Part 2 Flashcards
1
Q
What are scaffolds?
A
provide support for cell to proliferative and maintain their function
deliver and retain cells and bioactive materials
2
Q
What are design considerations for scaffolds?
A
Allow musculature to penetrate scaffold to bring 02 and nutrients to the cells
3
Q
scaffold design 1
A
Biomaterials- biodegradable
- scaffold and cells
- scaffold cellularisation
- tissue formation
- 3D artificial tissue formed
- pos feeback
* degradation kinetics= not too fast or slow, non toxic materials produced
4
Q
Scaffold design 2
A
Cell attachment and function - surface properties - ability to attach to matrix and start signalling - interact with cells (Non fouling don't attach- RGD domain)
5
Q
Acellular tissue matrices as scaffolds
A
Not biomaterials
already made tissue matrices
Decellurisation (appears white- avid of cells)- recellularization
Cellular components of tissue removed so they can repopulate - removed all components and antigens so wont induce an immune response- no immunosuppressants needed
6
Q
What is the importance of complete decellularization?
A
Incomplete decellularization could lead to endotoxin/bacteria contamination- cross linking
leads to scar tissue and encapsulation
Proper complete cellularisation= proper sterilization and non crossing linking= appropriate tissue decomposition
7
Q
What happens after a tissue is implanted?
A
Absorption of plasma and blood pressure haemostasis immune response cell infiltration tissue remodelling
8
Q
How do you decellularize?
A
Mechanical physical scraping
biological process- enzyme digest cellular components
9
Q
Advantages and limitations of Acellular tissue matrices?
A
+ exploit of intact 3D structure of ECM, accessibility, commercially available
10
Q
How to check that acellular matrices have been properly decellularized
A
Stain for DNA or any cellular components
Use PCR method
11
Q
Scaffold design 3
A
Mechanics and architecture
final shape of the tissue engineered construct is defined by the scaffold
Examples
- trachea= measured to fit specific person using CT and mRNI
- human bone= appears solid but actually porous
Adequate mechanical properties, and nutrient supply, and accessible (upscaling)
12
Q
Porosity
A
pore= space within scaffold
Porosity= a collection of pores
too many pores= leaky
13
Q
Pore examples
A
- 100% Acessible and 100% interconnecting
- 100% accessible and <100% interconnecting
- <100% accessible and <100% interconnecting
14
Q
Examples of methods of scaffold fabrication
A
- Porogen leaching
- phase separation
- electrospinning
- addictive manufacturing
15
Q
Porogen leaching
A
- easy and accessible
use polymer, dissolve solvent, mix, evaporate solvent, polymer solidifies (salt particles inside and salt with leach out), left with porous scaffold
*where salt was left pore formed
16
Q
Phases separation
A
Scaffold challenged thermodynamically
1. homogenous solution
2. 2 separate phases- polymer rich and solvent rich
3. don’t freeze dry we will end up with solid object but inside like honey comb
Mix, phase separate, freeze dry, nanofibrous hybrid, optical picture, SEM image
17
Q
Electrospinning
A
high voltage power, polymer supply (using syringe), put in jet initation and electric field- collection target with rotation and produce fine fibres
- make ecm really well
- Slowly injected polymer field, fibres displayed
18
Q
Fibres types produced from electrospinning
A
1 random
2 parallel
3 perpendicular
*change polymer layout by height and speed of plate
19
Q
Additive manufacturing- advantages and disadvantages
A
process of joining materials to make object from 3D model data usually layer upon layer
+ scaffold with precise morphology, combination medical imaging to fabricate anatomically shaped implant
- limited number of biomaterials
20
Q
3D printing
A
Used lots
Difficult to apply to cell biology
Roller deliver layer of material onto platform, cartilage filled with adhesive, ink inject printer, controlled by computer
polymer not bound can be shaken off at the end
21
Q
Cell encapsulation
A
Cells put onto scaffold- form porous network
issues with delivery- some may sit in pores need for vasculature rather than go in- cell seeding
22
Q
Study of 3D printing 2016
A
3d print ear
make relevant shape, size, structural integrity
“integrated tissue organ printer” ITOP overcome limitations of currently used bioprinting technologies
23
Q
Issues with 3D printing
A
Number of cartridge
Dispenses different things
cells encapsulated in polymer already
printing it harder than material due to keeping cells alive
24
Q
Skeletal muscle reconstruction
A
3D mouse construct 15x15x1mm dimension containing mouse myoblasts
Designed fibre bundle structure- pcl pillars maintain structure
crosslinked with thrombin solution to induce gelatin of fibronegin and unlinked material was removed
High cell viability
25
Biological factors in tissue growth
1. small molecules
2. proteins and peptides
3. oligonucleotides
26
Bone morphogenic protein
BMP
- extracted from bone matrix
- membrane of TGFb
- made by osteoblasts
- osteoinductive- BMP2/7 place ectopically and form bone
27
Bone regeneration study
Repair bone defects using synthetic mimics of collagen and ECM
28
How did they do the bone stufy
- use BMP= locally acting factors
- Use biomaterials that retain and sequester BMP
small proteins dissipate and diffuse through tissue - if not right conc it wont work
- scaffold to deliver and keep right conc
29
BMP in PEG
BMP trapped in PEG
1. PEG synthetic material non fouling
2. crosslinked- added site for cleavage
30
Results of BMP
Fibroblast invasion
sensitivity of gel to proteolysis by cell secreted mmp's using as in vitro model system for cell invasion
scaffold can be degraded
release of BMP from MMP-sensitive scaffold
bone healing in rat
31
Micropcomputed tomography of rat calvariae
Shows mmp-sensitive and BMP bone rapair at site of injury
| took rats with critical size bones- no way body can regenerate it themselves
32
How does the bone repair?
