Sample collection and preparation

42 3.7. Quantification of sRANKL, osteocalcin and chordin by enzyme linked immunosorbent assay (ELISA)

repeated at 1500 x g for 6–10 minutes. The supernatant synovial fluid was aspirated, aliquoted as 300 µl and frozen at -80 °C.

3.6.2. Patients in surgery

All dogs of which samples were taken presented for stifle surgery. As usual blood was taken for a preanesthetic blood tests so that serum could be obtained for these investigations, too. Serum was prepared for the ELISA following instructions of BlueGene: the blood was kept at room temperature for 2 hours to clot. Then it was centrifuged at 1000 x g for 15 minutes. Serum was aspirated, aliquoted and frozen at -80 °C. Radiographs of the injured stifle were made to evaluate the grade of OA.

In surgery, the synovial fluid was aspirated by opening the joint. The bones, cartilage and synovium were examined to evaluate the degree of osteoarthritis. The synovial fluid was centrifuged at 1000 x g for 15 minutes to remove debris and blood. If the blood had not been slumped entirely the centrifugation was repeated at 1500 x g for 6–10 minutes. The clarified synovial fluid was aspirated, aliquoted and frozen at -80 °C.

3.7.1. Assay procedure

Commercially available quantitative competitive ELISA kits were used. All three kinds of ELISA to determine canine osteocalcin, canine sRANKL and canine chordin had been manufactured by BlueGene Biotech (Shanghai, China) and were done in the same procedure: after bringing all reagents to room temperature, 100 µl of standard or sample were added to each well (coated with polyclonal anti-sRANKL/

-osteocalcin/-chordin antibody), for blank control 100 µl PBS were used. Then 10 µl 3.7. Quantification of sRANKL, osteocalcin and chordin

by enzyme linked immunosorbent assay (ELISA)

of balance solution were dispensed into synovial fluid (not into serum) to buffer the reagents. Into samples and standards 50 µl of enzyme conjugate were added and they were mixed well. Enzyme conjugate solution contains a specific antigen-horseradish peroxidase (HRP) conjugate (meaning osteocalcin-, chordin- or sRANKL-specific) which can bind to the antibody binding site. This way it competes with sample antigen. The plate was then covered, incubated for 1 hour at 37 °C and was washed 5 times manually with a provided wash solution after incubation. 50 µl of substrate A and substrate B were added to each well and the plate was incubated for another 15 minutes. The substrates contain 3,3’,5,5’-tetramethylbenzidine (TMB) and citric acid. The bound HRP catalyzes the oxidation of TMB by hydrogen peroxide into a blue colored product. The more antigen samples contain, the less enzyme conjugate can bind to the antibody binding site and therefore less chromogenic turnover of the substrate can take place. Finally, 50 µl of stop solution containing sulfuric acid were added to each well, affectively stopping the enzymatic reaction and changing the color of TMB to yellow. The optical absorption of the product at 450 nm (with a wavelength correction of 570 nm) was then measured spectrophotometrically in a microplate reader (TECAN Group Ltd., Männedorf, Switzerland).

3.7.2. Calculation of sample concentrations

The standard series solutions contained predetermined concentrations of canine osteocalcin (0–25 ng/ml), canine chordin (0–10 ng/ml) and canine sRANKL (0–1000 pg/ml). Standards and samples were measured in duplicate (except when sample volume did not suffice for more than one well). Mean values of standards and samples were calculated and the average blank control value was subtracted from all values. Standard curves were generated by plotting the given concentrations of each standard against the measured absorption. For a possibly fitting curve a four-parameter logistic regression curve was generated using a commercial software (Curve Expert Professional, version 2.6 by Daniel G. Hyams). Because competitive ELISAs have been used, absorption and concentration presented inversely

44 3.8. Statistical Analysis

proportional. By means of the standard curve, antigen concentrations in samples could be calculated. Values that were out of range of the standard curve (higher absorption than the one of standard A) could not be calculated and were regarded as a concentration of 0 ng/ml or 0 pg/ml, respectively.

According to the manufacturers information, all kits showed intra- and interassay coefficients of variation (CV) of less than 10%. The sensitivity in chordin and osteocalcin assays was 0,1 ng/ml, in sRANKL assay it was 1,0 pg/ml. No significant cross-reactivity or interference between osteocalcin/sRANKL/chordin and analogues had been observed (for all analogues tested so far).

