Research in Veterinary Science 118 (2018) 317–323

Contents lists available at ScienceDirect

Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

Review

Concepts and challenges in the use of mesenchymal stem cells as a treatment T

for cartilage damage in the horse
Mohammed Zayeda,1, Steve Adaira, Tena Ursinia, James Schumachera, Nabil Miskb,
⁎
Madhu Dhara,
a
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
b
Department of Animal Surgery, College of Veterinary Medicine, Assuit University, 71526 Assuit, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: Osteoarthritis (OA), the most common form of joint disease affecting humans and horses, is characterized by the
Regenerative medicine advance and decline of cartilage and loss of function of the affected joint. The progression of OA is steadily
Mesenchymal stem cell accompanied with biochemical events, which interfere with the cytokines and proteolytic enzymes responsible
Cartilage for progress of the disease. Recently, regenerative therapies have been used with an assumption that me-
Osteoarthritis
senchymal stem cells (MSCs) possess the potential to prevent the advancement of cartilage damage and po-
Horse
Review
tentially regenerate the injured tissue with an ultimate goal of preventing OA. We believe that despite various
challenges, the use of allogenic versus autologous MSCs in cartilage regeneration, is a major issue which can
directly or indirectly affect the other factors including, the timing of implantation, dose or cell numbers for
implantation, and the source of MSCs. Current knowledge reporting some of these challenges that the clinicians
might face in the treatment of cartilage damage in horses are presented. In this regard we conducted two
independent studies. In the first study we compared donor matched bone marrow and synovial fluid – derived
equine MSCs in vitro, and showed that the SFMSCs were similar to the BMMSCs in their proliferation, expression
of CD29, CD44 and CD90, but, exhibited a significantly different chondrogenesis. Additionally, 3.2–21% of all
SFMSCs were positive for MHC II, whereas, BMMSCs were negative. In the second study we observed that
injection of both the autologous and allogenic SFMSCs into the tarsocrural joint resulted in elevated levels of
total protein and total nucleated cell counts. Further experiments to evaluate the in vivo acute or chronic re-
sponse to allogenic or autologous MSCs are imperative.

1. Introduction
The inflammation of the synovial membrane leads to synovial cell
proliferation, pain, chronic dysfunction, and it initiates the deteriora-
tion of the cartilage in horses (McIlwraith CW, 1996). If not treated or
controlled, cartilage degeneration progresses and results in osteoar-
thritis (OA). Currently, there are many options for treating OA in
equids. These include systemic or intra-articular administration of non-
steroidal anti-inflammatory drugs (NSAIDs), hyaluronan (HA), corti-
costeroids (CSs), polysulphated glycosaminoglycan, articular resurfa-
cing techniques, including microfracture, autologous chondrocytes
implantation, and osteochondral autologous graft procedures (Frisbie
et al., 2003; Litzke et al., 2004). The poor intrinsic capability of ar-
ticular cartilage to repair makes the effective treatment of cartilaginous
defects challenging (Buckwalter, 1998). Even though various cellular
transplant techniques and the use of drugs have been intended to aid in
the regeneration of cartilage, the outcome is not very effective. The
defects characteristically heal with fibrous tissue, rather than hyaline
cartilage, and hence, the biochemical and biomechanical qualities of
the repaired cartilage are suboptimal (Jackson et al., 2001; Buckwalter,
2002). As a result, there is still an unmet need to identify an efficient
and efficacious strategy to control the progression of cartilage de-
gradation and the subsequent treatment of OA in horses.
2. Articular cartilage
Articular cartilage, the most abundant type of cartilage found in
horses, is composed of flexible highly specified connective tissue. This
connective tissue composed of a dense matrix of collagen fibers and
elastic fibers embedded in a rubbery ground substance makes the car-
tilage perfect for bearing mechanical stresses without permanent de-
formation. Articular cartilage gets nourishment through diffusion of the

⁎
Corresponding author at: Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Drive Knoxville, TN 37996-4500, USA.
E-mail address: mdhar@utk.edu (M. Dhar).
1
Department of Animal Surgery, College of Veterinary Medicine, South Valley University, 83,523 Qena, Egypt.

https://doi.org/10.1016/j.rvsc.2018.03.011
Received 18 December 2017; Received in revised form 13 March 2018; Accepted 18 March 2018
0034-5288/ © 2018 Elsevier Ltd. All rights reserved.
M. Zayed et al.
synovial fluid (Brama et al., 2000). The chondrocyte is the inhabitant
cell type within articular cartilage and is responsible for synthesizing
and controlling the extracellular matrix. Chondrocytes depend on
anaerobic metabolism because they receive no direct supply of nu-
trients from blood vessels or lymphatics (Buckwalter, 1998); hence, it
has minimal potential to regenerate. It is suggested that injured carti-
lage and the associated synovial tissues cannot produce cartilaginous
tissue with the same morphologic and biomechanical properties of
healthy articular cartilage (McIlwraith, 1996; Buckwalter, 1998). The
development of a joint disease, such as OA, is associated with sig-
nificant alterations in the metabolism of cartilage, physiological im-
balance of degradation, and synthesis factors of chondrocytes
(Todhunter, 1996).
3. Treatment options for OA
The use of NSAIDs and CSs as a conservative treatment option can
be used to inhibit the inflammation but cannot prevent the progression
of OA or regenerate the lost tissues (Goodrich and Nixon, 2006). The
healing of cartilage through the regeneration of hyaline tissue, rather
than the production of fibrocartilage, should keep the damaged joint
with the best strength. Surgical approaches currently used to regenerate
damaged articular cartilage are complex and not very efficacious. One
of these techniques is bone marrow stimulation, which includes tech-
niques of abrasion chondroplasty (Edward et al., 2007), debridement
down into subchondral bone (Story and Bramlage, 2004), and sub-
chondral drilling (Lang et al., 2009). The clinical efficiency of the ar-
throscopy of microfracture for articular cartilage regeneration in horses
has also been evaluated in an equine model of cartilage healing (Frisbie
et al., 2003). Although the clinical functionality was improved, the
hyaline cartilage content was not optimal and methods to further en-
hance the quality of repaired cartilage are needed.
Autologous chondrocyte implantation is another procedure used to
treat full-thickness defects of articular cartilage. In an equine model,
this procedure was used to evaluate the effect of fixed chondrocytes
using a periosteal flap in a partial- and full-thickness cartilage defects.
Results indicated that the cartilage defects recovered with better car-
tilage healing scores (Nixon et al., 2011). Additionally, autologous os-
teochondral autografts may be substituted for small and medium-sized
focal chondral and osteochondral defects (Hangody et al., 2008).
However, clinical outcomes of regenerating fibrous tissue rather than
hyaline cartilage, the reduction in biochemical and biomechanical
qualities, the complex and invasive nature of those procedures, together
with the high costs are serious drawbacks for these procedures
(Buckwalter, 2002; Cokelaere et al., 2016).
