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The Effectiveness of Specific Collagen Peptides on Osteoarthritis in Dogs-Impact on Metabolic Processes in Canine Chondrocytes

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In clinical trials over the past decade, the beneficial effect of orally administered collagen peptides in osteoarthritic dogs has been clearly demonstrated [1] [2] [3]. Although a statistically significant improvement in the lameness and vitality of dogs in general has been documented, the mode of action of the collagen peptide treatment is still under discussion. A previous study [3] indicated that the reduction in lameness and increased mobility in dogs after collagen peptide treatment were associated with a statistically significantly lowered plasma content of MMP-3, which is involved in collagen degradation. In addition, the content of the MMP-antagonist TIMP-1 increased slightly after collagen peptide supplementation, suggesting a direct impact on the cartilage metabolism, particularly on the decrease of extracellular matrix degradation. Based on these findings, the impact of specific collagen peptides (PETA-GILE ®) on cartilage metabolism was tested in canine chondrocytes in the current investigation. In addition to the biosynthesis of various matrix molecules (type II collagen, aggrecan and elastin), the RNA profile of inflammatory cy-tokines and degenerative matrix molecules was investigated. The results showed clearly that the supplementation of specific collagen peptides reduced catabolic processes, as indicated by a statistically significant decrease in in-flammatory cytokines and proteases in canine chondrocytes compared with untreated control experiments. In addition, a statistically significantly enhanced biosynthesis of type II collagen, elastin, and aggrecan was observed. Hence, the current data supports the suggested anti-inflammatory effect of specific collagen peptides, but also clearly demonstrates a pronounced stimu-latory impact on matrix molecule synthesis. A combination of both observed effects might help to explain the previously reported clinical improvements after collagen peptide supplementation. Furthermore, the beneficial effect of How to cite this paper: Schunck, M., M. Schunck et al. 255 the specific collagen peptides was also confirmed in case reports on osteoarth-ritic dogs that demonstrated decreased lameness and increased vitality in the affected animals after PETAGILE treatment.
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Open Journal of Animal Sciences, 2017, 7, 254-266
http://www.scirp.org/journal/ojas
ISSN Online: 2161-7627
ISSN Print: 2161-7597
DOI: 10.4236/ojas.2017.73020 July 6, 2017
The Effectiveness of Specific Collagen Peptides
on Osteoarthritis in Dogs-Impact on Metabolic
Processes in Canine Chondrocytes
Michael Schunck1*, Helen Louton2, Steffen Oesser1
1Collagen Research Institute, Kiel, Germany
2Department of Veterinary Sciences, Institute of Animal Welfare, Animal Behavior, Animal Hygiene and Animal Husbandry,
Faculty of Veterinary Medicine, Ludwig-Maximilian-University Munich, Munich, Germany
Abstract
In clinical trials over the past decade, the beneficial effect of orally admini
s-
tered collagen peptides in osteoarthritic dogs has been clearly demonstrated
[1] [2] [3]
. Although a statistically significant improvement in the lameness
and vitality of dogs in general has been documented, the mode of action of the
collagen peptide treatment is still under discussion. A previous study [3] i
n-
dicated that the reduction in lameness and increased mobility in dogs after
collagen peptide treatment were associated with a statistically significantly l
o-
wered plasma content of MMP-3, wh
ich is involved in collagen degradation.
In addition, the content of the MMP-antagonist TIMP-1 increased slightly a
f-
ter collagen peptide supplementation, suggesting a direct impact on the cart
i-
lage metabolism, particularly on the decrease of extracellular matrix degrad
a-
tion. Based on these findings, the impact of specific collagen peptides (PETA
-
GILE®) on cartilage metabolism was tested in canine chondrocytes in the cu
r-
rent investigation. In addition to the biosynthesis of various matrix molecules
(type II collagen, aggrecan and elastin), the RNA profile of inflammatory c
y-
tokines and degenerative matrix molecules was investigated.
The results
showed clearly that the supplementation of specific collagen peptides reduced
catabolic processes, as indicated by a statistically significant decrease in i
n-
flammatory cytokines and proteases in canine chondrocytes compared with
untreated control experiments. In addition, a statistically significantly e
n-
hanced biosynthesis of type II collagen, elastin, and aggrecan was
observed.
Hence, the current data supports the suggested anti-
inflammatory effect of
specific collagen peptides, but also clearly demonstrates a pronounced stim
u-
latory impact on matrix molecule synthesis. A combination of both observed
effects might help t
o explain the previously reported clinical improvements
after collagen peptide supplementation. Furthermore, the beneficial effect of
How to cite this paper:
Schunck, M.,
Louton
, H. and Oesser, S. (2017) The Effec-
tiveness of Specific Collagen Peptides
on
Osteoarthritis in Dogs
-Impact on Metabol-
ic Processes in Canine Chondrocytes
.
Open
Journal of Animal Sciences
,
7
, 254-266.
https:
//doi.org/10.4236/ojas.2017.73020
Received:
May 4, 2017
Accepted:
July 3, 2017
Published:
July 6, 2017
Copyright © 201
7 by authors and
Scientific
Research Publishing Inc.
This work is licensed under the
Creative
Commons Attribution International
License (CC BY
4.0).
http://creativecommons.org/licenses/by/4.0/
Open Access
M. Schunck et al.
255
the specific collagen peptides was also confirmed in case reports on osteoarth-
ritic dogs that demonstrated decreased lamen
ess and increased vitality in the
affected animals after PETAGILE treatment.
