chondrotransplant disc
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1 Content
In vitro characterization – of co.don chondrotransplant® DISC
Animal trial - feasibility
Indications
Background
Manufacturing of co.don chondrotransplant® DISC
Application of co.don chondrotransplant® DISC
Autologous Disc-derived chondrocyte Transplantation (ADCT)
2 Background
Degenerative disc disease (DDD)
Early stage
Loss of disc tissue due to disc herniation Progression of degeneration Development of chronic back pain Significant recurrence rate
Late stage
FusionLoss of mobility Artificial discPersistent back painTherapy failure
3 Background
Degenerative disc disease (DDD)
Early stage
Loss of disc tissue due to sequestrectomy Progression of degeneration Development of chronic back pain Significant recurrence rate
Late stage
Loss of mobility Artificial discPersistent back painTherapy failure
? Transplantation of autologous disc-derived cells (ADCT) ?
4 Indication
Indications stage after sequestrectomy progressive degeneration of the Nucleus pulposus stage after decompression expansion of indication for discogenic pain and dark disc adults up to approx. 55 years of age
Transplantation of autologous disc-derived cells
For biological repair of disc tissue functional and structural regeneration prevention/delay progressive degeneration avoiding spinal fusion
5 Content
In vitro characterization – of co.don chondrotransplant® DISC
Animal trial - feasibility
Indications
Background
Manufacturing of co.don chondrotransplant® DISC
Application of co.don chondrotransplant® DISC
Autologous Disc-derived chondrocyte Transplantation (ADCT)
6In vitro processing of disc-derived chondrocytes
Sequester tissue
Isolation of disc
chondrocytes
Expansion of disc
chondrocytes
Disc cells in monolayer
Cell cultures
Sequestrectomy
7In vitro characterization of co.don chondrotransplant® DISC
low cell density
high cell density
Fig.: Proliferation curve.
Proliferation of disc-derived cells
8
Phenotype of disc chondrocytes
Anulus fibrosus (AF), Nucleus pulposus (NP) and Co-culture
(CC) cultures:
constant expression of biglycan, decorin,
aggrecan, CEP68,
collagen types I and III
decreasing expression of S100 in higher passages
no expression of collagen type II
Fig.: Decorin mRNA Expression in CC ML cultures.
M P1 P2 P3 P4 P5 P6 P7
In vitro characterization of co.don chondrotransplant® DISC
9
adhesion of cultured disc derived chondrocytes to native tissue cell migration into tissue fissures filling of fissures by de novo synthesized matrix
Adhesion and Integration of cultured disc chondrocytes
disc-derived cells
native disc tissue
In vitro characterization
10
• Improved differentiation of disc derived
chondrocytes in co-
cultures
• disc specific markers expressed and secreted
by co-cultures
Regenerative Capability of human disc chondrocytes
contribution to regenerative processes following disc-derived chondrocyte transplantation
In vitro characterization of co.don chondrotransplant® DISC
11
Experimental Model
• 15 mixed-bred dogs• lateral vertebral plate debridement• use of nucleus and lateral anulus tissue• autologous disc cells with own serum• no addition of antibiotics/fungistatics• L1-L2 damaged & no cell transplantation• L2-L3 control disc• L3-L4 damaged & disc cell transplantation (ADCT) after pressure measurement and
under fluoroscopic control
Animals: Disc damage: Cell transplants:
Experimental groups:
Animal trial
Pressure measurement
fluoroscopic control
• 12 months follow up
12
Progressive disc healing induced by ADCT
Damaged discs: • dense scar tissue, • marrow fibrosis & edema
ADCT-treated discs: • reduced marrow blanching, • lucent matrix appearance, • less scarred, • reestablishment of vertebral margin
L3-L4ADCT
L1-L2damaged
Animal trial
Disc height differences after 1year:• dam./control & dam./ADCT significant • control/ADCT not significant
13
Structural restoration of intervertebral disc induced by ADCT
- homogenous cell distribution
- complete fibrous reconstruction
- regenerate with disc-specific composition
- homogenous tissue structure
- no signs of vascularization and necrosis
12 months, damaged disc.
12 months, damaged & ADCT treated disc.
intact disc.
Animal trial
14
Positive BrdU-stained cell nuclei within ADCT-treated disc levels.
Identification of transplanted BrdU-labeled disc derived cultured chondrocytes
Animal trial
15
Damaged disc: ADCT-treated disc:
Pericellular positive SafraninO staining for
transplanted chondrocytes
Loss of SafraninO staining within defect
area. No pericellular
staining.
Intact disc:
Compositional restoration of the intervertebral disc
SafraninO staining
of disc interior
x25x100
x25x200x25
Animal trial
16
x200 x100
Strong staining for collagen type
II.
