Vanessa Aguilar Project Plan October 27, 2010 Natural Materials for Dural Replacement and Neuroprotection.

Vanessa AguilarProject Plan

October 27, 2010

Natural Materials for Dural Replacement and Neuroprotection

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

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

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

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

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

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

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

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

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

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

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

K. Hida et al. Surgical Neurology 65 (2006) 136–143

Manometer

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

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://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

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