09_FACCHINI [modalità compatibilità]

Cellule staminali nel trattamento delle lesioni
traumatiche e degenerative della cartilagine
Andrea Facchini
Laboratorio di Immunoreumatologia e rigenerazione tisssutale
Istituto ortopedico Rizzoli, Università degli Studi di Bologna
“Le cellule staminali e il loro impiego attuale nella clinica”
UniSalute,, Bologna, 30 Settembre 2011
UniSalute
Autologous chondrocyte transplantation
Cartilage sample
Chondrocyte suspension
Brittberg M et al. N Engl J Med, 331:889-95, 1994
Chondrocytes p=0
Expression of Collagen II
Proteoglycans (aggrecan)
Expression of Collagen I
FGF R3
BMP-2
Chondrocytes p=3/4/5/6…………..
Expression of collagen II
Expression of proteoglycans (versican)
Expression of Collagen I
Activin receptor-like kinase 1
De-differentiation process
IOR
Grigolo et al. Biomaterials 2002
Type I Collagen
Type II Collagen
2,0
Mean
1,5
1,0
0,5
0,0
1 Day
3 Days
7 Days
14 Days
21 Days
Grigolo B. et al. Biomaterials,23; 2002
Mean+/-SD
16000
Fold changes in gene expression
Fold changes in gene expression
Mean+/-SD
Mean
12000
8000
4000
0
1 Day
3 Days
7 Days
14 Days
21 Days
Clinical application of a biocompatible scaffold
(Hyalograft C) for cartilage defects of the knee
HYALOGRAFT C
TRANSPLANT THROUGH
MINI-ARTHROTOMY
DEVELOPMENT OF
ARTHROSCOPIC IMPLANT
TECHNIQUE
Marcacci M et al. Knee Surg Sport Art, 2002, 2007
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
Marcacci M et al. Knee Surg Sport Art, 2002; 2007
LESS INVASIVE NEW TECHNIQUE FOR
CARTILAGE REGENERATION
Marcacci M et al. Knee Surg Sport Art, 2002; 2007
STUDIO MULTICENTRICO ITALIANO
VALUTAZIONE SOGGETTIVA IKDC
FOLLOW UP MEDIO:
100
79.3±
±20.8
47.2
± 11
mesi
175 PAZIENTI
80
IKDC: mean
score
60
39.4±
±14.0
40
166 pazienti migliorati: 87%
20
p<0.0001
0
Basal
Follow-up
Marcacci M et al. “Articular Cartilage Engineering with Hyalograft® C: 3-year
Clinical Results” June 2005
IKDC patient evaluation (n=137):
Basal vs follow-up related to lesion size
Basal
IKDC: punteggio medio
100
90
79,6
78,1
78,3
80
Follow-up
70
60
50
43,0
35,9
37,6
40
30
20
10
0
<2 cm2
n=42
Improved patients:
92,9%
2-4 cm2
n=61
>=4 cm2
n=34
90,2%
91,2%
Marcacci M .et al , CORR, 435, 96-105, 2005
Mean IKDC score vs time (n=109)
36.6 ± 12.8
72.7 ± 20.2
17.2 ± 4.4 months
Mean IKDC score
Marcacci M.et al , CORR 435,96-105 2005
p< 0.05
78.1 ± 22.0
40.3 ± 8 months
p<0.0001
p<0.0001
Basal
Subjective evaluation IKDC:
basal vs follow-up related to osteoarthritis presence (classif.
Ahlback)
Improved
patients:
95.0
No osteoarthritis
n=100
90.9%
Ahlback I-II
n=22
68.8%
Ahlback III-IV
n=16
IKDC mean score
Graft stability - MRI results
Cartilage maturation: columnar re-organization
3) Columnar Organization
Integration with subchondral bone
Normal Cartilage
II° look Biopsy
Cartilage
Tidemark
Bone
STUDIO COMPARATIVO
M.MARCACCI
E. KON
S.ZAFFAGNINI
G. FILARDO
M. DELCOGLIANO
MICROFRATTURE
40 PAZIENTI
VS
A. GOBBI
vs HYALOGRAFT C
40 PAZIENTI
Kon E, Gobbi A, Filardo G, Delcogliano M, Zaffagnini S and Marcacci M
Arthroscopic Second Generation Autologous Chondrocyte Implantation Compared with Microfracture
treatment for chondral lesions of the Knee.
