Tissue engineering in the peripheral nervous system

Question 1

what is the structure of a nerve generally?

Answer 1

epinerium around the pernuerium which contain neurons and a perineum with its nerves is called a fasicle

Question 2

what supports the surrounding of the nerves?

Answer 2

collagen- ECM

Question 3

what is the role of the ECM around the nerves?

Answer 3

aligns the axons

Question 4

what does the perineurium do ?

Answer 4

has tight junctions between the cell and control fluid movement into and out of the endonuerium

Question 5

what is the collagen like in the epinerium

Answer 5

like a woven sheath

Question 6

what is biomechanical anatomy?

Answer 6

looking at the materials and the interactions between these materials of the nerve and tissue

Question 7

why is nerve biomechanisms important?

Answer 7

critical to repair choices and normal function- tension causes fibrosis, adhesion restricts movement

• is repair different depending on anatomical location?
• artificial replacement tissues need to match and restore mechanical integrity
• it is poorly understood

Question 8

what happens generally when damage in inflicted on the nerves of the peripheral nervous system?

Answer 8

• the distal tip generates
• the schwann cells change their phenotype and recruit the macrophages d start to digest the myelin and also become a repair schwann cell
• the form bands of bunger and pump out lots of neurotrophic factors
• the axon sprouts and then one will make it back to the muscle and you get a restored function
• but also a neuroma can form

Question 9

what generally causes here damage?

Answer 9

• bone breaks
• trauma
• accidents- road
• surgery
• compression syndromes

Question 10

what is the current procedure for nerve repair?

Answer 10

• if it is small, tweezers can be used to pull the epinerial together and then sowing up.
• if there is a gap then you will get tension when you do this and yo dont want tension so instead you can use an autograft where a surgion finds a spare bit of nerve and then this gets used to bridge the gap

Question 11

what is wrong with using an autograft approach?

Answer 11

there is limited availability of autograft material- if there is a big damage then you urn out of nerves that you can use. It provides schwann cells, tissue architecture, biomechanicsl properites and guidance cues. But it doesn’t provide neurons

Question 12

what does an autograft provide?

Answer 12

schwann cells, tissue architecture, biomechanicsl properites and guidance cues. But it doesn’t provide neurons

Question 13

what can be used to circumvent the problems with using autografts?

Answer 13

using a conduit that has been enriched

Question 14

?when are conduits good

Answer 14

for a shorter gap

Question 15

when are plain conduits bad?

Answer 15

when there is larger gap and the neurons need support

Question 16

what is a decellularised allograft?

Answer 16

• empty nerves that ave been deculluarisd and just have the ECM

Question 17

what is important to be involved in the autograft?

Answer 17

schwann cells

Question 18

what are the 5 things that need to be in an autograft?

Answer 18

• Biocompatible & bioresorbable
• Cell guidance substrate (core)
• Schwann cells (or alternatives)
• Doesn’t adhere to surrounding tissue (sheath) •Mechanical support/protection (sheath)

Question 19

how can you use a collagen hydrogel to create an alignment of the cells and the collagen?

Answer 19

put cells into a collagen hydrogel- they stat to attach to it and pull on it. This causes it to shrink.

• you can take advantage of this and anchor opposite ends so that it can only shrink in one direction and causes the cells to be aligned. cells and collagen becomes highly aligned.
• then take the water out- compression and results in a flat sheet

Question 20

how can you use the aligned sheet idea to produce a supportive construct for enrols?

Answer 20

• yo put cells into the collagen hydrogel and hey become aligned and then these can be rolled up and put into a tube and implant this . You create a 15 mm gap which is where a tube wouldn’t work. Use GFp to distinguish between cells pt in and cells already there. you can then look at the regeneration rate and cat promote nerve growth in a rat

Question 21

what cells can be put into engineered neural tubes and why?

