TE 3 Flashcards
(134 cards)
1
Q
Epidemiology of corneal disease/injuries?
A
Corneal disease/injuries is 2nd cause of vision loss.
10 million people across the world suffer from vision loss due to corneal disease (e.g. trachoma) or injury
2
Q
Third world countries vision loss due to? Devloped world?
A
Trachoma- bacteria in eye.
Developed world- due to injury- acid/alkali burns etc.
3
Q
What is the cornea?
A
Forms the outer surface of the eye over iris- protective role and refracts light (2/3 of eyes refractive power is the cornea)
4
Q
Layers of cornea?
A
Epithelium (bowmans membrane) corneal stroma, (descements membrane) endothelium.
5
Q
Function of the corneal epithelium?
A
10% of corneal thickness (50um of 500um total).
Highly innervated.
Prevent fluid loss, respond to environment, protection.
Rapid wound heeling
6
Q
Structure of the corneal epithelium?
A
Stratified non- keratinised epithelium.
Split into 3 layers:
Superficial: tight junctions- barrier. Microvilli with glycocalyx.
Wing cells:
Basal cells: Communicate through desmosomes.
Constant state of turnover, replacement every 5-7days
7
Q
TE potential of the corneal epithelium?
A
They are in a constant state of turnover every 5-7 days, so there must be stem cells in there which could be harnised and used.
8
Q
Function of the corneal stroma?
A
90% of corneal thickness.
Provides strength, allows transparency- mainly acellular.
9
Q
Structure of the corneal stroma?
A
-relatively acellular, molecular layer e.g. collagens, proteoglycans and glycoproteins.
-Very particular regulated molecular arrangement- colagen in regularly packed fibrils. This geometric arrangement reduces scatter when transmits light (allows transparency).
Main cells are keratinocytes- synthesise and maintain the collagen arrangement.
10
Q
Corneal endothelium function?
A
Metabolically active- lots of mitochondria for energy to actively pump water out of stroma (leaks across). This maintains collagen hydration- if water comes in, collagen swells and deshapes (more light scatter- less transparency).
Solutes, nutrients are allowed through into the stroma from tear fluid or aqueous humor.
11
Q
If inflamed endothelial cells of cornea?
A
The tight junctions become less tight and water is leaked- swollen cornea.
12
Q
Corneal Innervation?
A
6-10x more nerve endings than skin. Important for blink reflex, wound heeling, tear production.
13
Q
Why is cornea avascular?
A
So remains transparent. Nutrients diffuse from tear fluid and aqueous humor
14
Q
corneal substitute qualities would need?
A
- Transparent
- Refractive (2/3 of eyes refractive index)
- Tensile strength
- Avascular, but allow high innervation.
- O2, nutrients etc needs to diffuse through.
15
Q
Why TE corneal replacement need?
A
Can take cornea from cadaver, place and suture into patient- but sourcing is hard as only 7% of bodies cornea is suitable and available.
16
Q
When repeated cornea grafts fail use?
A
Keratoprosthesis. optical Polymethylmethacrylate (pilot spliter material) to replace cornea. But need life long regime of anti-biotics to control inflammation and prevent glaucoma.
17
Q
Example of design of Keratoprosthesis?
A
Boston- Keratoprosthesis (B-KPro).
The device consists of two main parts: an anterior plate of poly(methyl methacrylate) (PMMA) and a snap-on titanium back plate with 16 holes (1.3 mm diameter each) that facilitate the access of the corneal tissue to the aqueous humor. An artificial cornea is sandwiched between the plates, and the complex is then sutured into the patient’s own eye like a standard graft.
18
Q
B-KPro advances in the last 10 years?
A
- Introduction of a daily dose of a topical antibiotic prophylaxis to reduce the rate of infectious inflamed cornea and of tissues inside eye.
- To monitor chronic inflammation a smaller version has been inserted into rodents and their cytokine levels monitored. Have explored the use of tumor necrosis factor-alpha (TNF-α) inhibitors, such as Infliximab®, in decreasing the levels of inflammation in patients with B-KPro.
- Optic nerve can be damaged in surgery causing glaucoma- in a study on 106 eyes after insertion- 26% developed de novo glaucoma afterward and 31% developed Disc pallor.
- In order to ensure intraocular pressure to not rise too high to cause glaucoma- an ahmed glaucoma valve can be inserted with the keratoprosthesis and acts as a drainage implant to keep pressure down, so does not compress the optic nerve. Hard to measure intraocular pressure without normal cornea.
