tissue final presentation
TRANSCRIPT
Spinal Cord Injury Regeneration via Two Phase Tissue Engineered HA Scaffold and Decellularized Matrix with OECs and Growth Factors
Kelene Boyle, Xitlalic Soto-Sida, Zina Kurian
Agenda
▷ Problem: Spinal Cord Injuryo What, Why, How?
▷ Current Research Strategieso Overcoming Limitations
▷ Solution Strategyo Defense & Offense
▷ Future Directions▷ Ethical Considerations▷ Questions
Spinal Cord Anatomy▷ White/Gray matter▷ Dorsal/Ventral▷ Sections
o Cervical, Thoracic, Lumbar, Sacral
❖ Neurons and Glial Cells❖ Oligodendrocytes (OL),
progenitors (OPC):➢ CNS, myelin sheath
❖ Olfactory Ensheathing Cells (OEC): ensheath olfactory neurons
❖ Neurotrophic Factors:➢ BDNF➢ NT-3,-4➢ NGF
Spinal Cord Anatomy-ECM
▷ Structural proteins: elastin, collagen, fibronectin▷ Hyaluronic Acid, Proteoglycans▷ Laminin, Nidogen, Reelin, Netrin-1, Tenascin-C,-R▷ Growth Factors: FGF-2, EGF, NGF, BDNF▷ PNN-stabilize synapses and connections in
development
Acute Tissue Damageo Hemorrhage, ischemiao Macrophages, Neutrophils
& Leukocytes proteolytic enzymes
degrade ECM pro-inflammatory
cytokines and chemokines
o Mitochondrial dysfunction (Ca) Excitotoxicity and
glutamate❏ Problems:
❖ Inflammation❖ Cell death
Secondary Damage➢ Reactive Astrocytes
■ Chondroitin & Sulfate Proteoglycans, glial scar
➢ Myelin-associated inhibitory proteins
➢ Endogenous axon growth inhibitory factors
➢ Loss of OL➢ Cyst formation❏ Problems:❖ axonal
degeneration❖ apoptosis❖ loss of neural
circuits
12,500Number of new cases of Spinal Cord Injuries (SCI) each year (NSCISC)
<1%People who had complete neurologic recovery by time of hospital discharge
$3,398,426Lifetime Costs for Low Tetraplegia Injury at 25 years old (Economic Impact of SCI)
Current Treatments
❖ No way to reverse spinal damage
❖ Methods:▷ Medications
o methylprednisolone (inflammation)
▷ Immobilization▷ Surgery
o stabilization, remove compression
▷ Experimental Treatmentso cell death, inflammation,
nerve/axonal regeneration
Current Strategies▷ Synthesized Scaffold
▷ Decellularized Scaffold
▷ Scaffold seeded with Cells
o Cell types
Oligodendrocytes
Adult Stem Cells
Schwann Cells
OECs
▷ Growth Factors in situ injection
NeuraGen PNS repair
Synthesized ScaffoldIn Vivo Therapeutics uses FDA approved PLGA Poly-L-Lysine Scaffold in current clinical trial
● Must fit exact injury structure● Extensive Study, no cell
marking● Takes weeks to degrade- no
immediate relief
Scaffold PropertiesMechanical Characteristics of Natural Spinal Cord Tissue: ▷ Soft, Flexible, Collagen
Possible Scaffold Materials:▷ Chitosan▷ Starch/polycaprolactone▷ PLGA▷ Alginate▷ Agarose▷ Hyaluronic Acid
Limitations▷ Difficult Optimization Properties▷ Communication with body▷ Short residence time▷ Shape and conformation
A,B, show fibrous structure of SEM analysis of brain matrix gels
Decellularized Scaffold
▷ Proso Non-antigenic ECMo Native mechanical
properties preserved
o 3-D structureo Growth factorso Cell proliferation,
differentiation, adhesion and migration mechanisms
▷ Conso Decellularization
weakens ECMo Toxic crosslinkers
to strengtheno Properties not
controlledo Variationo Cell repopulation
not well understood
Scaffold Seeded with Cells
Why use cells?▷ CNS unable to regenerate damaged axons▷ Injured neurons lost synaptic connections▷ Low intrinsic potential to regenerate▷ Increase rate of regenerationLimitations: Low cell survival and limited regenerative properties
Cell Options
▷ Oligodendrocyte Precursor cells
▷ Neural Stem Cells▷ Schwann Cells▷ Olfactory Ensheathing Cells
Oligodendrocyte Precursor Cells
▷ Provide myelination for CNS▷ Difficulty in maintaining and passaging
cultures
Adult Stem Cells
▷ Difficulty of isolation and extraction
▷ Low efficiency of differentiation
▷ Low proliferation
Schwann Cells▷ Provide myelination for PNS▷ Harvested from sciatic nerve▷ Poor/no interaction with astrocytes▷ Secrete CTGF preventing differentiation of OPCs
OECs▷ Olfactory bulb
or olfactory mucosa
▷ Easy to purify & grow in numbers
▷ Interacts with astrocytes
▷ Myelination▷ Promotes regeneration, can grow into host
spinal cord, reform synapses
Limitations
▷ Initial Injury▷ Scar Tissue▷ Inflammation▷ Time Limit▷ Limited Axon Growth▷ Synthetic Polymers Lack of Biofunctionality▷ Negatively charged Gel hinders cell
