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Veterinary Therapeutics • Vol. 8, No. 4, Winter 2007
272
Effect of Adipose-Derived Mesenchymal Stem
and Regenerative Cells on Lameness in Dogs with
Chronic Osteoarthritis of the Coxofemoral Joints:
A Randomized, Double-Blinded, Multicenter,
Controlled Trial*
Linda L. Black, DVM, PhD
a
James Gaynor, DVM, MS, DACVA, DAAPM
b
Dean Gahring, DVM, DACVS
c
Cheryl Adams, DVM, CVA
d
a
Vet-Stem, Inc.
12860 Danielson Court, Suite B
Poway, CA 92064
b
Animal Anesthesia & Pain Management Center
5520 North Nevada Avenue, Suite 150
Colorado Springs, CO 80918
c
San Carlos Veterinary Hospital
8618 Lake Murray Boulevard
San Diego, CA 92119
CLINICAL RELEVANCE
Autologous stem cell therapy in the field of regenerative veterinary medicine in-
volves harvesting tissue, such as fat, from the patient, isolating the stem and re-
generative cells, and administering the cells back to the patient. Autologous adi-
pose-derived stem cell therapy has been commercially available since 2003, and
the current study evaluated such therapy in dogs with chronic osteoarthritis of the
hip. Dogs treated with adipose-derived stem cell therapy had significantly improved
scores for lameness and the compiled scores for lameness, pain, and range of mo-
tion compared with control dogs. This is the first randomized, blinded, placebo-con-
trolled clinical trial reporting on the effectiveness of stem cell therapy in dogs.
■ INTRODUCTION
Advances in understanding of the biology of
adult stem cells have attracted the attention of
the biomedical research community, including
those studying osteoarthritis (OA).
1
Autolo-
gous adult stem cells are immunologically
compatible, can be harvested from a variety of
sources, including bone marrow and adipose
tissue,
1
and have no ethical issues related to
*This study was sponsored by Vet-Stem, Inc., Poway,
California. Correspondence should be directed to
Dr. Black (LBlack@vet-stem.com).
Dennis Aron, DVM, DACVS
e
Susan Harman, AHT, BS
a
Daniel A. Gingerich, DVM, MS
f
Robert Harman, DVM, MPVM
a
d
Arboretum View Animal Hospital
2551 Warrenville Road
Downers Grove, IL 60515
e
Veterinary Surgical Specialists of San Diego
5610 Kearny Mesa Road, Suite B
San Diego, CA 92111
f
Turtle Creek Biomedical Consulting
2219 Wilmington Road
Lebanon, OH 45036
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273
L. L. Black, J. Gaynor, D. Gahring, C. Adams, D. Aron, S. Harman, D. A. Gingerich, and R. Harman
healing score, and collagen oligomeric matrix
protein scores in an equine tendonitis model.
15,16
Used commercially on more than 2,500 horses
with no significant systemic adverse events re-
ported and less than 0.5% local tissue reactions
(as of December 12, 2006), autologous AD-
MSC therapy has been shown to be reasonably
safe and therapeutically successful.
A number of recent publications provide ev-
idence of therapeutic success with stem cell
therapy in tendon or ligament injuries and de-
generative joint disease in other species.
1,17–19
Nathan and colleagues demonstrated that AD-
MSCs in a fibrin carrier were able to fill osteo-
chondral defects created in rabbit femoral
condyles better than fibrin carrier alone, and
the biomechanical performance of the AD-
MSC–treated group was clearly superior as
well.
19
In a model of OA in the goat, BM-MSC
therapy resulted in regeneration of the menis-
cal tissue and retardation of the normal pro-
gression of OA seen in the model.
17
Cell-treat-
ed joints had marked meniscal regeneration
with implantation of the BM-MSCs and a re-
duction in degeneration of the articular carti-
lage, osteophyte remodeling, and subchondral
sclerosis. Based on scientific evidence and the
therapeutic success in horses, veterinarians are
now beginning to use regenerative medicine to
treat similar conditions in dogs, including OA.