Keywords
Specific Collagen Peptides, Osteoarthritis, Dogs, Metabolism, Cartilage Tissue
1. Introduction
Osteoarthritis is a common degenerative joint disease in pets and humans. Dogs
are mainly affected as a consequence of dysfunctions of the ankles and breed-
ing-related ankle anomalies, as in German shepherd dogs [4]. Nowadays, dogs
are used to a lesser extent as working animals like sheepdogs, hunting or guard
dogs. Their role as an alternative life partner or pet dog leads to other urban-re-
lated alterations in these animals. An extended life span through optimized pri-
mary health care or, conversely, misconceived animal welfare with overfeeding
or an inappropriate diet often leads to a degenerative osteoarthritic joint disease
with symptoms of chronic pain and increasing lameness. Restricted vitality and
disability is a direct consequence of the osteoarthritic defect in the dogs [5]. The
American Pet Products Association (2011-2012 National Pet Owners Survey) es-
timated a canine population of 72 million dogs in the United States. Some 14
million of these dogs are estimated to be affected by osteoarthritis. Consequent-
ly, approximately 20% of dogs older than 1 year are suffering from osteoarthritis
[6] [7], which also implies a reduction in the quality of life for the dogs’ owners.
An improvement in animals’ health conditions is, therefore, of widespread in-
terest for pet owners. Current treatments tend to focus on osteoarthritic symp-
toms that alleviate inflammation and reduce pain by the use of Non-Steroidal
Anti-Inflammatory Drugs (NSAIDS), but pay no attention to the actual cause
[8]. In recent years, various so-called chondroprotective drugs for dogs and hu-
mans have appeared on the market. These nutraceuticals claim to provide a car-
tilage-protecting quality and anti-inflammatory characteristics. Although the ef-
ficacy of the chondroprotectives is still under discussion, they are promoted as
safe and effective components for managing canine osteoarthritis without ap-
preciable side-effects [6] [9] [10] [11] [12] [13].
A further supposed therapeutic approach for the treatment of osteoarthritis is
the restoration of cartilage tissue by the injection of mesenchymal stem cells into
the osteoarthritic affected joint. The idea behind this therapy is to enhance the
cell count in the cartilage tissue and so stimulate the regeneration of extra cellu-
lar cartilage matrix. To date, these methods have failed to demonstrate the res-
toration of a durable cartilage surface to an osteoarthritic joint [14] [15].
Nevertheless, stimulation of the extracellular matrix synthesis seems to be a
promising approach as it addresses not only disease-related symptoms, but fo-
cuses on the basic cause of osteoarthritis-the metabolic imbalance of the carti-
M. Schunck et al.
256
lage.
Findings with an imaging method by McAlindon
et al
. in 2011 indicated for
the first time that orally administered collagen peptides have the potential to
stimulate proteoglycan synthesis in the knee joints of people with mild forms of
osteoarthritis [16]. With this in mind, the aim of the current research was to in-
vestigate the effectiveness of specific collagen peptides on the metabolism of car-
tilage connective tissue in canine chondrocytes. Furthermore, the pre-clinical
findings with specific collagen peptides were to be investigated under
in vivo
conditions in a small number of dogs with mild to moderate osteoarthritis. The
investigation might help to introduce a new approach for the treatment of os-
teoarthritis in dogs and establish specific collagen peptides as an effective and
safe chondroprotective agent. This could, in turn, improve the quality of life for
millions of affected dogs.
2. Materials and Methods
2.1. Study Design
2.1.1. In Vitro Examination
The mechanism of action of specific collagen peptides for the gene expression
profile and biosynthesis of extracellular matrix molecules was to be investigated
in cell culture experiments on primary canine chondrocytes.
The canine chondrocytes (095Cn402-05, tebu-bio GmbH, Offenbach, Ger-
many) were seeded on 12-well culture plates. The cells were kept in monolayer at
37˚C, 5% CO2 and O2-reduced atmosphere (5%) and cultivated in Ham’s F12
medium, supplemented with 10% fetal calf serum, 20 U/ml penicillin-strepto-
mycin and 50 µg/ml ascorbic acid. The medium was renewed every second day
until a cell confluence of 80% was reached. The regular medium was then re-
placed with the stimulation medium supplemented with 0.5 mg/ml of the specif-
ic collagen peptides.
For the gene expression studies, the chondrocyte monolayer cultures were
treated for 24 hours in comparison with untreated control experiments. RNA
was purified by extraction with the phenol (PeqGoldTriFast, VWR, Erlangen,
Germany)-chloroform method. The RNA expression of matrix molecules and
other chondrocyte related genes was determined semi-quantitatively via Real-
time Polymerase-Chain-Reaction. The RT-PCR examination was performed
(DyNAmo Flash SYBR Green, Thermo Scientific, MA, USA) after the RNA
concentration had been photometrically determined. For each reaction, 10 to 40
ng of transcribed cDNA was added after individual optimization in preliminary
tests for all genes. 12.5 µl distilled water, 5.0 µl DyNAmo Flash Master Mix, and
1.25 µl of the designed primer pairs described in Table 1 were used for the sam-
ple. The following reaction profile was programmed: Singular 7’ 95˚C, 35 - 45
repeated cycles of denaturation for 10’’ at 95˚C followed by 15’’ at annealing
temperatures (view Table 1) and elongation 20’’ at 72˚C. The reaction was
stopped by a melting temperature of 60˚C for 60’’. The RNA expression was de-
termined in relation to the housekeeping gene GAPDH. For all the genes under
M. Schunck et al.
257
Table 1. Primer sequences used for RT-PCR analysis.
