Concentrations of markers

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been higher than in grade 2. Therefore, it is considered meaningful that sRANKL concentration in synovial fluid increased during the beginning of OA development until grade 2 was reached, and then decreased.

If these results reveal the initiating tissue of OA needs to be discussed. As RANKL is produced in subchondral bone as well as in synovial membrane, both tissues are a possibility. This could mean that either OA is initiated by subchondral bone changes or by synovial inflammation. Further investigations of sRANKL in bone tissue and synovial membrane during OA development are needed to be done.

The significant difference of sRANKL concentrations in synovial fluid of healthy (OA grade 0) and diseased (grade 1–3) dogs (Figure 3) needs to be evaluated critically because of the findings in the different stages of OA. The significant increase of sRANKL in diseased dogs gives the impression that sRANKL must be significantly higher in grade 1, 2 and 3. But truly, concentrations in grade 0 and grade 3 were similar. Nevertheless, the difference between healthy and OA patients was significant (p < 0,01). In conclusion, sRANKL concentrations need to be evaluated separately for each OA grade. For this use, the radiographic scoring system was elaborated. A comparison between healthy and osteoarthritic joints is misleading and should not be used.

There was no significant difference between synovial fluid sRANKL concentrations in different degrees of lameness (Figure 5). Nevertheless, higher medians have been found in dogs with lameness degree 1 and 2. This makes sense if the following theories are connected: sRANKL concentrations increase with the progression of OA (besides grade 3, see above) and with the duration of lameness (see below). The degree of lameness is worst right after injury and becomes better with time due to stabilization and habituation (CICCOTTI et al. 1994, BOERBOOM et al. 2001; cf.

5.3), so it must proceed inversely proportional to the duration of lameness and the progression of OA. Therefore, sRANKL concentrations should proceed inversely proportional to the degree of lameness. But again, the last group (degree 4) is rather small compared to the others so that statistical evaluation is vague. Additionally, the decrease of lameness behavior during OA development and the duration of lameness were not significant (cf. Figure 60 and Figure 62). In prospective studies,

the measurements should be repeated with more samples in each group to see if the differences become significant.

The duration of lameness showed no significant influence on the sRANKL concentrations in synovial fluid (Figure 6). It was expected to proceed similarly to the course of concentrations in different stages of OA. But results reveal a more bell-shaped curve with highest concentrations in dogs which have been lame for 2 to 8 weeks. This could mean that during this time span, local influence of RANKL on the progression of osteoarthritis is strongest.

The sex, age and weight showed no significant correlation to sRANKL concentrations in synovial fluid (Figure 7, Figure 8 and Figure 9). For the sex, a significant influence was not expected, and these expectations were fulfilled. The age was believed to contribute to marker concentrations as older dogs or humans suffer more from OA than younger patients. But as patients in this study presented for surgery of the stifle joint, the study population was not equal to a natural population of dogs with systemic OA. Thus, most patients were of middle age. As higher weight is believed to be a risk factor for OA in humans (MELE 2007), concentrations of RANKL were expected to increase with higher body weight. This theory was not confirmed; on the contrary, the correlation tended to be negative, although not significant. These findings concur with the distribution of weight in all study patients and its connection to osteoarthritis development: no connection of higher weight and higher grades of OA could be found as well (cf. 4.6.2).

In serum, sRANKL displayed no significant differences either between the radiographic grades of OA or healthy and osteoarthritis, the degree of lameness, the duration of lameness, the sex, the age or the weight (Figure 30–Figure 35). Not even clear tendencies could be seen. These findings are contradicting to the findings of PILICHOU et al. (2008), who found elevated RANKL concentrations in patients with primary OA. It illustrates that sRANKL concentrations in serum are not meaningful for osteoarthritis development in a single joint. This makes sense as study patients did not suffer from generalized OA (as far as it was known) but from local OA caused by

110 5.1. Concentrations of markers

RCCL or patellar dislocation. This also confirms the theory that sRANKL is produced locally in the joint, presumably by synovial fibroblasts (TAKAYANAGI et al. 2000).

The comparison of sRANKL concentrations in serum and synovial fluid showed a positive correlation (Spearman Rank Order correlation: r = 0,42, Figure 54), but serum concentrations were significantly higher (Figure 53). This demonstrates that despite of the local sRANKL expression in the joint, systemic and local expression are in some way depending. Either local and systemic expression are stimulated by the same factors or diffusion between synovial membrane or bone and blood vessels takes place. The latter theory seems more likely as synovial membrane and subchondral bone are highly vascularized, especially during inflammation and OA progression (GLYN-JONES et al. 2015). As the serum contains sRANKL which possibly originates from all over the body, e.g. from lymph nodes, thymus and bone (WONG et al. 1997, WANG et al. 2005), its concentration depends on far more unknown processes than the local one. Reactions that are independent of OA progression could be involved as well. This might explain why serum concentrations were significantly higher but showed no correlation to osteoarthritis development, whereas synovial concentrations did. Therefore, sRANKL concentration in synovial fluid seems to be more specific for local joint reactions like OA.

