1
Q
Biocompatability of TE construct
A
- biomaterials and scaffold immune response
2. cells- immune reponse
2
Q
Biomaterial tissue interactions
A
1, effect of biomaterials on body
- changes to wound healing
- toxicity
- infection
- tumorigenity
- embolism
- Effect of body on biomaterial
- calcification
- enzymatic degradation
- abrasion
- corrosion
3
Q
Healing process
A
Inflammatory- proliferative- remodelling
outcome of wound response depends on tissue impacted
4
Q
Foreign body reponse
A
- pro inflammatory- non specific serum protein absorption, immune cell infiltration, macrophage classical activation
- anti-inflammatory- macrophage alternative activation, macrophage fusion and fibrous encapsulation
5
Q
Macrophage mediated phagocytosis
A
- recognition
- adhesion
- phagocytosis
- digestion
6
Q
Giant macrophage engulfed
A
- recognition
- cell fusion/ adhesion
- engulfment and digestion
7
Q
Extracellular degradation
A
- recognition
- cell fusion and adhesion
- ec degradation
8
Q
How does the body responds?
A
Difficult to predict
depend on material
macrophages release of messengers- foreign body granulation- scar formation- PVDF optimal integration of implant- retained flexibility
9
Q
Fibrous Encapsulation
A
Tissue response to implanted biomaterials
Abundant deposition of extracellular matrix
Isolation of biomaterial from the local tissue environment
10
Q
Effects of Fibrous Encapsulation on TE device
A
Forms a Diffusion barrier
Stops Vascularization
11
Q
Device-related infections
A
Implant infections are relatively common Initially localized to the implant site Bacterial biofilm Staphylococci Clinical examples Biofilm-resistant materials
12
Q
Bacterial biofilms
A
Communities of bacteria grown on surfaces of abiotic materials and host tissues
The bacteria embed themselves in a matrix “EXTRACELLULAR POLYMERIC SUBSTANCE”
Ancient adaptation
Coordinated behaviour
Enhanced survival at the population level
13
Q
Stages of biofilm development
A
- attachment
- cell-cell adhesion
- proliferation
- maturation
- dispersion
- planktonic bacteria
14
Q
Biofilm formation by Staphylococci
A
Staphylococci are particularly prone to creating biofilms
Multidrug-resistant methicillin-resistant -strains
S. aureus (MRSA)
S. epidermis (MRSE)
15
Q
Examples of biofilm infections
A
Surgical repair materials (staples, sutures, mashes)
Orthopedic prosthetic joint infections
16
Q
The race to the surface
A
Interactions with biomaterials, host cells/proteins
- floating bacteria and host cells/ proteins
Continual cell layer
- no place for bacteria adhesion
- floating bacteria
17
Q
Cells do not interact directly with materials
A
A layer of protein (from growth media or plasma) adheres to the surface
Protein adsorption is affected by the surface properties of the material
18
Q
Bacterial with bacteria repelling proteins
A
No reaction with bacterial binding proteins
Bacteria repelling proteins
Anti-adhesive coating- metal/ polymer surface
19
Q
Toxicity and tumorigenity
Systemic and remote effects
A
By-products of physical/chemical wear can enter the blood stream and cause side effects in other parts of the body
Example: metal-on-metal hip replacements; releasing cobalt and chromium in the blood
20
Q
Thromboembolic complications
A
Exposure of blood to an artificial material can cause thrombosis, embolization and consumption of platelets and plasma coagulation factors
21
Q
Stent thrombosis
A
A rare but serious complication
Clinical consequences: death (20-48% cases) or myocardial infarction (60-70% cases)
Clinical approach to controlling thrombosis is the use of anticoagulant drugs
22
Q
Tumours associated with implants
A
Orthopedic implant-related osteosarcoma
not enough cases to establish relationship
dont know if its a direct cause
23
Q
Tumours associated with implants
A
- Potential tumorogenicity of some biomaterials demonstrated in animal models
- Neoplasms associated with therapeutic implants in humans are rare
- Difficult to prove causal relationship in some cases
- Mechanisms not clearly understood
24
Q
Biocompatability of TE construct
A
Cells- immune response
- HUMORAL IMMUNITY: mediated by soluble antibodies produced by B lymphocytes
- CELLULAR IMMUNITY: mediated by T lymphocytes
25
Immunoisolation as a TE strategy for treatment of Type I Diabetes
Type I Diabetes – autoimmune disease
Loss of insulin-producing islet cells of the pancreas
“The Edmonton Protocol” – successful islet transplantation from cadavers
Need for immunosupression
26
Immunoisolation
Does not use immunosupression
| Uses selectively permeable membranes
27
Different approaches for immunoisolation
1. Macroencapsulation
2. microencapsulation
3. ultra-thin coating
Immunoisolating membranes size decreases
Diffusion of nutrients increases
28
Development of stem cell-derived islet replacement therapies
Use encapsulation
allow insulin secretion
stops immune system from destroying them
*lots of companies trying to make this a viable approach
29
Other approaches
Hypothyroidism
Genetically engineered cells to secrete neurtropic or angiogenic factors
Enhancing tumor specific cellular immunity
30
Food news
Vegetarian meat aimed t replacing the real thing
| lab grown meat
31
Why engineering of food?