Statistical calculations were done using commercial software (SAS Enterprise Guide, version 7.1 by SAS Institute Inc., Cary, NC, USA and Statistica, version 13.3 by TIBCO Software Inc., Palo Alto, CA, USA). The data showed no normal distribution according to Shapiro-Wilk and Anderson-Darling tests. Consequently, for comparing two or more parameters like sample concentration and the grade of osteoarthritis (independent samples), Wilcoxon-two-sample-test (Mann-Whitney U test) or Kruskal-Wallis test were used. Wilcoxon matched pairs test was used to compare concentrations in synovial fluid and serum (dependent samples). For correlation analysis, Spearman’s correlation coefficient (r) was calculated together with its associated p-value. If the r-value was not significant (according to its p-value), it was not displayed.

P-values lower than 0,05 were defined as statistically significant, values below 0,01 were labeled separately.

For the graphic representation of data histograms, scatterplots, frequency plots, pie charts and box plots were used. In the latter, the coefficient for calculation of the non-outlier ranges, non-outliers and extremes was 1,5.

3.8. Statistical Analysis

3.9.1. Equipment

• Centrifuge: Eppendorf Centrifuge 5424, rotor: FA-45-24-11 (Eppendorf AG, Hamburg, Germany)

• Centrifuge: Hettich EBA 20, rotor: E1624 (Hettich, Tuttlingen, Germany)

• Vortex: Vortex-Genie 2 (Scientific Industries, Inc. Bohemia, New York, USA)

• Washer for microtiter plates: Nunc-Immuno™ Wash 8 (NUNC A/S, Denmark)

• Microplate reader: TECAN GENIOS Pro-Basic W/O FP (TECAN Austria GmbH, Groedig, Austria)

• Repeating pipette: Eppendorf Repeater 4780 (10 μl–5 ml; Eppendorf AG, Hamburg, Germany)

• Pipettes:

• Pipetman P200 (Gilson, Villers Le Bel, France)

• DISCOVERY comfort Variable Volume Single Channel Pipette, 100–1000 µl (HTL, Warsaw, Poland)

• Multichannel Pipettes:

• DISCOVERY comfort Variable Volume Multichannel Pipette, 20–200 µl (HTL, Warsaw, Poland)

• Eppendorf Research® plus, 8-channel, 0,5–10 µl (Eppendorf AG, Hamburg, Germany)

• X-ray unit: Siemens Opti 150/30/50 HC-100 (Siemens AG, Munich, Germany)

• X-ray film developer: Optimax x-ray film processor, Mod. 1170-1-0000 (PROTEC Medizintechnik GmbH & Co. KG, Oberstenfeld, Germany)

3.9. Equipment and consumables

46 3.9. Equipment and consumables

3.9.2. Consumables

• X-ray films: FUJIFILM Super HR-E Medical x-ray film, 18x24/24x30/30x40 cm (FUJIFILM Corporation, Tokyo, Japan)

• Serum tubes: SARSTEDT serum tubes 1,3 ml & 4,5 ml (SARSTEDT AG & Co, Nürnbrecht, Germany)

• Single Use Syringe 10 ml/12 ml (Henry Schein Services GmbH, Langen, Germany)

• Sterican® Hypodermic-needle, 0,9x40 mm / 20 G x 1 ½ ‘’ (B. Braun Melsungen AG, Melsungen, Germany)

3.9.3. Solutions and reagents

• PBS (phosphate-buffered saline), pH 7,1

• Distilled water (Milli-Q^® Biocel (Millipore SAS, Molsheim, FRANCE))

Solutions and reagents contained in canine osteocalcin / chordin / sRANKL-ELISA kits (BlueGene Biotech, Shanghai, China):

• microtiter plate precoated with polyclonal anti-sRANKL-/chordin-/OC-antibody

• Enzyme conjugate: conjugate of sRANKL/chordin/osteocalcin and horseradish peroxidase (HRP)

• Standard solutions A-F: recombinant sRANKL/chordin/osteocalcin in buffer solution containing PBS, glycerol and ProClin™ 300 (a commercial biocide).

(Given concentrations: sRANKL: A = 0 pg/ml, B = 50 pg/ml, C = 100 pg/ml, D = 250 pg/ml, E = 500 pg/ml, F = 1000 pg/ml.

