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Use of Mesenchymal Stem Cells for the Treatment of Canine Osteoarthritis
By
Stephanie Flansburg-Cruz, B.S.
A Scholarly Project Submitted in Partial Fulfillment
of the Requirements for the Degree of
Master of Arts in Education
Chadron State College
December 2015
Advisor:
Dr. Joyce Hardy
Committee:
Dr. Lara Madison Dr. Teresa Frink
Dr. Ann Buchmann
Copyright 2015, Stephanie Flansburg-Cruz
iii
Abstract
Canine osteoarthritis (OA) is a chronic degenerative joint disease with a
prevalence of 20% in adult dogs and 80% in geriatric dogs. Canine OA is a non-curable
disease; therefore current therapeutic approaches focus on preventing or delaying the
structural and functional changes of the affected tissues. The use of NSAIDs is associated
with gastric ulceration and renal and hepatic damages, for this reason, alternative
treatment modalities without these adverse side effects are highly desirable. Stem cells
represent a novel treatment alternative for canine OA, which has the potential of repairing
diseased cartilage. The purpose of this review was to discuss the current scientific data
regarding the efficacy of MSCs in the treatment of canine OA. Current scientific
evidence shows that the use of autologous and heterologous adipose derived
mesenchymal cells (ADMSCs) can improve the signs of canine OA without causing any
significant side effects. Stem cells can transform into chondrocytes in the site of injection
and therefore, aid in the reparation of diseased cartilage. In addition, stem cells can
promote the migration of endogenous repairing cells to injury sites and suppress
immunoreactions leading to an improvement in the signs of OA.
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Table of Contents
Abstract .............................................................................................................................. iii
List of Figures ..................................................................................................................... v List of Tables ..................................................................................................................... vi
Chapter 1: Introduction and Statement of Problem ............................................................ 1 Problem and Significance ............................................................................................... 1 Objectives ....................................................................................................................... 1 Definition of Terms ......................................................................................................... 2
Chapter 2: Literature Review .............................................................................................. 4 Anatomy and Physiology of Joints ................................................................................. 4 Canine Osteoarthritis .................................................................................................... 10
Pathophysiology of Canine Osteoarthritis ................................................................ 11 Current Treatments for Canine Osteoarthritis ........................................................... 14
Non-steroidal anti-inflammatory Drugs ................................................................ 15 Steroidal Drugs ..................................................................................................... 18 Nutraceuticals ....................................................................................................... 19
Glucosamine and Chondroitin Sulfate .............................................................. 19 Omega-3 Fatty Acids ........................................................................................ 21 Perna Canaliculus or Green-lipped Mussel ....................................................... 22 Avocado/Soybean Unsaponifiables .................................................................. 23 S-Adenosyl-L-methionine ................................................................................. 24
Stem cells .............................................................................................................. 24
Chapter 3: Procedures ....................................................................................................... 30 Chapter 4: Results and Discussion .................................................................................... 31
Effects of Mesenchymal Stem Cells in the Treatment of Osteoarthritis ...................... 31 Autologous AMSCs .................................................................................................. 34 Autologous AMSCs combined with PRGF .............................................................. 36 Heterologous AMSCs ............................................................................................... 37 Safety ........................................................................................................................ 38
Discussion ..................................................................................................................... 38 Conclusions ................................................................................................................... 41
References ......................................................................................................................... 42
v
List of Figures
Figure 1. Synovial joint ...................................................................................................... 5!
Figure 2. Diffusion of glucose and oxygen into articular cartilage .................................... 7!
Figure 3. Electron microscope view of an aggrecan aggregate molecule and Schematic
representation of the aggrecan aggregate molecule .................................................... 7!
Figure 4. Zonal organization in normal articular cartilage ................................................. 8!
Figure 5. The ligaments and menisci of the canine stifle joint ........................................... 9!
Figure 6. Lateral and cranial-caudal radiographic views of the stifle of a dog with OA.. 15!
Figure 7. Biosynthetic pathway of prostanoids ................................................................ 16!
Figure 8. The multipotentiality of MSCs.. ........................................................................ 28!
vi
List of Tables
Table 1. Factors that contribute to articular cartilage degradation ................................... 12!
Table 2. Classification of NSAIDs based on their selectivity for COX-1 and COX-2 .... 17!
Table 3. Summary of studies evaluating the therapeutic potential of mesenchymal stem cells in dogs with spontaneous osteoarthritis….................………………………………32
1
Chapter 1: Introduction and Statement of Problem
Problem and Significance
Osteoarthritis (OA) is a group of mechanical abnormalities involving degradation
of joints, including articular cartilage and subchondral bone. In North America, the
prevalence of canine OA is of 20% in adult dogs and 80% in geriatric (more than 8!years
old) dogs (Railland et al., 2012). This disease is characterized by progressive
degeneration of articular cartilage. When articular cartilage degenerates, bones and
nerves become exposed causing pain and inflammation. No cure is known for
osteoarthritis; current therapeutic approaches focus on preventing or delaying the
structural and functional changes of OA. Current treatments include non-steroidal anti-
inflammatory drugs (NSAIDs), steroidal drugs and dietary supplements. The use of stem
cells represents a novel alternative for the treatment of OA. Stem cell therapy has been
demonstrated to induce profound healing activity in animals with various forms of
arthritis. For example, the company Vet-Stem routinely utilizes stem cells in horses with
various joint deformities to accelerate healing. In addition to healing of damaged tissues,
stem cells have the ability to modulate the immune system to decrease pathological
responses while preserving ability to fight disease. The purpose of this project is to
review the pathophysiology of canine osteoarthritis and the effects of stem cells for its
treatment.
Objectives
o Understand the pathophysiology of canine osteoarthritis.
o Describe the current treatments of canine osteoarthritis
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o Analyze and discuss the current scientific data regarding the efficacy of
mesenchymal stem cells for the treatment of canine osteoarthritis.
o Compare the efficacy of mesenchymal stem cells treatment with the
efficacy of treatment with nutraceuticals and NSAIDs
Definition of Terms Joint: the area where two bones are united for the purpose of permitting body parts to
move. Joints are also known as articulations and are composed of fibrous connective
tissue and cartilage.
Glycosaminoglycans (GAG): any of the carbohydrates containing amino sugars
occurring in proteoglycans, for example, hyaluronic acid or chondroitin sulfate.
Aggrecan aggregate: is a proteoglycan, or a protein modified with large carbohydrates,
which is part of the extracellular matrix of cartilagenous tissue. It is also known as
cartilage-specific proteoglycan core protein and chondroitin sulfate proteoglycan 1.
Osteoarthritis: degenerative disease of the entire joint involving the cartilage, joint
lining, ligaments, and underlying bone. The degeneration of these tissues leads to pain
and stiffness.
Non-steroidal anti-inflammatory drugs: class of drugs that provides analgesic,
antipyretic and anti-inflammatory effects through the inhibition of the activity of
cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2), and thereby, the
synthesis of prostaglandins and thromboxanes.
Nutraceuticals: oral compounds that are neither nutrients nor pharmaceuticals but are
claimed to have certain health benefits. The North American Veterinary Nutraceutical
Council was formed in 1996 and defined a nutraceutical as a substance that is produced
3
in a purified or extracted form and administered orally to patients to provide agents
required for normal body structure and function and administered with the intent of
improving health and well being of animals.
Stem cells: an undifferentiated cell of a multicellular organism that is capable of giving
rise to indefinitely more cells of the same type, and from which certain other kinds of cell
arise by differentiation.
Multipotent cells: cells limited to forming specialized cells within their own group.
Cellular differentiation: the process by which an unspecialized cell develops special
functions, for example, when a stem cell becomes a cartilage-making cell. This process is
controlled by internal signals like genetic factors and external signals like cytokines and
other substances secreted by neighboring cells.
