Chondrotransplant DISC

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



Indications

Background




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



Indications

Background




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


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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


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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


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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


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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)


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


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)


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 )

Economy & Finance

Chondrotransplant DISC