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

2. Dural Replacement Therapy Needs
Dura lesion complications:
Meningitis
Cerebral spinal fluid leak
Pseudomeningocele
Arachnoiditis
Epidural abscess
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
Current dural replacement market
Gore-Tex (ePTFE)
Neuropatch (polyester urethane)
Duragen (Collagen)
DuraSeal (PEG-based spray)
Tisseel (Fibrin/trombin solutions)
Preclude (PTFE/ elastomeric fluoiropolymer)
Problems : pseudomeningocele- abnormal collection of CSF, arachnoiditis, epidural absess
History: 1926, was the first successful surgical repair of a dural lesion.
before 1970s they used allographt but due to Creutzfeldt-Jacob disease there was a need to get an alternative
Since 1970’s biosynthetic graft were investigated, such polyvinyl, polyethylene, sylicon Dracon,
In 1978 firs intruduced the collagen isponge in clinical practice
Nasser, R. et al. Covidien
1. Stendel et al. J Neurosurg, Cammisa et al, Spine. 2000

3. Anatomical Dural Overview
Dura matter composition and function
Arachnoid surface of the dura ( facing the CSF) or meningeal dura mater and epidural (facing out) or periosteal
compose of a rich network of collagen fibers with intermingles fibroblast and Is covered by subdural neuroepithelium
arachnoid has some parallerl layers oberlaping and horizxontal
epidural is more compact dense and ireegular collagenous fibers
Problem : Dura matter is rupture in neural surgeries
in Tumor resection, craniotomy dura is cromopromized
most injuries are acidential
Runza et al, Anesth Analg, 1999
Stendel R et al.J Neurosurg 2008,
Narotam P. et al, Spine 2004

4. Dural Replacement / Cranial Adhesion Barriers
Barrier 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% 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
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
What is CE???? FDA approval in europe
DuraGen
1. Spotnitz, W and Burks, S. Transfusion. 2008

5. 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
Biocompatible
Non-immunogenic
Non cell-adhesive / cytotoxic
Inhibits protein absorption
Mechanically robust
Watertight / sealing
Anti-fibrotic
AIM 2: Develop bilayer biofunctionalized HA-based film
Biocompatible
Bioabsorbable
Non-immunogenic
Dual functioning
Regenerative
Anti-adhesive
Mechanically robust
Cost effective
Clinically sized
Repositionable
AIM 3: Drug release studies
Biocompatible
Effective at reducing adhesions
Encapsulate aspirin or ibuprofen
Tunable release rates
Aim 1: determine the characterize, fibrotic response - demonstrate that you don’t have protein absorption - develop the anti - adhesive component.
Show Aim page overview of 3 aims….not so detailed. “Plan of Work”. WHAT IS MY GOAL? To develop composite, dual-functioning materials at the interface of healing tissue and the surrounding tissues. improving upon anti-adhesive biomaterial barriers, to aid in wound healing (angiogenic), and decrease - healing tissue and rest of body, prevent scaring and adhesions. Control inflammatory response.

6. 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
Biocompatible
Non-immunogenic
Non cell-adhesive / cytotoxic
Inhibits protein absorption
Mechanically robust
Watertight / sealing
Anti-fibrotic
AIM 2: Develop bilayer biofunctionalized HA-based film
Biocompatible
Bioabsorbable
Non-immunogenic
Dual functioning
Regenerative
Anti-adhesive
Mechanically robust
Cost effective
Clinically sized
Repositionable
AIM 3: Drug release studies
Biocompatible
Effective at reducing adhesions
Encapsulate aspirin or ibuprofen
Tunable release rates

7. Jeon et al, Biomaterials, 2009
Material Properties
Hyaluronic Acid
Alginate
Jeon et al, Biomaterials, 2009
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
Antifibrotic (Massie, 2005)
Reduce adhesions formation in animal and human Zawaneh, 2008; Diamond, 2006; Wiseman, 2010; Rajab, 2010)
1,4-linked b-D-mannuronic (M) and
a-L-guluronic acid (G) units
1. Massie et al, The Spine Journal, Zawaneh et al, Tissue Eng Part B Dusseault et al. Wiley InterScience, 2005

8. Anti Cell-Adhesion Properties
1. Well and film
2. Culture fibroblast cells
hours in culture
4. Fix and stain for DAPI.
5. Validate cell-adhesion / non cell-adhesions

9. Results Alginate /GMHA Alginate Alginate /GMHA /HA
There is significant difference between control and films (p < 0.005)
Alginate /GMHA /HA
Control

10. 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

11. Results Alginate Alginate /GMHA
There is no statistical difference between control and films in live and dead assay
Control

12. Antifibrotic studies (using laminectomy model)
1. Collect the tissue
2. Dehydrate in ethanol
3. Acid decalcify
HA coating instead of visual detection of HA
4. Wait for 3 days
5. Slice every 50 um
6. Stain with H./E and Masson’s trichrome staining and analyze

13. K. Hida et al. Surgical Neurology 65 (2006) 136–143
Watertight Studies
Manometer
Intracraneal pressure is 5 mmHG and can increase up t 50 mmHG . Yammada 1997
K. Hida et al. Surgical Neurology 65 (2006) 136–143

14. Protein Adsorption Studies
Film
Human serum albumen and human plasma fibronectin
1. Shake for 24 hours at 37˚C
2. Rinse with PBS
SDS remove protein absorvee on the surface
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

15. Acknowledgments PI: Dr. Christine Schmidt,
Graduate Students: Sarah Mayes

16. 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)
Synthetic are fairly rigid. Need something that is flexible. Have shown strong foreing body reaxtions
Stendel R et al. 2008, J Neurosurg 209:

17. Results Alginate Alginate /GMHA
There is no statistical difference between control and films in live and dead
Alginate /GMHA/HA
Control