Interleukin 1 receptor antagonist protein (IRAP) that neutralizes the
destructive effects of IL-1 (DeForge et al., 1992). IRAP was developed to
counteract IL-1 that is produced in the traumatized joint. IRAP works by
preventing IL-1 binding to the IL-1 receptors, therefore blocking the
damage and inflammation caused by IL-1 (Hannum et al., 1990;
Eisenberg et al., 1990). Frisbie et al. injected equine IRAP gene into
experimentally-induced OA, resulting in significant improvement of
clinical and histological signs, whereas placebo-treated horses did not
(Frisbie et al., 2002). IRAP however, cannot regenerate lost tissues.
Platelet-rich plasma (PRP) is plasma enriched with platelets relative
to the whole blood. It has been demonstrated in vitro that when platelets
within the PRP are stimulated by collagen (Harrison et al., 2011), they
release growth factors including, transforming growth factor beta (TGF-
β), platelet-derived growth factor, epidermal growth factor, and in-
sulin-like growth factor, all of which can significantly affect cartilage
regeneration (Amable et al., 2013). These growth factors act synergis-
tically to enhance the access of healthy inflammatory cells to the area of
injury and speed the formation of connective tissue at the site of injury
(van Buul et al., 2011). PRP has also been suggested to regulate the
action of matrix metalloproteinases to activate signal transduction
pathways (Patel et al., 2013). Most of the animal or human studies
318
Research in Veterinary Science 118 (2018) 317–323
using PRP have limitations due to small sample sizes or nonrandomized
methodology (Sampson et al., 2008; Mishra et al., 2009; Rowden et al.,
2015). Hence, the use of PRP in the clinic has been limited (Sheth et al.,
2012). It is difficult, however, to identify if the failure is due to the
actual treatment or the lack of standardized protocols, platelet se-
paration techniques, and consequence measures.
4. MSCs
MSCs are plastic-adherent cells with a fibroblast-like morphology,
have the capability of renewing themselves through cell division, and
have ability to differentiate into bone, cartilage, and fat in vitro. MSCs
originally are considered to be a type of pericyte or adventitial cells
(Krampera et al., 2007; Caplan, 2017). When implanted in vivo, MSCs
provide an immunomodulatory and anti-inflammatory effects on the
injured tissue (Asari et al., 2009). MSCs supply growth factors, re-
generative cells, and the extra-cellular matrix necessary for cartilage
repair. When MSCs are administered intra-articularly, MSCs will adhere
to injured tissues, where they may potentially undergo differentiation
or may induce the endogenous progenitor cells to differentiate, thereby,
regenerating the injured articular cartilage (Koch et al., 2008).
Bone marrow is a common source of MSCs (BM) (Govoni, 2015), but
MSCs have also been derived from adipose tissue (AT) (Vidal and Lopez,
2011), umbilical cord blood (UCB) (Tessier et al., 2015), peripheral
blood (Dhar et al., 2012), synovium and synovial fluid (Murata et al.,
2014). Synovial fluid-derived MSCs (SFMSCs) have been reported to
have features similar to the MSCs isolated from the bone marrow and
the adipose tissue, and have become a valuable source of MSCs for the
treatment of cartilage injuries (Murata et al., 2014; Prado et al., 2015;
Mak et al., 2016; Zayed et al., 2017; Yao et al., 2018).
Synovial fluid lubricates and nourishes articular cartilage. A de-
crease in the quantity of synovial fluid within a joint is attributed to
inflammation, especially at the early stage of OA, which leads to the
wearing of cartilage (Elsaid et al., 2005; Antonacci et al., 2012). Ad-
ditionally, synovial fluid contains important growth factors, such as
TGF-β1 and fibroblastic growth factor, which help in chondrogenesis
(Okazaki et al., 2001). Studies have illustrated that ovine BMMSCs co-
cultured with normal synovial fluid express chondrogenic markers after
2 weeks (Chen et al., 2005). Synovial fluid from injured human knee
joints significantly affects MSCs in an in vitro model of chondrogenesis
(Zhang et al., 2008), indicating that synovial fluid is important for MSC
differentiation.
Primary cultures of cells are classified as MSCs based on the prop-
erties displayed after they are cultured and expanded in vitro (Dominici
et al., 2006). One of these properties is the ability to adhere to poly-
styrene surfaces and establish colonies of cells (Friedenstein et al.,
1970). Another is their capacity to self-renew through mitosis (Qiu
et al., 2016). Finally, under specific cell culture medium conditions,
MSCs can differentiate into cell types of the mesenchymal origin (bone,
fat, and cartilage) (Vidal et al., 2006; Barberini et al., 2014). At the
same time, MSCs may differentiate into a cell type of a non-mesodermal
origin (Song et al., 2006). Because MSCs are able to differentiate into
chondrocytes, they are a potential source of cells for regenerating in-
jured cartilage tissue in clinical cases (Seo et al., 2014).
Differentiation of MSCs into chondrocytes is a complex process.
After MSCs are subjected to conditions that induce chondrogenesis,
they begin to condense and form high-density, cellular aggregates,
which progress into the deposition of cartilaginous extracellular matrix
and cartilage formation (Yamashita et al., 2010) (Fig. 1). TGF-β1, β2,
and β3 are the most commonly used isoforms, which serve as inducers
of chondrocytes, and exposing MSCs to these growth factors leads to
differentiation i.e. accumulation of proteoglycan and collagen type II
(Tuli et al., 2003; Wang et al., 2014). Other inducers of chondrogenesis
include BMP7 or BMP9 and IGF1 (Murphy et al., 2015). SRY-type HMG
box9 (Sox9) is a master regulator for chondrogenesis and stimulates
transcriptional activation of genes that develop the components of the
M. Zayed et al. Research in Veterinary Science 118 (2018) 317–323

Fig. 1. The phases of chondrogenesis. Mesenchymal stem cells (MSCs) were stimulated to undergo chondrogenesis in monolayer culture for 14 days in chondrogenic
medium containing TGF-β1. (A) The diagram shows the principal stages of chondrogenesis and indicates the role of Sox9, TGF-β1, FGF-2 and BMP-2 as a stimulant at
different stages during differentiation. (B) Expression of cartilage-specific marker (collagen type II) was detected by immunofluorescence in cultured MSCs; the image
demonstrates that collagen II is present throughout the section (green) and is merged with DAPI. The images illustrate the different phases of chondrogenesis
described in the referenced paper (Yamashita et al., 2010), are generated from our laboratory work. Scale bar: 100 μm. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

cartilage matrix, including collagen type II (Dy et al., 2012). It has been
shown that the relative expression of collagen II, a specific marker of
hyaline cartilage, depends on the expression of the Sox9 gene (Bi et al.,
1999; DeChiara et al., 2000).
5. Clinical application of MSCs
Clinical application or implantation of MSCs for the treatment of
cartilage damage can be determined by a number of factors, including
the dose, time of implantation, source, and the number of injections
required (Fortier et al., 2011). Since controlled studies investigating
each of these parameters is lacking, the clinicians use their judgement
with the hope of obtaining a positive clinical outcome. Using the pa-
tient's own MSCs (autologous) to treat an injury or disease is believed to
be safer than using MSCs from other donors (allogenic). One of the
major challenges in using autologous MSCs, however, is the timing.