Gene Fwd. Seq (5'-3') Rv. Seq (3'-5') Tm (˚C) Accession
GAPDH GTGACACCCACTCTTCCACC GAGGCCATGTAGACCATGAGG 55.0 NM_001003142.1
Collagen type II TGGCTTCAAAGGCGAACAAG TTTGGACCTGCCAGACCATC 51.7 XM_847773.3
Aggrecan CACTCAGGAGAGGAGACCCA GCAGGTGGCTCCATTCAAAC 55.0 NM_001113455.1
Elastin GGCCTGGGAATTGGTGGTAA CTCTTCCGGCCACAGGATTT 53.2 XM_005620958
TNF
α
GGCCTCCAACTAATCAGCCC CTGGCTTGTCACTTGGGGTT 55.0 XM_001003244.4
IL-1
β
CAGCCAGTCTTCACTATTCAGGT AGACTTGCAACTGGATGCCC 50.0 XM_005630074.1
IL-6 ACCACTCACCTCTGCAAACAA GTGCCTCTTTGCTGTCTTCAC 55.5 NM_001003301.1
MMP-13 ACAACCACCAAGATGCTCCC CCAGCAGGATTCAGGGGATAG 61.8 XM_536598.3
ADAMTS4 CATGTGCAACGTCAAGGCTC CGACTCAGTGAAGCAAAGCG 50.0 XM_545768.4
ADAMTS5 TGTGGGAAGGGGAGGATCTG GCGACCATTGTTTCTGGGTG 64.6 XM_846025.3
investigation, the annealing temperature (Tm ˚C) was optimized in a twelve-step
gradient PCR amplification ranging from 50˚C to 70˚C. PCR products were
electrophoretically separated on a 2% agarose gel (3 g agarose diluted in 150 mL
buffer of 0.05 M EDTA, 40 mM Tris, 57.1 mL acetic acid) stained with 10 µL
GelRed (41003, BIOTREND Chemikalien GmbH, Cologne, Germany). Amplifi-
cated PCR products were analyzed in an Alpha Innotech Fluorchem Imager
(Bio-Rad Laboratories GmbH, Munich, Germany). The PCR products were spe-
cified by a 50 bp-DNA-Fluoro-Ladder (8263.1, Carl Roth GmbH, Karlsruhe,
Germany).
In the case of the biosynthesis of the main extracellular matrix molecules,
commercial assay kits were used. For the collagen type II metabolism investiga-
tions, the amount of newly generated collagen was determined after three weeks
of stimulation. Synthesized collagen was isolated by the Sircol assay (tebu-bio,
Offenbach, Germany) in accordance with the manufacturer’s instructions. In
brief, the culture medium was discarded and adherent cell layers were digested
in 0.1 mg pepsin solution in 0.5 M acetic acid at 4˚C overnight. Cell suspensions
were neutralized by the addition of 100 µl acid neutral reagent, and synthesized
collagen was subsequently separated by the supplementation of 200 µl isolation
& concentration kit solution and vigorous agitation at 4˚C overnight. After cen-
trifugation (12,000 rpm, 10’) and discarding of the supernatants, isolated colla-
gen was re-suspended in 1 ml Sircol dye reagent. After 30 min of agitation and
further centrifugation, the collagen pellet was overloaded with 750 µl of cold ac-
id-solid wash reagent. After centrifugation, the supernatant was again discarded
and the enriched collagen was resolved in 250 µl of alkali solution. 200 µl of
these sample solutions were taken for photometrical quantification of the syn-
thesized collagen. The extinction was measured at 492 nm. The amount was de-
termined on the basis of standard measured collagen solutions.
The Blyscan glycosaminoglycan assay (tebu-bio, Offenbach, Germany) was
used for the detection of aggrecan. The biosynthesis of proteoglycans was de-
termined after a two-week stimulation. In accordance with the manufacturer’s
instructions, the cell layers were overloaded with 1 ml papain extraction solution
M. Schunck et al.
258
after the cell culture medium was discarded and incubated for 3 h at 65˚C with
vigorous agitation. The cell suspensions were centrifuged (10,000 g, 10’) and the
supernatants were collected. After the addition of 1 ml Blyscan dye solution and
agitation (30’), the supernatants were again centrifuged (12,000 rpm, 10’) and
discarded. Isolated proteoglycan pellets were re-suspended in 500 µl dissociation
solution. 200 µl per sample was determined photometrically at 656 nm in com-
parison to untreated control experiments.
The synthesis of elastin was determined by the Fastin elastin assay kit (te-
bu-bio, Offenbach, Germany). Chondrocytes were stimulated for 2 weeks with
the specific collagen peptides or left untreated as a control experiment. After
discarding the culture medium, the cell monolayers were solubilized with 250 µl
trypsin. Trypsin solution was removed after centrifugation (3000 rpm, 10’). Cell
pellets were resolved in 100 µl oxalate acid solution (1 M) and boiled for 1 h in a
water bath. After cooling, 300 µl elastin precipitation solvent was added to pre-
cipitate dissolved elastin within 15 minutes. After centrifugation (10’), the su-
pernatant was discarded and the elastin pellet was resolved in 1 ml dye solution
by 90 minutes’ agitation. After a repeated centrifugation and discarding of the
supernatant the elastin pellet was re-suspended in 260 µl dye dissociation solu-
tion. 200 µl of the resolvents were used for photometrical elastin measurement at
a wave length of 492 nm.
3.1.2. In Vivo Examination
The pre-clinical findings on primary canine chondrocytes were validated on a
small number of selected osteoarthritic dogs (n = 22). In these case reports, the
efficacy of the specific collagen peptides was evaluated under in vivo conditions.
All the dogs in our current research had a treatment-resistant history. All pre-
viously performed treatment options had failed. The observational treatments
conducted on these animals in our investigation were performed in accordance
with guidelines for the care and use of laboratory animals [17] as well as the nu-
tritional assessment guidelines for dogs and cats [18], and with the full consent
of the pet owners.