Chordin: A = 0 ng/ml, B = 0,5 ng/ml, C = 1 ng/ml, D = 2,5 ng/ml, E = 5 ng/ml, F = 10 ng/ml.

Osteocalcin: A = 0 ng/ml, B = 1 ng/ml, C = 2,5 ng/ml, D = 5 ng/ml, E = 10 ng/ml, F = 25 ng/ml.)

• Stop solution: containing 1M H2SO4 (sulfuric acid)

• Wash solution: containing PBS and Tween^® 20, an emulsifying agent

• Substrate A: containing citric acid

• Substrate B: containing TMB as chromogen

• Balance solution: containing SDS (sodium lauryl sulfate) and NP-40 (Nonoxynol-40), both anionic surfactants.

48 4.1. Analysis of synovial fluid

4. Results

4.1.1. sRANKL

For the detection of canine sRANKL, synovial fluid of 44 dogs was analyzed. 40 dogs were stifle surgery patients, 4 were deceased. The radiographic OA scoring accounted for 9 dogs with OA grade 0, 21 dogs with OA grade 1, 10 dogs with grade 2 and 4 dogs with grade 3. In these 44 samples, canine sRANKL could be detected in half of them, the rest were regarded as a concentration of 0 pg/ml. Detected concentrations ranged from 2,87 pg/ml to 152,7 pg/ml. If all dogs with visible osteoarthritic changes (radiographic grade 1–3 or the illness group) are compared to OA grade 0 (the healthy group), a significant increase of sRANKL in synovial fluid can be seen once OA progression has started (Mann-Whitney U-test: p = 0,004, Figure 3).

Except for one extreme value of 4,58 pg/ml, every sample of healthy stifles was without detectable sRANKL, and in no sample of OA grade 3 sRANKL could be found as well. In OA grade 1, higher concentrations with a median of 6,13 pg/ml could be seen and dogs with OA of grade 2 had highest concentrations in their synovial fluid with a median of 14,66 pg/ml. These concentrations distinguished significantly (Kruskal-Wallis test between all grades: p = 0,003). There also was a significant increase from grade 0 to grade 2 (p = 0,02, Figure 4).

4.1. Analysis of synovial fluid

Median 25%-75%

Non-Outlier Range Outliers

Extremes

Healthy Osteoarthritis

-20 0 20 40 60 80 100 120 140 160 180

SF sRANKL (pg/ml)

**

Figure 3: Concentration of canine sRANKL in synovial fluid (SF) of healthy and osteoarthritic dog stifles categorized by the radiographic scoring system.

**

= p < 0,01

50 4.1. Analysis of synovial fluid stages of osteoarthritis categorized by the radiographic scoring system.

*

= p < 0,05

Degree of lameness, duration of lameness and sex had no significant influence on the concentration of sRANKL in synovial fluid (Kruskal-Wallis test: p = 0,46, Figure 5, p = 0,41, Figure 6 and p = 0,70, Figure 7). Nevertheless, concentrations seemed to be higher in lower degrees of lameness. According to Spearman rank order correlation, there was no significant correlation between concentrations in synovial fluid and the age or the weight of the dogs, although weight tended to have a negative influence on concentration while age and concentration tended to be correlated positively (Figure 8, Figure 9).

Median lameness (according to ARNOCZY and TARVIN 1981).

Median lameness (wks = weeks, mths = months).

52 4.1. Analysis of synovial fluid

Figure 8: Correlation analysis of sRANKL concentration in synovial fluid (SF) of dogs and their age in years.

0 10 20 30 40 50 60 Weight

-20 0 20 40 60 80 100 120 140 160

SF sRANKL (pg/ml)

Figure 9: Correlation analysis of sRANKL concentration in synovial fluid (SF) of dogs and their weight in kg.

The group of 44 dogs for the analysis of sRANKL in synovial fluid consisted of 11 hybrids and 33 purebred dogs, involving 23 different breeds. Comparing sRANKL concentrations in synovial fluid of purebreds and hybrids, significantly higher concentrations could be found in the latter group (Mann-Whitney U-test: p = 0,004, Figure 10).

54 4.1. Analysis of synovial fluid

Median 25%-75%

Non-Outlier Range Outliers

Extremes

Purebred Hybrid

-20 0 20 40 60 80 100 120 140 160 180

SF sRANKL (pg/ml)

**

Figure 10: Concentration of sRANKL in synovial fluid (SF) of purebreds and hybrids.

