1
Q
Define morphogens
A
Inductive signals (e.g. growth factors) which initiate/govern tissue morphogenesis
2
Q
Define stem cells
A
Primordial progenitors
Have enormous potential
3
Q
What are biomaterial scaffolds?
A
They mimic ECM
4
Q
What are major prostheses used for?
A
- load transmission (e.g. tendon/ligament replacements)
- bearing surfaces (total joint replacement)
- controlling fluid flow (heart/vascular/urethral replacements, ventricles valves for CSF control)
- passive space filling (cosmetic/rhinoplasty)
- functional space filling (cranial plates to protect the brain)
- generation/application of external stimuli (pacemakers)
- light transmission (intra-ocular prostheses)
- sound transmission (ossicular replacement)
5
Q
Requirements for biomaterials
A
- non-toxic/bioinert/biostable
- bioactive/biodegradable
6
Q
What happens if a biomaterial is not bioinert/biostable?
A
Toxic -> inflammation when implanted -> repair -> restimulation of inflammation -> resorption + cell necrosis + de-differentiation
7
Q
How do biomaterials become bioactive/biodegradable?
A
- form chemical bonds with tissue stabilizing them
- get resorped in the body when no longer required (sutures, drug capsules)
- use biological components (cells/biomolecules) within implants
8
Q
Limitations of transplantation
A
Donor shortage Immunological rejection Ethical issues Not available for all tissues/organs Transmissible infective agents in animals and humans
9
Q
What is the difference between tissue engineering and tissue regeneration?
A
Tissue Engineering = in vitro (outside)
Tissue Regeneration = in vivo (inside the organism)
10
Q
What are the 3 aims of tissue engineering?
A
To repair, replace and regenerate tissues
11
Q
What do we mean by tissue repair?
A
Manipulate
Deliver cells/materials
12
Q
What do we mean by replacing tissues?
A
Transplantation
13
Q
What do we mean by tissue regeneration?
A
Stimulating new growth
14
Q
What are the principle steps of tissue engineering?
A
1 - cell isolation from a source 2 - cultivate in 2D 3 - seeding in 3D porous scaffold 4 - tissue organization 5 - engineered tissue transplantation
15
Q
What are the pros of using isolated cells or cell substitutes?
A
- avoids surgery
- allows replacement of only those required functional cells
- allows manipulation of cells before infusion
16
Q
What are the cons of using isolated cells or cell substitutes?
A
- failure of infused cells to maintain their function in the recipient
- immunological rejection
17
Q
What are the pros of tissue inducing substances?
A
- development of methods to target molecules
- large scale production of signal molecules
- purification of signal molecules such as growth factors
18
Q
What are the pros of cell seeded on or in scaffolds?
A
- open or closed systems
- natural materials such as collagen and/or synthetic materials
- immunological acceptance with use of immuno-suppressive drugs or autologous cells
19
Q
What is a tissue engineered biological substitute comprised of?
A
- cells
- ECM
- scaffold
- signals
20
Q
What are some issues with cells?
A
- finite lifespan then expire
- they generate newly differentiated cells through multi-step lineage pathways
- newly differentiated cells replace expired ones
- they then fabricate ECM and new tissue
21
Q
What do we need to consider when choosing a cell type?
A
Availability of cells:
- embryonic stem cells? (but a lot of ethical issues)
- autologous (donor to donor)
- allogenic (donor to recipient)
Cell Phenotype
Cell capacity to redifferentiate and produce ECM
22
Q
How is cell signalling a challenge?
A
- cells exchange mechanical and chemical signals
- these are essential in determining cell fate/phenotype and tissue homeostasis
- the signals are mediated by cell surface molecules
- cell surface molecules transduce them across compartments of the cell
- communication between cells and ECM (scaffold) needs to facilitate this and consider how porosity and diffusion will play a part
23
Q
How can scaffolds control cell signalling?
A
Can be engineering to be a source of signaling molecules promoting cell differentiation
24
Q
What are some features of intelligent scaffolds?
