Natural Materials for Dural

Replacement and Neuroprotection

Vanessa Aguilar
Project Plan
October 27, 2010

Dural Replacement Therapy Needs

Dura lesion
complications:

Meningitis
Cerebral spinal fluid
leak
Pseudomeningocele
Arachnoiditis
Epidural abscess

http://www.meningitistrust.ie/Meningitis.html

Current dural
replacement
market

History of dural
replacement

1895 first dural

replacement1
Mid 90s xenograph
and allograph were
used
Since 70s biosynthetic
graft were investigated
14% of spinal surgeries
requires a dural
replacement
technology2

http://www.nlm.nih.gov/medlineplu
s/ency/imagepages/17146.htm

Gore-Tex (ePTFE)
Neuropatch (polyester
urethane)
Duragen (Collagen)
DuraSeal (PEG-based
spray)
Nasser, R. et al. Covidien
Tisseel (Fibrin/trombin
http://www.emmgraphics.com/projects/covidien/spineseal
solutions)
/pdfs/09_0924jallocase.pdf
http://www.medcompare.com/details/16911/DuragenDural-Graft-Matrix.html
Preclude (PTFE/
elastomeric
1. Stendel et al. J Neurosurg, 2008. 2. Cammisa et al, Spine.
fluoiropolymer)
2000

Anatomical Dural Overview

Runza et al, Anesth Analg, 1999

http://members.cox.net/injections/images/esi_images/roo
ts.jpg

Stendel R et al.J Neurosurg 2008,

Narotam P. et al, Spine 2004

Dural Replacement / Cranial Adhesion Barriers

Barrier Device

DuraGen
(Integra Life Sciences)

Synthecel Dura
(Synthes)

DuraSeal
(Covidien)

Adherus
(HyperBranch)

Description

DuraGen/DuraGen
Plus is an innovative
matrix designed to
prevent peridural
fibrosis and adhesions

Cellulose
microbially grown
cellulose

PEG hydrogel

Synthetic surgical
sealant PEG
hydrogel blend

Properties

Collagen based
Added cellulose layer
for suturable
performance

Thick, very strong

sheets of cellulose

100% synthetic
Water-tight sealant
to be applied during
cranial or spinal
surgeries for dura
repair

CE approved for
spinal applications

Advantages

FDA approved for

neural applications
Natural-based
material
Bioresorbable but
degradation resistant

FDA approved for

dural replacement
and wound dressing
Phase III clinical
trials

FDA approved for

cranial and spinal
surgeries.
Injectable and easy
to use

Spray-use, easy to
use

Disadvantage
s

Not easy to handle

Not an adhesion
barrier
Attracts adhesions

Very expensive
Timely to grow
Cannot be grown
mass-scale

Set up required
Synthetic
Can be
procoagulant
Nerve compression
may ocur1

Set up required
Synthetic
Can be
procoagulant

Model

1. Spotnitz, W and Burks, S. Transfusion.

Plan of Work
GOAL: To develop composite, dual-functioning materials that would
serve to encourage healthy cell growth, wound healing and inhibits
post-surgical scar tissue formation for neural applications. We aim to
develop an all-in-one product to replace dural tissue as well as
support healthy healing.
AIM 1: Develop and
AIM 2: Develop bilayer
characterize suturable biofunctionalized HAanti-adhesion film /
based film
foam
Biocompatible
Biocompatible
Bioabsorbable
Non-immunogenic
Non-immunogenic
Non cell-adhesive /
Dual functioning
Regenerative
cytotoxic
Inhibits protein
Anti-adhesive
Mechanically robust
absorption
Mechanically robust Cost effective
Watertight / sealing Clinically sized
Anti-fibrotic
Repositionable

AIM 3: Drug release

studies
Biocompatible
Effective at reducing
adhesions
Encapsulate aspirin
or ibuprofen
Tunable release
rates

Plan of Work
GOAL: To develop composite, dual-functioning materials that would
serve to encourage healthy cell growth, wound healing and inhibits
post-surgical scarred tissue formation for neural applications. We
aim to develop an all-in-one product to replace dural tissue as well as
support healty healling.
AIM 1: Develop and
AIM 2: Develop bilayer
characterize suturable biofunctionalized HAanti-adhesion film /
based film
foam
Biocompatible
Biocompatible
Bioabsorbable
Non-immunogenic
Non-immunogenic
Non cell-adhesive /
Dual functioning
Regenerative
cytotoxic
Inhibits protein
Anti-adhesive
Mechanically robust
absorption
Mechanically robust Cost effective
Watertight / sealing Clinically sized
Anti-fibrotic
Repositionable