19
Q
WHat structure can be used for regenerative approaches for corneal epithelium?
A
limbal epithelial stem cells in the corneal rim at the border between the cornea and sclera. Undulated niches seen near pupils.
20
Q
limbal stem cells divide how?
A
Asymmetric division- one will stay as a stem cell in niche and one daughter cell will move out.
Transit amplifying cells- as proliferate and differentiate in the basal layer.
Most mitotic in wing cell layer.
When cells terminally differentiated move up to the squamous layer on top of epithelium.
21
Q
When have Limbal stem cell deficiency?
A
By: Anaridia, sclerocornea, thermal, alkali or acidic burns.
Results in: conjectiva overgrowth compensation but this results in tissue loss due to loss of transparency.
22
Q
Fix for limbal stem cell deficiency?
A
Direct transplant from patients healthy eye to diseased/injured.
or
take healthy limbal epithelium to seed a culture to produce a sheet to then implant.
23
Q
Disadvanatge of Direct transplant from patients healthy eye to diseased/injured for LSC deficiency?
A
This relies on the other eye being healthy. Donor site damage, two surgeries.
24
Q
How can LSCs be cultured to implant?
A
Cultured on plastic (adherent). Then removed by adding trypsin or scraping. But want them to stay in one sheet and therefore best to use temperature responsive dishes. Cells adhere at 37degrees, but if placed at 20degrees can be lifted off. Move from a hydrophobic to phillic state of plastic.
25
First stem cell theapy to be approved in western world?
In 2015 Europe approved use of stem cell therapy for stem cell therapy (Holoclar) - Expanded autologous LSCs for patients with LSC deficiency eg ocular injuries. (Corneal epithelia replacement)
26
Holoclar what is it?
Biopsy taken from the patients healthy eye limbus, to extract limbus stem cells from. Thesse can be frozen until ready for surgery (primary cell culture) and proliferated into secondary for a corneal epithelial replacement.
Holoclar = cells + fibrin matrix.
27
Holoclar advantages?
75/104 patients study showed:
- stable corneal surface with no surface defects.
- Little or no in grown blood vessels (good- avascular)
- reduction in pain and inflammation.
- Vision improvements.
- Autograph- no rejection.
28
Holoclar disadvantages?
-If Genetic disease all cells will be affected so may not be able to take from the other eye.
-Delay to expand own cells.
-Graft from healthy eye- risk damage to this eye.
HENCE WHY OTHER SOURCES EXPLORED
29
Other source of stem cells explored for stem cells for corneal epithelia replacement?
Mucousal stem cells from mouth- epithelial sheet formed, when culture for 2 weeks on temp sensitive culture surface. No damage to the other eye involved or scarring in mucosa. Risk of neovascularisation though as different stem cells.
30
Mucosal stem cells for corneal replacement study: results? 2004
2004 study: Oral mucosa taken from patient cultivated and implanted into 4 human patients.
Complete reepithelialization of the corneal surfaces occurred within one week in all four treated eyes. Corneal transparency was restored and postoperative visual acuity improved remarkably in all four eyes. During a mean follow-up period of 14 months, all corneal surfaces remained transparent. No complications.
31
Pathological wound healing of corneal stroma?
Normally keratocytes are quiencence but under TFG-B and IL-1 after stromal injury they differentiate into fibroblasts and under TGF-B into myofibroblasts Physiologically should apoptose after wound healing but in pathological conditions the Myofibroblasts lay down EXCM proteins in an irregular pattern and the cornea loses its transparency.
32
Corneal Stromal graft options?
E.g. Biomaterial based- human collagen scaffold, or self-assembling peptides, or decellularised cornea.
or using limbal stromal stem cells.
33
Human collagen as corneal graft?
Recombinant human collagen cell free implants. Acellular when implanted and cells invade and remodel it. Endogenous cell recruitment has been shown (stromal cells populated and nerve repopulation)
34
Sucess of human collagen as corneal graft? (+) (-)
+Endogenous cell recruitment has been shown (stromal cells populated and nerve repopulation)
+No cells so no need for immunosupressents.
+Regenerated neo-corneas stably integrated.
+ transplanted into 7 patients, vision improved and pain relived.
-Visual acuity could be improved, add a synthetic lipid> Not appropriate mechanical structure- refractive power less.
35
How can limbal stem cells be used for stroma?
Also have for stroma.
Isolate and culture, these implanted remodel scarring in model animals, supress fibrotic scar formation, have anti-inflammatory properies- prevent neutrophil recruitment.
