TodaysVeterinaryPracticeArticle8-2011.pdf

dncesinsemcellhery

Musculoskeletal

disorders open

wounds

eerreviewed

Donna Alderman, DO, and Robert W. Alexander, MD, DMD, FICS

rticleverview

•Inveterinarymedicine,thereareoptionsavailableforbothsmallandlargeanimalpractitionerstoutilize

autologousadipose-derivedstem/stromalcells(AD-SCs)topromotehealingforinjuriesanddegenerativejoint

•Byprovisionofalivingbioscaffoldingtoencouragestemcelladherence–proliferation,theadditionalcell

availabilitycanbefurtherenhancedwithadditionofhigh-densityplatelet-richplasma(HDPRP).

•Thepotentialofstem/stromalcells,coupledwithimportantinflammatorypromotion(HDPRP),isrecognizedas

safeandefficaciousinbothopenwoundsurgicalcareandguidedplacement.

•TheabilitytoprepareasiteforskingraftbyplacementofAD-SCsinrecalcitrantfull-thicknesswoundsspeeds

thehealingandrecuperationofsmallandlargedefectsinanimals.

•AD-SCsareofsignificantvalueinmusculoskeletaltissueinjuryordiseasebecausethereisgradualdepletionof

nativestem/stromalcellsinchronicinjuryordegenerativestates.

•MultiplestudiessupporttheeffectivenessofAD-SCsforuseinconnectivetissueandjointrepair,amongother

potentialuses.

•Controlledveterinaryclinicaltrialsarecontinuing,whichwillprovidestatisticaldocumentationofthesafetyand

efficacyofAD-SCs,aswellascomparisonsofdifferentprotocolsforadministration.

•UtilizationofAD-SCs,withorwithoutHDPRPconcentrates,haveprovenveryeffectiveinseveralthousand

injectionsinpreclinicalandclinicalusebybothhumanandveterinaryphysiciansintheU.S.andelsewhere.

Reference

dJ Prolother

dncesinsemcellhery

O steoarthritis afflicts 10

to 12 million dogs in the

United States and is the

most common cause of chronic pain

in dogs. 1 Tendon and ligament inju-

ries are common in performance

horses and potentially more threat-

ening than a fracture to the horse’s

athletic ability. 2

Clinical evidence is growing that

autologous adipose-derived stem/

stromal cells (AD-SCs) can dramati-

cally improve healing of injuries and

decrease degenerative processes. 3

eiveterireicie

Veterinarians have used autologous

adipose-derived mesenchymal stem

cells to treat tendon and ligament

injuries and joint disease in horses

on a commercial basis since 2003. 4

This procedure involves:

•Extractionofafatsamplefrom

the animal

•Samplesenttoalaboratoryfor

stem/stromal cell processing

•Processedsamplereturnedto

treating veterinarian for direct placement or injection

into the injured tissue or joint.

Successful outcomes from this treatment have been

shown in horses treated from 2003 to 2008, with: 5-8

•77%returningtopriorlevelofperformance

•94%stable1yearormoreaftertreatmentforacute

and chronic suspensory ligament injuries

•57%withjointinjuriesreturningtopriorlevelofper-

formance.

No systemic adverse events were reported and less than

0.5%hadlocaltissuereactions.

Figure 1. Flow chart elucidating possible commitment, lineage progression, and

maturation of adipose-derived mesenchymal stem cells

animal veterinarians for dogs and cats, with more than

1000 cases of connective tissue and joint repair reported

through2009,

andagreaterthan80%successratein

blinded, placebo-controlled canine clinical trials. 10

Recently in-office stem cell procedure kits have become

available, allowing for treatment at the clinic or on the

farm, depending on the species of animal receiving therapy.

tecellcrcteritic

There are 2 kinds of stem cells:

•Embryonic(prenatal)stemcells

•Adult(postnatal)stemcells.

Although most lay people recognize the term embryonic

stem cells, the important potentials of adult stem cells have

been recognized in the veterinary and human medical lit-

eraturesince1963,whenBecker,etal,reportedonthe

regenerative nature of bone marrow. 12

In2007,thistechnologywasmadeavailabletosmall

efiigtec

• tistheabilityofasinglecelltodivide

andproduceallthedifferentiatedcellsinanorgan-

ism,includingextraembryonictissues.