Cellular transdifferentiation: the process by which stem cells from one tissue
differentiate into cells of another tissue.
Mesenchymal stem cells: are multipotent stromal cells that can differentiate into a
variety of cell types, including: osteoblasts, chondrocytes, myocytes and adipocytes.
Plasma rich in growth factors: is also known as plasma rich in platelets. Is a
concentrated source of autologous platelets, which release several different growth
factors and other cytokines that stimulate healing of bone and soft tissue.
4
Chapter 2: Literature Review
Anatomy and Physiology of Joints
Bones meet each other at joints or articulations, some of which unite bones firmly
while others allow free movement. Joints are classified into fibrous, cartilaginous and
synovial. Synovial joints are of clinical importance and their anatomy and physiology
will be discussed. Synovial articulations act as buffers that absorb pressure changes in the
body during movement. Examples of synovial joints in the dog are the stifles, elbows and
carpus. These joints consist of two bone ends covered by articular cartilage, which are
surrounded by a joint capsule (see Figure 1). In this type of joints the articulating bones
are separated by a fluid-filled space, the joint cavity (Dyce, Sack & Wensing, 2010).
Since the articulating bone surfaces are not directly connected to each other with fibrous
connective tissue or cartilage, synovial joints have a greater range of mobility in
comparison with fibrous or cartilaginous joints. The combination of a smooth articular
cartilage surface and the lubrication of both the articular cartilage and the synovial fluid
provide frictionless motion in healthy joints. Shock absorption to the joint is provided by
a combination of structures, including articular cartilage, subchondral bone, and the soft
tissue structures such as the joint capsule and ligaments (McIlwraith, 2015).
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Figure 1.A, Synovial joint with articular disk. B, Synovial joint with meniscus. 1, Compact bone; 2, periosteum; 3, fibrous layer of joint capsule; 4, synovial membrane; 5, articular disk; 6, meniscus; 7, joint cavity (Dyce, Sack & Wensing, 2010).
The articular surface of each bone in a synovial joint is covered by articular
cartilage that is generally of the hyaline variety, although fibrocartilage or dense fibrous
tissue is substituted in a few locations. The articular cartilage is smooth and resilient and
permits frictionless movement of the joint (Dyce, Sack & Wensing, 2010). Because of its
resilient nature and ability to compress, articular cartilage is a good shock absorber.
However, the primary shock absorbing function relies on soft tissues and bones of a joint.
Any disease that affects bone or soft tissue, like fractures or inflammation, will interfere
with this shock absorption (McIlwraith, 2015). In the dog, the articular cartilage is only a
few millimeters thick and it accentuates the curvature of the underlying bone, being
thickest in the center of convex surfaces and about the periphery of concave surfaces.
Articular cartilage is insensitive and avascular. This insensitivity explains why a
joint lesion may progress far before the dog owner becomes aware of its existence. In
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addition, this tissue has a limited capacity for intrinsic healing and repair. For this reason,
the preservation and health of articular cartilage are paramount to joint health (Fox, Bedi
and Romeo, 2009). The oxygen and nutritive requirements of the articular cartilage are
met by diffusion from three sources: fluid within the joint cavity, vessels in the tissues at
the periphery of the cartilage, and vessels in subjacent marrow spaces (see Figure 2).
Diffusion is assisted by the porosity of the cartilage matrix, which soaks up and releases
fluid as the cartilage is alternately unloaded and compressed during movement (Dyce,
Sack & Wensing, 2010).
Knowing the composition of articular cartilage allows us to understand the
mechanism of action of various drugs prescribed for the treatment of canine osteoarthritis
(McIlwraith, 2015). Articular cartilage is composed of chondrocytes and a specialized
extracellular matrix (ECM). Although cartilage ECM contains many molecular
components, its main components are fibrils of type II collagen, a cartilage-specific
proteoglycan (aggrecan) and water. The collagen II fibrils account for up to 60% of
articular cartilage dry weight and aggrecan proteoglycans comprise approximately 35%
of articular cartilage dry weight. On the other hand, water accounts for 80% of the wet
weight of articular cartilage. Each individual aggrecan molecule consists of a polypeptide
core protein from which numerous covalently linked glycosaminoglycan (GAG) side
chains extend, specifically, chondroitin sulfate and keratan sulfate polysaccharides
(Izadifar, Chen and Kulyk, 2012). Aggrecan molecules form very large aggregates where
each aggrecan molecule is non-covalently bound to the long molecule of hyaluronan (see
Figure 3). GAGs are chains of sugars that have negative charges and repel each other but
attract water. Since collagen is a positively charged molecule, GAGs are trapped within a
7
collagen framework that contains them. Loss of proteoglycans or breakdown of collagen
means that the articular cartilage cannot function normally (McIlwraith, 2015).
Figure 2. Diffusion of glucose and oxygen into articular cartilage. Articular cartilage is avascular, which means that chondrocytes must obtain nutrients and oxygen via diffusion from the synovial fluid. Thus, these two compounds show a gradient of concentrations in the cartilage, being lower in the deeper layers than at the surface (Blanco, Rego, & Ruiz-Romero, 2011).
Figure 3. A, Electron microscope view of an aggrecan aggregate molecule (Buckwalter, 1997). B, Schematic representation of the aggrecan aggregate molecule (Ternopil State Medical University, n.d.).
8
Four distinct zones or layers can be identified in the articular cartilage: the
superficial zone, the middle zone, the deep zone, and the calcified zone (see Figure 4).
Gillogly, Voightm and Blackburn (1998, as cited in Izadifar, Chen & Kulyk, 2012)
mention that the cell density of chondrocytes decreases from the superficial zone to the
deep zone, and their morphology changes from a flattened discoidal shape in the
superficial zone, to a more spherical shape in the middle zone, to a slightly elongated
form in the deep zone. The calcified zone provides a transition between the hyaline
cartilage tissue of the overlying zones and the basal subchondral bone, within this zone
the cartilage ECM is mineralized and type II collagen is replaced by a distinct type X
collagen (Athanasiou, Darling & Hu, 2009; Izadifar, Chen & Kulyk, 2012).
Figure 4. Zonal organization in normal articular cartilage, the black lines and the red solids represent collagen fibrils and chondrocytes, respectively (Doulabi, Mequanint & Mohammadi, 2014).
9
The stability of an articulation is maintained by a fibrous joint capsule, which is
composed of an outer fibrous layer and an inner synovial membrane. The outer fibrous
layer attaches around the margins of the articular surface and presents local thickenings,
which are named individually as ligaments when well developed and discrete. The
fibrous layer and ligaments are provided with proprioceptive and pain nerve endings
(Dyce, Sack & Wensing, 2010, p. 19). Ligaments are strong bands of fibrous connective
tissue that strengthen and support the joint. Ligaments allow for normal movements at a
joint, but limit the range of these motions, thus preventing excessive or abnormal joint
movements (Betts, 2013, p. 333). Ligaments are classified based on their relationship to
the fibrous articular capsule as intra-capsular or extra-capsular. A good example of
extra-capsular ligaments is the collateral ligaments, which maintain the stability in
joints such as the fetlock, carpus, elbow and stifle. The cruciate ligaments are the best
example of intra-capsular ligaments (McIlwraith, 2015); these ligaments maintain the
integrity of the femorotibial compartments of the stifle joint (see Figure 5).
Figure 5. The ligaments and menisci of the canine stifle joint (Palmer, 2005).
