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The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; A dream for tendon repair?

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Tendon injuries represent even today a challenge as repair may be exceedingly slow and incomplete. Regenerative medicine and stem cell technology have shown to be of great promise. Here, we will review the current knowledge on the mechanisms of the regenerative potential of mesenchymal stem cells (MSCs) obtained from different sources (bone marrow, fat, cord blood, placenta). More specifically, we will devote attention to the current use of MSCs that have been used experimentally and in limited numbers of clinical cases for the surgical treatment of subchondral-bone cysts, bone-fracture repair and cartilage repair. Based on the recently emerging role in regenerative mechanisms of soluble factors and of extracellular vesicles, we will discuss the potential of non-cellular therapies in horse tendon injuries.
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212 Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221
Review article
Ciro Tetta1,
Anna Lange Consiglio2,
Stefania Bruno3,
Emanuele Tetta4,
Emanuele Gatti1,
Miryana Dobreva1,
Fausto Cremonesi2,
Giovanni Camussi5
1Center of Translational Regenerative Medicine, Frese-
nius Medical Care Deutschland GmbH, Torino, Italy
2Reproduction Section, “Polo Veterinario di Lodi”, Faculty
of Veterinary Medicine, University of Milan, Italy
3Department of Molecular Biotechnology and Health Sci-
ences, University of Turin, Italy
4Faculty of Science of Animal Production, University of
Bologna, Italy
5Department of Medical Sciences, University of Turin,
Italy
Corresponding author:
Giovanni Camussi
Department of Medical Sciences University of Turin
Corso Dogliotti 14, 10126 Turin, Italy
e-mail: giovanni.camussi@unito.it
Summary
Tendon injuries represent even today a challenge as
repair may be exceedingly slow and incomplete. Re-
generative medicine and stem cell technology have
shown to be of great promise. Here, we will review the
current knowledge on the mechanisms of the regen-
erative potential of mesenchymal stem cells (MSCs)
obtained from different sources (bone marrow, fat,
cord blood, placenta). More specifically, we will devote
attention to the current use of MSCs that have been
used experimentally and in limited numbers of clinical
cases for the surgical treatment of subchondral-bone
cysts, bone-fracture repair and cartilage repair. Based
on the recently emerging role in regenerative mecha-
nisms of soluble factors and of extracellular vesicles,
we will discuss the potential of non-cellular therapies
in horse tendon injuries.
Key words: horse tendinopathies, microvesicles, regen-
erative medicine, soluble factors, stem cells.
Introduction
Stem cells have evoked considerable excitement in vet-
erinary medicine because of the promise that stem cell
technology could deliver tissue regeneration for injuries for
which natural repair mechanisms do not deliver func-
tional recovery and for which current therapeutic strate-
gies have minimal effectiveness. Tendon injuries have
represented an area of particular interest since conven-
tional treatments often lead to an unsatisfactory healing
process that usually results in a relatively high recur-
rence rate. In recent years, regenerative medicine has
emerged as an attractive field for new cellular and non-
cellular approaches to tissue repair. Here, we will review
the current knowledge on the mechanisms of the regen-
erative potential of mesenchymal stem cells (MSCs) ob-
tained from different sources (bone marrow, fat, cord
blood, placenta). More specifically, we will devote atten-
tion to the current use of MSCs that have been used ex-
perimentally and in limited numbers of clinical cases for
the surgical treatment of subchondral-bone cysts, bone-
fracture repair1and cartilage repair2,3.However, by far the
most frequent clinically use has been the treatment of
overstrain-induced injuries of tendons in horses. We will
discuss the hypothesis that also soluble factors and ex-
tracellular vesicles, also called microvesicles (MVs), re-
leased by MSCs may have a relevant regenerative poten-
tial and may open new therapeutic perspectives.
The paracrine effect of stem cells
Increasing experimental evidence indicate that the active
factors exert effects on neighbouring cells. Indeed, MSCs
express high levels of transcripts of hematopoietic stem
cells maintenance factors, including CXCL12 chemokine,
stem cell factor, angiopoietin-1 (Ang-1), interleukin-7,
vascular cell adhesion molecule 1 and osteopontin4. Sup-
port for the hypothesis of paracrine action of MSCs de-
rives from in vivo studies indicating that, although MSCs
exhibit multilineage differentiation potential and can mi-
grate to injured sites after systemic administration, the dif-
ferentiation of MSCs in cells of injured tissues contributed
little to their therapeutic benefits. A growing number of ev-
idence indicates that the in vivo effects of MSCs depend
primarily on their capacity to secrete bioactive soluble fac-
tors. This bioactive molecules may inhibit fibrosis and
apoptosis, enhance angiogenesis, stimulate mitosis
and/or differentiation of tissue-intrinsic progenitor/stem
cells5and modulate the immune response6.
In different pre-clinical animal models, MSCs administra-
tion have been shown to improve perfusion and restore
cardiac function after myocardial infarction7; MSCs accel-
erates recovery in acute kidney injury (AKI) induced by
toxic agents or ischemia reperfusion and induces func-
tional improvement in chronic kidney disease8-13. In addi-
tion, MSCs have been studied in several in vivo models
The role of microvesicles derived from
mesenchymal stem cells in tissue regeneration;
a dream for tendon repair?
of lung disease14,15. For example, in the bleomycin in-
duced lung injury and fibrosis, MSCs improve lung inflam-
mation and survival when given intravenously. These ef-
fects are not accounted to lung engraftment rates (< 5%)
but rather to a paracrine mechanism16.
