Racing statistics indicate tendinitis is the most frequent
cause of breakdown and is often a career-ending event
in Thoroughbreds.1,2 Injuries to the SDFT alone ac-
count for an estimated 8% to 30% of all racing inju-
ries.3–5,a Moreover, recurrence of tendinitis after return
to competition can be as high as 43%.6,7 These issues
have been recognized for many years, yet few major ad-
vances in treatment have improved the proportion of
horses that resume sustained racing.
endinitis of the digital flexor tendons remains a
catastrophic condition in all types of sport horses.
Effect of adipose-derived nucleated cell fractions
on tendon repair in horses
with collagenase-induced tendinitis
Alan J. Nixon, BVSc, MS; Linda A. Dahlgren, DVM, PhD; Jennifer L. Haupt, BS; Amy E. Yeager, DVM;
Daniel L. Ward, PhD
Results—Ultrasonography? revealed? no? difference? in? rate? or? quality? of? repair? between?
Conclusions and Clinical Relevance—ADNC?injection?improved?tendon?organization?in?
ranted.?(Am J Vet Res?2008;69:928–937)
Traditional modes of treatment for tendinitis have
included medical and surgical options.7–14 Most treat-
ment modalities attempt to decrease inflammation
within the tendon, prevent further trauma to damaged
tissues, stimulate the healing process, and prevent re-
currence of the injury after the horse resumes training.
Regardless of the type of treatment selected (ie, medi-
cal or surgical), extended rest with gradual increases in
controlled exercise has been the cornerstone for a suc-
cessful outcome.14–16 Retrospective studies2,17 have re-
vealed that approximately 40% to 60% of injured horses
return to athletic soundness for at least a short period
after injury of the SDFT; however, recurrence of injury
High rates of tendon reinjury have stimulated in-
vestigations of methods for modulating tendon heal-
ing that provide better repair consistent with the
original tendon architecture.2,9,11,13,18 Cells, growth
Received July 31, 2007.
Accepted November 15, 2007.
From the Comparative Orthopaedics Laboratory, Department of
Clinical Sciences, College of Veterinary Medicine, Cornell University,
Ithaca, NY 14853-6401 (Nixon, Dahlgren, Haupt, Yeager); and
the Veterinary Medicine Experiment Station, Virginia-Maryland
Regional College of Veterinary Medicine, Virginia Polytechnic
Institute and State University, Blacksburg, VA 24061 (Ward). Dr.
Dahlgren’s present address is the Department of Large Animal
Clinical Sciences, Virginia-Maryland Regional College of Veterinary
Medicine, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061.
Supported in part by Vet-Stem Incorporated.
The authors thank Mary Lou Norman for assistance with histologic
preparation of tissues.
Address correspondence to Dr. Nixon.
Adipose-derived nucleated cell
Cartilage oligomeric matrix protein
Insulin-like growth factor-1
Mesenchymal stem cell
Superficial digital flexor tendon
AJVR, Vol 69, No. 7 , July 2008 929
factors, and scaffolds to support mixtures of these
components have been described. Adult tissue–
derived stem cells have emerged as injectable potentially
multipotent cells that have the capability of differentiat-
ing and participating in the healing of various muscu-
loskeletal tissues, including tendon.19–29 In adults, these
multipotent cells originate from numerous mesenchy-
mal tissues, such as bone marrow, perivascular tissues,
blood, tendon, muscle, and adipose tissue,30,31 all of
which can be used as a source of autogenous multipo-
tent cells for transplantation. Mesenchymal stem cells
were identified in bone marrow stroma > 25 years ago32;
however, potential drawbacks to the use of bone mar-
row aspirates to provide a pool of MSCs for therapeutic
purposes have included pain associated with the collec-
tion process, low cell yield, and pericardial laceration
during collection of sternal bone marrow.33,34 Addition-
ally, bone marrow–derived MSCs require several weeks
of culture after marrow harvest until they are available
as a substantial pool of cells for reimplantation.35
Adipose tissue can be harvested from several sites in
horses, including pericoccygeal, sternal, and inguinal fat
depots. Coccygeal fat at the base of the tail represents the
most accessible site in standing horses and can be surgi-
cally excised in horses administered sedatives and local
anesthesia. Isolation of the nucleated cell fraction from fat
provides a pool of cells for immediate injection into sites
of injury or for use in further culture propagation to yield
isolated adipose-derived stem cells. The value of ADNC
fractions or adipose-derived multipotent stem cell cultures
for repair of acute and subacute tendinitis and suspensory
desmitis are largely unknown.
