Part 1 Flashcards
What is regenerative medicine?
Umbrella term for tissue engineering and cell therapy- incorporates research on self healing
Tissue engineering?
Uses biomaterials- design tissues and ECM/scaffold
Biological substitutes to restore, maintain or improve tissue functions
Cell therapy?
Take cells and manipulate and place back into patient, sometimes cells transplanted or need support
Major causes of organ failure
Injury
disease
ageing
Current treatments for organ failure
- Surgical reconstruction- suture
Limitations= surgical complications, morbidity at the donor sites
-mechanical devices- pace maker, hip replacement, dialysis machine
limitations= only mechanical support, do not grow with the tissues (children)
-transplantation- of organs or tissues
Limitations= immunosuppressants, transplantation rejection
What is the transplantation crisis?
Three people die each day in the UK because a suitable organ can not be found
Problem with supply verses demand
Problem with some donors
Died from encephalitis
NOT kidney failure
Both got same donor, got the same disease as the donor that died- didn’t screen for it before
New releases in medicine
NHS blood and transplant statement about inquest into deaths of 2 transplant recipients after kidney transplant from the same donor
New Solutions for treatments for treatments of organ failure
Why is it needed?
- donor tissues and organs are in short supple
- we want to minimise immune system response
Historical perspective of tissue engineering
Made in 1987
1990s research accelerates and industry begins to emerge. Stem cells started- derivation of pluripotent embryonic stem cells
How do we build a tissue?
Cells in tissues and interlinked with ECM component
ECM- protein fibres- elastin, collagen, reticular and ground substitutes
Resident cells- mesenchymal cells, macrophages, adipocyte, fibroblast
What are the building blocks of tissue engineering?
- cells
- biomaterial scaffolds
- Bioactive molecules
the first Tissue engineering cartilage
Plastic and reconstructive surgery
- total reconstructive of ear is difficult
- elevated the feasibility of growing tissue engineered cartilage in the shape of a human ear
How did they make a cartilage in the shape of a human ear?
- A plaster mould of a ear of a 3 year old child was cast from an impression of he ear- used as a SCAFFFOLD for seeding cells
- Cartilage CELLS from a calf were seeded onto scaffold
- After 12 weeks the constructs were explanted sectioned and stained
What were the drawbacks of early years in TE
- Skin coverage is missing
- bovine chondrocytes were used
- Scaffolds had to be reinforced for mechanical stability
- Implications on the growth rate of the artificial ear
Misconceptions of human ear tissue engineering
Not a genetically engineered mouse with a human ear on its back
- caught lots of media attention
- false
not human ear on back but calf
3D bioprinting system to produce human scale
doctors 3D print of living body parts
Since then the field has moved on
show ears have been 3D printed and the structures are more relevant
Steps of Red medicine and tissue engineering
Step 1-Research 1-5 years= Laboratory testing to establish cellular biology, scaffold engineering and action to provide proof of contact
Step 2- Development 3-5 years= preclinical and clinical testing to determine safety efficacy and production
Step 3- regulatory 3-5 years= Regulatory review of results in small and large populations
Step 4- commercial= product registration
General principles in tissue organisation
You know how this happens in nature - regeneration
in vitro= make tissues from scratch, need to know how function and their organization
Structure and components existing in cells and ECM
Wound heeling facts
30 days after injury new skin formed
injure protective barrier to body
open wound- incidence- no visible scar
Phase 1 of wound heeling
- Inflammatory phase- primary objective is to stop bleeding
- clear out dead cells
- stop injection- phagocytosis
- Redness, swelling, clotting
1 dilate promoting connection
2 increase viscosity allowing blood to flow more slowly near the site of clotting
3 leukocytes/WBC phagocytes go inflamed tissue engulfing bacteria
4 GF production results in fibroblasts
Phase 2 of wound heeling
proliferative phase
- new tissue formation
- disorganised tissue
- focus moves to building new tissue to fill wound space, fibroblasts secrete collagen and cause angiogenesis
- form granulation tissue which is a scaffold for tissue scar, soft so bleeds easily and is leaky
- epithelization= regeneration, migration and organization of the epithelial cells at the wound edge
Phase 3 of wound heeling
Remodelling phase
- remodelling new collective tissue
- can take a while
- collagen forms final scar tissue- may achieve 70/80% if normal tensile strength by 3 months
The healing process steps
- cut blood vessels bleed onto wound
- blood clot forms and leukocytes clean the wound
- blood vessels regenerate and granulation of tissue form
- epithelization regenerates and scar tissue forms
What does the outcome of injury depend on?