The beneficial effects of MSCs infusion in different animal
models are interpreted as not dependent on a direct sub-
stitution of injured cells, but rather on paracrine effectors
that facilitate endogenous repair processes. In this way,
a paracrine role of MSCs in renal tissue repair has been
supported by experiments showing that conditioned
medium (CM) from MSCs mimics the beneficial effects of
the cells of origin, when intra-peritoneal injected in mice
with cisplatin induced AKI17. Moreover, intravenous admin-
istration of CM from MSCs induces significant survival im-
provement in fulminant hepatic failure18,19.
MSCs have been also investigated as a new therapeutic
strategy for graft-versus-host disease, Chron’s disease
and for the prevention of organ transplantation rejection.
The mechanism by which MSCs modulate the immune re-
sponse is still under investigation, but it is evident that it
involves also the release of soluble factors and not only
the cell-to-cell contact. MSCs may suppress several
T-lymphocyte activities both in vitro and in vivo and may
alter the cytokine expression profile of dendritic cells
(DCs), naïve and effector T cells and natural killer cells
(NK) to induce a more anti-inflammatory or tolerant phe-
notype and to increase the proportion of regulatory T
(Treg) cells. Prostaglandin E2 (PGE2) is implicated in the
immunomodulatory effects of MSCs. Indeed, PGE2 pro-
duction is up-regulated after co-culture of human MSCs
with peripheral blood mononuclear cells20 and the in-
hibitors of PGE2 production diminish MSC-mediated im-
munomodulation in vitro21. Indoleamine 2, 3 deoxyge-
nase (IDO), PGE2 and TGF-?1 can represent relevant
mediators of MSC inhibition of NK functions21-23. MSCs
also secrete IL-6, that is involved in the reversion of mat-
uration of DCs to a less mature phenotype24. Blockade of
PGE2 synthesis in MSCs reverts the inhibitory effects on
DC differentiation and function. PGE2 and IL-6 can me-
diate the effects of MSCs on DCs, thus leading to T-cell
suppression25.
Regenerative medicine and tendinopathies
Tendon repairs are often weak and susceptible to re-in-
jury. Given the frequency and increasing cost of these in-
juries, mainly in sport horse, as well as the relatively poor
result of surgical intervention, it is not surprising that new
and innovative strategies like tissue engineering have
become more appealing.
Several lines of evidence suggest that multipotent stem
cells are present also in tendons and ligaments. First, both
human and mouse tendons develop fibrocartilage and os-
sification in response to injury26,27. Second, tendon-derived
immortalized cell lines or human tendon derived fibrob-
lasts express genes of adipogenic, osteogenic and chon-
drogenic differentiation pathways, suggesting that they
possess multiple differentiation capacities in vitro28,29. Fi-
nally, postnatal stem cells capable of differentiating into
adipocytes and osteoblastic cells have been identified in
human periodontal ligaments30 while human and mouse
tendons harbor a unique cell population, termed tendon
stem/progenitor cells (TSPCs), that has universal stem
cell characteristics such as clonogenicity, multipotency
and self-renewal capacity31. Recently, Lovati et al.32 iden-
tified TSPCs specifically in the horse SDFT with the abil-
ity to be highly clonogenic, to grow fast and to differenti-
ate in different induced cell lineages as well as bone
marrow derived progenitor cells (BM-MSCs). The hy-
pothesis that TSPCs possess a mesenchymal stem cell
behavior opens a new prospective for tendon regenera-
tive medicine approaches because TSPCs could repre-
sents an important tool to study basic tendon biology. The
exact site for TPSCs cells within tendon is not known, but
they are most likely to reside in the endotenon tissue be-
tween the collagen fascicles and adjacent to the vascu-
lature33. Although this might be true in young growing ten-
don, mature equine tendon, however, does not appear to
possess a substantial subpopulation of these cells capa-
ble of differentiating into multiple cell lines, as reported for
adult tissue34,35, and this may explain why this component
of the repair process is limited and hence natural repair
is inferior to normal tendon.
During the repair process, there is a large influx of cells
into the lesion. Kajikawa et al.36 showed that at 24 h af-
ter the injury, the wound contained circulation-derived
cells but not tendon-derived cells. Tendon-derived cells
appeared in the injured area at 3 days after the wound,
and significantly increased in number with time and main-
tained a high level of proliferative activity until 7 days
after the injury, whereas the circulation-derived cells de-
creased in number and are replaced by the tendon-de-
rived cells. These findings suggest that circulation-derived
and tendon-derived cells contribute to the healing of ten-
dons in different periods as part of a biphasic process but
that the cells mainly involved in the synthesis of new tis-
sue are believed to be tendon derived cells36,37. For this
reason some authors hypothesized that the implantation
of far greater numbers of progenitor stem cells, than are
present normally within tendon tissue, would have the
potential of regenerating or improving the repair of the
tendon. Fibroblasts derived from tendon or other
sources could be used38, but the removal of sections of
tendon to recover cells leads to the formation of a sec-
ondary lesion in the horse that is unacceptably. Alterna-
tive cell sources under investigation (Tab. 1) include
dermal fibroblasts, which were shown to be capable of
functionally bridging a tendon defect and to have simi-
lar histological and tensile properties to the tenocyte-
seeded scaffold39 although in vitro these cells behave
differently from tenocytes40. By contrast, an optimal in
vivo regenerative response could be accomplished by
MSCs of different sources (Tab. 1).
