Evidence for enhanced collagen type III deposition focally in the territorial matrix of osteoarthritic hip articular cartilage

Abstract

Objective: To determine if type III collagen is concentrated in the chymotrypsin-extractable collagen pool from osteoarthritic articular cartilage to assess its potential as a biomarker of Osteoarthritis (OA) pathogenic mechanisms.

Methods: Full thickness articular cartilage from grossly normal surfaces was analyzed from femoral heads, obtained at hip replacement surgery, from OA (n = 10) and fracture (n = 10) patients. Collagen, extracted by α-chymotrypsin, was characterized by SDS-PAGE/Western blot analysis, ELISA and immunohistochemistry using monoclonal antibodies specific to collagens types II and III.

Results: α-Chymotrypsin extracted more collagen from OA than control cartilage. The extractable pool included collagen types II and III from both OA and control hips. Importantly, OA cartilage contained 6-fold more collagen type III than control cartilage, based on ELISA. The estimated total tissue ratio of collagen III/II was in the 1-10% range for individual OA cartilage samples, based on pepsin-solubilized collagen using SDS-PAGE densitometry. Collagen type III N-propeptide trimers were the main molecular fragments seen on Western blot analysis of OA and control extracts. The chymotrypsin-extracted type II collagen gave primarily full-length α1(II) chains and chain fragments of α1(II) on Western blot analysis from both OA and control tissues. Immunohistochemistry showed that type III collagen was more concentrated in the upper half of OA cartilage and in the territorial matrix around individual chondrocytes and chondrocyte clusters.

Conclusions: The findings confirm that collagen type III deposition occurs in adult articular cartilage but significantly more pronounced in osteoarthritic joints, presenting a potential marker of matrix repair or pathobiology.

Keywords: Articular cartilage; Collagen type II and type III; Hip osteoarthritis.

Fig. 1

Western blot analysis of collagen type III degradation products in α-chymotrypsin extracts of OA and control femoral head cartilage samples (A). Extracts of full thickness tissue samples from individual hips were run on SDS-6%PAGE, blotted to PVDF membrane and Westerns developed using anti-type III collagen antibody (A) or stained with Coomassie blue to visualize total protein (B). Serum albumin is the only prominent protein by staining (confirmed by mass spectrometry) and is more prominent in the OA cartilage, presumably because of increased penetration from synovial fluid. Collagen type III N-propeptide trimers are consistently enriched in α-chymotrypsin extracts of OA cartilage compared with controls. Western blot analysis of collagen type II degradation products in chymotrypsin extracts of OA and control femoral head cartilage samples (C). A replicate SDS-6%PAGE gel to that shown in Fig. 1(A) was blotted to PVDF for Western development using mAb 1C10, which recognizes a sequence-specific epitope in the α1(II) chain. The main bands are essentially an intact α1(II) chain and large fragments of it.

Fig. 2

Electrophoretic resolution of pepsin-solubilized collagen types II and III from OA and control femoral head cartilage samples. SDS-6%PAGE was run using delayed DTT addition to separate α1(III) and α1(II) chain components. Protein was stained using Coomassie blue, revealing only type II and III collagen chains. Collagen type I is absent based on the lack of α2(I) which would run below α1(II).

Fig. 3

Immunohistochemical localization of collagen type III in full-thickness sections of femoral head cartilage from OA and fracture control hip joints. The mAb 4G9 was used as primary antibody to detect collagen III in full depth sections of frozen articular cartilage from OA and control joints. Representative sections from two OA samples, A (84 yr, female), B (62 yr, male) and two controls, C (59 yr, male), D (79 yr, female) are shown. Collagen type III is more prominent in cartilage from OA than control femoral heads consistent with the extracted collagen findings. In both it is most concentrated in territorial matrix surrounding cells in the upper half of the tissue.

Fig. 4

Illustrated molecular concept of polymeric pN type III collagen filaments deposited between and covalently attached to the surface of type II collagen fibrils (modified from reference).