• referstoastemcellthathasthepoten-

tialtodifferentiateintoanyofthe3germlayers:

1.Endoderm(interiorstomachlining,gastrointes-

tinaltract,lungs)

2.Mesoderm(muscle,bone,blood,urogenital)

3.Ectoderm(epidermaltissues,nervoussystem).

• referstocellsthatretainthecapa-

bilitytodifferentiateintoavarietyofcellularphe-

notypesderivedfrom1germlayer.

Embryonicstemcellsare,intheory,abletotransform

into any type of tissue; they are totipotent when an egg

is fertilized; then after several divisions are pluripotent . 13

Although less of an issue in veterinary than human medi-

cine, there are religious, political, and ethical issues that

limit the use of fetal stem cells.

Postnataladultstemcells:

•Retainregenerativeorreparativecapabilitiesasundif

ferentiated cells

•Maintainhomeostasisinalltissues

dncesinsemcellhery

•Arecapableofrepairorregenerationinatissueor

organ system.

These multipotent cells are locally activated to pro-

liferate and differentiate into some, or all, of the major

specialized cell types of tissue when required for main-

tenance or repair. They facilitate tissue maintenance,

regeneration, growth, and wound healing through-

out life with the capability of differentiation to a wide

variety of types of adult cells, such as muscle, bone,

cartilage, tendon-ligament, and adipose tissues. 14

Adult stem cells are found in all tissues in the

body 15 in varying quantities, with major reservoirs

in adipose tissue 16

differentiate into tissue derived from ectodermal and

endodermal origins, such as organ tissue, nerves, and

skin, suggesting that they have pluripotent and multi-

potent capabilities (see Figure 1 ). 23-32

Recent studies have determined the safety and effi-

cacy of implanted/administered AD-SCs in various

animal models as well as human clinical trials. AD-SCs

also meet certain criteria described for the ideal stem

cell for regenerative medicinal applications: 33,34

•Foundinabundantquantities

•Harvestedwithaminimallyinvasiveprocedure

•Canbedifferentiatedalongmultiplecelllineage

pathways in a regulatable and reproducible manner

•Canbesafelyandeffectivelytransplanted.

As a result, adipose tissue has become an important

resource for research and patient care applications in

human and veterinary medicine.

and,toalesserextent,bone

ma r row. 17

Bothbonemarrowandadiposetissueare

derived from embryonic mesodermal tissues and

containamicrovascularnetwork,includingextra-

cellularmatrixandextensiveperivascularstroma,

which is credited for clinical promise in regenerative

medicine applications. 18

igeitlteletricl

ccetrte

Enhancedhealingcapabilityispossiblewhenplatelet

concentrations are increased within injured or dam-

aged tissue. 35

Adipose stroma contains large numbers of undiffer-

entiated stem/stromal cells capable of producing all

tissue types derived from the mesodermal layer. 19,20

These AD-SCs have the potential to differentiate to

tenoligamentous, skeletal muscle, smooth muscle, and

cardiac muscle. 21,22 AD-SCs also have the potential to

cessfully as a treatment modality in both veterinary

(equine) and human medicine.

High-densityplatelet-richplasma(HDPRP)isdefined

as plasma with platelet concentrations > 4 times the

levels found in circulating blood. It is isolated and

concentrated from a peripheral venous blood sample

by bidirectional centrifugation. 36

Various portable commercial centrifugation units

existforin-officeuse;however,thereareonlyafewthat

Platelet-richplasmahasbeenusedsuc

igieerivete

trlcell

Therehasbeensomevariationandquestion

regardingthecorrectterminologyforstem/stromal

adiposecells.

Atfirst,mesenchymalstemcells(discovered

tohaveanactiveroleinconnectivetissuerepair

intheearly1990s

)werethoughttobethemost

importantcontributortotissueregeneration;

however,itbecameevidentthatwithintheadipose

tissuecomplexisakeyextracellularmatrix,which

includedmatureadipocytesandadipocyticprecur-

sors(knownasprogenitorcells).Inaddition,there

wasavarietyofadditionalnucleated,undifferenti-

ated,multipotent,andpluripotentcells,includ-

ingpericytesandendothelialcells,whichareall

thoughttoplayimportantrolesinmesenchymal/

stromal-derivedtissueregeneration(f).