Responding cells adhere to RGDs in matrix
cells secrete MMPs that degrade MMP cleavable bonds serving as crosslinks for the matrix
BMP liberated from matrix diffuses from site, signalling osteoblast precursor cells
Osteoblasts secrete bone matrix
bone defect is regenerated
33
Classical bioreactors
Enable ED tissue development
big tanks full of medium for eukaryotes under control conditions
grow cells to produce high amounts to use clinically
34
Key challenges of TE
ability to grow 3D tissue structures of relevant clinically sizes
growth and 3x ability of cell types required for complex cell function
35
Static cultures and the problems
relatively cheap and easy
cells grown without mixing, conc gradient occur
issues with growing 3D
Unequal distribution
36
Dynamic culture systems
mixing gives rise to homogeneous concentrations of nutrients, toxins and other components
1. medium tank- pump through
2. peristatic pump
3. fermentor
4. hollow fiber module
5. gas liquid separator- recycle
6. gas meter
* mixes media
37
Role of TE bioreactors
1. establish spatially uniform cell distribution of 3D structures
2. overcome mass transport limitations in 3D constructs 3, expose the developing tissue to physical stimuli
38
what is a good scaffold in terms of cell distribution?
high seeding efficacy
short inoculation period
uniform distribution
3D scaffold+ cell types- scaffold cellularization
39
Issue with seeding?
Place on top of scaffold- uneven distribution
| autologous sample- hard to get more so needs to be efficient
40
How seeding works
Pipette cells onto scaffold
24 hole to place scaffold
static culture= small nutrient distribution, cells accumulate at the top
Non-static culture= media flow through- cells distribute along biomaterials surface
41
Example of cell seeding of 3D scaffold
Bone marrow stromal cells seeded onto scaffolds
after 18hrs MTT assay USED
MTT- bromide is converted by mitochondria from a soluble yellow salt to insoluble purple formazan salt
MTT= indicator to see where the cells are and whether they are living
42
Resuluts of 3D scaffold seeding
As expected, static has dark staining on top and not widely distributed - dense at top
Cells need 02 and have had metabolites removed
43
Mass transfer of 3D constructs
external or internal transfer- 02 and nutrients to cell
- can only diffuse 200mm away from a capillary in the body
removal of metabolites and CO2
44
experiment where they looked at mass transfer
chrondytes seeded using perfusion cell seeding
cultured for 2 weeks with or without perfusion
Hydrostatically= no mixing of media= only survived at ECM is at edges, cells inside didn't survive
Perfusion= mixing, seeding with cells, get 02 and created well organised functional cells
45
Physically conditioning
tissues and organs in the body are subject to complex biomechanical environment
Physical forces= hydrodynamic, mechanical, electrical
46
Physically conditioning of the blood vessels, lungs and arm muscle
Endothelial cells line blood vessels
Undergo hydrodynamic forces
Lungs- Air pressure on lungs, inhalation and exhalation
Muscles- withstand tension
47
System approach
Bioreactor on outside
Appropriate environmental control and nutrient delivery/ waste removal- O2,PH,CO2
Specific scaffold- migration or attachment, proliferation or degradation
mechanical stimulation
intracellular signalling or activity
48
Design considerations for bioreactors
Diversity in bioreactor reflects rang of signals for various tissue formation
general requirements:
- biocompatibility- made of material that's not toxic
- sterility and sterile containment
- sterile environment
49
Types of reactors
1. Spinner flask bioreactor- magnetic stirrer, help transfer nutrients from outside to inside- cheap and easy
2. rotating wall bioreactor-centrifugal forces generated by rotation of cylinder balance the gravitational pull of the scaffold, as tissues grow speed increases to counteract gravity forces (remain in suspension)
3. Perfusion bioreactor- scaffold static, medium flow through, continuous flow through construct and most mass transfer limitations are mitigated- depends on flow rate
4. Compression bioreactor- press down to mimic mechanical stimulation
50
Why we need new bioreactors
- mass transfer in flask, not enough to deliver homogenous cell distribution throughout scaffold and cells at periphery
51
What bioreactor would you used for decellularizing a tissue, tissue engineering articular cartilage and studying near zero gravity?
