Role of Mesenchymal Stem Cells in Orthopedic Treatment of Tendon and Ligament Damage_LinkedIn

The Role of Mesenchymal Stem Cells in Orthopedic Treatment of Tendon and Ligament
Damage
Daniel Wojciech Janisz
University of Toledo
2015

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Dedication
I would like to dedicate this work to my best friend and sister, Dominika whose
unwavering fortitude has served as my inspiration and motivation throughout the course of my
life. As your big brother, I promise to always protect you, and as your best friend, I promise that
you’ll never walk alone.

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Acknowledgements
Although only my name appears on the cover of this project, a number of individuals
have significantly contributed to its completion. Words cannot express the debt of gratitude that I
owe to all those that have stood by me and served as mentors, guides, and as my support system.
Along with making this project possible, the selfless contributions of these individuals have
made my graduate experience one that I will cherish forever.
I would like to express my deepest gratitude to my mentor, Dr. Patrick Siparsky. I have
been very fortunate to have an advisor who gave me the freedom to explore on my own, and at
the same time offered invaluable guidance when my steps faltered. Apart from his support on
this project, I am thankful Dr. Siparsky gave me the opportunity to experience the clinical side of
medicine. His dedication, passion, and care has shown me what it means to be a great physician.
I hope that one day I would become a physician of Dr. Siparksy’s caliber. His mentorship goes
beyond this project as I truly value his guidance in my future endeavors. I am truly grateful for
all that he has done.
I would also like to thank my parents, Voytek and Joanna, and my sister Dominika. They
have been my strength and my foundation for as long as I can remember. No matter how many
times I may have fallen they have always been there to pick me up, dust me off, and give me the
push I needed to keep going. This project would not have been possible without their love and
support, and I doubt that I will ever be able to convey my appreciation fully, but I owe them my
eternal gratitude.

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Table of Contents
Introduction 1
Background: Stem Cells 2
Background: Tendon and Ligaments 7
Current Therapeutic Strategies 12
Mesenchymal Stem Cells 16
Mesenchymal Stem Cells to Treat Tendon and Ligament Damage 26
Tissue Engineering Concepts 34
Clinical State and Perspectives for Tendon and Ligament Healing 37
Conclusion 40
References 42
Figures 55
Abstract 57

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List of Figures
Figure 1: Two strategies of stem cell applications in regenerative medicine 7, 55
Figure 2: The mesengenic process 19, 56

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Introduction
Tendon and ligament injuries constitute a significant portion of cases in a variety of
medical fields and in particular orthopedics. It is estimated that tendon injuries account for 30%-
50% of all sports related injuries 1,2
. For example, over 300,0003
Anterior Cruciate Ligament
(ACL) injuries occur annually in the United States. Tendons and ligaments are particularly
susceptible to overuse and stress injuries that span three grades of severity2,4,5
. Grade I injuries
constitute tissue stretching or minor tearing. Grade II injuries constitute partial tissue tears,
presenting with pain and possible joint instability. Grade III injuries constitute complete tissue
tears. Despite advancements in prevention, detection, and conventional treatments, outcomes of
clinical interventions for tendon and ligament injuries are still highly variable. Treatments to
repair the injured tissue include non-steroidal anti-inflammatory drugs (NSAIDs) and
corticosteroids, implantation of autografts, allografts, and synthetic prostheses, as well as
surgical interventions such as direct tendon suturing and ligament reconstruction6,7
. Current
treatments are often inadequate in providing consistent and definitive resolution of tendon and
ligament damage8,9
. Surgical interventions often produce the best outcomes5
; however many
current modalities offer more symptomatic relief instead of fixing the underlying condition5-7
.
The details pertaining to the specific limitations of each modality will be discussed in a later
section.
The term “regenerative medicine” was first introduced in 1992 by Dr. Leland Kaiser
when he predicted that “a new branch of medicine will develop that attempts to change the
course of chronic diseases and in many instances will regenerate tired and failing organ systems
10
.” Regenerative medicine is an interdisciplinary field of research combined with clinical
applications that focuses on understanding the human body’s natural repair, replacement, and
regeneration mechanisms. This fundamental scientific knowledge is then translated into various

2
materials, devices, systems, and treatment strategies in order to replace, engineer, or regenerate
human cells, tissues, and organs in an effort to restore normal physiological function 11,12
.
Research has identified adult stem cells known as mesenchymal stem cells (MSCs) as the most
promising candidates for use in regenerative therapies. MSCs possess self-renewal potential,
multi-lineage differentiation potential towards cartilage, tendon, and bone cells, and they can be
harvested from a variety of tissues 6,11,13,14
. Pre-clinical and clinical trials with MSCs suggest
these cells play critical roles in repairing injured body tissues 13,15
which indicates potential for
application in orthopedic treatment of tendon and ligament damage. Although these results are
promising, researchers must now address more complex obstacles before MSCs are a widely
utilized therapy. The inherent MSC multi-lineage potential, self-renewal potential, and
presentation in multiple body tissues creates a significant amount of variables that must be
accounted for and evaluated in order to determine the safest and most effective treatment
options.
This manuscript focuses on the suitability of MSCs in regeneration and repair of tendon
and ligament tissue. Current research of MSC-based therapies is evaluated in order to address the
mechanisms of action, the advantages and disadvantages of various harvest sites, as well optimal
harvesting and in vitro expansion techniques. Furthermore, this review will discuss specific
tissue engineering methods of introducing MSCs into the human body, as well as the challenges
that need to be overcome in future research and the development of clinical orthopedic
applications.
Background: Stem Cells
Stem cells became of particular interest to regenerative medicine research due to several
unique characteristics distinguishing them from other cell types.13
In order for a cell to be

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considered a stem cell, it must be an unspecialized cell that possess two defining characteristics:
the capacity to renew itself for long periods of time (self-renewal) and the ability to differentiate
into specialized cell types (potency)16
. The intrinsic abilities of self-renewal and potency make
stem cells a major focus of regenerative orthopedic medicine.
Stem Cell “Niche”
A stem cell niche is simply an extracellular microenvironment in which the cell resides
responsible for regulating the biochemical and physical signals a stem cell receives 17
. These
signals are critical in stem cell survival, proliferation, and differentiation 17-19
. The defining
attributes of a stem cell niche include tissue mechanics, composition and structure of the
extracellular matrix, and cell-cell interactions.
Within the various extracellular microenvironments of the human body researchers have
identified what are known as “resident stem cells.” Resident stem cells are undifferentiated cells
that reside in the developed tissues and organs of infants, children, and adults. Researchers have
identified distinct populations of resident stem cells in organs such as the epithelia of the skin
and digestive system, bone marrow, blood vessels, brain, skeletal muscle, and adipose tissue,
among others 16,20
. These cells function to maintain tissue-specific cell populations, as well as
participate in tissue remodeling and repair of tissue/organ in which they reside 20,21
.
Preferential differentiation of resident stem cells toward certain cell types is largely
dependent on the biochemical and physical signals that arise from the stem cell niche in which
they reside. Furthermore, resident stem cells possess a certain affinity for certain niches which
define their localization within the body and impact the ability of cells to mobilize and implant
correctly 16,22
. The therapeutic potential of stem cells is impacted by the niche requirements
making it important to engineer artificial systems conducive to specific stem cell niches. This

4
proves challenging since a stem cell niche is dynamic and changes during development and
varying physiological states19
.
Self-Renewal and Proliferation
The abilities of self-renewal and potency result from a stem cell’s unique capability of
undergoing asymmetric division. Unlike symmetric cell division that produces two identical
daughter cells, asymmetric cell division produces two different daughter cells. One daughter cell
is identical to the parental stem cell, whereas the other daughter cell is a progenitor or
differentiated cell 16
. Asymmetric cell division serves to maintain, not increase the number of
stem cells. Stem cells are capable of undergoing symmetric cell division and rapid proliferation
seen during fetal development or in disease/repair states 22
.
Due to resident stem cells being relatively quiescent (dormant, non-dividing) it is
desirable to culture and expand stem cells in vitro to obtain an adequate quantity that can be used
in research and therapy. Cultured stem cells derived from resident stem cells are known as adult
or somatic stem cells. Establishing cultured stem cell lines has been a topic of dispute in research
due to difficulties in assessing the specific characteristics of individual stem cell types.
Hierarchy of Potency
The ability of a stem cell to differentiate into multiple specialized cell types is known as
potency. Multiple levels of potency exist, each defining the number and types of progenitor cells
that can arise from a certain stem cell. Totipotent stem cells are the most potent cell type,
possessing the ability to autonomously form an entire organism. Totipotent stem cells can only
be found in a zygote at, or before the morula stage (3-4 days post-fertilization, preimplantation).
As an embryo continues to divide, stem cell potency becomes more restricted. At the blastocyst
stage, the cells divide into two pluripotent stem cell populations: embryonic and extraembryonic.

