cell therapy sciences · “caregiver placebo effect for dogs with lameness from...
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Effect of a Dog’s Age on Stem Cell Culture Times and Efficacy Outcomes in Dogs with Osteoarthritis Treated with Intra-articular Injections of Autologous Mesenchymal Stem Cells.
REFERENCES:1. Conzemius,MichaelG.,andRichardB.Evans.“Caregiverplaceboeffectfordogswithlamenessfromosteoarthritis.”JournaloftheAmericanVeterinaryMedicalAssociation241.10(2012):1314-1319.2. Guercio,Annalisa,etal.“Productionofcaninemesenchymalstemcellsfromadiposetissueandtheirapplicationindogswithchronicosteoarthritisofthehumeroradialjoints.”Cellbiologyinternational36.2(2012):189-194.3. Lee,Jienny,etal.“Effectofdonorageontheproliferationandmultipotencyofcanineadipose-derivedmesenchymalstemcells.”Journalofveterinaryscience18.2(2017):141-148.4. Perry,K.www.vettimes.co.uk/article/using-cellular-therapies-for-canine-joint-treatment-part-1/5. Harmanetal.“AProspective,Randomized,Masked,andPlacebo-ControlledEfficacyStudyofIntraarticularAllogeneicAdiposeStemCellsfortheTreatmentofOsteoarthritisinDogs.”FrontVetSci.3(2016):816. Kuahetal.“Safety,tolerabilityandefficacyofintra-articularProgenzainkneeosteoarthritis:arandomizeddouble-blindplacebo-controlledsingleascendingdosestudy.“JTranslMed16(2018):49 AFFILIATIONS AND CONFLICT OF INTEREST: AuthorsworkinResearchandDevelopmentatCTSLACKNOWLEDGEMENTS: ToallofthesmallanimalvetclinicsintheUKwhohavekindlysentussamplesandcasesforthisstudy.
OBJECTIVES:
To evaluate whether a dog’s age influences either the growth rate of adipose-derived Mesenchymal Stem Cells (MSCs) during culture-expansion, or clinical efficacy outcomes following treatment of the dog’s osteoarthritis using intra-articular injections of autologous MSCs.
cell therapy sciencesDeveloping Regenerative Medicine for Vets
CLINICAL DATA:
RESULTS:
INTRODUCTION: MATERIALS AND METHODS:
Osteoarthritis isa leadingcauseofpainand lameness indogs.Regenerativemedicineand inparticularMSCtreatmentsareincreasinglybeingusedtotreatOAbecausenotonlydotheyalleviatepain,theyalsodecreaseinflammation,improvestructuraldamageandenhancetissuerepair.MSCtherapyprovidesanimportantoptionwhenconventionaltherapiesdonotprovideadequatecontrolofarthriticpainorwherethedogcannottoleratehighlevelsofpainrelievingmedicationandincaseswherethereisnosuitable,provensurgicaloption(forexampleinelbowdisease)or todelay theneed forsalvagesurgeries (Perry2016).MSCculture-expansionselects forhighnumbersofviable,adhesiveMSCsandremovesresidualconnectivetissuedebris,thusprovidingaquality-controlled,standardised,viability-testedstemcellinjectiontoensurethehighestlevelofsafetyandefficacyintheclinic.TherearesomereportsparticularlyinhumanmedicinethatMSCsextractedfromolderpeoplearelessabletoproliferateandarelessefficaciousthanthosefromyoungerpeople.CellTherapySciencesisinauniquepositiontobeabletostudytheculturecharacteristicsofMSCsfromawiderangeofdogsofdifferentagesandtofollowthisupwithacaseseriesofdogstreatedwiththeseMSCs.
Dogs of various ages, each with a documented history of moderate to severe OA inadequately controlled onconventionaltherapy,underwentadiposetissueharvestforMSCextractionandculture-expansion.MSCswereseparatedfromtheadiposetissueusinganoptimised,standardprotocol(Guercio2012)andtheadherentcellsfromtheSVFwereculture-expandeduntil90%confluent.Theculturedcellswereharvestedtopreparequalitycontrolled,autologousstemcellinjectionsforeachdog,whichwerecheckedforstemcellnumbers,morphology,point-ofcareviability(>90%)andconfirmedaspathogen-free.Cell-cultureswerephotographeddailyusingthesameopticalfield,cellswerecountedandpopulationdoubling-timecalculated.CellTherapySciencesuseonlyearlypassagestemcells(P0-P2)toprepareinjectionsforclinicaluseandthereforeP0-P2cellsweretestedinthisstudy.Dogsweremonitoredforupto6monthsfollowingintra-articularMSCinjectionsintotheiraffectedjoints.Aglobalscore,basedonclinicalexaminationsandowners’feedbackonpainandmobility,wasusedbythetreatingveterinariantoevaluatetreatmentresponse.Resultswereanalysedusingatwotailedstudentt-testforthepopulationdoublingtimecomparisonandaMann-WhitneyU-testfortheclinicaloutcomedata.
