values. [A] Mean whole joint microCT score^† was 4.00 in iron overloaded animals (n = 10) compared to 1.60 in dextran control animals (n = 10; 95% CI 0.12 – 4.68). Whole joint microCT score was determined by analyzing radiographic changes typically used in evaluating human OA, such as the presence and location of osteophytes, subchondral bone changes, and articular bone lysis. [B] Mean whole joint OARSI score^† was 19.00 in iron overloaded animals (n = 7) and 3.875 in dextran control animals (n = 8; 95% CI 13.29 – 16.96) (possible range of scores 0 – 84). Five animals (3 from the iron overload group and 2 from the dextran control group) were unable to be evaluated for whole joint OARSI histologic grading due to appropriate tissue sections being unavailable. [C-D] Representative microCT images of [C] a control knee joint with minimal to no radiographic evidence of OA and [D] a knee joint from the iron overload group. Arrows indicate an irregular surface with an enthesophyte forming on the femur and another, more prominent osteophyte on the tibia. [E-F] Photomicrographs of Toluidine blue stained sections from medial compartments of knee joints. [E] Representative image from the control group with a relatively smooth articular cartilage surface, minimal proteoglycan loss, and expected cellularity. [F] Representative image from the iron overload group, which displays a disrupted articular cartilage surface on the tibia and some focal loss of proteoglycans (as evidenced by lighter staining), with chondrocyte loss observed in the same area. Normally distributed data with similar variance were compared using parametric t tests^†. No significant sex differences were present for total joint microCT score and total joint OARSI score.

Figure 4.

Normalized mRNA counts for iron trafficking proteins in articular cartilage and the IFP. Black lines on graphs represent mean values. [A] TFR^†◊ [B] DMT1^† [C] ZIP14^× [D] CD163^× [E] FPN^× and [F] FTH-1^†◊. One cartilage sample from an animal in the iron overload group did not pass initial quality control for the assay and, as a result, was not analyzed for mRNA expression. As such, the number of animals from the iron overload group for cartilage gene expression analysis was 9, while all other gene expression analyses included 10 animals per group. Normally distributed data with similar variance were compared using parametric t tests^†. Normally distributed data with significant differences in variance were compared using parametric t tests with Welch’s correction^◊. Data with non-Gaussian distribution were compared using non-parametric Mann-Whitney tests^×. No significant sex differences were present for the genes analyzed.

Figure 5.

Normalized mRNA counts for select genes in articular cartilage and the IFP. Black lines on graphs represent mean values. [A] collagen type II^× [B] aggrecan^†× [C] IL-1β^× [D] TNF^× [E] IL-6^× and [F] TGFß1^×◊. One cartilage sample from an animal in the iron overload group did not pass initial quality control for the assay and, as a result, was not analyzed for mRNA expression. As such, the number of animals from the iron overload group for cartilage gene expression analysis was 9, while all other gene expression analyses included 10 animals per group. Normally distributed data with similar variance were compared using parametric t tests^†. Normally distributed data with significant differences in variance were compared using parametric t tests with Welch’s correction^◊. Data with non-Gaussian distribution were compared using non-parametric Mann-Whitney tests^×. No significant sex differences were present for the genes analyzed.