On going trials for.
36
Study on how stromal LSCs can be made and inserted into the eye?
scaffold-free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs. . Human corneal stromal stem cells (CSSC) were cultured on substrates with aligned surface microgrooves which directed parallel cell alignment and matrix organization, similar to the organization of native corneal stromal lamella.
They secrete collagen which acts as the EXCM.
After being transplanted into mouse corneal stromal pockets, the engineered corneal stromal tissues became transparent.
CELL GENERATED BIOMATERIAL
(not tested in humans yet)
37
Struggle with the corneal endothelium for TE?
It doesnt regenerate. At birth there are 3500-4000 cells per mm2 but these gradually die by 85 around 2300/mm2 (only need 500 to function).
They have a finite lifespan and limited proliferative ability.
38
Corneal endothelial TE replacement?
2018 study: Induced pluripotent stem cells explored (iPSCs).
Derived from human with normal ocular history. cells were drived towards corneal endothelial fate by adding differentiation factors to the media. Took 25 days.
They displayed a hexagonal, tightly-packed morphology and express transcripts and proteins that are established markers of mature corneal endothelial cells.
Future studies needed on their ability to pump water and meet metabolic demands before implantation.
39
What part of the body is most challenging to engineer?
The nervous system, especially CNS.
40
Difference between regeneration in PNS and CNS?
PNS promotes repair, whereas CNS inhibits it. Whereas Schwann cells line up to guide axon repair, myelin cells inhibit regeneration (Nogo?) and macrophages infiltrate a lot slower into the CNS (BBB v tight). Astrocytes also in the CNS assume 'reactive phenotype' and produce glial scars inhibiting regeneration.
41
Epidemiology of PNS injuries?
9000 cases in UK each year. Mainly young populations e.g. car accidents. Not working etc- healthcare, financial and societal burden.
30% cases are lacerations (tear) or compressions als common.
42
PNS anatomy?
Individual axons surrounded Schwann cells and by endoneurium.
Have the axons bundled together- surrounded by Perineurium.
These fascicles bundle to be surrounded by epineurium to create the nerve fibres.
43
Why is there a critical gap length the body can repair severed nerves?
The gap at which regeneration occurs at 50% of the time. At longer length the fibrin cable thins and isn't robust enough to provide a platform for regeneration. If too large gap fibrin cable wont form at all.
Also the CT of nerves allows 10-20% elongation, but for every 8% stretch, there is a 50% reduction in blood flow (complete ischaemia at 15%).
44
3 degrees of nerve damage?
1) Neuropraxia- reversible conduction block (little structural damage)
2) Axonotmesis- complete interruption of axon and myelin (peri and epi in tact)
3) Neurotmesis-nerve and surrounding stroma are disconnected, no spontaneous recovery- atrophy.
45
If axon damage what will happen to the distal end?
WALLERIAN DEGENERATION
protease degeneration as metabolic activity etc in proximal end in cell body.
Debris is created which macrophages will engulf and clear up (2 weeks) Schwann cells clear up and take on the 'progenitor phenotype'- they proliferate and align in tracts to guide regenerating axons- Bands of bungner. (3 weeks).
Regenerated by 3 months.
46
Neural tissue repair methods? (3)
surgical reconstructions
Grafts (autologous/ allogenate)
Nerve conduits.
47
Direct surgical reconstruction of nerves?
Reattaching the proximal and distal nerve bundles by suturing. This can only be done if they are very close to each other. If too far away- decrease blood flow e.g. for every 8% stretch, there is a 50% reduction in blood flow (complete ischaemia at 15%).
48
Autologous grafts for PNS injury? + and -?
```
Gold standard.
+Low immune risk
-LOF at donor site
- 2 surgeries required
-Limit of size and type of nerve can use e.g. sensory as just below skin.
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49
Allogenate grafts for PNS injury? + and -?
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Same species donor.
+no second surgery for patient
+ no LOF at donor site
-high immune rejection risk
-Limitations in availability.
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50
What are nerve conduits?
Hollow structures that form between the proximal and distal nerve stumps to guide axon regeneration- Prevent infiltration of scar tissue and increased concentration of cytokines and growth factors and support cells here.
Natural but can TE.
51
Sequence of events of nerve conduits guiding axon regeneration?