Stem cell therapies in tendons
MSCs have been implanted into surgical defects in ten-
dons in multiple in vivo experiments in laboratory animals
with mostly positive outcomes. Most of these models
have used surgically created defects in rabbit or rat ten-
dons and have variously shown some improvement in
The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221 213
C. Tetta et al.
214 Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221
Cell source Advantages Disadvantages Ref
EMBRYO Embryonic stem cells - pluripotent - teratoma formation [88]
(ESC)
EXTRA-FETAL Amnion-derived cells no invasive collection - strict surveillance [72]
TISSUED high plasticity and of parturition
proliferative capacity
high number of
immediately available
cells for therapy
well-tolerated by horses
MSCs from umbilical - no invasive collection - strict surveillance [89-90]
cord tissue - greater multipotent than of parturition
BM-MSCs
- possibility to obtain more
rapidly proliferating cells
by cell sorting
- no immune response
ADULT TISSUES Concentrated bone - minimal manipulation invasive aspiration [91]
marrow aspirate (BMC) - no cell expansion procedure with risk of
pneumopericaridium
no reports on the use
of BMC on tendonitis
Stromal vascular fraction - minimal manipulation - invasive collection [92]
from adipose tissue - no cell expansion
- well-tolerated by horse
ADULT MSCs from bone - multipotenti - invasive aspiration [93-95]
STEM/PROGENITOR marrow (BM-MSCs) - no immune response procedure with risk of
CELLS pneumopericaridium
- limited potential than ESC
in terms of expansion (delay
of 2-4 weeks to obtain
a sufficient number of cells
to in vivo implant)
MSCs from adipose tissue - higher proliferative - invasive collection [94-97]
potential and less
senescence of BM-MSCs
- multipotent
Tendon stem/progenitor - possible activation of this - invasive collection [31]
cells endogenous population (removal of sections of
- multipotent tendons leads to the
formation of secondary lesion)
- mature equine tendon
do not posses a substantial
population of these cells
ADULT Tenocytes - appropriate tendon - invasive collection [38]
DIFFERENTIATED matrix synthesis - age-related reduction
CELLS in synthesis of matrix ability
Fibroblasts derived - appropriate tendon matrix - invasive collections [37]
from tendon synthesis
Dermal fibroblasts - easy to recover, with - different protein-matrix [39]
acceptable donor site lesion synthesis than tenocytes
- similar histological and
tensile properties than
tenocyte
Table 1 - Sources for cell therapy of tendinopathies.
The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221 215
structure and strength of defects implanted with MSCs in
a biodegradable scaffold (collagen gel, Vicryl knitted
mesh or fibrin glue) compared to controls implanted with
just the scaffold, as assessed by histology or simple bio-
chemical assays41-45. In other studies using a rat patellar
defect model, MSCs implantation has been associated
with both greater ultimate tensile stress and improved
quality of reparative tissue determined by an increased
collagen I/III ratio46,47. Thus, MSCs-seeded constructs
implanted in vivo have shown the ability to integrate into
the tissue and induce the synthesis of tissue-specific ex-
tracellular matrix. In the horse, tendon injuries are mostly
located in the superficial digital flexor tendon (SDFT),
which represents the strongest tendon in the equine body.
The SDFT displays several similarities to the human
Achilles tendon concerning anatomy, biomechanics and
pathogenesis of tendinopathy. In different species, path-
omorphology of tendinopathy differs in lesion size. In the
horse, one typical so-called “core lesion” is usually cen-
trally located within the tendon, extended in length and still
surrounded by intact tendon tissue. The equine SDFT in-
jury lends itself to cell therapy because provide many of
the additional elements required for tendon tissue engi-
neering. The lesion manifests within the central core of the
tissue provides a natural enclosure for implantation that,
at the time of stem cell implantation is filled of granulation
tissue, which acts as a scaffold (Fig. 1)48. This enables the
application of MSCs without any artificial scaffold mate-
rial, merely by injecting a cell suspension directly into the
lesion49; thereby, MSCs are exposed to a natural environ-
ment providing collagen fibers and growth factors. In ad-
dition, during rehabilitation with controlled exercise, there
is an ideal mechanical stimulation allowing the newly
created tissue to organize itself in the direction of the force
application, hence this approach can be referred to as “in
vivo tissue engineering”50. Unfortunately, in the horse,
the efficacy of these treatments is difficult to determine,
since the use of control animals is rarely reported and of-
ten the stem cell treatment is combined with other biolog-
ical factors, such as bone marrow supernatant, autolo-
gous serum, or platelet-rich plasma. In any case, since
this treatment regime was first published in 200349 there
have been several experimental and clinical studies with
encouraging results, giving evidence of the benefit and
safety of MSCs application for tendon regeneration. Fur-
thermore, unfortunately, it is still unclear whether the ma-
jor contribution of the MSCs to the healing process is to
differentiate into tenocytes and thus produce extracellu-
lar matrix molecules, whether it is rather to supply growth
factors and thus stimulate the residing cells within the ten-
don51,52 or whether a combination of the two mechanisms
occurs6,53. Mononuclear cells could represent an exo-
genic stimulus for induction of pro-inflammatory mediators
in tendon54. In addition, recent studies have suggested an
anti-inflammatory role of implanted stem cells. In this
context animal model studies have demonstrated that
MSCs are hypo-immunogenic and inhibit the activation of
T and B lymphocytes and NK cells55,56. The precise mech-
anism of the anti-inflammatory effect of these cells is
largely unknown. The role of soluble factors and extracel-
lular vesicles as effectors in paracrine effect is described
below. In essence, the paracrine effect results in the
combination of different, biological activities: anti-apopto-
sis, additional recruitment of resident multipotent stem
cells, stimulation of angiogenesis, and the release of
growth factors48.