Mesenchymal stem cells derived from equine bone
marrow have been evaluated experimentally for carti-
lage repair in horses36 and for treatment of a clinically
affected horse with tearing of the SDFT.22 These reports
support the use of multipotent cells in repair of connec-
tive tissue diseases in horses. Treatment with immediate
reinjection of bone marrow aspirates have been used for
adult horses with tendon and ligament injuries (chron-
ic suspensory disease) for many years.37 In reality, bone
marrow aspirates contain few stem cells and these are
extensively diluted by the large volume of bone mar-
row–derived blood. Culture expansion increases the
number of stem cells available for injection22,35,38; how-
ever, this technique requires a delay of several weeks.
Adipose tissue provides an alternative source of mul-
tipotent cells for culture expansion but may also pro-
vide benefit in the form of concentrated nucleated cell
populations, many of which may be clinically relevant
when compared with bone marrow.39,40 Overnight di-
gestion, separation, and concentration of the nucleated
cell content in fat provides an injectable source of po-
tentially stimulatory cells. However, the effectiveness of
concentrated nucleated cells prepared by digestion and
centrifugation, compared with the effectiveness for cul-
tured multipotent stem cells, is unknown.
Adult-derived stem cells can participate in the
regeneration of damaged tissues via 2 distinct mecha-
nisms. Direct contribution through differentiation into
tissue-specific cell phenotypes and the production
of tissue-appropriate extracellular matrix products is
obvious. To this end, adipose-derived adult stem cells
have the capability of differentiating into osteoblasts,
myoblasts, chondroblasts, and tenocytes in vitro24,30,41
and in vivo.42,43 Potentially of equal importance, adult
tissue–derived stem cells contribute to tissue healing
indirectly through the production of bioactive proteins,
such as growth factors, antiapoptotic factors, and che-
motactic agents.44 These secreted proteins have a pro-
found effect on local cellular dynamics, stimulation of
vascular ingrowth, and recruitment of additional adult
stem cells capable of further stimulation of healing.
The objective of the study reported here was to
evaluate the short-term efficacy of ADNC fractions for
the treatment of horses with tendinitis. Collagenase
was used to induce tendinitis of the SDFT. Clinical,
ultrasonographic, morphologic, biochemical, and gene
expression effects of ADNC injection were compared
with results for vehicle-injected control tendons. The
hypothesis was that intralesional injection of ADNC
fractions would improve healing of tendon core lesions
through an increase in cell numbers, reduction of ten-
don inflammation, and increases in collagen and other
matrix components, which would lead to improved tis-
sue architecture in repaired tendon.
Materials and Methods
Animals—Eight clinically normal young adult (2
to 6 years old) horses of various breeds were used in the
study. Horses were examined clinically and ultrasono-
graphically to ensure there was no preexisting tendon
damage. The project was approved by the Cornell Uni-
versity Institutional Animal Care and Use Committee.
Induction of tendinitis—Tendinitis was induced
in the midmetacarpal region of the SDFT of 1 randomly
selected forelimb of each horse. Tendinitis was induced
by use of a method described and characterized else-
where.45–51 Each horse was medicated with NSAIDs
before injections to induce tendinitis. Regional nerve
blocks were used for pain management, and local anes-
thesia of the skin allowed precise administration of col-
lagenase. Filter-sterilized bacterial collagenase type Ib
diluted in sterile water was prepared, and a small vol-
ume (0.3 mL) containing 2,097 units of collagenase
was divided equally and injected into 2 sites in the cen-
ter of the SDFT approximately 2 cm apart. Collagenase
was injected 13 and 15 cm distal to the accessory carpal
bone. The injected limb was supported by application
of a sterile bandage, and each horse was confined to a
stall without exercise for the initial 4 weeks after col-
Ultrasonographic examinations—Development of
a focal tendinitis core lesion was monitored by use of
repeated ultrasonographic examinations. Ultrasonogra-
phy was also performed weekly after ADNC injection.
Ultrasonographic examinations with a 12- to 15-MHz,
38-mm linear probec were performed in transverse and
longitudinal planes to evaluate the SDFT. Measured
variables were derived from cross-sectional images
obtained 15 and 18 cm distal to the accessory carpal
bone. Cross-sectional area of the tendon core lesion and
cross-sectional area of the total tendon were assessed.
Echoic characteristics of the core lesion on transverse
images and the linear fiber pattern on longitudinal im-
ages were graded.52 Lesion grade was scored on a scale
from 1 (normal tendon) to 4 (extensive damage), and
linear fiber pattern was graded on a scale from 1 to 4 (1
= loss of 0% to 25%, 2 = loss of > 25% to 50%, 3 = loss
of > 50% to 75%, and 4 = loss of > 75% to 100%).
Collection of adipose tissue and preparation of
ADNC fractions—Formation of tendinitis core lesions
was verified ultrasonographically 4 days after collagenase
injection. On day 5 after collagenase injection, adipose
tissue was harvested. Adipose tissue harvest and ADNC
preparation were performed for all horses to ensure inves-
tigators remained unaware of treatments for each horse.