1. conduit fills with plasma from the nerve stumps- fibrin EXC matrix proteins etc to help regeneration (hours)
2. Fibrin cable forms which creates a scaffold to enable schwann cell migration and guides axon regeneration. (days)
3. Cell migration and axon regeneration (months)
4. resulting tissue is noticeably thinner not at 100% function, but working (years)
52
Ideal nerve conduits should be..?
- Porous to allow plasma through
- Malleable- too stiff can injury nerve/tissue
- Biocompatible
- Biodegradable- within years (don't want a removal surgery but takes a long time to fix)
- suturable into the nerves
- sterilisable.
53
Types of TE nerve conduits? (2)
Decelluarised nerve conduits- top down
| Bioengineered- bottom up
54
Decellularised nerve conduits + and -?
```
+Will have pores
+retain 3D structure/ architecture.
+Hollow
+remove antigens (no immune reaction)
+can be xenograft as well as allograft
+Clear channels for axons to follow ensure good distribution throughout the nerve.
```
-From cadavers-may have limited supply.
55
Decellularized nerve conduits in pre-clinical study?
Decellularized (detergents) sciatic nerve (biodegradable, collagen-based nerve conduit) has outer wall to prevent scar tissue infiltration and a porous inner structure to allow axonal growth.
56
Decellularised nerve conduits on humans?
Avance® grafts of 0.5–3cm- Evaluation based on infection, rejection, and graft extrusion revealed that nerve sensations were restored within 9 months.
57
Bioengineered nerve conduit natural options? + or -?
Chitosan (weak degradability, low structural integrity)
Collagen (Trials partly recovered axon function)
fibrin (saw axon regrowth, easy manipulation, angiogenic, glial cell invasion)
Silk- (nervous tissue colonisation, mildly inflammatory, biocompatible/ degradable, good integrity)
Gelatin- (economical, schwann cell proliferation, but low integrity, unmyelinated axons)
58
First Collagen nerve conduit success?
FDA approved. First trial NeuraGen advantage was seen as no compression neuropathy was seen like other more rigid materials used at the time. nerve regrowth in 4 weeks.
59
collagen nerve developments since NeuraGen?
newer alternatives are: resorbable (4-8months), flexible, non-friable, semipermeable tubular matrices with a pore size in the range 0.1–0.5 μm.
Can be electrospun, and gradually release neurotrophic factors.
Crosslinking with Chitosan- also improved cell carrying ability.
60
Synthetic nerve conduits?
PCL- sheffield research, from CAD file can design exactly what want- 3D UV printing. As good as graft.
Regenerated length correlation with width of conduit, and improved with aligned schwann cells encapsulated. Also cell adherance better if make less hydrophobic (O2 or acid).
Balance complexity with cost- want to keep below £1000.
61
How can we increase the critical gap length?
Instead of using a hollow tube to enhance nerve regrowth, use a solid tube which will mimic the nerve so it doesn't need to regrow.
E.g. combinatory approach- EXCM components, cell grafts that promote regen, interluminal support, neutotropic factors.
62
How can we increase the critical gap length?
Instead of using a hollow tube to enhance nerve regrowth, use a solid tube which will mimic the nerve so it doesn't need to regrow.
63
Examples of the 4 approaches to increase the critical gap length?
```
E.g. combinatory approach of :
- EXCM components,
cell grafts that promote regen,
interluminal support,
neutotropic factors.
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64
EXC M components help to bridge the critical gap how?
In preclinical studies Matri-gel promotes regeneration: This is taken from mice carcinoma cells and contains a mix of EXC proteins e.g. collagen, laminin, fibronectin etc, but this will have to be remade for use in humans as this varies too much batch to batch for clinic use.
65
EXC M components matrices to bridge critical gap should be?
Weak, Viscoelastic hydrogels with high water content (too viscous would prevent growth cones of axons penetration)
66
Intraluminal supports help to bridge the critical gap how?
Create hollow scaffold tubes for the axons to follow. These can be highly functionalised to guide axons optimally.
67
Intraluminal supports help to bridge the critical gap how?
Create hollow scaffold tubes for the axons to follow. These can be highly functionalised to guide axons optimally e.g. functionalise inner surface, electrospun for pores, intraluminal guidances, microgrooved luminal design etc.
68
Success of an intraluminal support?
2012 patient 3cm median nerve defect in arm (previous critical gap length 2cm) regenerated by implantation of a chitosan–PGA nerve guidance conduit. Could redo fine movements e.g. holding chop sticks etc. Muscle AP's measured- present. Sterilisable. Made by liquidifcation over a mould.
