Cellule staminali nel trattamento delle lesioni traumatiche e degenerative della cartilagine

Andrea Facchini

“Le cellule staminali e il loro impiego attuale nel la clinica”“Le cellule staminali e il loro impiego attuale nel la clinica”UniSaluteUniSalute, Bologna, 30 Settembre 2011, Bologna, 30 Settembre 2011

Andrea Facchini

Laboratorio di Immunoreumatologia e rigenerazione tisssutale

Istituto ortopedico Rizzoli, Università degli Studi di Bologna

Autologous chondrocyte transplantation

Cartilage sample

Brittberg M et al. N Engl J Med, 331:889-95, 1994

Chondrocyte suspension

Chondrocytes p=0 Chondrocytes p=3/4/5/6…………..

De-differentiation process

Expression of Collagen II Expression of collagen IIProteoglycans (aggrecan) Expression of proteoglycans (versican)Expression of Collagen I Expression of Collagen IFGF R3 Activin receptor-like kinase 1BMP-2

IOR

Grigolo et al. Biomaterials 2002

Type I Collagen

Fol

d ch

ange

s in

gen

e ex

pres

sion

1,0

1,5

2,0

Mean+/-SD

Mean

Type II Collagen

Fol

d ch

ange

s in

gen

e ex

pres

sion

12000

16000Mean+/-SD

Mean

Fol

d ch

ange

s in

gen

e ex

pres

sion

0,0

0,5

1,0

1 Day 3 Days 7 Days 14 Days 21 DaysF

old

chan

ges

in g

ene

expr

essi

on0

4000

8000

1 Day 3 Days 7 Days 14 Days 21 Days

Grigolo B. et al. Biomaterials,23; 2002

Clinical application of a biocompatible scaffold(Hyalograft C) for cartilage defects of the knee

HYALOGRAFT C TRANSPLANT THROUGH MINI-ARTHROTOMY

HYALOGRAFT C TRANSPLANT THROUGH MINI-ARTHROTOMY

DEVELOPMENT OF ARTHROSCOPIC IMPLANT

TECHNIQUE

DEVELOPMENT OF ARTHROSCOPIC IMPLANT

TECHNIQUE

Marcacci M et al. Knee Surg Sport Art, 2002, 2007

LESS INVASIVE NEW TECHNIQUE FOR CARTILAGE REGENERATION

LESS INVASIVE NEW TECHNIQUE FOR CARTILAGE REGENERATION

Marcacci M et al. Knee Surg Sport Art, 2002; 2007

Marcacci M et al. Knee Surg Sport Art, 2002,2007

LESS INVASIVE NEW TECHNIQUE FOR CARTILAGE REGENERATION

LESS INVASIVE NEW TECHNIQUE FOR CARTILAGE REGENERATION

Marcacci M et al. Knee Surg Sport Art, 2002; 2007

LESS INVASIVE NEW TECHNIQUE FOR CARTILAGE REGENERATION

LESS INVASIVE NEW TECHNIQUE FOR CARTILAGE REGENERATION

Marcacci M et al. Knee Surg Sport Art, 2002; 2007

60

80

100

IKDC:

mean 39.4±±±±14.0

79.3±±±±20.8

VALUTAZIONE SOGGETTIVA IKDCVALUTAZIONE SOGGETTIVA IKDC

FOLLOW UP MEDIO: 47.2 ±±±± 11 mesi FOLLOW UP MEDIO: 47.2 ±±±± 11 mesi

175 PAZIENTI175 PAZIENTI

STUDIO MULTICENTRICO ITALIANOSTUDIO MULTICENTRICO ITALIANO

0

20

40

60

Basal Follow-up

IKDC:

mean

scor

e

p<0.0001

39.4±±±±14.0

166 pazienti migliorati: 87%166 pazienti migliorati: 87%

Marcacci M et al. “Articular Cartilage Engineering with Hyalograft® C: 3-year Clinical Results” June 2005Marcacci M et al. “Articular Cartilage Engineering with Hyalograft® C: 3-year Clinical Results” June 2005

35,9

79,6

43,0

78,3

37,6

78,1

50

60

70

80

90

100IK

DC: pu

ntegg

io m

edio

Basal

Follow-up

IKDC patient evaluation (n=137): Basal vs follow-up related to lesion size

35,9 37,6

0

10

20

30

40

50

IKDC: pu

ntegg

io m

edio

<2 cm2 2-4 cm2 >=4 cm2

n=42 n=61 n=34

Improved patients: 92,9% 90,2% 91,2%

Marcacci M .et al , CORR, 435, 96-105, 2005

Basal

17.2 ± 4.4 months 72.7 ± 20.2

p<0.0

001

36.6 ± 12.8

p<0.0

001

Mean IKDC score vs time (n=109)

17.2 ± 4.4 months

40.3 ± 8 months

Marcacci M.et al , CORR 435,96-105 2005

72.7 ± 20.2

78.1 ± 22.0

p<0.0

001

p< 0

.05

Mean IKDC score

p<0.0

001

Subjective evaluation IKDC: basal vs follow-up related to osteoarthritis presence (classif.