An opportunity to use blood samples arose in three of the case subjects. These
blood samples remained after routine blood tests in the veterinary practices and
could be analyzed for potential biomarkers of cartilage metabolism. Commercial
canine-specific ELISA kits were used for the cartilage biomarker analysis: In
plasma samples, ELISA’s for procollagen type II (CP II, 60-1003-001) and ag-
grecan chondroitin sulfate (CS846, 60-1004) from Ibex Pharmaceuticals, Mon-
treal, Canada, and for the detection of hyaluronic acid (HA, MBS737480), carti-
lage oligomeric matrix protein (COMP, MBS012790), matrix metalloprotease-3
(MMP-3, MBS703093), and tissue inhibitor of metalloproteases-1 (TIMP-1,
MBS006398), ELISA’s were provided by My Biosource, Eching, Germany. All
investigations were performed according to the manufacturers’ instructions.
The specific collagen peptides used in the current research are commercially
available under the brand name PETAGILE®, provided by GELITA AG (Eber-
bach, Germany). The test product is composed of different specific collagen pep-
M. Schunck et al.
259
tides with a high safety profile derived from a complex multistep procedure by
the degradation of bovine type I collagen. It is classified as a safe food by the
European Food Safety Authority (EFSA, 2005) and by the Food and Drug Ad-
ministration (FDA, 2003).
The dogs received 5 g of the specific collagen peptides with their regular pet
food once a day for a period of 12 weeks. Their usual veterinarians accompanied
the course of treatment and provided information on the medical history of the
animals by means of an orthopedic examination. The degree of osteoarthritis
was assigned a lameness score on a 5-point scale from 0 = no lameness to 4 =
high degree of lameness with relieving posture of the affected limb (Table 2).
Lameness-related parameters, “pain at activity”, “joint swelling”, “instability”
and “restriction of mobility” were evaluated on a 4-point scale from 0 = without
symptoms, 1 = low, 2 = moderate and 3 = severe symptoms. The validation of
“pain at activity” was based on the characterization of step-wise abnormal beha-
vior of the dogs in the palpation inspection. A low degree of pain at activity was
characterized by licking flews, twitching the ears, looking in the direction of
pain, and increased panting. A moderate degree was defined by anxiousness,
loud panting, straining in the muscles of the OA affected limb, turning around
to the painful ankle, and limping. A severe degree of pain at activity was deter-
mined if the dog was trembling and the whole skeletal musculature was strained.
Also if the dog barked, whimpered, growled, or snapped.
Joint swelling was characterized by an increased amount of synovia fluid,
which led to a graduated extension of the ankle volume.
Instability of the affected ankles was classified by the so-called drawer test.
Finally, with regard to the restriction of mobility, the maximum deflection
possibility of the affected ankles was determined. The instability of the ankles
and movement restriction was graded according to the expertise of the attending
veterinarians.
The pet owners were asked to evaluate changes in the dogs’ behavior regard-
ing their everyday routine, such as “discomfort when climbing stairs” or “prob-
lems when standing up”, and of their “vitality” in general. These parameters
were evaluated on a 6-point scale with 1 = near complete immobilization (90%)
to 6 = no pain or restriction in mobility. In addition, “tenderness on palpation”,
which often occurs when combing, for example, is common in dogs suffering
from backbone pain. Changes in this pain were evaluated in 3-categories because
of the smooth transition between the degree of pain levels. It was classified as 1 =
Table 2. Degree of lameness.
Score Description
0 No lameness
1 Indistinct, mild symptoms (only at trot)
2 Deranged, consistent wearing (at step & trot)
3 Moderate degree, inconsistent wearing (only tiptoeing)
4 High degree of lameness with relieving posture of the affected limb
M. Schunck et al.
260
no pain on palpation, 2 = moderate contact pain, and 3 = severe contact pain
with defensive reactions (biting, snapping).
4. Statistical Analysis
Statistically significant differences in the RNA expression of cartilage matrix
molecules, inflammatory cytokines, and matrix metalloproteases were analyzed
by the One-Student’s T-test in comparison to untreated control cells. For a
p-value < 0.05, the difference was accepted as statistically significant.
Statistically significant changes in the degree of lameness in the case subjects
were calculated with an analysis of variances with repeated measurement. For a
p-value < 0.05, the difference was accepted as statistically significant. Improve-
ments in the dogs’ vitality were also calculated with a repeated measurement
ANOVA. P-values < 0.05 were accepted as statistically significant. Changes con-
cerning everyday routine or contact pain were calculated with the Wilcoxon
signed-rank test in comparison to the baseline situation. For a p-value < 0.05,
the difference was accepted as statistically significant.
Alterations in the biomarkers’ plasma content and in the physiological, os-
teoarthritic-related parameters of the case subjects were statistically validated by
sign-tests in comparison with the baseline situation. For completeness of the re-
sults, this data is listed in a table format.
5. Results and Discussion
5.1. Mechanism of Action of Specific Collagen Peptides
A clearly increased RNA expression (p < 0.05) of the main extracellular cartilage
matrix molecules (type II collagen, aggrecan, elastin) compared to the control
cells was induced (Table 3) after the PETAGILE treatment of canine chondro-
cytes. The positive impact of the specific collagen peptides on cartilage metabol-
ism was also demonstrated by an increased accumulation of new synthesized
collagen type II (7.5%), aggrecan (5.5%) and elastin (6.3%) at the end of the sti-
mulation period of 2 - 3 weeks.
