lecture 28: tissue engineering Flashcards
1
Q
What is tissue engineering?
A
- the loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care
- a new field, tissue engineering, applies the principles of biology and engineering to development of functional substitutes for damaged tissue
- R Langer and JD Vacanti
- aims to generate new functional tissue to repair or replace tissues missing due to disease, genetic defects or trauma
- promise of:
- alleviating tissue shortages
- superior results
- customised implants
- new treatments where non currently suffice
- great promise by limited outcomes to date… why?
2
Q
What is the Vacanti Mouse?
A
- 1997
- transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear
3
Q
What are hurdles for TE success?
A
- technical
- commercial
- regulatory
4
Q
What are tissue components?
A
- cells
- matrix
- blood supply
5
Q
What is the traditional TE approach?
A
- create a scaffold
- add cells and growth factors to that scaffold
- implant in patient
6
Q
On what do strategies depend?
A
- tissue type
- design criteria based on specific tissue properties
- in vitro construct development feasible for avascular/small/2D tissues
- 3D human scale tissues need blood supply early
- in vivo bioreactors allow construct development concurrently with vascularisation
7
Q
What are challenges in TE?
A
- blood supply for 3D vascularised tissues
- suitable biomimetic matrix materials
- delivery of biological signals
- infection control
- → tailored design of systems for tissue engineering
8
Q
What are criteria for biomaterials in TE?
A
- biocompatibility
- mechanical properties for target tissue and implantation site
- biodegradability profile (time, strength and by-products)
- suitable in vivo responses e.g. inflammation, FBR
- ability to be fabricated into desired structures
- cost-effective, available, regulatory approval
- ability ot be sterilised safely
- adequate stability and shelf-life
- promote desired cellular responses e.g. proliferation, differentiation, gene expression
9
Q
What is mechanical characterisation of tissue and cell microenvironment?
A
- micropipette aspiration: cells
- AFM: cells, tissues, biomaterials
- instron microtester: tissues, biomaterials; stress-strain relations for explants (incubated, rate controlled, cyclic)
10
Q
What are examples of tailoured porous biomaterials?
A
- polymers
- hydrogels
- ceramics
- composites
- consider based on:
- chemical and physical properties
- architecture
- stiffness
- degradation
11
Q
How can cell surface-interactions be viewed?
A
- in 2D (morphology and migration rates) and 3D
- visualise how cells interact with material
12
Q
What are quantitative models?
A
- modified fisher equation
- non-linear parabolic PDE with travelling wave solutions
- captures main mechanisms of wound healing: diffusion and proliferation
- includes cell density dependent diffusivity
- u(x,t) = cell density at x and t
- D0 = diffusivity for isolated cells
- D(u/u*) = dimensionless diffusivity function with D(0) = 1 and dD/du less than 9
- u* = confluent cell density
- a = cell growth rate
- very complex equation required to characterise the change in cell density as a function of time
13
Q
What are surface engineering strategies?
A
- Layer-by-layer (LbL) assemblies (Tristan Croll, Dewi Go)
- layering of hyaluronic acid, chitosan and carbodiimide (EDC) (cross-links layers)
- cells often don’t like materials used
- but these materials are important so develop ways to prevent cells coming in direct contact with the material
- e.g.
- chemically modify hydrophobic polymer to have some hydrophilic groups e.g. hydroxyl and amino
- add on large biological molecules (hyaluronic acid, positively charged, will bind onto positive amino groups leaving excess negative charge)
- add positively charge polyelectrolyte (chitosan)
- many layers to cover up the cell surface
14
Q
What is seen with biodegradable PLGA subcutaneous in rat 2 weeks?
A
- if you just put PLGA
- very little tissue growth
- lots of macrophages i.e. immune response
- LbL
- number of macrophages much less
- gave some power to regulate the response
15
Q
How can we deliver bioactive molecules?
A
- growth factor delivery:
- gelatin microspheres
- size/shape easy to control
- cross-linking allows control of chemical and mechanical properties
- electrostatically bind many GFs
- achieved GF release over ~3 weeks
16
Q
What are potential delivery vehicles?