a) perfusion- complex nature makes difficult- used for heart, lungs pancreas etc
b) compression
c) rotating wall
52
Porcine heart decellularization
retrograde coronary perfusion
attach tube to heart (barbed end to aorta) and secured with host clamps
use appropriate solution to decellularize
As solutions go through it loses colour
53
Mauck et al 2000
Auricular cartilage is loadbearing tissue
Four week culture with loading applied five days per week
improved biochemical properties
Clinical Stress and mechanical loading exposure
54
Tamma et al 2009
Spaceflights represent the best environment of near zero gravity effects
Rotating wall bioreactor created by NASA
Studies on osteoclasts in the spaceflight conditions- microgravity stimulation- suggest microgravity stimulates osteoclastogenesis
55
Tissue engineering of heat valves- case study 1
4 heart valves- enable forward flow
certain illness valves are open or closed- no medical intervention- repair or replace
Aortic valve stenosis
56
Issues with heart valves
need antithrombotic drugs which can cause bleeding or blood clotting problems
don't grow with child
57
TE heart valve
Autologous would be ideal
Biophysical forces that the heart valve cells are exposed to in vivo- mechanical stretch, hydrodynamic shear- withstand forces of body
58
Flex stretch flow bioreactors study
TE heart valve tissue mechanbiology
mimic the mechanical stimulation and perfusion in vitro could lead to further improvement in TE
1. Enabled flexing and stretching of scaffold- scaffold fixed on one side and other side moved from flexed to stretch position
2. Enabling flow by allowing media to flow over it to mimic blood flow- recirculates within the bioreactor by magnetic coupled paddle wheel to provide laminar flow
59
Mesenchymal sc differentiation
MSCs derived form sheep bone marrow cultured on PGA/PLA scaffolds
test group under mechanical and hydrodynamic sheer add increased collagen content and effective stiffness of engineered valves
By 3 weeks engineered valves had modulus comparable with smc
60
How do they know mesenchymal sc worked?
tested with and without bioreactor for several weeks
forces necessary to mimic function in body and get functional valve
*cyclic flexure and laminar flow synergistically accelerate MSC-mediated tissue formation
61
Blood vessel study case 2
pulsatile pump
flow direction to engineered vessel and medium reservoir
mimic blood flow
62
Blood vessel study 3
Vocal folds
no easy to design out of body
speakers to mimic sounds and create vocal folds
63
What is skin?
