research article smooth muscle cell alignment and...

Research ArticleSmooth Muscle Cell Alignment and Phenotype Controlby Melt Spun Polycaprolactone Fibers for Seeding of TissueEngineered Blood Vessels

Animesh Agrawal1 Bae Hoon Lee1 Scott A Irvine1 Jia An2 Ramya Bhuthalingam1

Vaishali Singh3 Kok Yao Low4 Chee Kai Chua2 and Subbu S Venkatraman1

1Materials and Science Engineering Nanyang Technological University N41-01-30 50 Nanyang Avenue Singapore 6397982School of Mechanical amp Aerospace Engineering Nanyang Technological University 50 Nanyang Avenue Singapore 6397983Department of Polymer and Process Engineering Indian Institute of Technology Roorkee Roorkee Uttarakhand 247667 India4School of Biological Sciences Nanyang Technological University 60 Nanyang Drive Singapore 637551

Correspondence should be addressed to Scott A Irvine sairvinentuedusg

Received 19 April 2015 Revised 4 August 2015 Accepted 11 August 2015

Academic Editor Rosalind Labow

Copyright copy 2015 Animesh Agrawal et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A method has been developed to induce and retain a contractile phenotype for vascular smooth muscle cells as the first steptowards the development of a biomimetic blood vessel construct with minimal compliance mismatch Melt spun PCL fibers weredeposited on a mandrel to form aligned fibers of 10120583m in diameter The fibers were bonded into aligned arrangement throughdip coating in chitosan solution This formed a surface of parallel grooves 10120583m deep by 10120583m across presenting a surface layerof chitosan to promote cell surface interactions The aligned fiber surface was used to culture cells present in the vascular wall inparticular fibroblasts and smooth muscle cells This topography induced ldquosurface guidancerdquo over the orientation of the cells whichadopted an elongated spindle-like morphology whereas cells on the unpatterned control surface did not show such orientationassuming more rhomboid shapes The preservation of VSMC contractile phenotype on the aligned scaffold was demonstrated bythe retention of 120572-SMA expression after several days of culture The effect was assessed on a prototype vascular graft prosthesisfabricated from polylactide caprolactone VSMCs aligned longitudinally along a fiberless tube whereas for the aligned fiber coatedtubes the VSMCs aligned in the required circumferential orientation

1 Introduction

As research in implantable biomaterial advances the under-standing andmanipulation of cell-substrate interactions haveincreased in importance One approach is to produce amore biomimetic construct that can recruit and control thepatterning of functional cells to mimic the native tissueorganization For example the aligned orientation of cells onextracellular matrix (ECM) plays an important role in severaltissues including corneal stroma tendons bones skeletalmuscle and with significance to the present study thevasculature [1] Here we demonstrate an intrinsically effectivecell aligning surface fabricated from the biodegradable andcytocompatible polymers PCL chitosan and gelatin [2]

The development of a small diameter vascular prosthesis(gt6mm diameter) for arterial disease has been hampered bythe mechanical compliance mismatch of the prosthesis andthe native blood vesselThemismatch is the key factor for therelatively rapid loss of patency compared to larger-diameterprostheses In turn the mismatch is due to the fact thatartificial prostheses do not mimic the layered structure of thenative vessel in which one of the layers has circumferentiallyaligned vascular smooth muscle cells (VSMCs) as well asextracellular matrix (ECM) [3]

The two predominant cell types within blood vesselsfibroblasts and VSMCs could both functionally benefit cell-seeded prosthesis if recruited in an aligned orientation [14] Fibroblasts produce extracellular matrix such as collagen

Hindawi Publishing CorporationInternational Journal of BiomaterialsVolume 2015 Article ID 434876 8 pageshttpdxdoiorg1011552015434876

2 International Journal of Biomaterials

fibrils and elastin [5] that confer to blood vessels most oftheir mechanical and structural properties In the context ofa vascular prosthesis it is beneficial to align the growth ofcells to control the pattern of ECM deposition If the scaffoldof the prosthesis is biodegradable then the construct will beeventually replaced by ECM with the desired orientation [6]

Of particular interest to this study is the alignment ofVSMCs These cells are integral to the vascular functioningthrough regulation of vessel tone and lumen diameter Inter-estingly these cells exist as two very distinct and changeablephenotypes the contractile characterized by a spindle shapeand the abundant presence of alpha-smooth muscle actin(120572-SMA) and the secretory (also referred to as synthetic)recognizable by rhomboid shape and reduced 120572-SMA [7]The contractile VSMC allows the changes that mediate bloodpressure by altering the vessel luminal diameter and is notproliferative whereas the secretory phenotype is associatedwith tissue remodeling inflammation and proliferationThesecretory phenotype is central to the pathology of neointimalhyperplasia and artery bypass failure There is plasticitybetween the two states as they are not differentiation end-points [7 8]

During the culturing of VSMC freshly seeded VSMCsexist primarily in the contractile state but over time thepopulation shifts predominantly towards the secretory phe-notype For long-term patency it is important to preserve thecontractile phenotype as only VSMCs in the contractile stateare beneficial in fabricating cellularized tissue engineeredblood vessel (TEBV) because this phenotype minimizesthe compliance mismatch Furthermore the cells must becircumferentially orientated to direct their function [9]

