4. Results
4.2. Analysis of serum
Figure 28: Concentration of sRANKL, chordin and osteocalcin in synovial fluid (SF) compared in deceased dogs and stifle surgery patients separated into different grades of osteoarthritis.
4.2.1. sRANKL
To investigate serum concentrations of canine sRANKL in dogs, 59 blood samples of patients who came for stifle surgery have been analyzed. According to the radiographic scoring system, 7 stifles were free of OA, 29 were defined as grade 1,
4.2. Analysis of serum
18 patients had OA grade 2 in their stifle and 5 patients had a grade 3 stifle. Only in 26 of these 59 samples sRANKL could be detected, so in 33 samples the concentration was noted as 0 pg/ml. Detectable concentrations ranged from 11,45 pg/ml to 768,3 pg/ml. Comparison of grade 0 dogs and OA dogs showed no significant difference (Mann-Whitney U: p = 0,94, Figure 29).
No significant differences of concentrations were found between osteoarthritic grades, as well (according to Kruskal-Wallis test p = 0,27), although highest concentrations and the highest median were seen in beginning OA (grade 1, Figure 30).
Median 25%-75%
Non-Outlier Range Outliers Extremes
Healthy Osteoarthritis
-100 0 100 200 300 400 500 600 700 800
Se sRANKL (pg/ml)
Figure 29: Concentration of canine sRANKL in serum (Se) of healthy and diseased dogs categorized by the radiographic scoring system.
72 4.2. Analysis of serum
Median 25%-75%
Non-Outlier Range Outliers Extremes
0 1 2 3
Radiographic grade of OA -100
0 100 200 300 400 500 600 700 800
Se sRANKL (pg/ml)
Figure 30: Serum concentration (Se) of canine sRANKL in dogs with different stages of osteoarthritis categorized by the radiographic scoring system.
The degree of lameness, the duration of lameness and the sex did not have any significant influences on the concentrations of sRANKL in serum (Kruskal-Wallis test:
p = 0,59, p = 0,47 and p = 0,3, Figure 31–Figure 33).
There was no correlation between the concentrations and the age or the weight of the patients (Figure 34 and Figure 35).
Median
Figure 31: Concentration of sRANKL in serum (Se) of dogs with different degrees of lameness (according to ARNOCZY and TARVIN 1981).
Median
Figure 32: Concentration of sRANKL in serum (Se) of dogs with different durations of lameness (wks = weeks, mths = months).
74 4.2. Analysis of serum mn = male neutered, f = female, fn = female neutered).
0 2 4 6 8 10 12 14
Figure 34: Correlation analysis of sRANKL concentration in serum (Se) of dogs and their age in years.
0 5 10 15 20 25 30 35 40 45 50 55 Weight
-100 0 100 200 300 400 500 600 700 800
Se sRANKL (pg/ml)
Figure 35: Correlation analysis of sRANKL concentration in serum (Se) of dogs and their weight in kg.
The group of patients whose blood was analyzed for sRANKL consisted of 20 hybrids and 39 purebreds which in turn contained 29 different breeds. Comparing concentrations in purebreds and hybrids, hybrids had significantly higher concentrations of sRANKL in their serum than purebreds (Mann-Whitney U:
p = 0,044, Figure 36).
76 4.2. Analysis of serum
Median 25%-75%
Non-Outlier Range Outliers
Extremes
Purebred Hybrid
0 200 400 600 800 1000
Se sRANKL (pg/ml)
*
Figure 36: Concentration of sRANKL in serum (Se) of purebreds and hybrids.
*
= p < 0,054.2.2. Chordin
In order to measure serum concentrations of canine chordin, 55 blood samples of stifle surgery patients were analyzed. The radiographic OA scoring defined 7 patient stifles as free of OA, 27 stifles as grade 1, 15 stifles as grade 2 and 6 as grade 3. In 6 of 55 samples, no chordin could be detected. The remaining concentrations ranged from 0,19 ng/ml to 3,94 ng/ml. When comparing healthy (grade 0) to diseased dogs (grade 1–3), serum concentrations of canine chordin did not differ significantly (Mann-Whitney U test: p = 0,28). Nonetheless, it seemed that chordin concentrations tended to be higher in diseased dogs than in healthy dogs (Figure 37).
Generally, the concentrations tended to be higher in patients with beginning osteoarthritis (grade 1 and grade 2 stifles), but it was not significant (Kruskal-Wallis test: p = 0,45, Figure 38).