adhesion▷ Irregular Shaped Cavities▷ Injection fails to localize molecules▷ Cell Transplantation Therapies
o Cell death, lack of matrix support
Our Solution vs. Current Strategies
▷ Synthetic Scaffoldo Prevent secondary damage
▷ Decellularized Scaffoldo Encourage cell survival and
growth ▷ Scaffold with Cells
o Prevent secondary damageo Encourage cell survival and
growth❖ 2 Steps towards Walking
➢ Prevent secondary damage➢ Encourage cell survival and
growth➢ Structural guidance and
regeneration
Two STEPS Towards WALKING
1.DEFENSE: Biodegradable biomimetic hydrogel scaffold with growth factors
2.OFFENSE: A decellularized scaffold seeded with Olfactory Ensheathing Cells and Growth Factors
DEFENSE: Biodegradable Hydrogel Scaffold
▷ Biodegradable HA scaffold lasting ~ 2 weeks▷ Provide stabilization, prevent effects of scarring
and inflammation▷ Maintain blood-spinal cord barrier
o prevent infections and spread of cellular damage
▷ Growth Factors:o PDGF-A, FGF-2, IGF, CNTF (OPC proliferation)
Limitations addressed:❖ Scarring, inflammation❖ Create microenvironment for 2nd phase
Scaffold Properties
▷ Compressive Strength Modulus
▷ Crosslink Density▷ Composition▷ Porosity▷ Degradation
Temperature▷ Molecular Diffusion▷ In vivo cell
attachment, growth, proliferation
▷ Degradation▷ Biocompatibility▷ Compression
▷ Inhibition of scar formation▷ Hydrogel microenvironment:
Cell Receptor / Ligand Density▷ Cell Sensitivity from
Environmental Triggers
Scaffold Properties
▷ Nerve Guidance Channelso Concentrate moleculeso Collagen with crosslinking agentso Prevents collapse
OFFENSE: Decellularized Matrix with OECs and Factors
▷ Decellularized allogenic/xenogenic spinal cord (ECM)
▷ OECs grown for 2 weeks before seeded to decellularized scaffold ECM and transplanted
▷ Growth Factors: BDNF, Neurotrophin-3,-4, CNTF, NGF
▷ ECM and factors provide conditions for cell survival, growth, and regenerationo replenish ECM after degradation from
inflammation
Limitations addressed:❖ Axonal Growth, Regeneration (Growth Factors)❖ Remyelination of injured neurons (OEC)❖ Cell survival (Decellularized ECM, BDNF & NGF)
Olfactory Ensheathing Cells
▷ Show neurogenesis throughout life▷ Form fascicles for axon growth
o Support and guide axons▷ Ensheath non-myelinated axons▷ Phagocytose axonal debris▷ Grow through glial scars▷ Secrete neurotrophic factors▷ CNS neuroglia
Our Process is Simple
Implantation
Cell Connectio
ns
White indicates decellularized ECM
Stabilization
In Vitro Growth
Future Directions
Possibilities of further research:▷ 3D printed spinal cord▷ Gene therapy for
regeneration▷ Peripheral Nerve Transplant▷ Complete SCI solution▷ Long-term SCI patients
Issues▷ Animal testing, injuring animals▷ Animal source for decellularized scaffold▷ Failure of cell culture protocol
o Side effects of brain surgery▷ Invasive surgeries▷ Effectiveness▷ Societal Impact: Selectivity in Patient▷ $$$
ReferencesLang, Bradley T., Jared M. Cregg, Marc A. Depaul, Amanda P. Tran, Kui Xu, Scott M. Dyck, Kathryn M. Madalena, Benjamin P. Brown, Yi-Lan Weng, Shuxin Li, Soheila Karimi-Abdolrezaee, Sarah A. Busch, Yingjie Shen, and Jerry Silver. "Modulation of the Proteoglycan Receptor PTPσ Promotes Recovery after Spinal Cord Injury." Nature 518.7539 (2014): 404-08. Web.
https://www.nscisc.uab.edu/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084613/Why olfactory ensheathing cells?http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089736/
Straley, Karin S., Cheryl Wong Po Foo, and Sarah C. Heilshorn. “Biomaterial Design Strategies for the Treatment of Spinal Cord Injuries.” Journal of Neurotrauma 27.1 (2010): 1–19. PMC. Web. 30 May 2015.
Xu, Xiao Ming, Véronique Guénard, Naomi Kleitman, Patrick Aebischer, and Mary Bartlett Bunge. "A Combination of BDNF and NT-3 Promotes Supraspinal Axonal Regeneration into Schwann Cell Grafts in Adult Rat Thoracic Spinal Cord." Experimental Neurology 134.2 (1995): 261-72. Web.
Mindan Wang, Peng Zhai, Xiongbiao Chen, David J. Schreyer, Xiaodan Sun, and Fuzhai Cui. Tissue Engineering Part B: Reviews. June 2011, 17(3): 177-194. doi:10.1089/ten.teb.2010.0648.
http://www.researchgate.net/publication/236226623_Using_extracellular_matrix_for_regenerative_medicine_in_the_spinal_cord