Horses were sedated, the paraxial caudodorsal glu-
teal region was clipped, and the skin was aseptically
prepared. An inverted L pattern of local anesthetic in-
filtration was used for regional desensitization. A linear
incision 10 to 15 cm in length was made approximate-
ly 10 cm abaxial and 10 cm cranial to the tail head;
the incision was centered in the groove formed by the
proximal origin of the biceps femoris and semitendino-
sus muscles. Approximately 15 to 20 g of subcutaneous
adipose tissue was dissected by use of curved scissors.
The adipose tissue specimen was placed in a 50-mL
polypropylene centrifuge tube containing sterile PBS
solution and maintained at 4°C. The skin incision was
closed with 0 polypropylene in a simple interrupted or
Ford interlocking suture pattern. Adipose tissue was har-
vested from 1 site of 4 horses and 2 sites (second specimen
was from an additional identical site in the contralateral
gluteal region) of 4 horses. All horses received NSAIDs for
3 days after harvest of adipose tissue.
Harvested adipose tissue was shipped chilled by
overnight courier to a commercial laboratoryd for isola-
tion of nucleated cells. Collagenase digestion (0.1% in
Dulbecco minimal essential medium) achieved by incu-
bation for 50 minutes and concentrating of cells by serial
centrifugation were performed by use of modifications
of techniques described elsewhere.40,53 After isolation
and purification, the ADNC pellet was resuspended in
sterile PBS solution as 0.6-mL aliquots, each of which
was loaded into 3 sterile syringes labeled with the date
and the identification of the donor horse. Additionally,
a similar number of syringes were loaded with PBS so-
lution and labeled with the date and syringe contents.
The investigator who injected the horses was not aware
of the contents of the syringes. The syringes containing
PBS solution and ADNC fractions were shipped chilled
overnight to the investigators for immediate injection.
ADNC injection—The 8 horses were randomly as-
signed to an ADNC-treated group and a control group
(4 horses/group). Power analysis was used to establish
the number of horses on the basis of the coefficient of
variation for biochemical assays of tendon samples from
other experiments50,51 in which the same collagenase
technique was used. Significance (probability of type
I error) was set at 5% (α = 0.05), and power analysis
was used to calculate a sample size that minimized the
probability of a type II error to ≤ 15% (ie, maximized
the statistical power to ≥ 85%). The operating charac-
teristic was iteratively calculated by use of the follow-
ing equation: φ2 = nΣαδ2/aσ2 = (n/a) X (Σδ2/σ2), where
φ2 is the operating characteristic, a is the number of
main treatments (a = 2 [ADNC and PBS solution]), n is
the sample size per group, and Σδ2/σ2 is the ratio of the
sum of squares of the treatment effect to the variance de-
termined on the basis of other studies with this technique.
The equation was solved for sample size by use of operat-
ing characteristic curves. Power analysis was based on the
SD of collagen, DNA, and proteoglycan values of 12.2%
to 29.2% that have been reported in other studies, which
resulted in an approximate mean SD = 17.7% and Σδ2/σ2 =
1.02/0.1772 = 31.92; hence, φ2=15.96 X n. A sample size of
4 horses/group yielded φ = 7.9 for a statistical power of >
95%. Similar analyses for collagen response genes yielded
power values of ≥ 90%.
Two days after harvest of adipose tissue (ie, 7 days
after collagenase injection), the midmetacarpal area
over the tendinitis core lesion was prepared aseptically
for injection. Three 22-gauge needles were inserted in
each horse. Ultrasonographic guidance was used to en-
sure needles entered the core lesion from the lateral as-
pect of the SDFT. Needles were inserted approximately
1 cm apart starting 15 cm distal to the accessory carpal
bone and progressing in a distal direction. The 0.6-mL
contents of each syringe (ADNCs or PBS solution for
ADNC-treated and control groups, respectively) were
injected into the center of each tendon. The same in-
vestigator performed all intratendinous injections. A
sterile bandage was then applied. Day of injection of
ADNCs or PBS solution was designated as day 0.
