acid polilactic

Journal of Membrane Science 371 (2011) 117126

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Journal of Membrane Science

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Development of poly(l-lactic acid) hollow ber mvasculature in tissue engineering scaffolds

N.M.S. Bettahalli, H. Steg, M. Wessling1, D. Stamatialis

MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Membrane TechnolP.O. Box 217, 7500 AE Enschede, The Netherlands

a r t i c l

Article history:Received 3 AuReceived in reAccepted 15 JaAvailable onlin

Keywords:Hollow berPoly(l-lactic acid)Phase separationBiodegradableTissue engineering scaffolds

ularizselvecells limits(l-la

tro caeloptem

and spongy sub-layer. Porogens like poly-vinylpyrrolidone (PVP) and poly (ethylene-glycol) (PEG) areadded to the dope solution. Subsequent treatment with sodium hypochlorite produces open ber sur-face and increased pore interconnectivity. The produced bers have good mechanical properties. Thetransport of bovine serum albumin (BSA) and of cell culture medium supplement with 10% fetal bovineserum (FBS) through the produced PLLA ber is high (permeance 1963L/(m2 hbar)) with low protein

1. Introdu

Tissue eas a thrivinIt is an inteneering andrestore, maago most sc

Abbreviatioalbumin; C2C1medium; EDTAliquid inducedpyrrolidone; PPLGA, poly(lacparts per millining electron m

Corresponnology and TeTechnology, FThe Netherlan

E-mail add1 Present ad

52064 Aachen

0376-7388/$ doi:10.1016/j.retention. In vitro static and dynamic cell culturing for 3 and 7 days using mouse pre-myoblast (C2C12)cells suggests that the fabricated PLLA hollow bers are suitable for delivery of nutrients to the cells in atissue engineering scaffold.

2011 Elsevier B.V. All rights reserved.

ction

ngineering is a cell based therapy that has emergedg and complimentary new eld of medical science.rdisciplinary eld that applies the principles of engi-the life sciences to develop biological substitutes thatintain, or improve tissue function [1,2]. Just a decadeientists believed that human tissue could be replaced

ns: 3D, 3 dimensional; BCA, bi-cinchoninic acid; BSA, bovine serum2, mouse pre-myoblast cells; D-MEM, Dulbeccos modied eagles, ethylene-di-amine-tetra-acetic acid; FBS, fetal bovine serum; LIPS,phase separation; NaOCl, sodium hypochlorite; NMP, N-methyl-2-BS, phosphate buffer solution (pH=7.3); PEG, poly(ethylene-glycol);tic-co-glycolic acid); PLLA, poly(l-lactic acid)/poly(l-lactide); ppm,on; PVA, poly(vinyl alcohol); PVP, poly-vinyl-pyrrolidone; SEM, scan-icroscope.

ding author. Present address: MIRA Institute for Biomedical Tech-chnical Medicine, University of Twente, Biomaterials Science andaculty of Science and Technology, P.O. Box 217, 7500 AE Enschede,ds. Tel.: +31 53489 4675; fax: +31 53489 4611.ress: [email protected] (D. Stamatialis).dress: RWTHAachenUniversity, ChemischeVerfahrenstechnik (CVT),, Germany.

only with direct transplants from donors or with fully articialsubstitutes made of plastic, ceramics, metal etc. However, inno-vative and imaginative work in laboratories around the worldis demonstrating that creation of bio-hybrid organs is feasible.But before this research will begin to pay off in terms of reli-able tissues, tissue engineering must surmount some importanthurdles.

In nature, blood vessels carry blood from the heart to the tissuesand organs and back to the heart. They form a branched systemof arteries and veins which then branch into smaller arteriolesand capillaries. In active tissue, sufcient diffusion is conned to150200m from the nearby capillary [3]. In 3D tissue formation,vascularization of in vitro cultured constructs is one of the majorchallenges apart from cell differentiation and proliferation up toscaffold design and bioreactor culture.

The mass transfer limitation within the in vitro 3D culturedconstructs could be addressed by utilization of porous biodegrad-able hollowbersmimicking the blood arterieswhen incorporatedwithin the traditional tissue scaffold. As incorporated porousber wall itself act as barrier between the proliferating cellsand owing media, perfusion bio-reactor systems could be usedto develop 3D tissue of clinical relevance. Hence, implantablemechanically strong porous biodegradable hollow bers with high

see front matter 2011 Elsevier B.V. All rights reserved.memsci.2011.01.026e i n f o

gust 2010vised form 10 January 2011nuary 2011e 22 January 2011

a b s t r a c t

In tissue engineering constructs, vasclems, as proliferating cells act themnutrient and oxygen delivery to therelevant dimensions, which in-turn lhighly permeable biodegradable polywith tissue engineered scaffolds in viious ber spinning parameters to devand ethanol as non-solvent at very low/ locate /memsci

embranes for articial

ogy Group, Faculty of Science and Technology,

ation within in vitro cultured tissue is one of the major prob-s as a barrier for mass transfer. Issues related especially toimit tissue construct development to smaller than clinicallythe ability for in vivo integration. In this work, we developctic acid) (PLLA) hollow bers (HF) which when integratedn improve nutrient supply to the cells. In fact, we study var-the optimum ber structure. By using 1,4 dioxane as solventperature (6 C), we develop bers with thin dense top-layer

118 N.M.S. Bettahalli et al. / Journal of Membrane Science 371 (2011) 117126

cell culture medium permeance and rather low cell adhesion arenecessary to deliver nutrient to the cells in tissue engineeringscaffolds.

Recently, an interesting study reported the development ofbiodegradafor tissuebiodegradaery [57]the latter,but also hmedium cowould notcells.

In this swe developcally stablewater andlow proteinditions showhereashigcan be usedscaffolds.

2. Materia

2.1. PLLA ho

Initially,(PLLA, MolD. GrijpmaUniversitytions: 14, 1purity). Theincrease poto the polym(Mol wt. 4(Mol wt. 3.poly(ethyleSince PVPwere initial(ACROS 99%Inmost casein to the potion. Detaildiscussed inthrough a 2spinning.

2.2. Spinnin

A speciaonly smalldegassed sinto the syrneret wereheater. Theof 1bar to esisted of ourespectivelynon-solvencoolant throused as borThe preparesolvent bat72h to rem

Schematic representation of miniaturized hollow ber spinning set-up.

embrane treatment

Gas plasma treatmentncrease the membrane surface porosity, some hollow bersreated with oxygen plasma (PlasmaFAB-508, Electrotech) atg power input (1030W) and etching time (1030min)withediate relaxation or cooling steps. For the plasma treatment,lowberswerehunghorizontally in the center of theplasmaer to expose complete surface of the ber to the generated.

Sodium hypochlorite treatmentium hypochlorite (NaOCl) treatment was used to removeom the ber matrix and increase membrane porosity. Ine hollow bers after extensive washing in ethanol were rstrred to running tap water bath for 24h and then transferrede 4000ppmNaOCl solution for 4 and 24h. Subsequently, theble poly(lactic-co-glycolic acid) (PLGA) hollow bersengineering application [4]. Others also developedble PLLA hollow ber membranes for drug deliv-or ultra ltration membrane applications [8]. Inthe PLLA membranes have high clean water uxigh protein retention (80%). Since the cell culturentains high amount of proteins, these membranesbe suitable for delivering the medium to the

tudy by tailoring the hollow ber spinning conditionsvia liquid induced phase separation (LIPS) mechani-biodegradable PLLA hollow bers with very high cleanmedium uxes (in the microltration range) and veryretention. Cell culture experiments under static con-

w that cells attach and proliferate very well on our HFhmediumperfusion through themsuggests that theHFas articial vasculature for in vitro tissue engineering

ls and methods

llow ber polymer solution

hollow bers were fabricated using poly(l-lactic acid)wt. 1.6105 g/mol, kindly provided by Prof. Dr.(Biomaterials Science and Technology (BST) group,

of Twente, The Netherlands), of different concentra-7 and 20wt% dissolved in 1,4-dioxane (Merck, 99%se hollow bers had low porosity. Subsequently torosity and wettability different porogens were addeder solution: 2.5wt% poly-vinylpyrrolidone (PVP K-30)104 g/mol), 2.5wt% poly-vinylpyrrolidone (PVP K-90)

6105 g/mol) (both Fluka, analytical quality) and 5%ne-glycol) (PEG-400) (Mol wt. 400g/mol) (MERCK).is sparingly soluble in 1, 4 dioxane, these additivesly dissolved in 10wt% N-Methyl-2-pyrrolidone (NMP)purity) and then added to the PLLA polymer solution.s non solvent ethanol (MERCK 99.9% purity)was addedlymer solution to decrease the time for phase separa-s about the selection of the spinning compositions areSection 3. In all cases the polymer dopes were ltered

5m metal lter and degassed for at least 1day before

g set-up

l miniaturized spinning system was developed sinceamounts of polymers were available to process. Thepinning dope was maintained at 60 C before loadedinge of the spinning setup. The syringe and the spin-also maintained at 60 C during spinning, using a tapesyringe was pressurized using nitrogen gas at pressurextrude the dope solution through spinneret, which con-ter and inner needles of diameter 0.5mm and 0.2mm(Fig. 1). Ethanol (MERCK 99.9% purity) was used as

t maintained at various temperatures by circulatingugh the jacketedwalls of coagulationbath. Ethanolwase liquid at room temperature at a ow rate of 1ml/min.d bers were rst collected at the bottom of the non-h and later were soaked in ethanol at 4 C for at leastove residual solvent.

Fig. 1.

2.3. M

2.3.1.To i

were tvaryinintermtheholchambplasma

2.3.2.Sod

PVP frfact, thtransfeinto th

N.M.S. Bettahalli et al. / Journal of Membrane Science 371 (2011) 117126 119

Table 1Screening of PLLA spinning solvents.

Spinning dope Mixing temperature (C) Hollow ber morphology Remarks

Components Concentration (wt%)

PLLA:NMP in witPLLA:CHCl3 in witPLLA:dioxan in wit

bers werein ethanol.

2.4. Hollow

2.4.1. ScannSEM ima

scanning eldried overnSEM analysples were sSCD-040).

2.4.2. WateFor the

water wasber (insidin modulespolyethylenwere carefupolyurethanplaced to cowater ux,their wettamodules foThe ux (Jtrans-mem202 C. Thsured by coleast 60minmeance (L/(pressure plhollow beber spun.

2.4.3. ProteProtein

ried out usiof 0.5 g/l (m(containingteins extensBoth proteiat pH 7.3 ahollow bemeability edifferent homembraneand thecoll30min for 6

BSA concter (Varianusing BCA (assay readteric readerprotein wer

Mechmecle tesof hotubelly bseparess/min5 bet theand sond

Cell cuse pm cosuppml pere iheyizededC2thetic o

. Stareateethasteriallyept i2C12cubte da

. Dynwit

ne inin t. Thl, PBre (cellstailsar wactor svisucopyic cu12:88 80 Dense sk18:82 52 Dense sk

e 24:76 75 Dense sk

washed with running tap water for 24h before storing

ber characterization

ing electron microscopy (SEM) analysisges of dry PLLA bers were obtained using JeoL 5600LVectronmicroscope at accelerating voltage of 5 kV. Fibersight in vacuum oven at 30 C were directly used foris after carefully fractured in liquid nitrogen. All sam-puttered with gold (1520 nm-thick, Balzer-Union

r permeabilitypure water permeability experiment, de-mineralizedpressurized through the lumen of the PLLA hollowe-out permeation). The experiments were carried outcontaining single hollow ber prepared using 6mme tube housing of approximately 10 cm long. The berslly inserted into the polyethylene tube and sealed withe glue. In the middle of the housing, a T-junction wasllect the owing permeate. Before measuring the cleanthe bers were rst ushed with ethanol to improvebility. Subsequently, water was pumped through ther at least an hour to remove/exchange the ethanol.) through the membrane was measured at differentbrane pressure ranging between 100 and 500mbar ateux (L/(m2 h)) at eachpressurewas successivelymea-llecting permeatingwater (L) with respect to time (h, at) and active ber surface area (m2). The purewater per-m2 hbar)) was calculated using the slope of ux versusot. The data presented in this work is an average of 5r modules prepared from different sections of the total

in permeabilitypermeation experiments through PLLA ber were car-ng model protein bovine serum albumin (BSA) solutionolecular weight 66kDa) and fetal bovine serum (FBS)a cocktail of albumin 65% and other small chain pro-ively used as a supplement with cell culture medium).ns were dissolved in phosphate buffer solution (PBS)nd the ltration experiments was carried out usingr modules prepared similar to that used for water per-xperiments. The data presented here is an average of 5llow ber modules tested for each protein type. Trans-pressure of 0.1 bar was applied across the hollow berectedpermeatewasanalyzed forprotein rejectioneveryh of continuous ltration.entrationwas analyzedusingUV-VIS spectrophotome-

2.4.4.The

a tensitestinged forvertica(Z020)stant p50mmage ofslope astresscorresp

2.4.5.Mo

mediuGibco)100U/Cellswuntil ttrypsinobtainface ofthe sta

2.4.5.1PLLA tof 70%insideand nwere kwith Cin an inalterna

2.4.5.2treatedurethasurfaceerationethanopressuC2C12(see de0.1bbiorea

FormicrosdynamCARY 300 Scan) at 278nm. Whereas FBS was analyzedbi-cinchoninic acid) assay kit, which is a colorimetricat 562nm using 96 micro-well plate spectrophotome-. In all cases the feed and permeate concentration ofe monitored, too.

entwells anof total DNto the manKit, Invitrog(Perkin Elmh closed pores Sparingly soluble + low mechanical stabilityh closed pores High viscosity + low mechanical stabilityh closed pores Gelation at RT

anical propertieshanical properties of hollow berweremeasured usingting machine (Zwick, Static Material Prufung unit). Thellowberswas carried out using an ISO standard speci-s/cylinders. A dry 10 cm long hollowberwas clampedetween the holders/clamps of the Zwick test machinerated by 5 cm. 500N load cell was used to apply con-ure on the hollow ber elongated at a constant rate ofuntil break point. The data presented here is an aver-rs. The E-modulus of the ber was estimated using theinitial part of the stress strain curve. The maximum

tress at break were noted from the stress strain curveingly.

ulturingre-myoblast, C2C12 cells were cultured in proliferationntaining Dulbeccos Modied Eagles Medium (D-MEM,lemented with 10% fetal bovine serum (FBS, Cambrex),enicillin (Gibco) and 100g/ml streptomycin (Gibco).nitially plated in T-ask for expansion at 2000 cells/cm2

reached 7080% conuence, after which they wereusing 0.05% Trypsin contained in 1mM EDTA. TheC12 cellswere subsequently seededon to the outer sur-hollow ber and were allowed to attach for 6h beforer dynamic culture experiments.

tic culture. Hollow bers 2 cm long, fabricated of 17%d with NaOCl for 24h were rst sterilized in excessnol and iso-propanol, after evaporation of the alcoholsle ow-hood, the bers were washed 3 times with PBSneutralized in proliferation medium. Five such bersnside the tissue culture plate (non-treated) and seededcells. These samples were cultured for 0, 3 and 7 days

ator with proliferation medium being exchanged everyy

amic culture. Hollow bers fabricated of 17% PLLAh NaOCl for 24h were potted at both ends using poly-side 6mm silicon tubing, such that 2 cm long berhe middle was exposed for cell adhesion and prolif-e modules prepared were sterilized by pumping 70%S and proliferation medium correspondingly at low0.1bar). The sterilized ber modules were seeded withand cultured in a recirculation perfusion bioreactorlater) for 0, 3 and 7 days. Trans-membrane pressure ofs maintained across the cell seeded ber module in theystem with an average cross ow velocity of 1ml/min.alization of cell adhesion and proliferation using light, 4000 cells/cm2 were cultured in both static andlture. For the DNA analysis, bers were placed in differ-

d 2106 C2C12 cells/berwere seeded. QuanticationA concentration per sample was measured accordingufacturers protocol (CyQuant Cell Proliferation Assayen/Molecular probes) using a uorescent plate readerer).


Table 2PLLA spinning dopes used in this study to fabricate hollow ber at 6 C.

Dope solution code PLLA (wt%) Additives (wt%) Dioxane (wt%)

PVP K30 PVP K90 PEG NMP Ethanol

D1 14 2.5 2.5 5 10 66D2 14 2.5 2.5 5 10 1 65D3 17 2.5 2.5 5 10 1 62D4 20 2.5 2.5 5 10 1 59

Fig. 2. Effect of air gap and non solvent in the dope solution on PLLA hollow ber morphology.


3. Results

3.1. Prepara

Initiallyration of PLstability ofresults obtamer concenber. In allas has been[9]. Only thcally stableexperimentwith thin sk

Earlier sthat PVP (K1can increaseTo identifyporous andsive screeniconcentrati(K15, K30, KFig. 3. Effect of PLLA polymer concentration in the dope solution o

and discussion

tion of PLLA hollow ber

various organic solvents were screened for the prepa-LA bers. Especially, the morphology and mechanicalthe ber were evaluated. Table 1 presents the bestined for 3 different solvents with corresponding poly-trations and mixing temperature to extrude hollowthese cases ethanol was used as non-solvent at 4 C,reported for fabrication of PLLA porous atmembranese PLLA bers prepared in 1,4 dioxane were mechani-and could be used for further tests (SEM, permeations etc.). Despite this, these bers were also rather densein layer and closed pores.tudies (some of them performed in our group) showed5, K30, K90) [1013] and PEG (200 and 400g/mol) [14]theporosity andpore interconnectivity ofmembranes.

the optimum cocktail of porogens for fabricating highlymechanically stable bers we also performed exten-ng. Hence, we fabricatedHFwith 14wt% PLLA, differentons of PVP (2.5, 5, 7.5wt%) of variousmolecularweights90) and PEG (molecular weight 200 and 400g/mol) of

different cocerning mewith PVP (2in NMP (10porogens inspinning pa

Furthermthe hollowtemperaturThe most pnon-solven

In the fousing PLLAages of nonPVP and 2.5porogens. Inconstant at

3.1.1. EffectFig. 2 sh

for ber sp(PVP and PEand low pon PLLA hollow ber morphology.

ncentrations (2.5, 5 and 10wt%). The best result con-chanical stability was obtained for the bers fabricated.5wt% K30 and 2.5wt% K90) and PEG (5wt%) dissolvedwt%) in the dope solution. Hence results with thesethe dope solution of different PLLA concentrations andrameters are presented further in this work.ore, to minimize the formation of dense skin layer of

ber we also investigated the effect of the non solvente. Besides 4 C, we also spun bers at 0, 4, 6, 10 C.romising bers with open surface were obtained witht bath temperature of 6 C.llowing sections, the spinning tests were performed

of various polymer concentration,with various percent-solvent (ethanol) in the polymer dopewith 2.5wt% K30wt% K90 PVP, PEG (5wt%) dissolved in NMP (10wt%) asall cases the temperature of non solvent (ethanol) was

6 C. Table 2 presents the polymer dopes investigated.

of air gap and non-solvent in dope solutionows the effect of the air gap on membrane morphologyun with dope solution D1 (14wt% PLLA) and additivesG). For air gap of 1 and 2 cm the bers had outer skin

rosity. An increased air gap results in a longer residence


time in air,average veland the takeformed (seethe volatileskin format

Fig. 2 alsthe 14wt% Papproachesated [13,16in faster dedemixing isturized setu

3.1.2. EffectDifferen

Table 2 weity and meof ber sputions of theporous struhave poorpared for mincreasingmechanicalmembrane.polymer coto be the m(pore size rFig. 4. SEM images of PLLA hollow ber (D3, 17% PLLA dope solution

whose magnitude can be roughly estimated using theocity of the polymer solution as it exits the spinneret-upvelocity [15]. Althougha spongyporousmatrixwascross sectional SEM images in Fig. 2) the evaporation ofdioxane and the delayed demixing at the surface causesion.o shows the effect of adding 1% non-solvent ethanol toLLA dope solution (D2).With this the polymer solutionthe binodal demixing and nucleus growth is acceler-]. Thus slight change in the solvent composition resultsmixing and a more open porous structure. In fact, rapiddesired also due to the short non-solvent bath inminia-p.

of polymer concentration in dope solutiont PLLA concentrations along with additives as listed inre tested to prepare optimum ber with high poros-chanically stable structure. Fig. 3 shows SEM imagesn with dope solution D2, D3 and D4. The cross sec-bers spun with D2 and D3 show a well-distributedcture, but the bers have dense outer skin. D4 bersinterconnectivity and a dense skin layer. When com-echanical strength, the order is directly proportional topolymer concentration i.e., the D4 bers are the mostly stable, but with closed pores across the wall of theThis decrease in porosity is expected due to the higherncentration. Thebers spunwithdope solutionD3seemost optimal concerning to wall and surface porosityange 0.55m) and mechanical stability. Hence, fur-

ther optimiD3.

3.2. Hollow

3.2.1. PlasmOxygen

skin of theboth contaogy and insthe hydropconditionsing time. SELow intensisure for 3 tthe ber sk10min explayer becombers are eplasma treastructure oered for fur

3.2.2. SodiuAnother

pore-interca porousmlar level (heof the pore) after oxygen plasma treatment.

zation was carried out on ber spunwith dope solution

ber post treatment

a treatmentplasma etching was carried out to remove the outerhollow ber. Plasma treatment causes an increase inct angle and roughness, altering the surface morphol-ertion of polar groups, which consequently, enhancinghilicity of the polymer [17]. Optimization of the etchingwas performed to evaluate the etching rates and etch-M pictures of a few positive results are shown in Fig. 4.ty of 10W does not open the ber skin even after expo-imes of 10min each. Intensity of 30W etching opensin but the bers melt due to heating (see Fig. 4, 3 timesosure at 30W). Although the surface opens, the sub-es denser due to polymer melting. Most of the etchedxtremely fragile to handle. Based on these results, thetment does not offer promising results concerning berptimization. Hence plasma treated HF were not consid-ther characterization.

m hypochlorite treatmentpost treatment to increase membrane porosity andonnectivity is removal of PVP using NaOCl. In preparingembrane PVP is notmixedwith polymer on themolecu-terogeneous blend), but it will be located at the surfacewall [10]. The removal of PVP can be achieved with


Fig. 5. SE

4000ppmNwere treateshows thatthose bers(permeanceing to earlibers with(K90 and Kremain in ththe PEI andPLLAmembipate havintreatment. Tandpermea[1921].

3.3. Mechan

Mechanforces encoversus strausing dopeForce per uwere subjethis ber, tstressstraiandPLGA-PM images of surface and cross section of PLLA hollow ber (D3, 17% PLLA dope solution)

aOCl treatment. Therefore the bers fabricatedwithD3d by immersing in the NaOCl bath for 4 and 24h. Fig. 5the bers treated for 24hwith NaOCl are very open andare further characterized in the following paragraphstesting, mechanical testing and cell culture). Accord-

er studies performed in our laboratory for PEI hollow25wt% PVP (K-30) [10] and PES bers with 33wt% PVP30) in the membrane [18], only small amounts of PVPemembrane after NaOCl treatment for 24h (


Fig. 7. (a) Clea applied trans-membranepressure. (b) Permeation of BSA and FBS (dissolvedin PBS at pH 7 d concentration, at 0.1 bar trans-membrane pressure.

linearwithing pressurin Fig. 7a)of a microbioreactor sphysiologicto human sthese low tr

Formimber shouldbovine seruother smallbovine seruthe lumen osure. Fig. 7bthe membr1963L/(m2

For the pturemediumproblems da solution onormal cultation resultpermeabilitthan 10% rethe suitabilmedium to

3.5. Cell cul

3.5.1. StaticWe perf

pared fromNaOCl) thastability. Fi(stained wi7 days. Theobserved virial. In add(dioxane, Nbrane afterper cm2 ofing cell numbers.nwater ux through PLLAhollowber (D3, 17%PLLAdope solution)with respect to) through PLLA hollow ber (D3, 17% PLLA dope solution) with respect to initial fee

no compaction (several cycle of increasing and decreas-e ux were tested). The clean water permeance (slopeis 2094L/(m2 hbar), which indicates that the ber isltration type. The maximum allowable pressure in aystem for tissue engineering applicationmimicking theal condition for cell culture is 0.15bar correspondingystolic blood pressure. Our ber performs very well atans-membrane pressures.icking vasculature in tissue engineering application, thedeliver to the cells culture medium containing fetal

m (FBS) to the cells. FBS is a cocktail of 65% albumin andchain proteins. Hence the permeation ofmodel protein,m albumin (BSA, molecular weight 66kDa) throughf the ber was tested at 0.1 bar trans-membrane pres-shows that more than 90% of BSA permeates through

ane (feed concentration=0.5 g/l) and at permeance ofhbar) over a period of 6h.ermeation experiments, we could not use D-MEM cul-due to the presence there of phenol red which causes

uring BCA analysis of the proteins. Instead, we usedf 10% FBS dissolved in PBS of pH 7.3 (comparable toure medium composition). Fig. 7b shows the perme-s obtained for 5 different ber modules tested. Highy of 1939L/m2 hbar (>90% of pure water ux) and lesstention of FBS molecules over a period of 6h indicatesity of produced PLLA ber for delivering of the culturethe cells in a perfusion bioreactor system.ture

cultureormed static cell culture experiments using bers pre-D3 (17% PLLA in dope solution, treated for 24h with

t have very high medium permeance and mechanicalg. 8a shows light microscopy images of C2C12 cellsth methylene blue) cultured for 0 (seeding day), 3 andcells attach and proliferate well on HF surface (as

sually) showing high afnity of the cells to the mate-ition, these results suggest that no traces of solventsMP and ethanol) remain within the porous HF mem-the membrane preparation. Fig. 8b presents the DNAPLLA ber. As expected, the DNA (and the correspond-ber) increases in time due to cell proliferation on our

Fig. 8. C2C12 cells cultured statically for 0, 3 and 7 days on PLLA hollow ber (D3,17%PLLAdope solution, 24hNaOCl treatedHF). (a) Lightmicroscopic images stainedwith methylene blue stain (4 magnication). (b) DNA results.


Fig. 9. Schematic illustration of re-circulating hollow ber pe

Fig. 10. Light microscopic images of C2C12 cells cultured dynamically in a per-fusion bioreactor for 0, 3 and 7 days on PLLA hollow ber (D3, 17% PLLA dopesolution, 24h NaOCl treated HF), stained with methylene blue stain, seeding den-sity =4000 cells/cm2 (4 magnication).

3.5.2. DynaDynami

for 24h wiactor systeStatically sebioreactor aof the ber.1ml/min) wcells on thescopic imagmethyleneIn contrastsurface dueberswereSince the c(see earlierconditionsthe mediumdynamic ceble with lothese bersneed to beout causingscaffold.

4. Conclus

In this wnectedporoBSA and FBsuitability fengineeringbers showing that theto inuenceture, the cethe mediumfouling duron the inconeering scaconditions.rfusion bioreactor system.

mic culturec cell culturing on D3 ber surface (17% PLLA, treatedth NaOCl) was tested in recirculation perfusion biore-m (Fig. 9) which was built inside a sterile incubator.eded hollow ber module was placed inside the glassnd cultured by perfusing medium through the lumenSteady trans-membrane pressure of 0.1 bar (ow rate as maintained to deliver nutrients to the proliferatingouter surface of the ber. Fig. 10 shows light micro-es of dynamic cultured, ber-cell module stained withblue after 0 (seeding), 3 and 7 days culturing period.to the static culture very few cells adhere on the berto the ow of the medium. The DNA results for thesealso very low (below thedetection limit of our analysis).ell attach and proliferate well under static conditions) this low cell adhesion on the ber under dynamiccan only be due to de-attachment of the cells due toow. This phenomenon may be advantageous during

ll culture as the ber permeance would remain sta-w pressure drop and low fouling. Nonetheless whenare integrated into the scaffold, the ow conditions

adapted carefully to keep the ber pores open with-de-attachment of the cells in the surrounding tissue

ion outlook

ork, PLLA microltration hollow bers with intercon-us structurehavebeendeveloped. Thehighcleanwater,S protein permeation through the ber indicates itsor delivering medium to the cells in perfusion tissuebioreactor systems. The static culture studies onto ourthat the cells attach and proliferate there well show-

re are no solvent/non-solvent extractable from the berthe cell attachment and proliferation. In dynamic cul-lls de-attach from the ber surface probably due toow but this could be favourable for avoiding ber

ing bioreactor studies. Our future work will be focussedrporation of the developed PLLA ber into tissue engi-ffolds and the optimization of tissue culture bioreactor


Acknowledgements

The authors would like to acknowledge the nancial supportfrom Technology Foundation STW (Project number TKG. 6716).We also thank Prof. Dr. D. Grijpma (Biomaterials Science and Tech-nology (BST) group, University of Twente, The Netherlands) forkindly providing the PLLA polymer and Prof. Dr. C.A. van Blitter-swijk (Tissue Regeneration (TR) group University of Twente, TheNetherlands) for allowing performing cell culture experiments inhis lab. Thanks are also due to S. Schuller and J.B. Bennink forassistance with mechanical testing equipment and set-up artworkrespectively.

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Development of poly(l-lactic acid) hollow fiber membranes for artificial vasculature in tissue engineering scaffoldsIntroductionMaterials and methodsPLLA hollow fiber polymer solutionSpinning set-upMembrane treatmentGas plasma treatmentSodium hypochlorite treatment

Hollow fiber characterizationScanning electron microscopy (SEM) analysisWater permeabilityProtein permeabilityMechanical propertiesCell culturingStatic cultureDynamic culture

Results and discussionPreparation of PLLA hollow fiberEffect of air gap and non-solvent in dope solutionEffect of polymer concentration in dope solution

Hollow fiber post treatmentPlasma treatmentSodium hypochlorite treatment

Mechanical propertiesHollow fiber permeabilityCell cultureStatic cultureDynamic culture

Conclusion outlookAcknowledgementsReferences

acid polilactic

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