2006 69% increase to 2050
required increase in food calories 9.6 billion by 2050
Meat= most valuable livestock products
- all essential aa, bioavailable minerals and bitamins
- more people eating plant based
32
Agriculture has impact on environment 2010
70% water consumption
24% greenhouse gas emissions
37% earth landmarks
33
Current meat production is not sustainable
maturing complex organisms
| using them for basic muscle and fat
34
Cultured meat concept
Growing animal parts for food production separate 1932 Churchill
not much else change
35
Meat production on spaceships
2002 benjaminson et al
culture skeletal muscle of fish
2013- lab grown burger , not good in terms of taste and texture NOT the point could be viable approach to lab grown food
36
Building blocks
Cells, biomaterials/ scaffolds, bioactive molecules, bioreactor
skeletal muscles
37
What do you need to know to culture meat
1. Skeletal muscle structure- muscle cells- myofiber= grouped into muscle fibres, long dividing cells dont degenerate normally
2. Satelite cells regenerate muscle upon injury
- satelite cells= Pax 7/3 myf5
- myoblasts= MyF5, Pax7/3
- myotubes= MyoD, myogen
- de novo fibres
38
Satelite cells, culture expand
Satelite cells - isolate+ culture - proliferative phase - differentiation phase
39
What other cell types fir cultured meat
Embryonic stem cells
| source= some animals
40
Properties of biomaterials/ scaffolds for TE meat process
Edible, non toxic, non-allergenic biomaterial, supportive of full differentiation
41
Source of allernative cells for cultured meat production
Embryonic or induced pluirpotent stem cells
- unlimited proliferation
- need for differentiation optimisation
Alternative adult stem cells
42
Scaffolds and bioreactors for cultured meats
1. Stem cells
2. Animal free growth serum + myoblast proliferate + porous collagen microspheres
3. Myoblasts form myotubes on collagen microspheres
4. - bioreactors
5. Myotubes differentiate into myofibres
6. meat products
43
Maturation of muscle tissue
Anchorage
mechanical stimulation
electrical stimulation
44
Alternative fabrication strategies
Currently fabrication strategies- no hgihly organised structure
3D printing muscle tissue- high cell viability after the printing process
45
Technical challenges of food TE
Lab grown meat gets rare funding boost
all research lies with conventional companies
- academic research lags behind
- copyright this protocol= difficult to build research
not completely transparent, protect
46
Specifics of food TE
Scale
Efficacy
customer acceptancy
47
Scale
Global meat production= 293 million tons/ years
1 bioreactor per 10 million
culture media
48
Efficacy
Optimise scale up
| optimize differentiation
49
Customer acceptancy
no benefits so why would they do it?
| would you eat a chicken if grown in a lab?