Moreover, the statistically significant (p < 0.05) down regulation of inflame-
matory cytokines and of degenerative matrix metalloproteases (Table 3) indi-
cated that the metabolic processes of the chondrocytes are directly influenced by
Table 3. Gene expression profile in canine chondrocytes by specific collagen peptides supplementation. An increment of the bio-
synthesis of ECM-molecules is shown in relation to the baseline situation (+ %). (n = 46, x-fold of control, Student’s T-test, p <
0.05 statistically significant, Coll. Type II = collagen type II, TNFα = tumor necrosis factor α, IL = interleukin, MMP = matrix
metalloprotease, ADAMTS = a disintegrin and metalloprotease with thrombospondin-1-like domains).
Extracellular matrix Inflammatory cytokines Degenerative proteases
Gene Coll.
type II Aggrecan Elastin TNFα IL-1
β
IL-6 MMP-13 ADAMTS4 ADAMTS5
x
± SEM
1.62 ± 0.33
(+7.5%)
3.23 ± 1.01
(+5.5%)
13.0 ± 4.33
(+6.3%) 0.44 ± 0.15 0.15 ± 0.05 0.55 ± 0.16 0.23 ± 0.05 0.07 ± 0.03 0.01 ± 0.01
p-value 0.034 0.018 0.024 0.003 <0.001 0.003 <0.001 <0.001 <0.001
M. Schunck et al.
261
the specific collagen peptides.
5.2. Efficacy of Specific Collagen Peptides in Osteoarthritic Dogs
In the current investigation, the beneficial effect of PETAGILE on joint cartilage
was validated in a small number of osteoarthritic dogs that had previously not
reacted to osteoarthritic treatments. The supplementation of 5 g of PETAGILE
per day to the regular diet resulted in a statistically significant (p < 0.001) reduc-
tion in lameness compared to the situation at the beginning of the treatment
(Figure 1). Only minor symptoms were described by the attending veterinarians
after the 12-week treatment period.
The veterinary findings were confirmed by a continuous increase in the vitali-
ty of the animals (p < 0.001) as observed by the dogs’ owners (Figure 1). The
positive influence of the specific collagen peptides was supported by a high effect
size level for both assessments (
η
2 > 0.5).
Pet owners also reported on improvements in their dogs’ daily routines. After
12 weeks of treatment, a statistically significant decreased discomfort in standing
up (p = 0.007, Figure 2) and a clear reduction in contact pain was reported by
the dog owners (p = 0.001, Figure 3). The beneficial effect of the specific colla-
gen peptides was also demonstrated by the large effect size of
η
2 = 0.2125 for
these parameters. In contrast, discomfort at climbing stairs only showed a
smaller improvement by trend (p = 0.089,
η
2 = 0.0296). This might be due to the
less pronounced symptoms at the beginning of treatment among the dogs when
climbing stairs.
A more detailed analysis of various blood parameters was performed on three
dogs in which the attending veterinarians had observed a statistically significant
improvement in the degree of lameness and lameness-related discomfort after 12
Figure 1. The lameness of the osteoarthritic dogs decreased continuously in the course of
the collagen peptide treatment as stated by the attending veterinaries (orange line). Dur-
ing the study period, the pet owners observed an increased vitality in their dogs (blue line,
x
± SEM, ANOVA, p < 0.05 statistically significant,
η
2 = effect size).
M. Schunck et al.
262
Figure 2. Evaluation of the everyday routine of the osteoarthritic dogs. Comparison of the
evaluated parameters “discomfort at standing up” and “discomfort at climbing stairs” at the
end of the treatment period (12th week) in comparison with the baseline situation (1st week,
x
± SEM, Wilcoxon rank-test, p < 0.05 statistically significant,
η
2 = effect size).
Figure 3. Evaluation of “contact pain” by pet owners at the end of
the treatment period (12th week) in comparison with the baseline
situation (1st week,
x
± SEM, Wilcoxon rank-test, p < 0.05 statis-
tically significant,
η
2 = effect size).
weeks of treatment (p = 0.006). All the dogs were crossbreed, older (8, 9 and 11
years) and a relieving posture in one or both front limbs was diagnosed during
the orthopedic examination. Moreover, the dogs were characterized on a 5-point
scale of the body conditioning score (BCS). As osteoarthritis is often associated
with overweight [18] [19], the body weight of the animals was also checked. Two
female dogs were slightly overweight with 10% above the ideal body weight,
while the one male dog had a normal BCS (Table 4).
The blood-sample analyses revealed a positive impact on the cartilage matrix
molecule synthesis after supplementation with the specific collagen peptides
(Table 5). The amount of the metabolic biomarkers CP-II, CS846, HA, and
M. Schunck et al.
263
Table 4. Demographic values of three case subjects and validation of osteoarthritic-related parameters by the attending veterina-
rians at baseline and end of treatment (12th week, y = year, kg = kilogram).
Case subject 1 2 3
Breed Crossbreed Crossbreed Crossbreed
Age 11 y 9 y 8 y
Gender Female Female Male
Weight 14 kg 33 kg 37 kg
Body
Conditioning Score 10% above ideal body weight 10% above ideal body weight Ideal body weight
Orthopedic
examination
Relieving posture at
right front limb
Relieving posture at
both front limbs Relieving posture at both front limbs
Validation baseline 12th week baseline 12th week baseline 12th week
Pain at activity moderate-severe low moderate low moderate-severe moderate
Joint swelling low low low-moderate none low-moderate low
Instability low low low low moderate low
Restriction of mobility low-moderate low low-moderate moderate moderate low-moderate
Lameness 3 2 1 - 2 1 2 1
Table 5. Cartilage metabolic biomarkers in routine blood samples of the case at baseline and end of treatment (12th week, ng/ml
plasma, y = year, = female, = male, Δ = difference 12th week to baseline, CP-II = procollagen type II, CS846 = chondroitin
sulfate 846, HA = hyaluronic acid, COMP = cartilage oligomeric matrix protein, MMP-3 = matrix metalloprotease-3, TIMP-1 =
tissue inhibitor of metalloproteases-1).