A
- hold/attract cells
- influence cell development
- reserve space for regeneration
- inhibit inflammation events/immunoprotective
- breakdown into active factors
- encapsulate morphogens, cytokines and MMPs
- provide mechanical stability
- facilitate integration
- contribute to final events
25
What are some challenges with scaffolds?
- porosity is essential for repair/regeneration
| - 3D is essential to allow cellular function without compromising mechanical integrity
26
What are some examples of scaffolds?
```
Gel
Sponges
Fibres
Natural (collagen, fibrin)
Synthetic (PEG)
Carbohydrate based (PLA, PLGA, agarose, alginate
```
27
What does is it mean to model something?
To test this prostheses/material onto something
- this may be in vivo or in vitro
- may be on a organ-on-a-chip
- may be on an animal
28
What are some challenges with modelling?
- in vivo or in vitro
- need to maintain cell viability via GF
- may get large necrotic regions as there is limited transport in the centre of 3D scaffolds
- need to enhance mass transport with mechanical conditions and creating fluid environments in a bioreactor
29
What are bioreactors?
Establish and maintain 3D culture
Provide mechanical conditioning
Enhance tissue regeneration
Provide transport system for nutrients to cells
Allow withdrawal of toxic/inhibitory metabolic products
Allow mechanical conditions to a 3D scaffold
Enhance cell differentiation and tissue development
30
What are some mechanical challenges?
- little is known about mechanical forces required to maximally stimulate matrix formation
- complexity of mechanobiology
- effects of mechanical signals on matrix synthesis will vary greatly with each different scaffold system
31
What do manufacturing processes require?
- tested cells from working cell banks
- automatic injection into tissue bioreactors
- computerized system to monitor growth and conditions
- frozen tissue
- quality control for cells and matrix
- processed in bioreactors until clinical use
32
Define tissue engineering
In vitro
| Seed cells into a biomaterial, grow matrix and then implant into body/model
33
Define regenerative medicine
In vivo
| Implant biomaterial matrix with or without seeded cells into body to facilitate regeneration of the tissue
34
What are the key principles in tissue engineering?
Cells + biomaterial + bioreactors + factors ->
Engineered tissue ->
Transplantation
35
What are some general considerations when choosing tissue engineering methods?
- choice of cells/starting material (do you need to make some genetic modifications? do you need to isolate and purify?)
- choice of culture media
- phenotypic characterisation
- seeding logistics
- growth conditions (static on a plate or dynamic with a bioreactor)
- 2D vs 3D models
36
What are simple tissue types?
FLUID
- bone
- bone marrow
37
What are complex tissue types?
Not fluid => TISSUE
38
What are some conditions for growth?
- serum
- vitamins
- glucose
- glutamine, methionine, serine
- antibiotics
39
What is a primary culture?
Coming from a living organism (e.g. adult, embryo, gametes)
40
What are some methods for cell isolation from a primary culture?
- you have your primary culture
- aim is end up with single stem cells to grow on a dish
- may do this by mechanical dissection to break up major tissue organisation so you can get the cells you want (hepatocytes from a liver)
- rendering cells from soft tissue
- rendering specific cells non soft tissues is more complex
- then finally compose growth media (micronutrients + serum/serum free)
41
Pros of autologous cells
- promote tissue regeneration
- no inflammation/immunogenic response
- 10 fold expansion with primary cells
42
Cons of autologous cells
- limited availability
- low yield (cell concentration)
- further expansion without reprogramming is difficult
43
Pros of allogenic cells
- potentially more cells at isolation
- could be used to treat more than 1 patient (cell bank)
- 10 fold expansion (even more with reprogramming)
44
Cons of allogenic cells
- inflammation
| - immunogenecity failure
45
What does anchorage independent cells mean?
- are anchored to a culture for example (haematopoietic cells are expanded in suspension culture)
- vs. anchorage dependent are cultured in monolayer (most human cells)
46
What are the 3 phases during growth in a monolayer
1) Lag phase
2) Exponential (log) phase
3) Plateau phase
47
What happens during the lag phase?
- adhesion to the surface
- recovery from isolation/subculture
SO NEED TO
- precoat to enhance adhesion
- gentle subculture
48
What happens during the exponential (log) phase?
- rapid division
SO NEED TO
- feed regularly
49
What happens during the plateau phase?