The clinical and not experimental nature of the use of
MSCs for horse tendinopathies preclude the routine post
mortem analyses but some experimental works has been
carried out to monitor the fate of injected MSCs in horses
and the structural aspect of the healing. Guest et al.57
studied the fate of autologous and allogeneic MSCs trans-
fected with green fluorescent protein (GFP) following in-
jection into the SDFT and revealed that GFP labeled
cells located mainly within injected lesions, but with a
small proportion integrated into healthy tendon. Further-
more, the authors showed that both autologous and allo-
geneic MSCs may be used without stimulating an unde-
sirable cell mediated immune response from the host.
Other postmortem examinations have shown that MSCs
application improved the extracellular matrix structure of
damaged tendons. In histological sections of MSC-treated
tendon lesions, compared to non-treated tendon lesions,
increased tendon fiber densities, increased organization
Figure 1. Severe SDFT core lesion in a forelimb SDFT. Arrows show anechoic area in transverse (A) ultrasound scans, and slightly
ipoechoic area in transverse (B) ultrasound sections, respectively, in the same lesion 50 days after amniotic derived cells implant.
C. Tetta et al.
216 Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221
of the collagen fibers and a reduced vascularity have
been found58-60. The beneficial effect of MSCs seems to
be due to the improvement of structural organization
rather than of matrix composition. However, it has been
shown that MSCs treatment can enhance expression
levels of cartilage oligomeric matrix protein (COMP)58,59,
a glycoprotein that is known to be important for tendon
elasticity and stiffness62. Ultrasonographic follow-up ex-
aminations showed significant improvements in fiber
alignment and echogenicity scores at 1, 3 and 6 months
after MSCs treatment63, supporting the histological find-
ings in the above-mentioned studies. In these studies, au-
tologous adult progenitor cells have been used, either ex-
panded bone marrow-derived MSCs60,64-66, or adipose
derived MSCs59,67 or adipose-derived mononuclear cells
(ADNCs)58,68. Furthermore, the effects of autologous bone
marrow derived expanded MSCs and bone marrow-de-
rived mononuclear cells on tendon healing have been
compared revealing a similar improvement, in both treat-
ment groups compared to the control group, which was
demonstrated by significantly improved ultrasonography
and histology scores, higher COMP expressions and rel-
atively lower type III collagen contents61,70.
If stem cells are truly immunomodulatory, allogeneic trans-
plantations should be possible. Safe and efficacious ap-
plications of allogeneic stem cells would imply that off -
the-shelf stem cell products could be developed for
increased availability and rapid implementation of stem
cell therapies early in a disease course54. Indeed, not only
autologous progenitor cells but also allogeneic bone mar-
row-derived MSCs57, allogeneic adipose-derived MSCs67
and allogeneic amniotic derived MSCs72 have been ap-
plied for treatment of equine tendon injuries and no evi-
dence of immune rejection were detected.
Extracellular vesicles released from MSCs as an
emerging paracrine mechanism
Recent studies have shown that beside soluble factors
small vesicles released from cells, named extracellular
vesicles or MVs, are instrumental in cell-to-cell commu-
nication73,74 (Fig. 2). MVs are an heterogeneous popula-
tion of small vesicles constituted by a circular fragment of
membrane containing cytoplasm components which are
released by different cell types. The two major classes of
MVs released in the extracellular environment are the ex-
osomes and shedding vesicles75. Exosomes originate
from inward of endosomal membrane, accumulate within
multivesicular bodies, are secreted by a process of exo-
cytosis and exhibit a 30-120 nm size. At variance, shed-
ding vesicles take place from direct budding of plasma
membrane surface and are more heterogeneous in size
ranging from 80nm to <1mm depending from the cell of
origin and on stimuli75. The released MVs can be up-taken
by neighbouring cells either as result of surface receptor
mediated interaction or by a process of membrane fusion.
After interaction MVs can be internalized by the recipient
cells and deliver their content73,74. Therefore, MVs have
been uncovered as a new mechanism of inter-cellular
communication that involves direct receptor mediated
stimulation of the target cells and delivery of bio-active
lipids, proteins and nucleic acids. The content of MVs and
their biological action not only depends on the cell of ori-
Figure 2. Schematic representation of the potential anti-inflammatory action of microvesicles (MVs) released by mesenchymal stem
cells (MSC) on horse tendon.
The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221 217
gin, but also on the metabolic state of the cells. Therefore,
different stimuli may modify not only the amount of MVs
release, but also their content. One of the most exiting
findings is that MVs were found to be a vehicle for ex-
change of genetic information capable to induce transient
or permanent phenotypic changes in the recipient
cells73,74. This observation has deep implications in differ-
ent physiological and pathological conditions. In the con-
test of stem cell biology it has been suggested that sig-
nals shuttled by MVs are an integral component of the
stem cell niche and may be critical in the differentiation de-
cision of stem cells76. In particular, the signals between in-
jured cells and stem cells are bi-directional73. Indeed,
MVs derived from injured cells are able to induce tissue
specific differentiation of bone marrow cells and MVs de-
rived from stem cells are capable to activate regenerative
programs in cells survived to injury. The first possibility is
proved by the observation that MVs released from injured
lung cells induce expression of specific lung transcripts
and phenotypic changes in bone marrow cells77. The hor-
izontal transfer of genetic information from stem/progen-
itor cells to differentiated cells was firstly shown for MVs
derived from human endothelial progenitors (EPC). These
MVs shuttle mRNA to quiescent endothelial cells via in-
teraction with specific adhesion molecules (?4- and ?1-
integrins) and activate an angiogenic program78. The mo-
lecular analysis of mRNA indicate that MVs derived from
EPC contain specific subset of cellular mRNA, including
mRNA associated with pathways relevant for angiogen-
esis such as the PI3K/AKT and eNOS signalling path-
ways78. This mRNA are functional as are they are trans-
lated into proteins within the recipient cells. Besides
mRNA, MVs may transfer microRNAs (miRNAs) to target
cells79. Since miRNAs are naturally occurring regulators
of protein translation, this observation opens the possibil-
ity that stem cells can alter the expression of gene prod-
ucts in neighbouring cells by transferring miRNAs con-
tained in MVs80.
