Aligned poly(L-lactic-co-e-caprolactone) electrospun microfibers and knitted structure: A novel...

Aligned poly(L-lactic-co-e-caprolactone) electrospun microfibersand knitted structure: A novel composite scaffold forligament tissue engineering

Cedryck Vaquette,1,2 Cyril Kahn,1 Celine Frochot,3 Cecile Nouvel,4 Jean-Luc Six,4 Natalia De Isla,1

Li-Hua Luo,1 Justin Cooper-White,2 Rachid Rahouadj,1 Xiong Wang1,5

1Group of Cell and Tissue Engineering, LEMTA, Nancy-Universite, CNRS, 2 Avenue de la foret de Haye,

Vandoeuvre 54 500, France2Tissue Engineering and Microfluidics Laboratory, Australian Institute for Bioengineering and Nanotechnology,

The University of Queensland, Building 75—Cnr of College and Cooper Roads, Brisbane 4072, QLD, Australia3Departement de Chimie Physique des Reactions, DCPR, Nancy-Universite, CNRS, 1 rue Granville, Nancy 54 000, France4Laboratoire de Chimie Physique Macromoleculaire, LCPM, Nancy-Universite, CNRS, 1, rue Granville, BP 20451,

Nancy, F-54001, France5Laboratoire Physiopathologie et Pharmacologie et Ingenierie Articulaires, LPPA, UMR 7561, Nancy-Universite, CNRS,

9 Avenue de la foret de Haye, Vandoeuvre 54 500, France

Received 1 September 2008; revised 30 October 2009; accepted 23 December 2009

Published online 10 May 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32801

Abstract: We developed a novel technique involving knitting

and electrospinning to fabricate a composite scaffold for liga-

ment tissue engineering. Knitted structures were coated with

poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a

rotating cylinder and a PLCL solution was electrospun onto

the structure. Highly aligned 2-lm-diameter microfibers cov-

ered the space between the stitches and adhered to the knitted

scaffolds. The stress–strain tensile curves exhibited an initial

toe region similar to the tensile behavior of ligaments. Com-

posite scaffolds had an elastic modulus (150 6 14 MPa) similar

to the modulus of human ligaments. Biological evaluation

showed that cells proliferated on the composite scaffolds and

they spontaneously orientated along the direction of micro-

fiber alignment. The microfiber architecture also induced a

high level of extracellular matrix secretion, which was charac-

terized by immunostaining. We found that cells produced col-

lagen type I and type III, two main components found in

ligaments. After 14 days of culture, collagen type III started to

form a fibrous network. We fabricated a composite scaffold

having the mechanical properties of the knitted structure and

the morphological properties of the aligned microfibers. It is

difficult to seed a highly macroporous structure with cells,

however the technique we developed enabled an easy cell

seeding due to presence of the microfiber layer. Therefore,

these scaffolds presented attractive properties for a future use

in bioreactors for ligament tissue engineering. VC 2010 Wiley

Periodicals, Inc. J Biomed Mater Res Part A: 94A: 1270–1282, 2010.

Key Words: ligament tissue engineering, electrospinning,

knitting, composite scaffolds, polyesters

INTRODUCTION

Anterior cruciate ligament (ACL) is a band of a dense con-nective tissue that binds the tibia and the femur. Its role isessential for normal movement and assures joint stability.1

ACL is predominantly composed of fibroblastic cells withina web of aligned collagen fibers.2 The architecture of thistissue confers to the ligament specific mechanical propertiessuch as elasticity and important tensile strength. Because ofits highly heterogenic nature and poor vascularization, ACLhas little or limited healing ability and a surgical procedureis often required in the event of an injury.1

Injury of the ACL frequently occurs in the young andphysically active population and is responsible for a loss ofmobility and life’s comfort. Numerous therapies have beendeveloped to restore or replace the ACL and can be classi-

fied as either biological or synthetic grafts. Among the bio-logical restoration treatments, autograft has the best out-comes in term of improved mobility, and enhanced ability toparticipate in sport activities. Allograft is another alternativesince the replaced tissue already possesses good mechanicalproperties.3 Limitations of biological grafts include; donorsite morbidity, pain, lack of donors and immunogenicproblems.4

To overcome these limitations, synthetics grafts weredeveloped. These grafts mainly utilized braiding technologyand materials such as polyethylene terephthalate,5 poly-tetrafluoroethylene,6 polypropylene.7 However, these graftsare confronted to an increase in the knee laxicity over thetime and catastrophic failures often occurred after severalyears of implantation.8 In addition, wear particles induced

This article is dedicated to our colleague and friend Luc Marchal.