**

= p < 0,01

4.1.2. Chordin

To measure canine chordin concentrations in synovial fluid, 47 samples were analyzed. 6 of them were from deceased dogs, 41 from surgery patients. The radiographic OA scoring system classified 10 of the sampled stifles as free from OA, 21 as grade 1, 12 as grade 2 and 4 as grade 3. In 17 of all 47 samples, canine chordin could be detected, the others were noted as a concentration of 0 ng/ml.

Measured concentrations varied from < 0,1 ng/ml to 3,13 ng/ml. Comparing OA grade 1–3 as one osteoarthritis group to the healthy group (grade 0), the variation of concentrations seemed to be bigger in the OA group. Highest concentrations could be seen here. But as in both groups medians were still at 0 ng/ml, the difference was not significant (Mann-Whitney U: p = 0,21, Figure 11).

Except of two extreme values of which the highest concentration was 0,33 ng/ml, all samples from grade 0 stifles were without detectable chordin. In no sample of grade 3 chordin could be found as well. In grade 2, the highest concentration of 3,13 ng/ml could be measured and in grade 1 extremes were seen up to 1,88 ng/ml, but still the median of both groups was at 0 ng/ml. According to Kruskal-Wallis test, the concentrations did not vary significantly (p = 0,20, Figure 12).

56 4.1. Analysis of synovial fluid

Figure 11: Concentration of canine chordin in synovial fluid (SF) of healthy and osteoarthritic dog stifles categorized by the radiographic scoring system.

Median stages of osteoarthritis categorized by the radiographic scoring system.

Concentrations of chordin in synovial fluid did not differ significantly with regards to the degree of lameness and the sex (Kruskal-Wallis test: p = 0,19 and p = 0,30, Figure 13 and Figure 14), although highest concentrations seemed to be in lower degrees of lameness, again. The subdivision into the duration of lameness showed highest concentrations in patients who have been lame for 2 weeks until 2 months, but the difference was not significant either (p = 0,053, Figure 15).

Median 25%-75%

Non-Outlier Range Outliers Extremes

1 2 3 4

Degree of lameness -0,5

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

SF chordin (ng/ml)

Figure 13: Concentration of chordin in synovial fluid (SF) of dogs with different degrees of lameness (according to ARNOCZY and TARVIN 1981).

58 4.1. Analysis of synovial fluid lameness (wks = weeks, mths = months).

No significant correlation was found between the concentration of chordin in synovial fluid and the age (Figure 16), but there was a significant negative correlation between concentrations and the weight of dogs (Spearman Rank Order Correlation: r = -0,31, Figure 17).

-2 0 2 4 6 8 10 12 14 16

Age -0,5

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

SF chordin (ng/ml)

Figure 16: Correlation analysis of chordin concentration in synovial fluid (SF) of dogs and their age in years.

60 4.1. Analysis of synovial fluid

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

SF chordin (ng/ml)

r = -0,31

Figure 17: Correlation analysis of chordin concentration in synovial fluid (SF) of dogs and their weight in kg.

13 of all 48 dogs were hybrids and 35 dogs were purebred, consisting of 27 different breeds. Comparing chordin concentrations in synovial fluid of hybrids and purebred, significantly higher concentrations were found in hybrids (Mann-Whitney U test:

p = 0,006, Figure 18).

Median

Figure 18: Concentration of chordin in synovial fluid (SF) of purebreds and hybrids.

** = p < 0,01

4.1.3. Osteocalcin

Canine osteocalcin was measured in synovial fluid of 44 dogs. 6 of them were deceased, 38 were stifle surgery patients. The radiographic arthritis scoring system identified 9 of them as OA grade 0 or healthy, 18 of them had grade 1 OA, 13 had grade 2 and 4 of them had OA grade 3 in their stifle joint. Only in 2 of the 44 samples, no osteocalcin could be detected. Both had been classified as OA grade 0.

The measured osteocalcin concentrations ranged from 0,25 ng/ml to 4,06 ng/ml.

Comparing OA grade 1–3 as one group of OA dogs to the healthy group (OA grade 0), a significant elevation of osteocalcin in synovial fluid of diseased dogs was found (Mann-Whitney U test: p = 0,004, Figure 19).