OA is the most common cause of chronic
pain in dogs, with more than 20%, or 10 to
12 million dogs, afflicted in the United States
at any time.
20–22
OA is characterized by degen-
eration of the articular cartilage, with loss of
matrix, fibrillation, and formation of fissures,
and can result in complete loss of the cartilage
surface.
23
Chondrocytes, the only cells of ar-
ticular cartilage, maintain homeostatic synthe-
sis and degradation of the extracellular matrix
via the secretion of macromolecular compo-
nents (collagen, glycosaminoglycans, and
hyaluronic acid) and modulation of the extra-
their use. Mesenchymal stem cells (MSCs) de-
rived from bone marrow (BM-MSCs) and adi-
pose tissue (AD-MSCs) are the most highly
characterized and are considered comparable.
2
Both have demonstrated broad multipotency
with differentiation into a number of cell line-
ages, including adipo-, osteo-, and chondro-
cytic lineages.
2
However, the easy and repeat-
able access to subcutaneous adipose tissue, the
simple isolation procedure, and the approxi-
mately 500-fold greater numbers of fresh
MSCs derived from equivalent amounts of fat
versus bone marrow provide a clear advantage
in using AD-MSCs over BM-MSCs.
3,4
Isolated
AD-MSCs can also be easily cryopreserved.
3
The area of AD-MSC use for regenerative
medicine has been the focus of many recent re-
views, underlining the rapid pace of this
field.
2–8
Isolation of cells from adipose tissue
entails mincing and washing, followed by col-
lagenase digestion and centrifugation.
8,9
The
pellet formed from centrifugation is deemed
the stromal vascular fraction (SVF), which is
resuspended and used as the treatment modal-
ity. The SVF contains a heterogenous mixture
of cells including fibroblasts, pericytes, en-
dothelial cells, circulating blood cells, and AD-
MSCs.
8,10–12
As a result of the cells’ “minimally
manipulated” nature, many autologous stem
cell therapies do not require an FDA drug ap-
proval application.
Veterinarians have used autologous AD-
MSCs to treat tendon and ligament injuries and
joint disease in horses on a commercial basis
since 2003.
13–15
Studies and anecdotal clinical
experience with more than 2,500 horses demon-
strate that autologous AD-MSC therapy helps
horses with tendon and ligament injuries.
13–16
In
a blinded, placebo-controlled study, Dahlgren
and Nixon and colleagues demonstrated statisti-
cally significant improvement in inflammatory
cell infiltrate, collagen fiber uniformity, polar-
ized collagen fiber crimping, overall tendon
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Veterinary Therapeutics • Vol. 8, No. 4, Winter 2007
274
cellular matrix turnover. Chondrocyte secre-
tion and release of lytic and tissue-damaging
mediators (cytokines, free radicals, proteases,
prostaglandins) are controlled by a balance of
anabolic and reparative substances (growth
factors, inhibitors of catabolic cytokines) and
inhibitors of degradative enzymes.
2
3
In OA,
there exists an overproduction of destructive
and proinflammatory mediators relative to the
inhibitors, resulting in a balance in favor of ca-
tabolism rather than anabolism, which in turn
leads to the progressive destruction of articular
cartilage.
23
Scientific studies and clinical experience
with OA therapy in dogs suggest that NSAIDs,
the current cornerstone of care, often do not
provide complete pain relief.
24–28
In contrast
to drug therapy, cellular therapies such as
AD-MSC therapy do not rely on a single tar-
get receptor or pathway for their action. Cel-
lular therapy functions trophically by secret-
ing cytokines and growth factors
29
and by
recruiting endogenous cells to the injured
site, and it may promote cellular differentia-
tion into the resident lineages.
8
MSCs “com-
municate” with the cells of their local envi-
ronment, can suppress immunoreactions, and
inhibit apoptosis, and new data now demon-
strate that BM-MSCs can deliver new mito-
chondria to damaged cells, thereby rescuing
aerobic metabolism.