Roughly 3 weeks are needed to expand and generate sufficient cell
numbers for implantation. This is highly dependent on the tissue har-
vest, isolation, characterization, and cell culture propagation cap-
abilities. The time necessary to isolate the cells and implant them
clinically might hinder the use of autologous MSCs. In addition, age,
gender, and disease can also significantly affect the biological proper-
ties and the function and thus, the efficacy of MSCs derived from spe-
cific tissue sources (Siegel et al., 2013). Using allogeneic, “off-the-
shelf,” in vitro characterized MSCs alleviates some of these challenges.
Using allogenic MSCs that have been expanded in culture, proven to
undergo tri- lineage differentiation, and immunophenotyped to express
specific protein markers (CD29, 44, 90, 105) may be more efficacious
than using autologous MSCs, which are rarely characterized prior to
therapeutic application. MSCs have immunosuppressive properties,
which primarily result from the lack of major histocompatibility class II
(MHC II) antigens and the inhibition of T helper cytokines (Machado
et al., 2013). Even though it has been shown that MSCs lack MHC II, a
slight increase in MHC II has been observed when MSCs undergo
chondrogenesis, in vitro (Le Blanc et al., 2003), which may or may not
affect the in vivo function. It has been demonstrated that equine MSCs
from different tissues modulate the function of immune cells by unique
mechanisms (Paterson et al., 2014; Carrade et al., 2014) and hence, if
MSCs lack the MHC II proteins and can inhibit T cell activation and
proliferation, they can potentially be implanted allogenically.
Despite the potential for increased immunogenicity, certain limita-
tions of autologous cells have allowed allogenic cells to remain as a
treatment option. As an animal or a human ages, the differentiation
potential, expansion potential, and the overall number of stem cells will
decrease (Fafián-Labora et al. 2015). Isolation and expansion of
autologous cells normally takes roughly three weeks; however, it may
take longer in some patients (Watts, 2014). Due to the wide variety of
patients and injuries being treated with stem cells, it is impossible to
compare optimal treatment protocols. There is no appreciable literature
determining the ideal time, number of doses, or number of cells per
injection. Twenty million stem cells have become the current “cell
dose” based on evidence of improved results (Smith, 2014); however,
doses can range from 10 to 50 million cells (Frisbie, 2014). Recently, it
has been suggested to inject joints four to six weeks post injury or
surgery to allow the inflammatory phase to pass (Frisbie, 2014); which
is dependent on the clinician's judgement. More work must be done in
controlled models to determine the ideal dose and timing of injections.
Two studies showed that administration of allogeneic UCB or AT-
derived MSCs into normal joints did not cause a greater local in-
flammatory response than autologous administration (Carrade et al.,
2011; Pigott et al., 2013b). In contrast, Pezzanite et al., 2015 showed
that intradermal injection of allogenic MSCs produced antibody re-
sponses and could reduce the effectiveness of allogeneic MSCs injected
repeatedly into the same horse (Pezzanite et al., 2015). In view of these
conflicting reports, the specific in vitro and in vivo immunomodulatory
effects of equine MSCs should be more thoroughly evaluated before
allogeneic MSCs are used to treat horses for OA or other diseases. In
summary, several restrictions must be overcome before MSCs can be
used extensively for the treatment of articular cartilage damage. Vari-
able reports regarding safety, limited clinical data to support efficacy,
and excessive differences in patient reaction must be addressed (Fink,
2009).
6. Potential mechanism(s) of action of MSCs in cartilage repair
As described above, MSCs can differentiate into multiple cell-
lineages and strategies exist to grow them in culture; hence, the interest
in a cell-based therapeutic approach for regenerating equine cartilage
using MSCs has increased in the past few years (Wilke et al., 2007; Koch
et al., 2008; De Schauwer et al., 2013). Despite the increase in the use of
MSCs in the clinic, the exact mechanism(s) by which MSCs speed the
healing of the damaged tissue has not been determined. Several the-
ories have been put forward. Research suggests that MSCs are attracted
to the site of the injury, adhere to the damaged tissue, and differentiate
into specific cellular phenotypes, such as chondrocytes, osteocytes, or
tenocytes, (Gruh and Martin, 2009; Prockop and Oh, 2012). Alter-
natively, MSCs might function by recruitment of anti-apoptotic factors
to inhibit cell death, formation of new blood vessels, recruiting growth
factors, and anti-inflammatory proteins to the site of injury to re-
generate and suppress inflammation in the injured tissue (Yagi et al.,

319
M. Zayed et al.
Fig. 2. Outline of the main molecular, synovial and chondral changes that occur in the synovial joint during osteoarthritis (OA). This schematic underlines the
consequence of actions which direct to chondral degradation. Due to external stimuli on the synovial joint, pro- inflammatory cytokines, inflammatory meditators
and proteolytic enzymes will be released. Subsequently, chondral changes include cartilage degradation and loss of Aggrecan, COMP and collagen type II (Col II). The
figure was generated using the information provided in the referenced paper (Bonnet and Walsh, 2005).
2010; English, 2013).
Synovitis, inflammation of the synovial membrane, is one of the
earliest changes in the pathophysiology of OA of horses and is asso-
ciated with changes in cartilage (McIlwraith, 1996). Pro-inflammatory
cytokines, such as IL-β1 and TNF-α, and extracellular matrix–degrading
enzymes, such as matric metalloproteinases, aggrecanases, and neuro-
peptides,are generated by the inflamed synovium. These substances
modify the balance of catabolic and anabolic factors, leading to the
breakdown of cartilage (Bonnet and Walsh, 2005) (Fig. 2). Therefore,
controlling or resolving synovitis may inhibit the catabolic processes
within the joint, subsequently protecting cartilage damage. Therapy to
resolve non-infectious synovitis of horses involves systemic adminis-
tration of NSAIDs, PSGAGs, and intra-articular administration of CSs,
HA, or ACS (Doucet et al., 2008; Frisbie et al., 2007). Suppression of
lymphocytes by intra-articularly implanted MSCs has been suggested to
be one of the mechanisms by which MSCs act to suppress inflammation.
MSCs were found to secrete soluble factors that suppress proliferation
of mononuclear cells in the peripheral blood, alter expression of cyto-
kines, and immunosuppress lymphocytes (Carrade Holt et al., 2014;
Paterson et al., 2014; Ranera et al., 2016). Recently, intra-articular
implantation of cord blood MSCs in an equine in vivo model of synovitis
resulted in a significant decrease in the concentration of neutrophils
and mononuclear cells within the synovial fluid, supporting the concept
that MSCs may have therapeutic potential for treating horses for OA
(Williams et al., 2015).
7. Delivery and source of MSCs
Two other factors that might be important in using MSCs in the
clinic are the method of implantation and the source of the tissue from
which MSCs are isolated. Frisbie et al. assessed the clinical, biochem-
ical, and histologic effects of intra-articularly administered BMMSCs
and AT stromal vascular fraction for treatment of horses with OA. A
greater improvement was seen in joints with BMMSCs relative to the AT
stromal vascular fraction or placebo treated joints (Frisbie et al., 2009).