69
Neurotrophic factors in nerve conduits?
Nerve GFs and neurotrophin-3 also.
Support axonal growth, migration and proliferation of schwann cells.
Best with controlled release e.g. encapsulation in scaffold- like BMP example.
70
Cells use in nerve conduits?
GE from stem cells? Then don't need to encapsulate neurotrophic factors as the cells make them.
Take bone marrow MSCs and can differentiate into schwann cells. Autologous Schwann cells for the repair of segmental peripheral nerve defects. The cells can be delivered inside a hollow tube, seeded in a hydrogel and subsequently filled into the lumen of the conduit, or placed inside a conduit containing a supportive structure. Last two have improved outcomes but more technical and expensive. BUt very high numbers of cells needed- donor site LOF.
71
Neurolac?
Peripheral Nerve conduit able to bridge the gap upto 2cm. Made of PLCL, but thoought to induce nerve damage as too rigid. Addition of more pores inhibited the nerve regeneration as the scaffold degraded too quickly.
72
Trade off with drug testing model?
Trade off between physiological relevenace (e.g. human body best model) and experimental tractibilit (e.g. single cells easiest to work with).
73
Current limitations of drug testing? (6)
- 10years average
- Inefficient, 5-10,000 chemicals in drug discovery, to only 5 in clinical trial to maybe only 1 approved.
- Very expensive $2.5billion per drug
- Drying pipeline- less drugs getting licenced
- adverse reactions- 20% of acute kidney injuries due to drug nephrotoxity. Drug testing models for humans aren't humans so unpredictable
- Drug testing not personalised unknown patient impacts
74
Example of bad reactions after drug marketted? (2)
Vioxx- for Angina- 88,000 had heart attacks and 38,000 died in US.
TGN1412- MAB for leukemia/arthritis. Phase 1 trial 6 patients died, despite being tested on primates at 500x dose. Major organ failure.
75
Main causes of drug bad reactions?
Hepatotoxicity or cardiotoxicity
76
Three models used for in vitro drug testing? (+ or -)
1. Primary cells- (-) Batch to batch variability- e.g. taken from biopsies e.g. patients. (+) e.g. can take hepatocytes to test hepatotoxity- but may not be representative of organ
2. Transformed/immortilised cells- (+) less batch to batch varaibility- can proiferate indefinitely. (-) transformed primary cells may change behaviour/ phenotype
3. Stem cells- (-) ethics, sourcing, not cell type specific (+) Can differentiate into any desired cell
77
What are immortilised/transformed cells?
Immortilised cells are cells that have been mutated to be able to proliferate indefinitely. Transformed cells are genetically altered e.g. trasefectio with AB resistance and GFP etc in a virus.
78
In vivo drug testing?
Animal models- not humans can't be sure of toxicity e.g. TGN1412 drug even on primates.
79
Organoids criteria?
1. Contain Organ specific cell types.
2. Capable of recapitulating some specific functions of the organ e.g. excretion, filtration, neural activity, contraction etc dependant upon organ
3. Grouped cells spatially organised similar to an organ.
80
components/how make an organoid?
Stem cells or progenitor cells aggregated into a 3D shape, with soluble cues (GF and cytokines) coaxed into self organising into an organoid.
81
WHat is an organ- on-a-chip?
Device for culturing cells in continuously perfused, micrometer-sized chambers that incorporates minimal units that mimic tissue- and organ-level functions. Used for drug disovery and testing.
82
How are organs-on-a-chip classically made?
1. Polymer like PDMS used and mixed with a crosslinking agent.
2. Liquidified and poured over a mold (specifically designed, and has channels pours).
3. Cooled to a solid and pealed off/
4. Put on a glass slide.
5. Puncture holes in the glass slide so can add liquid in and out- perfuse.
83
Tests for the organ-on-a-chip manufacture?
- Check perfusion- food dye- if want even, or gradients created etc.
- MOnitor cell growth and interactions under microscope
- Check biocompatable- no affecting the cells.
84
Organ on a chip fluid movement?
Microfluids- normally linear due to such small scale, have more control over. WHereas at macroscale can get turbulent flow.
85
Typical components of an organ on a chip? (4)
1. Geometrical confinement and patterning- control where cells placed and how they interact.
2. Presence of flow.
3. Environmental control- PH, O2 etc
4. an add different parameters e.g. sensors, electrodes, contractile tissue etc depending on the organ trying to replicate.
86
Functional unit for lungs used for organ on chip? Layers?