Answer 21

• can put rat adipose tissue derived stem cells because these will upregulate lots of trophic factors when put in the collagen gel and they aligned nicely. The guide neuronal growth in vitro and in vivo when compared to the neural tube
• pulp derived stem cells can also be used- they are neural crest derived which means that these are geared towards making neurons and schwann cells and you can differentiate them into something that behaves like a schwann cell

Question 22

how can engineered NT be made using ‘off the shelf’ allogeneic stem cells?

Answer 22

• with the stem cell idea from adipose and death pulp, this would have to be autologous but you can get clinicl grade human neural stem cels that have been developed which do not promote an immune response

Question 23

what is the importance of stiffness and how are human neural stem cells engineered tube good for this?

Answer 23

• the stiffness of a cells environment can influence how it differentiates and there are methods for controlling the the stiffness of the neural stem cell tubes

Question 24

what are the remaining problems in nerve repair?

Answer 24

• Neuroma formation
• Muscle atrophy
• Damage due to sensory loss
• Fibrosis
• Cortical remodelling
• Poor functional outcome

Question 25

what is the micro-channel electrode array method?

Answer 25

• ant silicie fitting is sutured to the proximal and distal endings of the injured nerve. The implant length from 0.5 to 5mm.

Question 26

what is the biological relay approach

Answer 26

build a structure which has living neuron sin and try to connect at each end- put a bridge in to get function back immediately

Question 27

what factors have been shown to promotee survival of axons after injury?

Answer 27

• NAC

- ALCAR and phosphoisesterase inhiitor- sildenafil

Question 28

what different types of topography have been used in order to promote scaffold success?

Answer 28

• grooves
• electrospun fibres
• gels
• films

Question 29

what scaffold promotes SC elongation?

Answer 29

polycaprolactone (PCL) films

Question 30

why should ECM molecules be futon scaffolds?

Answer 30

whilst ECM molecules support SC attachment and stimulate excretion of neurite promoting factors

Question 31

what growth factors have been suggested to promote regeneration of axons?

Answer 31

• NGF, BDNF and CTNF improves te refeneration distance
• regeneration speed can be improved by IGF-1, FGF and GDNF
• NRG1 signalling is a key player for axonal myelination during development

Question 32

what is difficult about applying growth factors?

Answer 32

• different levels or different factors have actually been linked to inhibited growth so getting the levels right is paramount

Question 33

as well as SCs, which other cell can be used to support regrowth potentially?

Answer 33

• olfactory ensheathing cells

Question 34

what is the problem with using schwann cells or OECs?

Answer 34

the sourcing of these requires the sacrifice of a functional nerve and both OECs and SC have limited expansion capailites.

Question 35

give an example of an experiment using stem cells to promote neural regrowth (3)

Answer 35

• Mouse ESC-derived neural progenitor cells promoted nerve repair in a rat sciatic nerve 10 mm gap as shown by histological, molecular and electrophysiological studies
• SC-like precursors can be generated from human ESC and have been shown to express myelin protein in models of peripheral nerve regeneration in vitro
• NSC showed results comparable to au- tografts when seeded in chitosan nerve guides to repair a 10 mm nerve gap [78], and they can be genetically engineered to overexpress GDNF or NT-3, which can potentially improve their regenerative potential
• iPSC can efficiently generate functional neural crest cells and have been used, in combination with FGF-incorporated gelatin micro- spheres, to repair 10 mm nerve gaps with 50% poly l-lactide (PLA) and 50% poly ε-caprolactone porous nerve conduits

Question 36

rather than ESC or iPSC, what stem cell can be used? why arteries maybe preferable?

Answer 36

MSCs-Compared to ESC and iPSC, the use of MSC in regenerative medicine comes with less ethical implications regarding the sourcing of the cells, and the risk of teratoma formation or undesired cell differentiation.

Question 37

why are ASCs preferable to MSCs?

