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

1.PROBLEM

Spinal Cord Injuries and Treatments

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

Spinal Cord Injury

▷ Complete vs. Incomplete

▷ Site of injury

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

2.METHODS

Current Tissue Engineering Strategies and Limitations

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

3.SOLUTION

A Modification of Tissue Engineering Strategy and Future Possibilities

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

4.ETHICS

Ethical Implications to Consider

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▷ $$$

Thanks!Any questions?

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/

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http://www.researchgate.net/publication/236226623_Using_extracellular_matrix_for_regenerative_medicine_in_the_spinal_cord