8,30
Taken together, AD-
MSCs respond to the local microenvironment
in a manner that in many cases is demon-
strated to enhance healing. The purpose of
this blinded, randomized, placebo-controlled,
multicenter study was to evaluate the clinical
effect of a single intraarticular injection of
adipose-derived stem and regenerative cells in
dogs with lameness associated with OA of the
coxofemoral joints.
■ MATERIALS AND METHODS
Study Population
Four companion animal regional referral
veterinary practices in the San Diego area,
Chicago, and Colorado Springs participated in
this randomized, double-blinded, placebo-
controlled trial that included outpatient dogs
with OA of the coxofemoral joint. Twenty-one
dogs (14 females and 7 males) ranging in age
from 1 to 11 years were recruited based on the
presence of bilateral coxofemoral joint OA
with a minimum duration of 6 months. The
breeds included Akita, boxer, German shep-
herd mix, Gordon setter, Great Pyrenees,
Labrador retriever, rottweiler, schnauzer mix,
standard poodle, Aussie mix, collie mix, gold-
en retriever, puli, and Weimaraner; body
weights ranged from 25 to 110 lb.
Before enrollment, all dogs underwent rou-
tine clinical chemistry and hematology evalua-
tion to ensure overall health. Study animals
demonstrated gait changes characteristic of
OA, including persistent lameness at a walk
and trot, pain on passive manipulation of the
affected joint(s), and limited range of motion
with pain at less than full range of passive mo-
tion. Finally, dogs demonstrated functional
Veterinarians have used autologous adipose-derived
mesenchymal stem cells to treat tendon and ligament
injuries and joint disease in horses on a commercial
basis since 2003.
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275
L. L. Black, J. Gaynor, D. Gahring, C. Adams, D. Aron, S. Harman, D. A. Gingerich, and R. Harman
disabilities, including level of stiffness as meas-
ured by willingness to walk and run.
Each qualified case demonstrated pretreat-
ment radiographic evidence of degenerative
joint disease of grade 2 or higher on the fol-
lowing radiographic scoring scale:
0 = Normal joint
1 = Radiographic evidence of instability; no
degenerative change
2 = Mild degenerative change (occasional os-
teophytes)
3 = Moderate degenerative change (osteo-
phytes, subchondral sclerosis)
4 = Severe degenerative change (osteophytes,
subchondral sclerosis, bone remodeling)
Dogs were excluded from the study if they
had a history of coxofemoral joint surgery; very
severe hip dysplasia with functional luxation; a
history of spontaneous luxation or a likelihood
of spontaneous luxation during the 6 months
of the study; concurrent disease, such as a fun-
gal, bacterial, or viral infection; malignant neo-
plasia; or any severe systemic disease that would
confound interpretation of treatment effects.
Dogs on concomitant therapy, such as
NSAIDs, were required to be on these medica-
tions for at least 14 days before enrollment in
the study and to remain on the drugs at the
same level throughout the study. Hyaluronic
acid and polysulfated glycosaminoglycan injec-
tions and such alternative treatments as chiro-
practic and acupuncture, if used, were discon-
tinued in all dogs in both groups for 10 days
before enrollment in the study and were not
administered during either phase of the study
period. Two dogs were disqualified during the
study because of inadvertent administration or
removal of NSAIDs, which would preclude
evaluation.
To be eligible, the dogs had to be cared for by
attentive owners who agreed by informed con-
sent to participate in this clinical study, to follow
a set schedule of veterinary appointments, and
to observe their dog for the entire study period.