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Embryonic stem cells (ESCs) are found in the inner mass cell mass of the blastocyst and can
form almost all of the bodies cell lineages (endoderm, mesoderm, and ectoderm), including germ
cells. The trophectoderm of the blastocyst contains the extraembryonic (trophoblast) stem cells,
which can populate the placenta 16,22
.
In 2006 researchers successfully dedifferentiated somatic adult fibroblast cells into
pluripotent ESC-like cells, more commonly known as induced pluripotent cells (iPS) 23
. The
adult fibroblast cells underwent in vitro transfection with four transcription factors normally
expressed in ESCs. The overexpression of these transcription factors changed the adult fibroblast
cells into cells similar to ESCs in regards morphology, proliferation, surface antigens, gene
expression, and pluripotency 23,24
. The ability of iPS cells to differentiate into cell types of all
three germ layers shows promise for future development of autologous therapies, however
significant complications prevent clinical applications in present-day 25,26
. One of the major
difficulties associated with iPS cells is controlling lineage-specific differentiation and overall cell
growth. The injection of undifferentiated iPS cells into animal tissue is associated with teratoma
formation and mice generated from iPS cells show high rates of tumorigenicity 13,24,27,28
. The
oncogenic (tumor development) risk alone is enough to prevent the application of ESCs and iPS
cell therapy in humans 13,29
.
The need for alternative stem cell sources lead researchers to investigate multipotent stem
cells. As pluripotent stem cells continue to divide they undergo further specialization which
gives rise to multipotent stem cells. Multipotent stem cells are more restricted than pluripotent
stem cells in their ability to differentiate. Whereas pluripotent stem cells can differentiate into
cells of all three germ layers, differentiation of multipotent stem cells is limited to closely related
cell types within a particular tissue and/or organ. Multipotent stem cells will often differentiate

6
along specific cell lineages. For example, hematopoietic stem cells (HSCs) are multipotent blood
stem cells found in red bone marrow. The HSCs are the progenitor cells uniquely capable of
giving rise to all of the different mature blood cell types and tissues. The stem cells undergo
directed differentiation towards myeloid (red blood cells, platelets, macrophages, neutrophils,
etc.) and lymphoid (T-cells, B-cells, NK-cells) cell lineages. Other than HSCs, multipotent stem
cells have been identified for many different types of human tissues including neural,
endothelial, muscle, mesenchymal, gastrointestinal, and epidermal 18,24,30,31
. These multipotent
stem cells are known as adult stem cells. An adult stem cell is identified as an undifferentiated
cell present in a differentiated tissue. The primary function of adult stem cells is to maintain
tissue homeostasis by replacing damaged cells with the help of self-proliferation and
differentiation.
Oligopotent stem cells such as neural stem cells can form more than one cell lineage but
are more restricted than multipotent cells. Lastly, there are unipotent (monopotent) stem cells
that can only form a single differentiated cell lineage.
Application in Regenerative Medicine
There are two primary strategies of cell-based therapy in regenerative medicine: cell-
therapy and tissue engineering (Figure 1). The cell-therapy approach involves the application of
healthy cells and/or cellular components to substitute the damaged tissue cells. This usually
involves the injection of a cell suspension into the damaged tissue or into blood circulation 13
.
Stem cells are isolated from the patient (autologous transplant) or from a donor (allogenous
transplant) and then expanded in vitro 13
. One of the most successful examples in cell therapy is
the transplantation of hematopoietic stem cells in patients with severe hematological diseases 32-
34
. For bone marrow transplants, hematopoietic stem cells are injected into the blood circulation

7
of the recipient and find their way to the bone marrow with specific chemokines in a process
known as homing 13
. One of the major limitations of cell therapy is that it is often not sufficient
to regenerate large tissue defects or even replace whole organs.
Tissue engineering shows promise in addressing this limitation. The process of tissue
engineering involves seeding tissue-specific cells on a scaffold that imitates the architecture of
the tissue-specific extracellular matrix. Researchers have created multiple different functional
tissue structures in vitro 35-37
. One of the major challenges that is currently limiting clinical
applications of tissue engineering is the yet unsolved problem of vascularization 38,39
.
Figure 1. Two strategies of stem cell applications in regenerative medicine. Stem cells are
isolated from the patient (autologous) or from other donors (allogenous). Once the cells are
expanded in vitro they are directly applied to the patient (cell therapy) or applied to a scaffold
(tissue engineering) to generate an implantable tissue. Reprinted from 13
Background: Tendons and Ligaments
Structure and Function
Tendons and ligaments are specialized connective tissues that are essential to the proper
biomechanical function of joints and limbs. Tendons connect muscle to bone and are responsible
for transmitting the mechanical force from muscle contractions to bones which produces joint
movement. Tendons are composed of dense fibrous connective tissue organized in a hierarchical

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collagen structure. The primary cell types of tendons are spindle-shaped tenocytes (fibrocytes)
which are mature tendon cells anchored to collagen fibers. Bundles of collagen fibers come
together to form primary, secondary, and tertiary fiber bundles that run longitudinally. These
bundles are surrounded by sheathes of connective tissue that facilitate tendon movement. As a
result of their structure, tendons possess one of the highest tensile strengths found among soft
tissues 40
.
Ligaments are connective tissue structures that connect two or more bones together.
Ligaments are found in every joint in the body and are primarily responsible for providing joint
stability. Joints define the range of motion for joints, protect joints and bones by absorbing stress,
and function in maintaining proper proprioception. Like tendons, ligaments are also composed of
dense bundles of collagen fibers. There are two major types of ligaments: white ligaments rich in
inelastic collagen fibers and yellow ligaments rich in elastic fibers that permit joint movement.
At the joint itself, ligaments enclose the articulating bone ends in a joint capsule lined with a soft
tissue known as the synovial membrane. This serves to lubricate the joint and facilitate bone
articulation 41
.
Pathophysiology
When discussing tendon disorders, two common terms are tendinitis and tendinosis.
Tendinitis is defined as tendon inflammation resulting from micro-tears caused by overload of
the musculotendinous unit with a force that is too heavy or too sudden42
. Tendinosis is defined as
the degeneration of the collagen within a tendon as a result of chronic overuse that doesn’t allow
the tendon time to heal and rest42
. Improper distinctions in diagnosing these two disorders has led
to widespread confusion about the differences between tendinitis and tendinosis. In order to
mitigate any confusion, the term tendinopathy will be used as it is a generic descriptive term for

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clinical conditions arising from tendon overuse 43
.
Tendon and ligament injuries can be acute or chronic and have a variety of associated
factors. In general, tendinopathy is associated with disordered tendon healing that can result in
pain, weakness, and future re-injury of the tendon. Tendon rupture is a complete or partial tear
due to overstretching of the tendon. Rapid acceleration-deceleration of the ankle joint is reported
in approximately 90% of sports-related tendon ruptures 43
. Research has found that the most
common histopathological change in tendon ruptures, both chronic and acute, is degeneration
present before the rupture 44-46
. Exposing ligaments to tensions greater than what they can sustain
will also result in partial and/or complete tears. Ligament tears are most often the results of blunt
trauma, planting or pivoting, or anterior knee dislocations that overstretch the ligament 9
.
Tendons and ligaments possess a limited capacity for self-healing primarily due to poor
vascularization. Tendons and ligaments consume 7.5 times less oxygen than other skeletal
muscles 47
. A low metabolic allows tendons and ligaments to carry and maintain tension for long
periods of time, but also results in slow healing after an injury 43
. Tendon and ligament healing
occurs in three phases. The initial inflammatory phase involves the infiltration of red and white
blood cells into the injury site. White bloods cells phagocytize necrotic material and various
cytokines and growth factors are released. During this phase, type III collagen synthesis is
initiated 43
. It is important to note that in healthy tendons and ligaments there is a high proportion
of thick type I collagen. Type III collagen is thinner than type I but is capable of rapid production
of stabilizing cross-links 41,48,49
. The second phase of healing is known as the proliferative phase.
During this phase the synthesis of type III collagen peaks due to increased activity of
hypertrophic fibroblasts. The newly formed tendon and ligament tissue is significantly
disorganized compared to healthy tissue. Reorganization of the tissue requires the third phase of