```
alveolar to capillary unit at the air blood interface.
3 layers were created.
Epitheliium cells (alveolar) were placed on top of a pourous membrane, with enodthelium (capillaries) below. The upper chamber represents the air and lower filled with medium to represent the blood.
The side chambers were connected to a vacuum connected to a computer, to vary and cause cyclicial stretching (couldnt be done to 2D structures)
```
87
What tests were done on the alveolar organ on the chip?
epithelium marker of occludin was used and endothelium cadherin- formed distinct bands- shows separation.
Check viability of cells- close to 90% over 20 days.
And the epithelial cells secreted their own surfactant like in vivo.
88
Steps for pulmonary inflammation? (and what)
(Acute- pneumonia or chronic asthma)
1. Pathogens, toxins or allergens cause the epithelium to release TGF alpha.
2. Activation of endothelium causes upregulation of adhesion receptors like ICAM1.
3. Leukocyte infiltration into the alveolar after rolling along surface and adhering.
89
How was organ on a chip used to model pulmonary inflammation? Checks?
Add TGF alpha to the epithelia and infuse leukocytes into the lower 'blood' chamber.
Monitor: Immunocytochemistry for ICAM1 expression on endothelial cells. And GFP label leukocytes to see their infiltration into the top chamber through the pourous membrane into the 'alveolar'
90
What was the problem with pulmonary inflammation that they were trying to prevent in the organ on a chip? Work?
IL-2 administration to cancer patients- caused pulmonary oedema as a side affect, leading to respiratory failure.
Added IL2 to the lower capillary chamber, Noticed cells became more permeable and a menicus of fluid appeared in the top chamber.
Tested Ang-1 which had previoisly been shown to stabilise junctions between endothelial cells. The Ang-2 reduced permeability back down to levels of no IL-2 addition.
91
More advanced Organ on a chip alternative?
Body on a chip. Have all the organs/ tissues represented and linked. Can have this personlised to the patient also- cells from them and their plasma biomarker levels in the compartments etc, and exercise level (For cyclic stretch breathing pattern) More accuritely predict a patients response to drugs.
92
One of the pitfalls of TE of large organs?
Vascularisation. Cells ususally only located 100-200um from capillaries.
93
Differences in cells vascular need?
May be more/less sensitivities to oxygen. E.g islet cells in pancreas dysfunction if further than 100um from vasculature. Whereas Cartilage can be as far as 1mm or the cornea is avascular (easier to engineer)
94
First observation that showed the importance of vascularisation?
Tumor growth needed new vascular growth (angiogenesis). Saw that only the periphery of cancer tumour were alive when not vascularised and the centre was necrotic. Noticed diffusion was enough when the tumor was small but as grew needed angiogenesis.
95
Role of Vascularisation in TE?
1. Avoids graft necrosis (if no O2)
2. Allows generation of thicker tissues not limited by diffusion distance.
3. Endothelial cells in blood vessels secrete factors that help promote graft innervation
4. Improve graft function
96
Blood vessels structure?
Macrovessels (arteries and veins)- microvessels (arterioles/venules)-capillaries.
Endothelial cells line- important as secrete anti-thrombotic factors preventing clotting.
97
Three types (brief) of blood vessel formation?
1. Vasculogensis: De novo new blood vessel formation from progenitor cells during development
2. Angiogenesis: New blood vessel formation from existing blood vesself by extension/remodelling.
3. Arteeriogenesis: maturation of blood vessels via increase increase in the lumen size.
98
Vasculogenesis process?
Mesoderm differentiates into hemangioblasts which move to the surface under GF's as the embryo grows and patterns. These line up to form tubes creating the primary capillary network.
99
Angiogensis process?
1. Hypoxia drives. Induces the production of pro-angiogenic factors e.g. VEGF (vascular endothelial growth factor).
2. VEGF signals to endothelial cells, which secrete factors like matrix metalloproteinase (MMP).
3. MMP degrades the Basement membrane to create punctures.
4. Activated endothelial cells proliferate and create vascular sprouts.
5. sprouts elongate towards VEGF hypoxic signal.
6. Formation of a new lumen.
7. Mature blood vessels SMC and pericytes are recruited by PDGF.
8. blood flow to hypotoxic tissue so VEGF is decreased.
100
Physiological angiogenesis vs pathological?
Physiological: Wound heeling, ovarian cycle
Pathological: Cancer, rhumatoid arthiritis
101
VEGF to which receptors?
