TE Flashcards
(162 cards)
1
Q
Definition of Regenerative medicine?
A
Umbrella term incompassing tissue engineering and research into self-healing, where the body uses its own systems, sometimes with help [biomaterials] to recreate cells and rebuild tissues and organs.
2
Q
Definition of Tissue engineering?
A
Multidisciplinary field aiming to develop biological substitutes to restore, maintain or improve tissue functions.
e.g. specific cornea or trachea replacements, or lab grown food or heart etc.
3
Q
Definition of cell therapy?
A
Where cellular material is injected, grafted or implanted into a patients e.g. in tact living cells e.g. T cells capable of fighting cancer cells or stem cells to a wound.
May need support by biomaterials (scaffold)
4
Q
Mechanical device examples used in the body? (3)
A
- Pacemaker
- Dialysis machine
- Hip replacements
5
Q
What is an artificial pacemaker?
A
generates electrical impulses delivered by electrodes to contract the heart muscles and regulate the electrical conduction system of the heart. Replaces the SAN for patients with arrhythmias.
6
Q
Diadvantages to mechanical devices use in the body?
A
Cant replace tissues- dont perform all the functions e.g. Hip replacements- may offer support as a bone alternative, but bone has other roles for example calcium homeostasis or bone marrow. Also doesn’t grow with patient if child.
7
Q
Transplantation disadvantages?
A
- Immunosupression tablets to avoid rejection.
- Transplantation crisis- ageing pop always demand over supply waiting lists v long.
- Due to demand may have to use less suitable alternatives e.g. from elderly, or less vigorous checks- in 2013 kidney given carried encephalitus and 2 donors infected and killed.
HENCE THE GROWTH OF TE SINCE 1990’s.
8
Q
Why growth of TE field?
A
- Transplantation demand etc,
- 1998 IPSCs derived which excited scientists and prompted a lot of study.
9
Q
3 components of TE?
A
Cells+ EXCM/ Biomaterial Scaffold + Bio-active molecules. (direct cellular behaviour)
10
Q
Stats on the Transplantation crisis?
A
3 people in the UK die everyday waiting for an organ for transplantation(2010) with 55,000 on the waiting list for kidneys alone, and only 17,000 kidney transplantations happening a year-
11
Q
Mouse ear study aim?
A
- Wanted to create an off the shelf preformed flexible yet structural slicone scaffold as an alternative to using autogenous costal cartilage- where the shape was less similar and involves host site mobidity and long operating times.
Evaluated feesibility of this, monitoring rejection, cell take over etc.
12
Q
Mouse ear study method?
A
1997.
Chondrocytes (calf cartilage cells) were seeded onto the polymer scaffold mold (3 year old ear). This was implanted subcutaneously onto a mouse and fixed with or without an external stent.
13
Q
Mouse ear study results?
A
1997
No implants were extruded or infected significantly. Without the stent the tissues lost their shape, but if kept for 4weeks maintained shape for the trail duration. Neocartilage grew over the structure. BruD labelling- proliferation of chondrocytes- still alive and AB to collagen 11- specific to cartilage.
(+) Scaffolds were populated by cells
(-) Skin Coverage was missing, Bovine Chondrocytes are poor model for human cells, Scaffold not v stable needed stent or collapsed. Growth rate was slower.
14
Q
Current treatments for organ failure?
A
surgical reconstruction
mechanical devices
transplantation
15
Q
From bench to bedside timescale?
A
Research: 1-5 years
Development: 3-5 years- preclinical and clinical testing safety.
Regulation: 3-5 years- regulatory review of testing results in small and large scale patient populations.
Commercialisation: Registration and post marketing survelliance.
16
Q
4 types of tissue are?
A
Epithelial (carry out functions) Connective tissue (support and nourishment) Muscle (support and movement) Nervous (instructions)
17
Q
3 phases of tissue repair in body?
A
- Inflammatory phase- stop bleeding, clear up dead cells and microbe protection.
- Proliferative stage- Day 4 to weeks - make new tissue but disorganised.
- Remodelling phase- week 4 to 2-3 years. Reorganse the new tissue.
18
Q
Inflammatory stage of tissue repair?
A
Leukocytes migrate through vessel wall into tissue (WBCs recruited by inflammatory mediators and chemoattractants e.g. cytokines). Leukocyes engulf cellular debris to prevent infection.
GF release by CT and attract fibroblasts and angiogenesis.
19
Q
Proliferative stage of tissue repair?
A
Resolution stage clears up the inflammation e.g. IL-1B macrophages engulf apoptosed neutrophils which have engulfed cellular debris.
Blastema is made of proliferating fibroblasts. New tissue is built to fill the wound space. Fibroblasts secrete collagen and GFs- promotes EC proliferation and angiogenesis. Epithelialization- proliferation, differentiation and migration at wound edge. Granulation tissue (EC+fibroblasts)
20
Q
Remodelling stage of tissue repair?