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healing, matrix remodeling. During this phase the collagen fibers begin to align in the direction
of stress and form crosslinks, improving overall organization. The newly formed tissues have
some similarities in regards to healthy tendon and ligament tissue, but many differences persist.
Resulting tendon tissue is often thickened, fibrotic, and less resistant to tensile strength
when compared to health tendon tissue 43,48
. Differences in the newly formed ligament tissue
include abnormal type III collagen with small fibril diameter that fails to properly generate and
mature stabilizing crosslinks. This leads to weakness in ligament strength and stiffness leaving
the ligament more susceptible to future injury 9,41
. The biological and mechanical properties of
healed tendon and ligament tissue are significantly inferior to those of healthy tendons and
ligaments. Clinical therapies must address the intrinsic and extrinsic factors that limit natural
healing in order to better treat tendinopathy and damage to ligamentous structures in the human
body.
Epidemiology
The successful treatment and prevention of tendon and ligament injuries presents a
significant clinical challenge. It is estimated that in the U.S. alone, tendon and ligament injuries
accounted for approximately 40% of the 65 million health care visits made for musculoskeletal
injuries in 2010 50
. A trend in recent decades of increased interest in competitive and recreational
sporting participation has contributed to a rise in sports related musculoskeletal injuries 51,52
.
Two very commonly injured tendons are the rotator cuff 53,54
and the Achilles tendon 1,2,55
.
Sprains of ankle ligaments and ACL ruptures are the most commonly reported ligament injuries
56,57
.
Rotator cuff tears are a common cause of pain, weakness, and shoulder disability
especially in the aging population. Age-related degenerative changes in the tendon tissue likely

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contribute to rotator cuff tearing 52
. Increasing prevalence of full thickness tears has been
observed in patients as they age. Full thickness tears will be present in approximately 13% of
patients in their 50s 58
, 25% of patients in their 60s, and 50% of individuals in their 80s.
Approximately 40% of patients older than 60 years will be affected by either a partial
thickness tears (fraying of tendon) or full thickness tears (holes/complete detachment) 59
. The
rate of rotator cuff injury has increased 115% 53
with over 75,000 rotator cuff repairs performed
annually in the United States 59,60
.
Although the Achilles tendon is the largest and strongest tendon in the body, it is the
most likely to be injured accounting for 30-50% of all sports-related injuries 61,62
. Achilles
tendon injury rates have increased from 2/100,000 to approximately 12/100,000 on average,
52,55,63
with a majority of injuries occurring in males in the third to fifth decades of life 2,52
. The
Achilles tendon is also the most injured lower extremity tendon in athletes and is cited as the
most common tendon to spontaneously rupture 64,65
.
Ankle sprains are among the most common ligamentous injuries seen in athletic and
general populations. In a systematic review of ankle sprains in sports, the ankle was the most
common site of injury in 24 of the 70 sports that were studied 66
. More specifically, of 43 sports
with ankle injury information, 33 sports (76.6%) reported ankle sprain as the major ankle injury
66
. Ankle sprains were also the most common injury seen in 15 collegiate sports, accounting for
14.8% of all reported injuries 51
. High incidence of ankle sprain injuries are also seen in the
general population. Ankle sprains have an average incidence of 215/100,000 person-years in the
United States. The peak incidence of ankle sprain, 720/100,000 person-years is seen in
individuals between 15 and 19 years of age 67
. Persistent symptoms such impaired function and
pain have been shown to remain in up to 30% of individuals that have suffered an ankle sprain 68
.

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Ankle sprains are also the most common predisposing factor for the occurrence of future ankle
sprains and the development of chronic ankle instability years after an initial injury 66,69-71
.
The ACL is reported as most commonly injured ligament in the knee joint and second
leading cause of injury among athletes 9,51,72
. The reported incidences of ACL injury vary
significantly throughout the literature ranging from 8.1/100,000 per year 52
to over 30/100,000
per year 72,73
making it difficult to provide an accurate incidence of ACL tears among the general
population. Epidemiological studies analyzing trends of ACL reconstruction surgeries in the U.S.
from the mid-1990s to mid-2000s indicate increases of over 30% in ACL reconstruction
surgeries 74,75
. Between 1988 and 2004 the rates of ACL tears among collegiate athletes
exhibited an average annual increase of 1.3% 51
which corresponds to the increased overall
musculoskeletal injury rates.
Current Therapeutic Strategies
There are a variety of options available to clinicians for the management and treatment of
tendon and ligament injuries. An initial clinical evaluation of an individual’s injury permits
clinicians to determine the grade of injury and establish initial management strategies. For
tendon and ligament injuries, initial management strategies are aimed at controlling swelling,
decreasing pain and inflammation, and increasing stability for the patient5
. Clinicians often use
radiological imaging such as ultrasound and magnetic resonance imaging (MRI) to obtain an
internal view of the injury which assists in the diagnosis. Once the injury is diagnosed the patient
is considered for conservative or surgical treatments, each possessing certain advantages and
disadvantages.
Diagnostic Imaging
A standard tool used for the diagnosis of tendon and ligament injuries is an MRI scan.

13
The MRI scan uses a magnetic field and pulses of radio wave energy to create radiographic
pictures of structures within the body. Current research indicates that MRI scans may be rather
ineffective in the diagnosis of tendon and ligament injuries 76
. Although MRI scans have been
shown to be somewhat effective in recognizing major tissue disruptions such as partial tears and
full ruptures, the MRI lacks the ability to detect tendons and ligament stretch or laxity 9,77,78
.
Studies have also shown that MRI scans can have a tendency to inaccurately diagnose false-
positive ligament lesions 79
. This means that the MRI indicates the presence of a ligament injury
when in fact there isn’t one. It has also been discovered that MRI scans can be ineffective in
detecting pathologic lesions in tendon tissue 77,78,80
. For both tendon and ligament injuries,
arthroscopic examination has often been shown as more effective than MRI scans in detecting
pathological lesions 81,82
. The limitations of diagnostic imaging for tendon and ligament injuries
can significantly impact the course of treatment and patient outcomes. Effective diagnosis is the
first step in proper management of tendon and ligament injuries and cannot be ignored.
Conservative (Non-Surgical) Treatments
Conservative treatment options are used for small tears or overuse injuries that do not
require surgical intervention. Non-surgical treatments for tendinopathy include combinations of
rest and immobilization, physical therapy, application of heat and/or ice, and modalities such as
cryotherapy, electrotherapy, and shock wave therapy 8,83
. Pharmaceutical agents such as non-
steroidal anti-inflammatory drugs (NSAIDs) and various peri-tendinous injections of drugs such
as corticosteroids are also used for the conservative management of injury. The use of NSAIDs
and corticosteroid injections appears to be effective for short-term (7-14 days) pain relief 8,84
.
There is inconsistent evidence that NSAIDs and corticosteroid injections are effective in long-
term treatment of tendinopathy. Furthermore, long-term use of NSAIDs may impair healing of

14
damaged tendon and ligament tissue. NSAIDs are known to block cyclooxygenase enzymes that
normally act during the inflammatory phase of injury 85,86
. These enzymes play a significant role
in initiating healing of damaged tendons and ligaments 9
.
Another very common conservative treatment approach is physical therapy. A common
initial intervention for tendon and ligament injuries is to prevent further damage by immobilizing
and resting the injured tissue. Research has shown that immobilization of injured tendons and
ligaments for too long can have a detrimental impact on healing. Physical therapy that
encourages movement of the tendon and ligament is critical for proper organization of collagen
fibrils. Mobilization and exercise has also been shown to preserve tissue range of motion as well
as decrease pain, swelling, and stiffness 8,9,87,88
. The general consensus of many researchers is
that clinicians must exhaust conservative treatment options before proceeding to surgical
interventions 89
. Due to a lack of research into the effectiveness of certain conservative
treatments it is not possible to suggest one over another at this time.
Surgical Treatments
Surgery is most often considered as the last treatment option to manage tendon and
ligament injuries once all conservative options have been exhausted. The effectiveness of
surgical treatments varies and is largely dependent on the specific tendon or ligament and to
what extent the tissue is injured 8
. Other risks associated with surgery include infection, nerve
damage, and adhesion of scar tissue to healthy tissue which restrict motion 5,90
.
The effectiveness of surgical interventions varies among individual tendons and
ligaments and this must be taken into consideration when exploring treatment options. For
example, the rate of rotator cuff re-tearing after surgical repair varies from 11% to 94% 91
. A
study of 1000 patients that underwent rotator cuff repair surgery revealed an overall re-tear rate

