Biologic Minimally Invasive Spinal Surgery :-

Feasability of embryonic Stem Cells for Intervertebral

Disc Regeneration

Dr Ajay Popli
Fellow Minimally Invasive Spine surgery MHSI, Providence Hospital,Southfield , Michigan, USA

Consultant:- Minimally Invasive Spine & Orthopedics Surgery

Pushpanjali Crossaly Hospital ,
Vaishali, GZB (NCR),
 Introduction

A stem cell can

renew itself
through cell
division and can
be induced to
develop into many
different
specialized cell
types with ability to
migrate and
engraft within
various tissues
The Major Types of Stem
Cells
Embryonic Stem Cells
• From blastocysts left over
from In-Vitro Fertilization in
the laboratory
• From aborted fetuses
Adult Stem Cells
• Stem cells have been
found in the blood, bone
marrow, liver, kidney,
cornea, dental pulp,
umbilical cord, brain, skin,
muscle, salivary gland
Rationale for this study

• Disc degenerative
disease is a painful
condition in humans
with a progressive
course over years.

• This finally end up

with a variety of
anatomic correlates
and potentially
physical disability
 Rationale for study
• Treatment options

“palliative”
• We are involved in an
animal model to
promote disc
Regeneration
using murine embryonic
stem cells.
 Degenerative Cascade of IVD
Degeneration

• Loss of Notochordal Cells (Bubble Cells)

& Loss of Osmo-regulatory Mechanism
• Decreased Proteoglycan and GAG
content
– Loss of H2O
  Type II/Type 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
Thompsons Stages of
Aging Disc
Grade I - Normal

Grade 2 – Fibrous tissue I I

Extending into nucleus +
Chondroid in annulus
II II
Grade 3, Chondroid
delamination in annulus +
Extensive fibrous tissue in
nucleus III III

Grade 4, Nuclear cleft +

focal annular disruption IV IV

Grade 5, Large cleft

Extending through nucleus V V
and annulus
Notochordal Cell Retention in Various
Mammalian Species

Animal Age at skeletal Age at loss of Age at onset of disk

maturity notochordal cells degeneration

Rat 2 months 12 months 12 months

Rabbit 6 months 6 months NA

Horse 5 years birth NA

Cat 2 years 18 years Rare

Human 20 years 10 years 30-50 years

Hunter et al, 2003

Can cell-based tissue engineering
methods be a realistic choice for nucleus
pulposus regeneration?

• Embryonic stem cells (ESCs) grow indefinitely in vitro

and differentiate into different cells originating from all
three 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 notochordal cells for

normal function and structure.
 Establishing a flouroscopic guided percutaneous
animal model of disc degeneration in the rabbit spine

•Animal under general

anesthesia AP

•Using AP and Lateral

louroscopy

•Disc punctured
Percutaneously with a 16G
needle

•No morbidity Lateral

•Reproducibility

needle
Rabbit Spine
Human Spine
Murine Embryonic Stem
Cell Preparation
• stem (ES) cell culture: Mouse 7AC5/EYFP ES cells
Embryonic
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)
Harvesting and sectioning of rabbit
intervertebral disc

40X

H&E
Notochordal cells and
chondrocyte staining
differentiated cells seen
in ES cell post-injected
nucleus pulposus, 100X
10X
 Degenerated disc
In rabbit spine

Post Stem
Cell implantation
Disc
In rabbit spine

Low power magnification High power magnification

3-D integration of implanted
murine embryonic stem cells in
intervertebral disc confirmed with
confocal microscopy
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 the

intervertebral disc space is an immunologically privileged site.

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

Degenerative disc disease.
What next ?

• Long term analysis

• Analysis with specific cell markers
• Quantitative analysis of proteoglycan
synthesis and GAG contents of implanted
disc
 Experimental Design

• 20 New Zealand Rabbits were used.

• 4 sites grouped (L1-2, L2-3,L3-4,L4-5)
A. Naïve disc
B. Lesion discs
C. 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
 Experimental Design

Group 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
Experimental Design

• H & E staining
• Confocal Microscopy
• Immunoflouroscopy /Immunihistology using
• SSEAs (Stage Specific Embryonic Antigens)
• PCR Analysis
• Proteoglycan synthesis rate and GAG content
estimation
– radioactive sulphate incorporation and liquid
scintillating counter
– 1,9-dimethylmethylene blue assay
 ANTICIPATED
OUTCOME
• Histologically similar chondrocyte differentiation
and notochoradal cells will be seen in the
implanted embryonic stem cells
• QUALITATIVE AND QUANTITATIVE increase in
Proteoglycan and GAG (glycosaminoglycan)
content in the regenerating discs.
Thank you