Intercellular and pericellular positive collagen type II
staining
Low expression of
collagen type I
Compositional restoration of the intervertebral disc
ADCT-treated disc:Intact disc:
Low staining for collagen type I.
x25 x25
x25
x25
Animal trial
17
Feasibility:
• human nucleus and anulus cells can be propagated in vitro• freezing/thawing does not affect cell viability• transplantation is technically feasible
– pressure validation– minimally invasive
Outcome:
• no progressive disc degeneration• maintenance of disc height• de novo matrix generation through transplanted cells • well integrated de novo synthesized matrix• complete fibrous reconstruction • maintenance of segmental mobility • contribution of transplanted cells to observed clinical success of pilot trials
Ganey et al., 2004, Spine
Induction of intervertebral disc regeneration by ADCT
Conclusion
Animal trial
18 Content
In vitro characterization – of co.don chondrotransplant® DISC
Animal trial - feasibility
Indications
Background
Manufacturing of co.don chondrotransplant® DISC
Application of co.don chondrotransplant® DISC
Autologous Disc-derived chondrocyte Transplantation (ADCT)
19
co.don chondrotransplantDISC®
• Autologous manufacturing using the Integrated Isolator Technology (IIT)
• Regulated as a biological therapeutic:
-Manufacturing permission according
to the German drug law §13 since 1997
-Quality-Management-System DIN EN ISO
9001:1994/2000 guideline 91/356/EWG
-Good Manufacturing Practices (GMP) for
pharmaceutical products WHO 1993
-Guidelines for pharmaceutical Companies in
Germany-ISO 14644-1 for the clean room -PIC/S-guideline (Pharmaceutical
Inspection- Convention)
Integrated Isolator Technology (IIT)
Regulation of human TE products in Germany
20
Agreement co.don® AG
Biopsy and Blood obtaining
Xmedika
Biopsy kit
Surgeon
Biopsy kit
Xmedika new Biopsy kit
Shipment via courier, within 48h to co.don® AG
Manufacturing of co.don chondrotransplant® DISC
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21
Isolation of disc-chondrocytes by enzymatical digestion,
monolayer culture, In-Process control
Lock in of Biopsy and Serum into the isolator
Quality control
co.don® AG ProductionIIT (Integrated Isolator
Technology)
Preparation of Biopsy and
SerumReceiving
control
Manufacturing of co.don chondrotransplant® DISC
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22
Expansion ofdisc- chondrocyte transplants
If necessary freezing of monolayer cells &
recultivation
Arrangement of the transplantation appointmentbetween co.don® AG, Xmedika and surgeon
Expansion of disc-chondrocytes in monolayer
Manufacturing of co.don chondrotransplant® DISC
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23
Completion of the product including packing and labeling Quality
control
Final inspection &
Product release
Shipment of
co.don chondrotransplant® DISC and under LHO condition using an courier organized by co.don® AG, Xmedika Durability : 43h
Xmedika / Surgeon
Transplantation
Manufacturing of co.don chondrotransplant® DISC
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24
Biopsy Kit
Biopsy Kit
• Evaluated and standardized shipment conditions• Standardized packaging• Qualified supplier for raw materials
25
Disc biopsy obtaining
- Discectomy
- Discectomy combined by nucleotomy
- Endoscopy (e.g. endoscopic alligator forceps d=1.3mm)
- Decompression (e.g. using a decompressor, avoiding heating and only by short application)
Manufacturing of co.don chondrotransplant® DISC
26
Transplant Kit
Transplant Kit
• Evaluated & standardized shipment conditions• Standardized packaging• In time delivery (limited durability)• theoretical and practical training of surgeons
27
Placement of the needle for application of chondrotransplant DISC. under fluoroscopic
control; application of chondrotransplant DISC without fluoroscopic control
Vial with co.don chondrotransplant® DISC
co.don chondrotransplant® DISC shipment under LHO conditions.
Transplantation (ADCT)
- under local anesthesia - placing injection needle under into nucleus area under fluoroscopic control- percutaneous injection of chondrotransplant® DISC contralateral to the site of disc herniation
Application of co.don chondrotransplant® DISC
28Application of co.don chondrotransplant® DISC
Injection needle
-puncture needle = injection needle: e.g. 21Gor
-use of puncture needle (e.g. 19G) and injection needle (22G)
29
- 1 ml tube containing cell transplant storage on ice till injection needle was placed
- Re-suspending cell solution by gentle shaking
- Transferring into a e.g. 1ml syringe using a cannula (> 22G)
Application of co.don chondrotransplant® DISC
Transplantation
30 Indication – Contraindication
Indications
stage after sequestrectomy progressive degeneration of the Nucleus pulposus stage after decompression expansion of indication for discogenic pain and dark disc adults up to approx. 55 years of age
Contraindications
chronic inflammations or chronic degenerative spine diseases (i.e. spondylarthrosis) previous chemo- and thermonucleolysis of the affected intervertebral disc uncontained anulus fibrosus Modic Changes >2 pregnancy infectious diseases (Hep C,HIV I+II )
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