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