A
Granulation tissue is the foundation tissue for scar tissue. This is formed by synthesis and lysis of collagen simultaneously. Scar tissue is avascular and is 70-80% tensile strength by 3 months.
21
Q
How is the wound gap blocked?
A
- Cut blood vessels bleed into the wound.
- Blood clots to fill the gap and leukocytes in the blood clean up wound.
- Granulation tissue is layed down (collagen) by fibroblsts.
- Epithelium regeneration causes scar tissue formation
22
Q
When is no scar tissue formed?
A
If its a mild superficial injury that only damages the epithelium. If only proliferation stage needed, no remodelling. REGENERATION.
23
Q
WHen does scar tissue form?
A
If its a more severe injury with damage ti the tissue framework. Connective tissue deposition is needed due to a disrupted matrix. REPAIR.
Or Peristant tissue damage= FIBROSIS
24
Q
Regeneration vs repair?
A
Regeneration- only proliferation- no scar tissue.
Repair- need to lay down new structures, proliferationa differentiation and reorganisation- scar tissue.
E.g. repair a bike- change parts etc may be blue bike with red handles. Whereas to regenerate it- it will magically extend itself to make red handles.
25
Liver regeneration vs repair?
Regeneration: Proliferation of hepatocytes.
Repair of liver: If damage to cells and matrix- deposition of connective tissue- Hepatic fibrosis. If persistant tissue damage.
26
Fibrous encapsulation?
A fibrous CT layer that forms between the implant and surrounding tissue. Tissue response to implanted biomaterals/ medical devices results in injury to tissue and organs. Collagen deposition isolates biomaterial from local tissue environment, as body tries to protect itself.
27
sources of cells? (4)
Autologous cells: Cells from patients own body. NO rejection risk, but host damage.
Allogenic cells: Cells from same species but different person, try to match, can transfer disease
Xenogenic cells: From another animal e.g. Bovine.
Syngenic/isogenic cells: Genetically identical e.g. twins or very similar e..g mother.
28
Advantages and disadvantages of using Adult stem cells?
Bone marrow or tissue specific (more limited).
+ can get from the patient- no immune supressors.
-Low proliferative capacity, and no fully pluripotent.
-If genetic disease may not be appropriate.
29
Advantages and disadvantages of using induced stem cells?
Induced pluripotent stem cells- adult stem cells that are treated with signals to reverse their differentiation. +pluripotent. can keep cells and proliferate indefinitely.
30
Advantages and disadvantages of using embryonic stem cells?
+Pluripotent from ICM.
| -Ethics
31
Advantages and disadvantages of using differentiated cell types?
- terminally differentiated- low proliferative capacity.
+may be hard to get to S.C for bone marrow etc.
+ already functional e.g. fibroblasts, keratinocytees etc. from patient biopsy after proliferating.
32
How are cells cultured before TE?
Growth medium- GF's, nutrients, glucose, sugars, AA's salt etc- everything that would be in the blood.
Microbes also can grow in this so need asceptic technique.
Incubator at 37 degrees, let gaseous exchange so not sealed- but asceptic solution at bottom of fridge.
33
GMP?
Good manufacturing practice.
| For medicinal products- highly controlled and consistantly produced quality. Problem with batch to batch variability.
34
Roles of the EXCM in native tissues?
Most cells are adherent so need a structure to grow on In the body the EXC Matrix is the scaffold which cells grow on,and this needs to be replicated with biomaterial scaffolds.
Also provides the bioactive cues which need mimicing, acts as a reservoir for growth factors, contributes to mechanical properties and keeps renewal ordered.
35
Extracellular matrix proteins for strength?
Collagen and elastin.
36
Extracellular matrix proteins for lubrication?
Water hydrated gels e.g. proteoglycans, hyaluronan
37
Extracellular matrix proteins in EXC M to connect cells?
Adhesive glycoproteins fibronectin and laminin.
38
Collagen structure?
Most abundant protein in animal kingdom.
Proline, hydroxyproline, glycine repeats. Bonds between create a helix to make a collagen fibril, which come together to make a fibre.
Stronger than steel
39
Proteoglycan structure?
highly hydrophillic. Forms hydrated compressible gels.
| Composed of glycosaminoglycan chains linked to a specific protein core. Necessary at joints
40
How do adhesive molecules connect EXCM to cells?
adhesive molecules have a RGD sequence (Arg-gly-Asp) which cells recognise on fibronectin and bind to it. Cells ahve adhesion receptors which can bind to these adhesion molecules.
They can signal between then ie if stressed.
41
What receptors are found on cells which enable them to bind to the EXCM?
Integrin receptors.
42
Structure of integrins?
heterodimers. 19alpha and 8 B subunits varieties where the Composition determines what binds. Determines their specificity. Most integrins recognise several ECM proteins.
One Beta for every 2 alpha- which are bound together by disulphide bonds.
43
Two different integrin activation ways?
Inside out signalling: Cue inside the cell signals with an intrinsic ligand to cause a conformational change to open the integrin up enabling ligand binding.