Treatments
The in-house laboratory at Vet-Stem pre-
pared the test treatment material for each study
dog. Lab technicians isolated autologous AD-
MSCs and regenerative cells from a minimum
of 23 g of fat collected from each dog by the
investigator. Adipose was collected from both
treatment and control dogs to maintain blind-
ing. Laboratory personnel provided the test
and control material to the investigator in two
covered, sterile 1-ml syringes. Each dog re-
ceived either 0.6 ml of phosphate buffered
saline (PBS; control dogs) or a suspension of
4.2 million (MM) to 5 MM (depending on
cell yield) viable cells prepared from the dog’s
own fat tissue in 0.6 ml PBS/joint. The veteri-
narians injected the hip joints at the midpoint
of the proximal edge of the greater trochanter
of the femur. One dog received 4.2 MM viable
cells/joint; all other dogs received 5 MM
cells/joint. The adipose samples from the con-
trol animals were also processed, and the viable
nucleated cells were cryopreserved for use later.
Laboratory technicians also prepared and
archived a sample of the cell preparation from
each case for additional study and prepared
two saline syringes to flush the test or control
article through the needle. The Vet-Stem clini-
cal document coordinator prepared random-
ization sheets that were stratified by investiga-
tional center to ensure balance between treated
(Group A) and control (Group B) dogs within
centers. Dogs were assigned to a group during
the receiving process for the sample according
to the randomization sheet for the investigator.
The Vet-Stem clinical document coordinator
maintained the administration code through-
out the study until the day 90 examination was
concluded or in the event an animal was with-
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Veterinary Therapeutics • Vol. 8, No. 4, Winter 2007
276
drawn from the study.
Dogs in Group A received a single intraar-
ticular injection of the fresh test treatment ma-
terial in each hip joint on day 0, and dogs in
Group B received a single intraarticular injec-
tion of the placebo material in each hip on day
0. Neither the owners nor the investigators had
knowledge of group assignments.
Owners were counseled to leash-walk their
dogs twice daily. However, one dog in the test
group was allowed to run and one had free run
of a large pen. One control dog was leash-
walked and allowed to swim.
Stem and Regenerative Cell Preparation
Adipose Tissue Collection
Adipose tissue was collected from the ab-
dominal, inguinal, or thoracic wall regions of
the dog. A small surgical incision (5 cm) was
made aseptically after the patient was anes-
thetized. The adipose tissue was resected by
scalpel or surgical scissors and placed into a la-
beled sterile tube containing 15 ml of PBS.
The sample tube was placed in a validated,
temperature-controlled 2˚C transport box spe-
cially fitted with a frozen cold pack and
shipped by overnight express courier to the
Vet-Stem laboratory for processing.
Tissue Processing and Stem
and Regenerative Cell Isolation
Adipose tissue was washed with PBS, then
minced and washed several times with PBS to
remove debris and excess blood. The minced tis-
sue was mixed well. Enzymatic digestion was
performed by use of a combination of collage-
nase and hyaluronidase at 37˚C for 50 minutes
with agitation. The mixture was centrifuged at
400 ×g for 15 minutes, and the cell pellet was re-
suspended in PBS a total of four times. An
aliquot of the final cell suspension was assessed
for viability (trypan blue exclusion method) and
total nucleated cell yield. This constitutes the
SVF preparation. Vet-Stem internal data
demonstrate that the mean CFU–fibroblast
(CFU-F) percentage for canine regenerative cells
is 1.72%, which is within the reported range of
1% to 4% CFU-F
4
for human AD-MSCs and
far greater than the 0.001% to 0.01% CFU-F
reported for human bone marrow.
12
Recent phe-
notypic cluster of differentiation (CD) marker
analysis data reported from three independent
laboratories demonstrate an approximate mean
of 30% AD-MSC (CD34
+
, CD31
–
, CD146
–
)
in human SVF and approximately 1% to 10%
other “progenitor” cell types, including a peri-
cyte cell fraction.
10–12
Canine fresh SVF contains
approximately 85.6% mononuclear cells that
do not fall into the hematopoietic lineage. Fur-
ther characterization of canine SVF will be com-
pleted as canine CD marker reagents become
available.