Am J Sport Med, 2009
VALUTAZIONE SOGGETTIVA IKDC a 5 ANNI
MICROFRATTURE vs
p=0,0003
HYALOGRAFT C
Cellule per medicina rigenerativa
osteoarticolare
Cellule
•
•
Condrociti
Cellule Mesenchimali
Staminali/Stromali
Staminali/
Stromali
(MSC)
Variabili
•
•
•
•
Autologhe
Allogeniche
Espanse in vitro
Non espanse in vitro
Growth Factors
Immunosuppressive
Mesenchymal Stem Cell (MSC)
MSC Proliferation
Proliferation
Osteogenesis
Chondrogenesis
Myogenesis
Marrow Stroma
Transitory
Osteoblast
Transitory
Chondrocyte
Myoblast
Transitory
Stromal Cell
Tendogenesis/
Ligamentogenesis
Other
Commitment
Bone Marrow/Periosteum
THE MESENGENIC PROCESS
Transitory
Fibroblast
Osteoblast
Mesenchymal Tissue
Lineage
Progression
Chondrocyte
Myoblast Fusion
Differentiation
Unique
Micro-niche
Maturation
Osteocyte
Hypertrophic
Chondrocyte
Myotube
Stromal
Cells
T/L
Fibroblast
BONE
CARTILAGE
MUSCLE
MARROW
TENDON/
LIGAMENT
Adipocytes,
Dermal and
Other Cells
CONNECTIVE 23
TISSUE
Main sources for human MSC, e.g.:
Bone Marrow
Pittenger M, et al.
Science 1999
Adipose Tissue
Zuk P, et al.
Tissue Eng. 2001
Umbilical Cord Blood/
Amnios/Placenta
Erices AA et al.
Brit. J. Haematol. 2000
+
+
+
Best characterised
(Pre-) Clinically used
High MSC Frequency
(Pre-) Clinically used
Youngest Adult Stem Cells
Easily Accesible
-
-
-
Invasive procurement
Age related detriments
Invasive procurement
Age related detriments ?
From: K.Bieback ,2007
MSC present in term CB?
Low frequency
Fresh UCB units needed
H-MSC s isolation and culture expansion
Bone marrow
aspirate
Plate cells at
interface
Colony formation
Centrifugation
Primary Culture
1.073g/ml Percoll
hMSCs markers
•
There is not a single marker for their identification
•
They are generally positive for
for:: CD29 (fibronectin R),
CD44 (hyaluronic acid R), CD90 (Thy
(Thy--1,HSC) CD105/SH2
(endoglin ,TGF
,TGFβ
β1-3R), CD73/SH3
CD73/SH3;; CD147 (neurotelin
neurotelin),
),
CD166,, CD271 (NGFR) and STRO
CD166
STRO--1.
•
They are generally negative for
for:: CD14 (M
(MØ)
Ø),, CD31
(endotelials cells
cells),
), CD34 (HSC), CD45 (CLA),
CD117(SCFR).
CD117
(SCFR).
Geni modulati nel differenziamento di MSC
Chondrocytes
MSCs
Osteoblasts
Collagene tipo I
Fosfatasi
Alcalina
Collagene tipo II
Collagene
tipo I
SOX-9
Aggrecano
Collagen type IX
Osteocalcina
Bone
sialoproteina
CBFA-1
CELL PHENOTYPE
PHENOTYPE AND DIFFERENTIATION
FACS Analysis
CD 3434-/ CD45
CD45-- /CD 105+
In vitro differentiation potential
Chondrogenic
Osteogenic
CD 45
45--
IOR
“One step” procedure
Bone marrow concentrated stem cells + scaffold (Hyaff
(Hyaff
11)
1) Platelet gel production (growth
(growth factors
factors))
2) Marrow concentration
(dedicated centifuge)
centifuge)
3) Concentrated BM Stem cells loaded on
biomaterial
29
4) Arthroscopic implant
IOR
ACI vs “One step” procedure
Similar trend of improvements
93 patients with 6 months minimum FU (all
(all cases 112)
IOR
FU evaluation in “One step”
MRI T2 mapping
Multiecho sequence to evaluate the presence of water in
cartilage according to colour scale
normal
cartilage
(3D SPGR)
T2T2-mapping:
45 msec
mean
ONE-STEP,
ONE18 Months FU
HighT2 (45
msec)) areas
msec
hyline
cartilage
expression
Histology
Safranin O staining
Hyaline like cartilage in remodelling