- how long
- type of tissue damaged
- amount of damage
Injury pathway
- mild, superficial injury= regeneration
2. severe injury= scar formation
Injury- cellular and vascular response
- Stimulus removed acute injury either:
- Cell death, intact tissue framework- Regeneration restitution of normal structure
- Cell death, framework of tissue damaged- repair scar formation - Persistent tissue damage- fibrosis, tissue scar
What is acute injury
Intact matrix
Some loss of cell but will regenerate
Cells + matrix=
Scar
Deposition of connective tissue, proliferation of residual cells within
Pulmonary fibrosis
Results of infection in lungs
persistent
connective tissue scar
lead to lung organ failure
Fibrous encapsulation
Hip replacement
- body sees foreign object in the body it will try to protect by laying down collagen
- deposit ECM- get fibrous encapsulation
Granulation tissue
formation of scar
soft so bleeds easily
new tissue forms 3/4 days post wound healing process and called this as looks granular
Sources of cells
- autologous- patients own cells
- allogenic- cells from same species
- xenogeneic- different species
- syngeneic or isogenic- genetically isolated
What are autologous cells?
Tissue matching not required
no graft and host response
engraftment faster
Disease transmission not needed
What are allogenic cells?
Tissue matching required
host response needed
slower engraftment
Disease transmission possible
Cell types for tissue engineering
differentiated mature cells
mixture of differentiated cells
stem cells
Advantages of stem cells
Adult= multipotent, derive different cell types, get from patient
Embryonic= kept in culture for long periods
induced pluripotent stem cells= halogenate (differentiate into any cell types)
Disadvantages of stem cells
Adult= get from patient so depends on which patient is suffering from (genetic) hard to multiply
Embryonic and IPS= hard to get specific cell type as so many trivial, done in vitro
Differential cells
Advantages - already functional Disadvantages - specific - already permanently differentiated - limited proliferation
How does adult stem cells work?
Biopsy from bone marrow or adult tissue
bone marrow derived mass
How does embryonic cells work?
Egg and sperm
fertilisation
inner cell mass human biopsy
embryonic stem cell colony
How does induced pluripotent stem cells work?
Adult somatic cells
lentiviral delivered + transcription factors
IPSC colony
Example of differentiated cell types
Fibroblasts, keratinocytes, osteoblasts, endothelial cells, chondrocytes, preadipocytes, adipocytes
Steps of culturing cells
- Growth medium contains necessary components- GF, nutrients, glucose
- laminar hood- Stops infection
- incubator- 37 degrees, not sealed as need gas exchange, stop evacuation using solution at bottom of incubator
What is GMP
Good manufacturing practise
Ensures that medicinal products are consistently produced and controlled to the quality standards appropriate to their intended use
Outcomes of GMP
- control environment
- everything documented
- correct regulation
Importance of studying cells- material interaction
In tissue engineering, cells are in contact with biomaterials
Use of materials in TE requires understanding of cells with materials
Most cells require attachment to a solid surface
What does the ECM do?
Provides structural support mechanical properties provides bioactive cues for cells scaffold for tissue renewal Act as the reservoir of GF and potentiates their actions
Components of ECM
Collagen
glycosymonaglycons
Fibronectin
Composition varies depending on tissues
Muscle= high tensile strength
- fibrous structural proteins- high tensile strength
- water hydrated gels- resilience
- adhesive glycoproteins - connect cells to ECM
Structural proteins collagen
80/90% collagen 1,2,3
Polypeptides chains- twist helices, helical structures
High abundance of 3 aa- proline, hydroxyproline + glycine
Proteoglycans
Composed of glycosaminoglycan chains linked to a specific protein core
very hydrophilic- form highly hydrated compressive gel
lubricated, resistant, found in joints
Adhesive molecules
Fibronectin
Attach cell to ecm
Stress activated mechanical pathway
Cell adhesion to ECM/biomaterials
What are integrins?
heterodimers made up of 19a + 8b subunits
Which subunits depends on what is recognised- composition determines specifically
extracellular, transmembrane and intracellular domain
most integrins recognise several ECM proteins
What is conformational integrin activation
Inactive (bent)
- active (extended)= intrinsic ligand, inside out- ligand in short cytoplasmic
- active (clustered)= extrinsic ligand, outside in
What does activated integrin activate
Activates the FA complex
- talin kindlin
- vinculin
- a-actinin
- FAK cos Src paxillin
- ILK
The goes onto assembly of the actin cytoskeleton, activation of signalling pathway
What is the major signalling pathways integrins use?
Focal adhesion kinase
- integrins a and b
- activates FAK
Mechanotransduction
process by which external mechanical stimuli are transmitted into the nucleus
Modulate biomaterials to determine cell fates
Contains
- ecm- Laminin fibres, collagen fibronectin
- binds integrins
- cytoskeleton meditated signals- cytoplasmic signal transduction
- proliferation, differentiation, protein synthesis, attachment, migration, shape change
What is EBSC?
derived retinal pigment epithelium patch in age related macular degeneration (replace missing cells from eye)
- widely publicised
- Phase 1 clinical trial
- used biomaterials, synthetic basement membrane
What are biomaterials?
Non viable materials used in a medical device intended to interact with biological systems
Used to develop scaffolds
NIH definition of a biomaterial
National institute of health
Any substance 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 tissues, organs or function of the body
What is the historical perspective on Biomaterials
examine eyes of spit fighter plane pilots- had splinters in eyes from cockpit but did not produce an immune response
- catalax= clouding of lens
- could be used as a synthetic substance implanted in body without immune response
What is biocompatibility of materials
Ability of materials to perform with an appropriate host response in a specific application
Examples of appropriate host response
resistance to blood clotting
resistance to bacterial colonization
normal healing
What is the evolution of biometerials?