- primary cells have finite life span
- division slows/stops after 50 divisions when you reach the Hayflick limit or if there is contact inhibition
- cells start to show transformation
SO NEED TO
- subculture
50
What are some properties of transformed cells?
- altered morphology
- loss of contact inhibition (cells grow over each other)
- proliferate with attachment (anchorage independent)
- proliferate indefinitely (immortalisation)
- reduced requirement for mitogenic GF
- tumorigenicity
51
What culture conditions are key for tissue engineering?
- defined culture media (induce differentiation of progenitor stem cells)
- biomaterial scaffolds (provide 3D cell culture environment + influence mechanical properties of engineered tissue)
- bioreactor systems (promote spatially uniform cell seeding, mass transport, biophysical stimulation)
52
What culture conditions determine cell fate?
```
Physiological = temp, pH, osmolarity, oxygen, 3D environment, cell to cell contact
Biochemical = nutrients, small molecules, GF, nanoparticles
Physical = compression, tension, hydrostatic pressure
```
53
List some scaffold requirments
- stability (size, shape, biomechanics)
- porosity
- biodegradable/remodellable
- absorbable
- biocompatible
- cell attachment
- bioactive/gene delivery
- surgical application
54
Which scaffold materials have the highest (+ lowest) structure stability?
Hydrogel (high)
Woven Mesh (intermediate)
Sponge (medium)
Non-woven mesh (low)
55
Which scaffold materials have the highest (+ lowest) porosity?
Non-woven mesh (high)
Woven mesh (intermediate)
Hydrogel + Sponge (low)
56
Which scaffold materials are naturally absorbable?
- agarose
- alginate
- hyaluronic acid
57
Which scaffold materials are biodegradable/synthetic?
- polyglycolic acid
| - PLGA
58
Which scaffold materials are remodelable?
Calcium phosphate
59
Advantages of biopolymers
- similar size to proteins
- large SA with functional groups/ligands
- rapid absorption and release behaviour
- rapid diffusion and volume change
- tuneable degradation kinetics and mechanical properties
60
What do innovative 3D models for cell culture involve?
- microfabrication techniques
| - microfluidics (deliver nutrients/agents, generate dynamic fluid flows)
61
What are the 3 principles of tissue engineering?
CELL TYPE
SCAFFOLD
MECHANICS
62
What is a rocking bioreactor?
Automated
Just need to provide nutrients and chemicals
Load cells and culture medium into cellbag bioreactor via ports on bag surface
Motorized base, rocking motion induces waves in cell culture for efficient mixing and gas transfer to respiring cells
Also want them to respond to changes (e.g. give more glucose when cells are producing lots of lactic acid due to respiration for example)
63
What are some examples of mechanics?
Compression -> dynamic and static
Dynamic is better to create
Then
Intermittent dynamic or continuous dynamic
Need to determine which is best for the type of tissue you are using.
64
What molecules can we use to influence cells?
```
GF
Chemokines
Cytokines
Hormones
(can cause growth, be protective etc.)
```
65
What is the effect of passage?
Also called splitting, method of culturing to keep cells alive.... BUT:
- cells change morphologically
- adhesion rate increases
- cell proliferation reduces
- collagen, proteoglycan and mRNA content is reduced
66
What does cell immortalisation mean?
Keep expanding + passaging indefinitely until eventually passaged so many times that morphology is very different
67
What is confluency?
When cells proliferate to cover 70% of the plate
68
How do we culture cells?
- isolate cells in a plate with culture medium (allows them to grow)
- cells expand
- cells reach a state of confluence
- need to split the cells into separate plates using trypsin
- cells now float up into the culture medium
- spin the cell population down and culture them
- continue to passage them forming 2 cell populations
69
What does trypsin do?
Breaks down integrins which make up the receptors allowing the cells adhering to each other and the plate
70
What phase do we want the cells to be in, to work best at?
Exponential Phase
71
What are the affects of cell passaging?
- increases cell concentration
| - negatively affects cell's ability to maintain phenotype
72
What type of model is best to maintain cell phenotype?
3D model!
Tissue Engineering & Regenerative Medicine
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