Case subject Biomarker CP-II CS846 HA COMP MMP-3 TIMP-1
11 y
Baseline 139.35 47.99 286.20 16.58 3.11 3.30
12th week 547.79 53.11 406.09 20.80 2.57 3.57
(ng/ml) +408.44 +5.12 +119.89 +4.22 0.54 +0.27
9 y
Baseline 576.67 44.06 334.61 13.41 1.33 13.41
12th week 712.28 46.54 193.09 15.37 1.26 132.57
(ng/ml) +135.61 +2.48 141.52 +1.96 0.07 +119.16
8 y
Baseline 493.95 53.59 136.53 13.26 1.44 not detectable
12th week 1326.6 59.35 413.96 15.97 1.17 9.68
(ng/ml) +832.65 +5.76 +277.43 +2.71 0.27 -
COMP increased after 12 weeks of collagen peptides supplementation compared
to the baseline situation. Biomarkers associated with degenerative cartilage
processes were also positively influenced by the specific collagen peptides. The
MMP-3 plasma content decreased at the end of the treatment whereas the
amount of its antagonist, TIMP-1, increased. A statistically significant difference
(p < 0.001) was determined for alterations in all the osteoarthritis-related bio-
markers tested in comparison with the baseline situation.
The modified plasma levels of MMP-3 and TIMP-1 in particular confirmed
the findings described by Weide [3] for collagen peptide treatment in osteoarth-
ritic dogs.
Our findings suggest that the efficacy of specific collagen peptides for the
M. Schunck et al.
264
treatment of osteoarthritis is not only based on its anti-degenerative and an-
ti-inflammatory potential, but instead, seems to be primarily a consequence of
its positive impact on the metabolism of extracellular cartilage matrix molecules.
This could be demonstrated for the first time, to the best of our knowledge, for
the same specific collagen peptides on primary canine chondrocytes and under
in-vivo
conditions. The correlation with the results of the cell-culture experi-
ments and of the treatment success in osteoarthritic dogs clearly demonstrate the
effectiveness of the specific collagen peptides, PETAGILE, in the current re-
search.
The stimulation of the chondrocyte matrix metabolism and reduced lameness
with a resulting increased vitality in osteoarthritic dogs are accompanied by an
appropriate blood plasma profile of biomarkers, which directly corresponded
with metabolic processes in cartilage tissue. That biomarkers such as hyaluronic
acid (HA) correlate very well with an osteoarthritic disease pattern in dogs was
already described by Plickert in 2011 [20].
Our experimental set-up for the treatment of osteoarthritic dogs further indi-
cated that PETAGILE is an optimized mixture of specific collagen peptides for
the treatment of pets. In recent trials on osteoarthritic dogs of similar age and
body weight, a daily dosage of 10 or 20 g of collagen peptides were required to
achieve a positive impact in osteoarthritis treatment [1] [2] [3]. In our current
research, the dosage of specific collagen peptides could be reduced to 5 g per day
to achieve an osteoarthritic treatment success.
The current investigation clearly demonstrated the effectiveness of specific
collagen peptides that focus mainly on the regeneration of the extracellular car-
tilage matrix. Our data suggests that inflammatory and degenerative processes
may be reduced after PETAGILE supplementation and, as a consequence, an
improvement in cartilage structure could be determined.
6. Conclusions
The effectiveness of PETAGILE is clearly demonstrated in primary canine
chondrocytes as shown by a statistically significant stimulation of the extracellu-
lar cartilage matrix synthesis and the decreased expression of inflammatory cy-
tokines and matrix metalloproteases.
Osteoarthritic dogs benefit from PETAGILE administration, as the decrease in
lameness and increased vitality illustrates. The data suggest that specific collagen
peptides can improve cartilage extracellular matrix structure, as demonstrated
by the positive impact of the cartilage-related plasma biomarkers as well as by
the stimulated biosynthesis of matrix molecules in canine chondrocytes.
The beneficial effect of PETAGILE with respect to degenerative processes in
cartilage was moreover demonstrated by a decrease in MMP-3 content and by a
concurrent increased level of TIMP-1 in the plasma samples of the case subjects.
Although the current results confirm the anti-inflammatory effects and pro-
tection against degenerative processes of collagen peptides for the treatment of
osteoarthritis in dogs, the stimulation of the cartilage matrix synthesis seems to
M. Schunck et al.
265
be even more important in the treatment of osteoarthritis, as illustrated by the
increased synthesis of collagen type II (+7.5%), aggrecan (+5.5%), and elastin
(6.3%) in canine chondrocytes.
The current investigation demonstrated the positive impact of the specific
collagen peptides PETAGILE on osteoarthritis in dogs and suggests a possible
mode of action.
On the basis of these results, a randomized, double-blind and placebo-con-
trolled clinical study is currently planned to confirm the efficacy of PETAGILE
for the treatment of osteoarthritic dogs.
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... We analyzed structure-function relationships and focused on molecular modeling calculations that would allow us to better understand the details of intermolecular interactions between sialic acids and relevant proteins. The collagen hydrolysate under study [32][33][34][35][36][37] was examined with high resolution NMR experiments, especially, DOSY NMR [4,7]. Additionally, molecular modeling approaches such as molecular docking and molecular dynamics simulations were carried out in order to obtain more information about the interaction potency of collagen fragments and sulfated GlcNAc with receptor structures. ...