High proteoglycan expression (red)
Low evidence of Col I
Good osteochondral integration
RISULTATI
TIBIOTARSICA
GINOCCHIO
EXPERIMENTAL ANIMAL MODEL OF
OSTEOARTHRITIS
New Zealand male adult rabbits
12 months old
ANTERIOR CRUCIATE LIGAMENT
TRANSECTION (ACLT)
SHAM
8 weeks
8 weeks
3 months
6 months
EXPERIMENTAL MODEL
Bilateral ACLT
Bone marrow aspiration
8 weeks
Implant
Cell Seeding
Right Knee
HA
8 weeks
Expansion
Left Knee
CFU
2 x106 MSCs-HA
Right Knee
HA
Left Knee
Primaryculture
2x106 MSCs-HA
MSCs isolation
Animal sacrifices
3 Months
Implant
TGFβ-1
Hyaff®-11 seeding
Animal sacrifices
6 Months
HISTOLOGICAL ANALYSIS: SAFRANIN-0
40 X
40 X
LATERAL CONDYLE
MEDIAL CONDYLE
8 weeks
Safranin-O
8 weeks
3 months
6 months
Sham operation group
40 X
40 X
40 X
100 X
ACLT (OA) group
40 X
40 X
40 X
100 X
HA treated group
40 X
40 X
40 X
100XX
40
MSCs-HA treated group
Collagen II
8 weeks
3 months
6 months
Sham operation group
40 X
40 X
40 X
40 X
40
40 XX
40 X
40 X
40 X
40 X
40 X
100
X
40 X
ACLT (OA group)
HA treated group
MSCs-HA treated group
Collagen I
8 weeks
3 months
6 months
Sham operation group
40 X
40 X
40 X
40 X
40 X
40 X
40 X
40 X
40 X
40 XX
100
ACLT (OA group)
HA treated group
MSCs-HA treated group
MMP-3
8 weeks
3 months
6 months
Sham operation group
40 X
40 X
40 X
40 X
40 X
40 X
40 X
40 X
40 X
40 X
ACLT (OA group)
HA treated group
MSCs-HA treated group
100 X
Cartilage Thickness (µm)
8 Weeks
ACLT
3 Months
ACLT
HA
MSCs - HA
6 Months
380 ± 32
SHAM
ACLT
HA
MSCs - HA
P< 0,0005 ACLT versus MSCs-HA
P < 0,0005 HA versus MSCs-HA
312 ± 16
301 ± 25
340 ± 11
324 ± 25
280 ± 13
301 ± 17
471 ± 25
Subchondral Bone Thickness (µm)
8 Weeks
3 Months
SHAM
ACLT
ACLT
HA
MSCs-HA
325 ± 34
389 ± 49
403 ± 65
362 ± 32
324 ± 30
504 ± 26
ACLT
6 Months
HA
MSCs-HA
P< 0,0005 ACLT versus MSCs-HA
P < 0,0005 HA versus MSCs-HA
551 ± 48
349 ± 20
Conclusions
•
The deliveriy of MSCs on a hyaluronan (Hyaff
11) scaffold can positively interfere with OA
progression
Open question
•
Is the repair activity induced by MSCs + HA
due to MSC differentiation to chondrocytes
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
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.
44
ADIPOA
ASC isolation
Fragmentation
Adipose
tissue
Pellet seeding
ASC
Enzymatic
treatment
Centrifugation
Fenotipo e potenzialità differenziative delle ASC
Miocytes
Analisi in citometria a flusso di
ASC evidenzia che sono positive
CD105
105,, CD
CD73
73,, CD
CD29
29,, CD
CD44
44,,
a CD
CD
CD90
90 e negative a CD
CD106
106,, CD
CD45
45,,
CD
CD14
14,, CD
CD34
34,, and CD
CD31
31
Adipocytes
Osteocytes
Chondroytes
Adipose derived stromal cells for
osteoarthritis 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
CONTROL
100 µm
500 µmc
2x106 ASC
100 µm
500 µmc
6x106 ASC
100 µm
500 µm
Acknowledgements
Laboratory of Immunology and Genetics
Dr.ssa Brunella Grigolo
Dr.ssa Gina Lisignoli
Dr. ssa Giovanna Desando
Dr.ssa Carola Cavallo
Laboratory of Experimental Surgery
Prof. Roberto Giardino
Dr.ssa Milena Fini
Dr. Gianluca Giavaresi
Dr.ssa Matilde Tschon
Orthopaedic Divisions
Prof. Sandro Giannini
Dr. Roberto Buda
Prof. Maurilio Marcacci
Dr.E.Kon
Grants from:
Grazie per l’attenzione
Istituto Ortopedico Rizzoli (Biblioteca))