15
of 17% six months after surgery 91
. Among the study population, re-tears occurred in 27% of
patients that had full-thickness tears. Although specific re-tear rates can largely vary, surgical
repair of rotator cuff injuries exhibits high rates of re-injury. Surgical repair of the Achilles
tendon also proves to be controversial. Non-operative treatment of Achilles tendon rupture
exhibits similar re-rupture rates when compared to surgically repaired tendons 92
.
Surgical repair of ligament tears has generally exhibited a high success rate relative to
tendon repairs. ACL reconstruction has become the standard treatment for ACL tears, but certain
problems do exist. Estimates show that on average only 65% of patients that receive ACL
reconstruction will return to the same level of physical activity as before the surgery 93
. The
overall incidence rate of re-injuring the ACL within 2 years of ACL reconstruction surgery is
approximately 6 times greater than that in healthy participants 94
. Another problem associated
with ACL repair surgeries is that of graft failure. Patients that undergo allograft (tissue from
another person) reconstruction have an increased risk of ACL graft rupture when compared to
autograft (using one’s own tissue) reconstruction patients. Specifically, the odds of a graft
rupture due to allograft reconstruction are four times higher than autograft reconstructions 95
.
One of the commonly reported long term complications of treating ACL ruptures is the
development of osteoarthritis (OA) 96,97
. A meta-analysis of 9 long-term studies compared the
incidence of moderate to severe radiologic changes indicative of OA between ACL-injured knees
and healthy controls. ACL-injured knees exhibit a 20% incidence of OA changes compared to a
5% incidence in uninjured knees 98
. In a 14-year follow-up study from ACL reconstructions, a
reported incidence of 57% of OA was significantly greater than the 18% incidence in healthy
controls 99
. A long-term retrospective case series similarly concluded a greater incidence and
severity of OA in knees that underwent ACL reconstruction compared to controls 100
.

16
Current therapeutic strategies for the management of tendon and ligament injuries are
often unable to provide significant improvement in patient outcomes. The relative inability of
current treatments to effectively restore the native structure and function of tendons and
ligaments is an ongoing challenge for clinicians and researchers. The need for effective
therapeutic strategies has led to developments in cell therapies aimed at enhancing native repair
mechanisms with biomaterials and stem cells, in particular mesenchymal stem cells.
Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are a major
candidate for cell-based and tissue engineering therapies. These cells possess an intrinsic ability
of self-renewal and differentiation into functional cell types of the tissues in which they reside.
Current research is focused on developing an understanding and controlling the mechanisms that
govern the ability of these cells to self-renew and differentiate. The following sections present a
detailed view of MSCs and their potential clinical applications.
History
The first major breakthrough in stem cell research occurred in 1961 with the publication
of a series of experiments by Till and McCulloch on radiation sensitivity of bone marrow in
response to cancer treatments 101
. These experiments resulted in the identification of self-
renewing and multi-potent cells responsible for generating the blood cell components, more
commonly known as hematopoietic stem cells (HSCs)101
. Improvements in the ability of
researchers to isolate and assay HSCs in vitro and in vivo has resulted in HSCs being the
paradigm and best-characterized example of tissue-specific stem cells 102
.
Alexander Friedenstein is largely credited with discovering MSCs as well as describing
the associated key biological properties 103
. A study conducted by the research group in 1963

17
demonstrated that pieces of bone marrow transplanted under the renal capsule in mice formed an
osseous tissue invaded by hematopoietic cells 104
. In a follow-up study researchers transplanted
the marrow cells in a closed system known as a diffusion chamber. Diffusion chambers are
implantable structures that house a chamber lined with microporous filters. Prior to implantation
the chambers are filled with the cell suspension of interest. Upon implantation, the microporous
filters allow for interaction between the cell suspension and external environment. These
diffusion chambers are then harvested and the interior cell suspension is evaluated. The
transplantation of the diffusion chambers filled with the bone marrow suspension resulted in the
formation of osseous tissue that lacked colonization of hematopoietic cells105
. These studies
revealed an inherent osteogenic property of bone marrow that differentiates it from other tissues.
These studies also suggested the existence of distinct non-hematopoietic bone marrow precursor
cells 103
.
In order to demonstrate that bone marrow contained separate populations of osteogenic
and hematopoietic precursor cells, Friedenstein took bone marrow cells from a parental mouse
strain and transplanted them into a hybrid F1 mouse strain. Karyotype analysis of the resulting
bone tissue revealed it was genetically identical to the parental strain. Analysis of the
hematopoietic tissue that colonized the newly formed bone revealed that it was of host (hybrid
mouse) origin106
. This confirmed that osteogenic and hematopoietic cells were derived from
distinct precursor marrow cells.
Friedenstein then went on to develop a simple method of isolating stromal cells from the
bone marrow which led to the successful identification of a subpopulation of osteogenic (bone
producing) precursor cells106
. Friedenstein was able to isolate these stromal cells from the bone
marrow based on their relatively high adhesion to tissue culture plastic compared to other cells.

18
Researchers then demonstrated the ability of these stromal cells to give rise to the colony-
forming unit-fibroblast (CFU-F) in vitro 107,108
. Colony-forming units are used as a measure of
the number of microorganisms that are present on the surface of a sample. In this case, the
colony-forming units consisted of fibroblastic cells, or cells that synthesize the extracellular
matrix and collagen of connective tissue. Later studies demonstrated the ability of these
clonogenic clusters to undergo differentiation into osteogenic cells109
.
In 1991, Arnold Caplan proposed the use of the term “mesenchymal stem cells” for the
cells described by the Friedenstein group 110
. Caplan’s work in the 1990s, focused MSC research
toward tissue regeneration, particularly along mesodermal lineages (connective tissue, bone,
cartilage, muscle, blood vessels, etc.) 110
. Caplan is also credited with creating the concept of the
“mesengenic process” (Figure 2) to highlight the differentiation of mesodermal tissues from a
single population of precursor mesenchymal stem cells. The mesengenic process involves a
progressive pattern of transcriptional and phenotypic transitions that ultimately lead to
differentiated cells. The first study to show that clonal populations of human bone marrow
stromal cells were multi-potent was published in 1999 by Pett et al. 111
ultimately led to the wide
adoption of the term “mesenchymal stem cell.”

19
Figure 2. The mesengenic process. This figure depicts the self-renewal, proliferation, and
potential lineage differentiation pathways of MSCs. The differentiation of MSCs is dictated by
lineage commitment of progenitor cells which then mature in lineage specific patterns. Reprinted
from 112
Defining MSCs in vitro
The in vivo identity and characteristics of MSCs have been debated since their discovery
by Alexander Friedenstein in the late 1960s. MSCs were originally defined by Friedenstein as
colony forming unit-fibroblasts and osteogenic stem cells. Since then, several names and
definitions have been proposed to reflect the ever changing understanding of MSC properties.
The term “mesenchymal stem cells” was proposed by Caplan in 1991 as hypothetical postnatal
cells that were multi-potent and self-renewing, with the primary function of giving rise to
functionally and phenotypically mature tissues 110
. The term was widely adopted but

20
considerable variability in the methods used to isolate, expand, and characterize MSCs has
resulted in the use of several terms to describe the same cell type 113,114
.
In an effort to limit confusion and discrepancies among studies, in 2006 the
Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy
(ISCT) proposed minimal experimental criteria to define human MSCs 113
:
• MSCs must be plastic-adherent in standard culture conditions
• ≥95% of the MSC population must express the surface markers CD105, CD73, and CD90
• ≤2% of the MSC population must express CD34,CD45, CD14 or CD11b, CD79 or
CD19, and HLA-Class II
• MSCs must differentiate to osteoblasts, adipocytes, and chondroblasts in vitro
The publication of the minimal criteria by the ISCT created a quality control step for
researchers to verify MSC identity, which decreased the variability and increased the
transparency of published data. New research data published since the establishment of these
criteria suggests that the minimal criteria be revised to account for newly identified criteria by
which researchers can evaluate MSCs. Although the functional definition of MSCs is likely to
remain rather broad, the development of specific phenotypic definitions accounting for tissue and
species specific markers is likely 102,115
.
For the purposes of this manuscript, the term “mesenchymal stem cells” is used as a
generic term for a variety of heterogeneous cell populations, isolated from a variety of tissues,
that are multipotent, self-renewing, and possess the ability to differentiate into a variety of
progenitor cells including adipocytes, chondrocytes, and osteoblasts.
Fundamental Properties
Researchers have yet to identify MSCs within their tissue of origin in vivo. Therefore, the