50
Examples of companies producing tissue engineered meat
- memphysis
- FM
- cleanmeat
- Alephform
51
Public sentiment is everything
Abraham lincoln 1858
1. tastes and texture
2. ethics and attitudes
52
Benefits
1. Healthier meat- adding vitamins, reduce fat, increase levels of conjugated inoleric acid
2. create new products
3. control over texture and flavour
53
Method for making in vitro meat
Skeletal meat- isolate- muscle sc- essential cues- expand- essential cues niche for differentiation- maturate towards functional myofibers- essential cues 3D model system- culture in bioreactors= in vitro meat
54
Other animal product substituted for TE
lab grown leather and fat
55
Method for making TE leather
1. take small sample
2. isolate/ culture
3. deposit cells in sheets and induce collagen
4. layer sheets and continue culture
5. fuse layers to form hide
6. tan hide using fewer chemicals
7. finish and dye
8. fashion into finished designer items
56
Regulatory framework: a historical perspective
‘Elixir Sulfanilamide’ incident (1937)
The Nuremberg Code (1946)
The Softenon (thalidomide) incident (1956)
The Declaration of Helsinki (1964)
A statement of ethical principles for medical research
57
Conventional drug developement
Discovery
preclinical study
clinical trials 1,2,3
authorization
58
Regulatory agencies
European Medicines Agency (EMA) [EU]
Food and Drug Administration (FDA) [USA]
Ministry of Health, Labour and Welfare [Japan]
59
What are the different phases for
1. healthy volunteers
2. people with disease
3. largest group- risk benefit ratio
60
Legislation framework
Advanced therapies- have their own
guidelines- ATMP (advanced tissue medicincal product) advanced product that is either gene therapy, somatic cell therapy or TE product
61
Concerns related to the use of cells
```
Survival
Interaction
Evolving functionality
Migration
- no standard
```
62
Regulatory requirements
preclinical test- each TE method different
disease
The diversity and inherent properties of TE products require a case-by-case consideration
An overarching set of general considerations
63
Overarching regulatory requirements
1. The establishment of manufacturing process and controls
2. Preclinical safety and efficacy studies in clinically relevant models
3. Clinical trials on human participants
64
Manufactoring and scalability
Product consistency
product stability
- functional= cells can change over time
producible process- people trained in same way
65
Preclinical studies
```
Testing in animal models
expectations from proclincal data
consider
- safety
- functionality/engraftment
-immune response
```
66
Animal models
No default species
chosen based on scientific reasoning
NEED to understand limitations
67
Phase 1 trial- Age related macular degradation
Embryonic stem cells use to device retinal pigment epithelium patch
quality control required for each step of manufacture- eg medium for cells see appearance and viability of cells
68
Design preclincial studies for hESC-RPE derived cells
Main concern= tumors- dont expect too much proliferation but could end up with teratoma
ensure no remaining stem cells to give rise to tumours
69
Model of choice for preclinical hESC-RPE derived cells
Mice
Demonstrate safety of teratoma
immunocomprimised and can ensure no rejection
70
What did they use for clinical studies on eyes?
had to transplant 100s into human eye
Used pigs as same size eye
Similar
71
Exploratory trial
Phase 1 not appropriate as not stages of TE method often include surgery
not appropriate to perform surgery on healthy volunteers
Use small study with small group of patients
primary concern= safety
72
What does the exploratory trial do?
Evaluates feasibility of treatment in humans but may not be statistically significant
- starting doses based on realistic possibility of therapeutic benefit
- threshold dose- max dose not limited by potential toxicity
73
Defining comparator
The currently accepted treatment – but this may not always be available or proven to work better
A device performance can be surgeon-dependant!- skills of surgeon dictate success
74
Randomization
Each participant has the same chance of receiving treatment
standardized and independent of investigator
issues/solutions for TE products
blinding a trial impossible these pretty much
75
Follow up/ patient care
Important for determining the efficacy
Either one person follows up all patients or all care givers should be appropriately trained
The trial should be designed to minimize the effect of the procedure on patient care
76
Risk- benefit
Hospital Exemption
ATMPs exempted from the centralized marketing authorization procedure
Individual case studies
77
Healthcare industry
translation of therapies to patient
user vs payer in healthcare industry-you as the consumer decide to buy product based on own opinions
in this case doctor decides this and patients doesnt directly pay
goverment biggest payer/inserter- heavy influence in politics
78
Valley of death
The gap between discoveries in the lab and therapeutic leads that enter clinical trials
Academic research – early phases of therapeutic development
make breakthrough
Big Pharma- make investments= no guaranteed promise of success
79
GO GO TE
Integrin dermal regeneration template
trancyte, dermograft (advanced tissue science)
- named for TE by this company but company went bankrupt- company founded in 87
- 40 patients praise from the doctors
- substitutes for skin wounds produced but no profit made
80
Case study for skin replacement in burn victims
Large market- 100,000 per year
25,000 require skin replacement
- treatment for skin ulcers= potential profits in the billions
- got approval for dermograft for FDA 1991
81
Why didnt it work
Unanticipated delays
price dropped dramatically
manufacture challenging
82
How did they try to pay it off?