Outside in: Lingand outside the cell binds to the integrins whch activates and causes a sgnalling cascade inside the cell- pathways or assembly of the actin cytoskeleton.
44
Which Integrin downstream pathways are there?
FAK- focal adhesion kinase.
Auto phosphorylate, so other adapters can bind to form a focal adhesion complex. Macro-assembly of proteins at the site where the cell is attatched to the EXC marix.
activate proliferation etc
45
Different conformations of integrins?
```
Bent= closed inactivated.
Exended= activated can bind ligands.
Clustered= active- ligand bound and extrinsic ligand (laminin, fibronectin, collagen) bound with many signalling proteins making a FA complex.
```
46
Why are biomaterials used in cell therapy?
Biomaterials are used as a scaffold as most cells are adherent and need a surface to grow on like the EXC Matrix in the tissue.
47
Definition of a biomaterial?
```
Nonviable materials used in a medical device, intended to interact with biological systems for example as a scaffold in Tissue engineering.
Or
any substance (other than a drug) or combination of substances synthetic or natural in origin, which can be used for any period of time, as a whole or part of a system which treats, augments, or replaces tissue, organ, or function of the body.” NIH
```
48
First example of a biomaterial?
Sir Herold Ridley was a pilot in the royal airforce and he noticed that pilots got splinters in their eyes from plastic of the plane canopy, but they had no immune reaction.
Developed the intra-occular lens from this for use in patients with cataracts from 1949. 10million now have every year.
Not only was this hugely helpful for this field, but also showed people could have a synthetic substance in the body without rejection- Opened up the TE field.
49
biocompatability definition?
Ability of a material to perform with an appropriate host response in a specific application.
e.g. Catheter- needs no rejection for a few days, whereas hip replacement needs to be viable for life.
Examples of ‘appropriate host response’:
Allow normal healing, clotting response and resistance to bacterial colonization.
50
Generations of biomaterials- first?
First generation: Bioinertness- Didnt interact with the tissue but no immune response, non-toxic, non-carcinogenic.
51
Generations of biomaterials- second?
Bioactivity e.g. Bioglass, interracts with native tissue by binding but doesnt respond to cues.
52
Generations of biomaterials- third?
Functional tissue-strive to induce appropriate cellular signalling and respond to cues. e.g. polymers.
53
Bioglass?
First biomaterial found to chemically bond with tissue in the body
54
Bioglass?
Hench. First biomaterial found to chemically bond with tissue in the body e.g bone. Also avoids an immune reaction and fibrous encapsulation. But they dont respond to cues.
similar composition to that of hydroxyapatite, the mineral component of bone. The high calcium content of the glass enables the binding with bone.
first use: replacement of ossicles in the middle ear, as a treatment of conductive hearing loss.
55
Bioglass more recent developments? (bone still)
Bioactive glass offers good osteoconductivity and bioactivity, it can deliver cells and is biodegradable. The formation of neocartilage can also be induced with bioactive glass by using an in vitro culture of chondrocyte-seeded hydrogels and can serve as a subchondral substrate for tissue-engineered osteochondral constructs.
56
Bioglass more recent developments? (dentistry)
Investigated as an enamel replacement for dentin hypersensitivity lesions. Compared to normal inactive glass, bioglass has antibacterial properies, and studies have shown it inhibits the growth of pathogens such as E-coli, which could benefit post-surgical periodontal wound healing.
GSK put in toothpaste- NovaMin, which can help repair tiny holes and decrease tooth sensitivity
57
Polymers structure as a biomaterial?
Large molecules made of chains or rings of liinked monomeric units. MW 200,000 Da. Different monomers can be used.
58
Range of use of polymers?
From car tyres which are linked by sulphur, to cataracts (PMMA)
59
How can different polymer properties be given?
Use of differnt monomer types.
Can be homopolymer (same monomer in chain),
Block copolymer (one monomer chain, then another, then that first again in runs)
Alternating co-polymer- alternating
Random copolymers
Graft copolymers- all same monomers in a chain but perpendicular a different polymer chain.
Can also have 2 differnt chains of monomers then bound together.
60
Examples of monomers used in polymers?
PMMA- cataracts
PE (polyethylene)
PVC,
PP (polypropylene)
61
What are hydrogels?
Cross-linked polymer networks that are insoluble but swell in aquous medium, resembling the highly hydrated state of natural tissues.
e.g. contact lenses.
62
Three cateogries of biomaterals?
1. Natural- from nature
2. synthetic- synthesised in lab
3. Semi-synthetic -mix- (incorporation of bioactive macromolecules to the backbone of a synthetic polymer)
63
Examples of natural biomaterials?
Protein based natural polymers: Silk, collagen, gelatin, fibrin, elastin,
Polysaccharide natural polymers-
e.g. Chitosan (fungi cell wall component)
e.g. alginates (algi and pseudobacteria)
e.g. hyaluronan-(skin, cartilage etc)
e.g. Chondroitin sulfate- cartilage
64
how to get collagen/gelatin as a natural biomaterial?