Collection of tendon specimens—At the comple-
tion of the 6-week study, horses were euthanatized by
administration of a barbiturate overdose. The SDFT from
the carpus to the proximal reflection of the digital sheath
was harvested by use of RNase-free conditions and stored
on ice for immediate dissection. In the injected tendons,
the region from 13 to 18 cm distal to the accessory carpal
bone was isolated from the remainder of the SDFT; the
central region was then further trimmed, and the cross-
section was photographed. The affected portion of the
tendon was then divided on the midline to provide a lon-
gitudinal sagittal block of tissue for histologic examina-
tion. Longitudinal tissue sections were harvested from the
axial portion of the tendon, and the remaining portions
of the lesion were allocated equally as diced fragments for
biochemical and gene expression assays. Samples for his-
tologic examination were fixed in 4% paraformaldehyde
at 4°C. Diced samples for gene expression assays were
snap-frozen in liquid nitrogen and stored at –80°C until
processed, whereas diced samples for biochemical analy-
sis were rinsed in protease inhibitorse and snap-frozen in
longitudinal tissue segments were dehydrated, cleared
in xylene, infiltrated with paraffin, sectioned at a thick-
ness of 6 µm, and mounted on microscope slides. Tissue
morphologic characteristics were examined on H&E-
stained sections, and collagen architecture was exam-
ined by use of polarized light microscopy on sections
stained with picrosirius red F3B. A composite score of 2
investigators was assigned by use of a scale of 1 (normal
tissue) to 4 (severe changes) for each histologic vari-
able (Appendix). A cumulative score was obtained for
each horse by summing the score for each variable.
AJVR, Vol 69, No. 7 , July 2008 931
Tissue location of mRNA expression for collagen
types I and III was performed by in situ hybridization
with equine-specific sequences corresponding to the 5′
regions for equine collagen type I and type III.50 The [35S]-
uridine triphosphate–labeled sense and antisense probes
were synthesized, applied to serial tissue sections on glass
slides, and incubated overnight in humidified cham-
bers at 43°C, as described elsewhere.50 Slides were then
washed repeatedly, dried, and coated with photographic
film emulsionf for autoradiography. Slides were developed
after 10 to 21 days and counterstained with hematoxylin.
Location of gene expression was assessed and scored on
the basis of defined criteria (Appendix). Similarly, IGF-I
expression in tissue sections was identified by use of ri-
boprobes targeting the 5′ region of equine IGF-I.50 Scores
for IGF-I gene expression were assigned from 1 (> 90%
tenocytes expressing IGF-1 mRNA) to 4 (< 10% tenocytes
expressing IGF-1 mRNA).
Immunohistochemical analysis was used to as-
sess formation of collagen type I and type III proteins
in tissue sections. Primary antibodies were generated
in the Comparative Orthopaedics Laboratory at Cor-
nell University (collagen type I) or were purchased
from commercial vendors (collagen type III).g Non-
immune serum and secondary biotinylated antibody
were obtained from relevant species to allow binding
to the primary antibody. This was followed by the use
of streptavidin-conjugated peroxidase to catalyze color
production from the chromogen diaminobenzidine tet-
rachloride, and sections were then counterstained with
hematoxylin. Abundance and intensity of immunoreac-
tion were graded on a scale of 1 to 4 (Appendix). Nega-
tive procedural control samples consisting of serial ten-
don sections were included on each slide, except the
primary antibody was replaced by nonimmune serum.
Positive and negative tissue control samples consisting
of sections of normal tendon, articular cartilage, and
neonatal costochondral junctions were incubated with
collagen type I or type III primary antibodies.
Gene expression—Tendon specimens for gene ex-
pression assays were pulverized in a freezer millh with
liquid nitrogen. Pulverized tendon was then homog-
enized, and total RNA was extracted by use of the guani-
dinium chloride–phenol extraction processi; total RNA
was purified by use of an affinity column.j Purity and
concentration of RNA were assessed by agarose gel elec-
trophoresis and UV spectrophotometry at 260 and 280
nm. A fluorescence-based real-time quantitative PCR as-
sayk was used to determine gene expression for collagen
types I and III, decorin, and COMP by use of primers and
dual-labeled fluorescent probes generated on the basis of
published equine sequences and designed with a com-
mercially available software program.l Equine sequences
for the primer and probe sets and their use in quantita-
tive PCR assays with an absolute copy number derived
from a standard curve have been described in other stud-
ies49,50,54,55 conducted by our laboratory group. Total copy
number for mRNA of each horse was standardized on
the basis of the total RNA loaded and to expression of
18S ribosomal RNA used as a housekeeping gene.
Biochemical analysis—Frozen tendon specimens
that had been immersed in protease inhibitors were
pulverized in liquid nitrogen and lyophilized. Ten mil-
ligrams of lyophilized tissue was digested by incubation
in 0.5% papainb for 4 hours at 65°C, and the glycosami-
noglycan content was then determined by use of the
dimethylmethylene blue dye–binding assay.56 Residual
papain digest was incubated for another 20 hours at
65°C, and DNA concentrations were then quantified by
use of the bisbenzimide fluorometric assay.57
Concentrations of soluble collagen were measured
by use of the picrosirius red F3B dye–binding method.m
Pulverized, lyophilized tissue samples were weighed;
resuspended in lysis buffer that contained detergent,n
protease inhibitors, and glycerol; homogenized; and
centrifuged at 15,000 X g for 10 minutes. The precipi-
tate was digested by incubation in acid-pepsin at 4°C
overnight with stirring, which was followed by centrif-
ugation at 30,000 X g for 45 minutes. Supernatant was
harvested and used for the dye-binding spectrophoto-
metric assay. Values were standardized on the basis of
the number of milligrams in the dry weight of the origi-
nal tissue specimen.