62 4.1. Analysis of synovial fluid

Statistical analysis also showed a significant difference of concentrations between all osteoarthritis grades (according to Kruskal-Wallis test p = 0,018) with a significant increase of osteocalcin from OA grade 0 to grade 2 (p = 0,04, Figure 20).

Median 25%-75%

Non-Outlier Range Outliers

Extremes

Healthy Osteoarthritis

0 1 2 3 4 5

SF osteocalcin (ng/ml)

**

Figure 19: Concentration of canine osteocalcin in synovial fluid (SF) of healthy and osteoarthritic dog stifles categorized by the radiographic scoring system.

**

= p < 0,01

Median 25%-75%

Non-Outlier Range Outliers

0 1 2 3

Radiographic grade of OA 0

1 2 3 4 5

SF osteocalcin (ng/ml)

*

Figure 20: Concentration of canine osteocalcin in synovial fluid (SF) of dog stifles with different stages of osteoarthritis categorized by the radiographic scoring system.

*

= p < 0,05

The degree of lameness or the sex did not have any significant influence on the concentration of osteocalcin in synovial fluid (Kruskal-Wallis test: p = 0,37 and p = 0,12, Figure 21 and Figure 22). Similar to the radiographic grade of OA, the duration of lameness showed a clear positive coherence to the concentrations of osteocalcin, but here it was not significant (Kruskal-Wallis: p = 0,059, Figure 23).

64 4.1. Analysis of synovial fluid

Figure 21: Concentration of osteocalcin in synovial fluid (SF) of dogs with different degrees of lameness (according to ARNOCZY and TARVIN 1981).

Median

Median 25%-75%

Non-Outlier Range

< 1 week 1 - 2 wks 2 - 8 wks 2 - 12 mths > 1 year Duration of lameness

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

SF osteocalcin (ng/ml)

Figure 23: Concentration of osteocalcin in synovial fluid (SF) of dogs with different durations of lameness (wks = weeks, mths = months).

The concentrations of osteocalcin in synovial fluid showed no significant correlation to the age or the weight of the dogs, but both seem to be associated with a negative influence on the concentrations (Figure 24 and Figure 25).

66 4.1. Analysis of synovial fluid

Figure 24: Correlation analysis of osteocalcin concentration in synovial fluid (SF) of dogs and their age in years.

Figure 25: Correlation analysis of osteocalcin concentration in synovial fluid (SF) of dogs and their weight in kg.

Of all 44 dogs from which osteocalcin was analyzed in their synovial fluid, 10 were hybrids, the other 34 dogs were purebred dogs (consisting of 24 different breeds).

Like the other markers, osteocalcin showed significantly higher concentrations in synovial fluid of hybrids when compared to purebreds (Mann-Whitney U-test:

p = 0,0002, Figure 26).

Median 25%-75%

Non-Outlier Range Outliers

Purebred Hybrid

0 1 2 3 4 5

SF osteocalcin (ng/ml)

**

Figure 26: Concentration of osteocalcin in synovial fluid (SF) of purebreds and hybrids.

**

= p < 0,01

68 4.1. Analysis of synovial fluid

4.1.4. Deceased dogs and surgery patients

To get a more distinct comparability between all OA grades and not just between healthy deceased dogs and arthritic surgery patients, both deceased dogs and patients were included in OA grade 0 and in every other OA grade (if samples were available). Nevertheless, concentrations of each biochemical marker were compared between deceased and living dogs to see if there are any differences that could distort the results.

Comparing pooled concentrations of each marker in deceased dogs and surgery patients, significantly higher concentrations of chordin and osteocalcin could be seen in surgery patients (Mann-Whitney U test: p = 0,044 and p = 0,002). sRANKL concentrations also seemed to be higher in surgery patients, but the increase was not significant (Figure 27).

A separation into all grades of osteoarthritis showed that most low values in deceased dogs belonged to OA grade 0 (Figure 28).

Deceased Patients

Figure 27: Concentration of sRANKL, chordin and osteocalcin compared in synovial fluid (SF) of deceased dogs and stifle surgery patients.

*

= p < 0,05

**

= p < 0,01

70 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 < 0,05

4.2.2. 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

Figure 37: Concentration of canine chordin in serum (Se) of healthy and diseased dogs

Im Dokument Analysis of the pathogenesis and progression of osteoarthritis in canine stifle joints considering three bone healing markers (Seite 41-0)