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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.
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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 • History of dural replacement • 1895 first duralreplacement1 • Mid 90’s xenographand 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) http://www.nlm.nih.gov/medlineplus/ency/imagepages/17146.htm http://www.meningitis-trust.ie/Meningitis.html Nasser, R. et al. Covidien http://www.emmgraphics.com/projects/covidien/spineseal/pdfs/09_0924jallocase.pdf http://www.medcompare.com/details/16911/Duragen-Dural-Graft-Matrix.html 1. Stendel et al.J Neurosurg, 2008. 2. Cammisaet al, Spine. 2000
Anatomical Dural Overview Runza et al, AnesthAnalg, 1999 http://members.cox.net/injections/images/esi_images/roots.jpg Stendel R et al.JNeurosurg2008, Narotam P. et al, Spine 2004
Dural Replacement / Cranial Adhesion Barriers 1. Spotnitz, W and Burks, S. Transfusion. 2008
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
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
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 Alginate /GMHA Alginate Alginate /GMHA /HA There is significant difference between control and films (p < 0.005) 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 Alginate Alginate /GMHA There is no statistical difference between control and films in live and dead assay Control
1. Collect the tissue Antifibrotic studies (using laminectomy model) 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 trichromestaining and analyze http://freepages.genealogy.rootsweb.ancestry.com/~gomery/gomorigeo.html
Watertight Studies Manometer K. Hida et al. SurgicalNeurology 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-Graft-Matrix.html http://medgadget.com/archives/2005/04/duraseal.html Stendel R et al. 2008, J Neurosurg 209:215-221
Results Alginate Alginate /GMHA There is no statistical difference between control and films in live and dead Alginate /GMHA/HA Control