lecture 13: tissue engineering - introduction Flashcards
1
Q
What is a stem cell?
A
- undifferentiated cell
- capable of self-renewal
- ability to differentiate into multiple cell types
- operational definition: stem cells maintain tissue and organ integrity by sustaining life long production of mature, functional cells in the steady state and in response to occasional stress
2
Q
Why is stem cell research important?
A
- basic biology - cell fate decisions
- development - tissue formation
- homeostasis - tissue maintenance and turnover
- understand how alterations to steady state can result in disease
- cancer
- potential to use cells as therapeutics
3
Q
What are classic stem cell properties?
A
- renewal
- high proliferative potential
- clonal repopulation
- multi-lineage differentiation
- present in low numbers - rare
- often morphologically unrecognisable
- quiescent in niche
4
Q
What is the classical stem cell hierarchy?
A
- stem cells living in niche
- in response to cues in the microenvironment we get a triggering of proliferation (self renewal) or differentiation
- series of events associated with becoming more specialised
- transit amplifying progenitors
- eventually there is a range of differentiated cells
- increasing lineage restriction + decreasing proliferative potential
- can get transdifferentiation
- can also get dedifferentiation
5
Q
What determines stem cell behaviour?
A
- microenvironment or niche
- restrictive
- permissive
- local or systemic factors that may influence stem cell behaviour
- stromal cells
- ECM
- combination of soluble factors, ECM, cell-cell interactions that the give the stem cell in its niche cues on how to behave
6
Q
What is the extracellular matrix?
A
- the extracellular space is comprised of a complex and dynamic network of macromolecules which constitute the ECM
- the ECM provides structural support, regulating cell-cell communication, sequestering of growth factors, and signalling molecules
- proteins and polysaccharides which assemble into an organised meshwork
- distribution and composition of the ECM in different tissues is unique
- seemingly equal cells can behave differently depending on the microenvironment to which they are introduced
- the microenvironment includes biomechanical and biochemical components in addition to the ECM
- cartilage: collagen, elastin, proteoglycans, hyaluronan, fibrinogen, laminin, fibronectin
7
Q
What are clues from ontogeny?
A
- what determines stem cell fate?
- how can we grow and control stem cells in the lab?
- study ontogeny:
- gives us clues as to cells
- cell-cell interaction
- matrix
- and soluble factors required for proliferation and differentiation
8
Q
What do we do when tissues or organs fail?
A
- transplantation
- require human donor (low incidence of organ donors)
- organ rejection - require immunosuppressants
- transplant rejection can damage other functional and healthy tissues
- prostheses
- requires replacement
- provides structural support but often limited function
- what about making the tissue from scratch?
9
Q
What is tissue engineering?
A
- exciting, new, innovative, multidisciplinary field
- scientists, clinicians, engineers
- bioengineering/material science, chemistry, biology, medicine
- alternative to traditional surgical procedures including organ transplantation, reconstructive surgery, and prosthesis
- biological approach
- tissue engineering is the process of growing new tissues and organs for the maintenance/repair/improvement/replacement of damaged, diseased, or poorly functioning tissues or organs
- trauma/birth defects/cancer/disease
- regenerative medicine more specifically refers to the application of stem cells to regrow tissues and organs
10
Q
What is the history of tissue engineering?
A
- genesis: the lord, breathed a deep sleep on the man and while he was asleep he took out one of his ribs and closed up its place with flesh. The lord god then built into a woman the rib that he had taken from the man.
- few thousand years later: pioneering studies in 1980s
- first studies developing skin grafts for tissue engineering
- epicel: layer of keratinocytes
- dermal regeneration template: combination of two naturally occuring ECM molecules, chondroitin and collagen, with a silicone membrane, requ ired to close large wounds that can’t heal themselves
- apligraf: combination of a collagen matrix with dermal fibroblasts
- 1990s: application of tissue engineering approaches for regenerating or repairing cartilage surfaces
- cartilage is avascular
- low cell density
- chondrocytes are poor at regenerating
- one approach to take some cartilage from a non weight-bearing area of the joint, grown up in the lab, periosteal patch, injected under surface → relativley successful
- more recent approach: use of combining chondrocytes with some kind of ECM / scaffold in order to repair weight bearing surfaces
- similarity between skin and cartilage transplants is that they are thin and flat and comprised of one cell type
11
Q
What occurs with tissue complexity?
A
- increasing functional parameters
- increasing metabolic requirements
- increasing cellular interactions
- increasing inter-organ interactions
- increasing engineering complexity: flat tissue structures (e.g. cornea) → hollow structures (e.g. trachea) → hollow, viscous structures (e.g. bladder) → solid organs (e.g. kidney)
12
Q
What are limitations in tissue engineering?
A
- vasculature
- attempts to tissue engineer skin and cartilage have been more successful because they are less dependent on the generation of a vascular supply
- cartilage is avascular
- skin is thin enough for diffusion
- one of the major challenges to the field in the development of strategies for other tissues is the establishment of a vascular supply
- why do tissues need vascularisation?
- nutrients
- oxygen
- removal of CO2 and cellular waste products
- any tissue thicker than 400µm must be vascularised. Oxygen transport is limited to 150-200µm
- needs to be established while constructs grown or assembled or implanted
- how are tissue engineers addressing this?
13
Q
What are approaches to dealing with the vascularisation challenge?
A
- provide biochemical signals within transplant to signals within transplant to stimulate endogenous angiogenesis (new vessels from pre-existing) and vascularisation (new vessels in absence of pre-existing – embryogenesis)
- VEGF/PDGF/FGF
- the generation of well distributed blood vessels within engineered tissue in vitro remains a major challenge
- currently vascularisation of engineered tissue is most successful when preimplantated within body at a different site (or animal)
14
Q
What are the three key factors of tissue engineering?
A
- soluble factors/biomolecules
- cells
- scaffold/biomaterials
15
Q
What cells can be used in tissue engineering?
A
- autologous (from own body)
- proliferative capacity of adult stem cells may not be sufficient to meet that need
- allogenic (from another individual of same species)
- expand up a large number of cells from an individual in the lab
- can have them ready to go as a product on the shelf
- use when required
- immune response
- transmission of disease
- less variable
- short turn around time is advantageous
- differentiated
- lower proliferative capacity so harder to expand up the number of cells to use
- functionally mature cells that will behave in the way that we want them to
- undifferentiated (stem cell)
- going to need to differentiate these cells in the dish - how?
- can’t transplant stem cells in because we are putting them into an environment where we don’t know how they are going to behave
- how do we isolate? where from? enrichment?
- non-invasive (autologous)
- purified (only desired cells)
- how generate enough cells for transplantation?
- turn-around time from harvest to transplantation?
- cell number/density is tissue dependent
- how do we guide cells to differentiate and maintain desired phenotype?
- how do we deliver cells to the correct location?
- how do we ensure survival, maturation, and function?
- need immediately
- yes:
- large organ:
- allogenic adult SCs, ESCs
- banked iPSCs
- small organ/substructure:
- autologous adult SCs, allogeneic SCs
- large organ:
- no:
- large organ:
- autologous adult cells and iPSCs
- small organ/substructure:
- autologous adult primary cells or SCs, iPSCs
- large organ:
- yes:
19
Q
What is bioprinting/3D printing?
A
- techniques for the manufacture of scaffolds are becoming more sophisticated including computer assisted 3D fabrication of custom scaffolds and tissues i.e. robotic printing
- precise control of architecture
- control density, functionality, shape to mimic organs
- controlled gradients in mechanical properties
- controlled gradients of biologically active factors
