lecture 28: tissue engineering

Question 1

What is tissue engineering?

Answer 1

• 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?

Question 2

What is the Vacanti Mouse?

Answer 2

• 1997
• transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear

Question 3

What are hurdles for TE success?

Answer 3

• technical
• commercial
• regulatory

Question 4

What are tissue components?

Answer 4

• cells
• matrix
• blood supply

Question 5

What is the traditional TE approach?

Answer 5

• create a scaffold
• add cells and growth factors to that scaffold
• implant in patient

Question 6

On what do strategies depend?

Answer 6

• 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

Question 7

What are challenges in TE?

Answer 7

• blood supply for 3D vascularised tissues
• suitable biomimetic matrix materials
• delivery of biological signals
• infection control
• → tailored design of systems for tissue engineering

Question 8

What are criteria for biomaterials in TE?

Answer 8

• 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

Question 9

What is mechanical characterisation of tissue and cell microenvironment?

Answer 9

• micropipette aspiration: cells
• AFM: cells, tissues, biomaterials
• instron microtester: tissues, biomaterials; stress-strain relations for explants (incubated, rate controlled, cyclic)

Question 10

What are examples of tailoured porous biomaterials?

Answer 10

• polymers
• hydrogels
• ceramics
• composites
• consider based on:
  
  • chemical and physical properties
  • architecture
  • stiffness
  • degradation

Question 11

How can cell surface-interactions be viewed?

Answer 11

• in 2D (morphology and migration rates) and 3D
• visualise how cells interact with material

Question 12

What are quantitative models?

Answer 12

• 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

Question 13

What are surface engineering strategies?

Answer 13

• 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

Question 14

What is seen with biodegradable PLGA subcutaneous in rat 2 weeks?

Answer 14

• 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

Question 15

How can we deliver bioactive molecules?

Answer 15

• 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

Question 16

What are potential delivery vehicles?

Answer 16

• scaffolds
• microspheres, nanospheres, nanofibres, nanoporous materials
• other 2D or 3D biomaterials

Question 17

What is the production of porous PLGA microspheres?

Answer 17

• 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

Question 18

What is the characterisation of porous PLGA microspheres?

Answer 18

• 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

Question 19

What is the multilayered polyelectrolyte biomolecule delivery strategy?

Answer 19

• 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

Question 20

What are in vitro release kinetics?

Answer 20

• 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

Question 21

What is controlled release to modulate inflammation?

Answer 21

• 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

Question 22

What was insight into peptide surface interactions?

Answer 22

• 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

Question 23

What was the in-vivo reponse to aMSH coated PLGA microspheres?

Answer 23

• reduced inflammatory response
• aMSH coated PLGA microspheres appeared to reduce the influx of inflammatory cells

Question 24

What is dual biomolecule delivery?

Answer 24

• 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

Question 25

How does biomolecule size affect release rate?

Answer 25

• 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

Question 26

What are in vivo tissue responses at 6 weeks?

Answer 26

• PLGA vs PLGA + aMSH vs PLGA + aMSH-(HA-CS)5-HA
• significant decrease in tissue response for LbL-aMSH

Question 27

What is happening towards clinical application?

Answer 27

• 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

Question 28

conclusions

Answer 28

• TE strategies need to be tailored to tissue targets
• nano-/micro-engineering can enhance outcomes
• success requires technical developments closely linked to clinical realities