Collagen- vertebrates 25% of body weight is - can use enzyme treatment to separate. Gelatin is derived from collagen by denaturing.
65
3 steps in Natural biomaterial separation for use?
Extraction-purification-concentration
66
Synthetic polymers for scaffold building examples?
polylactric acid, polyglycolic acid, poly (lactic-co-glycolic) acid (PLGA) bound together.
67
Semi-synthetic biomaterial examples?
PEG-Fibrinogen, where PEG controls density, stiffness, and biodegradability and Fibrinogen offers biofunctional domains.
68
Advantages of Natural biomaterals? (3)
More likely to act like native tissue.
Dont need to engineer them.
Biofunctional- already proven functionality, and may already have RGD domans for exmaple.
69
Disavantages of natural biomaterials (3)
Sourcing the biomaterial- A lot available? Cost? Ethics?
Immune response if foregn tissue/ Disease pass on?
Batch to batch variability.
70
Advantages of synthetic biomaterials? (2)
Can mix different properties to personalise it to the use.
| Industrial sized production- for off the shelf use, can be stored, limitless supply.
71
disadvantages of synthetic biomaterials? (3)
Immune response-foreign material
Biogradability- needs tailoring to purpose.
New unpredictable, toxic?
72
Biomaterial properties to think about?
Physical/mechanical: Strength, elasticity, architechture. Fatigue resistant, stable.
Chemical: Degradability, resorption (reabsorbed), water content.
Biological: Interactions with cells, release of bioactive signals
73
Why does biodegradability need to be considered in biomaterials?
Because biomaterials are often used as scaffolds for cells to adhere and grow on but degrade overtime as the body replaces them.
E.g. In body covalent bonds are broken by hydrolysis of polymers into monomers. All biproducts need to be non-toxic.
74
Bioglass example of biodegradability?
1. Growth factors adsorb to the surface of Bioglass due to its structural and chemical similarities to hydroxyapatite.
2. These activate M2 macrophages, which promote wound healing and migration of osteoprogenitor cells and mesenchymal stem cells attatch to the bioglass.
3. These differentiate into osteoblasts and bone cells.
4. Osteoblasts deposit EXCM e.g. type 1 collagen (main bone component).
5. A the collagen becomes mineralised like native bone, the bioglass degrades being replaced by bone.
75
M2 vs M1 macrophages in biomaterials?
M2 promote wound healing and migration of progenitor cells in biocompatable materials, whereas in non-biocompatable materials M1 macrophages are activated which trigger an immune response.
76
What does reabsorbable mean in terms of biomaterials?
Total elimination of the initial foreign material and its biproducts. (not all degradable materials are fully)
77
Evidence of Matrix elasticity directs stem cell lineage specification ?
2006
Engler 2006
Took mesenchymal stem cells and put them on 3 types of biomaterials with different stiffness
e.g. 1KPa (stiffness of brain), 10kPa (muscle) or 100 (bone)
- Different differentiation of S.C's according to the stiffness of the scaffold. Cells sense their environment and respond.
78
Surface modifications to biomaterials that can be done?
Chemically or physically altering the atoms/molecules in the existing surface. Functionalisation (RGD)
Overcoating the existing surface with a material having a different composition
Creating surface textures or patterns.But layer needs to be kept thin.
79
WHy do biomaterials need to be kept thin? How thin?
diffusion limit of 100–200 μm diffusion distance of O2 from blood vessels so useful to be as thin as possible.
80
How do cells adhere to a petridish for example?
Indirectly.
A layer of protein (from growth media or plasma) adheres to the surface, and the cells then adhere to these adsorbed proteins via integrins(cell) to focal adhesions (RGD domains in fibronectin/victrinectin).
The integrins then signal to nucleus and the cell responds- if high density of RGD domains adheres.
81
What are non-fouling surfaces?
Surfaces that resist adsorbtion of proteins and therefore adhesion to cells to this. Typically want cells to interact with the biomaterial.
82
Examples of non-fouling surfaces?
PEG, zwitterionic polymers.
| Benefit or medical devices- this may inhibit bacterial colonization.
83
How can biomaterials be functionalised?
If non-fouling and so resistant to adsorbtion can add an RGD domain to the material by chemical binding.
This is also advantagous as can control where the cells will attatch depending on where attatch to domains.
84
How can RGD domains be added to biomaterials? (5)
Using microstamping or microcontact printing.
1. Take a liquidified prepolymer and pour over a structured master mold.
2. The prepolymer is cured and removed from mold.
3. Stamp is cut into smaller pieces and inserted into 'ink' (fibronectin)
4. The ink is stamped onto the biomaterial.
5. The patterned substrate is complete and cells can now adhere to the inked parts.
85
Study showing how functionalising a biomaterial differently affects outcome?
Chen et al 1997.
Method: microstamped fibronectin at differnt sized squares from 5um to 40um and 80um dotted.
```
Results:
5um- no cell adherence
30um- apoptosis
50um-quiescence
80um dotted- proliferation.