21
definition and understanding of MSCs ultimately relies on in vitro culture-expanded cell
populations 108,116
. In vitro analysis has allowed for the identification of key properties that help
define MSCs and their functions. It is known that MSCs are non-hematopoietic stromal cells that
are capable of differentiating into a number of mesenchymal tissues such as bone, cartilage,
muscle, ligament, tendon, adipose, as well as neurons 117
. The profile used to identify MSCs
includes the functional characteristics of self-renewal and multipotent differentiation as well as
the presence or absence of certain accepted cell-markers. Phenotypically, MSCs are often
defined as positive for the cell markers CD29, CD44, CD71, CD73, CD90, CD105, CD106,
CD120, CD124, and CD166. MSCs are also identified by a lack of certain hematopoietic cell
markers that can include CD11, CD14, CD18, CD31, CD34, CD40, CD45, CD56, CD79 CD80,
and CD86 14,108,118
. It is important to note that the presence or absence of these cell markers is not
absolute. The expression of MSC markers has been shown to differ between the various sources
of MSCs as well as between culturing methods 119-123
.
A defining characteristic of MSCs is their self-renewal potential, or ability to generate
identical copies of cells through mitotic division over extended time periods. MSCs are capable
of a considerable number of cell divisions but an absolute number is still unknown. It has been
determined that single-cell-derived colonies of MSCs can be expanded to over 50 population
doublings 14,118,124
. The rate of cell division and number of divisions an MSC can complete varies
between tissue sources, culturing conditions, and the age of the donor 124-126
. After a certain
number of cell divisions MSCs will become senescent and will be unable to divide.
Another important feature of MSCs is the ability for differentiation along a number of
mesenchymal cell lineages. MSCs possess the ability to acquire characteristics of cells such as
osteoblasts, chondrocytes, adipocytes, tendon cells, as well as cells with neuronal properties 14
.

22
MSC differentiation largely depends on the tissue from which cells are harvested as well as
donor and experimental conditions. This leads to significantly variation in the multi-lineage
differentiation potential of MSCs 124
. Evaluation of the various sources and the differentiation
abilities are addressed in a later section. It is believed that multipotent MSCs undergo
transcriptional modification that is regulated by the environment in which they reside. This leads
to the generation of more committed progenitor cells and ultimately to fully differentiated cells14
.
Mechanism of Action
Apart from the key properties that are used to identify MSCs, the cells exhibit unique
functions that make them likely candidates for cell-therapies and tissue engineering. A major
concern associated with tissue engineering or allogeneic organ and tissue replacement is
transplant rejection or graft-vs.-host disease 127
. Various studies have provided evidence that
MSCs possess immunomodulatory capabilities that prevent an immune response 128
. MSCs are
able to avoid triggering an immune response since MSCs do not express major
histocompatibility complex (MHC) II antigens 129-131
. This allows MSCs to evade the host
immune system by not provoking the host’s T-cell response after implantation. Another
immunogenic mechanism used by MSCs is that of immunosuppression, specifically by the direct
modulation of T-cell activation and proliferation. Further immunosuppressive properties of
MSCs impair the maturation of dendritic cells which in turn limits T-cell activation 13,128
.
Studies have demonstrated the ability of MSCs to migrate throughout the body
chemotactically to tissues that are inflamed or injured. This ability has been well documented in
animal models for myocardial infarction and cerebral ischemia118
. Certain chemokine receptors
and adhesion molecules known to be involved in leukocyte migration to sites of inflammation
have also been found to be expressed by MSCs129
. This permits the MSCs to hone in on injured

23
tissues and ultimately contribute to endogenous tissue repair 132
. Once the MSC travels to an
injured tissue it can secrete numerous proteins such as cytokines, chemokines, and growth
factors that act on the surrounding injured tissue 133
. Secretions from the MSC have also been
shown to exhibit anti-inflammatory activity, inhibition of apoptosis 134
, and promotion of
vascularization 135
, all which contribute to a healing or trophic effect136
.
The participation of MSCs in tissue regeneration occurs in three different ways. First,
MSCs directly differentiate into the tissue-specific cells acting as substitutes for injured and/or
missing cells. Second, the MSCs indirectly impact tissue regeneration by the secretion of soluble
factors. Third, MSCs modulate the inflammatory responses of injured tissue allowing for
directed control of endogenous repair mechanisms.
Sources of Mesenchymal Stem Cells
One of the most attractive features of using MSCs is that they can be harvested from a
patient’s own tissues, allowing for the development of allogenic treatment methods. MSCs were
originally isolated from bone marrow as precursors to stromal elements in the 1960s 106
, but
since then researchers have been investigating a number of alternative sources. It is consistently
reported that MSCs can be isolated from almost every tissue in the human body 137,138
. The
sources include bone marrow 138
, trabecular 139
and cortical bone 140
, synovial membranes 141,142
,
adipose tissue 143-145
, tendons, skeletal muscle 146
, peripheral blood 147,148
, umbilical cord blood
149
, Wharton’s jelly 150
(gelatinous substance within the umbilical cord), skin, and the nervous
system 124
. The optimal source of MSCs is still under debate since each source has various
advantages and disadvantages that must be taken into consideration.
Bone Marrow MSCs. Bone marrow MSCs (BM-MSCs) were discovered as a
subpopulation of the stromal cells lining the endosteal surface of the marrow space 124
. These

24
cells were identified as non-hematopoietic cells that can differentiate into a variety of connective
tissues. Cells that are almost identical to BM-MSCs have also been isolated from trabecular and
cortical bone 151
and from non-hematopoietic marrow sites such as the femoral or humeral head
124
. In general, BM-MSCs exhibit a stable undifferentiated phenotype, high proliferative activity,
and good chondrogenic and osteogenic differentiation potential 124,152
. These stem cells can be
utilized in two major forms; culture-expanded BM-MSCs and bone marrow concentrate (BMC)
without expansion 28
. Culture-expanded BM-MSCs require the harvesting of stem cells from a
site such as the iliac crest via aspiration. The BM-MSCs must then have their numbers increased
with in vitro laboratory procedures in order to obtain a therapeutic amount of stem cells. BMC
does not involve in vitro expansion which permits a same-day procedure. The preparation of
BMC requires centrifugation of the bone marrow aspirate to remove red cells and plasma cells.
This leaves nucleated cells with a small stem cell component 153,154
. In fact, MSCs yields from
bone marrow have accounted for only 0.001-0.01% of all nucleated cells 111
. In small human
trials, both BM-MSCs and BMC exhibit beneficial results when compared to controls 28
.
There are certain disadvantages to the use of BM-MSCs. Studies have found that number
of cells harvested from bone marrow are highly variable. Technique related differences in
harvest site and syringe size have yielded wide ranges of BM-MSCs 155
. There is also substantial
variability in yield amount between patients with counts ranging from 8 million to 45.6 million
cells 28
. It has been shown that cell numbers, proliferative potential, and ability to differentiate
decreases with age 126
. Furthermore, cells harvested from older patients exhibit greater rates of
apoptosis (programmed cell death) and senescence than cells harvested from younger
patients126,156
.
Synovium-derived MSCs. Researchers have discovered that MSCs can be isolated from

25
both the synovial membrane as well as synovial fluid 141
. Synovium-derived MSCs exhibit high
proliferative activity and possess the best chondrogenic potential when compared to MSCs
derived from bone marrow, periosteum, adipose tissue, and muscle tissue 152
. Due to their
chondrogenic potential, these stem cells have been shown to accelerate the early remodeling of
tendon-bone healing in rats 142
. Synovium-derived MSCs are also advantageous for clinical use
because they can be obtained arthroscopically with limited invasiveness and donor site morbidity
124,157
. Although synovium-derived MSCs exhibit good chondrogenic potential, they are limited
in their osteogenic potential when compared to BM-MSCs152
making synovium-derived MSCs a
better candidate for treating cartilage defects.
Adipose-derived MSCs. Adipose-derived MSCs were first discovered in 2001158
. Since
then, adipose tissue has been determined to be one the richest sources of MSCs144
. Harvesting of
MSCs from lipoaspirate of adipose tissue has been shown to be relatively easy and without
significant morbidity 159,160
. A success rate of 100% has been observed in isolating MSCs from
adipose tissue161
with yields as high as 100,000 cells per 100ml of lipoaspirate159
. Adipose-
derived stem cells have become an attractive alternative to BM-MSCs primarily due to the high
yield of cells. For example, one gram of adipose tissue can yield over 50 times more stem cells
than one gram of aspirated bone marrow162
. Compared to BM-MSCs, adipose-derived MSCs
exhibit superior adipogenic ability but lack in chondrogenic and osteogenic ability152
. In contrast
to BM-MSCs that decrease in number as a person ages, adipose-derived MSC numbers decrease
with obesity 163
.
Umbilical Cord Stem Cells. Stem cells can be isolated and harvested from various
compartments of the umbilical cord including the umbilical arteries, umbilical vein, cord lining,
Wharton’s jelly, and umbilical cord blood164
. These regions are capable of giving rise to a large