Previous studies already have demonstrated that thealigned orientation and phenotypic characteristics of cells canbe guided by surface cues generated throughmicropatterninga surface with channels [10]These channels are often consid-erable wider and deeper than the dimensions of the cell forexample 60 120583m deep and 300 120583m wide [9] Although thesescaffolds encourage cell alignment the channel depth oftenprevents cell interaction across the interval Cells are able toalign on grooves as shallow as 150 nm [11] however deeperchannels aremore effective for cell alignment down to depthsof approximately 25 120583m [12] whereas for groove width as itincreases cellular alignment decreases as cells lying centrallycan longer sense the edges [13 14]

Here we construct a scaffold of parallel melt spun PLCfibers As the fiber width is 10 120583m a scaffold of side-by-side fibers can be formed to orientate the cells within agroove of 10 120583m width with no overly large groove wallsnor any extended interval between grooves thus enablingintercellular contact

Early alignment studies employed nondegradable poly-mers such as channels onPDMSfilms to produce aligned cell-orientating scaffolds More recently however biodegradablepolymers have been studied since such scaffolds can beslowly replaced by native tissue and ECM which mini-mizes the issue of thrombosis Such polymers have includedP[(LLA-CL)] [15] PCLLGA [9] Here we use the biodegrad-able polymers polycaprolactone and polylactide caprolac-tone These polymers have been promoted as suitable for

Melt holder

Mandrel

Slide rail z-stage

(a)

(b) (c) (d)

Figure 1 Melt spinning apparatus depositing fiber on a turningmandrel demonstrating the slide rail z-stage melt holder andmandrel formore details seeAn et al [21] (a) SEM image of the PCLfiber removed from the mandrel (b) PCL fibers without adhesive tomaintain alignment (c) PCL fibers adhered into aligned orientationusing chitosan solution as an adhesive (d)

fabricating a vascular conduit and such an application isactively investigated by several groups [16ndash20]

In this work we have successfully produced a highlyalignedmelt spunPCLfiber scaffold that allows fibroblast andVSMC aligned attachment and also preserves the contractile120572-smoothmuscle actin (120572-SMA) expressing phenotype of theVSMCThis techniquewas also shown to be applicable for cellalignment on a prototype synthetic PLC vascular conduit

2 Materials and Methods

21 Production of Melt Spun PCL Melt spinning of poly-caprolactone was performed as described in An et al [21]using a customized apparatus as shown in Figure 1(a) Inbrief powdered PCL (50 kDa) was melted at approximately120∘C and then was drawn by gravity to mandrel at 750 rpm[21]

The fibers were adhered into place by dip coating in 5chitosan (wv) in acetic acid and then incubated for 5 days atroom temperature to allow solvent evaporation As a controlchitosan films were formed by coating the base of 6-wellplateswith 500 120583L of 5 (wv) chitosan in acetic acid followed

International Journal of Biomaterials 3

by incubation for 5 days at room temperature in a fume hoodto allow solvent evaporation

22 A Dip Coated Prototype PLC Vascular Prosthesis A PLCvascular graft was formed by dip coating a 5mm diameterstainless steel mandrel in a 12 (wv) PLC solution dissolvedon chloroformusing aNimaDC-Mono 300 dip coating appa-ratus The PLC coating was incubated at room temperaturefor 48 hours to allow solvent evaporation Subsequently themandrel was placed into the melt spinning apparatus and alayer of circumferentially aligned PCL fibers was appliedThefibers were adhered using 5 chitosan solution as an adhesiveas described above following solvent evaporation the scaffoldwas coated in 1 gelatin to enhance cell attachment on thetubular surface

23 Fourier Transform Infrared Spectroscopy FTIR analysiswas used to qualitatively characterize the functional groupsintroduced presented on the surface of the construct FTIRspectra were collected with Frontier FT-IR spectrometer(PerkinElmer) at resolution of 4 cmminus1 and signal average of 16scans in each interferogramover the range of 4000ndash600 cmminus1Two measurements were retrieved on two random locationsper sample for each group Results were analyzed by plotting transmission against wavelength (nm)

24 Atomic Force Microscopy (AFM) Intermittent contactmode atomic force micrographs were obtained on JPKInstruments Nanowizard III (Aufgang C Germany) with ananoprobe of 100 120583m length and a rotated monolithic siliconnarrow cantilever with a force constant of 40Nmminus1 anda tapping frequency of 300KHz The tapping mode AFMconditions were as follows scan rate was 04Hz set pointwas 15 V drive amplitude was set to 05 V and the drivefrequency was approximately 280KHz Adjustments of theintegral gain proportional gain and set point were done tominimize contact force and electronic noise as well as tomaximize the features of the sample The AFM micrographswere evaluated by flattening the images (second-order) withthe help of Nanowizard Data Processing JPK Software