Concerning the regenerative potential of MSC-derived
MVs experiments have been performed in different animal
models of tissue injury81-85. In models of acute renal injury
MSC-derived MVs were found to be able to mimic the
beneficial effects of the cells. In particular MVs acceler-
ate the recovery in models of toxic and ischemia-reperfu-
sion injury of the kidney and significantly enhance survival
in a lethal model of cisplatin induced acute renal in-
jury81,82. The mechanism was related to the delivery of
mRNA derived from the MSCs and to its translation in the
recipient cells. Through this mechanism MSC-derived
MVs can limit the injury by inhibiting apoptosis and stim-
ulate regeneration by inducing cycle re-entry of injured tu-
bular epithelial cells. Therefore, the recovery for acute re-
nal injury promoted by MSCs, mainly take place from the
renal resident cells that undergo transient de-differentia-
tion, proliferation to reconstitute the loss cell mass and fi-
nally re-differentiation. Similar results were observed in a
model of ischemic hearts treated with MVs derived from
embryonic MSCs84,85.
Based on these observations, we can speculate that MVs
released from MSCs may act also in different context of
regenerative medicine such as the tendinopathies (Fig. 2).
MVs, released by MSCs, may interact with and stimulate
tendon-resident cells to initiate an anti-inflammatory, anti-
apoptotic and angiogenic response, and to reprogram so-
matic cells toward a regenerative response. In particular,
MVs derived from MSCs may counteract the action of in-
flammatory cells accumulated at the site of injury.
Perspectives
In recent years, regenerative medicine has emerged as
an attractive field for new cellular and non-cellular ap-
proaches to tissue repair. The current knowledge on the
mechanisms of the regenerative potential of MSCs put at-
tention on the role of soluble components released by
cells in the conditioned media. Soluble components, or
growth factors, are used indirectly in equine medicine, as
before discussed, in cases where stem cells are com-
bined with platelet rich plasma, bone marrow supernatant,
or autologous serum.
Growth factors are peptide signaling molecules that reg-
ulate many aspects of cellular metabolism including the
cell cycle, cell growth and differentiation, and the produc-
tion and destruction of extracellular matrix products. Their
effects are mediated primarily via autocrine and paracrine
mechanisms, which provides the rationale for local admin-
istration of exogenous growth factors to influence cellu-
lar metabolism59. Of the growth factors influencing tendon
metabolism, platelet derived growth factor, insulin-like
growth factor-I (IGF-I), and transforming growth factor β
show the most promise for enhancing tendon healing86.
Although exogenous IGF-I has been shown to stimulate
tendon healing in vivo in an equine model86 it has a short
half-life, which necessitates repeated dosing, making
clinical application challenging and costly. For this reason
Schnabel et al.59 examined the effects of MSCs, as well
as IGF-I gene enhanced MSCs (AdIGF-MSC) on tendon
healing in vivo showing that both MSC and AdIGF-MSC
injection resulted in significant histological tendon healing
with minimal added value of IGF-I gene-enhanced MSC
implantation compared to native MSC injection. This min-
imum added value would confirm the hypothesis that in it-
self the stem cells secrete growth factors and that the
therapeutic effects of MSCs are mediated by paracrine
factors secreted by the cells to stimulate the residing
cells within the injured tissue rather than differentiate
themselves. These paracrine factors could be exploited
to extend the therapeutic possibilities of MSCs for the
treatment of a variety of diseases. In this context MVs
have a potential therapeutic application, as they mimic
several of the biological actions of stem cells and may limit
the concern of using of active replicating cells that may
undergo mal-differentiation or mutation. In addition, MVs
may be engineered to express and deliver molecules
that favor reprogramming of resident cells toward regen-
eration.
Conclusions
Use of the cells and technologies presented here in the
horse are likely to continue and expand in the near future.
The horse has been advocated as an animal model of
tendon and ligament injuries, since many of the sponta-
neous injuries seen in horses are similar to those seen in
human athletes but other equine tissues and diseases,
such as recurrent airway obstruction (asthma) and vari-
ous hypoxic ischemic injuries, seem like straightforward
candidates for equine stem cell research.
It is hoped that experience gained from treating naturally-
occurring tendon injury in horses will provide sufficient
supportive data to encourage the translation of this tech-
nology into the human field where large randomized con-
trolled trials will lead to a higher level of clinical evi-
dence87.
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... Exosomes are found as the main agent mediating the therapeutic effect of many factors secreted by MSC for several diseases and injuries. Tetta C et al. discuss the exosomal potential to treat tendon injuries in the horse animal model and described it as an attractive option (Tetta et al., 2012). ...
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The knee joint is one of the largest, most complex, and frequently utilized organs in the body. It is very vulnerable to injuries due to activities, diseases, or accident, which lead to or cause knee joint injuries in people of all ages. There are several types of knee joint injuries such as contusions, sprains, and strains to the ligament, tendon injuries, cartilage injuries, meniscus injuries, and inflammation of synovial membrane. To date, many drug delivery systems, e.g. nanoparticles, dendrimers, liposomes, micelles, and exosomes, have been used for the treatment of knee joint injuries. They aim to alleviate or reverse the symptoms with an improvement of the function of the knee joint by restoring or curing it. The nanosized structures show good biodegradability, biocompatibility, precise site-specific delivery, prolonged drug release, and enhanced efficacy. They regulate cell proliferation and differentiation, ECM synthesis, proinflammatory factor secretion, etc. to promote repair of injuries. The goal of this review is to outline the finding and studies of the novel strategies of nanotechnology-based drug delivery systems and provide future perspectives to combat the challenges of knee joint injuries by using nanotechnology.