... Articular cartilage destruction is mediated by the loss of collagen type II and proteoglycans and this loss is a characteristic feature of osteoarthritic (OA) symptoms. Our results show that it is possible to correlate the influence of collagen hydrolysates on cartilage tissue [32][33][34] through specific biochemical pathways and cell-biological processes [4,7,8]. We found that collagen hydrolysates were able to alter the levels of MMP-3 without changing the level of TIMP-1 (Tissue Inhibitors of MetalloProteinases-1; data not shown). ...
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... We analyzed structure-function relationships and focused on molecular modeling calculations that would allow us to better understand the details of intermolecular interactions between sialic acids and relevant proteins. The collagen hydrolysate under study [32][33][34][35][36][37] was examined with high resolution NMR experiments, especially, DOSY NMR [4,7]. Additionally, molecular modeling approaches such as molecular docking and molecular dynamics simulations were carried out in order to obtain more information about the interaction potency of collagen fragments and sulfated GlcNAc with receptor structures. ...
... Articular cartilage destruction is mediated by the loss of collagen type II and proteoglycans and this loss is a characteristic feature of osteoarthritic (OA) symptoms. Our results show that it is possible to correlate the influence of collagen hydrolysates on cartilage tissue [32][33][34] through specific biochemical pathways and cell-biological processes [4,7,8]. We found that collagen hydrolysates were able to alter the levels of MMP-3 without changing the level of TIMP-1 (Tissue Inhibitors of MetalloProteinases-1; data not shown). ...
Preprint
The bioactivities of collagen-hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen-hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics were evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceuticals data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameters were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen-hydrolysates or sulfated glucosamines. Collagen-hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen-fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen-hydrolysates, sulfated glycans (i.e. sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.
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Osteoarthritis (OA) is a chronic, painful, degenerative and inflammatory disease that affects the synovial joints and leads finally to the loss of mobility. It is highly prevalent in dogs. Nowadays, no cure exists, and the pharmacological treatment is limited to clinical signs alleviation. Some positive beneficial effects have been highlighted with dietary supplements in the course of dog OA. The goals of this narrative review are to summarize the scientific data available in the literature on dietary supplements assessed in dog OA and to discuss some trails about how to improve several aspects of research and issues with dietary supplements, such as bioavailability and dosage regimen. Chondroitin sulphate, glucosamine, undenaturated type II collagen, avocado-soya bean unsaponifiables, curcumin and polyunsaturated fatty acids were studied in dog OA and therefore discussed in the present review. Most of them showed anticatabolic and anti-inflammatory effects. Unfortunately, few data exist concerning their pharmacokinetics. Their bioavailability is low, but new formulations are developed to enhance their gastrointestinal absorption. The clinical relevance of these new formulations compared to native forms should be demonstrated in good clinical trials. Even if further investigations are needed, dietary supplements should be considered in OA management. © 2015 John Wiley & Sons Ltd.
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Osteoarthritis can be defined as a disorder of movable joints characterized by deterioration of articular cartilage, osteophyte formation and bone remodeling, changes in periarticular tissues, and a low-grade, nonpurulent inflammation of variable degree. This article reviews normal joint anatomy and function, as well as biochemical, biomechanical, and pathologic changes that are characteristic of osteoarthritis.
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S23 expression of all MMPs was greater in meniscus than AC. In general MMP expression in the outer meniscus was more responsive to TNFα. TIMP-1 and -3 were generally decreased in all tissues by IL-1α and TNFα. There was more pro and active MMP-2, and pro-MMP-13 in control cultures of outer meniscus than AC or inner meniscus. Unlike AC, both meniscal zones released active MMP-13 after IL-1α and TNFα treatment, and active MMP-2 after IL-1α (inner) or both cytokines (outer). Conclusions: Aggrecan processing differed between outer versus inner meniscus, with the later being similar to AC. Aggrecan proteolysis in the outer meniscus rather than AC, may be responsible for MMP-generated neoepitopes detected in OA human knee synovial fluid. The meniscus was generally more responsive to TNFα than AC, particularly the outer zone. Expression of MMPs and ADAMTS was higher in meniscus than AC, and unlike AC active MMPs were released from meniscus. The meniscus may contribute significantly to metalloproteinase levels in the joint, and therefore to cartilage breakdown in OA. Differential regulation of MMPs and ADAMTS in the knee joint meniscus compared with AC, and between inner and outer meniscal zones has important implications in OA therapy. Purpose: Lameness associated with osteoarthritis (OA) is highly prevalent in dogs. Typically, OA dogs have a decrease in their limb function, demon-strate discomfort and disability with an unwillingness to perform daily life activities. To alleviate those clinical signs, management of OA based on diet enriched in polyunsaturated omega-3 fatty acids (omega-3 PUFAs) is broadly considered by clinicians despite the lack of published clinical trials supporting their efficacy. The aim of this study was to measure functional outcomes in lame client-owned dogs afflicted by naturally occurring OA before and after a 13 week period of feeding with either a control diet (CTR) or a veterinary therapeutic diet (VTD) containing high levels of omega-3 PUFAs. Hypothetically, the functional outcomes would be significantly improved from Baseline only in VTD-treated dogs. The magnitude of those changes would exceed CTR-treated dogs. Methods: A randomized, double-blinded, placebo-controlled trial was per-formed in 45 lame client-owned dogs with OA. Dogs were included in the study when they had hind limb lameness observed by a veterinary surgeon, signs of OA upon orthopedic examination and low dynamic weight bearing (peak vertical force, PVF) recorded on force platform gait analysis. The presence of OA was confirmed on radiographs (hip and/or stifle). The initial condition (Baseline) of dogs was determined by the recording of PVF (primary study outcome) and by client-specific outcome measures (CSOM). Dogs were randomly allocated to 3 groups (15/group) and received a control diet (CTR1 or CTR2) or the therapeutic VTD diet. Assessment by CSOM was performed twice weekly by the owner, and PVF was repeated after 13 weeks of treatment. Statistical analyses were performed using nonparametric tests at p<0.05. Results: Median PVF values increased significantly in VTD-treated dogs (+9.4%, p=0.049). Neither CTR1-(+2.4%,p=0.582) nor CTR2-treated (+2.6%, p=0.187) dogs revealed such significant improvement. In addition, at week 13, CSOM assessment denoted a significant decrease in score (meaning improvement) for VTD-treated dogs (-20%, p=0.021), but not for CTR1 (0%, p=0.216) or CTR2 (-5.8%, p=0.267). The magnitude of the changes of PVF and CSOM in VTD-treated dogs was not significantly different from CTR-treated dogs. Conclusions: The use of a high quality diet and good nutritional habits may have prompted the monitoring of a trend toward a better function in CTR dogs, despite the absence of omega-3 PUFAs in these control diets. Nutrition-based OA management improved functional outcome and performance in daily life activities when dogs were fed with VTD over a short-term period. This veterinary diet containing omega-3 PUFAs should be considered as a therapeutic modality to alleviate the clinical signs in dogs afflicted by OA as a unique treatment or as part of a multimodal approach in the management of OA.