Thereforethetermadipose-derivedstem/stromalcells(AD-SCs),ratherthansimply“mesenchymal

stemcell,”isusedtodescribethispopulationofcells.

Figure 2. Adipose tissue with (A) adipose cells, (B) extracellu-

lar matrix (most stem cells there), (C) pericytes (surround ves-

sels; important in angiogenesis), (D) mesenchymal stem cells

(small cells), and (

) pre-adipocytes (progenitor cells)

Reference

didwThe Anterior Cruciate Ligament: Current and

FutureConceptsr

dncesinsemcellhery

In many human stem/stromal cell protocols and some

veterinarystemcellprotocols,HDPRPiscombined

with AD-SC for placement into specific injury sites.

TherearealsomanyexamplesofutilizationofHDPRP,

with and without stem/stromal cell additives, that

have proven effective in acute and chronic full-thick-

ness wound defects. Stimulation of the inflammatory

cascade, coupled with the addition of AD-SC, remark-

ably shortens the healing process by promoting tissue

regeneration (see

tcgf

Fibroblastgrowthfactor

Insulin-likegrowthfactor i

Interferons:Alpha,Gamma I-A,I-G

Interleukin1 IL-1

Platelet-derived

angiogenesisfactor

Figure 3

). 41

Platelet-derivedepidermal

growthfactor

reerc

Multiplestudies,bothinthehumanandveterinary

literature, have clearly demonstrated the ability of

AD-SCs to actively participate in tissue homeostasis,

regeneration, and open wound healing. 42-65 AD-SCs

also differentiate into and repair musculoskeletal con-

nective tissue, including:

•Ligament

•Tendon

•Cartilage

Platelet-derivedgrowth

factoraa,bb,ab

Plateletfactor4 PF-4

P-selectin GMP-140

Transforminggrowthfactor

B1,B2

TGF-B1,TGF-B2

are capable of consistently concen-

trating platelets to high-density

therapeutic levels, including the

HarvestTechnologiesSmartPReP2

centrifugation system (harvest-

tech.com) , which has been cleared

bytheFDA.

tic

atelets contain a significant num-

ber of growth factors, key signal

proteins, chemokines, cytokines,

and other proinflammatory bioac-

tive factors that initiate and reg-

ulate most basic aspects of the

inflammatory cascade, resulting in

natural wound healing (

). 37

•Elevatedplateletconcentra

Table

tions are known to stimu-

late proliferation and differ-

entiation, recruitment, and

migration of mesenchymal

and stromal repair cells to an

injury site. 38

•Circulatingplatelets,whenacti

vated, begin a degranulation

process that secretes a variety

of important growth factors

and cytokines/chemokines. 39

Figure 3. Use of high-density platelet-rich plasma in equine open wound defect: (A)

Pre-operative avulsion, 4-degree tissue loss, bone surface exposed, wound 8 days old;

(B) postoperative wound at 48 hours with 1 sharp debridement and thrombin-activated

HDPRP topical concentrate; (C) postoperative wound on day 8 with 2 debridements

plus second activated HDPRP topical concentrate; (

•

Activated platelets also secrete

stromal cell-derived factor 1

alpha(SDF-1a),whichsup

ports primary adhesion and

migration of mesenchymal

stem/stromal cells. 40

) split-thickness skin graft taken

from left chest, suture fixed, and meshed, with activated HDPRP under graft and plate-

let-poor plasma as fibrin gel on surface.

dncesinsemcellhery

•Disk

•Muscle

•Nervetissue

•Bone

•Hematopoietic-supportingstroma.

mesenchymal and stromal vascular stem-like cells

existinadiposeascomparedtobonemarrow.

For

further information on ongoing stem cell research,

go t o todaysveterinarypractice.com .