Evaluations
Veterinary evaluation incorporated history,
physical examination, and lameness examina-
tion including joint mobility and notation of
pain on manipulation, a modified version of
Based on the scientific evidence and the therapeutic
success in horses, veterinarians are now beginning
to use regenerative medicine in similar conditions
for dogs, including OA.
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277
L. L. Black, J. Gaynor, D. Gahring, C. Adams, D. Aron, S. Harman, D. A. Gingerich, and R. Harman
published criteria.
27
Clinical outcome measures
were based on veterinary orthopedic examina-
tion evaluation using a numerical rating scale
based on a standardized questionnaire (Figure
1). Baseline results for both owner and veteri-
nary evaluations were recorded between 2 and
14 days before the dogs received either the test
or control preparation by intraarticular injec-
tion. Follow-up visits to the veterinary clinic
were required at 30, 60, and 90 days after the
dog’s intraarticular injection. At each visit,
owners were also asked to complete a numeric
rating scale (1 [best] to 5 [worst]) as part of a
standard questionnaire adapted from the
Cincinnati Orthopedic Disability Index,
31
which included evaluation of the following 13
parameters: walk, run, jump, turning sudden-
ly, getting up from lying down, lying down
from standing, climbing stairs, descending
stairs, squatting to urinate or defecate, stiffness
in the morning, stiffness in the evening, diffi-
culty walking on slippery floors, and willing-
ness to play voluntarily.
Statistical Evaluation
The statistical significance of changes in
scores over time for each parameter in each
treatment group was tested by one-way repeated
measures analysis of variance (ANOVA). In the
rare instances in which the data were not nor-
mally distributed, results were substantiated by
the nonparametric Friedman repeated measures
ANOVA on rank sum test. Comparisons of re-
sponses between treatment and placebo groups
Persistent Ambulatory
non–weight- only with Non-
Not detectable Intermittent Persistent bearing assistance ambulatory
Lameness—
123456
walk
Lameness—
123456
trot
Mild pain Severe pain
No pain (attempts to withdraw limb) (immediate limb withdrawal)
Pain on
123
manipulation
Pain only at Pain at less than Pain at any attempt
No limitation full range of motion full range of motion to manipulate joint
Range
1234
of motion
Dog does not
Slightly stiff Stiff, dog has Very stiff, dog want to walk,
gait, only noticeable does not want must be helped
noticeable difficulty walking to walk or run up, and
Normal activity on running or running unless coaxed will not run
Functional
1 23 45
disability
Figure 1. Veterinary orthopedic examination assessment score sheet submitted at days 0, 30, 60, and 90.
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Veterinary Therapeutics • Vol. 8, No. 4, Winter 2007
278
were made by two-way repeated measures
ANOVA with treatment and time as grouping
variables. Post hoc comparisons were made by
the Tukey test. The data were analyzed using
commercially available statistical software (Sig-
maStat 3.5, Systat Software, Point Richmond,
CA) at the nominal .05 level of significance.
To determine the magnitude of the response,
the “effect size,” defined as the difference be-
tween the treatment and placebo response di-
vided by the standard deviation of the placebo
response,
32
was calculated for each parameter at
each posttreatment evaluation time. Originally
developed for the behavioral sciences,
33
effect
size is a unitless measure of the degree to which
the apparent treatment effect exceeds the
placebo effect, wherein an effect size of 0.2 or
less is considered a “small” effect and 0.8 or
more is a “large” effect.
■ RESULTS
Eighteen dogs completed the 90-day study.
Two dogs were removed from the study as a re-
sult of either receiving or discontinuing an
NSAID, and one dog was lost from the study
because of other compounding medical issues.
Both test article and control article were well
tolerated by the dogs, with the exception of two
placebo-treated dogs, both from the same in-
vestigator, that demonstrated biting and
scratching at the injection sites of short dura-
tion. Each case resolved within 48 hours, and the
cause was postulated to be possible joint over-
extension during injection. There were no other
adverse events reported and no further issues
with these control dogs throughout the study.
Veterinary Evaluation
There were no significant (P > .05) differ-
ences by investigator for any outcome variable;
therefore, the data were pooled for further
analysis. At baseline, there were no significant
differences between the test and control groups
in terms of veterinarian or owner scores. After
treatment, veterinary orthopedic examination
scores for all parameters decreased over time in
the stem cell group and, to a lesser extent, in
the placebo group (Table 1).
The improvement in clinical scores was sta-
tistically significant in the stem cell group at all
posttreatment evaluation times for lameness at
walk and trot, pain on manipulation, and pain-
free range of motion (Table 1). Functional dis-
ability, a highly subjective evaluation (Figure 1),
added variance to the data that caused a lack of
significance at later time points. Control ani-
mals did not significantly improve over time for
lameness, pain on manipulation, or range of
motion. Veterinary assessment revealed greater
improvement from baseline for lameness at a
trot, pain on manipulation, and range of mo-
tion in test animals compared with placebo
controls (Figure 2). The combined scores for all
parameters measured are also shown in Figure
2. There was no correlation between an animal’s
weight and its improvement score.
To determine which of the orthopedic ex-
amination parameters were most responsive to
The improvement in clinical scores was statistically
significant in the stem cell group at all posttreatment
evaluation times for lameness at walk and trot, pain on
manipulation, and pain-free range of motion.
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279
L. L. Black, J. Gaynor, D. Gahring, C. Adams, D. Aron, S. Harman, D. A. Gingerich, and R. Harman
stem cell therapy and the timing of the re-
sponses, the effect size was calculated for each
parameter at each time point. By this analysis,
lameness at the trot, pain on manipulation,
and range of motion were the most responsive,
with large effect sizes (>0.8) at all posttreat-
ment times. Comparison of average effect sizes
by parameter are presented in Figure 3. The
overall effect size was 1.34, which is considered
a large effect in orthopedics.
Owner Evaluation
Overall, owners with treated dogs evaluated
their dogs to be more improved in the param-
eters scored, relative to owners with control
dogs (Figure 4). Five dogs were eliminated as a
result of multiple owner evaluations or com-
pounding clinical conditions that made it im-
possible for owners to accurately evaluate their
animals. Figure 4 demonstrates that treated
dogs had a higher percentage of improvement
overall (all scores combined) relative to control
animals, although data did not reach statistical
significance. Owners evaluated the dogs for the
parameters that affected their dog most. There-
fore, not all parameters were applicable to all
dogs. In review of the 13 parameters scored on
a 5-point scale, the control dogs had 1.9 pa-
rameters that improved by 2 or more points,
whereas the test dogs had almost 4.7 parame-
ters that improved by 2 or more points.
■ DISCUSSION
Effective therapies for OA have been slow in
developing, and many dogs continue to suffer
with chronic pain associated with OA, even
with multimodal treatment protocols. Results
of this double-blind, placebo-controlled study
demonstrate that AD-MSC therapy resulted
in improved orthopedic examination scores as
assessed by skilled veterinarians. Treated dogs’
lameness, range of motion, and pain on ma-
nipulation, as well as their overall combined
scores, significantly improved over time and
TABLE 1. Stem Cell Therapy Improves Orthopedic Examination Scores
in Dogs with Bilateral Hip Osteoarthritis (mean ± SEM; n = 18)
Parameter