```
Cells assume the shape of the island and their adherence size affects their fate- cells like to be stretched- increases proliferation.
86
Different properties needed for internal sutures, soft contact lenses or artifical hip bones? All biomaterials for.
Internal sutures: Need to biodegrade and be replaceable by the body, as don't want another surgery to remove. Also provide support and hold the tissues together until heals.
Soft contact lenses: Correct vision- hold shape, transparent, refractive. Don't need cells to incorporate to, no response from body needed.
Artifical hip bone: Strong, support, mechanical stretch, incorporate into tissues, not biodegrade quickly.
87
Another example of how cellular geometry changes fate?
Differentation fate of human embryonic stem cells. In response to BMP4 colonies reproducibly differentiated into an outer ring of ectoderm and inner ring of endoderm with mesoderm between.
Showing that simple confinement of hESCs to a disk-shaped region is sufficient to recapitulate much of germ layer patterning. GEOMETRY V IMPORTANT.
88
design considerations for biomaterial scaffolds?
What material? Biodegradable etc
Architechture? Pores, Mechanical structure
Interact with cells? function, non-fouling
Up-scaling for mass production? Accesibility
3D shape? Anatomically reproducible
Nutrient supply? Accessibility-porous. Angiogenesis to
89
Brief life cycle of a scaffold?
3D scaffold, cellularisation of scaffold, forms the tissue, then as the scaffold starts to degrade, the cells lay down extracellular matrix e.g. collagen to repair the tissue.
90
Forth type of biomaterial scaffolds (along with natural synthetic etc)
Acellular tissue matrices. Natural scaffold tissue which has been decellularised. E.g. if from own patient, no immune response, which can be recellularised by seeding cells in.
91
Example of decellularised scaffold?
2013. Pigs heart- been decellularised completely- turned white- which creation of 3D extracellular matrix (ECM) templates that mimic nature's design to a degree that-as for today-is not reproducible with any synthetic materials. Using automated coronary perfusion with standard decellularization detergents
92
WHy is complete decellularisation important for an acellular scaffold?
If the decellularisation is incomplete there may be endotoxin/bacteria contamination. Scar tissue is layed down and cross-linking of EXCM occurs , which means the cells cannot degrade or remodel it.
Whereas if completely decelluarised- sterile and non-cross linked, enables remodelling and appropriate collagen deposition.
93
Exploiting of the intact acellular 3D structure of the ECM availibility?
Availibility:
Easily get from: dermis of the skin, submucosa of the small intestine and urinary bladder, pericardium, Achilles tendon, but organs etc would need from a donor.
- Still relies on donors/patients tissues
94
Commerically available decellularised scaffold?
Alloderm. (skin graft)
95
How can one check that a tissue has been decellularised? Methods of decellularisation?
Enzymes, mechanically physically scraping, acids/bases.
Can stain for any cellular components e.g. DNA bnding dyes, histology under microscope or PCR
96
3D printed biosynthetic scaffold example? Aim?
2016- Ear matrix in vitro.
A challenge for tissue engineering is producing three-dimensional (3D), vascularized cellular constructs of clinically relevant size, shape and structural integrity. Testing ITOP (integrated tissue organ printer) as opposed to the previous LIFT bioprinting which can only have a low hydrogel content and that is thin due to lack of structural support.
97
3D printed biosynthetic scaffold example? Printing/ material level Method?
Use integrated tissue–organ printer (ITOP) that can fabricate stable, human-scale tissue constructs of any shape. Printed hydrogels within the biodegradable polymers using Multi-cartridge module nozzles that can dispense different materials.
Microchannels (pores with no material in) were incorportated into the tissue constructs facilitates diffusion of nutrients to printed cells, thereby overcoming the diffusion limit of 100–200 μm for cell survival in engineered tissues.
CELL ENCAPSULATION into the scaffold.
98
3D printed biosynthetic scaffold example? Anatomical level Method? What tissues were made?
MRI scans of structures imported into CAD to make anatomically correct- PERSONLISATION. Some had PCL sacrificial supports left while others didnt.
e.g. .mandible and calvarial bone, cartilage and skeletal muscle.
Ear used rabbit chondrocytes, instead of bovine- better model and were implanted onto the dorsal of mice.
Muscle: The printed construct was cross-linked with thrombin solution to induce gelation of fibrinogen and uncross-linked sacrificial material was removed by dissolving in cold medium.
The bioprinted muscle constructs were implanted in the subcutaneous space over the gluteal muscle, separated by fascia and subcutaneous tissue. (no direct contact)
99
3D printed biosynthetic scaffold example Results EAR?
The constructs with microchannels showed enhanced tissue formation as evidenced by the production of new viable cartilaginous matrix throughout whole ear, whereas those without channels had limited tissue formation and restricted to periphery only. Type II collagen present also.
Ears implanted onto mice dorsum: The shape was well maintained, with substantial cartilage formation upon gross examination. Outer regions were vascularised, but inner still avascular as in native tissue, but cartilage cells still viable. Resilience from bending increased after 1month vs before implantation- cartilage reinforcement.