10
The synovial membrane is a vascular and sensitive sheet of connective tissue,
which, unlike mucus membranes, does not have a continuous covering of cells. The more
cellular parts produce the lubricant component of the synovial fluid (GAGs), while the
other components are derived from the blood plasma. Synovial fluid consists of a
transudate of plasma from synovial blood vessels, supplemented with high molecular
weight saccharide-rich molecules, notably hyaluronans, produced by type B
synoviocytes. Type A synoviocytes are phagocytes that remove debris from the synovial
fluid. Normal synovial fluid is an ultra-filtrate of plasma, has no clotting factors, is
viscous, is acellular, and is aparticulate (Denton, 2012). The synovial fluid has lubricant
and nutritive functions; it reduces friction so that there is no wear in healthy joints and
helps nourish the articular cartilage and any intra-articular structure (Dyce, Sack &
Wensing, 2010).
Some joints have intracapsular discs or menisci, which are fibrocartilage
structures, located between the articulating bones and have several functions, depending
on the specific joint. In the canine stifle, the disc provides shock absorption and
cushioning between the bones. In addition, an articular disc can serve to smooth the
movements between the articulating bones, as seen at the temporomandibular joint. Some
synovial joints also have a fat pad, which can serve as a cushion between the bones
(Betts, 2013).
Canine Osteoarthritis
Canine osteoarthritis (OA) is a chronic degenerative joint disease that affects dogs
of all ages, breeds and sexes. In North America, the prevalence of canine OA is of 20% in
11
adult dogs and 80% in geriatric (more than 8!years old) dogs (Railland et al., 2012).
Different primary lessons may lead to the development of OA but the molecular
pathophysiology of the disease is the same (Plickert, Bondzio, Einspanier, Tichy &
Brunnberg, 2013). In dogs, the most common causes for the development of secondary
OA are osteochondrosis, fragmented coronoid process, patellar luxation and anterior
cruciate ligament rupture (Plickert, Bondzio, Einspanier, Tichy & Brunnberg, 2013).
Canine OA is characterized by progressive degeneration of articular cartilage. When
articular cartilage degenerates, bones and nerves become exposed causing pain and
inflammation. Affected animals present pain and stiffness that affects their mobility and
quality of life. No cure is known for osteoarthritis. Treatment and management of canine
osteoarthritis usually consists of the use of prescribed anti-inflammatory drugs, dietary
supplements and life style changes.
Pathophysiology of Canine Osteoarthritis
In degenerative joint diseases such as OA, impairments in mechanical function are
associated with changes in the structure and biochemistry of articular cartilage
(Desrochers, Amrein & Matyas, 2012). Under normal conditions, the ECM of articular
cartilage is subjected to a dynamic remodeling process in which low levels of degradation
and synthesis exist so that the volume of cartilage is maintained. In canine OA, matrix-
degrading enzymes, mainly matrix metalloproteinases (MMPs), are overexpressed,
shifting this balance in favor of net degradation with resultant loss of collagen and
proteoglycans from the articular cartilage (Ling, 2012). Synoviocytes and chondrocytes
secrete both MMPs and its inhibitors (see Table 1). In patients with OA the synthesis of
12
MMPs is greatly enhanced and the amount of inhibitors produced is not enough to
maintain an adequate balance.
Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine, which plays a major
role in articular cartilage degeneration. IL-1 induces the synthesis of MMPs by
chondrocytes and synoviocytes, suppresses the synthesis of type 2 collagen and
proteoglycans, induces apoptosis of chondrocytes and stimulates production of nitric
oxide (NO) and prostaglandins. The presence of IL-1 RNA and IL-1 protein has been
confirmed in joints with OA. Thus, IL-1 may not only actively promote cartilage
degradation, but may also suppress attempts to repair articular cartilage. Under normal
conditions, an endogenous IL-1 receptor antagonist regulates IL-1 activity. A relative
excess of IL-1 and/or deficiency of the IL-1 receptor antagonist could result in cartilage
destruction. It is likely that other cytokines or particulate material from damaged cartilage
may also contribute to this inflammatory process. In addition, increased synthesis of NO
has been associated with cartilage degradation, inhibition of cartilage matrix synthesis
and the apoptosis of chondrocytes (Ling, 2012; Fox, 2014).
Table 1 Factors that contribute to articular cartilage degradation (Ling, 2012; Fox, 2014). Factors Actions Cyclooxygenase 2 (COX-2) Stimulates production of prostanoids. Prostaglandin E2 (PGE2) Play a major role in inflammation. Interleukin-1 (IL-1) Increases production of prostaglandins and
NO, induces the synthesis of MMPs, suppresses the synthesis of type 2 collagen and proteoglycans, induces apoptosis of chondrocytes.
Inducible nitric oxide synthase (iNOS)
Stimulates production of NO.
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Nitric oxide (NO) Cartilage degradation, inhibition of cartilage matrix synthesis and chondrocytes apoptosis.
Tumor necrosis factor alpha (TNF- α) Increases secretion of prostaglandins, COX-2 and iNOS and promotes cartilage degradation.
Aggrecanase Degrades aggrecan aggregate. Matrix Metalloproteinases (MMPs)
• Collagenases
Degrade native collagen in the triple helix region.
• Stromelysins
Degrades collagen types II, III, IV, IX, and X, proteoglycans, fibronectin, laminin, and elastin.
• Gelatinases Hydrolyze gelatin.
Traumatic joint injuries, abnormal joint loading, and degenerative joint diseases
can all cause defects in articular cartilage. Izadifar, Chen and Kulyk (2012) classify
cartilage lesions as chondral lesions (affect the articular cartilage), osteochondral lesions
(affect the articular cartilage and the subchondral bone), and microfractures (are not be
visible to the naked eye but affect the collagen network and can lead to further matrix
destruction). Because of the inability of articular cartilage to repair itself, the initial focal
cartilage damage leads to abnormal compressive loading and increased mechanical stress
in the surrounding healthy cartilage, which gradually expands the area of articular
damage. Over a period of years, this leads to a gradual erosion of the articular cartilage
layer of the joint, resulting in osteoarthritic disease (Izadifar, Chen & Kulyk, 2012). In
the end stages of osteoarthritic disease, the articular cartilage is totally destroyed thus
exposing the subchondral bone (see Figure 6), resulting in debilitating joint pain and
severely reduced joint mobility.
14
Canine OA has a considerable genetic component and it is considered a polygenic
disease. Genes associated with the development of OA tend to be related to the process of
synovial joint development. Mutations in these genes might directly cause OA and
determine the age of onset of the disease, the joint involved and the severity of the
disease. Sandell (2012) proposes that genetic mutations associated with OA can be placed
on a continuum. Early-onset OA is caused by mutations in matrix molecules often
associated with chondrodysplasias, whereas less destructive structural abnormalities or
mutations confer increased susceptibility to injury or malalignment that can result in
middle-age onset. In addition, Sandell (2012) mentions that mutations in molecules that
regulate subtle aspects of joint development and structure lead to the late-onset OA.
Current Treatments for Canine Osteoarthritis Joints function like buffers that absorb the pressure changes in the body. The
articular space contains the synovial fluid, which has a great amount of
glycosaminoglycans like chondroitin 4-shulphate and hyaluronates. GAGs are made of
repeated units of disaccharides, which, are composed of N-acetylgalactosamine, or N-
acetylglucosamine combined with uronic, D-glucuronic or L-iduronic acid, which are
essential for the proper functioning of joints. For this reason, one of the most widely used
treatments for canine OA consists of administering dietary supplements containing
glucosamine and/or chondroitin sulfate. Treatment of OA also includes activity
modification, physical therapy, weight loss, NSAIDs and steroidal drugs. Current
treatments for OA mainly alleviate the pain and discomfort in the arthritic joint without
correcting the underlying pathology.
