NANOFIBER TECHNOLOGY: DESIGNING THE NEXT GENERATION OF TISSUE ENGINEERING SCAFFOLDS C.P. Barnes 1, S.A. Sell 1, E.D. Boland 1, D.G. Simpson 2, G.L. Bowlin 1 1 Department of Biomedical Engineering, 2 Department of Anatomy and Neurobiology Virginia Commonwealth University, Richmond, VA MARK 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-immunogenic promote growth maintain 3-D structure only 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 regeneration drug delivery Current Focus - Nanofiber synthesis TISSUE ENGINEERING SCAFFOLDS - BACKGROUND EFFECTIVE SCAFFOLD DESIGN BEGINS WITH ACCURATE SCALING

NANOFIBERS - INTRODUCTION ECM fibers ~ nm in diameter Cell ~ several-10 um Fibers 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, 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 D- electric field overcomes solution surface tension; polymer stream generated E- fibers 1) collected and 2) patterned on plate A- polymer solution in syringe B- metal needle C- voltage applied to need

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 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 hydrophilic predictable 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 um HFP (alcohol) solvent (R) ~ 780 nm Both fibers randomly collected

PGA+PLA = PLGA - tested composition at 25-75, 50-50, 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 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 - 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 PCLPDO 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 PCLPDO 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 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 nm (not consistent) - almost identical to native collagen (TEM) - present is most tissues COLLAGENS: FIBRIL FORMING Type II 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 ( 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 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