Ahlback)Improved

patients:

95.0

90.9%

No osteoarthritis

Ahlback I-II

n=100

90.9% Ahlback I-II

Ahlback III-IV

n=22

n=1668.8%

IKDC mean score

Graft stability Graft stability -- MRI resultsMRI results

Cartilage maturation: columnar re-organization

3) Columnar Organization

Cartilage

Normal Cartilage II° look Biopsy

Integration with subchondral bone

Tidemark

Bone

MICROFRATTURE vs HYALOGRAFT CMICROFRATTURE vs HYALOGRAFT C

STUDIO COMPARATIVOSTUDIO COMPARATIVO

40 PAZIENTI VS 40 PAZIENTI40 PAZIENTI VS 40 PAZIENTI

M.MARCACCIE. KONS.ZAFFAGNINIG. FILARDOM. DELCOGLIANO

M.MARCACCIE. KONS.ZAFFAGNINIG. FILARDOM. DELCOGLIANO

A. GOBBIA. GOBBI

Kon E, Gobbi A, Filardo G, Delcogliano M, Zaffagnini S and Marcacci MArthroscopic Second Generation Autologous Chondrocyte Implantation Compared with Microfracture treatment for chondral lesions of the Knee. Am J Sport Med, 2009

Kon E, Gobbi A, Filardo G, Delcogliano M, Zaffagnini S and Marcacci MArthroscopic Second Generation Autologous Chondrocyte Implantation Compared with Microfracture treatment for chondral lesions of the Knee. Am J Sport Med, 2009

MICROFRATTURE vs HYALOGRAFT CMICROFRATTURE vs HYALOGRAFT C

VALUTAZIONE SOGGETTIVA IKDC a 5 ANNIVALUTAZIONE SOGGETTIVA IKDC a 5 ANNI

p=0,0003

Cellule per medicina rigenerativa Cellule per medicina rigenerativa osteoarticolareosteoarticolare

CelluleCellule

•• CondrocitiCondrociti

VariabiliVariabili

•• AutologheAutologhe•• CondrocitiCondrociti

•• Cellule Mesenchimali Cellule Mesenchimali Staminali/Staminali/StromaliStromali(MSC)(MSC)

•• AutologheAutologhe

•• AllogenicheAllogeniche

•• Espanse in vitro Espanse in vitro

•• Non espanse in vitroNon espanse in vitro

Tendogenesis/Ligamentogenesis

Marrow Stroma

TransitoryFibroblast

TransitoryStromal Cell

Osteogenesis Chondrogenesis Myogenesis

MSC Proliferation

TransitoryOsteoblast

TransitoryChondrocyte Myoblast

Mesenchymal Stem Cell (MSC)

Other

Bon

e M

arr

ow/P

erios

teum

Proliferation

Commitment

Lineage

THE MESENGENIC PROCESSTHE MESENGENIC PROCESSGrowth FactorsImmunosuppressive

23

Myoblast Fusion

UniqueMicro-niche

ChondrocyteOsteoblast

Mese

nchymal Tissu

e

LineageProgression

Differentiation

Maturation

OsteocyteHypertrophicChondrocyte

MyotubeStromalCells

T/LFibroblast

Adipocytes,Dermal andOther Cells

BONE CARTILAGE MUSCLE MARROWTENDON/LIGAMENT

CONNECTIVETISSUE

Main Main sourcessources forfor human MSC, e.g.:human MSC, e.g.:Bone Marrow Umbilical Cord Blood/

Amnios/Placenta

Adipose Tissue

Pittenger M, et al.Science 1999

++

Best characterised(Pre-) Clinically used

--

Invasive procurementAge related detriments

Erices AA et al.Brit. J. Haematol. 2000

++

Youngest Adult Stem CellsEasily Accesible

--

MSC present in term CB?Low frequency

Fresh UCB units needed

Zuk P, et al.Tissue Eng. 2001

++

High MSC Frequency(Pre-) Clinically used

--

Invasive procurementAge related detriments ?