25 Cell-Seeded PCL Fiber Film Smoothmuscle cells (SMCsLonza) or human fibroblasts were cultured up to the 8thpassage in smooth muscle cell basal medium (SmGM-2Media (Lonza Bioscience)) or FibroGRO Complete Media(Millipore) respectively PCL fiber films (1 times 1 cm3) weresterilizedwith 70 ethanol for 1 h and thenwerewashed awaywith PBS (three times)The cells were seeded on the PCLfiberfilms and a control surface at a density of 5 times 104 cellscm2Cell-seeded fibers were cultured in a flat bottom 24-well platefor 7 days

26 Cell Seeding Prototype PLC Vascular Prosthesis A 5 cmsection of either uncoated or aligned PCL fiber coated con-duits was placed in a 6 cm diameter dish Subsequently 3mLsof a concentrated SMC suspension (5 times 106 cellsmL) wasslowly applied to the upper surface after 10min incubationat room temperature the tube was turned over and another3mLs of the cell suspension was applied The constructs

were incubated over night at 37∘C in 5 CO2 then the cells

morphology was examined using fluorescence microscopy

27 Immunofluorescence Imaging with Confocal MicroscopyThe seeded fibroblasts were recorded after 14 days Thefluorescence was generated by 30-minute incubation with2 120583M calcein AM in PBS

SMCs in cell-seeded fibers at day 3 and day 7 werefixed in 4 paraformaldehyde for 30min in room temper-ature Following fixation fibers were washed 3x with PBSpermeabilized with 01 Triton X-100 and blocked using2 BSA in PBS for 1 h at 4∘C After washing 3x at roomtemperature in PBS and immunohistochemistry labeling onfibers and control (chitosan film) samples was performedapplying primary antibody against alpha-smooth muscleactin (monoclonal mouse anti-human) at 1 100 dilutions inPBSBSAbuffer at room temperature for 2 h After washing3 times for 10min in PBSBSA buffer secondary antibod-ies (AF 488 goat anti-mouse Invitrogen) were applied at1 200 dilutions in PBSBSA buffer at room temperature for1 h Cell nuclei were stained with DAPI (410158406-diamidino-2-phenylindole) and PCL fibers were then imaged via confocalmicroscopy (Leica Wetzlar Germany)

3 Results

31 Characterization of Aligned Melt Spun Fiber Mat Themelt spinmethod produced a thin layer of highly aligned PCLfibers of 10 120583m width on a mandrel within approximately 15minutes creating aligning fibers of 10 120583m diameter (Figures1(b) and 1(c))The PCL is melt spun into nonbonded distinctfibers that readily unravels (Figure 1(c)) Hence the chitosandip coating allows for an adhesive effect for the fibers tobe removed from the mandrel as an aligned fiber mat(Figure 1(d))

The fiber mat was examined by AFM (Figure 2(a)) andFTIR (Figure 2(b)) The FTIR demonstrated a strong signalfor the characteristic amide groups chitosan hence thechitosan coats the fibers and becomes the principle substancecells will then interact with The chitosan amide groups areknown to help provide for a cytocompatible surface to thedevice From the AFM it appears the fibers form curvedgrooves approximately 10 120583m and 10 120583m deep a depth whichallows cells tomake contact with cells growing in neighboringgrooves as seen in Figures 3(a) and 3(c) In addition thechitosan gives the scaffold a roughened surface feature unlikethe relative smooth surface of pristine PCL [22]

32 Aligned Orientation of Fibroblasts and Smooth MuscleCells Fibroblasts were seeded onto the fiber surface andreadily aligned with the parallel fibers and became noticeablyelongated compared to the control (Figures 3(a) and 3(b))These cells continued to proliferate until reaching 100confluency producing a continuous aligned monolayer (notshown) hence the fibroblasts were able to sense each otheracross channels Similarly for SMCs it was observed thatafter 7 days the cells adopted an elongated morphologyfollowing the orientation of the fiber resembling the spindle-like appearance of the contractile cells (Figure 3(c)) In


10

0

0 10 20

Slow

(120583m

)

Fast (120583m)

10

08

06

04

02

00

minus02

minus04

minus060 5 10 15 20 25

Offset (120583m)

Hei

ght (120583

m)

(a)

Chitosan film

chitosan film

Chitosan amides I and II

amides I and IIcarbonyl

T (

)

4000 3000 2000 1500 1000 650

(cmminus1)

Chitosan film

chitosan film


amides I and IIcarbonylyyPCL fiber with ChitosanPCL

(b)

Figure 2 Surface analysis of the aligned chitosan covered fibers AFM of fibers bonded by chitosan (a) FTIR of the chitosan film and PCLcoated chitosan (b)

comparison the SMCs seeded on chitosan film grew withoutobvious orientation and present a rhomboid appearance akinto the secretory phenotype (Figure 3(d)) Interestingly unlikethe fibroblasts the fiber seeded SMCs were not observed toproliferate towards confluency

33 Expression of 120572-Smooth Muscle Actin (120572-SMA) Therecently passaged smooth muscle cells (up to day 3) demon-strated positive staining by immunochemistry for the con-tractile phenotype marker protein 120572-SMA on both alignedfiber and nonaligning surface (Figures 4(a) and 4(c)) How-ever after several days the protein was only detectable incells on the aligned fiber surface This observation indicatesthat the fiber induced both cellular orientation and prolonged120572-SMA expression and thus promoted the retention of thecontractile phenotype (Figures 4(b) and 4(d))

34 Application of Technology to a PLCVascular Conduit Themelt spun fibers could be readily used to pattern the surfaceof a prototype PLC vascular conduit (Figure 5(a)) It wasobserved 48 hours after seeding the fiber covered andfiberlessPLC tubes that VSMCs aligned longitudinally (Figure 5(b))on the fiberless construct whereas on the fiber decoratedtubes the VSMC had orientated along the fibers giving a cir-cumferentially aligned elongated morphology This patternwas still observed 5 days after seeding (Figure 5(c))

4 Discussion

PCL based conduits are seen to have potential for the futurefabrication of cellularized vascular prosthesis with some

successful clinical implantation [23] SMCs are critical for theproper functioning of a blood vessel however the syntheticphenotype is often associated with inflammation and graftfailure Hence the functional contractile phenotype shouldbe promoted in a TEBV Few attempts have been made tocontrol SMC behavior on a cell-seeded tube [24 25] Here wetest techniques of surface guidance known to influence cellbehavior on 2D surfaces for their circumferential applicationaround a conduit tube as required on a PCL vascularprosthesis We are able to create cell aligning grooves usinga method of melt spinning PCL which successfully alignedthe orientation of both fibroblasts and SMCs and influencedthe phenotype of SMC Furthermore we have demonstratedthat polymer tubing (fabricated from PCL) on a mandrel canbe decorated with circumferentially aligned melt spun PCL

Fibers removed from the mandrel without the chitosanor gelatin bonding separated very readily Furthermore poly-caprolactone without modification has poor cell adheringproperties whereas chitosan with high deacetylation (85ndash95 in this case) has very good cell recruiting qualitiesThe higher the deacetylation the greater the amount of freecationic amino groups that encourage cell adhesion to thesurface [26 27] The smooth pristine PCL also lacks thesurface roughness that usually facilitates the establishment ofcellular adhesion points whereas the chitosan coating gives aconsiderably more roughened surface feature Furthermorethe AFM revealed that the chitosan covering does not fill inthe space between fibers but instead leaves a distinct curvedgroove that facilitates the aligned orientation of the cells

Previous studies have demonstrated that SMCs are oftenseeded in a strongly contractile phenotype then following


(a) (b)

(c) (d)

Figure 3 Fibroblasts cultures on aligned fiber (a) and on tissue culture plastic (b) for 14 days cells are stained with calcein AM SMCsorientated on melt spun aligned fiber (c) and cultured on chitosan film (d) for several days The cells are immunostained for F-actin andDAPI nuclear stain

a prolonged period of culture the cells gradually becomepredominantly synthetic [7 28] Similarly in the presentstudy we found SMCs seeded on chitosan films also beganwith notable expression of the contractile marker 120572-SMAThis expression is considerably diminished after several daysimplying a move towards the secretory The cells seededon the aligned fibers also showed substantial expressionof 120572-SMA 24 hours after seeding and this expression wasretained after several days indicating the preservation of thecontractile state [28 29]

The contractile phenotype in this study was assessed bythe elongated cell morphology and 120572-SMA expression thelatter is an important marker due to its dramatic loss duringphenotype change during in vitro culture There are severalother key characteristics andmarker genes that determine theSMC contractile phenotype Chang et al demonstrated thatSMCs seeded on and elongating along similarmicropatternedgrooves expressed a greater number of contractile relatedgenes and less proliferative related ones than SMCs grownon flat surface [30] thus proving the grooves promote theretention of the contractile phenotype

The intracellular mechanism by which the contractilephenotype can be promoted in SMC elongating with cell

aligning channels has been examined There is a milieu ofsignal transduction pathways affecting SMC phenotype (asreviewed by [31]) However studies have been carried outon the mechanism of phenotype determination by surfacefeatures Chang et al detected strong activation of ERK andFAK downstream signaling molecules from integrins thesurface adhesion proteins which ldquosenserdquo the surface featuresThakar et al [32] examined the effect of SMC shape onproliferationThey found a significant decrease in the expres-sion of both the mRNA and protein of the nuclear receptorNOR-1 by elongated SMCs NOR-1 appears to be associatedwith the characteristic proliferation of synthetic SMC Theknockdown of NOR-1 reduces SMC rate of proliferation [32]

Fibroblasts were able to align and become confluent onthe fibers whereas SMCs differentiating towards contractilephenotype have a greatly reduced rate of proliferation [78 28] To achieve confluency on the construct we expectthe requirement to seed SMCs at a high cell density henceconferring immediate confluency

Several groups have created aligning scaffolds by electro-spinning polymers For example Xu et al [15] electrospunPLLA-CL to create an aligned scaffold for cell ldquocontact guid-ancerdquo However the method presented here has substantial


(a)

(b)

(c)

(d)

Figure 4 SMC expression of 120572-SMA SMCs cultured on chitosanfilms for 3 days (a) and 7 days (b) SMCs on aligned chitosan coatedPCL after 3 days (c) and 7 days (d) All cells stained for 120572-SMA andwith DAPI nuclear stain

advantages over electrospinning such as the absence ofthe whipping action characteristic of electrospinning whichreduces exact fiber alignment as compared to melt spinning[33] To get a highly aligned structure by electrospinning anarrow rotating mandrel collector is used for fiber collectionthus generating aligning surfaces of limited width and withmultiple layers of piled fibers whereas our melt spinningmethod produces a scaffold of 10 cm length and 1-fiberthickness However an important advantage of electrospin-ning over melt spinning is the delivery of cells within thefibers for immediate and simultaneous deposition onto thegraft within cytocompatible polymers and solvents [34 35]This will become an important technique if it is found thatthe SMCs adopt a contractile phenotype following deliverywithin circumferentially aligned electrospun fibers

Unlike most work on cell aligning fiber scaffolds thecurrent study employed micron-diameter scaffolds It hasbeen demonstrated that PCL can be melt spun into fibers

(a)

(b) (c)

Figure 5 SMC seeding of a PLC prosthesis A PLC tube wascoated with PCL melt spun fiber (a) SMCs were seeded at highconcentration (5 times 106 cellsmL) on fiberless (b) and fiber coated (c)tubes and images were recorded 5 days after seeding

with range of diameters from 10 to 200120583m [33] The meltspun fibers we fabricate here have a diameter of 10120583mthese generate interfiber grooves 10120583macross whichmatchesthe width of an elongated SMC In a comparable study onmicrosized fibers produced by wet spinning of PLGA it wasfound that as the diameter of fibers increases from 10 120583mto 242120583m the degree of orientation decreases The larger-diameter fibers conferred cellular alignment similar to planarfeatureless surfaces [36] Hence the fibers we created by meltspinning are in the favorable width range for promoting cellalignment In addition the relatively low depth for the grooveallows fibroblast and SMCs to grow closely together as seenin Figure 3 unlike other methods that produce much deeperchannels or wider intervals which keep cells separated acrosschannels

The melt spun fibers can be used to circumferentiallyalign the SMCs on a prototype PLC vascular prosthesis in anelongated contractile-like phenotype thus demonstrating theeffectiveness of the technique to orientate the cell both on flat2D film and also circumferentially on the outer surface of atube

This ability of fabricating and controlling alignmentpatterns on tissue engineering scaffolds will aid the pro-duction of a more physiologically relevant representation ofnatural tissue [4] Moreover since the melt spun techniqueis delivered to a mandrel it can be adapted to introducecircumferentially aligned pattern on the outer surface of asynthetic polymer TEBV when placed in the position ofthe rotating mandrel This allows the alignment of cells thatcomprise the outer vasculature layers that is fibroblasts andSMCs


We aim to eventually achieve the coating of fully func-tional vasoresponsive SMCs for the structure HoweverSMCs can exist at various points of differentiation betweenthe two phenotypes Through contact guidance features wehave successfully promoted a more contractile-like pheno-type To achieve fully functional contractile SMCs we expectto involve additional steps [37] including seeding at highdensity optimized cell harvesting techniques (enzyme diges-tion rather than outgrowths) growth factors (such as TGF-120573) serum optimization SMC source and stimulation withpulsatile force It is unlikely that only one of these approachesin isolation can achieve the formation of a vasoresponsiveSMCmedial layer hence combinationsmay bemore effective[28 38]

In summary the combination of aligned melt spunmicron-sized fibers with a polymeric ldquoadhesiverdquo (chitosan)is a very promising substrate that enables the retention ofthe desired contractile phenotype of smooth muscle cellsSuch a construct may be advantageously applied onto tubularconstructs for forming a biomimetic blood vessel that willpotentially have superior compliance matching with thenative vessel

Disclosure

The technology described in this paper is included in apatent application filed by authors Scott A Irvine AnimeshAgrawal Jia An Chee Kai Chua and Subbu S Venkatraman

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Animesh Agrawal and Bae Hoon Lee contributed equally tothis paper

Acknowledgments

Theauthorswould like to thank ProfessorMark FeatherstoneSchool of Biological Studies NTU for his support Thisresearch is supported by the Singapore National ResearchFoundation under CREATE programme (NRF-Technion)The Regenerative Medicine Initiative in Cardiac RestorationTherapy Research Program and the AlowastSTAR SERC PublicSector Research Funding (PSF)

References

[1] Y Li G Huang X Zhang et al ldquoEngineering cell alignment invitrordquo Biotechnology Advances vol 32 no 2 pp 347ndash365 2014

[2] L S Nair and C T Laurencin ldquoBiodegradable polymers as bio-materialsrdquo Progress in Polymer Science vol 32 no 8-9 pp 762ndash798 2007

[3] X Wang P Lin Q Yao and C Chen ldquoDevelopment of small-diameter vascular graftsrdquo World Journal of Surgery vol 31 no4 pp 682ndash689 2007

[4] J-M Bourget M Guillemette T Veres F A Auger and LGermain ldquoAlignment of cells and extracellular matrix withintissuemdashengineered substitutesrdquo in Advances in BiomaterialsScience and Biomedical Applications chapter 14 InTech RijekaCroatia 2013

[5] H P Rodemann and H O Rennekampff ldquoFunctional diversityof fibroblastsrdquo inTumor-Associated Fibroblasts and theirMatrixvol 4 of The Tumor Microenvironment pp 23ndash36 SpringerBerlin Germany 2011

[6] S W Shalaby and W S W Shalaby ldquoSynthetic vascular con-structsrdquo in Absorbable and Biodegradable Polymers CRC Press2003

[7] N F Worth B E Rolfe J Song and G R Campbell ldquoVas-cular smooth muscle cell phenotypic modulation in culture isassociated with reorganisation of contractile and cytoskeletalproteinsrdquo Cell Motility and the Cytoskeleton vol 49 no 3 pp130ndash145 2001

[8] S S M Rensen P A F M Doevendans and G J J M van EysldquoRegulation and characteristics of vascular smooth muscle cellphenotypic diversityrdquo Netherlands Heart Journal vol 15 no 3pp 100ndash108 2007

[9] Y Cao Y F Poon J Feng S Rayatpisheh V Chan and M BChan-Park ldquoRegulating orientation and phenotype of primaryvascular smooth muscle cells by biodegradable films patternedwith arrays of microchannels and discontinuous microwallsrdquoBiomaterials vol 31 no 24 pp 6228ndash6238 2010

[10] J Y Shen M B Chan-Park B He et al ldquoThree-dimensionalmicrochannels in biodegradable polymeric films for controlorientation and phenotype of vascular smooth muscle cellsrdquoTissue Engineering vol 12 no 8 pp 2229ndash2240 2006

[11] J Heitz T Peterbauer S Yakunin et al ldquoDynamics of spreadingand alignment of cells cultured in vitro on a grooved polymersurfacerdquo Journal of Nanomaterials vol 2011 Article ID 41307910 pages 2011

[12] A Curtis and C Wilkinson ldquoTopographical control of cellsrdquoBiomaterials vol 18 no 24 pp 1573ndash1583 1997

[13] J D Glawe J B Hill D K Mills andM J McShane ldquoInfluenceof channel width on alignment of smooth muscle cells by high-aspect-ratio microfabricated elastomeric cell culture scaffoldsrdquoJournal of Biomedical Materials ResearchmdashPart A vol 75 no 1pp 106ndash114 2005

[14] P Clark P Connolly A S G Curtis J A T Dow and C D WWilkinson ldquoTopographical control of cell behaviour II Mul-tiple grooved substratardquo Development vol 108 no 4 pp 635ndash644 1990

[15] C Xu R Inai M Kotaki and S Ramakrishna ldquoElectrospunnanofiber fabrication as synthetic extracellular matrix and itspotential for vascular tissue engineeringrdquo Tissue Engineeringvol 10 no 7-8 pp 1160ndash1168 2004

[16] E Pektok B Nottelet J-C Tille et al ldquoDegradation and healingcharacteristics of small-diameter poly(epsilon-caprolactone)vascular grafts in the rat systemic arterial circulationrdquo Circula-tion vol 118 no 24 pp 2563ndash2570 2008

[17] W Mrowczynski D Mugnai S De Valence et al ldquoPorcinecarotid artery replacement with biodegradable electrospunpoly-e-caprolactone vascular prosthesisrdquo Journal of VascularSurgery vol 59 no 1 pp 210ndash219 2014

[18] J Lee J J Yoo A Atala and S J Lee ldquoThe effect of controlledrelease of PDGF-BB from heparin-conjugated electrospunPCLgelatin scaffolds on cellular bioactivity and infiltrationrdquoBiomaterials vol 33 no 28 pp 6709ndash6720 2012


[19] G Pitarresi C Fiorica F S Palumbo S Rigogliuso G Ghersiand G Giammona ldquoHeparin functionalized polyaspartamidepolyester scaffold for potential blood vessel regenerationrdquoJournal of Biomedical Materials Research Part A vol 102 no 5pp 1334ndash1341 2014

[20] L Ye X Wu H-Y Duan et al ldquoThe in vitro and in vivobiocompatibility evaluation of heparin-poly(120576-caprolactone)conjugate for vascular tissue engineering scaffoldsrdquo Journal ofBiomedical Materials Research Part A vol 100 no 12 pp 3251ndash3258 2012

[21] J An C K Chua K F Leong C-H Chen and J-P ChenldquoSolvent-free fabrication of three dimensionally aligned poly-caprolactonemicrofibers for engineering of anisotropic tissuesrdquoBiomedical Microdevices vol 14 no 5 pp 863ndash872 2012

[22] A P Khandwekar D P Patil Y Shouche and M Doble ldquoSur-face engineering of polycaprolactone by biomacromoleculesand their blood compatibilityrdquo Journal of Biomaterials Applica-tions vol 26 no 2 pp 227ndash252 2011

[23] T Shinrsquooka G Matsumura N Hibino et al ldquoMidterm clinicalresult of tissue-engineered vascular autografts seeded withautologous bone marrow cellsrdquo The Journal of Thoracic andCardiovascular Surgery vol 129 no 6 pp 1330ndash1338 2005

[24] J S Choi Y Piao and T S Seo ldquoCircumferential alignment ofvascular smoothmuscle cells in a circularmicrofluidic channelrdquoBiomaterials vol 35 no 1 pp 63ndash70 2014

[25] Y Wang H Shi J Qiao et al ldquoElectrospun tubular scaffoldwith circumferentially aligned nanofibers for regulating smoothmuscle cell growthrdquo ACS Applied Materials and Interfaces vol6 no 4 pp 2958ndash2962 2014

[26] I Aranaz M Mengıbar R Harris et al ldquoFunctional characteri-zation of chitin and chitosanrdquo Current Chemical Biology vol 3no 2 pp 203ndash230 2009

[27] T Freier H S Koh K Kazazian and M S Shoichet ldquoCon-trolling cell adhesion and degradation of chitosan films by N-acetylationrdquo Biomaterials vol 26 no 29 pp 5872ndash5878 2005

[28] JH Campbell andG R Campbell ldquoSmoothmuscle phenotypicmodulationmdasha personal experiencerdquoArteriosclerosisThrombo-sis and Vascular Biology vol 32 no 8 pp 1784ndash1789 2012

[29] J H Campbell O Kocher O Skalli G Gabbiani and GR Campbell ldquoCytodifferentiation and expression of 120572-smoothmuscle actin mRNA and protein during primary culture ofaortic smooth muscle cells Correlation with cell density andproliferative staterdquo Arteriosclerosis vol 9 no 5 pp 633ndash6431989

[30] S Chang S Song J Lee et al ldquoPhenotypic modulation ofprimary vascular smooth muscle cells by short-term culture onmicropatterned substraterdquo PLoS ONE vol 9 no 2 Article IDe88089 2014

[31] C PMack ldquoSignalingmechanisms that regulate smoothmusclecell differentiationrdquo Arteriosclerosis Thrombosis and VascularBiology vol 31 no 7 pp 1495ndash1505 2011

[32] R G Thakar Q Cheng S Patel et al ldquoCell-shape regulation ofsmooth muscle cell proliferationrdquo Biophysical Journal vol 96no 8 pp 3423ndash3432 2009

[33] S J Park B-K LeeMHNa andD S Kim ldquoMelt-spun shapedfiberswith enhanced surface effects fiber fabrication character-ization and application to woven scaffoldsrdquo Acta Biomaterialiavol 9 no 8 pp 7719ndash7726 2013

[34] S N Jayasinghe S Irvine and J R McEwan ldquoCell electrospin-ning highly concentrated cellular suspensions containing pri-mary living organisms into cell-bearing threads and scaffoldsrdquoNanomedicine vol 2 no 4 pp 555ndash567 2009

[35] S N Jayasinghe ldquoCell electrospinning a novel tool for func-tionalising fibres scaffolds and membranes with living cellsand other advanced materials for regenerative biology andmedicinerdquo Analyst vol 138 no 8 pp 2215ndash2223 2013

[36] C M Hwang Y Park J Y Park et al ldquoControlled cellularorientation on PLGA microfibers with defined diametersrdquoBiomedical Microdevices vol 11 no 4 pp 739ndash746 2009

[37] R GThakar F Ho N F Huang D Liepmann and S Li ldquoReg-ulation of vascular smooth muscle cells by micropatterningrdquoBiochemical and Biophysical Research Communications vol 307no 4 pp 883ndash890 2003

[38] J A Beamish P He K Kottke-Marchant and R E MarchantldquoMolecular regulation of contractile smooth muscle cell phe-notype implications for vascular tissue engineeringrdquo TissueEngineering Part B Reviews vol 16 no 5 pp 467ndash491 2010

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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NanoparticlesJournal of



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Biomaterials


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fibrils and elastin [5] that confer to blood vessels most oftheir mechanical and structural properties In the context ofa vascular prosthesis it is beneficial to align the growth ofcells to control the pattern of ECM deposition If the scaffoldof the prosthesis is biodegradable then the construct will beeventually replaced by ECM with the desired orientation [6]

Of particular interest to this study is the alignment ofVSMCs These cells are integral to the vascular functioningthrough regulation of vessel tone and lumen diameter Inter-estingly these cells exist as two very distinct and changeablephenotypes the contractile characterized by a spindle shapeand the abundant presence of alpha-smooth muscle actin(120572-SMA) and the secretory (also referred to as synthetic)recognizable by rhomboid shape and reduced 120572-SMA [7]The contractile VSMC allows the changes that mediate bloodpressure by altering the vessel luminal diameter and is notproliferative whereas the secretory phenotype is associatedwith tissue remodeling inflammation and proliferationThesecretory phenotype is central to the pathology of neointimalhyperplasia and artery bypass failure There is plasticitybetween the two states as they are not differentiation end-points [7 8]