Fortier et al., using an equine model, showed that delivery of a con-
centrated BM aspirate improves 15-mm diameter full-thickness carti-
laginous defects and is better than the microfracture technique (Fortier
et al., 2010). In another study, intra-articular injection of BMMSCs with
HA in chondral defects of horses, created experimentally, was eval-
uated. Results confirmed a significant increase in firmness and a trend
for better overall quality of repaired tissue after 12 months. Im-
munohistochemical analysis showed a significantly greater
320
Research in Veterinary Science 118 (2018) 317–323
concentration of aggrecan in repaired tissue treated with BMMSCs
(McIlwraith et al., 2011).
Even though intra-articular injection of cells is the implantation
method, it may not be effective. Implantation of cells loaded onto
scaffolds can be considered a more realistic way to deliver and “hold”
cells within cartilage defects. Natural or synthetic materials can be used
to generate scaffolds (Moutos and Guilak, 2008; Nöth et al., 2008).
Wilke et al. (2007) implanted MSCs suspended in autogenous fibrin into
15-mm experimentally created osteochondral defects in the femor-
opatellar joints of six young mature horses. The joints were examined
arthroscopically, and the defect was biopsied at 30 days. Repaired
cartilaginous tissue and surrounding cartilage were evaluated by his-
tochemical examinations, immunohistochemical analysis of collagen
type I and type II, and biochemical assay of the cartilage's matrix at
8 months. Results showed that arthroscopic scores for defects implanted
with MSCs had improved significantly after one month, and biopsies
revealed that several of the defects predominantly contained collagen
type II. Goodrich et al., (2016) recently compared the effects of intra-
articular administration of BMMSCs delivered in a scaffold of auto-
logous platelet-enriched fibrin (APEF) versus administration of APEF
alone on healing of full-thickness chondral defects. When the experi-
mentally created defects were evaluated one year later, the arthroscopic
findings, MRI scores, histologic scores, and scores for material stiffness
were found to be similar between the groups. The authors concluded
that adding BMMSCs to APEF did not improve repair of cartilage and
had an unexpected effect of stimulating bone formation in cartilaginous
defects. In summary, the method of implantation and the source of
MSCs are debatable and are dependent on the clinician's assessment.
8. Challenges facing the clinical use of MSCs in horses
Some specific safety concerns of MSCs include teratoma-like masses,
aberrant migration of cells to other parts of the body, and im-
munogenicity against implanted cells (Fink, 2009). When treating the
equine synovial joint, immune responses are of significant concern.
Reports have shown both immunomodulatory and immunogenic re-
sponses in equine synovial joints (Pigott et al., 2013a; Pigott et al.,
2013b). Fifteen million allogenic, autologous, or xenogeneic BMMSCs
injected into normal fetlock joints caused acute synovitis in all horses
tested (Pigott et al., 2013a). Evaluation of synovial fluid from these
joints revealed an increased nucleated cell count, increased total pro-
tein, and increased IL-6 concentrations. Allogenic and xenogeneic
treated joints showed a 25% greater degree of inflammation than
M. Zayed et al.
Table 1
Physical joint parameters, nucleated cell counts (cells/μl) (TNCC), total protein (TP), differential cell count 0–24 h after intra-articular administration of allogeneic
SFMSCs. Total is the mean ± SD.
Hours Lameness Circumference
0 0 35 ± 1.5
8 0 37 ± 1.5
24 3–4 39 ± 0.5
7.47 Fold
autologous treated joints; however, all BMMSCs treated joints exhibited
multiple clinical signs of synovitis. The same group reported sig-
nificantly greater numbers and distribution of mononuclear in-
flammatory cells in BMMSCs treated fetlocks compared to the control
synovium after 60 days (Pigott et al., 2013b). This brings into question
the use of MSCs in diseased osteoarthritic joints, which are by definition
already inflamed. Despite the use of multiple sensitive techniques no
BMMSCs were detected 60 days post injection. In another study, when
horses with stifle injuries were injected with autologous BMMSCs, 75%
returned to some level of athletic performance, reportedly higher than
previous reports of 60–63% after arthroscopy alone. However, this in-
cluded only 33 horses of which almost 10% showed evidence of post
injection synovitis (Ferris et al., 2014).
Intra-articular injections of culture-expanded BMMSCs to a normal
horse joint were performed at week 0 and week 4 by Joswig's group.
Results showed that repeated intra-articular injections of allogeneic
MSCs resulted in an undesirable clinical response, suggesting there is
immune recognition of allogeneic MSCs upon a second exposure
(Joswig et al., 2017). In contrast, Ardanaz et al. administrated single
and repeat doses of autologous or of pooled allogeneic MSCs in healthy
equine joints, and showed an absence of hypersensitivity response to
the second allogeneic BMMSCs injection (Ardanaz et al., 2016). Re-
cently, Colbath and colleagues showed that allogeneic and autologous
BMMSCs appear to modulate inflammatory processes related to acute or
chronic musculoskeletal injuries in the horse with equal potency
(Colbath et al., 2017). Thus, the use of autologous or allogenic MSCs is
still under debate and more studies are required.
9. Intra-articular administration of autologous versus allogenic
SFMSCs
As described above, allogeneic versus autologous MSCs is a hot topic
in veterinary regenerative medicine, debating whether there is a dif-
ference in efficacy for tissue healing and if there is a correlation with
the immune system. It has been recently suggested that assays to
evaluate cell-mediated and humoral immune responses against allo-
geneic MSCs should be performed to improve the efficacy of MSCs
while maintaining their safety (Berglund et al. 2017). While these as-
says are important, it defeats one of the main objectives of having an
allogenic source of cells, which is to provide the clinic with char-
acterized usable MSCs in a timely manner. Hence, further investigations
are required to fully understand the allogenicity of MSCs.
We recently carried out two independent studies to understand the
biological role of MSCs by evaluating some of the challenges described
in the above sections. In the first study, we carried out a donor-matched
comparison of chondrogenic potential of BM and SFMSCs (Zayed et al.,
2017). MSCs were evaluated in vitro for their proliferation, trilineage
differentiation, and expression of cell surface markers. Results showed
that even though the SFMSCs are considered more chondrogenic re-
lative to BMMSCs, there was considerable donor-to-donor variation in
the expression of MHC II. In the second study, we compared the in vivo
response when the SFMSCs were administered in an autologous or an
allogenic manner in normal joints. Three horses free of significant la-
menesswere involved in the study. One horse received autologous
MSCs, while the remaining two horses received allogenic MSCs.
Research in Veterinary Science 118 (2018) 317–323
TP TNCC Monocytes Neutrophils
< 2.5 100 ± 0 46 ± 12 9.5 ± 0.5
< 3.5 28,050 ± 5750 27 ± 9 71 ± 8.5
Lameness examination and SF analysis were assessed before and
after injection of three millions of cells (in 2 mL PBS) inside the tarso-
crural (TC) joint for 72 h. Lameness examinations were performed at
the walk and trot on hard ground every 24 h. Lameness was subjectively
graded (0–5) according to the AAEP lameness scale. Joints were sub-
jectively graded every 12 h for effusion (0 = none, 1 = mild,
2 = moderate 3 = severe). Joint circumference (cm2) was measured
every 12 h. Each SF sample was examined for routine cytological ana-
lysis, which included total nucleated cell count (TNCC) (Coulter Z2
nucleated cell counter), total protein (TP), and differential cell count.