26
number of MSCs that possess the ability to differentiate into osteoblasts, adipocytes,
chondrocytes, hepatocytes, neural cells, and cardiac cells165
. It is reported that the Wharton’s
jelly contains the highest concentrations of MSCs that can be isolated and harvested166
. Similar
to MSCs from other sources, MSCs from the umbilical cord possess immunomodulatory
characteristics and lack tumorigenic potential making them an attractive candidate for allogenic
based cell therapies 149,167
. Umbilical cord MSCs appear to have a faster population doubling
time and greater expansion ability than adult MSCs. This is most likely due umbilical cord stem
cells being less differentiated than adult MSCs168
. The success rate of isolating MSCs from the
umbilical cord has been reported as 63% which is significantly lower than the 100% success rate
of bone marrow and adipose tissue161
. However, it is also reported that umbilical cord MSCs
could be cultured in vitro for the longest amount of time and exhibited the highest proliferative
capacity, whereas BM-MSCs exhibit the shortest culture time and a limited proliferative capacity
149,161
.
Mesenchymal Stem Cells to Treat Tendon and Ligament Damage
The properties of self-renewal and multi-potency have made MSCs a topic of research
and development in orthopedic medicine. To date, MSCs have been used for treating various
musculoskeletal ailments such as osteochondral injury, osteoarthritis, rheumatoid arthritis,
osteogenesis imperfecta, and chondromalacia patellae25,28,169-171
. Recent clinical application of
MSCs has been shown as an effective treatment for graft-versus-host disease as well 172,173
.
These observed successes have made MSCs a candidate to potentially improve the current
orthopedic interventions for tendon and ligament damage. MSC-based clinical applications are
presently being studied in various animal models as well as in humans.
From a regenerative standpoint, tendons and ligaments are considered to be structurally

27
similar174
. Both consist primarily of type I collagen as well as fibroblasts, ground substance,
elastin, and water. Ligaments are noted as having a slightly reduced collagen fibril percentage,
but a higher elastin and proteoglycan component when compared to tendons. The major
difference between tendons and ligaments is functional, not compositional. The goal of this
researcher is to design cell-therapies that can be used efficiently to heal the collagen fibers
similarly in tendons and ligaments 50
.
The cells responsible for the generation of collagen fibers in tendons are elongated
fibroblast cells known as tenocytes. Under normal conditions, these cells are relatively dormant.
Upon sustaining a tendinous injury, the tenocytes are activated to promote collagen deposition by
the inflammatory response. The tenocytes are assisted by a population of cells known as tendon
progenitor stem cells (TPSCs), sometimes referred to as tendon-derived stem cells (TDSCs).
These cells have been identified in both humans and mice 175
. TPSCs possess a multi-
differentiation potential as they can differentiate into tenocytes, chondrocytes, osteocytes, and
adipocytes176
. Tenocytes derived from TPSCs would represent the most opportune cell choice to
treat injured tendons if it weren’t for two primary complications. First, tendons are relatively
acellular which limits the number of TPSCs and tenocytes that could be harvested. Second,
tenocytes proliferate for a short period of time in in vitro culture, and during this time period
tenocytes have a tendency to de-differentiate177
.
In comparison studies of BM-MSCs and TPSCs, TPSCs were found to have a higher
clonogenicity and proliferation rate as well as greater expression of soft tissue-specific mRNA
178
. The presence of the soft tissue-specific mRNA was also found in MSCs, indicating the
capacity of MSCs to act like TPSCs 179,180
. Direct evidence has shown to ability of MSCs to
differentiate into tenocytes 181,182
, making MSCs an attractive option for tendon healing.

28
Animal Studies
Numerous animal models have been used to explore the possibility of using MSCs in the
treatment of tendon and ligament injury in humans. Many of these studies have shown promising
results. One of the major complications related to surgical repair of tendons and ligaments is the
relatively slow tendon-bone healing that often results in scar tissue formation. MSCs have been
shown to have a regenerative effect on the bone-tendon junction. In a study using rats, the
researchers surgically repaired the Achilles tendon in which the tendon-bone junction was
destroyed. Along with the surgery, researchers injected either chondrocytes or MSCs into the
tendon. The group that received the injection of MSCs showed a tendon-bone junction similar to
the pre-injury state183
. Similar results were obtained in a study of ACL repair in rabbits, with the
reconstructed ACL initially exhibiting higher failure loads and less stiffness than controls184
.
Another study using the rabbit model for ACL regeneration found that MSCs seeded in a
collagen type I scaffold enhanced ACL regeneration, with 33% of specimens exhibiting tissue
comparable to a pre-injury ACL185
. Utilizing a rat model of partial ACL tear, researchers have
shown that intra-articular (joint) injections of BM-MSCs can accelerate healing 186
. The rats that
received the BM-MSCs injection exhibited healed ligaments with superior histological scores
and greater load failures when compared to controls.
In a study that explored the application of a MSC-loaded mesh onto a damaged Achilles
tendon-gastrocnemius/soleus junction in rats, there was reported enhanced early tendon healing,
particularly related to better quality collagen bundles187
. The use of human MSCs was tested in
rats with collagenase-induced injury to the Achilles tendon. Human MSCs were injected into the
site of injury and the tendon was then assessed for a variety of factors. The rats treated with
human MSCs exhibited a significantly better extracellular matrix structure and a larger amount

29
of type I collagen. Furthermore, the tendons treated with human MSCs also exhibited better
vascularization and improved tendon stiffness 188
.
The use of an equine model has granted some insight into MSC use in treatment of
tendon and ligament damage. The most frequently injured tendon in the horse is the superficial
digital flexor tendon which shares many similarities with Achilles tendinopathy in humans189
.
Due to the tendon similarities, the equine model has provided the proof of principle that MSCs
can produce tendon tissue and contribute to healing that resembles a pre-injury state190
. Proof of
principle studies take place once a compound has demonstrated potential in safety testing and
animal models. Acting as a link between Phase I and Phase II trials, the goals of these studies
include identification of compound activity on a relevant pathophysiological mechanism (i.e.
tendon tissue regeneration), as well as efficacy evaluation at a clinical endpoint191
. In an in vivo
collagenase-induced tendinopathy study, researchers demonstrated that intratendinous injections
of autologous BM-MSCs produced effective tendon regeneration192
. Further studies utilizing the
equine model have shown that intratendinous implantation of autologous BM-MSCs reduced re-
injury rates from 56% to 18% with no other reported adverse effects189
. A study of adipose-
derived MSCs used in the treatment of equine tendinitis has shown that injection of MSCs can
prevent the progression of a tendon lesion, greater organization of collagen fibers, and decreased
tendon inflammation193
. In a study of naturally occurring equine tendinopathy, researchers found
that tendons treated with BM-MSCs exhibited statistically significant improvement in structural
stiffness and histological organization compared to control tendons194
.
One possible disadvantage of MSC therapy for tendon and ligament damage is the
potential for ectopic bone formation (intratendinous ossification) and cartilage formation at the
repair site. In studies of BM-MSC treated rabbit tendons, the formation of intratendinous

30
ossifications has been reported in up to 28% of the treated specimens 195,196
. Similar
intratendinous ossification as well as the formation of cartilage at the implantation site was
reported in the Achilles tendon of rats 188
. In contrast to this, no calcification or ectopic bone
formation has been reported in similar equine tendinopathy studies 48,189
.
Human Clinical Studies
Since the first clinical trial of BM-MSCs in 1995197
over 2,000 patients have been
administered with autologous or allogenic MSCs. These studies have attempted to use MSCs to
treat a variety of diseases, including hematologic malignancies, cardiovascular diseases,
neurological diseases, autoimmune diseases, graft-versus-host diseases, organ transplantation,
refractory wounds, and bone/cartilage defects 198
. In the world’s largest clinical trials database
hosted by the United States National Institutes of health (www.clinicaltrials.gov), a broad search
of “mesenchymal stem cells” yields 433 total studies as of July 2015. Of these 433 studies, 116
are completed, 72 are active, and 245 are currently recruiting.
Despite the wide spread interest for the clinical application of MSCs and numerous
animal studies, only a limited number of orthopedic studies investigating MSC use for tendon
and ligament healing have been published to date. One of the most recent studies completed by
Hernigou et. al.199
investigated the impact of augmenting rotator cuff repair with BM-MSCs at
the time of arthroscopy. The study group included 45 patients receiving BM-MSC injections that
were then compared to 45 controls. Researchers aspirated bone marrow from the anterior iliac
crest of each study patient prior to the arthroscopy. The bone marrow aspirate was concentrated
into a buffy coat containing BM-MSCs and other progenitor cells. An average number of
51,000±25,000 BM-MSCs were returned to each patient via injections into the tendon at the
tendon-bone junction, as well as into the bone at the footprint of the greater tuberosity. The