15
Figure 6. A, lateral and B, cranial-caudal radiographic views of the stifle of a dog with OA. 1. Osteophytes are present on the proximal aspect of the femoral trochlea. 2. Proximal and distal aspects of the patella. 3. Femoral condyles. 4. Proximal tibia. 5. Sclerosis of the proximal tibia (Newton & Nunamaker, 1985)
Non-steroidal anti-inflammatory Drugs
The most frequent signs of canine OA are pain, lameness, inflammation and
articular movement difficulties. Pain and inflammation are mediated by prostanoids,
including prostaglandins and thromboxanes. Prostaglandins sustain homeostatic functions
and mediate pathogenic mechanisms, including the inflammatory response. When
inflammation is present, cell membrane phospholipids are converted to arachidonic acid a
20-carbon unsaturated fatty acid, by the action of phospholipase A. Then cyclooxygenase
isoenzymes (COX-1 and COX-2) convert arachidonic acid to prostaglandin H2, which in
turn in converted to thromboxane A, prostaglandin E2, prostacyclin, prostaglandin D2
and prostaglandin F2α by tissue specific enzymes (see Figure 7).
16
Figure 7. Biosynthetic pathway of prostanoids (Ricciotti, E., & Fitzgerald, 2011). PLA2: Phospholipase A2, TxA2: Thromboxane A2, PGD2: Prostaglandin D2, PGE2: Prostaglandin E2, PGI2: Prostacyclin, PGF2α: Prostaglandin F2α.
COX-1 is expressed constitutively in most cells and is the dominant source of
prostanoids in the organism. Prostanoids serve housekeeping functions, such as gastric
epithelial protection. Inflammatory stimuli, hormones, and growth factors induce the
expression of COX-2. COX-2 is the most important source of prostanoid formation
during inflammation and in proliferative diseases, such as cancer. However, both
enzymes contribute to the generation of autoregulatory and homeostatic prostanoids, and
both can contribute to prostanoid release during inflammation (Ricciotti & Fitzgerald,
2011).
PGH2 is produced by both COX isoforms and it is the common substrate for a
series of specific isomerase and synthase enzymes that produce PGE2, PGI2, PGD2,
PGF2α and TXA2. The profile of prostanoid production is determined by the differential
17
expression of these enzymes within cells present at sites of inflammation. For example,
mast cells predominantly generate PGD2 while macrophages produce PGE2 and TXA2.
In addition, alterations in the profile of prostanoid synthesis can occur upon cellular
activation. While resting macrophages produce TXA2 in excess of PGE2, this ratio
changes to favor PGE2 production after bacterial lipopolysaccharide activation (see
Figure 7).
NSAIDs like carprofen, ibuprofen and aspirin, block the action of cyclooxygenase
enzymes and their biosynthesis. Since the enzyme COX-1 plays an important role in the
production of endogenous prostaglandins in the gastric mucosa and it has important
homeostatic functions, drugs such as NSAIDs, have several potential side effects,
including the development of gastric ulcers. On the other hand, since COX-2 primarily
regulates the synthesis of inflammatory prostanoids, NSAIDs that selectively inhibit
COX-2 have fewer side effects (see Table 2). However, it should not be assumed that
complete COX-2 inhibition has no potential side effects because research data has
suggested that COX-2 can be expressed constitutively in various organs, including the
brain, spinal cord, ovary, and kidney (Asghar & Jamali, 2014; Hétu & Riendeau, 2005;
Zidar et al., 2008; Edwards, 2014).
Table 2 Classification of NSAIDs based on their selectivity for COX-1 and COX-2 (Riviere & Papich, 2009; Edwards, 2014). COX-2 Selective*
COX-2 Preferential**
COX Nonspecific***
COX-1 Selective****
Firocoxib Carprofen Flunixin Aspirin Deracoxib Celocoxib Ketoprofen Mavacoxib Deracoxib Phenilbutazone Robenacoxib Etadolac Tolfenemic acid
18
Luminacoxib Meloxicam Vedaprofen Nimesulide Flunixin *COX-2 selective: drugs that inhibit COX-2 (but not COX-1) at label dose **COX-2 preferential: drugs that inhibit COX-2 at lower drug concentrations ***COX-nonspecific: Both COX-1 and COX-2 are inhibited at label doses ****COX-1 selective: drugs that inhibit COX-1 (but not COX-2) at label dose
Steroidal Drugs
Two classes of steroid hormones, mineralocorticoids and glucocorticoids, are
naturally synthesized in the adrenal cortex from cholesterol. Mineralocorticoids
(aldosterone) are so named because they are important in maintaining electrolyte
homeostasis. However, mineralocorticoids also trigger a broader range of functions in
non-classic target cellular sites, including some effects on wound healing after injury. In
addition, a chronic and inappropriate increase in aldosterone secretion evokes a wound
healing response in the absence of tissue injury. Glucocorticoids suppress various
components of the inflammatory process; they inhibit phospholipase A, decrease
synthesis of interleukins and other pro-inflammatory cytokines, suppress cell-mediated
immunity, reduce complement synthesis, and decrease production and activity of
leukocytes. Glucocorticoids are the most effective and commonly used anti-inflammatory
drugs. However, because their pharmacologic and physiologic effects are so broad, the
potential for adverse effects is considerable.
Injections of corticosteroids like dexamethasone and methylprednisolone and are
often recommended to manage OA. Treatment with dexamethasone decreases joint
inflammation and joint tissue degradation and is chondroprotective (Huebner, Shrive, &
Frank, 2013). Vandeweerd et al. (2015) reviewed the current scientific evidence about the
19
effects of corticosteroids on articular cartilage. The review included 35 studies that
assessed the effects of corticosteroids on either normal cartilage, or on either induced OA
or synovitis, performed from 1965 to 2014. The findings of this investigation suggest that
in dogs, methylprednisolone acetate and triamcinolone hexacetonide have beneficial
effects in the treatment of OA. On the other hand, in horses, methylprednisolone acetate
was mostly deleterious, while triamcinolone acetonide had positive effects. Dvorak,
Cook, Kreeger, Kuroki, & Tomlinson (2002) found that dexamethasone caused
significant decreases in the production of prostaglandins, however this drug does not
inhibit the production of IL-1, therefore, even when pain is reduced, chondrocytes are not
being protected against the negative effects of IL-1.
Nutraceuticals
Glucosamine and Chondroitin Sulfate
The most commonly recommended supplements for the treatment of osteoarthritis
are glucosamine and chondroitin sulfate. There are various research articles that support
the idea that these supplements are effective in decreasing clinical signs (e.g. lameness
and pain) of osteoarthritis in dogs (Comblain, Serisier, Barthelemy, Balligand &
Henrotin, 2015). In 2007, McCarthy et al. reported that dogs treated with glucosamine
and chondroitin sulfate for 70 days showed statistically significant improvements in
scores for pain, weight bearing and severity of the condition. However, the onset of
significant response was slower than for carprofen-treated dogs. In addition, McCarthy et
al. (2007) showed that highly purified glucosamine and chondroitin sulfate are effective
chondroprotective agents for the treatment of osteoarthritis in dogs.
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The mechanism of action of glucosamine and chondroitin is not well-known,
however, their chondroprotective action can be explained by a dual mechanism; they act
as basic components of cartilage and synovial fluid, which stimulate the anabolic process
of cartilage metabolism and they have anti-inflammatory action that can delay many
inflammation-induced catabolic processes in the cartilage. Glucosamine enhances the
production of cartilage matrix components in chondrocyte culture; such as aggrecan and
collagen type II. In addition, glucosamine increases hyaluronic acid production and
prevents collagen degeneration in chondrocytes by inhibiting lipoxidation reactions and
protein oxidation. It is able to inhibit the MMP synthesis, and further proteoglycan
degeneration is therefore prevented. Glucosamine also inhibits aggrecanase by
suppression of glycosylphosphatidylinositol-linked proteins (Jerosch, 2011).