5.1.2. Chordin

Chordin is an important regulator of chondrocyte maturation and limb growth (ZHANG et al. 2002) and as an antagonist of bone morphogenetic proteins like BMP-2, it is also involved in fracture healing and was found to increase during bone remodeling (KLOEN et al. 2012, DEAN et al. 2010). It was also found in areas of cartilage formation (KWONG et al.2009a). Because of its different tasks and as no investigations in synovial fluid of chordin were known, there were arguments for either increase or decrease of chordin. Decrease was expected during osteophyte growth because of elevated functions for its antagonistic BMPs during this process,

increase was expected during bone remodeling (DEAN et al. 2010). This way, analysis of chordin levels was anticipated to contribute to the knowledge of triggers and biochemical reactions during osteoarthritis development.

The measurements of chordin in synovial fluid showed no significant differences between concentrations in healthy and osteoarthritic joints, although concentrations in OA dogs tended to be higher (Figure 11).

There was no significant difference between radiographic OA grades 0 to 3 as well, although the progress was reminiscent of the sRANKL variations in synovial fluid (increase from grade 0 to grade 2). But as most samples had no detectable chordin, all medians were at 0 ng/ml (Figure 12). This demonstrates that either chordin is rarely present in synovial fluid or the concentrations were lower than the detectable range of the ELISA. Trial should be repeated with tests of a smaller detection limit to see if it can be of diagnostic relevance. For this study, no ELISA with a smaller detection limit was available.

Neither the degree of lameness nor the duration of lameness, nor the sex, nor the age had a significant influence on chordin concentrations in synovial fluid (Figure 13–Figure 16). Nevertheless, concentrations seemed to run inversely proportional to the degree of lameness. This fits the theory that concentrations rise with time after injury, the progression of OA and the decrease of lameness intensity (cf. 5.1.1). But as individuals have varying lameness behavior based on the individual intensity of trauma, the involvement of the medial meniscus and the pain tolerance, the results are not as clear as hoped. The duration of lameness again showed highest concentrations during week 2–8, but as this group is the largest this could be coincidental.

Chordin concentrations in synovial fluid showed a significant negative correlation to the weight of the dogs (Spearman rank correlation: r = -0,31, Figure 17). This was unexpected as higher weight was thought to contribute to OA progression (cf.

discussion in 5.1.). But as chordin concentrations did not reveal a correlation to OA grades, either, this theory was not confirmed. Why smaller dogs tended to have higher concentrations of chordin in synovial fluid remains unknown. Possibly it is due

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to higher metabolism in smaller animals (VON ENGELHARDT 2010, p. 1). But as highest concentrations were found in dogs of middle weight and as these make up the biggest group, the tendency could be coincidental.

In summary, synovial chordin concentration does not seem to be of diagnostic or prognostic relevance for OA progression.

In serum, chordin concentrations have not been influenced significantly by the radiographic grade of OA, the degree of lameness, the duration of lameness, the weight, age or sex (Figure 38–Figure 43). Altogether, chordin concentrations in serum cannot be correlated to OA progression as well. Therefore, it is not a useful biochemical marker for OA, at least in serum and synovial fluid.

The comparison of chordin concentrations in serum and synovial fluid revealed significantly higher concentrations in serum (p < 0,01, Figure 55). This confirms the finding that chordin is barely produced in synovial fluid. In prospective studies, it should be analyzed in tissues like cartilage or synovial membrane.

Nevertheless, concentrations in synovial fluid and serum are correlated positively (r = 0,44, Figure 56), so systemic and local concentrations seem to be connected (cf. 5.1.1).

5.1.3. Osteocalcin

Osteocalcin takes part in bone mineralization and bone resorption (HAUSCHKA and CARR 1982; GLOWACKI and LIAN 1987) and is thought to rise especially during processes of bone formation and remodeling. During fracture repair, an increase of OC concentration can be seen (TANIGUCHI et al. 2003; ÅKESSON et al. 1995). It is also associated with accelerated fracture healing (ROZEN et al. 2007), higher bone mineral content and higher bone mineral density (NAKAJIMA et al. 2002). Therefore, it was expected to rise during the progression of OA if it is responsible for osteophytic growth and subchondral bone remodeling. But during endochondral ossification,

when the mineralization status of subchondral bone becomes less, OC concentrations could also decrease.

