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BMS354 Tissue engineering > Part 2 > Flashcards

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

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2
Q

What are design considerations for scaffolds?

A

Allow musculature to penetrate scaffold to bring 02 and nutrients to the cells

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3
Q

scaffold design 1

A

Biomaterials- biodegradable

  1. scaffold and cells
  2. scaffold cellularisation
  3. tissue formation
  4. 3D artificial tissue formed
  5. pos feeback
    * degradation kinetics= not too fast or slow, non toxic materials produced
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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)
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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

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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

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7
Q

What happens after a tissue is implanted?

A 
Absorption of plasma and blood pressure 
haemostasis 
immune response
cell infiltration 
tissue remodelling
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8
Q

How do you decellularize?

A

Mechanical physical scraping

biological process- enzyme digest cellular components

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9
Q

Advantages and limitations of Acellular tissue matrices?

A

+ exploit of intact 3D structure of ECM, accessibility, commercially available

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10
Q

How to check that acellular matrices have been properly decellularized

A

Stain for DNA or any cellular components

Use PCR method

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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)

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12
Q

Porosity

A

pore= space within scaffold
Porosity= a collection of pores
too many pores= leaky

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13
Q

Pore examples

A
  1. 100% Acessible and 100% interconnecting
  2. 100% accessible and <100% interconnecting
  3. <100% accessible and <100% interconnecting
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14
Q

Examples of methods of scaffold fabrication

A
  1. Porogen leaching
  2. phase separation
  3. electrospinning
  4. addictive manufacturing
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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
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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

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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
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18
Q

Fibres types produced from electrospinning

A

1 random
2 parallel
3 perpendicular
*change polymer layout by height and speed of plate

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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

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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

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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

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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

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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

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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

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25
Q

Biological factors in tissue growth

A
  1. small molecules
  2. proteins and peptides
  3. oligonucleotides
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26
Q

Bone morphogenic protein

A

BMP

  • extracted from bone matrix
  • membrane of TGFb
  • made by osteoblasts
  • osteoinductive- BMP2/7 place ectopically and form bone
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27
Q

Bone regeneration study

A

Repair bone defects using synthetic mimics of collagen and ECM

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28
Q

How did they do the bone stufy

A
  • 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
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29
Q

BMP in PEG

A

BMP trapped in PEG

  1. PEG synthetic material non fouling
  2. crosslinked- added site for cleavage
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30
Q

Results of BMP

A

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

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31
Q

Micropcomputed tomography of rat calvariae

A

Shows mmp-sensitive and BMP bone rapair at site of injury

took rats with critical size bones- no way body can regenerate it themselves

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32
Q

How does the bone repair?

A

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

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33
Q

Classical bioreactors

A

Enable ED tissue development
big tanks full of medium for eukaryotes under control conditions
grow cells to produce high amounts to use clinically

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34
Q

Key challenges of TE

A

ability to grow 3D tissue structures of relevant clinically sizes
growth and 3x ability of cell types required for complex cell function

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35
Q

Static cultures and the problems

A

relatively cheap and easy
cells grown without mixing, conc gradient occur
issues with growing 3D
Unequal distribution

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36
Q

Dynamic culture systems

A

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
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37
Q

Role of TE bioreactors

A
  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
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38
Q

what is a good scaffold in terms of cell distribution?

A

high seeding efficacy
short inoculation period
uniform distribution
3D scaffold+ cell types- scaffold cellularization

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39
Q

Issue with seeding?

A

Place on top of scaffold- uneven distribution

autologous sample- hard to get more so needs to be efficient

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40
Q

How seeding works

A

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
Q

Example of cell seeding of 3D scaffold

A

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
Q

Resuluts of 3D scaffold seeding

A

As expected, static has dark staining on top and not widely distributed - dense at top
Cells need 02 and have had metabolites removed

43
Q

Mass transfer of 3D constructs

A

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
Q

experiment where they looked at mass transfer

A

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
Q

Physically conditioning

A

tissues and organs in the body are subject to complex biomechanical environment
Physical forces= hydrodynamic, mechanical, electrical

46
Q

Physically conditioning of the blood vessels, lungs and arm muscle

A

Endothelial cells line blood vessels
Undergo hydrodynamic forces
Lungs- Air pressure on lungs, inhalation and exhalation
Muscles- withstand tension

47
Q

System approach

A

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
Q

Design considerations for bioreactors

A

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
Q

Types of reactors

A
  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
Q

Why we need new bioreactors

A
  • mass transfer in flask, not enough to deliver homogenous cell distribution throughout scaffold and cells at periphery
51
Q

What bioreactor would you used for decellularizing a tissue, tissue engineering articular cartilage and studying near zero gravity?