... Stem cells have been widely used in the treatment of musculoskeletal diseases (150,151). Current evidence suggests that stem cell therapy is highly effective on musculoskeletal disorders (152,153). Stem cells are considered as cells that have the ability to divide and self-renew over a long period of time and capable of differentiating in all cell lines (154,155). Stem cells have been assumed to promote regeneration during tendon healing (156,157). ...
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Tendon is a fibro-elastic structure that links muscle and bone. Tendon injury can be divided into two types, chronic and acute. Each type of injury or degeneration can cause substantial pain and the loss of tendon function. The natural healing process of tendon injury is complex. According to the anatomical position of tendon tissue, the clinical results are different. The wound healing process includes three overlapping stages: wound healing, proliferation and tissue remodeling. Besides, the healing tendon also faces a high re-tear rate. Faced with the above difficulties, management of tendon injuries remains a clinical problem and needs to be solved urgently. In recent years, there are many new directions and advances in tendon healing. This review introduces tendon injury and sums up the development of tendon healing in recent years, including gene therapy, stem cell therapy, Platelet-rich plasma (PRP) therapy, growth factor and drug therapy and tissue engineering. Although most of these therapies have not yet developed to mature clinical application stage, with the repeated verification by researchers and continuous optimization of curative effect, that day will not be too far away.
... These tissues contain progenitor cells able to differentiate into target cell lines or tissues in vitro [8]. However, in vivo, the improved outcomes can probably be attributed to immunomodulation and bioactive factors, such as cytokines and growth factors [7,9], either secreted directly or transferred alongside packaged genetic material in the form of exosomes [10,11]. In the context of musculoskeletal injuries, including sports injuries, mesenchymal stem cells (MSCs) are the most frequently studied cellbased therapy [12,13]. ...
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... The research on the mechanisms exerted by these vesicles and their potential clinical application is still an emerging field; however, a variety of clinical trials are already on their way, including treatment of dermal wounds, psoriasis, and type I diabetes. As EVs elicit several of the biological actions also observed for adult stem cells, they resemble an attractive option to treat diseased tendons, as they eliminate some of the potential disadvantages when using active, replicating cells that may undergo mal-differentiation or mutation (Tetta et al. 2012). ...
... Previous findings report promising results when stimulating DCs with growth factors such TGF-beta and BMP-3 and also with conditioned media derived from hMSCs [8,30,31] as these signaling molecules are believed to act either on themselves (autocrine) or on neighboring cells (paracrine) [32,33] to bring about the observed regenerative effects. In this study, it was demonstrated that with EV stimulation, an enhanced ECM production was observed as early as in day 7 in these 3D pellet culture models. ...
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Background: Extracellular vesicles (EVs) from human mesenchymal stem cells (hMSCs) are known to be mediators of intercellular communication and have been suggested as possible therapeutic agents in many diseases. Their potential use in intervertebral disc (IVD) degeneration associated with low back pain (LBP) is yet to be explored. Since LBP affects more than 85% of the western population resulting in high socioeconomic consequences, there is a demand for exploring new and possibly mini-invasive treatment alternatives. In this study, the effect of hMSC-derived small EVs (sEVs) on degenerated disc cells (DCs) isolated from patients with degenerative discs and chronic LBP was investigated in a 3D in vitro model. Methods: hMSCs were isolated from bone marrow aspirate, and EVs were isolated from conditioned media of the hMSCs by differential centrifugation and filtration. 3D pellet cultures of DCs were stimulated with the sEVs at 5 × 1010 vesicles/ml concentration for 28 days and compared to control. The pellets were harvested at days 7, 14, and 28 and evaluated for cell proliferation, viability, ECM production, apoptotic activity, chondrogenesis, and cytokine secretions. Results: The findings demonstrated that treatment with sEVs from hMSCs resulted in more than 50% increase in cell proliferation and decrease in cellular apoptosis in degenerated DCs from this patient group. ECM production was also observed as early as in day 7 and was more than three times higher in the sEV-treated DC pellets compared to control cultures. Further, sEV treatment suppressed secretion of MMP-1 in the DCs. Conclusion: hMSC-derived sEVs improved cell viability and expedited chondrogenesis in DCs from degenerated IVDs. These findings open up for new tissue regeneration treatment strategies to be developed for degenerative disorders of the spine.
... Previous ndings report promising results when stimulating DCs with growth factors such TGF-beta, BMP-3, and also with conditioned media derived from hMSCs [8,30,31] as these signaling molecules are believed to either act on themselves (autocrine) or on neighboring cells (paracrine) [32,33] to bring about the observed regenerative effects. In this study, it was demonstrated that with EV stimulation an enhanced ECM production was observed as early as in day 7 in these 3D pellet culture model. ...