Fundraising occured builiding a facility which couldnt be paid off
overall
finiancial planning needs to be thorough
communication important
HIG risk investment
83
What is the main aim?
How can product be developed and produced at an afforable price that still outweighs risk
84
Bottleneck TE products
```
Financial reward not garanteed
case by case pathway
manufacture usually expensive
complicated by biological nature of the products
no unique business
```
85
Risk in regenerative medicine revenues
906 reg medicine companies worldwide
13 billion raised
1028 clinical trials underway
86
Spectrum of models
Modelling systems appropriately- physiological tolerance but experimentally not viable
increase complexity= decrease experimental tractability
87
Drug attrition
Time from lab to market
~10 years
~2.6 billion
When clinical trial starts 5 drugs trialled from 5-1000 compound library
88
What should models do
Accurately predict the outcome in patients
89
Adverse reactions
4/1000 emergencies due to this
20% acute kidney
dying pipeline
90
When are drugs withdrawn from market
When the risk outweight the benefits
VIOXX 2004
- major causes hepatoxicity (liver) and cardiotoxicity (heart)
- current model not sufficient- otherwise would predict outcome more accurately
91
Details of model systems
In vivo= primary cells, transformed all lines, stem cells
| In vivo= animal models- even primates dont read out human outcome
92
Example of when model organism doesnt read out human outcome
PGN1412 immunotherapy leukemia
thought would revolutionise
6 patients died in phase 1 clinical trial caused by major organ killed
difference in aa between humans and primates
93
ORGANOIDS
resembling an organ
- organ on a chip
- enough physiological relevence and reasonably experimentally tractable
94
Stem cells prognosters aggregated into 3D ball and soluable cells
3D organoid produced through self organisation
huge field in stem cells atm
cells allowed to self organise to create stuctures- not in control of this
organs placed on chip to control this
95
ON A CHIP
Device for culturing cells in continuously perfused micrometer sized chamber
incorporates minimal units that mimic tissue and organ level functions
microfabrication and microfluidics combine with cell culture methods
96
Fabrication method
- polymer mixed with crosslinking agent and poured onto mould
- heated to solidify
- placed on glass slide- see through microscope
- puncture holes to cellular fluid through
add dye for tracability
97
Micro fluidics
Science underpinning small volume manipulation
fluid particles well organised
Follow laminar now normally
more control over fluid
means gradients can be created allowing case of analysis
98
Typical components
Geometric confinement and patterning- control our cell placement/ interactions
environment control- movement
sensors tested to give physiological readouts
99
Lung on a chip review
Human lungs gas exchange- large SA to vol ratio, alveolar epithelium in a singular layer- incontact with air and water
cyclical stretching is seen in aveolus and its epithelium#
Cells need mechanical stimualtion to mature properly
100
How is aleveolar primary functional unit mimicked on a chip
Chip design
- chip minature
- multilayered- larger porous membrane upper layer
- placed one side of porous membrane with alveolar epithelium
- other side endothelium
- air in top chamber and media representing blood in bottom layer- air liquid interspace achieved
- can use different dyes to show different cell tyeps and cell viability
- added air increased sufectant production
101
How do they mimic cyclincal stretching
Using vaccum attached to outer chambers
102
Modelling pulmonary inflammation
```
Caused by pathogens, toxins or allegens
pneuomia (acute) asthma (chronic)
- cytokines released by epithelium
- TNF-a
- activation of endothelium- ICAM1
- leukocytes infiltrate the alveoli
```
103
How would you modify the on the chip system for pulmonary inflammation?
TNF-a used to stimulate the cascade- add to endothelium
| staining used to measures levels of ICAM1- expression upregulated in cells stimulated with TNFa
BMS354 Tissue engineering
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