Authors: Joanna Miller1, Christine Standen1, Lucy Frost2 1CellTherapySciencesLtd,Coventry,UnitedKingdom.2UniversityofWarwick,Warwick,UnitedKingdom.
•Thisstudyprovidescompellingevidence from52dogsthatcanineadipose-derivedMSCscanbesuccessfullyandrapidlyexpandedincultureatearlypassagesregardlessoftheageofthedonordog.
•Thereisaccumulatingpublishedevidence,includingrecentdoubleblind,randomisedplacebo-controlled studies in OA ( Harman et al, 2016; Kuah et al 2018) that intra-articular injectionscontaininghighnumbersofMSCscanbringaboutsignificantimprovementsinclinicalsignsandsymptomssuchaspainandoverallmobility.
•Alltreateddogsinthecellculturestudywerefollowedupasacaseseriestodetermineresponsetotherapyintheopinionofthetreatingvet,basedonclinicalassessmentsandownerfeedback.Thisdataindicatesthat98%ofdogswithmoderatetosevereOAshowedclinicalimprovement,with60%demonstratinganexcellentimprovement.
•Thiswasacaseserieswithnoblindedcontrolgroupandwillbesubjectto“care-giverbias”;whilststudieshavereportedthatcare-giverplaceboeffectscanbeashighas40%incanineOAstudies(Conzemius2012)thisalonecannotexplainthe98%clinicalimprovementreportedhere.
•Efficacyresultsintheolderdogssuggestedthatwhilsttheoverallresponseratewashigh(94%),feweroftheolderdogsachievedanexcellentimprovement.Thisispossiblyduetotheoveralldisabilityleveloftheolderdogsandtheneedforcomplimentaryrehabilitationtherapy.Additionally,treatmentofonlyoneortwooftheworstaffectedjointsmayhighlightproblemsinotherjointsorsofttissues.
•AtCellTherapySciencesweonlypreparestemcelltherapiesculturedatearlypassage(P0-P2)andthereforetheseweretestedinthisstudy.Ithasbeenreportedinaprevioussmallstudy(5dogspergroup)thatdonoragecouldhaveanaffectoncumulativepopulationdoublingsatlaterpassages(Leeetal2017).”
• AgeofthedonordoesnotadverselyaffecttheabilityofMSCstogrownormallyinculture.
• Followingintra-articularinjectionsofMSCs,98%ofdogswithmoderatetosevereOAshowedaclinicalimprovement.
• Eveninolderdogs(10-15years)94%showedclinicalimprovements,withdogs<10yearsmostlikelytodemonstrateanexcellentresponse.
ABBREVIATIONS • OA: Osteoarthritis• MSC: MesenchymalStemCell• SVF: StromalVascularFraction
• FCS: FoetalCalfSerum• PDT: PopulationDoublingTime
ABSTRACT: OBJECTIVES:Toevaluatewhetheradog’sageinfluenceseitherthegrowthrateofautologousadipose-derivedMesenchymalStemCells(adMSCs)duringculture-expansion,orclinicalefficacyoutcomesfollowingtreatmentwithintra-articular(IA)adMSCinjections. METHODS:Dogsofvariousages,eachwithadocumentedhistoryofmoderate-severeOAinadequatelycontrolledonconventionaltherapy,underwentadiposetissueharvestforstemcellextractionandculture-expansion.Cell-cultureswerephotographeddailyusingthesameopticalfield,cellscountedandpopulationdoubling-timecalculated.Dogsweremonitoredforupto6monthsfollowingIAadMSCinjectionsintoallaffectedjoints.AGlobalScore,basedonclinicalexaminationsandowners’feedbackonpainandmobility,wasusedtoevaluatetreatmentresponse.RESULTS:Fifty-twodogswereincludedinthisanalysis.Nosignificantaffectofdog’sagewasseenonpopulationdoublingtimeoflowpassage(P0&P1)culturesofcanineadMSCs.Themajorityofdogs(98%)demonstratedaclinicalimprovement(51/52cases)followingadMSCinjections,with60%evaluatedasdemonstratinganexcellentresponse(31/52cases).Intheoldestdogs(10-15years)94%showedaclinicalimprovement,with6/18(33%)demonstratinganexcellentresponse.Inyoungerdogs(<10years)agreaternumbershowedanexcellentresponse(25/34;74%). STATEMENT:AgeofthedonordoesnotadverselyaffecttheabilityofadMSCstogrownormallyinculture.Followingintra-articularinjectionsofadMSCs,98%ofdogswithmoderatetosevereOAshowedaclinicalimprovement.Eveninolderdogs(10-15years)94%showedclinicalimprovements,withdogs<10yearsmostlikelytodemonstrateanexcellentresponse.