(Range) Treatment Baseline 30 Days P Value* 60 Days: P Value* 90 Days: P Value*
Lameness Stem cell 2.44 ± 0.34 1.7 ± 0.2 .037 1.56 ± .034 .015 1.56 ± 0.29 .015
at walk (1–6)
Control 2.11 ± 0.20 1.9 ± 0.3 NS 2.00 ± 0.33 NS 1.89 ± 0.26 NS
Lameness Stem cell 2.89 ± 0.20 1.8 ± 0.3 <.001 1.78 ± 0.32 <.001 1.56 ± 0.24 <.001
at trot (1–6)
Control 2.22 ± 0.15 1.9 ± 0.2 NS 1.89 ± 0.31 NS 1.89 ± 0.26 NS
Pain on manip- Stem cell 2.22 ± 0.15 1.2 ± 0.1 <.001 1.56 ± 0.18 .003 1.44 ± 0.18 .010
ulation (1–3)
Control 2.00 ± 0.17 1.9 ± 0.2 NS 1.89 ± 0.20 NS 1.89 ± 0.20 NS
Range of Stem cell 2.89 ± 0.20 1.7 ± 0.2 .001 1.89 ± 0.26 .009 1.89 ± 0.11 .009
motion (1–4)
Control 2.33 ± 0.17 2.0 ± 0.2 NS 2.11 ± 0.20 NS 2.33 ± 0.24 NS
Functional Stem cell 2.67 ± 0.24 1.4 ± 0.2 .011 1.78 ± 0.32 NS 1.89 ± 0.35 NS
disability (1–5)
Control 2.44 ± 0.18 1.8 ± 0.2 .033 1.78 ± 0.22 .033 1.89 ± 0.26 NS
*P value vs baseline; one-way repeated measures analysis of variance.
NS = not significant.
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Veterinary Therapeutics • Vol. 8, No. 4, Winter 2007
280
improved relative to control animals. In this
study, owner evaluation scores, although im-
proving, did not reach significance. Circum-
stances created difficulty in interpretation of
the owner data, including one household that
introduced a new puppy that reportedly en-
50
40
30
10
20
0
30
P
= .023*
P = .008*
P = .001*
P = .021*
P = .004*
P = .015*
P = .005*
P = .139*
P = .164*
P
= .099*
P = .029*
P
= .004*
0 60 90
Days after Treatment
Improvement in Score (%)
Lameness at Trot
Lameness and Composite Scores Were Significantly Improved in Treated Dogs
Pl
a
c
e
b
o
Stem Cell
*vs. Placebo
50
40
30
10
20
0
300 60 90
Days after Treatment
Improvement in Score (%)
Pain on Manipulation
50
40
30
10
20
0
300 60 90
Days after Treatment
Improvement in Score (%)
Composite Score
50
40
30
10
20
0
300 60 90
Days after Treatment
Improvement in Score (%)
Range of Motion
Figure 2. Percentage of improvement (mean ± SEM) in lameness at the trot, pain on manipulation, and range of mo-
tion and in all scores combined (composite score) for all parameters in stem cell–treated and control dogs. Treated dogs im-
proved significantly relative to control dogs in terms of lameness at the trot and composite score. P values are vs. controls.
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couraged more spontaneous
play on the part of a control dog
and one test dog with a concur-
rent elbow condition that made
isolation of the hip parameters
difficult for the owner.
Results of this study again il-
lustrate the value of systemati-
cally applied clinical scoring
systems in detecting therapeutic
effects. Effect size analysis,
which was possible because of
the double-blind, placebo-con-
trolled design of the study, is
particularly convincing. Al-
though there are few reports in
veterinary orthopedics with
which to compare the present
results, similar effect sizes were
reported in a clinical trial of another interven-
tion in orthopedic conditions in dogs.
31
It is
also useful to note that the overall effect size for
stem cell therapy under conditions described
was 1.34, which is much higher than the ag-
gregate effect size of 0.44 for glucosamine in
humans with OA.
32
The current study design employs a subjec-
tive numerical rating scale for veterinarians to
assess degree of lameness. Although controver-
sy exists as to how subjective scoring systems
compare with objective gait analysis, subjective
lameness score data have correlated well in cas-
es of acute and chronic lameness of the stifle
joint
34–37
and in dogs that underwent total hip
replacement for OA.
38
Quinn and colleagues
recently studied this question in detail and
demonstrated that subjective scoring scales are
not a replacement for force plate analysis.
