Vanessa Aguilar Project Plan October 27, 2010 Natural Materials for Dural Replacement and...
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Vanessa AguilarProject Plan
October 27, 2010
Natural Materials for Dural Replacement and Neuroprotection
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Dural Replacement Therapy Needs
Dura lesion complications:
• Meningitis• Cerebral spinal fluid
leak• Pseudomeningocele
• Arachnoiditis• Epidural abscess
Current dural replacement
market•Gore-Tex (ePTFE)• Neuropatch (polyester urethane)• Duragen (Collagen)•DuraSeal (PEG-based spray)• Tisseel (Fibrin/trombin solutions)• Preclude (PTFE/ elastomeric fluoiropolymer)
History of dural replacement• 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.nlm.nih.gov/medlineplus/ency/imagepages/
17146.htm
http://www.medcompare.com/details/16911/Duragen-Dural-Graft-Matrix.html
1. Stendel et al. J Neurosurg, 2008. 2. Cammisa et al, Spine. 2000
http://www.meningitis-trust.ie/Meningitis.html
Nasser, R. et al. Covidienhttp://www.emmgraphics.com/projects/covidien/
spineseal/pdfs/09_0924jallocase.pdf
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Anatomical Dural Overview
http://members.cox.net/injections/images/esi_images/roots.jpg
Narotam P. et al, Spine 2004
Stendel R et al.J Neurosurg 2008,
Runza et al, Anesth Analg, 1999
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Dural Replacement / Cranial Adhesion BarriersBarrier Device DuraGen
(Integra Life Sciences)Synthecel Dura
(Synthes)DuraSeal(Covidien)
Adherus(HyperBranch)
Model
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% syntheticWater-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 dressingPhase 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 expensiveTimely to growCannot be grown mass-scale
• Set up required• Synthetic• Can be procoagulant• Nerve compression may ocur1
• Set up required• Synthetic• Can be procoagulant
1. Spotnitz, W and Burks, S. Transfusion. 2008
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Plan of WorkGOAL: 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
• Biocompatible• Non-immunogenic• Non cell-adhesive /
cytotoxic• Inhibits protein
absorption• Mechanically robust• Watertight / sealing• Anti-fibrotic
AIM 3: Drug release studies
• Biocompatible• Effective at reducing
adhesions• Encapsulate aspirin
or ibuprofen• Tunable release
rates
AIM 2: Develop bilayer biofunctionalized HA-based film
• Biocompatible• Bioabsorbable • Non-immunogenic• Dual functioning• Regenerative• Anti-adhesive
• Mechanically robust• Cost effective• Clinically sized• Repositionable
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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
• Biocompatible• Non-immunogenic• Non cell-adhesive /
cytotoxic• Inhibits protein
absorption• Mechanically robust• Watertight / sealing• Anti-fibrotic
AIM 3: Drug release studies
• Biocompatible• Effective at reducing
adhesions• Encapsulate aspirin
or ibuprofen• Tunable release
rates
AIM 2: Develop bilayer biofunctionalized HA-based film
• Biocompatible• Bioabsorbable • Non-immunogenic• Dual functioning• Regenerative• Anti-adhesive
• Mechanically robust• Cost effective• Clinically sized• Repositionable
Plan of Work
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Material Properties
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
AlginateHyaluronic Acid
1. Massie et al, The Spine Journal,2005.. 2. Zawaneh et al, Tissue Eng Part B 2008. 3 Dusseault et al. Wiley InterScience, 2005
Jeon et al, Biomaterials, 2009
• Biocompatible• Low toxicity• Gels at physiological pH and
temperature• Very non-cell adhesive, polyanionic,
hydrophilic• Poorly immunogenic (depends on
alginate purification)3
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Anti Cell-Adhesion Properties
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
1. Well and film
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Results
Alginate Alginate /GMHA
Alginate /GMHA /HA Control There is significant difference between control and films (p <
0.005)
Control Alginate Alginate/GMHA
Alg/GMHA/HA
0
25
50
75
100
125
Cell Adhesion Studies
% C
ell A
dh
esio
n
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Cytotoxicity1. Culture fibroblast cells
2. Seed cells in PLL coated TC coverslips
4. Stain coverslips with calcein / ethidium to label live / dead cells.
5. Evaluate cytotoxicity
4. Wait 24 hours
www.biomedcentral.com
5. Place Alginate / HA film supernatant on top of cells
3. Place Alginate / HA film on cell medium
4. Wait 24 hours
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Results
Alginate Alginate /GMHA
Control
There is no statistical difference between control and films in live and
dead assay
Live Dead0
102030405060708090
100
Cytotoxicity
Control
Alginate
Alg-GMHA
% C
ells
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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
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Watertight Studies
K. Hida et al. Surgical Neurology 65 (2006) 136–143
Manometer
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Protein Adsorption Studies
2. Rinse with PBS
Huang and Yang, Polymers advanced technologies, 2009
Film
1. Shake for 24 hours at 37˚C
Human serum albumen and human plasma fibronectin
3. Addition of sodium dodecyl sulfate (SDS)
5. Measure absorbance at 562 nm with UV/Vis spectrometer
4. Stain with BCA protein assay reagent
6. Measure and analyze samples
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Acknowledgments
PI: Dr. Christine Schmidt, Graduate Students: Sarah Mayes
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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://medgadget.com/archives/2005/04/duraseal.html
http://www.medcompare.com/details/16911/Duragen-Dural-Graft-Matrix.html
Stendel R et al. 2008, J Neurosurg 209:215-221
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Results
Alginate Alginate /GMHA
Alginate /GMHA/HAControl
There is no statistical difference between control and films in live and
dead
Live Dead0
102030405060708090
100
Cytotoxicity
Control
Alginate
Alg-GMHA
% C
ells