In synovial fluid, OC concentrations were significantly higher in OA patients than in healthy dogs (p < 0,01, Figure 19). Furthermore, concentrations increased significantly from grade 0 to grade 2 (p < 0,05). Although not significant, they were even higher in grade 3 stifles (Figure 20). These results show that osteocalcin must be locally involved in osteoarthritis development of a stifle. As other studies pointed out that OC is involved in bone formation rather than in bone mineralization (LUMACHI et al. 2009; DUCY et al. 1996), it seems likely that OC increases in synovial fluid during OA progression because it triggers the degrading processes in subchondral bone. If these results imply that subchondral bone is the initiating tissue of OA needs to be discussed. As synovial osteocalcin increases during the development of OA, it could also mean that it takes its contribution to OA after the disease progression already has been started. Further investigations of OC in subchondral are needed to evaluate its role during the beginning of OA.

There was no significant influence on OC concentrations by the degree of lameness, the sex, the weight or age (Figure 21 to Figure 22, Figure 24 to Figure 25). Although not significant, the duration of lameness showed a distinct influence on OC concentration in synovial fluid (Figure 23). The more time had passed after trauma of the stifle, the higher concentrations tended to be. This coincides with the significant increase of OC during OA development: the passing of time after injury contributes to osteoarthritic processes and therefore OC increases. Nevertheless, these experiments should be repeated with a larger number of samples to see if this increase becomes significant.

In serum, OC concentrations had no significant relation to the radiographic grade of OA, the degree of lameness, the duration of lameness, the weight, the sex or the age (Figure 46–Figure 51). This elucidates the importance of local analysis of biochemical markers like OC to evaluate OA development in one joint because the systemic concentrations in serum are not diagnostic.

114 5.1. Concentrations of markers

The comparison of OC concentrations in serum and synovial fluid showed no significant difference in both body fluids, also no correlation of both fluids could be seen (Figure 57, Figure 58). This leads to the conclusion that osteocalcin expression in synovial fluid might be independent of systemic processes. Therefore, it is a biochemical marker of high interest for OA diagnosis and research.

5.1.4. Purebreds and hybrids

Interestingly, all markers revealed significantly higher concentrations in synovial fluid and serum of hybrids than of purebred dogs (except OC in serum: Figure 10, Figure 18, Figure 26, Figure 36, Figure 44 and Figure 52). Nevertheless, both groups did not show any significant differences considering the age, the weight, the duration of lameness or OA grades that could explain these results (Figure 72). As far as it is known, this is the first evidence of higher concentrations of sRANKL, chordin and osteocalcin in hybrids. Following facts need to be considered for this evaluation: all dogs which were presented at the clinic and which were not purely of one breed were classified as hybrid. Naturally, this included dogs which have been crossed from a purebred parent and one other breed. But these dogs were not mixed of as many breeds as other hybrids. Therefore, the group of hybrids was very widespread. Also, the group purebred consisted of very small breeds < 10 kg up to large dogs weighing more than 40 kg. Different breeds also have very different genetic backgrounds.

Altogether, many characteristics need to be taken into consideration which could explain the differences. For now, no logical explanation for these results can be found and further experiments with samples from different breeds should be done.

The radiographic scoring system for canine stifle osteoarthritis was developed for this study. Therefore, it needed to be compared to another evaluation technique to proof its reliability. The comparison of the radiographic results to the findings during surgery, meaning the intraarticular sight after opening the joint, revealed a significant positive correlation (r = 0,73, Figure 59). Interestingly, the radiographic evaluation tended to detect higher grades of osteoarthritis than the sight into the joint. This might be due to the fact that during surgery only a limited point of view is given so that the femoral condyles and the tibial plateau can only be examined on their front.

The medial and lateral sight are limited and the caudal sight is not given. Although structures like synovial membrane, cartilage and ligaments can be assessed far more detailed during surgery, it is mostly the osteophytic building that constitutes the osteoarthritis ranking on radiographs and therefore the mediolateral sight of femoral condyles on radiographs seems to be more sensitive. Altogether, the radiographic OA scoring system for dog stifles is useful for veterinary medicine and partly even more detailed than the evaluation during stifle surgery.

Evaluation of the relationship between the duration of lameness and the radiographic grade of osteoarthritis showed a significant positive correlation (Spearman rank order correlation, r = 0,55, Figure 61), which was expected as osteoarthritis is a disease that progresses with time after injury. The degree of lameness had no significant correlation to the radiographic grade of OA, although correlation tended to be negative (Figure 60). Similar results were found for the correlation of lameness degree and the duration of lameness (Figure 62). It was expected to be more distinct as the lameness is believed to be strongest right after injury when instability is worst and inflammation contributes to the pain. With time, inflammation usually decreases and the dog gets used to the instability. Also muscles like M. quadriceps femoris and