A
- scaffolds
- microspheres, nanospheres, nanofibres, nanoporous materials
- other 2D or 3D biomaterials
17
Q
What is the production of porous PLGA microspheres?
A
- combination of inkjet and thermally induced phase separation
- compressed air → pressure controller → PLGA solution reservoir → piezoelectric transducer → pulse controller → to computer
- drops into liquid nitrogen → polymer and solvent mixture → rapid cooling → polymer rich phase, solvent crystals → sublimation of solvent → voids
18
Q
What is the characterisation of porous PLGA microspheres?
A
- measure the effect of height on size of microsphere
- 5cm vs 15cm
- effect of nozzle size
- 25µm vs 59µm
- porous all the way through
- tuneable system
19
Q
What is the multilayered polyelectrolyte biomolecule delivery strategy?
A
- aminolysed microspheres → HA → Chi → HA → Chi → Heparin → bFGF → Heparin → Chi → HA → chi → HA
- crosslink during the buildup to ensure stability (EDAC/NHS)
- PLGA microsphere → PEI wash → aminolysed PLGA microsphere → HA wash → chi wash → HA, Chi, Heparin wash
- outside and inside of pores of those spheres
20
Q
What are in vitro release kinetics?
A
- long release times
- steady rate of release
- tunable via microsphere and LbL properties
- release conditions: 0.01M PBS, pH 7.4, 37 ± 0.5C, rotation at 90 rpm, sink conditions
- 100% released ~ 40-50 days
21
Q
What is controlled release to modulate inflammation?
A
- alpha-MSH
- aMSH can be uniformly absorbed on PLGA microspheres
- 94% of aMSH was released in 3 days
- incorporation of other molecules in the spheres
- only bind it to spheres lasts very little
- need some surface coating
22
Q
What was insight into peptide surface interactions?
A
- using molecular dynamic simulation
- modelling a-MSH peptide interaction with various surfaces
- aMSH binds strongly to hydrophobic surfaces
- positive residue arginine pointing upwards at pH 9
- optimise binding conditions
23
Q
What was the in-vivo reponse to aMSH coated PLGA microspheres?
A
- reduced inflammatory response
- aMSH coated PLGA microspheres appeared to reduce the influx of inflammatory cells
24
Q
What is dual biomolecule delivery?
A
- to incorporate both growth factor and anti-inflammatory hormone
- basic fibroblast growth factor - 17.2 kDa
- induces angiogenesis, cell division, chemo-attraction
- promotes the regeneration of adipose tissue, cartilage and nerves
- specific binding between heparin and bFGF help stabilise the GF
25
How does biomolecule size affect release rate?
* number of residues:
* aMSH: 13
* bFGF: 154
* MW (kDa)
* 1.6
* 17.2
* size (Å)
* 40 x 12 x 4
* 30 x 30 x 45
* increasing number of layers
* significant decrease
* decrease
* increasing crosslinker concentration
* no change
* significant decrease
26
What are in vivo tissue responses at 6 weeks?
* PLGA vs PLGA + aMSH vs PLGA + aMSH-(HA-CS)5-HA
* significant decrease in tissue response for LbL-aMSH
27
What is happening towards clinical application?
* 3D rapid prototyping to fabriate customisable constructs
* in vivo bioreactors
* the neopec solution for breast replacement
* a breast shaped o-brien chamber is inserted under the skin immediately after mastectomy. the synthetic chamber will act as a scaffold to support the growth of fat tissue
* surgeons redirection blood vessels from under the patient's arm into the chamber. a few grams of the patients fat cells are placed on the end of the vessels
* a special gel is placed in the chamber to stimulate the fat cells to multiply
* the fat grows in the shape of the chamber of the next 4-6 months to create a new 'breast'
* the chamber is removed or dissolves after the new 'breast' is formed
28
conclusions
* TE strategies need to be tailored to tissue targets
* nano-/micro-engineering can enhance outcomes
* success requires technical developments closely linked to clinical realities
