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Introduction

 A stem cell can

renew itself 

through cell

division and canbe induced to

develop into many

different

specialized cell

types with ability tomigrate and

engraft within

various tissues

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Rationale for this

study Disc degenerative

disease is a painfulcondition in humans

with a progressivecourse over years.

This finally end upwith a variety of 

anatomic correlatesand potentiallyphysical disability

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Rationale for study

Treatment options

³palliative´ We are involved in an

animal model to

promote disc

Regenerationusing murine embryonic

stem cells.

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Degenerative Cascade of IVD

Degeneration

Loss of Notochordal Cells (Bubble Cells)& Loss of Osmo-regulatory Mechanism

Decreased Proteoglycan and GAGcontent

 ± Loss of H2O

q Type II/oType I collagen

Disturbance of the O2 Transport

Lysozyme, Cathepsins increase

Free radicals Peroxidation of The Cell

Membranes and Complete Loss of Matrix

 ± Loss of Mechanical Competence ± 2o structural changes

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Thompsons Stages

of Aging Disc

II

IIII

IIIIII

IVIV

VV VV

Grade I - Normal

Grade 2 ± Fibrous tissue

Extending into nucleus +

Chondroid in annulus

Grade 3, Chondroid

delamination in annulus +

Extensive fibrous tissue in

nucleus

Grade 4, Nuclear cleft +focal annular disruption

Grade 5, Large cleft

Extending through nucleus

and annulus

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Notochordal Cell Retention in VariousMammalian Species

 Animal Animal Age at skeletal maturity   Age at skeletal maturity Age at loss of  Age at loss of 

notochordal cellsnotochordal cells

 Age at onset of disk  Age at onset of disk 

degenerationdegeneration

RatRat 2 months2 months 12 months12 months 12 months12 months

RabbitRabbit 6 months6 months 6 months6 months NANA

HorseHorse 5 years5 years birthbirth NANA

CatCat 2 years2 years 18 years18 years RareRare

HumanHuman 20 years20 years 10 years10 years 3030--50 years50 years

Hunter et al, 2003

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Can cell-based tissue engineeringmethods be a realistic choice fornucleus pulposus regeneration?

Embryonic stem cells (ESCs) grow indefinitely in vitro

and differentiate into different cells originating from allthree germlines (Mesoderm,Ectoderm,Endoderm).

Intervertebral disc consist of:

A. Outer annulus fibrosus - originating from mesoderm.

B. Central nucleus pulposus - originating from ectoderm with cells

embedded in a gelatinous material made of proteoglycan sulfate,GAG, hydrated hyaluronic acid and collagen type II.

Nucleus pulposus requires the presence of notochordalcells for normal function and structure.

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Establishing a flouroscopic guidedpercutaneous animal model of discdegeneration in the rabbit spine

Animal under general

anesthesia

Using AP and Lateral

flouroscopy

Disc punctured

Percutaneously with a 16G

needle

No morbidity

Reproducibility

needle

 AP

Lateral

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Murine EmbryonicStem Cell Preparation

Embryonic stem (ES) cell culture: Mouse 7AC5/EYFP ES cells

maintained on gelatin coated dishes with an irradiated MEF(mouse

embryonic fibroblasts) feeder layer in DMEM (Dulbecco¶s Modified

Eagle¶s Medium, optimized for ES cells) supplemented with (LIF)

leukemia inhibitory factor (Chaudhry et al. 2004).

The ES cells were then labeled with a mutant green fluorescent

protein (GFP) which serves as a useful tool to visualize the

implanted cells in the host nucleus pulposus.

The EBs (embryoid bodies) treated with 10-4 M cis-retionic acid and

cultured in a selective medium for differentiation into specific cell

types.

Stem cells labeled

With Green

Flourescent

Protein (GFP)

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Low power magnification High power magnification

Degenerated disc

In rabbit spine

Post Stem

Cell implantation

Disc

In rabbit spine

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3-D integration of implanted murineembryonic stem cells in

intervertebral discconfirmed with confocal

microscopy

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Conclusion

 A Degenerative Disc Disease model was established in a rabbit

through a percutaneous technique.

Preliminary MRI and histolgical studies demonstrated that murine

embryonic derived chondrogenic progenitor cells can integrate and

remain viable in degenerated discs

This model can further be used to investigate both the underlying

pathophysiology of Degenerative Disc Disease and various disc

regenerative strategies.

The lack of an immune response to the zenografts suggests that theintervertebral disc space is an immunologically privileged site.

The result of this study points to the future of biologic treatment of 

Degenerative disc disease.

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What next ?

Long term analysis

Analysis with specific cell markers

Quantitative analysis of proteoglycan

synthesis and GAG contents of implanteddisc

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Experimental Design

20 New Zealand Rabbits were used.

4 sites grouped (L1-2, L2-3,L3-4,L4-5) A. Naïve disc

B. Lesion discsC. Lesion discs with injected stem cells

D. Un lesion (naïve) disc with injected stem cells

Tissue sampling done at ± 6 weeks

 ± 3 months

 ± 6 months

 ± 12 months

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Experimental

DesignGroup A ± Control, normal disc

Group B ± Experimental control,

induced degenerated disc

Group C ± Experimental group,

degenerated disc with implanted

embryonic stem cells

Group D - Normal Disc with

implanted embryonic stem cells

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Experimental Design

H & E staining

Confocal Microscopy

Immunoflouroscopy /Immunihistology using

SSEAs (Stage Specific Embryonic Antigens) PCR Analysis

Proteoglycan synthesis rate and GAGcontent estimation

 ± radioactive sulphate incorporation and liquidscintillating counter 

 ± 1,9-dimethylmethylene blue assay

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ANTICIPATED

OUTCOM

E

Histologically similar chondrocyte differentiation

and notochoradal cells will be seen in the

implanted embryonic stem cells

QUALITATIVE AND QUANTITATIVE increase inProteoglycan and GAG (glycosaminoglycan)

content in the regenerating discs.

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Thank you