100
3D printed biosynthetic scaffold example Results MUSCLE?
Immunohistochemical staining for myosin heavy chain (MHC) was performed to evaluate myotube formation within the 3D printed constructs. After 7 d in differentiation media, muscle-like structures with aligned myotubes were observed.
The printed cells with the PCL support unwent cellular compaction and alignment, keeping the fibers taut during cell growth and differentiation (not without).
Electromyography was performed to see response of muscle to AP’s. Muscle constructs responded to electrical stimulation to an extent consistent with immature, developing muscle 2 weeks after implantation.
101
3D printed biosynthetic scaffold example general cell impacts?
≥95% cell viability from day 0-day 6. And cell proliferation increased over a 15 day period.
102
Why is the microarchitechture important when designing a scaffold?
Under an electron microscope can see that skin and bone are pourous. How many and how they are connected is important for vascularisation.
(pourosity= collection of pores)
103
Balance to weigh up with pores in a scaffold?
The more the better in terms of vascularisation, but this compromises the mechanical strength, so it depends on the use of the scaffold etc.
104
Two characteristics of pores?
Accesibility: So all the cells are accesible and the pores are sufficiently connected to allow vascularisation (e.g. not every pore needs to be connected together as long as the vessel can pass through (e.g. doesnt need connecting every direction but if in neither then accesibility will decrease)
Interconnecting- all pores connecting.
105
4 methods of scaffold making/fabrication with pours?
1. Porogen leaching
2. Phase separation
3. Electrospinning
4. Additive manufacturing
106
Method of porogen leaching? (3 steps)
1. Polymer dissolved in solvent and mixed with salt particles into mold.
2. Solvent evaporation- polymer with solidify with the salt particles dispersed in.
3. Add water, the salt leaches out leaving pours where the salt was.
107
Advantages/ disadvantages of porogen leaching?
Can change the size of the salt particles and so the pore size, but less control due to random distribution of the salt particles.
108
Method of phase separation? (3)
1. Gelatin solution (polymer) and silica (solvent) added to make a gelatin/silica hybrid.
2. Phase separation by altering the temperature or addition of a non- solvent to create a thermodynamically unstable gel which separates.
3. Solvent extracted through freeze drying leaving pores behind.
109
Electrospinning method? (2)
Micro or nano fibres created.
1. Have syringe with polymer solution in which is linked to a high voltage power supply, which creates an electric field.
2. The polymer is fired out of the syringe, and the electric field deflects and sprays the polymer onto a rotating collection target.
properties varied by distance between syringe, collection target and electric field.
110
Additive manufacturing method?(3)
the process of joining materials to make objects from 3D model data, usually layer upon layer, building it up.
3D printer:
1. cartridges filled with adhesive in nozzles add to the stage.
2. Roller delivers a layer of polymer on a platform and the polymer only sticks to where there is adhesive.
3. Unbound polymer shaken off
111
Advantages/ disadvantages of additive manufacting method?
+ Produce scaffolds with precise morphologies
+ MRI patient scans into CAD- combines medical images PERSONALISED.
- not all materials suitable for 3D printing.
112
Cell seeding vs cell encapsulation?
Cell seeding is simply putting the cells on top of the scaffold with a lack of control to whether the cells stay here or migrate into the desired locations.
Whereas cell encapsulation inserts cells as the scaffold is made.
113
BMP details?
Abundant in bone matrix, made by osteoblasts. Part of the TGFB family. Osteoinductive- can induce ectopic induction of bone. BMP also recruits osteoblasts promting tissue specific differentiation.
114
2003 BMP trapping scaffold paper aim?
Knew BMP induces bone formation in tissues, which would be a useful bioactive cue to use in scaffolds replacing bone. But problem, need to be in high concentration and is locally acting but the concentration decreases as diffuses through scaffold.
Would it work to trap it in the scaffold?
115
2003 BMP trapping scaffold paper method?
1. Used PEG (non-fouling so functionalised with RGD domain) as the gel scaffold.
2. Cross-linked the functionalised PEG with itself to add sites of cleavage by matrix metalloproteinases (MMP-cells use to remodel matrix later).
3. rhBMP-2 was physically entrapped into PEG gel pores by mixing with the PEG precursor before gelation.
4. Gradual BMP release as the scaffold is broken apart and degraded by the matrix metalloproteinases (MMPs).
116
2003 BMP trapping scaffold paper results?
In vivo critical bone defects (to big for body to regenerate on its own) in rats.
No BMP or MMP sites (control) - no repair
Scaffold MMP senstitive but no BMP (control)-no repair.
BMP treatment but no MMP sites (control)-no repair
BMP treatment with MMP sites- bone regeneration.
Osteoblasts recruited which secrete bone matrix and regenerate the bone.
117
Advantages of Silk:
Fully biogradable
Low energy input (sustainable- green)
Very strong vs weight
118
Why is silk so great?
it has independently evolved for use by arachnids, shrimp, silk worms etc. convergent evolution.