AIM 3: Drug release

studies
Biocompatible
Effective at reducing
adhesions
Encapsulate aspirin
or ibuprofen
Tunable release
rates

Material Properties
Hyaluronic Acid

Alginate

Jeon et al, Biomaterials, 2009

http://www.madsci.org/posts/archives/apr2001/986571103.Bc.1.gif

Biocompatible
Bioabsorbable / non-immunogenic
(non-animal)
Very non-cell adhesive, polyanionic,
hydrophilic
Antifibrotic1 (1% HMW HA)
Pro-angiogenic
Shown to reduce adhesion formation
in animals and humans2
Clinically used to reduce adhesions:
Seprafilm, most effective and widely
used anti-adhesion barrier on the
market

Biocompatible
Low toxicity
Gels at physiological pH and
temperature
Very non-cell adhesive, polyanionic,
hydrophilic
Poorly immunogenic (depends on
alginate purification)3

1. Massie et al, The Spine Journal,2005.. 2. Zawaneh et al, Tissue Eng Part B 2008. 3 Dusseault et al. Wiley InterScience, 2005

Anti Cell-Adhesion Properties

1. Well and film
2. Culture fibroblast cells

3. 1.5 hours in culture

4. Fix and stain for DAPI.

5. Validate cell-adhesion / non

cell-adhesions

www.biomedcentral.com

Results

Cell Adhesion Studies

125
100
75

Alginate

Alginate /GMHA

50
25 Adhesion
% Cell
0

There is significant difference

between control and films (p <
0.005)

Alginate /GMHA /HA

Control

Cytotoxicity
1. Culture fibroblast
cells
3. Place Alginate / HA
film on cell medium

2. Seed cells in PLL coated TC

coverslips

4. Wait 24
hours

5. Place Alginate / HA
film supernatant on top
of cells

4. Wait 24
hours
4. Stain coverslips with
calcein / ethidium to label live /
dead cells.

5. Evaluate
cytotoxicity

www.biomedcentral.com

Results

Cytotoxicity
100
90
80
70
60
50
% Cells
40
30
20
10
0

Control
Alginate
Alg-GMHA

Live

Alginate

Alginate /GMHA

Dead

There is no statistical difference

between control and films in live and
dead assay

Control

Antifibrotic studies
(using laminectomy model)
1. Collect the tissue

2. Dehydrate in ethanol
3. Acid decalcify
4. Wait for 3 days

5. Slice every 50 um

6. Stain with H./E and Massons

trichrome staining and analyze

http://freepages.genealogy.rootsweb.ancestry.com/~gomery/gomorigeo.html

Watertight Studies
Manometer

K. Hida et al. Surgical Neurology 65 (2006) 136143

Protein Adsorption Studies

Film

Human serum albumen and

human plasma fibronectin

1. Shake for 24 hours at 37C

2. Rinse with PBS

3. Addition of sodium dodecyl
sulfate (SDS)

4. Stain with BCA protein assay

reagent
5. Measure absorbance at 562 nm
with UV/Vis spectrometer
6. Measure and analyze samples

Huang and Yang, Polymers advanced technologies, 2009

Acknowledgments

PI: Dr. Christine Schmidt,

Graduate Students: Sarah Mayes

Current Technologies
Autologous grafts
Pericranium or temporal fascia
Xenografts and allografts
Menengitis and Creutzfeldt-Jacobs Disease
Natural and syntethic materials
Gore-Tex (ePTFE)
Neuropatch (polyester urethane)
Duragen (Collagen)
DuraSeal (PEG-based spray)
Tisseel (Fibrin/trombin solutions)
Preclude ( PTFE/ elastomeric fluoiropolymer)

http://www.medcompare.com/details/16911/DuragenDural-Graft-Matrix.html

http://medgadget.com/archives/2005/04/duraseal.html

Stendel R et al. 2008, J Neurosurg 209:215221

Results

Cytotoxicity
100
90
80
70
60
50
% Cells
40
30
20
10
0

Control
Alginate
Alg-GMHA

Live

Alginate

Alginate /GMHA

Dead

There is no statistical difference

between control and films in live and
dead

Alginate /GMHA/HA

Control