From: K.Bieback ,2007

HH--MSC s MSC s isolationisolation and culture and culture expansionexpansion

Bone marrowaspirate Plate cells at

interface Colony formation

Centrifugation

1.073g/ml Percoll

Primary Culture

hMSCshMSCs markersmarkers

•• ThereThere isis notnot a single a single markermarker forfor theirtheir identificationidentification

•• TheyThey are are generallygenerally positive positive forfor: : CD29 CD29 ((fibronectinfibronectin R), R), CD44 CD44 ((hyaluronichyaluronic acid R), acid R), CD90 CD90 (Thy(Thy--1,HSC) 1,HSC) CD105/SH2CD105/SH2CD44 CD44 ((hyaluronichyaluronic acid R), acid R), CD90 CD90 (Thy(Thy--1,HSC) 1,HSC) CD105/SH2CD105/SH2((endoglinendoglin ,TGF,TGFββ11--3R), 3R), CD73/SH3CD73/SH3; ; CD147 CD147 ((neurotelinneurotelin), ), CD166CD166, , CD271CD271 (NGFR) and (NGFR) and STROSTRO--11..

•• TheyThey are are generallygenerally negative negative forfor: : CD14 CD14 (M(MØ)Ø), , CD31CD31((endotelialsendotelials cellscells), ), CD34 CD34 (HSC), (HSC), CD45 CD45 (CLA), (CLA), CD117CD117(SCFR). (SCFR).

Geni modulati nel differenziamento di MSCGeni modulati nel differenziamento di MSC

MSCsChondrocytes Osteoblasts

Collagene tipo I

Collagene tipo II

Fosfatasi Alcalina

Collagene tipo ICollagene tipo II

SOX-9

Collagene tipo I

Osteocalcina

Aggrecano Bonesialoproteina

CBFA-1Collagen type IX

CDCD 4545--FACS FACS AnalysisAnalysis

CDCD 3434--/ CD45/ CD45-- //CDCD 105+ 105+

CELL PHENOTYPCELL PHENOTYPE AND DIFFERENTIATIONE AND DIFFERENTIATION

In vitro In vitro differentiationdifferentiation potentialpotential

ChondrogenicChondrogenicOsteogenicOsteogenic

IOR

IOR

IOR

IOR

2) 2) MarrowMarrowconcentrationconcentration

1) 1) PlateletPlatelet gel production (gel production (growthgrowth factorsfactors))

“One step” procedure“One step” procedureBone marrow concentrated stem cells + scaffold (Bone marrow concentrated stem cells + scaffold (HyaffHyaff

11) 11)

29

2) 2) MarrowMarrowconcentrationconcentration

((dedicateddedicated centifugecentifuge))

3) 3) ConcentratedConcentrated BM BM StemStem cellscells loadedloaded on on biomaterialbiomaterial

4) 4) ArthroscopicArthroscopic implantimplant

IOR

IOR

IOR

IOR ACI ACI vsvs “One step” procedure“One step” procedure

Similar trend of improvementsSimilar trend of improvements

93 93 patientspatients withwith 6 6 monthsmonths minimum FU (minimum FU (allall casescases 112)112)

IOR

IOR

IOR

IOR

MultiechoMultiecho sequence to evaluate the presence of water in sequence to evaluate the presence of water in cartilage according to cartilage according to colourcolour scalescale

normalnormalcartilagecartilage

(3D SPGR)(3D SPGR)

normalnormalcartilagecartilage

(3D SPGR)(3D SPGR)

FU evaluation in “One step” FU evaluation in “One step” MRI T2 mappingMRI T2 mapping

ONEONE--STEP, STEP, 18 18 MonthsMonths FUFU

T2T2--mapping: mapping: 45 45 msecmsec

meanmean

T2T2--mapping: mapping: 45 45 msecmsec

meanmean

HighT2 (45 HighT2 (45 msecmsec) ) areasareas

hylinehylinecartilagecartilage

expressionexpression

Safranin O staining

HistologyHistology

Safranin O staining

Hyaline like cartilage in Hyaline like cartilage in remodellingremodellingHigh High proteoglycanproteoglycan expression (red) expression (red)

Low evidence of Col ILow evidence of Col IGood Good osteochondralosteochondral integrationintegration

RISULTATIRISULTATI

TIBIOTARSICA GINOCCHIO

EXPERIMENTAL ANIMAL MODEL OF OSTEOARTHRITIS

ANTERIOR CRUCIATE LIGAMENT

New Zealand male adult rabbits12 months old

ANTERIOR CRUCIATE LIGAMENTTRANSECTION (ACLT)

3 months 6 months 8 weeks

SHAM

8 weeks

EXPERIMENTAL MODEL

Implant

Bilateral ACLT

8 weeks

Bone marrow aspiration

8 weeksCell Seeding Expansion

Animal sacrifices

Right KneeHA

Left Knee2 x106 MSCs-HA

3 Months

6 Months

Right KneeHA

Left Knee2x106 MSCs-HA

Animal sacrifices

Cell Seeding Expansion

Hyaff®-11 seeding

MSCs isolation

CFU Primaryculture

TGFβ-1

Implant

HISTOLOGICAL ANALYSIS : SAFRANIN-0

MEDIAL CONDYLE LATERAL CONDYLE

40 X 40 X

8 weeks

40 X

Sham operation group

ACLT (OA) group

Safranin -O

40 X 40 X 40 X

40 X 40 X

8 weeks 3 months 6 months

100 X

MSCs-HA treated group

HA treated group

40 X

40 X

40 X 40 X

40 X

40 X

40 X

100 X

100 X

40 X

40 X

Sham operation group

ACLT (OA group)

