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Tissue crosslinks concentrations in normal joints and chronic articular diseases
  1. Chair of Rheumatology
  2. University of Milan, Italy
  3. Pathology Department
  4. Istituto Ortopedico ‘Gaetano Pini’
  5. University of Milan, Italy
  6. LPB Research Institute
  7. Cinisello B Italy
  1. Dr L Sinigaglia, Cattedra di Reumatologia, Istituto Ortopedico ‘Gaetano Pini’, Piazza Cardinal Ferrari 1, 20122 Milano, Italy.
  1. Department of Orthopedic Surgery, Hamamatsu University School of Medicine, 3600 Handa, Hamamatsu, 431-31, Japan

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We read with interest the recent paper by Takahashi et al 1 concerning joint tissue concentrations of collagen crosslinks (Pyr, Dpyr) in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The main discovery of this study was the presence of Dpyr also in extra-osseous tissues, namely cartilage and synovium in patients with joint disorders and in the synovium of non- arthritic controls. These data suggest that extra-skeletal sources may contribute to the increased urinary excretion of the two crosslinks in joint disorders, as several observers have reported in recent years.2-4

We recently performed a similar study on samples of subchondral bone, cartilage, and synovium of six patients with OA (mean (SD) age 58.1 (10.7)) and six patients with RA (age 61.1 (18.1)) who underwent total hip arthroplasty. In contrast with the study by Takahashi, as a control group we studied joints from six young patients (aged 37.5 (13.5)) who underwent surgical amputation of an extremity for malignant osteosarcoma. All patients were physically active before surgery, permitting the sampling of normal ‘healthy’ joint tissues.

Briefly, tissue specimens of articular cartilage, subchondral bone, and synovium were collected, cleaned, dried, and immediately weighed and stored at −80°C. The assay of their crosslinks content was performed by high performance liquid chromatography according to a published method 5 after an overnight hydrolysis.

Data are expressed in nmol of crosslinks/gram of fresh tissue. Our data confirm the presence of both crosslinks in cartilage and in synovial samples from all the groups tested. The mean ratio Pyr/Dpyr was 30:1 in cartilage and 17:1 in synovium and was similar in the three groups. In both controls and in patients, cartilage was the tissue with the highest content of Pyr (table 1). Looking at the differences in tissue levels among groups (fig 1), we observed a sharp reduction of both crosslinks in both bone and in cartilage in patients with joint diseases compared with healthy controls. In contrast, mean levels of crosslinks found in synovium remained relatively constant. These results can be interpreted as a true ‘escape’ of crosslinks from subchondral bone and articular cartilage in the terminal phases of joint disorders and are in contrast with previous results by Takahashiet al 6 who performed a quantitative analysis of Pyr in articular cartilage of patients with different bone and joint disorders and concluded that cartilage Pyr content was not affected by articular diseases. This last study lacks a control group and a true comparison with healthy joints was not available. Moreover, changes in crosslinks tissue levels may be more representative of the true collagen content when data are expressed as absolute values (nmol/g of fresh tissue) rather than after normalisation for collagen moles. Our finding of a reduced Pyr content in articular tissues in joint disorders is in agreement with recently published experimental data demonstrating that a decrease in collagen crosslinks content is associated with a reduced bone strength.7 Finally, the lack of significant difference in synovial content of collagen crosslinks between controls and patients, which has been reported also by Takahashi et al 1 makes unlikely the contributory role of synovial membrane to urinary excretion of crosslinks in chronic joint diseases, which seems to be related to subchondral bone and articular cartilage increased turnover. This hypothesis is consistent with data from our previous study8demonstrating that crosslinks concentrations in synovial fluid are similar in two conditions with a highly different metabolic turnover of synovial membrane such as OA and RA.

Table 1

Mean (SEM) concentrations of collagen crosslinks (nmol/g of fresh tissue) in articular tissue of healthy controls and in patients with osteoarthritis (OA) and rheumatoid arthritis (RA)

Figure 1

Mean concentrations  (SEM) of PYR and DPYR in subchondral bone and cartilage in patients with osteoarthritis, rheumatoid arthritis, and in healthy controls.


Authors’ reply

As stated by Sinigaglia et al both pyridinoline (Pyr) and deoxypyridinoline (Dpry) have been recently found in more various tissues than previously expected.1-1 1-2 I would like to offer the following comments.

Firstly, the usefulness of a biochemical marker does not need to be related to change of the concentrations of materials for marker in tissues. For instance both of the above crosslinks in urine have been established as bone resorption markers.1-3 The urinary crosslinks considerably increase in metabolic bone diseases, however they do not increase in bone, but an increase in the bone resorption (bone turnover) leads to an increase of crosslinks in urine. For a biochemical marker reflecting tissue turnover, if the content of a marker material does not change in disease, change in urinary excretion of the marker reflects the net turnover of its distributed tissue. Therefore, the authors conclusion that no change in concentration of crosslinks in synovium does not contribute to urinary crosslinks excretion is not correct.

Secondly, Pyr and Dpyr are physiological crosslinks to maintain the structure of collagen fibril. Therefore, their reduction is expected to be responsible for the degeneration of collagen and also the fragility of extracellular matrix. Our paper did not concentrate on this issue, so does not give the solution because of the absence of normal control in the study for bone and cartilage. Our previous study showed that Pyr did not change in cartilage in OA and RA compared with that in osteoporosis where a significant degeneration of cartilage is not involved.1-4 There are two problems in methodology in the comments made by Sinigaglia et al. One is that the concentrations of crosslinks were expressed per gram of fresh weight. The authors maintain that crosslinks tissue levels per weight is more representative of the true collagen content, which however implies that content of collagen changes but not crosslinks in collagen. The other is that the control group is considerably younger than the OA and RA groups. I do not claim, however, that the crosslinks are constant among OA, RA, and normal groups. A reduction of crosslinks in cartilage in OA and RA seen by Sinigaglia et al may explain the degenerative change of cartilage in those diseases. However, the concentrations of Pyr and Dpyr in bone were considerably lower in OA and RA compared with the control group. In contrast, but in agreement with what I have previously mentioned, as Pyr and Dpyr are physiological crosslinks, the considerable change of these crosslinks induces diseases such as lathyrism. Therefore, the extreme reduction of crosslinks in bone is unlikely. The ideal way to solve this problem is to study the degenerated lesion and intact lesion of tissues mostly in cartilage in the same subjects, although I understand that it is difficult to critically distinguish those two lesions.


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