From these numbers, absolute numbers of neutrophils and mononuclear
cells were calculated.
Following injection of autologous and allogenic SFMSCs into the TC
joint, there was a moderate increase in lameness (range 3–4). Compared
to control joints, injection of both the autologous and allogenic SFMSCs
into the TC joint resulted in elevated levels of TP and TNCC. Most
importantly, there was a 7.47 fold significant increase in neutrophil
count in the allogenic cells (Table 1) and a 2.1 fold increase in the joints
that received autologous cells (Table 2). This result was unexpected,
and the results of this study didn't support our hypotheses that intra-
articular administration of equine SFMSCs could be implanted in an
autologous manner and would result in no adverse reactions, either at
the injection site or systemically in the animal. In addition, This pre-
liminary study reinforces the fact that it is preferable to characterize
MSCs in vitro before implantation. Even with the in vitro knowledge,
however, further work is needed to investigate allogenic or autologous
administration of equine SFMSCs in OA joints.
10. Conclusion
Despite recent advances in MSC biology in basic scientific and
clinical societies, there are still a lot of unanswered questions.
Optimization of these cell-based therapies will concentrate on cellular
origin, isolation, enrichment, and processing as well as on the timing,
route of administration, formulation, and dosing. The issue of safety of
allogeneic or even autologous MSCs is also very important.
Development of sound controlled clinical trials will be principal in in-
terpreting and identifying the ideal treatment regimens of MSC therapy
for joint disorders.
Acknowledgements
We acknowledge the assistance of Ms. Amanda Kelli Hand in
proofreading the article for any grammatical, typographical, or for-
matting errors. This study was funded, in a part, by the Egyptian
Table 2
Physical joint parameters, nucleated cell counts (cells/μl) (TNCC), total protein
(TP), differential cell count 0–24 h after intra-articular administration of au-
tologous SFMSCs. Total is the mean ± SD.
Hours Lameness Circumference TP TNCC Monocytes Neutrophils
0 0 36 < 2.5 200 16 32
8 0 37
24 3 38 < 4.4 17,700 30 70
2.1 Fold
321
M. Zayed et al.
Cultural and Educational Bureau (ECEB) and the University of
Tennessee's (R181710143) Center of Excellence in Livestock Diseases
and Human Health.
References
Amable, P.R., Carias, R.B., Teixeira, M.V., da Cruz Pacheco, Í., Corrêa do Amaral, R.J.,
Granjeiro, J.M., Borojevic, R., 2013. Platelet-rich plasma preparation for regenerative
medicine: optimization and quantification of cytokines and growth factors. Stem Cell
Res Ther 4, 67.
Antonacci, J.M., Schmidt, T.A., Serventi, L.A., Cai, M.Z., Shu, Y.L., Schumacher, B.L.,
McIlwraith, C.W., Sah, R.L., 2012. Effects of equine joint injury on boundary lu-
brication of articular cartilage by synovial fluid: role of hyaluronan. Arthritis Rheum.
64, 2917–2926.
Ardanaz, N., Vázquez, F.J., Romero, A., Remacha, A.R., Barrachina, L., Sanz, A., Ranera,
B., Vitoria, A., Albareda, J., Prades, M., Zaragoza, P., Martín-Burriel, I., Rodellar, C.,
2016. Inflammatory response to the administration of mesenchymal stem cells in an
equine experimental model: effect of autologous, and single and repeat doses of
pooled allogeneic cells in healthy joints. BMC Vet. Res. 12, 65.
Asari, S., Itakura, S., Ferreri, K., Liu, C., Kuroda, Y., Kandeel, F., 2009. Mesenchymal stem
cells suppress B-cell terminal differentiation. Exp. Hematol. 37, 604–615.
Barberini, D.J., Freitas, N.P., Magnoni, M.S., Maia, L., Listoni, A.J., Heckler, M.C.,
Sudano, M.J., Golim, M.A., da Cruz Landim-Alvarenga, F., Amorim, R.M., 2014.
Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical
cord: immunophenotypic characterization and differentiation potential. Stem Cell
Res Ther 5, 25.
Berglund, A., Fortier, L., Antczak, D., Schnabel, L., 2017. Immunoprivileged no more:
measuring the immunogenicity of allogeneic adult mesenchymal stem cells. Stem Cell
Res Ther 8, 288.
Bi, W., Deng, J.M., Zhang, Z., Behringer, R.R., de Crombrugghe, B., 1999. Sox9 is required
for cartilage formation. Nat. Genet. 22, 85–89.
Bonnet, C.S., Walsh, D.A., 2005. Osteoarthritis, angiogenesis and inflammation.
Rheumatology 44, 7–16.
Brama, P.A., Tekoppele, J.M., Bank, R.A, Barneveld, A., van Weeren, P.R., 2000.
Functional adaptation of equine articular cartilage: the formation of regional bio-
chemical characteristics up to age one year. Equine Vet. J. 32 (3), 217–221.
Buckwalter, J.A., 1998. Articular cartilage: injuries and potential for healing. J. Orthop.
Sports Phys. Ther. 28, 192–202.
Buckwalter, J.A., 2002. Articular cartilage injuries. Clin. Orthop. Relat. Res. 21–37.
Caplan, A.I., 2017. Mesenchymal stem cells: time to change the name!. Stem Cells Transl.
Med. 6 (6), 1445–1451.
Carrade, D.D., Owens, S.D., Galuppo, L.D., Vidal, M.A., Ferraro, G.L., Librach, F.,
Buerchler, S., Friedman, M.S., Walker, N.J., Borjesson, D.L., 2011. Clinicopathologic
findings following intra-articular injection of autologous and allogeneic placentally
derived equine mesenchymal stem cells in horses. Cytotherapy 13.
Carrade, D.D., Wood, J.A., Granick, J.L., Walker, N.J., Clark, K.C., Borjesson, D.L., 2014.
Equine mesenchymal stem cells inhibit T cell proliferation through different me-
chanisms depending on tissue source. Stem Cells Dev. 23, 1258–1265.
Chen, J., Wang, C., Lu, S., Wu, J., Guo, X., Duan, C., Dong, L., Song, Y., Zhang, J., Jing, D.,
Wu, L., Ding, J., Li, D., 2005. In vivo chondrogenesis of adult bone-marrow-derived
autologous mesenchymal stem cells. Cell Tissue Res. 319, 429–438.
Cokelaere, S., Malda, J., Weeren, R., 2016. Cartilage defect repair in horses: current
strategies and recent developments in regenerative medicine of the equine joint with
emphasis on the surgical approach. Vet. J. 214, 61–71.
Colbath, A.C., Dow, S.W., Phillips, J.N., McIlwraith, C.W., Goodrich, L.R., 2017.
Autologous and allogeneic equine mesenchymal stem cells exhibit equivalent im-
munomodulatory properties in vitro. Stem Cells Dev. 26, 503–511.