31
patients were evaluated by ultrasound on a monthly basis for the first 24 months. The rotator cuff
healing or re-tear was confirmed with post-operative MRI at 3 and 6 months and again at 1 and 2
years, with a final 10-year follow up. Forty-five (100%) of the 45 repairs that received the
injection of BM-MSCs had healed by 6 months, compared to 30 (67%) of the 45 repairs without
BM-MSC injection. At the 10-year follow up, intact rotator cuffs were found in 39 (87%) of the
45 BM-MSC patients. This is significantly better than the control group in which only 20 (44%)
of the 45 patients exhibited intact rotator cuffs.
A similar study investigated the outcomes of augmenting the surgical repair of rotator
cuff tears with an injection of autologous bone marrow mononuclear cells (BMMCs) into the
tendon borders200
. BMMCs are a heterogenous population that includes hematopoietic cells,
progenitor cells, and MSCs. The study consisted of 14 patients with complete rotator cuff tears.
Bone marrow aspirates were taken from the posterior iliac crest and then concentrated into a cell
suspension of BMMCs. Functional evaluation of each patient was done before and after the
surgery using The University of California-Los Angeles (UCLA) Shoulder Score. The UCLA
Shoulder Score is an objective rating scale used to assess pain, function, and range of motion,
strength, and patient satisfaction. The maximum score is 35, with higher scores indicating better
shoulder function201
. MRI evaluations of overall tendon integrity were also conducted. After a
12-month follow-up period, the mean UCLA Shoulder Score increased from 12±3.0 to 31±3.2
indicating significant improvement. MRI analysis after a 12-month follow-up period indicated
improvement in tendon integrity for all 14 cases200
.
The use of MSCs has also been shown to improve patient related outcome scores as well
as ultrasound tendon appearance in the treatment of refractory lateral epicondylitis (tennis
elbow). Researchers cultured skin-derived tenocyte-like cells to produce collagen-producing

32
cells which were then injected into 12 patients. The Patient-Rated Tennis Elbow Evaluation
(PRTEE) scale was used to assess pain and disability and tendon healing response was measured
via ultrasonography. At 6 months post-treatment, the median PRTEE score decreased
(improvement) from 78 to 12 and ultrasonography showed median decreases in number of tears,
number of new vessels, and tendon thickness. Of the 12 patients, 11 exhibited satisfactory
outcomes and 1 patient required surgery after treatment failure at 3 months202
.
Clarke et al. conducted a randomized control trial to investigate the use of skin-derived
tenocyte-like cells in the treatment of patellar tendinopathy. The study assessed a total of 60
patellar tendons in 46 patients. Patients were divided into a study group (n=33 tendons) that
received an injection of tenocyte-like cells cultured in plasma and a control group (n=27 tendons)
that received an injection of only plasma. In order to assess the patients’ pain and function
researchers utilized the Victorian Institute of Sports Assessment (VISA) questionnaire and
ultrasound imaging before and after receiving treatment. After a 6-month follow-up period, the
study results indicated an improvement in the VISA score in the treatment group from 44±15 to
75±17. The control group exhibited an improvement in the VISA score from 50±18 to 70±14.
The difference in VISA scores was found to be statistically significant. Patients that received the
injection of tenocyte-like cells experienced faster recovery times, exhibited a decrease in tendon
thickness, and showed normal histopathological tendon structure 203
.
In a long-term, 5-year follow-up study, researchers investigated the use of BM-MSCs in
treating chronic patellar tendinopathy204
. Eight patients with chronic patellar tendinopathy
underwent bone marrow aspiration of the anterior iliac crest. The BM-MSCs were separated
from the bone marrow aspirate via centrifugation. Under ultrasound guidance the researchers
injected the BM-MSCs into the patellar tendon lesions. Patient outcomes were assessed using

33
questionnaires administered preoperatively, one year postoperatively, and then on an annual
basis. The subjective measures assessed by these questionnaires were based on several scoring
systems including International Knee Documentation Committee (IKDC), knee injury and
osteoarthritis outcome score (KOOS), Short Form-12 (SF12), Lysholm, Tegner, and Cincinnati.
Objective evaluation of tendon healing was accomplished with ultrasound imaging that was
performed before treatment and at 6 months after the injection. At 5 years follow-up, the results
indicate statistically significant improvement for most of the subjective clinical scores. Seven of
the 8 patients were completely satisfied with the procedure. Pre-injection ultrasound evaluation
of the patellar tendons indicated grade II-III injuries in all 8 patients. At 6 months post-injection,
7 of the 8 patients were classified as grade I and 1 patient remained at grade III204
. It is important
to note that this study is limited by the lack of a control group and a small sample size, resulting
in a low level of evidence study.
Research that directly evaluates the use of MSCs to treat ligament injuries is primarily
limited to animal studies. However, the results of clinical trials evaluating MSCs and
osteoarthritis have the potential to impact future ligament treatments, particularly those for ACL
injuries. As previously discussed, there is a reported greater incidence of OA development in
patients that undergo surgical repair of ACL ruptures. Current OA treatment strategies focus on
exhausting conservative measures in order to delay major joint reconstruction (replacement)
surgery.
Conservative therapies aimed at decreasing joint pain and improving function can
include strength exercises, mechanical bracing, and pharmacological pain control. If these
measures are ineffective, clinicians can attempt intra-articular injections of corticosteroids or
hyaluronic acid, however the efficacy of these treatments is controversial170
. A number of small-

34
sized clinical trials evaluating MSC use in the treatment of OA have shown promising results
28,170
. In general, MSC use has been associated with decrease joint pain and improved function
(range of motion, walking time for onset of pain, swelling)205-210
. Although not directly related to
ligament repair, the results of these studies have the potential to impact the development of
therapies for the long-term management of ACL repairs.
Tissue Engineering Concepts
Studies in pre- and clinical settings have demonstrated the broad therapeutic efficacy of
MSCs which has made them attractive candidates for tissue engineering. MSCs are a focus of
regenerative medicine primarily because they present an autologous cell source that reduces the
likelihood of immune response. MSCs are also of interest due to their demonstrated extensive
proliferative capacity in vitro, multi-lineage potential, immunomodulatory effects, secretion of
trophic factors, and a demonstrated efficacy in treating tendon and ligament injuries211
. The wide
variety of interacting MSC properties allow for their use in many tissue engineering strategies,
include the use of scaffolds, growth factors, cell seeding, or a combination of the three212,213
.
Scaffolds
Scaffolds are materials designed to directly repair or augment repair of tendons and
ligaments by providing a structural support for cell attachment and tissue development. Previous
discussion of the pathophysiological healing outcomes, as well as the limited effectiveness of
current clinical therapies for tendon and ligament repair, highlight the need for a support system.
Scaffolds can provide specific structural support and act as vehicles for cells and new tissue
formation when applied appropriately.
Novel scaffold design is based upon the normal mechanics, matrix components and
organization, and cellular phenotypes of tendons and ligaments. The ideal scaffold would possess

35
the basic structure of the tendon/ligament, native extra cellular matrix, and cell seeding
capability212
. In general, a successful scaffold should support cell adherence, local growth factors
should accumulate and be released when appropriate, and the scaffold should be resistant to
matrix proteases5
.
Scaffolds are either classified as biological (derived from mammalian tissues) or
synthetic (manufactured from absorbable and non-absorbable polymers). Biologic scaffolds are
generated from xenogeneic and allogenic tissues and are chemically processed. Processing
ultimately yields a final scaffold composed mainly of naturally occurring collagen fibers
(primarily type I) and a bioactive surface structure/chemistry that can promote cellular
proliferation and tissue in growth212,213
. Biologic scaffolds are advantageous as they provide a
local environment for rapid cell attachment and proliferation and enhanced new tissue formation.
For example, seeding biologic scaffolds with MSCs has been shown to drastically improve
outcomes in animal-tendon models5
. A technique utilizing isoelectric focusing aligns the
collagen fibers of the scaffold to match the target tissue. The electrochemically aligned collagen
matrices (ELAC) have been shown to support a higher proliferation rate of MSCs compared to
random fiber orientation. Furthermore, the ELAC orientation upregulates scleraxis and
tenomodulin in MSCs, supporting a shift towards tenogenic differentiation 214
. Scleraxis is a
transcription factor and tenomodulin is a protein, both which regulate the proliferation of tendon
fibroblasts. Disadvantages include poor mechanical properties, poor suture retention strength,
risk of disease transmission, product variability, and the necessity to harvest from humans or
animals213
The risk of host immune response seen in biologic scaffolds led to interest in developing
synthetic scaffolds. Scaffolds can be made using a wide variety of polymers and fabrication

