Structural changes in the collagen network of joint tissues in late stages of murine OA

Abstract

Osteoarthritis (OA) is the most prevalent degenerative joint disease, resulting in joint pain, impaired movement, and structural changes. As the ability of joint tissue to resist stress is mainly imparted by fibrillar collagens in the extracellular matrix, changes in the composition and structure of collagen fibers contribute to the pathological remodeling observed in OA joints that includes cartilage degeneration, subchondral bone (SCB) sclerosis, and meniscal damage. Using the established OA model of destabilization of the medial meniscus (DMM) in C57BL/6J mice, we performed a comprehensive analysis of the content and structure of collagen fibers in the articular cartilage, subchondral bone, and menisci using complementary techniques, which included second harmonic generation microscopy and immunofluorescence staining. We found that regions exposed to increased mechanical stress in OA mice, typically closest to the site of injury, had increased collagen fiber thickness, dysregulated fiber formation, and tissue specific changes in collagen I and II (Col I and Col II) expression. In cartilage, OA was associated with decreased Col II expression in all regions, and increased Col I expression in the anterior and posterior regions. Col I fiber thickness was increased in all regions with disorganization in the center region. In the superficial SCB, all regions exhibited increased Col I expression and fiber thickness in OA mice; no changes were detected in the deeper regions of the subchondral bone except for increased Col I fiber thickness. In the menisci, OA led to increased Col I and Col II expression in the vascular and avascular regions of the anterior meniscus with increased Col I fiber thickness in these regions. Similar changes were observed only in the vascular region of the posterior meniscus. Our findings provide, for the first time, comprehensive insights into the microarchitectural changes of extracellular matrix in OA and serve as guidelines for studies investigating therapies that target collagenous changes as means to impede the progression of osteoarthritis.

Figure 1

Picrosirius red staining of the knee joint shows changes in the microarchitecture of knee joint tissues in OA mice. (A) Representative Safranin-O/Fast Green staining for comparison of Sham and DMM knee joints (× 10 magnification, scale bar = 100 μm, images acquired with OsteoMeasure software) Ant = anterior, Post = posterior, Cart = cartilage, SCB = subchondral bone. Black arrows indicate marrow space in the SCB. (B) Osteoarthritis cartilage histopathology assessment system (OARSI) scoring for samples analyzed (0 = no OA activity seen, stage 0, 5 = > 50% involvement OA, stage 4). (C) Uncalcified tibial cartilage area (UTC) in mm² calculated by subtracting calcified cartilage area (cartilage between chondro-osseous junction and tideline) from total cartilage area (total articular cartilage above the chondro-osseous junction). (D) Representative images of PSR for a comparison of the collagenous structure in cartilage, subchondral bone, and anterior/posterior menisci. Thicker fibers typically stain red while thinner fibers stain green. Blue arrows indicate marrow space in the SCB (× 10 magnification, scale bar = 100 μm). PSR staining quantification of the area percentage and mean intensity of red and green fibers in the (E) subchondral bone, (F) anterior meniscus, and (G) posterior meniscus. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using unpaired t-test with Welch’s correction, values are expressed as mean ± SEM; N = 5/group.

Figure 2

OA results in changes to collagen content, fiber width, and organization in the articular cartilage. (A) Immunofluorescence (IF) staining for Col I and (B) Col II expression in anterior, center, and posterior regions of articular cartilage, (× 40 magnification, scale bar = 5 μm, images acquired with Zeiss Zen Blue 2.6 edition software). Quantification of area percentage (area of positive signal normalized to total selected area) of (C) Col I and (D) Col II IF stains. (E) Volume fraction (volume producing SHG signal in the selected ROI normalized to the total volume) ratio between FSHG/BSHG VF in ROIs of articular cartilage. (F) Comparison of FSHG fiber width (μm) between sham and DMM samples in each ROI. *P < 0.05, **P < 0.01, ***P < 0.001 using unpaired t-test with Welch’s correction, values are expressed as mean ± SEM; 3 images were analyzed per ROI in each sample, N = 5/group.

Figure 3

OA results in increased collagen composition and increased fiber thickness in the superficial regions of subchondral bone. (A) Safranin-O/Fast Green staining depicting the superficial SCB (SSCB) (10 × magnification, scale bar = 100 μm, images acquired with OsteoMeasure software). IF staining for (B) Col I and (C) Col II expression in anterior, center, and posterior regions of the superficial SCB (× 40 magnification, scale bar = 20 μm, images acquired with Zeiss Zen Blue 2.6 edition software). Quantification of area percentage (area of positive signal normalized to total selected area) of (D) Col I and (E) Col II IF stains. (F) Comparison of FSHG fiber width (μm) between sham and DMM samples in each ROI. *P < 0.05, **P < 0.01, ***P < 0.001 using unpaired t-test with Welch’s correction, values are expressed as mean ± SEM; 3 images were analyzed per ROI in each sample, N = 5/group.

Figure 4

OA results in increased fiber thickness in the deep regions of subchondral bone. (A) Safranin-O/Fast Green staining depicting the deep SCB (DSCB) (× 10 magnification, scale bar = 100 μm, images acquired with OsteoMeasure software). IF staining for (B) Col I and (C) Col II expression in anterior, center, and posterior regions of deep SCB (× 40 magnification, scale bar = 20 μm, images acquired with Zeiss Zen Blue 2.6 edition software). Quantification of area percentage (area of positive signal normalized to total selected area) of (D) Col I and (E) Col II IF stains. (F) Comparison of FSHG fiber width (μm) between sham and DMM samples in each ROI. **P < 0.01 using unpaired t-test with Welch’s correction, values are expressed as mean ± SEM; 3 images were analyzed per ROI in each sample, N = 5/group.

Figure 5

OA results in changes to collagen composition and fiber thickness in the anterior meniscus. IF staining for (A) Col I and (B) Col II expression in vascular (peripheral) and avascular (internal) regions of the anterior meniscus (× 40 magnification, scale bar = 10 μm, images acquired with Zeiss Zen Blue 2.6 edition software). Quantification of area percentage (area of positive signal normalized to total selected area) of (C) Col I and (D) Col II IF stains. (E) Comparison of FSHG fiber width (μm) between sham and DMM samples in each ROI. *P < 0.05, **P < 0.01, ***P < 0.001 using unpaired t-test with Welch’s correction, values are expressed as mean ± SEM; 3 images were analyzed per ROI in each sample, N = 5/group.

Figure 6

Collagen composition and fiber thickness in the posterior meniscus are affected by OA. IF staining for (A) Col I and (B) Col II expression in vascular (peripheral) and avascular (internal) regions of the posterior meniscus (× 40 magnification, scale bar = 20 μm, images acquired with Zeiss Zen Blue 2.6 edition software). Quantification of area percentage (area of positive signal normalized to total selected area) of (C) Col I and (D) Col II IF stains. (E) Comparison of FSHG fiber width (μm) between sham and DMM samples in each ROI. **P < 0.01 using unpaired t-test with Welch’s correction, values are expressed as mean ± SEM; 3 images were analyzed per ROI in each sample, N = 5/group.