Statistical analysis—Quantitative data from bio-
chemical and molecular gene expression analyses for
ADNC-treated and control tendons were compared by
use of the Student t test. Histologic scores were com-
pared by use of the Wilcoxon rank sum test. To examine
changes in ultrasonographic cross-sectional area attrib-
utable to treatment and over time, a repeated-measures
ANOVA was conducted to test the main effects of treat-
ment and time and the treatment-by-time interaction.
For each response variable, values recorded before ad-
ministration of treatment were included in the model as
a covariant. Significance was set at values of P ≤ 0.05.
ADNC preparation and injection—Adipose tissue
was successfully harvested from all horses. Two sites
were required in 4 lean horses to obtain 20 g of tissue.
Skin incisions healed without complication in all hors-
es except one, which developed a seroma with subse-
quent dehiscence; the wound in that horse was allowed
to heal by second intention.
Yield of ADNCs ranged from 1.47 X 106 cells/g to
2.71 X 106 cells/g (mean ± SD, 2.30 ± 0.57 X 106 cells/
g). Cell viability after overnight digestion ranged from
83% to 91% (mean, 87.5%).
Tendon injection of ADNCs or PBS solution was
readily accomplished, and there were no adverse effects
that resulted from the injection. Cell viability at time
of injection was not determined. Range of elapsed time
from harvest of adipose tissue to injection of ADNCs
was 40 to 53 hours. Each horse received the 3-syringe
series (0.6 mL/syringe). Mean ± SD number of ADNCs
in each syringe was 13.83 ± 3.41 X 106 cells.
Clinical evaluation—Collagenase-induced disrup-
tion of tendon fibers developed within 5 days after col-
lagenase injection. Persistent enlargement of the SDFT
in the midmetacarpal region was visible throughout
the 6-week study. There were no apparent clinical dif-
ferences in the degree of swelling between the affected
limbs in the 2 groups of horses.
Gross morphologic characteristics—Surface epi-
tenon layers of ADNC-treated tendons appeared less
hemorrhagic and had a smoother surface, compared
with the appearance of control tendons (Figure 1).
Examination of transverse sections revealed the site
of tendinitis, which varied from brownish to reddish.
There were no differences of color in transverse sec-
tions between the 2 treatment groups.
Ultrasonographic examination—Quantitative analy-
sis of tendon ultrasonographic images revealed no differ-
ences between the 2 groups during the period preceding
ADNC or PBS solution injection or throughout the 6 weeks
after ADNC or PBS solution injection (Figure 2). Scores
for linear fiber pattern improved in both groups during the
course of the study. Scores for linear fiber pattern were ini-
tially higher (more deranged) in the ADNC-treated group,
but improved to become lower than scores for the control
group at 35 and 42 days after injection. Similarly, ADNC-
treated tendons revealed improvement on the basis of le-
sion grades for days 14 through 42 after injection.
Histologic examination—Longitudinal sections
stained with H&E and picrosirius red were analyzed and
scored. Normal tendon had a perfect score of 13, and
maximally damaged tendon had a score of 52. Mean ±
SD overall healing score for ADNC-treated horses (24.8
± 2.1) was significantly (P = 0.029) better, compared
with the overall score for PBS solution–treated horses
(33.5 ± 2.8). In general, scores for variables in ADNC-
treated tendons were significantly improved, compared
with scores for control tendons, including better orga-
sections? of? SDFTs? obtained? from? horses? with? collagenase-in-
AJVR, Vol 69, No. 7 , July 2008 933
Figure 3—Photomicrographs of tissue sections of SDFT from
representative horses obtained 42 days after injection of PBS
solution (A and B) or ADNCs (C and D). Sections were stained
with H&E and examined by use of light microscopy (A and C)
and polarized light microscopy (B and D) to reveal organization of
tendon fibers. Bar = 100 µm.
Figure 4—Photomicrographs of tendon sections from SDFTs in-
jected with ADNCs (A and B) and PBS solution (C and D) and ex-
amined by use of polarized light microscopy. Note the periodicity
for crimp of the tendon fibers and cocrimp organization. The edge
of each lesion (A and C) has a more normal coarse crimp and
cocrimping, whereas the center of each lesion (B and D) has bet-
ter crimp formation in the ADNC-injected tendons (B). Picrosirius
red F3B stain. Bar = 100 µm.
nization of tendon fibers (P = 0.08) and reduced WBC
infiltrates (P = 0.03). Significant improvement in scores
for organization of tendon fiber, which comprised lin-
earity of collagen fibers, uniformity, and crimp and
cocrimp appearances for polarized light microscopy,
was evident in ADNC-injected tendons (mean, 1.79 ±
0.6), compared with scores for control tendons (2.79 ±
0.4; Figure 3). Loss of cross linking of collagen fibers
was apparent in the central bundles of tendon fibers
from ADNC-injected SDFTs. Improvements in unifor-
mity of collagen fibers and crimping appearances were
apparent during polarized light microscopy of sections
stained with picrosirius red F3B stain (Figure 4).
Immunohistochemical analysis for collagen—Dis-
tribution of collagen type I and type III among longitu-
dinal sections of tendon was scored; these data were
included in the composite score for histologic grading.
There were no apparent differences in proliferation or
spatial arrangement of collagen type I within ADNC-
injected or control tendons. Formation of collagen type
III in ADNC-treated tendons was reduced throughout
the tendon sections, with concentrations around re-
sidual regions that had higher cellularity, which also
coincided with decreases in crimping and cocrimping
of collagen fibers (Figure 5).
Expression of collagen mRNA—Collagen gene ex-
pression by cells in the tendons was assessed by in situ
localization of collagen type I and type III mRNA. Expres-
sion of collagen type I was increased in areas of endotenon
proliferation within each section and in the epitenon. Ap-
parent differences were not evident between ADNC-treat-
ed and control tendons. Expression of collagen type III
Figure 5—Photomicrographs of collagen type III immunoreactivity in tissue sections of representative SDFT samples obtained from 3
horses injected with PBS solution (A through D) or ADNCs (E through H). Control sections (D and H) resulted from application of nonimmune
serum to serial sections. Collagen type III has formed throughout all sections, and intense deposition of collagen type III is apparent through-
out bundles of tendon fibers in tendons injected with PBS solution (A through C). Reaction used primary antibodies against collagen type III
and biotinylated secondary antibodies; diaminobenzidine tetrachloride chromogen; counterstained with hematoxylin. Bar = 100 µm.
934? ??AJVR,?Vol?69,?No.?7 ,?July?2008
was evident in areas of tendon healing and was reduced
in ADNC-treated tendons. Combined histologic scores for
collagen type III mRNA and collagen deposition revealed
significant (P = 0.03) improvement in ADNC-injected ten-
dons (mean ± SD, 2.13 ± 0.48), compared with scores for
control tendons (3.13 ± 0.25).
Expression of IGF-1—To evaluate other reasons
for the improvement in structural organization of
ADNC-treated tendons, spatial expression of IGF-1 was
assessed by use of in situ hybridization. Expression of
IGF-1 was evident at similar intensity and distribution
throughout tendon sections from ADNC-treated and
control tendons. Expression was evident in tenocytes
as well as endotenon layers (Figure 6). Scores for ex-
pression of IGF-1 in sections from ADNC-treated and
control tendons were similar.
Expression of matrix genes—Gene expression for
decorin and collagen type I and type III was not signifi-
cantly different between groups (Table 1). Expression
for COMP was significantly (P = 0.022) increased in the
ADNC-treated group, compared with expression for the
Biochemical assessment—The DNA content, gly-
cosaminoglycan content, and content of total soluble
collagen did not differ significantly between ADNC-
treated and control tendons (Table 2).
Intralesional injection of isolated ADNCs during
healing of tendinitis in the SDFT improved the outcome
for tendon structure. The short-term study reported here
was designed to assess the immediate impact of injec-
tion of multipotent cells into an acute tendinitis lesion.
Assay of DNA content and scoring of cell density during
histologic examination suggested that ADNC injection
had little impact on overall cell number. Rather, the prin-
cipal effect appeared to be an anti-inflammatory benefit
and improvement in structural organization in the heal-
ing tendon. Semiquantitative morphologic indices for col-
lagen organization and tendon fiber architecture revealed
improvement, and in combination with a reduction in in-
flammatory cell infiltrate, the composite histologic scores
for ADNC-injected tendons were superior to those for
control tendons injected with PBS solution. Both MSCs
and adipose tissue–derived stem cells have immunosup-
pressive effects,58,59 and the stem cell component (as well
as potentially other cell types) within ADNC injections
may reduce local tissue inflammation.
Surgical harvest of adipose tissue was technically
simple. When horses were particularly lean, adequate
samples of adipose tissue were obtained via bilateral ac-
cess to the dorsal gluteal region. Digestion of the adi-
pose tissues, separation of the lipid, and concentration
of the remaining nucleated cells provided the injectable
product. The technical aspects of limited incision and
dissection of subcutaneous adipose tissues were slightly
more complex than those for aspiration of bone marrow
from the sternum or tuber coxa. However, the ADNC
product differs from bone marrow aspirate. Digestion
and centrifugation of ADNC and removal of lipid from
the adipose sample results in a concentrated cell mass,
compared with the predominantly blood-diluted con-
stituents of a bone marrow aspirate. Achieving higher
cell numbers from marrow requires propagation of the
adherent population over a period of several weeks,
which does not compare favorably with the short in-
terval for generation of a nucleated cell pool following
digestion of adipose tissue.22,25 Additionally, regulatory
authorities (eg, the FDA Center for Veterinary Medi-
cine) allow autologous minimally manipulated cell
therapy in horses when the procedures do not apprecia-
bly change the cells (ie, differentiation), whereas more
manipulative methods, such as culture, differentiation,
or sorting, may require formal approval as a drug before
clinical use, which would add another dimension to the
choice of treatment.
Impact of the various cell types in adipose tissue di-
gests and bone marrow aspirates are largely unknown.60
Furthermore, the value or necessity for isolating and
proliferating specific adherent cell lines from each tissue
source, and the impact of additional growth factors con-
tained in bone marrow and at unknown quantities in adi-
pose tissue or digested derivatives, are poorly understood.
Harvest of multipotent cells from adipose tissue and bone
11.78??3.73? 14.70??4.62? 9.57??2.30? 1.10??0.48
11.98??1.95? 15.38??2.79? 7.62??3.27? 2.33??0.64*
by? use? of? fluorescence-based? real-time? PCR? assay? in? healing?
Figure? 6—Photomicrographs? of? expression? of? IGF-1? mRNA? in?
AJVR, Vol 69, No. 7 , July 2008 935
marrow aspirates results in a heterogenous population of
nucleated cells.35 Use of limited enzymatic digestion of
adipose tissue and collection of all nucleated cells broad-
ens the heterogeneity of cell types to include endothelial
cells, fibroblastic cells, pericytes, smooth muscle cells,
macrophages, and other formed nucleated blood cells.30 It
is not apparent from the literature which of these lineages
is potentially stimulatory to tendon healing. However, at a
minimum, pericytes are multipotent and may contribute
to the local tenocyte population.53,61 Other studies30,39,62
have revealed that approximately 80% of the cells isolated
from human lipid aspirates are mesenchymal in origin
and, when provided with appropriate in vitro factors, can
differentiate into adipose tissue, bone, cartilage, muscle,
and nerve cell lineages.
Use of adult tissue–derived stem cells to enhance
healing of connective tissue may provide more than cells
alone. Interest has been directed at the anti-inflamma-
tory, antiapoptotic, and growth factor stimulatory as-
pects of injection with multipotent cell mixtures.58,59,63
For tendon healing, a combination of anti-inflamma-
tory effects and bolstered cell numbers may minimize
the breakdown of propagating collagen fibers that re-
sult from the original injury. Moreover, the addition of
growth factors active in enhancing vascular ingrowth,
particularly vascular endothelial growth factor, may
play a role in revascularization of the poorly viable core
lesion within tendinitis regions in horses.64,65 The role
and temporal sequence characteristics of growth factors
and other bioactive peptides in tendon injury and the
acute phase of healing have been defined only for a few
growth factors.50 Injection of ADNCs has the propensity
to provide anti-inflammatory effects,58,59 add bioactive
peptides, and potentially transplant a multipotent cell
pool that can contribute to reformation and architec-
tural organization of tendon fibers. Results of the study
reported here suggest that the effect of ADNCs may
be more in maintaining or inducing organized tendon
architecture, rather than in provision of an increased
pool of cells to participate in the healing response. Ad-
ditionally, although DNA content was similar in treated
and control tendons, ADNC-injected tendons had a re-
duction in inflammatory cells within the lesion, which
suggests that other cell populations (such as endotenon
and tenocyte layers) may be enhanced in ADNC-treated
repair of tendons. This contradicts a study66 of MSC ef-
fects on repair of patellar tendons in rabbits in which
the investigators concluded that improvements in me-
chanical capabilities were not attributable to improve-
ments in tendon organization. However, that study in
rabbits did not include DNA data or histomorphomet-
ric quantitation of cell numbers, and few legitimate
comparisons can be made with results for our study.
However, without labeling of the autologous ADNCs
prior to injection, the study reported here lacks specific
information on cell survival to enable us to characterize
cell persistence and the impact on tendon repair.
Expression of COMP was significantly increased in
the ADNC-injected tendons. Originally identified in car-
tilage, COMP is a noncollagenous glycoprotein that pro-
vides structural integrity to the extracellular matrix by
binding to multiple collagen fibrils. It may play a role in
promoting formation of collagen fibrils, which could po-
tentially influence the quality of tendon and integrity of
cartilage matrix.67,68 Concentrations of COMP in equine
digital flexor tendons are positively correlated with ulti-
mate tensile strength and stiffness, which suggests that
COMP concentrations may be linked to organization
of the tendon matrix.69,70 An increase in expression of
COMP mRNA in the ADNC-treated tendons was con-
sistent with the histologic evidence of improvements in
tendon architecture and may be indicative of improve-
ments in tendon regeneration, or at the least, reductions
in degeneration of tendon fibers as a result of ADNC
treatment. This may be particularly relevant because
analysis of COMP concentrations in synovial fluid, ten-
don sheath fluid, and plasma are influenced considerably
more by tissue heterogeneity than COMP concentrations
in this focal tendinitis repair site.
Tendons injected with ADNCs had reductions in
inflammatory cell infiltrate. This suggested that ADNCs
may have had an anti-inflammatory effect on the re-
generating tendon or may have had some other anti-
inflammatory effect through stabilizing or minimizing
ongoing degeneration of tendon fibers. The mechanism
for the ADNC anti-inflammatory effects are largely un-
known, and the combination of an anti-inflammatory
effect, discrete antiapoptotic effects, and recruitment of
additional local multipotent stem cell pools to contrib-
ute to tendon healing are all possible.27 These mecha-
nisms likely contributed to the reduction in tissue
inflammation and improvements in tendon fiber archi-
tecture detected in the ADNC-treated tendons.
Adipose tissue provided a source of cells that
appeared to contribute to tendon repair in various
ways. Isolation of an injectable cell pool derived from
adipose tissue provided distinct advantages with regard
to timeliness, compared with the time frame for cultured
multipotent stem cells derived from other tissue sources.
Cost savings as a result of the reduction in the interval
from tissue collection until cell injection, simplicity
of laboratory procedures, and potential for a bioactive
mixture of cells and intrinsic peptides provided
additional advantages, compared with use of an injection
of cultured MSCs.71 Morbidity associated with harvest
of adipose tissue was mild, cell yield was high, and the
interval from tissue harvest until injection of the cellular
product was minimal, compared with results for cultured
MSCs derived from bone marrow.22,25 The improvements
in overall histologic appearance of the tendon tissue
and gene expression of a glycoprotein that correlated
with organized mature tendon tissue were important
variables for assessment of tendon repair. The short-term
morphologic results for the study support the need for
long-term studies and potential clinical trials of ADNCs
for the treatment of horses with tendon injuries.
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Shape of tendon cells
1 = Linear (normal)
2 = Slightly oval
3 = Moderately round
4 = Predominantly round
Density of tendon cells
Neovasculature (No. of vessels)
1 = Sparse (normal)
2 = Slight increase
3 = Moderate increase
4 = Sheets of cells
1 = None
2 = Sparse or patchy
3 = Multiple areas in each low-
4 = Predominant hemorrhage
1 = Normal
2 = Slight increase
3 = Moderate increase
4 = Severe increase
Inflammatory cell infiltrate
(leukocyte deposits in
endotenon and peritenon)
1 = None
2 = Slight increase
3 = Moderate increase
4 = Severe increase
Linearity of collagen fibers
1 = Linear
2 = 50% linear
3 = 20% to 50% linear
4 = No linear areas
Uniformity of collagen fibers
Crimping of collagen fibers*
Thickness of epitenon
Collagen type I†
Collagen type III†
mRNA for collagen type I‡
mRNA for collagen type III‡
1 = Uniform diameter of all
2 = 50% of fibers are uniform
3 = 20% to 50% of fibers are
4 = Complete disarray of fibers
1 = Coarse, even crimp
2 = Predominantly fine, even
3 = 50% with crimp formation
4 = No crimp formation;
1 = 1 to 2 cells (normal)
2 = 3 to 6 cells
3 = 7 to 15 cells
4 = Massive fibrosis
1 = 90% type I
2 = 50% to 90% type I
3 = 10% to 50% type I
4 = 10% type I
1 = 10% type III
2 = 10% to 50% type III
3 = 50% to 90% type III
4 = 90% type III
1 = 90% type I
2 = 50% to 90% type I
3 = 10% to 50% type I
4 = 10% type I
1 = 10% type III
2 = 10% to 50% type III
3 = 50% to 90% type III
4 = 90% type III
*Determined by use of polarized light microscopy. †Determined
by use of immunohistochemical analysis. ‡Determined by use of in
Histologic scoring system used for grading healing of tendon
tissues in horses.