Preprint
Full-text available
Background: Extracellular vesicles (EVs) from human mesenchymal stem cells (hMSCs) are known to be mediators of intercellular communication and has been suggested as possible therapeutic agents in many diseases. Their potential use in intervertebral disc (IVD) degeneration associated with low back pain (LBP) is yet to be explored. Since LBP affects more than 85% of the western population resulting in high socioeconomic consequences there is a demand for exploring new and possibly mini-invasive treatment alternatives. In this study, the effect of hMSCs derived small EVs (sEVs) on degenerated disc cells (DCs) isolated from patients with degenerative discs and chronic LBP was investigated in a 3D in vitro model. Methods: hMSCs were isolated from bone marrow aspirate and EVs were isolated from conditioned media of the hMSCs by differential centrifugation and filtration. 3D pellet cultures of DCs were stimulated with the EVs at 5x10¹⁰ vesicles/mL concentration for 28 days and compared to control. The pellets were harvested at day 7, 14, and 28 and evaluated for cell proliferation, viability, ECM production, apoptotic activity, chondrogenesis and cytokine secretions. Results: The findings demonstrated that treatment with sEVs from hMSCs resulted in more than 50% increase in cell proliferation and decrease in cellular apoptosis in degenerated DCs from this patient group. ECM production was also observed as early as in day 7 and was more than three times higher in the sEVs treated DC pellets compared to control cultures. Further, sEVs treatment suppressed secretion of MMP-1 in the DCs. Conclusion: hMSC derived sEVs improved cell viability and expedited chondrogenesis in DCs from degenerated IVDs. These findings open up for new tissue regeneration treatment strategies to be developed for degenerative disorders of the spine.
... Previous findings report promising results when stimulating DCs with growth factors such TGF-beta, BMP-3, and also with conditioned media derived from hMSCs [8,27,28] as these signaling molecules are believed to either act on themselves (autocrine) or on neighboring cells (paracrine) [29,30] to bring about the observed regenerative effects. In this study, it was demonstrated that with EV stimulation an enhanced ECM production was observed as early as in day 7 in these 3D pellet culture model. ...
Preprint
Full-text available
Background Extracellular vesicles (EVs) from human mesenchymal stem cells (hMSCs) are known to be mediators of intercellular communication and has been suggested as possible therapeutic agents in many diseases. Their potential use in intervertebral disc (IVD) degeneration associated with low back pain (LBP) is yet to be explored. Since LBP affects more than 85% of the western population resulting in high socioeconomic consequences there is a demand for exploring new and possibly mini-invasive treatment alternatives. In this study, the effect of hMSCs derived small EVs (sEVs) on degenerated disc cells (DCs) isolated from patients with degenerative discs and chronic LBP was investigated in a 3D in vitro model. Methods hMSCs were isolated from bone marrow aspirate and EVs were isolated from conditioned media of the hMSCs by differential centrifugation and filtration. 3D pellet cultures of DCs were stimulated with the EVs at 5 × 10¹⁰ vesicles/mL concentration for 28 days and compared to control. The pellets were harvested at day 7, 14, and 28 and evaluated for cell proliferation, viability, ECM production, apoptotic activity, chondrogenesis and cytokine secretions. Results The findings demonstrated that treatment with sEVs from hMSCs resulted in more than 50% increase in cell proliferation and decrease in cellular apoptosis in degenerated DCs from this patient group. ECM production was also observed as early as in day 7 and was more than three times higher in the sEVs treated DC pellets compared to control cultures. Further, sEVs treatment suppressed secretion of MMP-1 in the DCs. Conclusion hMSC derived sEVs improved cell viability and expedited chondrogenesis in DCs from degenerated IVDs. These findings open up for new tissue regeneration treatment strategies to be developed for degenerative disorders of the spine.
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Introduction: Chronic musculoskeletal pain is very prevalent, and accounts for major health-care expenses. Many of the present therapeutic modalities are only partially effective, and great interest is now posed on regenerative medicine. Areas covered: The authors discuss the role of a variety of regenerative medicine options to induce and favor regeneration and healing of tendon tissue, focusing on the role of mesenchymal stem cell therapy and their derivatives. Expert opinion: Stem cells, tissue engineering, and growth factors are new strategies for tendon repair and regeneration. MSCs not only can differentiate in tendon cells, but also secrete several cytokines that modulate inflammation and tissue healing. Future studies should be undertaken to overcome current obstacles to clinical translation. Further investigation of cell source, isolation, expansion, and differentiation methods, characterization of the tenogenic differentiation pathways, and clarifications of tendon-specific molecular markers are required. The role of donor variability, tendon type, and anatomic location also requires further understanding and research.
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Tendon injuries are among the most common orthopedic injuries in competition and race horses. It has already been reported that the intralesional injection of mesenchymal stem cells (MSCs) results in a better tendon healing and reduces re-injury rates compared to conventional therapies. The aim of this study was to evaluate success and safety of MSC-therapy in horses suffering from tendon and ligament injuries and to identify factors that influence the success of MSC-therapies. 98 horses had presented with tendon or ligament injuries, of which approximately two-thirds were suspensory ligament (SL) lesions and one-third superficial digital flexor tendon (SDFT) lesions. All horses had been treated with an intralesional injection of autologous bone marrow MSCs and follow-up information was obtained by the responsible practitioner. MSC-therapy associated complications and the MSC-treatment success rate were assessed based on the data obtained for all 98 horses. Treatment success was defined as return to intended use and/ or return to full training without re-injury. Followup information over a period longer than 12 months could be obtained for 58 horses. For these 58 horses, data was additionally analyzed with regard to the questions whether the disease stage at which the MSCs had been applied, the discipline in which horses used to perform, the age of the horses, or the injury localization (SL vs. SDFT) influence the treatment success rate. In 6 out of 98 cases treatmentassociated complications such as transient swellings had been observed; however, this had no effect on the clinical outcomes. The success rate after more than 12 months after treatment (n = 58) was 84.5%. Patients with acute or chronic disease showed overall better results (82.2% and 91.7% respectively) than horses that had presented with recurrent disease (50.0%). Generally, six to twelve year old horses showed a higher success rate (88.9%) compared to older horses (75.0%). Competition horses reinjured less frequently (10.0%) than flat (27.3%) and trot race horses (66.6%). The success rate did not vary between horses with SL or SDFT injuries. However, the localization of the lesion within the SL seems to play a role concerning the treatment success. The results of this case study confirm the positive results reported in previous studies on regenerative tendon therapy with MSCs. Furthermore it shows that MSC-therapy is not only successful in the treatment of SDFT lesions but also leads to promising results in the treatment of SL injuries.
