Natural Materials for Dural Replacement and
Neuroprotection
Vanessa Aguilar
Project Plan
October 27, 2010
Dural Replacement Therapy Needs
Dura lesion
complications:
History of dural
replacement
• Meningitis
• Cerebral spinal fluid leak
• Pseudomeningocele
• Arachnoiditis
• Epidural abscess
• 1895 first dural
replacement1
• Mid 90’s xenograph and
allograph were used
• Since 70’s biosynthetic
graft were investigated
•14% of spinal surgeries
requires a dural replacement
technology2
http://www.meningitis-trust.ie/Meningitis.html
http://www.nlm.nih.gov/medlineplus/enc
y/imagepages/17146.htm
Current dural
replacement market
•Gore-Tex (ePTFE)
• Neuropatch (polyester
urethane)
• Duragen (Collagen)
•DuraSeal (PEG-based spray)
• Tisseel (Fibrin/trombin
solutions)
• Preclude (PTFE/ elastomeric
Nasser, R. et al. Covidien
http://www.emmgraphics.com/projects/covidien/spineseal/pdfs/09
fluoiropolymer)
http://www.medcompare.com/details/16911/Duragen-Dural-GraftMatrix.html
1. Stendel et al. J Neurosurg, 2008. 2. Cammisa et al, Spine. 2000
_0924jallocase.pdf
Anatomical Dural Overview
Runza et al, Anesth Analg, 1999
http://members.cox.net/injections/images/esi_images/roots.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
Properties
• Collagen based
• Added cellulose layer for
suturable performance
Thick, very strong
sheets of cellulose
100% synthetic
CE approved for
Water-tight sealant to be spinal applications
applied during cranial or
spinal surgeries for dura
repair
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
Disadvantages
• 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. 2008
Synthetic surgical
sealant – PEG
hydrogel blend
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
characterize suturable
anti-adhesion film / foam
AIM 2: Develop bilayer
biofunctionalized HAbased film
AIM 3: Drug release
studies
• Biocompatible
• Non-immunogenic
• Non cell-adhesive /
cytotoxic
• Inhibits protein
absorption
• Mechanically robust
• Watertight / sealing
• Anti-fibrotic
• Biocompatible
• Bioabsorbable
• Non-immunogenic
• Dual functioning
• Regenerative
• Anti-adhesive
• Mechanically robust
• Cost effective
• Clinically sized
• Repositionable
• 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
characterize suturable
anti-adhesion film / foam
AIM 2: Develop bilayer
biofunctionalized HAbased film
AIM 3: Drug release
studies
• Biocompatible
• Non-immunogenic
• Non cell-adhesive /
cytotoxic
• Inhibits protein
absorption
• Mechanically robust
• Watertight / sealing
• Anti-fibrotic
• Biocompatible
• Bioabsorbable
• Non-immunogenic
• Dual functioning
• Regenerative
• Anti-adhesive
• Mechanically robust
• Cost effective
• Clinically sized
• Repositionable
• 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 (nonanimal)
• 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 celladhesions
www.biomedcentral.com
Results
Cell Adhesion Studies
% Cell Adhesion
125
100
75
Alginate
Alginate /GMHA
50
25
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
140
% Cells
120
100
Control
80
Alginate
60
Alg-GMHA
Alginate
Alginate /GMHA
40
20
0
-20
Live
Dead
-40
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 Masson’s trichrome
staining and analyze
http://freepages.genealogy.rootsweb.ancestry.com/~gomery/gomorigeo.html
Watertight Studies
Manometer
K. Hida et al. Surgical Neurology 65 (2006) 136–143
Protein Adsorption Studies
Film
Human serum albumen and
human plasma fibronectin
1. Shake for 24 hours at 37˚C
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/Duragen-Dural-GraftMatrix.html
http://medgadget.com/archives/2005/04/duraseal.html
Stendel R et al. 2008, J Neurosurg 209:215-221
Results
Cytotoxicity
140
% Cells
120
100
Control
80
Alginate
60
Alg-GMHA
Alginate
Alginate /GMHA
40
20
0
-20
Live
Dead
-40
There is no statistical difference between
control and films in live and dead
Alginate /GMHA/HA
Control