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. 2001 Mar;60(3):262–268. doi: 10.1136/ard.60.3.262

Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer

M Hyttinen 1, J Toyras 1, T Lapvetelainen 1, J Lindblom 1, D Prockop 1, S Li 1, M Arita 1, J Jurvelin 1, H Helminen 1
PMCID: PMC1753566  PMID: 11171689

Abstract

OBJECTIVE—To evaluate the influence of inactivation of one allele ("heterozygous knockout" or "heterozygous inactivation") of the type II procollagen gene (Col2a1) on the biomechanical properties and structure of the articular cartilage and subchondral bone in 15 month old mice.
METHODS—Indentation stiffness of the humerus head articular cartilage was measured by a microindentation method. Cartilage and subchondral bone were prepared for digital densitometry of proteoglycans (PGs), polarised light microscopy (PLM) of collagen, and osteoarthrosis (OA) grading.
RESULTS—Heterozygous inactivation of the Col2a1 gene softened articular cartilage (p=0.002) as measured by indentation stiffness ((mean (SEM) 0.50 (0.07) MPa v 0.94 (0.13) MPa in controls). Fibrillar collagen network exhibited lower birefringence in the intermediate (p=0.04) and deep zones (p=0.01) of cartilage by PLM, indicating either decreased collagen content or a lower degree of fibril parallelism in the knockout mice. The total and zonal thicknesses of articular cartilage were unchanged. Zonal PG contents did not differ significantly. In knockout mice, the prevalence of superficial fibrillation—that is, a sign of OA, was higher than in controls (73% v 21%, p=0.002). The collagen induced birefringence of the superficial zone was not reduced. The subchondral bone volume fraction was lower in knockout mice than in controls, 31% v 43% (p=0.01), and optical retardation values in PLM of bone collagen were slightly but significantly lower (p=0.01).
CONCLUSION—Heterozygous inactivation of the Col2a1 gene made articular cartilage softer, altered the collagenous network, reduced subchondral bone volume, and altered its microstructure. Changes in the cartilage collagen network probably contributed to increased susceptibility to OA.



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Figure 1  .

Figure 1  

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(A) Schematic presentation for the indentation measurement setup. (B) A typical series of four consecutive indentation stress relaxation steps (3 µm for each step with ramping speed of 1 µm/s) causing a total strain of 24% in mouse humerus articular cartilage (full thickness about 50 µm). After 200 s of relaxation the next step was taken. (C) Equilibrium stress-strain curve and determination of indentation stiffness from the stress-strain slope.

Figure 2  .

Figure 2  

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Indentation stiffness of articular cartilage (mean (SEM)) in the head of the humerus of the herterozygous Col2a1 knockout and control mice.

Figure 3  .

Figure 3  

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(A) Thickness (mean (SEM)) of superficial, intermediate, and deep zones in control and knockout mice. Absolute cartilage thickness is the sum of these three zones. Mann-Whitney U test. (B) Collagen birefringence expressed as area integrated retardation (AIR, mean (SEM)) of semicircularly polarised light in the superficial, intermediate, and deep zones of articular cartilage of heterozygous Col2a1 knockout and control mice. The three zones contain collagen fibrils oriented tangentially, obliquely, and perpendicularly to the surface. Mann-Whitney U test.

Figure 4  .

Figure 4  

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Safranin O absorbance expressed as area integrated optical density (AIOD) in superficial, intermediate, and deep zones of articular cartilage. Zones are the same as those in figs 3A and B. Mann-Whitney U test.

Figure 5  .

Figure 5  

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(A) Volume fraction of subchondral bone (mean (SEM)) in the proximal humerus. Mann-Whitney U test. (B) Collagen birefringence in subchondral bone expressed as area integrated retardation (AIR, mean (SEM)) of semicircularly polarised light. Mann-Whitney U test.

Selected References

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