77-79

In vitro , AD-SCs have demonstrated pluripotent

capabilities to differentiate into a variety of nonme-

sodermally derived tissues, including hepatic, pan-

creatic, and keratocytic tissue, and to be effective in

skin anti-aging and tissue regeneration, cardiovas-

cular muscle and vascular tissue repair, rheumatoid

arthritis, diabetes, and other diseases. 66-75

Formanyyearsinhumanmedicine,studieshave

focused on bone marrow-derived mesenchymal

and hematopoietic undifferentiated cells. It is well

documented that bone marrow possesses very few

true mesenchymal stem cells, compared to AD-SCs,

which are becoming the primary tissue source in

clinicalapplications.Adiposeisacomplexcellu-

lar–vascular tissue that is not only easier to harvest,

but offers markedly higher nucleated, undifferenti-

ated stem cell counts than bone marrow. 76 Research

has shown as much as 500 to 1000 times as many

tecellrvetigiitrti

The ability of AD-SCs to support and serve as

a cell reservoir for connective tissue and joint

repair is the basic theory for their use in joint

regenerative medicine.

Autologous AD-SC therapy involves:

1. Harvestingfatfromtheavailablesites

2. Isolating the stem and regenerative cells

3. Administering the cells back to the patient.

In veterinary practice, these cells are typically

found in the adipose deposits near the tail (horses)

and from the ventral fat pad (dogs). In these cases,

fat is either lipoaspirated 80 or, if taken surgically, en

bloc, morselized to permit separation of the stem–

stromalelementswithintheadiposecomplex.After

preparation,thistissue,combinedwithHDPRP,can

then be injected during open surgical intervention

or by guided injection.

It is important to understand that undifferenti-

ated stem/stromal cells must adhere to other cells

(cell-to-cellcontact)ortoextracellularmatrix/peri-

vascular tissues in order to proliferate effectively.

Within an injured or degenerative site, the stem/

stromalcellfateiscontrolledbyacomplexsetof

physical (cell-to-cell) and chemical signals (para-

crine-autocrine effects) dictated by the cellular and

chemical microenvironment (niche). 81

Further,thesecellsarebelievedtoproliferate

and differentiate from signaling based on the envi-

ronment in which they are placed. Therefore, if

AD-SCs are placed within and adherent to damaged

connective tissue, uncommitted progenitor and

stem/stromal elements within the AD-SC graft are

activated and differentiate toward the specific con-

nective tissue lineages for growth and repair.

teclgfrtecelll

ccetrtervetig

Currently,2companiesprovidestemcelltechnologyfor

veterinarymedicine.v (vet-stem.com), thefirst

companytocommerciallyofferstemcelluse,providesa

practical“asneeded”approach:Veterinarianscanremove

fatfromananimalandshipitovernighttotheVet-Stem

laboratory.There,thefatsampleisprocessedbychemical

digestion,isolation,andconcentration,thenreturnedtothe

veterinarianforinjectionintotheanimal’sinjurysite.

v (medivet-america.com) offersthe

abilitytodostemcellproceduresatpointofcare,within

clinicalsurgicalfacilities.Bothcompanieshavereported

favorableresultsintheirtreatmentprotocolsandreported

casestudies. 1,2

ccli

Stem cell therapy in regenerative veterinary medi-

cine is a viable option for the equine as well as the

small animal veterinarian, offering a safe and clin-

ically effective tool for the clinician to assist in his/

her treatment of the animal with difficult wounds

or unresolved musculoskeletal or joint pain. n

Variousportablecommercialcentrifugationunitsexistthat

processbloodsamples,resultinginplatelet-richplasma

concentrates.Theoneusedforourinvestigationwasthe

patentedt (harvesttech.com) Smart

PReP2centrifugationsystem:Thissystemusesasterilized

bloodcollectionkitthatallowsin-officephlebotomyand

processinginatabletopbidirectionalcentrifugationunit. 3

Itiscapableofconsistentlyconcentrating4to5times,or

more,thepatient’scirculatinglevelofplatelets,achieving

theneededtherapeuticlevelofHDPRP.

AD-SC=adipose-derivedstem/stromalcell;

HDPRP=high-densityplatelet-richplasma

TodaysVeterinaryPractice.com

References

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: