Transcript MICROPATTERNED CELL CO-CULTURES USING LAYER

NANOFIBER TECHNOLOGY: DESIGNING THE NEXT GENERATION
OF TISSUE ENGINEERING SCAFFOLDS
C.P. Barnes1, S.A. Sell1, E.D. Boland1, D.G. Simpson2, G.L. Bowlin1
1Department of Biomedical Engineering, 2Department of Anatomy and Neurobiology
Virginia Commonwealth University, Richmond, VA
MARK HWANG
EXTRACELLULAR MATRIX
Signalling
- cell adhesion
- migration
- growth
Components
- collagens
- elastin
- hyaluronic acid
- proteoglycans
- glycosaminoglycans
- fibronectin
- programmed cell death
- cytokine/growth factor activity
- differentiation
TISSUE ENGINEERING SCAFFOLDS - BACKGROUND
Premise
- ECM microenvironment key to tissue regeneration
- Cell not viewed as self-contained unit
Role of ECM
- ECM mediates biochemical and mechanical signalling
- Ideal ECM
non-immunogenic
promote growth
maintain 3-D structure
only native tissues remain post-treatment
Research emphases to-date
- Biocompatibility
- Degradability
TISSUE ENGINEERING SCAFFOLDS - BACKGROUND
Overall Goals
- Design scaffold with maximum control over:
biocompatibility (chemical)
biodegradability (mechanical)
- Utilize natural and synthetic polymers
- Future directions:
tissue regeneration
drug delivery
EFFECTIVE SCAFFOLD DESIGN BEGINS WITH ACCURATE SCALING
Current Focus
- Nanofiber synthesis
NANOFIBERS - INTRODUCTION
ECM fibers ~ 50-500 nm in diameter
Cell ~ several-10 um
Fibers 1-2 orders of magnitude < cell
Scale difference necessary
- single cell contacts thousands of fibers
- transmission of fine/subtle signals
3 techniques to achieve nanofiber scale
- self assembly
- phase separation
- electrospinning
NANOFIBERS: SELF-ASSEMBLY
Definition: spontaneous organization into stable structure without
covalent bonds
Biologically relevant processes
- DNA, RNA, protein organization
- can achieve small diameter
Drawbacks: more complex in vitro
- limited to 1) several polymers and
- 2) hydrophobic/philic interactions
- small size; larger = unstable
Example: peptide-amphiphiles
- hydrophobic tail
- cysteine residues  disulfide bonds
NANOFIBERS: PHASE SEPARATION
Definition: thermodynamic separation of polymer solution into
polymer-rich/poor layers
- similar to setting a gel
- control over macroporous architecture
using porogens, microbeads, salts
98% porosity achieved!
- consistent
Drawbacks:
- limited to several polymers
- small production scale
NANOFIBERS: ELECTROSPINNING
Definition: electric field used to draw polymer stream out of
solution
A- polymer solution in syringe
B- metal needle
C- voltage applied to need
D- electric field overcomes
solution surface tension;
polymer stream generated
E- fibers 1) collected and
2) patterned on plate
NANOFIBERS: ELECTROSPINNING
- simple equipment
- multiple polymers can be combined at
1) monomer level
2) fiber level
3) scaffold level
- control over fiber diameter
alter concentration/viscosity
- fiber length unlimited
- control over scaffold architecture
target plate geometry
target plate rotational speed
NANOFIBERS: ELECTROSPINNING
Drawbacks:
- natural fibers 50-500 nm; spun fibers closer to 500 nm
- architecture very random
LACK OF GOLD STANDARD
Current approaches combined techniques
- usually electrospinning + phase separation
- fibers woven over pores
NANOFIBERS: OVERVIEW
ELECTROSPINNING POLYMERS
Synthetics
- Polyglycolic acid (PGA)
- Polylactic acid (PLA)
- PGA-PLA
- Polydioxanone (PDO)
- Polycaprolactone
- PGA-polycaprolactone
- PLA-polycaprolactone
- Polydioxanone-polycaprolactone
Natural
- Elastin
- Gelatin collagen
- Fibrillar collagen
- Collagen blends
- Fibrinogen
POLYGLYCOLIC ACID (PGA)
- biocompatible
- consistent mechanical properties
hydrophilic
predictable bioabsorption (2-4 wks)
- electrospinning yields diameters ~ 200 nm
Parameters
- surface area to volume ratio
- spinning orientation affects scaffold elastic modulus
Drawbacks
- rapid hydrolitic degradation = pH change
tissue must have buffering capacity
POLYGLYCOLIC ACID (PGA)
Random fiber collection (L), aligned collection (R)
POLYGLYCOLIC ACID (PGA)
Fiber
collection
Orientation
affects
stress /
strain
POLYLACTIC ACID (PLA) – 200 nm
- aliphatic polyester
- L optical isomer used
by-product of L isomer degradation = lactic acid
- methyl group decreases hydrophilicity
- predictable bioabsorption, slower than PGA (30 wks)
- half-life ideal for drug delivery
Parameters (similar to PGA)
- surface area to volume ratio
- spinning orientation affects scaffold elastic modulus
Compare to PGA
- low degradation rate = less pH change
POLYLACTIC ACID (PLA) – 200 nm
Thickness controlled by electrospin solvent
Chloroform solvent (L) ~ 10 um
HFP (alcohol) solvent (R) ~ 780 nm
Both fibers randomly collected
PGA+PLA = PLGA
- tested composition at 25-75, 50-50, 75-25 ratios
- degradation rate proportional to composition
- hydrophilicity proportional to composition
Recent Study
- delivered PLGA scaffold cardiac tissue in mice
- individual cardiomyocytes at seeding
- full tissue (no scaffold) 35 weeks later
- scaffold loaded with antibiotics for wound healing
PGA+PLA = PLGA
PLGA modulus proportional to composition
POLYDIOXANONE (PDO)
- crystalline (55%)
- degradation rate between PGA/PLA
close to 40-60 ratio
- shape memory
- modulus – 46 MPa; compare:
collagen – 100 MPa
elastin – 4 MPa
Advantages
- PDO ½ way between collagen/elastin, vascular ECM components
- cardiac tissue replacement (like PLGA)
- thin fibers (180nm)
Drawbacks
- shape memory – less likely to adapt with developing tissue
POLYCAPROLACTONE (PCL)
- highly elastic
- slow degradation rate (1-2 yrs)
- > 1 um
- similar stress capacity to PDO, higher elasticity
Advantages
- overall better for cardiac tissue – no shape retention bc elastic
Previous Applications
Loaded with:
- collagen  cardiac tissue replacement
- calcium carbonate  bone tissue strengthening
- growth factors  mesenchymal stem cell differentation
POLYCAPROLACTONE + PGA
- PGA high stress tolerance
- PCL high elasticity
- optimized combination PGA/PCL ~ 3/1
- bioabsorption at least 3 mths (PCL-2 yrs, PGA 2-4 wks)
Clinical Applications – none yet
POLYCAPROLACTONE + PLA
- PLA highly biocompatible (natural by products)
- PCL high elasticity
- more elastic than PGA/PCL
- strain limit increases 8x with just 5% PCL
POLYCAPROLACTONE + PLA
- PCL elastic;
however, decreasing PLA/PCL ratios decreases strain capacity
- strain capacity optimized at 95:5
- still ideal in vivo – mostly PLA = natural by products
POLYCAPROLACTONE + PLA
Clinical Applications
- several planned
- all vasculature tissue
- high PLA tensile strength
react (constrict) to sudden pressure increase
- increased elasticity with PCL
passively accommodate large fluid flow
OVERALL – passive expansion, controlled constriction
= best synthetic ECM combination for cardiac application
POLYCAPROLACTONE + POLYDIOXANONE
PCL
PDO
Recall…
- PCL high elasticity
- PDO approx = PLA/PGA
- PDO shape memory – limits use in vascular tissue
Findings
- hybrid structure NOT = hybrid properties
- lower tensile capacity than PDO
- low elasticity than PDO
- larger diameter
- NOT clinically useful
“[This] will be further investigated by our laboratory”
In other wordsnot publishable, but 1 year’s worth of work and good enough for a master’s thesis
POLYCAPROLACTONE + POLYDIOXANONE
PCL
PDO
Principle Drawbacks
Large fiber diameter
Low tensile/strain capacity
Possible Cause?
PDO is the only crystalline
structure polymer
ELASTIN
- highly elastic biosolid (benchmark for PDO)
- hydrophobic
- present in:
vascular walls
skin
Synthesis of Biosolid?
- 81 kDa recombinant protein (normal ~ 64 kDa)
- repeated regions were involved in binding
- 300 nm (not as small as PDO ~ 180 nm)
- formed ribbons, not fibers – diameter varies
Findings:
- not as elastic as native elastin
- currently combined with PDO to increase tensile strength
- no clinical applications yet
COLLAGENS: GELATIN
- highly soluble, biodegradable (very rapid)
- current emphasis on increasing lifespan
COLLAGENS: FIBRIL FORMING
Type I
- 100 nm (not consistent)
- almost identical to native collagen (TEM)
- present is most tissues
Type II
- 100-120 nm (consistent)
- found in cartilage
- pore size and fiber diameter easily controlled by dilution
COLLAGENS: FIBRIL FORMING
Type I (inconsistent fibers)
Type II
easy to regulate 1) fiber
2) pore size
COLLAGENS: FIBRIL FORMING
Type III
- preliminary studies
- appears consistent ~ 250 nm
None of the electrospun collagens have clinical application yet
COLLAGENS BLENDS
In context: vasculature
- intima – collagen type IV + elastin
- media – thickest, elastin, collagen I, III, SMC
- adventia – collagen I
Scaffolds studied to-date
- reconstructing the media:
RECONSTRUCTING THE MEDIA
- SMC seeded into tube
- average fiber ~ 450 nm
slightly larger ECM fibers
- incorporation of GAG
carbohydrate ECM
collagen crosslinker
mediate signalling
- cross section of tube wall
- 5 day culture
complete scaffold infiltration
COMBINING COLLAGEN WITH PDO
Observations:
- collagen I highest tensile capacity
- 70:30 collagen-PDO optimal ratio for all collagens
FIBRINOGEN
- smallest diameter (both synthetic and bio)
80, 310, 700 nm fibers possible
- high surface area to volume ratio
increase surface interaction
used in clot formation
Stress capacity comparable
to collagen (80-100 MPa)
HEMOGLOBIN
- hemoglobin mats
- clinical applications:
drug delivery
hemostatic bandages
- fiber sizes 2-3 um
- spun with fibrinogen for clotting/healing
- high porosity = high oxygenation
OVERVIEW
- Electrospinning viable for both synthetic and biological scaffolds/mats
- Wide range of fiber sizes necessary and possible
ECM ideally 150-500 nm
cell mats 2-3 um
- Hybridizing polymers can, but not necessarily, lead to hybrid properties
Specifics:
- PGA, PLA, PLGA most commonly used scaffold materials
- PDO exhibits elastin+collagen functionality in 1 synthetic polymer
BUT inhibited by “shape memory”
- PCL most elastic synthetic – frequently mixed with other synthetics