119
Definition of Silk?
Structural proteins skun into fibres for use outside of the body.
120
Different silks different properties?
high tensile strength.auxillary spiral: making up the structural elements of the web-
Energy dissipation (e.g. when fly flies into web and cant get free).
Remote sensing- vibrations transmit location to spider.
(Both above at a nano scale hard to replicate).
121
Structure of Silk?
Silks are fibrous proteins, containing high proportions of glycine and alanine. May have many kinks, doesnt pack very well together hence its ability to stretch.
Hydrogen bonding and hydration important for features- can break and reform to disserpate energy.
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Silk vs Kevlar?
Kevlar is stronger but silk can withstand higher strain vs weight.
( Strain=stretching without breaking)
(strength=response (resistance) of a system to an applied stress.)
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How is silk made?
Stored as a liquid protein gel and spun into a fibre as the PH changes down the duct- phase transition from liquid into a solid. Proteins denature/flow induced crystallisation.
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Silk use as a biomaterial?
Can re-denature, turn back into a liquid protein to remould into a scaffold etc.
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Why have silk biomaterials been used for meniscus replacements?
Two alloplastic scaffolds for partial meniscal replacement are clinically available (CMI® and Actifit®) but their ability to protect the articular cartilage in the long-term remains unclear and their mechanical/viscoelastic properties are significantly different to that of human menisci.
HENCE SILK
(kinked, H bonds- energy disserpation, hydrated)
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First generation of silk for menisci?
2014 FibroFix first generation of silk for menisci- silkworm fibroin- extracted and processed into a porous matrix with the surface of the scaffolds being smooth, tough resillient matierial that felt like cartilage.
collagen wrapped around the silk fibres.
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second generation of silk fibroin for menisci study aim:
2019. Evaluate this second gen silk in sheep to restore meniscal function after excessive tissue damage, a silk fibroin implant for partial meniscal replacement was developed and investigated in an earlier sheep model. After 6 months implantation, it showed promising results in terms of chondroprotection and biocompatibility.
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second generation of silk fibroin for menisci study aim:
2019. To improve surgical fixation to the peripheral rim, the material was subjected to optimisation and a fibre mesh was integrated into the porous matrix.
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second generation of silk fibroin for menisci study results:
2019. On 9 sheep, no signs of inflammation after 6 months, no solid connection to the remaining peripheral meniscal rim and 3 had ruptures. After 6 months implantation, it showed promising results in terms of chondroprotection and biocompatibility.
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second generation of silk fibroin for menisci study conclusion:
2019. Compressive stiffness higher than native meniscal tissue. The material showed an increased stiffness in comparison to the first scaffold generation, which is likely to be associated with a fibre component integrated in the scaffold to enhance fixation strength to the meniscal host tissue. Increasing porous hydrogel content may make the stiffness more comparable.
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Besides silk for menisci what other use has there been?
Silk nerve condruit- Spidrex® fibres support neurite outgrowth.
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Silk nerve condruit spidrex method?
Nerve guidance conduits guide axon regrowth between two nerve stumps. Conduits consisted of a hollow highly porous cylindrical sheath composed of reconstituted (regenerated) mulberry silk fibroin and containing longitudinally orientated luminal Spidrex® fibres coated with hyaluronic acid (HA). For evaluation in vivo, silk conduits 10 mm in length were implanted to bridge an 8 mm gap in the rat sciatic nerve.
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Silk nerve condruit spidrex results?
(For evaluation in vivo, silk conduits 10 mm in length were implanted to bridge an 8 mm gap in the rat sciatic nerve.)
At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) as autologous grafts similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. No more inflammation.
ALSO SILK BIODEGRADABLE so just degrades after regrowth.different densities have different results- PN200 excellent and comparable to autologous
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Why people may need a silk menisci replacement?
Lost spongy meniscus and so the cartilage between the leg bones- this wears down causing arthritis so a metal knee replcement is given, but it would be less painful to replace the initial meniscus (fibrofix).
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What is the role of bioreactors?
Enables the growth of a high density of cells where one can control lots of factors to maximise yield.
e.g. stem cells, etc
Establish spatially uniform cell distributions on 3D scaffolds.
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Advantages/ disadvantages of static culture?
Just petridish
+Cheap and easy for labs.
-unevent distribution of metabolites etc, may form gradients.
- very little control of factors when in the incubator e.g. PH etc.
- Issues growing 3D structures in.
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Dynamic cell culture systems
Medium flows through a peristaltic pump and fermentor, and another pump (gas exits) before reaching the tissue chamber.
+ Gives rise to homogeneous concentrations of nutrients toxins etc by mixing.
+Can control/ monitor Ph etc also
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Considerations with seeding cells into a scaffold?
- distribution
- Seeding efficiency
- inoculation period
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What is seeded cell inoculation period?
cells need to attatch to a scaffold to survive so the faster the better.