Collagen II

40 X 40 X40 X

40 X40 X 40 X

8 weeks 3 months 6 months

40 X

MSCs-HA treated group

HA treated group

40 X

40 X

40 X40 X

40 X

40 X

40 X

100 X

Sham operation group

ACLT (OA group)

Collagen I

40 X 40 X

40 X40 X

40 X

40 X

8 weeks 3 months 6 months

MSCs-HA treated group

HA treated group

40 X

40 X

40 X40 X

40 X

40 X

40 X

100 X

Sham operation group

MMP-3

40 X 40 X 40 X

40 X 40 X 40 X

8 weeks 3 months 6 months

ACLT (OA group)

MSCs-HA treated group

HA treated group

40 X 40 X

40 X 40 X

40 X 40 X

40 X

100 X

8 Weeks

3 Months

SHAM

ACLT

HAACLT

380 ± 32

312 ± 16

301 ± 25

340 ± 11

Cartilage Thickness ( µm)

6 Months

HA

MSCs - HA

HA

ACLT

MSCs - HA

324 ± 25

280 ± 13

471 ± 25

301 ± 17

P< 0,0005 ACLT versus MSCs-HA P < 0,0005 HA versus MSCs-HA

325 ± 34

389 ± 49

403 ± 65362 ± 32

8 WeeksSHAM

ACLT

3 Months HAACLT

Subchondral Bone Thickness ( µm)

MSCs-HA 324 ± 30

504 ± 26

349 ± 20

551 ± 486 Months HA

ACLT

P< 0,0005 ACLT versus MSCs-HA P < 0,0005 HA versus MSCs-HA

MSCs-HA

MSCs-HA

ConclusionsConclusions

•• The deliveriy of MSCs on a hyaluronan (Hyaff The deliveriy of MSCs on a hyaluronan (Hyaff 11) scaffold can positively interfere with OA 11) scaffold can positively interfere with OA progression progression

Open questionOpen question

•• Is the repair activity induced by MSCs + HA Is the repair activity induced by MSCs + HA due to MSC differentiation to chondrocytes due to MSC differentiation to chondrocytes in situ or growth factor release (or both) ?in situ or growth factor release (or both) ?

Cellule mesenchimali da tessuto adiposo (ASC)

1. Nel tessuto adiposo la concentrazione di ASC è circa 500 volte superiore a quello delle BMSC nel midollo osseo

44

volte superiore a quello delle BMSC nel midollo osseo

2. Le ASC hanno un potenziale di espansione di circa 7 volte superiore alle BMSC.

3. Le ASC hanno le stesse potenzialità differenziative, anti-fibrotiche, anti-apoptotiche e anti- infiammatorie delle BMSC.

ADIPOAADIPOAADIPOAADIPOA

Fragmentation Enzymatic treatmentAdipose

tissue

ASC isolation

CentrifugationPellet seeding

tissue

ASC

Miocytes Adipocytes Osteocytes Chondroytes

Fenotipo e potenzialità differenziative delle ASC

AnalisiAnalisi inin citometriacitometria aa flussoflusso didiASCASC evidenziaevidenzia cheche sonosono positivepositiveaa CDCD105105,, CDCD7373,, CDCD2929,, CDCD4444,,CDCD9090 ee negativenegative aa CDCD106106,, CDCD4545,,CDCD1414,, CDCD3434,, andand CDCD3131

Adipose derived stromal cells forosteoarthritis treatment

(ADIPOA)

Collaborative Project Health-2009-1.4-3

Activation of endogenous cells as an approach to regenerative medicine

2 Months follow-up: Safranin-O staining

500 µmc100 µm

CONTROL

500 µm100 µm

100 µm500 µmc

2x106 ASC

6x106 ASC

AcknowledgementsLaboratory of Immunology and GeneticsDr.ssa Brunella Grigolo Dr.ssa Gina Lisignoli Dr. ssa Giovanna DesandoDr.ssa Carola Cavallo

Laboratory of Experimental SurgeryProf. Roberto GiardinoProf. Roberto GiardinoDr.ssa Milena Fini Dr. Gianluca GiavaresiDr.ssa Matilde Tschon

Orthopaedic DivisionsProf. Sandro GianniniDr. Roberto BudaProf. Maurilio MarcacciDr.E.Kon

Grants from:

Grazie per l’attenzione

Istituto Ortopedico Rizzoli (Biblioteca))