Chondroitin sulfate increases the production of hyaluronan by synovial cells,
which has a beneficial effect on maintaining viscosity in the synovial fluid. It has been
shown that chondroitin sulfate stimulates the chondrocyte metabolism, leading to the
synthesis of collagen and proteoglycan, the basic components of new cartilage.
Furthermore, chondroitin sulfate inhibits the enzymes leukocyte elastase and
hyaluronidase, which are found in high concentration in the synovial fluid of patients
with rheumatic diseases. Chondroitin sulfate also increases the production of hyaluronic
acid by synovial cells, which subsequently improves the viscosity and the synovial fluid
levels. In general, CS inhibits cartilage destruction processes and stimulates the anabolic
processes involved in new cartilage formation (Jerosch, 2011).
Glucosamine and chondroitin are safe for dogs and the only side effects to be
aware of are occasional diarrhea and a remote possibility of blood clotting problems. If a
21
dog is allergic to shellfish glucosamine should not be administered because these
supplements may have ingredients derived from shellfish (VCA Animal Specialty Group,
2014). In addition, high doses of glucosamine have been associated with polyuria and
polydipsia in dogs; however, the cause of these side effects has not been investigated
(McCoy & Bryson, 2013).
Omega-3 Fatty Acids
Fish oils are rich in polyunsaturated fatty acids like omega-3. The name of these
compounds comes from the fact that they have three double bonds at particular positions
in the hydrocarbon chain. Omega-3 fatty acids include: eicosapentaenoic acid (EPA),
docosahexaenoic acid (DHA) and alpha-linolenic acid (ALA). EPA and DHA are found
in fish oils and have beneficial effects on the health of humans and animals. ALA is
found in plants (e.g. flax-seed and canola oil) and it can be converted to EPA and DHA in
the body, however, this conversion is not efficient in dogs and cats (Lenox & Bauer,
2013). For this reason, it is best to supplement dogs and cats diets with fish oils instead of
plant oils. Moreau et al. (2012) found that dogs fed diets with high levels of omega-3
fatty acids from fish origin had improved loco-motor disability and the performance in
activities of daily living.
Currently, omega-3 fatty acids are used in managing many diseases including
neoplasia, dermatologic disease, hyperlipidemia, cardiovascular disease, renal disease,
gastrointestinal disease, and joint disease. The benefits of these compounds rely on the
fact that they can reduce the amount of arachidonic acid produced in the body.
Arachidonic acid is the precursor of prostanoids (see Figure 7), which are the mediators
of inflammation in the body, thus higher consumption of omega-3 fatty acids have been
22
related the decreased inflammation in the body. Target ranges for EPA and DHA vary
quite widely for different conditions, but typically fall between 50 and 220 mg/kg of
body weight. The higher dosages are often used to lower serum triglyceride
concentrations in patients with hypertriglyceridemia, whereas lower dosages are more
commonly used for inflammatory conditions, renal disease, and cardiac disease (Lenox &
Bauer, 2013).
In addition to mediating inflammation, prostanoids have several functions in the
body. They are needed for proper coagulation; wound healing and immune system
function. In addition, these molecules are needed to protect the gastric mucosa. For this
reason an excessive dose of omega-3 fatty acids can lead to several adverse effects. In
2013, Lenox & Bauer reviewed the potential adverse effects of omega-3 fatty acids in
dogs and cats and found that the most common adverse effects are: altered platelet
function, gastrointestinal adverse effects, detrimental effects on wound healing, lipid
peroxidation, potential for nutrient excess and toxin exposure, weight gain, altered
immune function, effects on glycemic control and insulin sensitivity, and nutrient-drug
interactions. These side effects are associated to the inhibition of endogenous
prostaglandins production.
Perna Canaliculus or Green-lipped Mussel
Green-lipped mussel (GLM) contains 61% protein, 13% carbohydrates, 12%
GAGs, 5% lipids (including omega-3 fatty acids), 5% minerals, and 4% water. Omega-3
fatty acids and GAGs are the key ingredient of this product. Omega-3 fatty acids provide
an anti-inflammatory effect and thereby the reduction of joint pain. GAGs are the main
components of articular cartilage and the synovial fluid and serve as building blocks that
23
promote the synthesis of articular cartilage. In 2003, Bui and Bierer investigated the
effects of green-lipped mussel in osteoarthritis. They added 0.3% of green-lipped mussel
powder to a dry diet for a period of 6 weeks to a group of dogs and found that there was
significant improvement in the test group versus the control group. Significant
improvements were observed in joint pain and swelling. However, crepitus and range of
joint movement were not significantly different between the test and control groups.
Their findings provide strong evidence that GLM incorporated into a complete dry diet
can help alleviate arthritis symptoms in dogs. Up to the date, no side effects have been
associated with this supplement. In 2002, Bierer showed that the following doses of
green-lipped mussel are effective in decreasing clinical signs of osteoarthritis: 1000 mg
GLM/day for dogs weighing more than 34 kg, 750 mg GLM/day for dogs weighing from
25 to 34 kg and 450 mg GLM/day for dogs weighing less than 25 kg.
Avocado/Soybean Unsaponifiables
Avocado/Soybean unsaponifiables (ASU) seems to have anti-catabolic properties
that prevent the degradation of cartilage and anabolic properties that promote cartilage
repair by stimulating collagen and aggrecan aggragate production. At the clinical level,
ASU reduces pain and stiffness while improving joint function, resulting in decreased
dependence on analgesics (Christiansen, Bhatti, Goudarzi, & Emami, 2014). No clinical
trials have been performed to test this supplement in dogs and cats; more research is
needed to determine its efficacy, mechanism of action and potential adverse effects in
these species.
24
S-Adenosyl-L-methionine
S-Adenosyl-L-methionine (SAMe) is produced in the body from methionine, an
amino acid found in foods. Early research showed that SAMe is involved in a variety of
body functions, especially in the brain and the liver. It is a common cosubstrate involved
in methyl group transfers, transsulfuration, and aminopropylation. Although these
anabolic reactions occur throughout the body, most SAM is produced and consumed in
the liver. More than 40 methyl transfers from SAM are known, to various substrates such
as nucleic acids, proteins, lipids and secondary metabolites. It is made from adenosine
triphosphate and methionine by methionine adenosyltransferase.
Given the actions of SAMe in the liver and brain, researches have investigated the
possible value of SAMe in the treatment of mental illnesses and liver diseases. During
clinical trials in people with depression, some study participants who also had
osteoarthritis reported that their joint symptoms improved when they were taking SAMe.
Therefore researchers began to investigate SAMe as a possible treatment
for osteoarthritis. The results of research on SAMe for osteoarthritis are mixed. Clinical
trials have compared oral SAMe with NSAIDs or placebos in patients with osteoarthritis
of the knee or hip. In general, trials that compared SAMe with NSAIDs showed that each
had similar pain relief and improvement in joint function, with fewer side effects in the
patients taking SAMe. The smaller number of trials that compared SAMe with placebo
did not consistently show SAMe to be beneficial (National Center for Complementary
and Integrative Health, 2012).
Stem cells Canine OA is a non-curable disease; therefore current therapeutic approaches
focus on preventing or delaying the structural and functional changes of the affected
25
tissues. The use of NSAIDs is associated with gastric ulceration and renal and hepatic
damages, for this reason, alternative treatment modalities without these adverse side
effects are highly desirable. Stem cells represent a novel treatment alternative for canine
OA. Stem cell therapy has the potential of repairing the tissue damage caused by the
disease. A stem cell is an undifferentiated cell of a multicellular organism that is capable
of giving rise to indefinitely more cells of the same type, and from which certain other
kinds of cell arise by differentiation. Not only do the stem cells form the new cells of a
structure, but also they serve in the repair of a damaged structure or in the process of
constant renovation as the structures decay with the passage of time (Global Stem Cells
Group, 2015).