During the culturing of VSMC freshly seeded VSMCsexist primarily in the contractile state but over time thepopulation shifts predominantly towards the secretory phe-notype For long-term patency it is important to preserve thecontractile phenotype as only VSMCs in the contractile stateare beneficial in fabricating cellularized tissue engineeredblood vessel (TEBV) because this phenotype minimizesthe compliance mismatch Furthermore the cells must becircumferentially orientated to direct their function [9]

Previous studies already have demonstrated that thealigned orientation and phenotypic characteristics of cells canbe guided by surface cues generated throughmicropatterninga surface with channels [10]These channels are often consid-erable wider and deeper than the dimensions of the cell forexample 60 120583m deep and 300 120583m wide [9] Although thesescaffolds encourage cell alignment the channel depth oftenprevents cell interaction across the interval Cells are able toalign on grooves as shallow as 150 nm [11] however deeperchannels aremore effective for cell alignment down to depthsof approximately 25 120583m [12] whereas for groove width as itincreases cellular alignment decreases as cells lying centrallycan longer sense the edges [13 14]

Here we construct a scaffold of parallel melt spun PLCfibers As the fiber width is 10 120583m a scaffold of side-by-side fibers can be formed to orientate the cells within agroove of 10 120583m width with no overly large groove wallsnor any extended interval between grooves thus enablingintercellular contact

Early alignment studies employed nondegradable poly-mers such as channels onPDMSfilms to produce aligned cell-orientating scaffolds More recently however biodegradablepolymers have been studied since such scaffolds can beslowly replaced by native tissue and ECM which mini-mizes the issue of thrombosis Such polymers have includedP[(LLA-CL)] [15] PCLLGA [9] Here we use the biodegrad-able polymers polycaprolactone and polylactide caprolac-tone These polymers have been promoted as suitable for

Melt holder

Mandrel

Slide rail z-stage

(a)

(b) (c) (d)

Figure 1 Melt spinning apparatus depositing fiber on a turningmandrel demonstrating the slide rail z-stage melt holder andmandrel formore details seeAn et al [21] (a) SEM image of the PCLfiber removed from the mandrel (b) PCL fibers without adhesive tomaintain alignment (c) PCL fibers adhered into aligned orientationusing chitosan solution as an adhesive (d)

fabricating a vascular conduit and such an application isactively investigated by several groups [16ndash20]

In this work we have successfully produced a highlyalignedmelt spunPCLfiber scaffold that allows fibroblast andVSMC aligned attachment and also preserves the contractile120572-smoothmuscle actin (120572-SMA) expressing phenotype of theVSMCThis techniquewas also shown to be applicable for cellalignment on a prototype synthetic PLC vascular conduit

2 Materials and Methods

21 Production of Melt Spun PCL Melt spinning of poly-caprolactone was performed as described in An et al [21]using a customized apparatus as shown in Figure 1(a) Inbrief powdered PCL (50 kDa) was melted at approximately120∘C and then was drawn by gravity to mandrel at 750 rpm[21]

The fibers were adhered into place by dip coating in 5chitosan (wv) in acetic acid and then incubated for 5 days atroom temperature to allow solvent evaporation As a controlchitosan films were formed by coating the base of 6-wellplateswith 500 120583L of 5 (wv) chitosan in acetic acid followed












3 Results





10

0

0 10 20

Slow

(120583m

)

Fast (120583m)

10

08

06

04

02

00

minus02

minus04

minus060 5 10 15 20 25

Offset (120583m)

Hei

ght (120583

m)

(a)

Chitosan film

chitosan film



T (

)

4000 3000 2000 1500 1000 650

(cmminus1)

Chitosan film

chitosan film



(b)





4 Discussion






(a) (b)

(c) (d)









(a)

(b)

(c)

(d)




(a)

(b) (c)








Disclosure






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














3 Results





10

0

0 10 20

Slow

(120583m

)

Fast (120583m)

10

08

06

04

02

00

minus02

minus04

minus060 5 10 15 20 25

Offset (120583m)

Hei

ght (120583

m)

(a)

Chitosan film

chitosan film



T (

)

4000 3000 2000 1500 1000 650

(cmminus1)

Chitosan film

chitosan film



(b)





4 Discussion






(a) (b)

(c) (d)









(a)

(b)

(c)

(d)




(a)

(b) (c)








Disclosure






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




10

0

0 10 20

Slow

(120583m

)

Fast (120583m)

10

08

06

04

02

00

minus02

minus04

minus060 5 10 15 20 25

Offset (120583m)

Hei

ght (120583

m)

(a)

Chitosan film

chitosan film



T (

)

4000 3000 2000 1500 1000 650

(cmminus1)

Chitosan film

chitosan film



(b)





4 Discussion






(a) (b)

(c) (d)









(a)

(b)

(c)

(d)




(a)

(b) (c)








Disclosure






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)

(c) (d)









(a)

(b)

(c)

(d)




(a)

(b) (c)








Disclosure






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

(b)

(c)

(d)




(a)

(b) (c)








Disclosure






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






Disclosure






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article smooth muscle cell alignment and...

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