Answer 37

some of the problems related to the use of BM-MSC include the painful procedure for the har- vest of marrow aspirates and the low yield of mononucleated colony forming units (CFU). By contrast, ASC obtained from the stromal vascu- lar fraction (SVF) following digestion of adipose tissue contain 600× more CFU, and these cells proliferate faster and for longer durations.
- Like BM-MSC they are multipotent and they can generate functional SC-like cells

Question 38

name a degradable conduit that degrades after 48 months?

Answer 38

• neuragen - collagen type -1 conduit

Question 39

what a synthetic conduit

Answer 39

eurolac - PDLLA conduit - 16 months to degrade

Question 40

what type of conduits are bad and why?

Answer 40

nonresorbable- chronic inflammation

Question 41

why are naturally derived materials sometimes good but risky?

Answer 41

Naturally-derived materials have advantageous properties such as cell binding domains encouraging neuronal and glial attachment and migration.20 However, these are usually from animal sources and carry a minor risk of disease transmission

Question 42

as well as collagen, what other material can be used and why?

Answer 42

Silk fibroin has been recently investigated, and is attrac- tive due to favorable degradation properties and low in- flammatory response.

Question 43

what are 2 synthetic materials used for nerve conduit?

Answer 43

PGA, PLLA

Question 44

what key ECM molecule can be added to a nerve conduit? what has it been shown to do

Answer 44

laminin - promote SC proliferation

Question 45

how can gradients be used in conduit construction?

Answer 45

Dodla et al.75 reported that gradients of laminin and NGF contained within anisotropic agarose hydrogels stimulate nerve regeneration in a 20 mm rat sciatic model comparable to syngeneic grafts.

Question 46

how can topology be used in conduit construction?

Answer 46

Britland et al.81 found that grooved surfaces 12mm wide and 6 mm deep were optimal for DRG neuronal alignment.

Question 47

how can intralumincal guidance be used? give one example.

Answer 47

• he presence of internal scaffolds either as synthetic polymers or as matrix proteins contained within the lumen of nerve guides for improved physical or chemical guidance.
• Cai et al. reported on improved distal regeneration when PLA or silicone con- duits contained 16 aligned PLLA microfilaments using a rat sciatic model.

Question 48

describe the principle behind micro-channel electrode arrays:

Answer 48

Peripheral nerve electrode interfaces are designed to capture and/or stimulate the APs via implanted electrodes in order to re-establish lost functional connectivity [1]. Ideally, they should reliably and efficiently interface with a large number of nerve fi- bres. It is hoped that high-resolution interfaces will eventually provide a means to restore function to nerve-injured patients by, for example, extracting signals from motor fibres proximal to an injury and using them to drive distal muscle stimulators to im- prove strength and precision. Interfaces might also provide an amputee with control and sensation over an artificial limb.

• the idea is that the micro channel electrode impact can promote the regeneration of axons while also detecting impasse fired and using these to stimulate the distal axons while the injured ones regenerate. The strategy we are using is to make the axons grow down microchannels, in which the electrical signal from action potentials is greatly amplified, making recording possible. This can circumvent the issues of axons growing randomly and connecting to the wrong muscles and skin regions.

Question 49

what is the general principle of using conduits containing live axons?

Answer 49

new strategy to restore damaged axon pathways using transplantable miniature constructs consisting of living neurons and axonal tracts internalized within hydrogel tubes. These hydrogel microconduits were developed through an iterative process to support neuronal survival and directed axon growth. The design included hollow agarose tubes providing a relatively stiff outer casing to direct con- strained unidirectional outgrowth of axons through a central soft collagen matrix, with overall dimensions of 250 mm inner diameter · 500 mm outer diameter and extending up to several centimeters. The outer casing was also designed to provide structural support of neuronal/axonal cultures during transplantation of the construct. Using neuron culture conditions optimized for the microconduits, dissociated dorsal root ganglia neurons were seeded in the collagen at one end of the conduits. Over the following week, high-resolution confocal microscopy demon- strated that the neurons survived and the somata remained in a tight cluster at the original seeding site. In addition, robust outgrowth of axons from the neurons was found,