Correspondence to: C. Vaquette; e-mail: [email protected]

Contract grant sponsors: French CNRS (ATIP/SPI), Region Lorraine, Agence Nationale de la Recherche (ANR, project TELiTeR)

1270 VC 2010 WILEY PERIODICALS, INC.

macrophage infiltration in the synovial cavity and inflamma-tion of the surrounding tissues.7 Poor tissue and cellularcolonization of the grafts is believed to be the reason oftheir unacceptable in vivo behavior. Although it has beenpossible to improve the biocompatibility and the biologicalintegration of these structures by surface treatments9,10

synthetic grafts are not commonly used for ACL replace-ment. There is currently no optimal treatment for ACLrestoration.

A third emerging alternative, called Tissue Engineering,aims to combine biodegradable or nonbiodegradable syn-thetic materials and autologous cells to overcome the afore-mentioned limitations of traditional treatments.11 In thisstrategy, autologous cells are seeded in a 3D scaffold andcultured in vitro to induce extracellular matrix synthesisand to allow neo-tissue formation. Ideally, the scaffold mustmeet well defined criteria such as biocompatibility, high sur-face area per volume for promoted cell adhesion, growthand proliferation, high and interconnected porosity for gasand nutrient supplying, and adequate mechanical properties(e.g., mechanical properties similar to healthy ACL).12 Scaf-folds materials for ACL tissue engineering are either fromdiverse natural sources, such as collagen gel,13–15 collagenfibers,16 porcine small intestinal submucosa,17–19 polysac-charides,20–22 silk,23,24 or from synthetic materials such asaliphatic polyesters.25,26

Braided structures of synthetic materials were amongthe first scaffolds to be developed for ligament tissue engi-neering due to their high tensile strength.23,25 However,their major drawback comes from poor cellular integrationin the scaffold. Knitting technology enables the fabricationof less dense structures with internal interconnected pores,essential for tissue growth, nutrient and gas supply. In addi-tion, the tensile mechanical properties of knitted structureshave been shown to be adequate for ligament tissue engi-neering.27,28 However, due to the high porosity of knittedstructures, a controlled and homogeneous cell seeding couldbe very difficult to achieve. To overcome this limitation, dif-ferent techniques were developed involving cell encapsula-tion in fibrin gel,28 elaboration of collagen fiber net-work,29,30 cell sheet tissue engineering,31 silk microporoussponge,32 nanofibers deposition by electrospinning.33 Elec-trospinning is a technique producing micro- or nano-fibersfrom a polymer solution forced through an electrostaticfield. Physical fiber properties, including diameter, morphol-ogy, and charge,34 can be adjusted when varying process pa-rameters. In addition, fiber orientation can be controlledusing a rotating wheel35,36 or a tailored collector geome-try.37,38 This study aims to demonstrate the possibility ofcoating knitted structures with aligned electrospun micro-fibers, which would spatially mimic the cell microenviron-ment encountered in ligament and tendon.

MATERIALS AND METHODS

The first part of this section describes the different testsperformed on fibrous membranes obtained by electrospin-ning. The fabrication and the evaluation of the composite

scaffolds, that is, knitted scaffolds coated by aligned micro-fibers, is reported in the second part.

Fibrous membranesElectrospinning procedure. Poly(L-lactic-co-e-caprolactone)[PLCL (LA/CL: 70/30), i.v. 1.4 dL/g, Boehringer Ingelheim,Germany] was utilized to produce nonwoven microfibersmats. A 15% (wt/vol) PLCL solution was prepared in asolvent mixture (90/10), (vol/vol) of chloroform (CF) andN,N-dimethylformamide (DMF) (Sigma-Aldrich, France) andelectrospun at a feed rate of 2 mL/h, at a distance of 15 cm,through a 19 G needle and at an initial voltage of 6 kV. Thevoltage was then gradually increased to 9 kV to avoid sec-ondary jets formation and to obtain a stable process. Analuminum cylinder (20 cm in diameter, and 5 cm wide)rotating at 1500 rpm (or 15.7 m/s) was used to align thePLCL microfibers.

Morphological evaluation. Aligned and randomly orien-tated fibrous membranes obtained by electrospinning wereobserved by Scanning Electron Microscopy (SEM, CambridgeS240). Before observation, samples were Gold-Palladiumcoated. The mean diameter was determined by measuringthe diameter of 20 microfibers.