Cellular differentiation is the process of a cell changing from a less specialized
type of cell to a more specialized cell type. Differentiation occurs numerous times during
the development of a multicellular organism as it changes from a simple zygote to a
complex system of tissues and cell types. Differentiation continues in adulthood as adult
stem cells divide and create fully differentiated daughter cells during tissue repair and
during normal cell turnover. Cellular differentiation almost never involves a change in
the DNA sequence itself. Thus, different cells can have very different physical
characteristics despite having the same genome. A cell that can differentiate into all cell
types of the adult organism is known as pluripotent. Such cells are called embryonic stem
cells in animals and meristematic cells in higher plants. A cell that can differentiate into
all cell types, including the placental tissue, is known as totipotent. In mammals, only the
zygote and subsequent blastomeres are totipotent (Global Stem Cells Group, 2015).
26
Until recently, scientists primarily worked with two kinds of stem cells from
animals and humans: embryonic stem cells and adult stem cells. Embryonic stem cells, as
their name suggests, are derived from embryos that develop from eggs that have been
fertilized in vitro. An adult stem cell is thought to be an undifferentiated cell, found
among differentiated cells in a tissue or organ. The adult stem cell can renew itself and
can differentiate to yield some or all of the major specialized cell types of a tissue or
organ. The primary roles of adult stem cells in a living organism are to maintain and
repair the tissue in which they are found (National Institutes of Health, 2015). Adult stem
cells have a much lower capacity than embryonic stem cells to self-renew and
differentiate; however, adult stem cells are immunocompatible, and their use is not
restricted by the ethical concerns associated with embryo-derived cells (Cuervo et al.,
2014).
The bone marrow contains at least two kinds of stem cells: hematopoietic stem
cells and mesenchymal stem cells (MSCs). MSCs, which are also known as stromal or
skeletal stem cells, make up a small proportion of the stromal cell population in the bone
marrow and can generate bone, cartilage (see Figure 8). On the other hand, hematopoietic
stem cells give rise to all blood cells through the process of haematopoiesis. They are
derived from mesoderm and located in the red bone marrow, which is contained in the
core of most bones. Hematopoietic stem cells give rise to both the myeloid and lymphoid
lineages of blood cells. Myeloid cells include monocytes, macrophages, neutrophils,
basophils, eosinophils, erythrocytes, dendritic cells, and megakaryocytes or platelets and
lymphoid cells include T cells, B cells, and natural killer cells (National Institutes of
Health, 2015).
27
MSCs were first identified in bone marrow but now are found in virtually all
tissues (Meirelles, 2006). The differentiation potential of MSCs was the initial reason for
using them as therapeutic agents for many diseases, however, scientific evidence has
shifted the therapeutic emphasis from differentiation to paracrine actions as the main
mechanism for MSCs therapeutic efficacy (Baraniak & Mcdevitt, 2010). When
transplanted into diseased cartilage, MSCs can transform into chondrocytes and help in
filling defects in cartilage. In addition, MSCs communicate with local cells through
secretion of a wide array of cytokines and growth factors. In addition, they can promote
the migration of endogenous repairing cells to injury sites and suppress immunoreactions
(Nöth, Steinert, & Tuan, 2008). Together, these actions help restore physiological balance
and enhance healing (Tsai, 2014).
In veterinary medicine, MSCs have been used to treat OA, tendon injury, bone
fracture, spinal cord injury, and liver disease (Ribitsch et al., 2010). Autologous stem cell
therapy in veterinary medicine involves harvesting tissue, such as fat, from the patient,
isolating the stem cells, and administering the cells back to the patient (Black et al.,
2007). The fact that stem cell yields are greater from adipose tissue than from other stem
cell reservoirs makes autologous adipose-derived mesenchymal (ADMSCs) stem cell
therapy an excellent option for the treatment of OA and other diseases. Routinely, 1 x
107 adipose stromal/stem cells are isolated from 300 ml of lipoaspirate, with greater than
95% purity. Subcutaneous adipose tissue samples can generally be obtained under local
anesthesia. Current methods used for isolating ADMSCs rely on collagenase digestion
followed by centrifugal separation to isolate the stromal vascular fraction from primary
adipocytes. They display a fibroblast-like morphology and lack the inter-cellular lipid
28
droplets seen in adipocytes. Isolated ADMSCs are typically expanded in monolayer
culture on standard tissue culture plastics with a basal medium containing 10% fetal
bovine serum (Mizuno, Tobita, & Uysal, 2012).
ADMSCs stem cell therapy has been commercially available since 2003. In 2007,
Black et al. evaluated this kind of therapy in dogs with chronic osteoarthritis of the hip.
Dogs treated with ADMSCs therapy had significantly decreased lameness and pain, and
increased range of motion when compared with the control group. In 2008, the same
group of researches, investigated the effects of this therapy in dogs with chronic
osteoarthritis of the elbow joints and found that lameness, pain and range of motion
improved in comparison with the control group. The purpose of this review is to discuss
the current scientific data regarding the efficacy of MSCs in the treatment of canine OA.
Figure 8. The multipotentiality of MSCs. Mesenchymal stem cells (MSCs) in the bone-marrow cavity have the ability to self-renew (curved arrow) and to differentiate (straight, solid arrows) towards the mesodermal lineage. The reported ability to transdifferentiate
29
into cells of other lineages is shown by dashed arrows, as transdifferentiation is controversial in vivo (Uccelli, Moretta, & Pistoia, 2008).
30
Chapter 3: Procedures
A systematic search of the electronic databases PubMed, ScienceDirect,
Springerlink was performed on August 13, 2015 and updated on October 16, 2015. The
key words used for this search were: canine, osteoarthritis, treatment and mesenchymal
stem cells. Original research articles were eligible for inclusion in the review if the
primary aim was to evaluate the effects of autologous or heterologous MSCs in the
treatment of canine OA and if they were published within the last 5 years. Six articles
met these criteria. The objectives, methods and results of these studies were summarized
on a table and are discussed below.
31
Chapter 4: Results and Discussion
Effects of Mesenchymal Stem Cells in the Treatment of Osteoarthritis
Autologous stem cell therapy involves harvesting tissue, such as fat or bone
marrow, from the patient, isolating the stem cells, and administering the cells back to the
patient (Vilar, 2014). The field of adipose-derived MSC therapy (ADMSCs) is a rapidly
growing area of research, and it has been shown that stem cells have affinity for damaged
joint tissue. Recent in vivo studies have confirmed that stem cells have the ability to
localize and participate in the repair of damaged joint structures, including cruciate
ligaments, menisci, and cartilage lesions (Vilar et al., 2013).
Several studies have described the use of plasma rich in growth factors (PRGF),
as an effective and safe method in the treatment of pain and joint dysfunction in OA
(Cuervo et al., 2014). PRGF, also known as plasma rich in platelets, is defined as the
volume of autologous plasma having a platelet concentration over baseline. Under normal
circumstances, platelets are the first cells to arrive at the tissue injury site and are
particularly active in the early inflammatory phases; for this reason PRGF represent a
good option for the treatment of OA. The effect PRGF has in the treatment of OA is due
to the behavior of the platelet concentrate, acting as a scaffold which through the various
growth factors promotes the stimulation of chondrogenesis, increases hyaluronic acid
production, stabilizes angiogenesis and differentiation of the existing cells in the area
treated. Platelets are cells that contain many important bioactive proteins and growth
factors, which are polypeptide substances, both soluble and diffusible, that regulate key
processes in tissue repair, including cell proliferation, chemotaxis, migration,
differentiation, and extracellular matrix synthesis. It has been hypothesized (Vilar et al.,
32
2014) that the combination of PRGF with ADMSCs is more effective for the treatment of
OA than ADMSCs alone. The effects of autologous AMSCs, autologous AMSCs
combined with PRGF and heterologous AMSCs for the treatment of OA have been
studied in the recent years (see Table 3). The results of these investigations and their
implications in veterinary medicine are discussed below.