36
methods, with the potential for optimizing desired scaffold features. Apart from the decreased
risk of immune response, advantages of synthetic scaffolds include the cost-effective mass
production of uniform scaffolds, often with improved mechanical strength. Disadvantages
include the lack of biologic function and reactions to scaffold degradation products 213,215
.
Successful tissue engineered scaffolds must be biocompatible, have mechanical properties
similar or greater to the regenerating tissue, promote tissue growth, and degrade in a way that
does not impact the new tissue216
. Scaffolds can come in the form of gels, membranes, or three-
dimensional structures. Materials such as collagen fibers, silk, and biodegradable polymers have
been used to manufacture fibrous scaffolds for tendon and ligament engineering.
Numerous biodegradable polymers play a pivotal role in tissue engineering. Some of the
most commonly used polymers for tissue and ligament scaffolds include, poly-1,8-octandediol-
co-citrate (POC), poly(lactide-co-glycolic acid) (PLGA), polyglycolic acid (PGA),
poly(urethanurea) (PUUR) and poly(ethylene glycol) diacrylate (PEG-DA)5,215,217
. The polymers
have variable degradation rates, MSC attachment profiles, and cell proliferation and adhesion
profiles allowing for customized generation of scaffolds to support cell growth218
.
Scaffolds made of polymers such as POC and PGA have shown significant potential for
use with MSCs-related tendon and ligament repair. These scaffolds are porous and biodegradable
making these scaffolds superior to non-degradable scaffolds5
. Non-degradable scaffolds are often
unable to reproduce the mechanical behavior of the target tissue and they require a second
surgery to remove the device. The polymers within the scaffolds can be modified to mimic the
tensile strength and Young’s modulus of tendons and ligaments for proper structural support.
These scaffolds also provide specific adhesion substrates for anchoring cells and delivering
growth factors through controlled release as the scaffold degrades5
. A scaffold can be seeded and

37
designed to degrade in a manner that provides a controlled delivery of various MSCs, progenitor
cells, growth factors, and cytokines to healing tissue at the appropriate time.
Studies evaluating the use of tissue engineering scaffolds to deliver MSCs and other
cellular factors in animal models of tendon and ligament injury have been promising. The
implantation of MSCs seeded on collagen delivery vehicle into a defective rabbit Achilles
tendon. Load related structural and material properties assessed at 4, 8, and 12 weeks were
greater than those of controls. The MSC-treated tendons had a significantly larger cross-sectional
area with better collagen alignment219
. Implantation of synovial-MSCs into bone tunnels at
Achilles tendon-bone junctions resulted in greater proportions of collagen fibers ultimately
accelerating the remodeling of the tendon-bone junction142
. In a study evaluating rabbit patellar
tendon injuries, MSCs were expanded in culture and suspended in a cell-collagen composite gel
and implanted. The MSC-treated patellar tendon demonstrated significant increases in maximum
stress, modulus, and strain energy density196
.
Clinical State and Perspectives for Tendon and Ligament Healing
The application of MSCs to clinical settings is a major topic of interest for researchers
and clinicians in a variety of medical specialties. Despite continued interest and advancements in
research, only a limited number of studies investigating orthopedic applications of MSCs have
been published. The major focus of orthopedic studies has been MSC applications for bone and
cartilage repair, with early clinical trials already published. In comparison, considerably fewer
studies evaluating the effects of MSCs on tendon and ligament healing in humans have been
published. Therefore, the majority of information pertaining to MSC therapy for tendon and
ligament damage is limited to animal studies220
. The results from pre-clinical animal studies
show a promising efficacy of MSCs to improve tendon and ligament healing. Recent clinical

38
trials attempting to replicate pre-clinical outcomes exhibited mixed results. These mixed results
served to highlight the challenges of applying MSC-therapy to clinical settings 115,220,221
.
Researchers are currently faced with significant challenges that must be addressed before
MSC therapy becomes clinically applicable. One of the most significant challenges of MSC-
therapy is the generation of a large number of cells needed for clinical cell transplantation of
MSCs. Harvested stem cells must undergo in vitro expansion to generate these large quantities of
cells. The significant variability in MSC classification and identification has led to the
development of multiple isolation and expansion protocols that can result in MSCs with
suboptimal or detrimental cellular properties 222,223
. It is possible that the variability of the
cellular properties of MSCs will lead to significant variability in study results. The variability
seen in present-day research impedes the ability of researchers to effectively compare research
outcomes, which can significantly impact further therapy development 222
. Establishing a gold
standard for MSC isolation and expansion will establish continuity among study protocols. This
will improve the ability of researchers to compare studies and draw conclusions that promote the
development of MSC therapy.
A number of questions pertaining to key aspects of MSCs must be addressed in order to
further develop effective MSC therapy options. Further research into the therapeutic and
immunomodulatory mechanisms of MSCs is necessary for the development of disease specific
therapies 115
. Further research dedicated to identifying the ideal source(s) of MSCs to be used for
particular conditions is also critical for the future of MSC-therapy220
. Lastly, the long-term safety
of MSCs is still relatively unknown. In initial assessments based on current clinical trials, the
therapeutic use of MSCs appears to be safe 198
. Large scale controlled clinical trials are needed
to better assess the long-term safety of MSC therapy.

40
Conclusion
The effective treatment of tendon and ligament damage has been a long-standing
challenge in orthopedic medicine. Due to the limited regeneration potential of tendons and
ligaments, healing often results in the formation of poor-quality tissue that is susceptible to
reinjury. Current treatment modalities are often inadequate in providing definitive resolution of
tissue damage, indicating the need for alternative therapies that augment tendon and ligament
healing. Regenerative medicine research has identified a population of adult stem cells known as
mesenchymal stem cells (MSCs) as the most promising candidates for use in regenerative
therapies.
MSCs are a subset of adult multipotent cells that are capable of self-renewal and multi-
lineage differentiation into cells such as osteocytes, chondrocytes, adipocytes, and
tendon/ligament fibroblasts. MSCs can be readily harvested from a variety of adult tissues
including bone marrow, adipose tissue, synovium, and the umbilical cord. Once harvested the
MSCs can be rapidly expanded in vitro to generate large quantities of potentially therapeutic
stem cells. Apart from self-renewal and multi-lineage differentiation, MSCs also display a
variety of unique functions that make them likely candidates for regenerative therapies. MSCs
possess immunomodulatory capabilities making them non-immunogenic. MSCs can further
regulate immune responses with direct and indirect immunosuppressive capabilities. MSCs are
also capable of chemotactic migration to inflamed/injured tissues. Upon reaching the injured
tissue, MSCs can replace damaged cells by differentiating into the appropriate cell. Furthermore,
MSCs can secrete numerous proteins that are anti-inflammatory, inhibit apoptosis, and promote
vascularization.
The ability of MSCs to differentiate into tendon and ligament fibroblasts has made them

41
an attractive option for potentially improving tendon and ligament healing. Numerous pre-
clinical animal models have been used to evaluate the impact of MSCs on tendon and ligament
healing. In general, the use of MSCs has been shown to improve the extracellular matrix
structure and functional outcomes of both tendons and ligaments. Furthermore, successful
regeneration of tendon tissue in equine superficial digital flexor tendons has provided the proof
of principle that MSCs can produce tendon tissue and contribute to healing that resembles a pre-
injury state. A limited number of relatively small human clinical trials investing the use of MSCs
in tendon and ligament healing have been published. The use of MSCs to augment surgical repair
of rotator cuff tears has been shown to increase healing rate, decrease re-tear rates, and improve
overall function. Studies of MSC injections to treat refractory lateral epicondylitis and patellar
tendinopathy generally resulted in faster healing times, improved histological appearance, and
improved functional outcomes. Although the initial results are promising, larger clinical studies
are needed to better assess the impact of MSCs on tendon and ligament healing in humans.
All together, the results from pre-clinical and initial clinical trials suggest that MSCs exhibit
therapeutic potential for tendon and ligament healing. Further research in animal models and
larger clinical trials are required to accurately determine if MSC therapies are effective in
treating tendons and ligaments. Researchers must also address questions regarding MSC
classifications, gold standard for isolation and expansion, ideal source, and long-term safety of
stem cells.

42
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