Articular cartilage superficial zone collagen birefringence reduced and cartilage thickness increased before surface fibrillation in experimental osteoarthritis

Abstract

Objectives: To investigate articular cartilage collagen network, thickness of birefringent cartilage zones, and glycosaminoglycan concentration in macroscopically normal looking knee joint cartilage of young beagles subjected to experimental slowly progressive osteoarthritis (OA).

Methods: OA was induced by a tibial 30 degree valgus osteotomy in 15 female beagles at the age of 3 months. Fifteen sisters were controls. Cartilage specimens were collected seven (Group 1) and 18 months (Group 2) postoperatively. Collagen induced optical path difference and cartilage zone thickness measurements were determined from histological sections of articular cartilage with smooth and intact surface by computer assisted quantitative polarised light microscopy. Volume density of cartilage collagen fibrils was determined by image analysis from transmission electron micrographs and content of glycosaminoglycans by quantitative digital densitometry from histological sections.

Results: In the superficial zone of the lateral tibial and femoral cartilage, the collagen induced optical path difference (birefringence) decreased by 19 to 71% (p < 0.05) seven months postoperatively. This suggests that severe superficial collagen fibril network deterioration took place, as 18 months postoperatively, macroscopic and microscopic OA was present in many cartilage areas. Thickness of the uncalcified cartilage increased while the superficial zone became thinner in the same sites. In operated dogs, glycosaminoglycan content first increased (Group 1) in the lateral tibial condyle and then decreased (Group 2) (p < 0.05).

Conclusion: In this OA model, derangement of the superficial zone collagen network was the probable reason for birefringence reduction. This change occurred well before macroscopic OA.

Figure 1

Sites of the polarised light microscopy analysis in the right knee (stifle) joint of valgus osteotomised dogs and controls. PSF = patellar surface of femur, P = patella, LF = lateral condyle of femur, MF = medial condyle of femur, LT = lateral condyle of tibia and MT = medial condyle of tibia. Samples from LF and LT were taken also for computer based digital densitometry and from LT for transmission electron microscopic analysis.

Figure 2

Polarised light micrographs taken from unstained sections of the articular cartilage from the lateral condyle of tibia (LT) from a valgus operated dog (seven months postoperatively) (A) and a littermate control (B). Birefringence in the superficial zone of the operated dog is markedly decreased compared with control. On the other hand, in the deep zone birefringence is increased in the operated animal (scale 125 µm).

Figure 3

A photomicrograph taken from the lateral tibial (LT) condyle of the right knee joint 18 months after valgus osteotomy. In the superficial and intermediate zones of articular cartilage, severe reduction in safranin O staining is evident. In this case, the thickness of uncalcified and calcified cartilage is reduced (A). A photomicrograph from the littermate control dog showing normal articular cartilage from the same area (B) (safranin O, fast green and iron haematoxylin, scale 250 µm).

Figure 4

Volume fraction (%) of collagen from the superficial zone of articular cartilage from the lateral condyle of tibia (LT) in operated and control dogs.

Figure 5

Histogram of the area integrated optical density (AIOD 1/µm²) of safranin O staining in articular cartilage from the lateral condyle of tibia (LT) and lateral condyle of femur (LF) divided to four zones 7 (Group 1) and 18 months (Group 2) postoperatively in valgus operated and control dogs. * p < 0.05 versus control, by Wilcoxon matched pairs signed ranks test. † p < 0.05 Group 1 versus Group 2, by Mann-Whitney U test.