De Schauwer, C., Van de Walle, G.R., Van Soom, A., Meyer, E., 2013. Mesenchymal stem
cell therapy in horses: useful beyond orthopedic injuries? Vet Q 33, 234–241.
DeChiara, T.M., Kimble, R.B., Poueymirou, W.T., et al., 2000. Ror2, encoding a receptor-
like tyrosine kinase, is required for cartilage and growth plate development. Nat.
Genet. 24, 271–274.
DeForge, L.E., Tracey, D.E., Kenney, J.S., Remick, D.G., 1992. Interleukin-1 receptor
antagonist protein inhibits interleukin-8 expression in lipopolysaccharide-stimulated
human whole blood. Am. J. Pathol. 140 (5), 1045–1054.
Dhar, M., Neilsen, N., Beatty, K., Eaker, S., Adair, H., Geiser, D., 2012. Equine peripheral
blood-derived mesenchymal stem cells: isolation, identification, trilineage differ-
entiation and effect of hyperbaric oxygen treatment. Equine Vet. J. 44, 600–605.
Dominici, M., Le, B.K., Mueller, I., Slaper-Cortenbach, I., Marini, F., et al., 2006. Minimal
criteria for defining multipotent mesenchymal stromal cells. The International
Society for Cellular Therapy position statement. Cytotherapy 8, 315–317.
Doucet, M.Y., Bertone, A.L., Hendrickson, D., Hughes, F., Macallister, C., McClure, S.,
Reinemeyer, C., Rossier, Y., Sifferman, R., Vrins, A.A., White, G., Kunkle, B., Alva, R.,
Romano, D., Hanson, P.D., 2008. Comparison of efficacy and safety of paste for-
mulations of firocoxib and phenylbutazone in horses with naturally occurring os-
teoarthritis. J. Am. Vet. Med. Assoc. 232, 91–97.
Dy, P., Wang, W., Bhattaram, P., Wang, Q., Wang, L., Ballock, R.T., Lefebvre, V., 2012.
Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth
plate chondrocytes. Dev. Cell 22, 597–609.
Edwards, R.B., Lu, Y., Uthamanthil, R.K., Bogdanske, J.J., Muir, P., Athanasiou, K.A.,
2007. Comparison of mechanical débridement and radiofrequency energy for chon-
droplasty in an in vivo equine model of partial thickness cartilage injury. Osteoarthr.
Cartil. 15, 169–178.
Eisenberg, S.P., Evans, R.J., Arend, W.P., Verderber, E., Brewer, M.T., Hannum, C.H.,
322
Research in Veterinary Science 118 (2018) 317–323
Thompson, R.C., 1990. Primary structure and functional expression from com-
plementary DNA of a human interleukin-1 receptor antagonist. Nature 343, 341–346.
Elsaid, K.A., Jay, G.D., Warman, M.L., Rhee, D.K., Chichester, C.O., 2005. Association of
articular cartilage degradation and loss of boundary-lubricating ability of synovial
fluid following injury and inflammatory arthritis. Arthritis Rheum. 52, 1746–1755.
English, K., 2013. Mechanisms of mesenchymal stromal cell immunomodulation.
Immunol. Cell Biol. 91, 19–26.
Fafián-Labora, J., Fernández-Pernas, P., Fuentes, I., De Toro, J., Oreiro, N., Sangiao-
Alvarellos, S., et al., 2015. Influence of age on rat bone-marrow mesenchymal stem
cells potential. Sci. Rep. 19, 16765.
Ferris, D.J., Frisbie, D.D., Kisiday, J.D., McIlwraith, C.W., Hague, B.A., Major, M.D.,
Schneider, R.K., Zubrod, C.J., Kawcak, C.E., Goodrich, L.R., 2014. Clinical outcome
after intra-articular administration of bone marrow derived mesenchymal stem cells
in 33 horses with stifle injury. Vet. Surg. 43, 255–265.
Fink, D.W., 2009. FDA regulation of stem cell–based products. Science 324, 1662.
Fortier, L.A., Travis, A.J., 2011. Stem cells in veterinary medicine. Stem Cell Res Ther 2,
9–14.
Fortier, L.A., Potter, H.G., Rickey, E.J., Schnabel, L.V., Foo, L.F., Chong, L.R., Stokol, T.,
Cheetham, J., Nixon, A.J., 2010. Concentrated bone marrow aspirate improves full-
thickness cartilage repair compared with microfracture in the equine model. J. Bone
Joint Surg. 92, 1927–1937.
Friedenstein, A.J., Chailakhjan, R.K., Lalykina, K.S., 1970. The development of fibroblast
colonies in monolayer cultures of Guinea-Pig bone marrow and spleen cells. Cell
Prolif. 3, 393–403.
Frisbie, D.D., 2014. How to choose cases and utilize mesenchymal stem cells in joint
injury, 1Proceedings of the 60th annual convention of the American Association of
Equine Practitioners, Salt Lake City, Utah, USA, 2014, (December 6-10).
Frisbie, D., Ghivizzani, S., Robbins, P., Evans, C., McIlwraith, C., 2002. Treatment of
experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1
receptor antagonist gene. Gene Ther. 9, 12–20.
Frisbie, D.D., Oxford, J.T., Southwood, L., Trotter, G.W., Rodkey, W.G., Steadman, J.R.,
Goodnight, J.L., McIlwraith, C.W., 2003. Early events in cartilage repair after sub-
chondral bone microfracture. Clin. Orthop. Relat. Res. 407, 215–227.
Frisbie, D.D., Kawcak, C.E., Werpy, N.M., Park, R.D., McIlwraith, C.W., 2007. Clinical,
biochemical, and histologic effects of intra-articular administration of autologous
conditioned serum in horses with experimentally induced osteoarthritis. Am. J. Vet.
Res. 68, 290–296.
Frisbie, D.D., Kisiday, J.D., Kawcak, C.E., et al., 2009. Evaluation of adipose-derived
stromal vascular fraction or bone marrow-derived mesenchymal stem cells for
treatment of osteoarthritis. J. Orthop. Res. 27, 1675–1680.
Goodrich, L.R., Nixon, A.J., 2006. Medical treatment of osteoarthritis in the horse - a
review. Vet. J. 171 (1), 51–69.
Goodrich, L.R., Chen, A.C., Werpy, N.M., Williams, A.A., Kisiday, J.D., Su, A.W., Cory, E.,
Morley, P.S., McIlwraith, C.W., Sah, R.L., Chu, C.R., 2016. Addition of mesenchymal
stem cells to autologous platelet-enhanced fibrin scaffolds in chondral defects: does it
enhance repair? J. Bone Joint Surg. 98, 23–34.
Govoni, K.E., 2015. Horses species symposium: use of mesenchymal stem cells in fracture
repair in horses. J. Anim. Sci. 93, 871–878.
Gruh, I., Martin, U., 2009. Transdifferentiation of stem cells: a critical view. Adv.
Biochem. Eng. Biotechnol. 114, 73–106.
Hangody, L., Vasarhelyi, G., Hangody, L.R., Sukosd, Z., Tibay, G., Bartha, L., Bodo, G.,
2008. Autologous osteochondral grafting—technique and long-term results. Injury
39, 32–39.