1st generation- bio inertness- do not interact with body fluid or tissues
2nd generation- bioactivity
3rd generation- function tissue
Bioglass
Started bioactivity
- exceptional material as it binds with bone
- when implanted into rats implants bound to bone and became as one, can be hit and wont come apart
- don’t respond to mechanical ques of body= non functional
What are polymers?
poly= many
meros= parts
- large molecules made up of chains or rings of linked monomeric links
mw- 200,000 Da
Different structures of a polymer?
Linear
branched
network- different polymers linked together
What is a car tire made up of?
Polymers linked together with sulfur and carbon black to give its colour
Repeat units in polymers
Polyethene, polyproline, polyvinylchloride
repeated units
single bonds between C atoms
Different types of polymers
- Homopolymer- Only one repeated functional group
- Block copolymer
- alternating copolymer
- graft copolymer
- random copolymer
What is mixing of polymers?
Mixing not chemically mixed
Hydrogels
Crosslinked polymer networks that are insoluble but swell in aqueous medicine
offer an environment that resembles the highly hydrated state of natural tissue
Basic classes of biomaterials
- Natural- nature
- synthetic- made in lab
- semi-synthetic- combo
Different types of natural polymers
- protein based natural polymer
- collagen (25% of body weight), silk (high strength weight ratio), gelatin (formed from collagen), fibrin (blood clot component), elastin and soybean - polysaccharide
- chitosan- extraskeleton of nuclei
- alginates- bacteria
- hyaluronan- umbilical cord cartilage and skin
- chondroitin sulfate- cartilage
How do we obtain materials?
- Extraction
- Purification
- concentration
What are synthetic polymers for scaffolds?
- polylatic acid
- polyglycolic acid
- poly (lactic to glycolic acid)
What are semi synthetic polymers?
Hybride molecules made by incorporation of biologically active macromolecules onto the backbone of synthetic polymers
example= PEG (polyethene glycol)
Benefits and disadvantages of natural
+ biofunctional, IDG domain, cheap
- mechanical stability, sourcing them, variation in branches
Benefits and disadvantages of Synthetic
+ industrial scale, tailored to suit needs
- don’t know if they work, immune response, toxicity, biodegradability
Properties of biomaterials
physical= strength, elasticity, architecture Chemical = degradability, reabsorption, water content Biological= interactions with cells, release of biological active signals
Degradable materials
Broken down overtime
use materials initially as scaffold and over time get replaces with scaffold in TE can be positive
break covalent bond
should produce non toxic biproducts
Resorbable materials
Total elimination of the initial foreign material and its biproducts
can be metabolised and secreted from the body, no trace
*products can be degradable and not reabsorbable
Properties of examples of biomaterials
- internal sutures= hold tissue together until heals and then degrade
- soft contact lenses= transparent, refractive and hold shape
- artificial hip joints= strong, not much wear and tear
Bulk properties of materials
Strength, toughness, fatigue and stability
How can mechanical properties of biomaterials influence how the cells can behave?
Stem cells placed onto 3 different types of materials with different stiffness
least stiff= blood/brain
medium= muscle
most= bone
Bulk verses surface properties
designing materials that fulfil bulk and surface
Suface modifications
- chemically/ physically alternating atoms/ molecules in the existing surface
- overcoating the existing surface with material that have different composition
- creating surface textures and patterns
Considerations for surface modifications
thin surface modification
delamination resistance
surface analysis
What do cells interact with?
Do not interact directly with materials
a layer of protein adheres to the surface
protein absorption is affected by the surface properties of the material
Recognise protein and form indirect link
Non fouling surfaces
Resistant to absorption of protein or adhesion of cells
PEG and zwitterionic polymers
How to make non fouling surfaces adhere?
Cell adhesion occurs through receptors in the cell membrane
RGD domain in fibronectin and vitronectin
Cellular response can vary with surface density of RGD
How to functionalise surfaces
- fibronectin= naturally occurring biometic biomaterials
- activation of fibronectin binding - adhesion complex- attach biomolecules to polymer surface
- immobilisation of integrin binding peptides or entire protein
- if resistant of cell binding- use RGD domains
- intracellular signalling modulation of events
Micrometer- scale chemical patterns
a) a prepolymer is poured on a structural master
b) the prepolymer is cured and the stamp peeled off the master
c) the stamp is cut into smaller pieces
d) the stamp is linked by soaking in ink solution
e) ink printed by contacting an inked stamp with a suitable surface
f) patterned substrate is obtained
Study for cell: material interaction
new blood vessel formation- angiogenesis
Endothelial cell growth is critical
EC increases in speed area are accompanied by increase of cell proliferation
Hypothesis for cell: material interaction
cell shape per SA controls cell fate of endothelial cells
Approach
micropatterning of fibronectin islands
cells assume the shape of the islands
Extent of cell spreading is determined whether a cell underwent proliferation or apoptosis
Conclusions
Same GF- genes- same ECM- different geometry- different fates
cells can filter the same set of chemicals input to produce different functional output