A

a) perfusion- complex nature makes difficult- used for heart, lungs pancreas etc
b) compression
c) rotating wall

52
Q

Porcine heart decellularization

A

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
Q

Mauck et al 2000

A

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
Q

Tamma et al 2009

A

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
Q

Tissue engineering of heat valves- case study 1

A

4 heart valves- enable forward flow
certain illness valves are open or closed- no medical intervention- repair or replace
Aortic valve stenosis

56
Q

Issues with heart valves

A

need antithrombotic drugs which can cause bleeding or blood clotting problems
don’t grow with child

57
Q

TE heart valve

A

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
Q

Flex stretch flow bioreactors study

A

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
Q

Mesenchymal sc differentiation

A

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
Q

How do they know mesenchymal sc worked?

A

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
Q

Blood vessel study case 2

A

pulsatile pump
flow direction to engineered vessel and medium reservoir
mimic blood flow

62
Q

Blood vessel study 3

A

Vocal folds
no easy to design out of body
speakers to mimic sounds and create vocal folds

63
Q

What is skin?

A

Largest organ in the human body
~10% of the body mass
functions of the skin- protection, regulation, sensation,

64
Q

Skin structure layers

A

Epidermis
Dermis
Hypodermis

65
Q

What is the epidermis

A 
Outermost layer 
thin 
protects body from environment no blood vessels 
only cells 
Eyelids= 0.05mm 
hands and feet= 1.5mm
66
Q

Cellular component of the epidermis

A

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
Q

Basement membrane

A

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
Q

Dermis

A

Bulk of skin
composed of collagen with some elastin and glycoaminoglycans
fibroblasts- wound heeling
blood vessels, hair follicles, sebaceous and sweat glands

69
Q

Hyperdermis

A

A network of adipose cells and collagen

functions as a thermal insulator, and shock absorber and stores fat as a energy store

70
Q

Injury of layers of skin and how they repair

A

Epidermis= regeneration- new cells to protect and dividing cells
epidermis and dermis= scar formation

71
Q

Contracture of skin

A

Fibroblasts lay down new collagen

myofibroblasts contract and pull wound together- too much skin= too tight, cant move their limb

72
Q

Clinical need for skin replacement

A

Acute trauma
chronic wounds
surgery
genetic disorders

73
Q

Thermal trauma of the skin

A

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
Q

Types of wounds

A

Classified based on layers it affects

  • epidermal
  • partial thickness- superficial or deep
  • full thickness
75
Q

Epidermis injuries

A 
Affect only the epidermis 
Characterised by arrhythmias and minor pain 
do not require surgery 
no scarring  
EXAMPLE= Sun burn
76
Q

Superficial partial thickness wounds

A

Affect the epidermis and superficial part of the dermis
wound appears wet, weeping and red
painful
heal spontaneously

77
Q

Deep partial thickness wounds

A

greater dermal damage
wound appears moist white/red/pink
results in few skin appendages remaining
scarring is pronounced

78
Q

Full thickness wounds

A

Complete destruction of epithelial regenerative elements
wound appears dry and leathery
No spontaneous healing

79
Q

Treatment of major skin injuries

A

Early excession of dry skin
wound closure- reduce mortality and mobility
skin graft

80
Q

Types of skin grafts

A

Split thickness- epidermis and part of dermis

full thickness- full epidermis and dermis

81
Q

Autologous skin graft

A

Gold standard for full thickness
skin obtained from non injured part
may not be enough so add mesh

82
Q

Graft take

A

Plasmic inhibition
inosculation
revascularization
* when skin is taken from one part and added to another it needs nutrients to take

83
Q

Preparation of wound bed

A

has to adhere to bed

needs a thin layer of connective tissue

84
Q

Skin allografts

A

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
Q

Problems with skin grafts

A

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
Q

Ideal skin graft

A 
readily available- most injuries acute not chronic 
cause no immune response 
cover and protect wound 
enhance healing 
lessen the pain 
leave no scars
87
Q

Different classifications of skin substitutes

A

layers to be substituted- epidermis, dermis and compound
durability- temporary, permanent
product origin- biologic, biosynthetic and synthetic

88
Q

Epidermal substitutes

A

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
Q

My skin

A

Subconfluent autologous keratinocytes
synthetic silicone delivery membrane
treated chronic wound of a diabetic foot= 80% successful

90
Q

+ and - of epidermal substitutes

A

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
Q

Dermal substitutes

A

Dermal alternative to facilitate wound healing process
acellular- synthetic or biological
after application to a prepared dermis, these substitute

92
Q

Integra dermal regeneration template q

A
  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
Q

Alloderm

A

Acellular human allogenic dermal matrix preserved by freeze drying
full thickness skin burns, alloplastic reconstruction, abdominal wall reconstruction, rhinoplasty

94
Q

Dermal substitute 2 step process

A
  1. applying a dermal substitute

2. epidermal cover

95
Q

Composite substitutes

A

Aim to mimic historical structure of normal skin allogenic skin incorporated into dermal scaffold
enable production of large batches
temporary bioactive dressings

96
Q

Aplifgraf

A

Composite skin consisting of bovine type I collagen cultured with allogeneic male neonatal fibroblasts and keratinocytes = resembles normal skin structure

97
Q

OrCell

A

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
Q

Limitations

A
  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!”

BMS354 Tissue engineering (5 decks)

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