Largest organ in the human body
~10% of the body mass
functions of the skin- protection, regulation, sensation,
64
Skin structure layers
Epidermis
Dermis
Hypodermis
65
What is the epidermis
```
Outermost layer
thin
protects body from environment no blood vessels
only cells
Eyelids= 0.05mm
hands and feet= 1.5mm
```
66
Cellular component of the epidermis
keratinocytes= Keratin which provides skin with toughness and waterproof
melanocytes= produce melanin, protects from UV
Langerhans cells= antigen presenting cells involved in immunity
merkel cells= mechanical receptors- touch stimuli
67
Basement membrane
Only not mature here
separate epidermis from dermis underneath
proliferate and daughter cells move upwards, leave cell cycle and differentiate
- produce ecm proteins which make BM
68
Dermis
Bulk of skin
composed of collagen with some elastin and glycoaminoglycans
fibroblasts- wound heeling
blood vessels, hair follicles, sebaceous and sweat glands
69
Hyperdermis
A network of adipose cells and collagen
| functions as a thermal insulator, and shock absorber and stores fat as a energy store
70
Injury of layers of skin and how they repair
Epidermis= regeneration- new cells to protect and dividing cells
epidermis and dermis= scar formation
71
Contracture of skin
Fibroblasts lay down new collagen
| myofibroblasts contract and pull wound together- too much skin= too tight, cant move their limb
72
Clinical need for skin replacement
Acute trauma
chronic wounds
surgery
genetic disorders
73
Thermal trauma of the skin
One of the most common reasons for major skin loss
burns and scolds can cause rapid and extensive wounds
damaging of large skin areas can cause death
74
Types of wounds
Classified based on layers it affects
- epidermal
- partial thickness- superficial or deep
- full thickness
75
Epidermis injuries
```
Affect only the epidermis
Characterised by arrhythmias and minor pain
do not require surgery
no scarring
EXAMPLE= Sun burn
```
76
Superficial partial thickness wounds
Affect the epidermis and superficial part of the dermis
wound appears wet, weeping and red
painful
heal spontaneously
77
Deep partial thickness wounds
greater dermal damage
wound appears moist white/red/pink
results in few skin appendages remaining
scarring is pronounced
78
Full thickness wounds
Complete destruction of epithelial regenerative elements
wound appears dry and leathery
No spontaneous healing
79
Treatment of major skin injuries
Early excession of dry skin
wound closure- reduce mortality and mobility
skin graft
80
Types of skin grafts
Split thickness- epidermis and part of dermis
| full thickness- full epidermis and dermis
81
Autologous skin graft
Gold standard for full thickness
skin obtained from non injured part
may not be enough so add mesh
82
Graft take
Plasmic inhibition
inosculation
revascularization
* when skin is taken from one part and added to another it needs nutrients to take
83
Preparation of wound bed
has to adhere to bed
| needs a thin layer of connective tissue
84
Skin allografts
cadaveric skin for temporary prevention of fluid loss or wound contamination
can be obtained from non profit skin banks
possibility of pathogen transmission
immunogenic rejection
85
Problems with skin grafts
cant use your own when more than 50% of your own body is injured
rejection
limited availability of skin grafts
Pain and scarring in the donor site area
further pain inflicted on the patient
86
Ideal skin graft
```
readily available- most injuries acute not chronic
cause no immune response
cover and protect wound
enhance healing
lessen the pain
leave no scars
```
87
Different classifications of skin substitutes
layers to be substituted- epidermis, dermis and compound
durability- temporary, permanent
product origin- biologic, biosynthetic and synthetic
88
Epidermal substitutes
isolation of keratinocytes from a skin biopsy
expanded in culture
cultured keratinocytes are delivered on the wound and form new epidermal layer
1. skin specimen
2. isolate keratinoyctes- form colonies that merge
3. apply by either cultured cell suspension in spray device, single cell suspension, cultured epithelium sheet
89
My skin
Subconfluent autologous keratinocytes
synthetic silicone delivery membrane
treated chronic wound of a diabetic foot= 80% successful
90
+ and - of epidermal substitutes
Only contain keratinocytes grown in vitro and applied ot wound- only replace epidermis
effective for treating foot ulcers
combination can be used with dermis substitutes- full thickness wound healing
*autologous limited but no immune response
91
Dermal substitutes
Dermal alternative to facilitate wound healing process
acellular- synthetic or biological
after application to a prepared dermis, these substitute
92
Integra dermal regeneration template q
1. dermal layer from bovine type 1 collagen and shark chondroitin 6 sulphate
2. epidermal layer made of silicone- regulate heat and fluid loss
93
Alloderm
Acellular human allogenic dermal matrix preserved by freeze drying
full thickness skin burns, alloplastic reconstruction, abdominal wall reconstruction, rhinoplasty
94
Dermal substitute 2 step process
1. applying a dermal substitute
| 2. epidermal cover
95
Composite substitutes
Aim to mimic historical structure of normal skin allogenic skin incorporated into dermal scaffold
enable production of large batches
temporary bioactive dressings
96
Aplifgraf
Composite skin consisting of bovine type I collagen cultured with allogeneic male neonatal fibroblasts and keratinocytes = resembles normal skin structure
97
OrCell
Includes cultured allogeneic fibroblasts and keratinocytes obtained from the same neonatal foreskin.
Fibroblasts are seeded into a bovine type I collagen sponge + keratinocytes on top
Cytokines and growth factors from the product promote host cell migration and wound healing
98
Limitations
1. wait time ranges from 3 to 12 weeks after the biopsy is taken.
2. only two cell types – keratinocytes and fibroblasts
3. “Clinical success but economic failure!"
