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NANOFIBER TECHNOLOGY: DESIGNING THE NEXT NANOFIBER TECHNOLOGY: DESIGNING THE NEXT GENERATION OF TISSUE ENGINEERING SCAFFOLDSGENERATION 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 NeurobiologyVirginia Commonwealth University, Richmond, VA
MARK HWANGMARK HWANG
EXTRACELLULAR MATRIX
Signalling- cell adhesion - programmed cell death- migration - cytokine/growth factor
activity- growth - differentiation
Components- collagens- elastin- hyaluronic acid- proteoglycans-
glycosaminoglycans
- fibronectin
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-immunogenicpromote growthmaintain 3-D structureonly native tissues remain post-treatment
Research emphases to-date- Biocompatibility- Degradability
Overall Goals- Design scaffold with maximum control over:
biocompatibility (chemical)biodegradability (mechanical)
- Utilize natural and synthetic polymers
- Future directions:tissue regenerationdrug delivery
Current Focus- Nanofiber synthesis
TISSUE ENGINEERING SCAFFOLDS - BACKGROUND
EFFECTIVE SCAFFOLD DESIGN BEGINS WITH ACCURATE SCALING
NANOFIBERS - INTRODUCTION
ECM fibers ~ 50-500 nm in diameterCell ~ several-10 umFibers 1-2 orders of magnitude < cell
3 techniques to achieve nanofiber scale- self assembly- phase separation- electrospinning
Scale difference necessary- single cell contacts thousands of fibers- transmission of fine/subtle signals
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, salts98% porosity achieved!
- consistent
Drawbacks:- limited to several polymers- small production scale
NANOFIBERS: ELECTROSPINNING
Definition: electric field used to draw polymer stream out of solution
D- electric field overcomes solution surface tension; polymer stream generated
E- fibers 1) collected and 2) patterned on plate
A- polymer solution in syringeB- metal needleC- voltage applied to need
NANOFIBERS: ELECTROSPINNING
- simple equipment- multiple polymers can be combined at
1) monomer level2) fiber level3) scaffold level
- control over fiber diameteralter concentration/viscosity
- fiber length unlimited
- control over scaffold architecturetarget plate geometrytarget plate rotational speed
NANOFIBERS: ELECTROSPINNING
Drawbacks:- natural fibers 50-500 nm; spun fibers closer to 500 nm- architecture very random
Current approaches combined techniques- usually electrospinning + phase separation- fibers woven over pores
LACK OF GOLD STANDARD
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
hydrophilicpredictable bioabsorption (2-4 wks)
- electrospinning yields diameters ~ 200 nm
Drawbacks- rapid hydrolitic degradation = pH change
tissue must have buffering capacity
Parameters- surface area to volume ratio- spinning orientation affects scaffold elastic modulus
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
Compare to PGA- low degradation rate = less pH change
Parameters (similar to PGA)- surface area to volume ratio- spinning orientation affects scaffold elastic modulus
POLYLACTIC ACID (PLA) – 200 nm
Thickness controlled by electrospin solvent
Chloroform solvent (L) ~ 10 umHFP (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 MPaelastin – 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 ApplicationsLoaded 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
- PLA highly biocompatible (natural by products)- PCL high elasticity- more elastic than PGA/PCL- strain limit increases 8x with just 5% PCL
POLYCAPROLACTONE + PLA
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 words-not publishable, but 1 year’s worth of work and good enough for a master’s
thesis
POLYCAPROLACTONE + POLYDIOXANONE PCL PDO
Principle DrawbacksLarge fiber diameterLow tensile/strain capacity
Possible Cause?PDO is the only crystalline structure polymer
ELASTIN
- highly elastic biosolid (benchmark for PDO)- hydrophobic- present in:
vascular wallsskin
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
Type I- 100 nm (not consistent)- almost identical to native collagen (TEM)- present is most tissues
COLLAGENS: FIBRIL FORMING
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 IIeasy to regulate 1) fiber2) 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 ECMcollagen crosslinkermediate 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 ratioincrease surface interactionused in clot formation
Stress capacity comparable to collagen (80-100 MPa)
HEMOGLOBIN
- hemoglobin mats- clinical applications:
drug deliveryhemostatic 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 possibleECM ideally 150-500 nmcell 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 polymerBUT inhibited by “shape memory”
- PCL most elastic synthetic – frequently mixed with other synthetics