Biological evaluationCell source. Bone marrow mesenchymal stem cells (b-

MSC) from rats (Wistar) were used to evaluate the biocom-patibility of the scaffolds. Rat b-MSCs were isolated byselective adherence on plastic culture plate. Rats were anes-thetized and bone marrow was aspirated from the tibia. Thebone marrow was mixed with heparin and sterile PBS toprevent coagulation and then centrifuged at 800 g for 10min. The nucleated cell layer was removed, suspended inculture medium and seeded in 25 cm2 culture flasks. Themedium was changed after 4 days of culture and then every2 days.

Culture medium. The culture medium used was Dulbec-co’s modified Eagle’s medium (DMEM) supplemented with10% fetal calf serum, penicillin (100 lm/mL), streptomycin(100 lg/mL), fungizone (2.5 lg/mL).

Cell metabolic activity. Fibrous membranes (1.2 � 1.2cm2) were sterilized by immersing the structures in ethanolfor 30 min. A 15 min-UV-irradiation was then performed oneach side of the membranes. They were rinsed three timesin culture medium to remove any residual ethanol. 104 ratb-MSCs between P4 and P6 were seeded by adding 500 lLof cell suspension on top of the fibrous membrane (n ¼ 3 �3) placed in a 48-well culture plate. After 4 h of incubation,500 lL of culture medium was added to the wells. The me-dium was then changed every 2 days. Polystyrene (PS) cul-ture plates were seeded at the same cell density were usedas a positive control.

The biocompatibility of the fibrous membranes wasassessed using the Alamar blue test (Serotec, UK). Mem-branes were transferred into a new 48-well plate and cell

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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | 15 SEP 2010 VOL 94A, ISSUE 4 1271

activity was measured at day 1, 3, 7, 10, and 14. Five hun-dred microliter of a 5% Alamar blue in DMEM without phe-nol red (Gibco, France), was poured into each well. The 48-well culture plate was then incubated at 37�C for 120 min.Absorbance was measured at 570 and 630 nm using a spec-trophotometer (DU 640, Beckman). The percentage ofreduction of Alamar blue was calculated according to thesupplier information. Student’s test was performed, and p <

0.05 was considered to be statistically significant.The seeded membranes were fixed and gradually dehy-

drated in ethanol solutions and finally gold-palladiumcoated for SEM-observation to evaluate cell distribution andmorphology (n ¼ 2 for each measurement).

Composite scaffoldsScaffolds fabrication

Knitted scaffolds. To demonstrate the versatility of thecomposite fabrication method two different sutures wereutilized; Vicryl poly(L-lactic-co-glycolic acid (10/90),(PLGA)) and silk sutures 4–0 were supplied by Ethicon,France. A Silver-reed ZK 270 (Suzhou, China) knittingmachine was utilized to fabricate the scaffolds. Knittedstructures were 12 stitches wide and 15 rows long with in-ternal stitch diameter of 1 mm.

PLCL coating. A 2.5% (wt/vol) PLCL solution in CF wasprepared to coat the knitted structures. The coating wasperformed using a silk brush immersed in the PLCL solu-tion. After solvent evaporation, knitted structures weremanually stretched to allow good sliding between thesutures. This procedure was repeated three timesconsecutively.

Deposition of aligned microfibers on the knittedscaffolds. Knitted structures were placed on the rotatingmandrel using metallic bars screwed in the wheel (Fig. 1),allowing the structures to be firmly gripped during themandrel rotation. Electrospinning was performed at a dis-tance of 8 cm between the collector and the needle toobtain wet microfibers. For the same reason, an initial 10min-deposition at 4 mL/h polymer solution feed rate wasperformed, followed by a 20 min-deposition at 2 mL/h feedrate to obtain smaller microfibers. The electrospinning volt-age was adjusted as described previously. Microfibers weredeposited on only one side of the scaffold, as we consideredone side deposition would be adequate for our tissue engi-neering application, since the scaffold will be rolled upbefore use in a bioreactor.

Scaffold evaluation. The scaffold evaluation consisted ofmorphological, mechanical and biological evaluations.

Morphological evaluation. The knitted scaffolds afterPLCL-brush-coating were observed using SEM whereas bin-ocular optical microscopy was used to investigate the mor-phology of the composites (knitted scaffold/microfibers).

Mechanical evaluation. The aim of the mechanicalstudy was to evaluate the influence of the PLCL microfibersdeposition on the tensile properties of the knitted scaffolds.As the aligned fibrous membrane was relatively thin, it wasassumed that it did not affect the overall tensile properties.Thus, the mechanical evaluation was performed on only onetype of composite scaffold. Silk scaffolds were chosen as amodel due to their slower degradation compared to theVicryl scaffolds. Tensile tests on the silk knitted scaffoldswith and without PLCL aligned microfibers were performedusing an Adamel-Lhomargy DY 22 (MTS, France) at a cross-head speed of 10 mm/min in a 37�C water bath. Stress inthe knitted structure was calculated using the diameter ofthe suture (290 lm) and the number of aligned sutures thatcomposed the scaffold section (25 sutures). Ultimate stressand strain were estimated and the modulus of elasticity wasdetermined from the linear part of the curve (strain >

10%). Student’s test was performed, and p < 0.05 was con-sidered to be statistically significant.