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In veterinary medicine, there are options available for both small and large animal practitioners to utilize autologous adipose-derived stem/stromal cells (AD-SCs) to promote healing for injuries and degenerative joint disease' • By provision of a living bioscaffolding to encourage stem cell adherence—proliferation, the additional cell availability can be further enhanced with addition of high-density platelet-rich plasma (HDPRP). • The potential of stem/stromal cells, coupled with important inflammatory promotion (HDPRP), is recognized as safe and efficacious in both open wound surgical care and guided placement. • The ability to prepare a site for skin graft by placement of AD-SCs in recalcitrant full-thickness wounds speeds the healing and recuperation of small and large defects in animals. • AD-SCs are of significant value in musculoskeletal tissue injury or disease because there is gradual depletion of native stem/stromal cells in chronic injury or degenerative states. • Multiple studies support the effectiveness of AD-SCs for use in connective tissue and joint repair, among other potential uses. • Controlled veterinary clinical trials are continuing, which will provide statistical documentation of the safety and efficacy of AD-SCs, as well as comparisons of different protocols for administration. • Utilization of AD-SCs, with or without HDPRP concentrates, have proven very effective in several thousand injections in preclinical and clinical use by both human and veterinary physicians in the U.S. and elsewhere. Reference 1. Alderman D, Alexander B, Harris G, Astourian P. Stem cell prolotherapy: Background, research and protocols. J Prolother, August 2011.
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Tendon injuries are among the most common orthopedic injuries in competition and race horses. It has already been reported that the intralesional injection of mesenchymal stem cells (MSCs) results in a better tendon healing and reduces re-injury rates compared to conventional therapies. The aim of this study was to evaluate success and safety of MSC-therapy in horses suffering from tendon and ligament injuries and to identify factors that influence the success of MSC-therapies. 98 horses had presented with tendon or ligament injuries, of which approximately two-thirds were suspensory ligament (SL) lesions and one-third superficial digital flexor tendon (SDFT) lesions. All horses had been treated with an intralesional injection of autologous bone marrow MSCs and follow-up information was obtained by the responsible practitioner. MSC-therapy associated complications and the MSC-treatment success rate were assessed based on the data obtained for all 98 horses. Treatment success was defined as return to intended use and/ or return to full training without re-injury. Followup information over a period longer than 12 months could be obtained for 58 horses. For these 58 horses, data was additionally analyzed with regard to the questions whether the disease stage at which the MSCs had been applied, the discipline in which horses used to perform, the age of the horses, or the injury localization (SL vs. SDFT) influence the treatment success rate. In 6 out of 98 cases treatmentassociated complications such as transient swellings had been observed; however, this had no effect on the clinical outcomes. The success rate after more than 12 months after treatment (n=58) was 84.5%. Patients with acute or chronic disease showed overall better results (82.2% and 91.7% respectively) than horses that had presented with recurrent disease (50.0%). Generally, six to twelve year old horses showed a higher success rate (88.9%) compared to older horses (75.0%). Competition horses reinjured less frequently (10.0%) than flat (27.3%) and trot race horses (66.6%). The success rate did not vary between horses with SL or SDFT injuries. However, the localization of the lesion within the SL seems to play a role concerning the treatment success. The results of this case study confirm the positive results reported in previous studies on regenerative tendon therapy with MSCs. Furthermore it shows that MSC-therapy is not only successful in the treatment of SDFT lesions but also leads to promising results in the treatment of SL injuries.
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In calcific tendinopathy (CT), calcium deposits in the substance of the tendon, with chronic activity-related pain, tenderness, localized edema and various degrees of decreased range of motion. CT is particularly common in the rotator cuff, and supraspinatus, Achilles and patellar tendons. The presence of calcific deposits may worsen the clinical manifestations of tendinopathy with an increase in rupture rate, slower recovery times and a higher frequency of post-operative complications. The aetiopathogenesis of CT is still controversial, but seems to be the result of an active cell-mediated process and a localized attempt of the tendon to compensate the original decreased stiffness. Tendon healing includes many sequential processes, and disturbances at different stages of healing may lead to different combinations of histopathological changes, diverting the normal healing processes to an abnormal pathway. In this review, we discuss the theories of pathogenesis behind CT. Better understanding of the pathogenesis is essential for development of effective treatment modalities and for improvement of clinical outcomes.
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Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures. Subsequently, tenocytes gene expression of extra-cellular matrix (ECM) components (type I collagen, decorin, fibronectin), the cell-ECM receptor β1-integrin, the angiogenic factor myodulin, ECM degrading matrix-metalloproteinase (MMP)1 and pro-inflammatory cytokines (interleukin [IL]-1β, tumour necrosis factor [TNFα] and IL-6) was analysed. The only significant effect of leukocytes on tenocytes ECM genes expression was a suppression of type I collagen by neutrophils combined with TNFα stimulation. The same effect could be observed analysing the β1-integrin and myodulin gene expression. However, PBMCs up-regulated significantly cytokine and MMP1 gene expression in tenocytes. These in vitro results suggest that mononuclear cells could present an exogenic stimulus for the induction of pro-inflammatory and catabolic mediators in tendon.