Hannum, C.H., Wilcox, C.W., Arend, W.P., Joslin, F.G., Dripps, D.J., Heimdal, P.L., Armes,
L.G., Sommer, A., Eisenberg, S.P., Thompson, R.C., 1990. Interleukin-1 receptor an-
tagonist activity of a human interleukin-1 inhibitor. Nature 343, 336–340.
Harrison, S., Vavken, P., Kevy, S., Jacobson, M., Zurakowski, D., Murray, M.M., 2011.
Platelet activation by collagen provides sustained release of anabolic cytokines. Am.
J. Sports Med. 39, 729–734.
Jackson, D.W., Lalor, P.A., Aberman, H.M., Simon, T.M., 2001. Spontaneous repair of full-
thickness defects of articular cartilage in a goat model. A preliminary study. J. Bone
Joint Surg. 83, 53–64.
Joswig, A.-J., Mitchell, A., Cummings, K.J., Levine, G.J., Gregory, C.A., Smith, R., et al.,
2017. Repeated intra-articular injection of allogeneic mesenchymal stem cells causes
an adverse response compared to autologous cells in the equine model. Stem Cell Res
Ther 8, 42.
Koch, T.G., Berg, L.C., Betts, D.H., 2008. Concepts for the clinical use of stem cells in
equine medicine. Can. Vet. J. 49, 1009–1017.
Krampera, M., Franchini, M., Pizzolo, G., Aprili, G., 2007. Mesenchymal stem cells: from
biology to clinical use. Blood Transfus. 5, 120–129.
Lang, H.M., Panizzi, L., Allen, A.L., Woodbury, M.R., Barber, S.M., 2009. Comparison of
three drilling techniques for carpometacarpal joint arthrodesis in horses. Vet. Surg.
38 (8), 990–997.
Le Blanc, K., Tammik, C., Rosendahl, Zetterberg, Ringén, O., 2003. HLA expression and
immunologic properties of differentiated and undifferentiated mesenchymal stem
cells. Exp. Hematol. 31 (10), 890–896.
Litzke, L.E., Wagner, E., Baumgaertner, W., Hetzel, U., Josimovic-Alasevic, O., Libera, J.,
2004. Repair of extensive articular cartilage defects in horses by autologous chon-
drocyte transplantation. Ann. Biomed. Eng. 32, 57–69.
Machado, C.D., Telles, P.D., Nascimento, I.L.O., 2013. Immunological characteristics of
mesenchymal stem cells. Rev. Bras. Hematol. Hemoter. 35, 62–67.
Mak, J., Jablonski, C.L., Leonard, C.A., Dunn, J.F., Raharjo, E., Matyas, J.R., et al., 2016.
Intra-articular injection of synovial mesenchymal stem cells improves cartilage repair
in a mouse injury model. Sci. Rep. 6, 23076.
McIlwraith, C.W., 1996. General pathobiology of the joint and response to injury. In:
McIlwraith, C.W., Trotter, G.W. (Eds.), Joint Disease in the Horse. WB Saunders,
M. Zayed et al.
Philadelphia, pp. 40–70.
McIlwraith, C.W., Frisbie, D.D., Rodkey, W.G., Kisiday, J.D., Werpy, N.M., Kawcak, C.E.,
Steadman, J.R., 2011. Evaluation of intra-articular mesenchymal stem cells to aug-
ment healing of microfractured chondral defects. Arthroscopy 27, 1552–1561.
Mishra, A., Jr, Woodall J., Vieira, A., 2009. Treatment of tendon and muscle using pla-
telet-rich plasma. Clin. Sports Med. 28, 113–125.
Moutos, F.T., Guilak, F., 2008. Composite scaffolds for cartilage tissue engineering.
Biorheology 45, 501–512.
Murata, D., Miyakoshi, D., Hatazoe, T., Miura, N., Tokunaga, S., Fujiki, M., Nakayama, K.,
Misumi, K., 2014. Multipotency of equine mesenchymal stem cells derived from sy-
novial fluid. Vet. J. 202, 53–61.
Murphy, M.K., Huey, D.J., Hu, J.C., Athanasiou, K.A., 2015. TGF-beta1, GDF-5, and BMP-
2 stimulation induces chondrogenesis in expanded human articular chondrocytes and
marrow-derived stromal cells. Stem Cells 33, 762–773.
Nixon, A.J., Begum, L., Mohammed, H.O., Huibregtse, B., O'Callaghan, M.M., Matthews,
G.L., 2011. Autologous chondrocyte implantation drives early chondrogenesis and
organized repair in extensive full- and partial-thickness cartilage defects in an equine
model. J. Orthop. Res. 29, 1121–1130.
Nöth, U., Steinert, A.F., Tuan, R.S., 2008. Technology insight: adult mesenchymal stem
cells for osteoarthritis therapy. Nat. Clin. Pract. Rheumatol. 4, 371–380.
Okazaki, R., Sakai, A., Uezono, Y., Ootsuyama, A., Kunugita, N., Nakamura, T., Norimura,
T., 2001. Sequential changes in transforming growth factor (TGF)-beta1 concentra-
tion in synovial fluid and mRNA expression of TGF-beta1 receptors in chondrocytes
after immobilization of rabbit knees. J. Bone Miner. Metab. 19, 228–235.
Patel, S., Dhillon, M.S., Aggarwal, S., Marwaha, N., Jain, A., 2013. Treatment with pla-
telet-rich plasma is more effective than placebo for knee osteoarthritis a prospective,
double-blind, randomized trial. Am. J. Sports Med. 41, 356–364.
Paterson, Y.Z., Rash, N., Garvican, E.R., Paillot, R., Guest, D.J., 2014. Equine mesench-
ymal stromal cells and embryo-derived stem cells are immune privileged in vitro.
Stem Cell Res Ther 5, 1–13.
Pezzanite, L.M., Fortier, L.A., Antczak, D.F., Cassano, J.M., Brosnahan, M.M., Miller, D.,
Schnabel, L.V., 2015. Equine allogeneic bone marrow-derived mesenchymal stromal
cells elicit antibody responses in vivo. Stem Cell Res Ther 6, 54.
Pigott, J.H., Ishihara, A., Wellman, M.L., Russell, D.S., Bertone, A.L., 2013a.
Inflammatory effects of autologous genetically modified autologous, allogeneic, and
xenogeneic mesenchymal stem cells after intra-articular injection in horses. Vet.
Comp. Orthop. Traumatol. 26, 453–460.
Pigott, J.H., Ishihara, A., Wellman, M.L., Russell, D.S., Bertone, A.L., 2013b. Investigation
of the immune response to autologous, allogeneic, and xenogeneic mesenchymal
stem cells after intra-articular injection in horses. Vet. Immunol. Immunopathol. 156,
99–106.
Prado, A.A., Favaron, P.O., da Silva, L.C., Baccarin, R.Y., Miglino, M.A., Maria, D.A.,
2015. Characterization of mesenchymal stem cells derived from the equine synovial
fluid and membrane. BMC Vet. Res. 11, 281.
