Developmental and osteoarthritic changes in Col6a1-knockout mice: biomechanics of type VI collagen in the cartilage pericellular matrix

Abstract

Objective: Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the "pericellular matrix" (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading.

Methods: Col6a1(-/-) mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1-month-old Col6a1(-/-), Col6a1(+/-), and Col6a1(+/+) mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique.

Results: In Col6a1(-/-) and Col6a1(+/-) mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild-type controls. With age, Col6a1(-/-) mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD.

Conclusion: These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1(-/-)-derived joint laxity, can lead to progression of OA.

Figure 1

Immunostaining revealed the pericellular distribution of type VI collagen in the cartilage of one-month-old Col6a1^+/+ and Col6a1^+/− mice (panel A and B). Collagen VI was also abundant in the ossification area (A). No type VI collagen was present in Col6a1^−/− mice (C). The pericellular distribution of type VI collagen was also present in 11-month old Col6a1^+/+ and Col6a1^+/− mice but not in Col6a1^−/− mice (D, E, and F).

Figure 2

Skeletal analysis of one-month-old Col6a1^+/+ (A) and Col6a1^−/− (B) mice using alcian blue (cartilage) and alizarin red (bone) staining. Homozygous mutants are smaller, with slower ossification progress in the upper (C–D) and lower (E–F) extremities (black arrows in A and B).

Figure 3

Lack of collagen VI results in delayed growth and ossification. (A–C) Toluidine blue staining of the femoral head of 3-month-old mice. (A) In the wild type mice, the secondary ossification process is almost complete by 3 months, while (B) Col6a1^+/− mice showed a delay with approximately 50% ossification at this age. (C) Col6a1^−/− mice exihbited delayed ossification Bars: 100 µm. (D) Quantitative measures of secondary ossification of the femoral heads showed that the extent of ossification depends significantly on the age and genotype (p<0.001). Col6a1^+/+, Col6a1^+/−, and Col6a1^−/− mice show similar secondary ossification at very early (1 month) and late stages (11 months) of their life. However, the rate of ossification is slower in mice lacking collagen VI.

Figure 4

Lack of collagen VI results in age-related osteoarthritis in the hip. (A–C) Histological sections of the femoral cartilage of 11-month-old mice stained with hematoxylin-eosin revealed significant progression of osteoarthritis in the knockout mice (C) when compared with their wild type counterparts (panel A). Scale bars = 100 µm. (D). Semi-quantitative scoring of histologic sections stained with Safranin O and H&E staining showing osteoarthritic degeneration of Col6a1^−/− mice as compared to Col6a1^+/− and Col6a1^+/+ mice. No significant differences were found for the heterozygous counterparts.

Figure 5

Bone mineral density of Col6a1^+/+, Col6a1^+/−, and Col6a1^−/− mice, as measured by micro-DXA. Bone mineral density depended on age (p<0.001) and was significantly lower in mice that lack type VI collagen (p<0.001).

Figure 6

Mechanical testing of articular cartilage and PCM. (A) A microindentation system comprised by a plane-ended glass indenter was employed to assess the mechanical properties of murine articular cartilage. (B) The micropipette aspiration technique was used to measure the mechanical properties of the PCM of isolated chondrons. (C) Young’s moduli of the PCM and cartilage specimens in Col6a1^+/+, Col6a1^+/−, and Col6a1^−/− mice. The Young’s moduli of the PCM was measured using the micropipette technique and significant differences were observed among all three groups. (D) Young’s moduli of the ECM of the cartilage. No differences were found in the Young’s modulus of the articular cartilage ECM as measured by the microindentation technique (n=18).