Table 3 Summary of studies evaluating the therapeutic potential of mesenchymal stem cells in dogs with spontaneous osteoarthritis.
Authors Objective Population
Sample (n) Treatment Findings
Vilar et al., 2014
Use a force platform to measure the efficacy of intra-articular administration of ADMSCs for limb function improvement in dogs with severe OA.
Treatment: 9 lame dogs with severe chronic hip OA.
Control: 5 healthy dogs.
Single intra-articular injection of autologous ADMSCs.
Treated dogs showed significant lower mean values of peak vertical force and vertical impulse within the first three months post-treatment than the control group. The duration of maximal effect was less than 3 months.
Vilar et al., 2013
Use a force platform to measure the efficacy of intra-articular administration of ADMSCs associated to PRGF for limb function improvement in
Treatment: 8 lame dogs with severe chronic hip OA.
Control: 5 healthy dogs.
Single intra-articular injection of autologous ADMSCs associated to PRGF.
Treated dogs showed significant lower mean values of peak vertical force and vertical impulse, reaching the maximal effect at 180 days after treatment. Intra-articular
33
dogs with severe OA.
ADMSCs associated with PRGF therapy resulted in reduced lameness due to OA.
Cuervo et al., 2014
Compare the efficacy and safety of a single intra-articular injection of ADMSCs versus plasma rich in growth factors (PRGF) as a treatment for canine OA.
39 dogs with symptomatic hip OA were assigned to one of two groups.
19 dogs received a single intra-articular injection of ADMSCs and 20 dogs received a single intra-articular injection of PRGF.
OA degree did not vary within groups. Dogs treated with ADMSCs and PRGF had a significant improvement of pain physical function.
Tsai et al., 2014
Test whether intra-articular injection of porcine ADMSCs can treat canine OA.
3 dogs presenting stifle joint OA that had lasted more than 3 months and had been treated without significant improvement.
Single intra-articular injection of porcine ADSCs.
The three patients had decreased pain and increased mobility. There were no radiological changes.
Zaragoza et al., 2015
Evaluate the effectiveness of the application of ADMSCs in the treatment of OA in the elbow, hip and knee of the dogs.
26 healthy dogs with hip, knee or elbow OA.
Dogs were treated with a single intra-articular injection of ADMSCs.
Dogs showed decreased pain and improved joint function without radiographic changes, with an increase in hyaluronic acid and collagen type II cleavage neoepitope concentration.
34
Cuervo-Serrato et al., 2014
Compare the effect of ADMSCs, PRGF and the combination of these two therapies in the treatment of canine hip OA.
66 dogs with hip OA
17 dogs received a single intra-articular injection of autologous ADMSCs
32 dogs received a single intra-articular injection of PRGF.
17 dogs received a single intra-articular injection of PRGF plus ADMSCs.
The three treatments were very effective in the control of pain and the recovery of joint functionality. The groups that received ADMSCs and ADMSCs plus PRGF had better results.
Autologous AMSCs
In 2014, Vilar et al. studied the efficacy of intra-articular administration of
ADMSCs for limb function improvement in 9 dogs with severe OA, using 5 healthy dogs
as the control group. Stem cells were extracted from subcutaneous fat tissue of the
inguinal region through a small surgical incision using general anesthesia. A biopsy of
twenty grams of adipose tissue and a 120 mL sample of blood were obtained. Meloxicam
0.1 mg/kg/ 24 h orally was administered during 3 days post-surgery. Adipose tissue was
sent to specialized laboratories for MSCs isolation and two weeks later the laboratory
returned the cultivated ADMSCs. Dogs in the treatment group received a single intra-
articular injection of ADMSCs into the hip joints. Peak vertical force (PVF) and vertical
impulse (VI) were measured using a force platform. PVF and VI represent maximal
35
weight bearing and distribution of forces through time, respectively. These variables
allow the objective measurement of the clinical impact of ADMSCs treatment on the
function of the limb during the stance phase of walking. PVF and VI were used to
evaluate the affected limbs at day 0, 30, 90, and 180 post-treatment. Improvement of the
degree of pain and lameness was observed up to 90 days post-treatment, with the best
results 30 days after treatment. After that a regression to the initial state was observed.
This study suggests that a single intra-articular administration of ADMSCs decreases
pain and lameness in dogs with OA during a period less than three months, at which point
PVF and VI values returned to being similar to the pre-treatment status.
Zaragoza et al. (2015) performed a similar study, which included twenty-six
healthy dogs with OA documented by radiological and clinical findings. The joints
affected were distributed in 17 hips, 4 knees and 5 elbows. The dogs were treated with a
2 ml intra-articular injection containing 30 millions of ADMSCs and were evaluated on
radiological changes of the affected joints, functional limitation, pain and serum
hyaluronic acid and collagen type II cleavage neoepitope concentration. There was an
improvement was observed from the first month to six months after treatment on all of
the measured parameters except the radiological changes. This study suggests that the
application of a single intra-articular injection of ADMSCs improves the clinical signs of
canine OA for up to six months.
Cuervo et al. compared the efficacy and safety of a single intra-articular injection
of ADMSCs versus PRGF as a treatment for canine OA in 2014. Thirty-nine dogs with
symptomatic hip OA were assigned to one of the two groups, to receive ADMSCs or
PRGF. The ADMSCs group received a 2 mL containing 30 million ADMSCs (n = 18)
36
and the PRGF group received 2 mL of PRGF (n = 19). As some animals were affected
bilaterally, the total numbers of joints studied were 40 and 38 for the ADMSCs and
PRGF groups, respectively. Throughout the study the owners could use meloxicam as an
analgesic. All the patients were evaluated at baseline (day 0) and 1, 3 and 6 months after
treatment. Passive manual mobilization of the joint, degree of atrophy of muscles, range
of movement, radiological changes and pain (subjective assessment by the veterinarian
and owner) were assessed. The results from this randomized trial showed that a single
intra-articular injection of ADMSCs is significantly more effective than one intra-
articular injection of PRGF in reducing pain and improving functional limitation and
quality of life in dogs with hip OA. Better results were subjectively observed at 6 months
in patients treated with ADMSCs. In the owner’s pain assessment, significant differences
were observed in the ADMSCs and PRGF groups between baseline and 1, 3 and 6
months post-treatment, with no statistically significant differences between them. In the
investigator assessment, significant differences were observed in both groups between
baseline and all other follow-up time points. Comparing both treatments, there were only
differences at 6 months post-infiltration, where patients treated with ADMSCs showed
more pain relief than those treated with PRGF.
Autologous AMSCs combined with PRGF
Vilar et al. (2013) evaluated the effects of autologous AMSCs combined with
PRGF for the treatment of canine OA. The study included eight dogs with lameness and
pain attributed to OA associated with hip dysplasia. The dogs were affected by chronic
OA confirmed with radiographs. A control group consisted of 5 healthy dogs. The
treatment group received a 4 mL injection containing 30 million ADMSCs and PRGF.
37
PVF and VI were evaluated. Mean values of PVF and VI were significantly improved
after treatment of the OA groups, reaching the maximal effect at six months after
treatment. Intra-articular ADMSCs associated with PRGF therapy resulted in reduced
lameness due to OA.
Cuervo-Serrato et al. (2014) compared the effects of ADMSCs, PRGF and the
combination of these two therapies in the treatment of canine hip OA. A total of sixty-six
dogs were included in this investigation. Seventeen dogs received a single intra-articular
injection of autologous ADMSCs, thirty-two received a single intra-articular injection of
PRGF and seventeen received a single intra-articular injection of PRGF plus ADMSCs.