39
However, subjective scoring systems are useful
in clinical settings in which force plate analysis
is impractical, such as the multicenter setting
of this trial. The blinded nature of the study
ensures that veterinary bias is negligible; al-
most all dogs were evaluated by one veterinar-
ian throughout all time points, and the scores
reported for all dogs were based on solid exam-
ination findings. We have follow-up studies
designed to evaluate the effects of AD-MSC
therapy using force-plate analysis in a con-
trolled environment.
This study was designed to evaluate the clini-
cal effects of AD-MSC therapy on OA and not
to determine the molecular mechanisms. How-
ever, many published in vitro and in vivo studies
have explored these mechanisms. The im-
munomodulatory effects of BM-MSCs are well
documented and represent one therapeutic
mechanism in which AD-MSCs may func-
tion.
8,40–42
AD-MSC therapy can ameliorate se-
vere graft-versus-host disease in people.
43
MSCs
are well known to secrete cytokines and growth
factors and may stimulate recovery in a paracrine
manner.
8,29
Specifically, Ortiz and colleagues re-
cently reported that BM-MSCs secrete inter-
leukin-1 (IL-1) receptor antagonist (IL-1ra),
which they determined to be the specific mech-
anism that reduced inflammation and fibrosis in
281
L. L. Black, J. Gaynor, D. Gahring, C. Adams, D. Aron, S. Harman, D. A. Gingerich, and R. Harman
Lameness
at Walk
“
Large” Effect
Lameness
at Trot
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Effect Size
Pain on
Manipulation
Range of
Motion
Functional
Disability
Composite
Score
Overall Effect Size Was 1.34, a Large Effect in Orthopedics
1
.34
0
.42
1
.45
1.57
1
.36
0.73
Figure 3. Mean effect size for all individual orthopedic examination meas-
urements and combined score for stem cell–treated dogs. Data are expressed as
the average effect size at three posttreatment time points for each parameter.
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282
a mouse model of lung injury.
43
IL-1 is known to
play a significant role in joint disease and is be-
lieved to be high in the cytokine cascade in all
animal species for which it has been studied.
44
Inhibiting IL-1 with IL-1ra has been shown to
play a beneficial role in equine OA
45–47
and,
therefore, is a likely mechanism by which AD-
MSCs may mediate their effect in canine OA.
Finally, cell-based tissue regeneration may play a
role similar to that seen in the rabbit model of
osteochondral defects.
19
One could imagine that
the AD-MSCs may engraft in synovium or in
cartilaginous lesions and either influence the lo-
cal cells to differentiate into cartilage or the AD-
MSCs themselves may differentiate into carti-
lage. Although the work by Nathan and
associates demonstrates that new cartilage was
formed,
19
other work reveals low levels of BM-
MSC engraftment in a model of spinal cord in-
jury.
47
Chopp and colleagues report that they
have no evidence that the clinical benefit of BM-
MSC therapy in the test rats was the result of dif-
ferentiation but suggest a different hypothesis;
that is, when these cells are placed in an environ-
ment of injury, they express cytokines and
growth factors that promote repair or activate
compensatory mechanisms and endogenous
stem cells within the tissue.
48
These trophic
mechanisms, rather than differentiation, are the
current prevailing theory regarding the clinical
benefits attributed to stem cell therapy.
29,48
■ CONCLUSION
Overall, dogs with OA of the coxofemoral
joint that were treated with intraarticular injec-
tion of AD-MSCs demonstrated statistically
significant improvement in lameness compared
with a blinded, saline-injected control group
and significant improvement over time from
baseline. Indeed, three of these dogs had own-
ers who were considering euthanasia because of
their animal’s pain and functional disability.
These dogs are now living relatively pain free.
The desired clinical outcome for a new treat-
ment modality is better control of patient dis-
comfort and increased functional ability. This
multicenter study shows that intraarticular ad-
ministration of adipose-derived stem and re-
generative cell therapy decreases patient dis-
comfort and increases patient functional ability.
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