Median 25%-75%
Non-Outlier Range Outliers Extremes
Healthy Osteoarthritis
-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5
Se chordin (ng/ml)
Figure 37: Concentration of canine chordin in serum (Se) of healthy and diseased dogs categorized by the radiographic scoring system.
78 4.2. Analysis of serum
Median 25%-75%
Non-Outlier Range Outliers Extremes
0 1 2 3
Radiographic grade of OA -0,5
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5
Se chordin (ng/ml)
Figure 38: Serum concentration (Se) of canine chordin in dogs with different stages of osteoarthritis categorized by the radiographic scoring system.
Neither the degree of lameness nor the duration of lameness nor the sex did have any significant influence on the concentration of chordin in serum (Kruskal-Wallis test: p = 0,85, p= 0,24 and p = 0,63, Figure 39–Figure 41). Furthermore, there was no correlation of the concentrations in serum with the age or weight of the patients (Figure 42 and Figure 43).
Median
Figure 39: Concentration of chordin in serum (Se) of dogs with different degrees of lameness (according to ARNOCZY and TARVIN 1981).
Median
Figure 40: Concentration of chordin in serum (Se) of dogs with different durations of lameness (wks = weeks, mths = months).
80 4.2. Analysis of serum mn = male neutered, f = female, fn = female neutered).
0 2 4 6 8 10 12 14
Figure 42: Correlation analysis of chordin concentration in serum (Se) of dogs and their age in years.
0 10 20 30 40 50 60 Weight
-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5
Se chordin (ng/ml)
Figure 43: Correlation analysis of chordin concentration in serum (Se) of dogs and their weight in kg.
Similar to the results in synovial fluid, in serum significantly higher concentrations of chordin were found in hybrids compared to purebred dogs (Mann-Whitney U test:
p = 0,028, Figure 44). Of all 55 dogs, there were 18 hybrids and 37 purebreds involving 28 different breeds.
82 4.2. Analysis of serum
Median 25%-75%
Non-Outlier Range Outliers
Purebred Hybrid
0 1 2 3 4 5
Se chordin (ng/ml)
*
Figure 44: Concentration of chordin in serum (Se) of purebreds and hybrids.
*
= p < 0,054.2.3. Osteocalcin
For osteocalcin, 54 blood samples of stifle surgery patients have been investigated.
In 23 of them, osteocalcin was measured, in 31 samples no OC could be detected.
Radiographic analysis of the operated stifles evaluated 7 of them as free of OA, 26 as grade 1, 16 as grade 2 and 5 stifles as OA grade 3.
The comparison of non-osteoarthritic patients (grade 0) to OA patients (grade 1–3) did not show any significant differences (Mann-Whitney U test: p = 0,69, Figure 45).
The highest serum concentration that could be detected was at 5,81 ng/ml (OA grade 2). No significant differences of concentrations were seen between the 4 grades of osteoarthritis (Kruskal-Wallis: p = 0,93, Figure 46).
Median 25%-75%
Non-Outlier Range
Healthy Osteoarthritis
-1 0 1 2 3 4 5 6 7
Se osteocalcin (ng/ml)
Figure 45: Concentration of canine osteocalcin in serum (Se) of healthy and diseased dogs categorized by the radiographic scoring system.
84 4.2. Analysis of serum
Median 25%-75%
Non-Outlier Range
0 1 2 3
Radiographic grade of OA -1
0 1 2 3 4 5 6 7
Se osteocalcin (ng/ml)
Figure 46: Serum concentration (Se) of canine osteocalcin in dogs with different stages of osteoarthritis categorized by the radiographic scoring system.
Neither the degree of lameness nor the duration of lameness nor the sex had any significant influences on the concentration of osteocalcin in serum (according to Kruskal-Wallis test: p = 0,38, p = 0,40 and p = 0,89, Figure 47 to Figure 49).
Furthermore, no significant correlations between the concentrations in serum and the age or the weight were seen (Figure 50 and Figure 51), although the age seemed to have a positive influence.
Median lameness (according to ARNOCZY and TARVIN 1981).
Median lameness (wks = weeks, mths = months).
86 4.2. Analysis of serum
Figure 49: Concentration of osteocalcin in serum (Se) of dogs according to their sex (m = male, mn = male neutered, f = female, fn = female neutered).
0 2 4 6 8 10 12 14
0 5 10 15 20 25 30 35 40 45 50 55 Weight
-1 0 1 2 3 4 5 6 7
Se osteocalcin (ng/ml)
Figure 51: Correlation analysis of osteocalcin concentration in serum (Se) of dogs and their weight in kg.