Functional limitation, joint mobility, range of movement, owner’s and veterinarians
perception of pain and owner satisfaction of the treatment, showed a clear improvement
after one month of treatment, maintaining for up to six months in the three groups,
without any differences between the ADMSCs and ADMSCs plus PRGF groups. Worse
results on joint mobility and range of movement were observed in the PRGF group
compared to the other two groups at 6 months post-treatment.
Heterologous AMSCs
In 2014, Tsai tested whether intra-articular injection of porcine ADMSCs can treat
canine OA. Three dogs with stifle joint OA that had lasted more than three months and
had been treated with OA medication without significant improvement were enrolled in
this study. ADMSCs were isolated from abdominal adipose tissue of a two month-old
female Yorkshire pig. Dogs received an intra-articular injection with 5 million ADSMCs.
The patients were observed for forty-eight hours after the injection to detect any sign of
inflammatory or allergic reaction. Patients were discharged to the owner and assessed at
38
two, six and twelve weeks after treatment. Injection of porcine ADMSCs into canine
stifle joints did not cause any inflammatory or allergic reactions. Significant
improvements were observed in all three dogs using a force-plate analysis. Orthopedic
evaluation found improvements in two dogs; particularly at the longest time point and
owners observed increased movement capacity and decreased pain in all the patients.
This study suggests that xenotransplantation of ADMSCs for the treatment of OA is
feasible; however, further studies are needed to validate this novel treatment modality.
Safety
ADMSCs represent a safe treatment option for canine OA. Cuervo et al. (2014)
observed two mild adverse effects events, one in the ADMSCs group and one in the
PRGF group. The owners of the two animals reported pain post-injection and prolonged
the NSAID treatment for 2 days in the ADMSCs group, and 4 days in the PRGF group,
with no significant differences between them. No adverse affects were observed any of
the other studies (Cuervo-Serrato et al., 2015; Tsai et al., 2014; Vilar et al., 2014; Vilar et
al., 2013; Zaragoza et al., 2015).
Discussion
Subjective and objective assessments have demonstrated that intra-articular
injections of ADMSCs can retard the progression of canine OA and decrease clinical
signs of disease such as lameness, range of motion and pain (Cuervo et al., 2014; Cuervo-
Serrato et al., 2015; Tsai et al., 2014; Vilar et al., 2014; Vilar et al., 2013; Zaragoza et al.,
2015). However, radiological changes were not observed on any of the above-mentioned
studies. Cuervo et al. (2014) suggest that these results do not necessarily mean that
changes did not exist at a cartilage structure level, and that further diagnostic studies such
39
as magnetic resonance imaging may be needed to detect such changes. On the other hand,
Tsai et al. (2014) mentions that the fact that there are no radiological changes after
treatment with ADMSCs suggests that the improved scores in force-plate, orthopedic,
and owner’s assessments were due to ADMSC’s anti-inflammatory and/or
immunomodulatory actions. Recent scientific reviews (Baraniak & Mcdevitt, 2010;
Sierra, Wyles, Houdek, & Behfar, 2015) support the idea that the functional
improvements attributed to stem cells may be due to paracrine actions in the host tissue
rather than to cell differentiation and repopulation. Whitworth and Banks (2014) mention
that it may be necessary to facilitate the engraftment of the new cartilage to the native
cartilage and bone in order to effect significant physical repair of the damaged cartilage.
Cuervo-Serrato et al., 2014 and Vilar et al. 2013 studied the effects of ADMSCs
combined with PRGF. PRGF increases the synthesis of chondrocytes, collagen and
proteoglycans and it acts as a coordinator to regulate homeostasis and proper functioning
of the cartilage. The effect PRGF has in the treatment of OA is due to the behavior of the
platelet concentrate, acting as a scaffold which through the various growth factors
promotes the stimulation of chondrogenesis, increases hyaluronic acid production,
stabilizes angiogenesis and differentiation of the existing cells in the area treated. In
addition, PRGF is believed to promote cell viability and extend cell life span (Cuervo-
Serrato et al., 2014). For these reasons, it was hypothesized that the combination of
PRGF with ADMSCs would be more effective for the treatment of OA than ADMSCs
alone; however, there was no statistically significant difference between the group treated
with ADMSCs alone and the group treated with ADMSCs and PRGF. Further studies are
needed to determine the possible synergic effects of these treatments.
40
The use ADMSCs for the treatment of OA into clinical practice faces many
challenges, including: most veterinary clinics lack the equipment and expertise for
ADMSCs isolation; excision of adipose tissue causes donor site morbidity; individually-
made ADMSCs isolation is costly and time-consuming; and, at least two veterinarian
appointments are needed for adipose tissue procurement and ADMSC injection (Tsai,
2014). The use of heterologous ADMSCs represents a good option to overcome these
challenges because veterinarians could purchase the isolated cells from specialized
laboratories instead of having to isolate them from the patient. The pilot study conducted
by Tsai et al. (2014) suggests that porcine ADMSCs provide similar effects to those
observed with autologous ADMSCs. The three dogs that participated in this study did not
show any adverse reaction to the xenotransplant, which suggests that the use of
heterologous ADMSCs for the treatment of OA is feasible. However, further studies are
needed to validate this novel treatment modality, which could be implemented for the
routine treatment of OA in veterinary medicine.
Currently, most dogs with OA receive a therapy based on NSAIDs and
nutraceuticals. NSAIDs, especially those selective for COX-2, provide a good analgesic
effect and improve the quality of life of the patient with few side effects. Nutraceuticals
like ome-3 fatty acids also provide anti-inflammatory effects with few side effects.
However, it is important to keep in mind that while these treatments provide pain and
inflammation control, the articular tissues continue to degenerate due to the continual
wear and tear caused by daily activities. For this reason, it is important to combine
analgesia with condroprotective therapy, which is usually achieved with the use of
nutraceticals. Some nutraceuticals like ASU seem to have anti-catabolic properties that
41
prevent the degradation of cartilage and anabolic properties that promote cartilage repair
by stimulating collagen and aggrecan aggragate production. On other hand, the effects of
glucosamine and chondroitin sulfate supplements are attributed to the fact that these
molecules are the building blocks for articular cartilage. However, there are many factors
involved in the degradation of articular cartilage (see Table 1) and the mere presence of
these components may not be enough to induce reparation of articular cartilage.
The lack of an effective, long term therapy for OA, in both human and veterinary
medicine, has drawn the attention of researchers to stem cells. It has been showed that a
single intra-articular injection of ADMSCs can decrease the clinical signs of canine OA.
However, contrary to what it was initially thought the effects of this treatment are
probably not due to cartilage regeneration by stem cells but to its paracrine
immunomodulatory and anti-inflammatory actions. Further studies are needed to
understand the mechanism of action of stem cells in this disease; however, it represents a
promising therapy for canine and human OA.
Conclusions
• A!single!intra,articular!injection!of!thirty!million!ADMSCs!improves!the!signs!
of!canine!OA!for!up!to!six!months.!!
• The!combination!of!ADMSCs!and!PRGF!is!equally!effective!for!the!treatment!
of!OA!as!ADMSCs!alone.!!
• Treatment!with!ADMSCs!alone!is!more!effective!than!treatment!with!PRGF!
alone.!!
• Heterologous!ADMSCs!provide!similar!effects!to!those!observed!with!
autologous!ADMSCs.!
42
• The!use!of!autologous!and!heterologous!ADMSCs!in!dogs!is!safe!since!only!
mild!adverse!reactions!have!been!observed.!!
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Athanasiou, K. A., Darling, E. M., & Hu, J. C. (2009). Articular Cartilage Tissue
Engineering. Synthesis Lectures on Tissue Engineering, 1(1), 1-182.
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