TISSUE ENGINEERING OF NERVOUS SYSTEM

Presented by

S.Shashank Chetty

2nd year M.Tech, NAST

Objectives: Introduction to

Nervous System.

Anatomy and Origin of Peripheral nerve System.

Autologous nerve grafts (autograft)

Nerve guides

critical gap length in peripheral nerves.

Matrices and scaffolds

selection of approaches

Summary

Introduction The loss of touch, sight, hearing or movement often

results from diseases or injuries to the nervous system. The retina, cochlear, spinal cord, brain and peripheral

nerve have very different molecular and cellular environments, and tissue engineering within each requires different strategies.

The limited regeneration in the central nervous system (CNS) is primarily due to the different cellular and molecular environment, and less to the internal properties of the corresponding neurons.

Scaffolds and matrices in the nervous system, therefore, provide a substrate for axonal growth that has efficacy even in the inhibitory CNS environment.

The most popular approach in peripheral nerve tissue engineering involves in vivo implantation of artificial scaffolds and substrates that will guide naturally regenerating axons to the distal segment.

Peripheral nerve Peripheral nerve injuries (PNI) can lead to lifetime loss of function and

disfigurement.

Figure: Anatomy of the peripheral nerve. The axons from the selected fascicle are indicated in yellow. The Schwann cell myelin sheath is drawn to demonstrate the wrapping of the cell body around the axon; however, the myelin is compact and only a single dark ring is typically seen with histology (right).

Astrocytes • create supportive framework for neurons• create “blood-brain barrier”• monitor & regulate interstitial fluid surrounding neurons• secrete chemicals for embryological neuron formation• stimulate the formation of scar tissue secondary to CNS injury

Oligodendrocytes• create myelin sheath around axons of neurons in the CNS. Myelinated axons transmit impulses faster than unmyelinated axons

Microglia• “brain macrophages”• phagocytize cellular wastes & pathogens

Ependymal cells• line ventricles of brain & central canal of spinal cord• produce, monitor & help circulate CSF (cerebrospinal fluid)

Schwann cells

• surround all axons of neurons in the PNS creating a neurilemma around them. Neurilemma allows for potential regeneration of damaged axons

• creates myelin sheath around most axons of PNS

Autologous nerve grafts (autograft) Figure: End-to-end suturing

of peripheral nerves.

Alignment of the fascicles is critical in successful regeneration and microsurgical techniques have been developed to optimize this surgery.

Use of tubes (nerve guides) in the PNS Figure: Sequence of events in empty silicone nerve

guides. Initially, fluid and cytokines fill up the nerve guide

(a), followed by the formation of a fibrin matrix inside the lumen which initially supports invading fibroblasts (blue)

(b), followed by Schwann cells (green) and axons (red)

(c), which eventually penetrate to the distal stump. The regenerated nerve usually is thinner in the middle of the bridge (yellow).Figure: The chance of

successful reinnervationwith nerve guides is dramatically reduced oncean injury gap reaches a certain value. This length,termed the critical gap length, L C, is where successful regeneration occurs 50% of the time.

Basic Nerve Guide Requirements• Sterilizable• Porous• Low antigenicity• Persists during regeneration• Biodegradable over long-term• Resists compression or collapse• Pliable (not too rigid)• Contains regeneration stimuli

In PNS, the proximal segment may be able to regenerate and reestablish nerve function. Proliferating Schwann cells, macrophages, and monocytes work together to remove myelin debris, release neruotrophins, and lead axons toward their synaptic targets, resulting in restored neuronal function.

In CNS, an impermeable glial scar composed of myelin, cellular debris, astrocytes, oligodendrocytes, and microglia formed. Regeneration neurons are blocked from reaching their synaptic target.

Matrices and scaffolds Peripheral nerve tissue engineering matrices have either

oriented or random structures, while scaffolds predominantly have an oriented morphology.

Figure: Selected examples of the critical gap length gap, LC, adapted from Yannas and Hill (2004) for the rat sciatic nerve. Empty silicone nerve guides

(a) have a L C of 9.7 mm and the introduction of a collagen–GAG matrix

(b) increases the L C to at least 14.8 mm with a L of 5.1 mm. When oriented fibrin matrices are used

(c), this results in a L of at least 4.7 mm when compared to the same, nonoriented matrices. Degradable collagen nerve guides

(d) have a L of at least 5.4 mm compared to silicone tubes. When polyamide or collagen fibers are introduced

(e), the L is at least 7.4 mm compared to empty nerve guides, while Schwann cells

(f) result in a similar L value of 7.4 mm, compared to nerve guides filled with phosphate-buffered saline.

Note: the shift length ( L ) is the result of the therapy – not necessarily compared to empty nerve guides .

First, there is the scaffold, this being a microporous, usually three dimensional, structure, within which a cell suspension can be placed and where the cells themselves have the opportunity to attach to the free surfaces of the material, and the media, with nutrients and other agents, can circulate.

Fibrin is commonly used as a matrix and can be modified with peptide sequences from laminin or used to deliver neurotrophic factors with a heparin-binding site

Secondly there are the matrices, which are gels, and more specifically hydrogels, involving a network of structural, usually cross-linked, molecules, within a water-based viscous matrix.

Guidance scaffolds/polymers based upon poly(-hydroxy acids) demonstrate the ability to stimulate axon growth

The principal advantages with the porous scaffold approach are the potential control over the morphology and mechanical characteristics, while the matrix has the very considerable advantage of resembling the extra cellularmatrix within which most cells normally reside.

Figure: A selection of approaches for delivering growth factors into peripheral nerve guides.

Growth factors can be incorporated into the nerve guide (a) or

introduced closer to the lumen with a rod(b) or

as growth factor containing microspheres (c).

The growth factors may be incorporated into the matrix as non bound (d) or as a

Non-covalent controlled release system(e).

Sequence of events after implantation of a fibronectin scaffold in the hemisection lesion of the spinal cord

Summary Neurotrophic factors play an important, but complicated, role in

regenerative therapies.

The limited regeneration in the central nervous system (CNS) is primarily due to the different cellular and molecular environment and less to the internal properties of the corresponding neurons.

A range of cell and scaffolds implanted into the injured spinal cord reduces astrocytic scarring, promotes tissue sparing, and facilitates axonal regeneration and myelination.

Age and gender differences may also need to be taken into account.

Tissue engineering therapies for treating traumatic injury (acute/chronic) versus degenerative diseases differ greatly (i.e. what to do and when to do it?).

Genetic modification of cells prior to transplantation into the injured brain and spinal cord can increase neuronal survival and enhance the regeneration response.

Neural tissue engineering strategies commonly adopt multifactorial strategies both in terms of diversity of therapies (i.e. scaffold and cells and drugs) and timing of interventions (parallel orsequential).

Thank you for Kind attention