VEGF A, B and placental have preference to VEGFR-1- for vasculogenesis.
VEGF C,D preference to VEGFR2 (tumor angiogenesis) or VEGFR3 lympohangiogenesis
102
Arteriogenesis process?
1. Occlusion of a blood vessel will increse the blood flow in this vessel and exert more sheer stress on the endothelial cells.
2. The endothelial cells release GFs e.g. TGF beta.
3. TGF beta signals the degredation of ECM and proliferation of endothelial cells and smooth muscle cells.
4. Matrix remodelling to create collaterals.
103
Two ways vascularisation can be promoted in 3D constructs? (vague)
1. Strategies that facilitate vascular ingrowth e.g. Scaffold design- pours, and functionalisation
2. Prevascularisation strategies.- In vitro or in vivo.
104
How can scaffolds be functionalised for vascularisation?
Deliver growth factors within e.g. VEGF, PDGF, controlled release e.g. BDGF experiment.
105
Functionalisation experiment for vascularisation method and results:
Scaffold to deliver both VEGF (inititated angiogenesis but vesels leaky) and PDGF (matures BVs).
Method: Admimcing VEGF and encapsulating PDGF into PLG microspheres which gradually degrade- maturing later.
Alpha smooth muscle staining
VEGF only- small vessels not mature
PDGF only-leaky vessels
Dual release: lots of BVs and more mature and larger
Highlighted the importance of multiple GF delivery and timings/kinetics.
106
Disadvantages to Functionalisation experiment for vascularisation?
Time consuming process.
Very slow microvessel growth rate at 5um/hr, which may not be sufficient to prevent necrosis in 3D constructs after implantation.
107
Prevascularisation in vitro? (+ and -)
The TE construct is cultured to build prevascularised structures which can then connect with existing blood vessels. (Anastomose) which is faster than angiogenesis.
(-) In vitro endothelial cells start to form BV structures but mature? Working with endothelial cells is problematic- finite proliferation.
(+) NO extra surgeries/ donor site morbidity.
108
Prevascualrisation in vivo?(+ and -)
Scaffold placed into highly vascularised tissues. Microvessels ingrow from the host. Can then excise this scaffold out and implant into the defect site. Use the body as the bioreactor.
(+) Mature organised vascularature.
Problem: Involves three surgeries for the patient. (Place scaffold, remove and then place at damaged site)
(-) Scaffold could be coered in fibrous tissue from first implantation.
109
Prevascualrisation in vivo? Flap technique? (+ and -)
Scaffold implanted into a muscle flap and microvessels in grow. After vascularisation the entire muscle flap is transferred to the site of damage.
(+) Can connect larger blood vessels so immediately join suture to host site.
(-) Sacrifice muscle flap.
110
Prevascualrisation in vivo? Arteriovenous AV loop technique? (+ and -)
Uses veins or synthetic graft to form a shunt loop between a host artery and vein in the hosts body, and vasculature will naturally sprout from. The loop can be placed in a protected chamber and when vascularised transferred to damged site-
(+) Tissue not embedded in surrounding tissue so no major donor site morbidity.
111
Three bottlenecks for translating TE constructs into the clinic?
Vascularisation and host response, and regulation and commercialisation.
112
To what in RM do you get an immune response, and when an inflammatory response?
Living cells- immune response. (e.g. antigens)
| Biomaterials- inflammatory response.
113
How can the biomaterial interact with the tissue?
Biomaterial can change tissue wound healing, cause infections, toxicity, tumorigenicity, hypersensitivity.
114
How can the body tissue interact with the biomaterial?
Body can try to degrade the tissue by enzymatic activity, calcification, abrasion, corrosion.
115
Three stages in the healing process?
Inflammation- Proliferation- Remodelling.
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Persistant tissue damage leads to?
Fibrosis
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The foreign body response to TE products?
1. Injury to tissue by surgery- blood flows out (ECM)
2. Adsorption of proteins immediately e.g. Fibrin layed down on the scaffold, adhering to the material.
3. Scaffold allows adherance of immune cells. Macrophage attraction and contact causes their activation.
4. Macrophages release cytokines to create a chemotactic gradient to attract others (IL-1B, TNF-a). Covered in couple of hours.
5. Macrophages want to clear up the debris but biomaterial so large they cant.If diameter 10um or less they phagocytose, but for larger than 100um, macrophages fuse together to form a foreign body cell.