Article
The aim of this randomized, placebo-controlled and double-blinded trial was to compare the effect of a veterinary therapeutic diet (VTD) rich in omega-3 fatty acids (omega-3) from fish origin to a regular diet used as control (CTR) over a period of 13 weeks in dogs afflicted by naturally occurring osteoarthritis (OA). Thirty privately owned dogs were selected. Dogs had lameness confirmed by an orthopaedic examination, had stifle/hip OA and had locomotor disability based on the peak of the vertically oriented ground reaction force (PVF) measured using a force platform. At Baseline, all owners were asked to determine 2-5 activities of daily living that were the most impaired. Activities were scores (0-4) in accordance with severity using case-specific outcome measures (CSOM). The PVF was also measured. Dogs (15/group) were then randomly assigned to receive either the CTR or the VTD. The CSOM was completed twice weekly. The recording of PVF was repeated at Week 7 and 13. The VTD-fed dogs showed a significantly higher PVF at Week 7 (p < 0.001) and at Week 13 (p < 0.001) when compared to Baseline. From Baseline to Week 13, VTD-fed dogs had a mean (± SD) change in PVF recording of 3.5 ± 6.8% of body weight (%BW) compared with 0.5 ± 6.1%BW (p = 0.211) in CTR-fed dogs. This change in primary outcome was consistent with an effect size of 0.5. Conversely, dogs fed the CTR did not show significant change in PVF measurements. At the end of the study, the CSOM was significantly decreased (p = 0.047) only in VTD fed dogs. In lame OA dogs, a VTD that contains high level of omega-3 from fish origin improved the locomotor disability and the performance in activities of daily living. Such nutritional approach appears interesting for the management of OA.
Article
Various treatments of osteoarthritis (OA) have been described, including use of nutraceuticals. To review systematically the literature about the effects of nutraceuticals on clinical signs of pain or abnormal locomotion in horses, dogs, and cats, and to discuss methodological aspects of trials and systematic reviews. A systematic search of controlled trials evaluating the impact of nutraceuticals on OA in horses, dogs, and cats was performed, using Medline, CAB Abstracts, and Google Scholar. Scientific evidence was evaluated by means of criteria proposed by the Food and Drug Administration (FDA), and a scoring system adapted from both the CONsolidated Standards of Reporting Trials (CONSORT) statement and recommendations for assessing trials by the Center of Evidence Based Medicine of Oxford. Twenty-two papers were selected and reviewed, with 5 studies performed in horses, 16 in dogs, and 1 in cats. The strength of evidence was low for all nutraceuticals except for omega-3 fatty acid in dogs. There were limited numbers of rigorous randomized controlled trials and of participants in clinical trials. The evidence of efficacy of nutraceuticals is poor, with the exception of diets supplemented with omega-3 fatty acids in dogs. Greater access to systematic reviews must be part of the objectives of the veterinary science in the future. Their reporting would be improved by internationally agreed-upon criteria for standards and guidelines.
Article
To determine whether either of two magnetic resonance imaging approaches - delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC), or T2 mapping - can detect short-term changes in knee hyaline cartilage among individuals taking a formulation of collagen hydrolysate. Single center, prospective, randomized, placebo-controlled, double-blind, pilot trial of collagen hydrolysate for mild knee osteoarthritis (OA). Participants were allowed to continue the prior analgesic use. The primary outcome was change in dGEMRIC T1 relaxation time in the cartilage regions of interest at the 24-week timepoint. Secondary endpoints included the change in dGEMRIC T1 relaxation time between baseline and 48 weeks, the change in T2 relaxation time at 0, 24 and 48 weeks, the symptom and functional measures obtained at each of the visits, and overall analgesic use. Among a sample of 30 randomized subjects the dGEMRIC score increased in the medial and lateral tibial regions of interest (median increase of 29 and 41 ms respectively) in participants assigned to collagen hydrolysate but decreased (median decline 37 and 36 ms respectively) in the placebo arm with the changes between the two groups at 24 weeks reaching significance. No other significant changes between the two groups were seen in the other four regions, or in any of the T2 values or in the clinical outcomes. These preliminary results suggest that the dGEMRIC technique may be able to detect change in proteoglycan content in knee cartilage among individuals taking collagen hydrolysate after 24 weeks.