DISCUSSION: CONCLUSION:
FIG 3 : CLINICAL EFFICACY OF MSC TREATMENT IN DOGS WITH OA FIG 4 : DOES AGE INFLUENCE THE CLINICAL EFFICACY OF MSC TREATMENT IN DOGS WITH OA ?
Veterinary Global Efficacy Score for dogs with OA treated with intra-articular culture expanded MSC injections (n=52). 60% achieved an excellent clinical improvement (31/52 dogs), 21% achieved a moderate clinical improvement (11/52 dogs), 17% achieved a mild clinical improvement (9/52 dogs) and only 2% had no clinical improvement (1/52 dogs).
Veterinary Global Efficacy Scores for dogs with OA treated with culture-expanded MSC therapy; responses presented according to the age of the dog. In the younger age group (< 10 years) , 100% of dogs were assessed as achieving a clinical improvement; 74% of these achieved an excellent clinical improvement (25/34 dogs), 21% achieved a moderate clinical improvement (7/34 dogs), 6% achieved a mild improvement (2/34 dogs). In the older age group (≥ 10 years) 94% were assessed as achieving a clinical improvement; 33% of these achieved an excellent clinical improvement (6/18 dogs), 22% achieved a moderate clinical improvement (2/34 dogs), 39% achieved a mild clinical improvement (7/18 dog) and 6% did not improve (1/18 dogs).
Clin
ical
Res
pons
e: %
of d
ogs
trea
ted
2%
17%
21%
60%
6%0% 6%
39%
22%21%
33%
74%<10 years (n=34) ≥10 years (n=18)
FIG 2 : MSC PDTs FOR YOUNG DOGS COMPARED WITH OLDER DOGS
Populationdoublingtimes(hours)foradMSCsculturedfromadiposesamplestakenfrom52dogsofdifferentages(range:1yearto15years).Thecultureswereearlypassage (P0-P2). No significant difference was found between the PDTs for youngdogscomparedwithdogsof10yearsandolder(Mean18.74+/-6.20and17.93+/-6.89respectively).
< 10 years (n= 34)
p = 0.665
5
00
10
15
20
25
30
35
40
≥10 years (n= 18)
Popu
latio
n D
oubl
ing
Tim
e ( H
ours
)
Photomicrographs of canine MSCs in culture taken using the same field of view on sequential days. Bar = 0.1mm.
FIG 1 : ESTIMATION OF MSC POPULATION DOUBLING TIMES (PDTS)
Day 2 Day 5 Day 6 Day 7 Day 8 Day 9
Table1.Thisdescribestherelationshipbetweenthenumberofcellscountedperfieldand theestimated totalnumberofcellson thecultureflask.Thebluefigureisthefinalharvestcellcountusingahaemocytometer.Populationdoublingtimeiscalculatedaccordingtotheformula:PDT=T(Log2/Log(Cf/Cs))whereTisthetimeinhoursbetweencellcounts,Cfisthefinalcellcount,Csistheinitialcellcount.
Growth rate of a typical case of canine MSCs
Population Doubling Time (hrs): 27.91
Days in culture
No cells in photo field
No MSCs (million)
2 6 0.695 34 3.936 48 5.557 99 11.448 216 24.969 389 44.950
A Typical Canine MSC Growth Curve
No of days in culture
Estim
ated
no.
of M
SCs
pres
ent e
ach
day
50
0 102 4 6 8
1015
2025
30354045
50
Clin
ical
Res
pons
e: %
of d
ogs
trea
ted