Prockop, D.J., Oh, J.Y., 2012. Medical therapies with adult stem/progenitor cells (MSCs):
a backward journey from dramatic results in vivo to the cellular and molecular ex-
planations. J. Cell. Biochem. 113 (5), 1460–1469.
Qiu, J., Li, D., Mou, X., Li, J., Guo, W., Wang, S., Yu, X., Ma, B., Zhang, S., Tang, W., Sang,
Y., Gil, P.R., Liu, H., 2016. Effects of graphene quantum dots on the self-renewal and
differentiation of mesenchymal stem cells. Adv. Healthc. Mater 5, 702–710.
Ranera, B., Antczak, D., Miller, D., Doroshenkova, T., Ryan, A., McIlwraith, C.W., Barry,
F., 2016. Donor-derived equine mesenchymal stem cells suppress proliferation of
mismatched lymphocytes. Equine Vet. J. 48, 253–260.
Rowden, A., Dominici, P., D'Orazio, J., et al., 2015. Double-blind, randomized, placebo-
controlled study evaluating the use of platelet-rich plasma therapy (PRP) for acute
ankle sprains in the emergency department. J. Emerg. Med. 49, 546–551.
Sampson, S., Gerhardt, M., Mandelbaum, B., 2008. Platelet rich plasma injection grafts for
musculoskeletal injuries: a review. Curr. Rev. Musculoskelet Med. 1, 165–174.
Seo, J.P., Tsuzuki, N., Haneda, S., Yamada, K., Furuoka, H., Tabata, Y., Sasaki, N., 2014.
Osteoinductivity of gelatin/beta-tricalcium phosphate sponges loaded with different
concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an
equine bone defect model. Vet. Res. Commun. 38, 73–80.
Sheth, N. Simunovic, Klein, G., Fu, F., Einhorn, T.A., et al., 2012. Efficacy of autologous
platelet-rich plasma use for orthopaedic indications: a meta-analysis. J. Bone Joint
323
Research in Veterinary Science 118 (2018) 317–323
Surg. Am. 94, 298–307.
Siegel, G., Kluba, T., Hermanutz-Klein, U., Bieback, K., Northoff, H., et al., 2013.
Phenotype, donor age and gender affect function of human bone marrow-derived
mesenchymal stromal cells. BMC Med. 11.
Smith, R.K.W., 2014. How to select cases and utilize stem cells in tendon injury. In:
Proceedings of the 60th Annual Convention of the American Association of Equine
Practitioners, Salt Lake City, Utah, USA. Lexington, American Association of Equine
Practitioners (AAEP) (December 6–10).
Song, L., Webb, N.E., Song, Y., Tuan, R.S., 2006. Identification and functional analysis of
candidate genes regulating mesenchymal stem cell self-renewal and multipotency.
Stem Cells 24, 1707–1718.
Story, M.R., Bramlage, L.R., 2004. Arthroscopic debridement of subchondral bone cysts in
the distal phalanx of 11 horses (1994–2000). Equine Vet. J. 36 (4), 356–360.
Tessier, L., Bienzle, D., Williams, L.B., Koch, T.G., 2015. Phenotypic and im-
munomodulatory properties of equine cord blood-derived mesenchymal stromal
cells. PLoS One 10, e0122954.
Todhunter, R.J., 1996. Anatomy and physiology of synovial joints. In: CW, McIlwraith,
Trotter, G.W. (Eds.), Joint Disease in the Horse. 1996. WB Saunders, Philadelphia,
pp. 1–28.
Tuli, R., Tuli, S., Nandi, S., Huang, X., Manner, P.A., Hozack, W.J., Danielson, K.G., Hall,
D.J., Tuan, R.S., 2003. Transforming growth factor-beta-mediated chondrogenesis of
human mesenchymal progenitor cells involves N-cadherin and mitogen-activated
protein kinase and Wnt signaling cross-talk. J. Biol. Chem. 278, 41227–41236.
van Buul, G.M., Koevoet, W.L., Kops, N., Bos, P.K., Verhaar, J.A., Weinans, H., Bernsen,
M.R., van Osch, G.J., 2011. Platelet-rich plasma releasate inhibits inflammatory
processes in osteoarthritic chondrocytes. Am. J. Sports Med. 39, 2362–2370.
Vidal, M.A., Lopez, M.J., 2011. Adipogenic differentiation of adult equine mesenchymal
stromal cells. Methods Mol. Biol. 702, 61–75.
Vidal, M.A., Kilroy, G.E., Johnson, J.R., Lopez, M.J., Moore, R.M., Gimble, J.M., 2006.
Cell growth characteristics and differentiation frequency of adherent equine bone
marrow–derived mesenchymal stromal cells: adipogenic and osteogenic capacity.
Vet. Surg. 35, 601–610.
Wang, X., Li, Y., Han, R., He, C., Wang, G., Wang, J., Zheng, J., Pei, M., Wei, L., 2014.
Demineralized bone matrix combined bone marrow mesenchymal stem cells, bone
morphogenetic protein-2 and transforming growth factor-beta3 gene promoted pig
cartilage defect repair. PLoS One 9, e116061.
Watts, A.E., 2014. How to understand regenerative medicine - what is it? Proceedings of
the 60th Annual Convention of the American Association of Equine Practitioners, Salt
Lake City, Utah, USA. pp. 494–498 (December 6-10).
Wilke, M.M., Nydam, D.V., Nixon, A.J., 2007. Enhanced early chondrogenesis in articular
defects following arthroscopic mesenchymal stem cell implantation in an equine
model. J. Orthop. Res. 25, 913–925.
Williams, L.B., Koenig, J.B., Black, B., Gibson, T.W., Sharif, S., Koch, T.G., 2015. Equine
allogeneic umbilical cord blood derived mesenchymal stromal cells reduce synovial
fluid nucleated cell count and induce mild self-limiting inflammation when evaluated
in an LPS induced synovitis model. Equine Vet. J. 48 (5), 619–625.
Yagi, H., Soto-Gutierrez, A., Parekkadan, B., Kitagawa, Y., Tompkins, R.G., Kobayashi, N.,
Yarmush, M.L., 2010. Mesenchymal stem cells: mechanisms of immunomodulation
and homing. Cell Transplant. 19, 667–679.
Yamashita, A., Nishikawa, S., Rancourt, D.E., 2010. Identification of five developmental
processes during chondrogenic differentiation of embryonic stem cells. PLoS One 5,
e10998.
Yao, Y., Yu Li, Z., Zhang, H., Zheng, Y., Mai, L., Liu, W., Zhang, Z., Sun, Y., 2018. Synovial
fluid-derived synovial fragments represent an improved source of synovial me-
senchymal stem cells in the temporomandibular joint. Int. J. Mol. Med. 41 (1),
173–183.
Zayed, M., Caniglia, C., Misk, N., Dhar, M.S., 2017. Donor-matched comparison of
Chondrogenic potential of equine bone marrow- and synovial fluid-derived me-
senchymal stem cells: implications for cartilage tissue regeneration. Front Vet Sci 3,
121.
Zhang, S., Muneta, T., Morito, T., Mochizuki, T., Sekiya, I., 2008. Autologous synovial
fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis
patients in tissue culture system. J. Orthop. Res. 26, 1413–1418.