6. Fibroblasts and lymphocytes attracted. 1 week macrophage inflammatory layer over.
7. FIBROUS ENCAPSULATION. Fibroblasts lay down collagen, causing an isolation of the biomaterial from the local environment of the hosts tissue 'foreign body granuloma'. BAD and unpredictable (depends on polymer)
8. Vascularisation after 2 weeks.
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What is fibrous encapsulation?
Tissue response to implanted biomaterials.
Abundant deposition of extracellular matrix e.g. fibroblasts laying down collagen. Isolation of biomaterial from the local tissue environment.
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Example of material bad for fibrous encapsulation/good for?
PVDF- less inflammatory layer so less fibrous capsule width, decreased macrophage stimulation. Pores not filled in. Minimal scar and granuloma and mesh flexibility retained.
PP- increase width of granuloma. Scar tissue bridges gaps between mesh pores- losing pore structure.
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When is fibrous encapsulation particularly bad?(and bad generally)
If implanting microelectrodes or have factors to release from the scaffold. Also impedes vascularisation and diffusion.
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Infection atfer biomaterial implantation?
5-10% of cases. Some localisalised but other progress e.g. sepsis. Bacterias tend to form a biofilm over materials.
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Slime that can form on biomaterials?
Bacterial biofilms, ancient adaptation that allows the bacteria to have coordinated behaviour and act as one. 'extracellular polymeric substance'.
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Stages of biofilm development?
1. Floating bacteria (Planktonic) attatch to the material.
2. Secrete slime.
3. adhere to each other and irreversibly to the material
4. Proliferate to create a multilayed structure.
5. Mature- some bacteria become planktonic again and dispurse to colonise other sites.
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WHy is biofilm so problematic>
Antibiotic resistant.
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Bacteria particularly prone to making biofilms>
Staphylococcus, or resistant strains like MRSA
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What determines whether biofilms form around TE materials? Idea?
What gets there first, whether bacteria populate before cells. if cells get there first, they populate and outcompete the bacteria, and vice versa.
Can we functionalise the material so human cells can attatch and not bacteria?
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Can we functionalise the material so human cells can attatch and not bacteria?
Add adsorbed proteins to non fouling surfaces (bacterial colonisation resistant)? Useful for medical devices to as dont need cell interacts. Add RGD domains to certain areas by microstamping to TE constructs?
Biomaterials with bacterial repelling proteins, but allow human cells to bind. SaNet
Differentially instructive matrices: E.g micronets
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MIcro-nets development to hinder bacterial growth? (ER)
SaNet (self assembling net) hinders bacterial adherance but promotes human cell adherance. SaNET lacks known cell recognition motifs but cell adherance was similar level to previously used materials, but the unique architechture promotes cell to mesh interactions. Had increased filapodia development.
The continuous arrangement of the domains can thus be viewed as an antimicrobial “carpet” potentially resistant to bacterial adhesion and colonization (e.g. P.aurginosa. Resistant to bacterial adherance in first few cruicial hours.
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How can biomaterials be tumourgenic/ toxic?
Bi-products of their wear can be released e.g. hip replacement- cobalt gradually released- bind to other proteins in the blood and presented to immune cells- trigger immune response. Need to ensure not carcinogenic etc- orthopaedic implant related osteocarcinoma- difficult to prove caused it
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Thromboembolic risk with biomaterials?
Exposure of blood to artifical material can cause thrombosis e.g. STENTS. BV lined with glycocaylx which biomaterials dont have. Need anti-coagulant drugs.
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humoral vs cellular immune system response?
HUMORAL IMMUNITY: mediated by soluble antibodies produced by B lymphocytes
CELLULAR IMMUNITY: mediated by T lymphocytes
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Examples (2) of cellular only regenerative medicine strategy?
Adding single cell that the patient needs.
E.g. Diabetes: “The Edmonton Protocol” – successful islet transplantation from cadavers. Immunosupressants needed.
Or Addition of dopinergic neurons for parkinsons.
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Technique of cell delivery where immunosupressants may not be needed?
Immunoisolation.
Add cells into a selectively permeable membrane. This allows metabolites out of the membrane and recieve nutrients, but immune cells and antibodies can't pass through. Can encapsulate around 100 cells or microencapsulate/ ultra thin encapsulation only a few cells- this increases diffusion of nutrients to cells.
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immunoisolation of cells in clincial trial?
Diabecell up to phase II
hES.Cs differentiation into B islet cells (pig). Used alginate- which was quite food at preventing fibrous encapsulation (prevent cells getting nutrients), but this is still the major bottleneck.