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Why are biorectors useful for cell seeding?
ensure even distribution, if cells placed on top in static media may just stay on top etc, but in non-static cell culture where media flows through the contruct the cells are distributed into it, avoiding hypoxia due to perfusion to them.
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Example of seeding static vs non study?
Bone marrow stomal cells seeded into a scaffold and after 18 hours MTT assay was used to show cell distribution (dye changes from yellow soluble salt to purple salt by mitrochondria) And in static cells all on top, whereas cells seeded by perfusion evenly dispersed.
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When may seeded cells get hypoxia?
If they have dispersed throughout the scaffold, but static culture is used so the cells do not get perfused.
200um O2 diffusion distance, no mass transfer.
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What is mass transfer in a 3D scaffold?
External mass transfer- removal of CO2 and metabolites and Internal mass tranafer- delivery of O2 and nutrients.
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Evidence of mass transfer of seeded cells in cultures?
2008- chondrocytes seeded using perfusion or without.
| Statically cultured cells survived only when at the edges (dont get O2 etc) whereas with perfusion survived throughout
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Evidence of mass transfer of seeded cells in cultures?
2008- chondrocytes seeded using perfusion or without.
| Statically cultured cells survived only when at the edges (dont get O2 etc) whereas with perfusion survived throughout
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Bioreactors also useful for physical conditioning- such as?
Tissues and organs in body subjected to complex biomechanicla evironments with physical forces e.g.
hydrodynamic (sheer stress of blood flow), hydrostatic (water force), mechanical stress- e.g. muscles under tension, lungs stretching and electrical.
Cells then need these pressures to differentiate/proliferate in response to. Bioreactors can provide these.
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Bioreactors need to be:
nontoxic to cells.
have diversity in design to suit the cells
sterile conditions (single use or autoclavable apparatus)
have matieral that withstan ds conditions
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Types of bioreactors? (5)
- Spinner flask bioreactor (magnetic stirring)
- Rotating wall bioreactor (scaffold in suspension)
- Perfusion bioreactor (media pushed through scaffold)
- Compression (applies mechanical pressures)
- Flex-stretch-flow bioreactor- (stretch scaffold mimic blood flow)
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Spinner flask bioreactor?
Magnetic stirrer helps with mass transfer of media. Not good enough to create a homogeneous cell distribution throughout as cells mainly reside on periphery of construct.
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What is homeogeneous cell distribution?
One cell population only.
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Rotating wall bioreactor?
The reactor is rotating- gravity pulls the scaffold down but centrifugal forces counterbalance it- scaffold in suspension. As tissue grows the rotational speed has to be increased (mg down increased)
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Perfusion bioreactors?
Scaffold held in place and media is pushed through- good for mass transfer but can decellularise the tissue, effects of perfusi on depend on flow rate.
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Compression bioreactors?
Applies mechancial pressure to cell-seeded constructs for varying time intervals.
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Which bioreactor if want to test zero gravity effects on bone?
Rotating wall bioreactor.
NASA developed to represent the environment in spaceflights. Osteoblsts cultured suggested that microgravity stimulates osteoclastogenesis (bone degeneration
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What bioreactor if want to decellularise a heart?
perfusion bioreactor at high flow rate. E.g. porcine heart
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Which bioreactor used if want to create articular cartilage?
Needs to be strong/ load bearing tissue so needs to grow under mechanical pressure- so compression bioreactor applies cyclical strss.
E.g. study took 4 weeks with loading pressure applied in one hour on off cycles for five days- significantly improved biomechanical properties.
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How was the porcine heart decellularised?
HEart aorta had tubing inserted in to perfuse the coronary arteries.
Heart was submerged intowater in 4l bucket (air bubbles were removed from the tubing)
The coronary arteries were perfused with a high flow rate- as reaches the heart tissue decellularises- turns white.
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Flex-stretch- Flow bioreactor?
Scaffold attratched to fixed post and moveable post. Linear motor is fixed and the moveable post can move, stretching the tissue cyclicially- this mimics blood flow e.g. like distole and systole.
Culture media flows over the scaffold- magnic wheel can provide laminar flow like shear stress.
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Heart valve replacement current problems?
Aortic valve stenosis (not opening fully or closing fully) so surgical repair or valve replacement.
This invovles prosthetic heart valves . Dont grow with childrena nd need anti-thrombotic drugs.
Tissue engineered alternative? But need to withstand high stress e.g. collagen crimps up in systole and expands in diastole
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TE heart valves? Method:
mesenchymas stem cells from sheep bone marrow cultured on PGA/PLA scaffolds.
FLEX-STRETCH-FLOW Bioreactor mimics blood flow (hydrodynamic shear stress but alsomechanical). Tested with and without.
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TE heart valves? Results:
After 3 weeks engineered valves exposed to flex-stretch-flow bioreactor accellerated MSC collagen formation, stiffness and an eleasticity modulus similar to SMC tissue.
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What bioreactor helps blood vessel tissue?
Pulsatile flow of medium through engineered blood vessel tissue to stimulate pulsatile flow- hydrodynamic stress.