88 4.3. Comparative analysis of synovial and serum values
The group of 54 dogs involved 16 hybrids and 38 purebreds (consisting of 28 different breeds). No significant differences could be seen between the osteocalcin concentrations in serum of hybrids and purebreds (Mann-Whitney U: p = 0,96, Figure 52).
Median 25%-75%
Non-Outlier Range Outliers
Purebred Hybrid
-1 0 1 2 3 4 5 6 7
Se osteocalcin (ng/ml)
Figure 52: Concentration of osteocalcin in serum (Se) of purebreds and hybrids.
4.3.1. sRANKL
Of some patients, blood could be stored but no synovial fluid was collectable because of too little volume (“dry joint”). On the other hand, from deceased dogs no blood could be taken. Therefore, it was not possible to collect both body fluids from every dog. For this reason, concentrations in synovial fluid and serum were looked
4.3. Comparative analysis of synovial and serum values
upon separately at first. Altogether, it was possible to investigate sRANKL in both body fluids of 34 patients so that concentrations could be compared directly.
Results showed significantly higher concentrations of sRANKL in serum than in synovial fluid (Wilcoxon Matched Pairs Test: p = 0,007, Figure 53). Nevertheless, a significant positive correlation was found between concentrations in serum and synovial fluid (Spearman Rank Order Correlation: r = 0,42, Figure 54).
Median 25%-75%
Non-Outlier Range Outliers
Extremes
Serum Synovial fluid
0 200 400 600 800
sRANKL (pg/ml)
**
Figure 53: Concentrations of sRANKL in serum and synovial fluid.
**
= p < 0,0190 4.3. Comparative analysis of synovial and serum values
-100 0 100 200 300 400 500 600 700 800
Se sRANKL (pg/ml) -20
0 20 40 60 80 100 120 140 160
SF sRANKL (pg/ml)
r=0,42
Figure 54: Correlation analysis of sRANKL concentrations in serum (Se) and synovial fluid (SF) (Spearman Rank Order Correlation Coefficient: r = 0,42).
4.3.2. Chordin
It was possible to analyze comparative chordin concentrations in serum and synovial fluid of 29 patients (for limitations see 4.3.1).
Measured concentrations in serum were significantly higher (Wilcoxon Matched Pairs test: p = 0,000005, Figure 55). Nevertheless, a significant positive correlation could be detected between elevations of concentrations in serum and synovial fluid (Spearman Rank Order Correlation: r = 0,436, Figure 56).
Median 25%-75%
Non-Outlier Range Outliers
Extremes
Serum Synovial fluid
0 1 2 3 4 5
Chordin (ng/ml)
**
Figure 55: Concentrations of chordin in serum and synovial fluid.
**
= p < 0,0192 4.3. Comparative analysis of synovial and serum values
-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5
Se chordin (ng/ml) -0,2
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0
SF chordin (ng/ml)
r=0,44
Figure 56: Correlation analysis of chordin concentrations in serum (Se) and synovial fluid (SF) (Spearman Rank Order Correlation Coefficient: r = 0,44).
4.3.3. Osteocalcin
For osteocalcin, it was possible to investigate comparative samples of serum and synovial fluid from 28 dogs. The juxtaposition of concentrations in serum and synovial fluid showed no significant differences, although the median is clearly higher in synovial fluid (= 1,24 ng/ml, median in serum = 0 ng/ml. Wilcoxon Matched Pairs Test: p = 0,54, Figure 57). Furthermore, no significant correlation could be seen between sRANKL concentrations in both body fluids (Figure 58).
Median
Figure 57: Concentrations of osteocalcin in serum and synovial fluid.
-1 0 1 2 3 4 5 6 7
Figure 58: Correlation analysis of osteocalcin concentrations in serum (Se) and synovial fluid (SF).
94 4.4. Comparing evaluation techniques of OA staging:
radiographic evaluation and intra-articular evaluation
The focus of osteoarthritis staging was the evaluation by studying radiographs. In order to see how this evaluation can be equalized to the three-dimensional sight of an opened joint during surgery, outcomes of each ranking were compared. The outcomes of those two evaluation techniques were similar, although not perfectly coinciding (significant positive correlation; Spearman’s correlation coefficient:
r = 0,74, Figure 59).
Grade of OA from intra-articular sight
r = 0,74
Figure 59: Comparison of osteoarthritis evaluation by radiographs vs. intra-articular sight during surgery (Spearman’s correlation coefficient: r = 0,74).