Biological evaluation. Similar to the mechanical evalua-tion, the silk scaffold was chosen as a model for the biologi-cal study. The composite scaffolds were sterilized accordingthe procedure described above. They were then dried usinga steritop vacuum filter and placed in a low adhesion 6-wellculture plate. The scaffolds were then extended until theyadopted a flat configuration. Rat b-MSCs between P4 and P6were seeded at density of 5 � 105 cells per scaffold by add-ing 100 lL of cellular suspension on the side of the scaffoldwith the microfibers (n ¼ 3 � 2). As the seeding solutionwas leaking from the scaffold after few minute, this solutionwas pipetted and added again on the top of the scaffold ev-ery 15 min. Every 30 min, 50 lL of culture medium wasadded. After 4 h, the scaffolds were transferred into another6-well-plate and 5 mL of culture medium was added to thewell, the medium being changed every second day. The cellviability was assessed using the Alamar Blue test accordingto the same procedure described in ‘‘Cell Metabolic Activity’’

FIGURE 1. Fixation of the knitted scaffold onto the 20 cm diameter

rotating drum. The collector was rotating at 1500 rpm and electrospin-

ning was performed. Electrospun fibers can be observed on the top

of the knitted scaffold.

1272 VAQUETTE ET AL. NOVEL COMPOSITE KNITTED/ALIGNED MICROFIBERS SCAFFOLDS

section. However, due to the sample size (4 cm long and 1.5cm wide), a 5% Alamar Blue solution was used and 5 mLwere added to the well to completely cover the scaffold.

The cell adhesion and proliferation were determined bymeasuring the DNA content of each scaffold 4 h after seed-ing and at day 14 of culture. Cellularised scaffolds wererinsed in PBS and placed in an eppendorf tube containing 1mL of a papainase lysis solution (1 mg/mL papainase, 100mM sodium phosphate, 5 mM EDTA dissodium salt, 5 mML-cysteine at pH 6.5 (Sigma Aldrich, Australia)). Cells weredigested at 60�C for 20 h. Then 20 lL of this solution wasstained with 80 lL of a 1/1000 Hoescht 33,342 solution.The excitation wavelength was 355 nm and the fluorescenceemission was collected at 460 nm with a plate reader (Spec-tramax M5, Molecular devices, Australia). The DNA standardcurve was obtained using calf thymus DNA (Sigma-Aldrich).To measure the cell seeding efficacy, three 25 cm2 cultureflask were seeded by 5 � 105 rat b-MSCs as a control andthe DNA was measured according to the above describedprocedure with the exception that 3 mL of papainase solu-tion was used. The efficacy of the cell seeding on the scaf-fold was determined by calculating the ratio of the DNAmeasured for the scaffold and the DNA measured for thecontrol.

Cell morphology was observed by SEM. The cellularsamples were fixed in a 2.5% glutaraldehyde solution andthen dehydrated in a concentration gradient of ethanol solu-

tions. The scaffolds were dried overnight in air and gold-palladium coated.

Immunostaining of collagen I and III. Immunostainingat day 7 and at day 14 was performed to detect collagentype I or type III produced by the cells onto the scaffold. Af-ter incubation in a blocking solution (PBS/BSA 0.5%) for 20min, scaffolds were incubated for 45 min in a solution con-taining the first antibody (dilution 1/50 in PBS; anti-rattype I collagen or anti-rat type III, both produced in rabbitand obtained from Calbiochem). After two washes in PBS,scaffolds were incubated for 45 min in a solution containingthe second antibody (dilution 1/50 in PBS; Alexa 488 anti-rabbit IgG, from Invitrogen). Scaffolds were rinsed twice inPBS and kept at 4�C. Scaffold observation was performedusing a confocal microscopy (LeicaSP2-AOBS).

RESULTS

Fibrous MembranesMorphology. The morphology of the fibrous PLCL mem-branes is shown in Figure 2. Electrospinning using a staticcollector produced randomly orientated microfibers [Fig.2(b)] whereas highly aligned microfibers were obtainedwhen the rotating cylinder was used [Fig. 2(a)]). Randomlyorientated mats were composed of 5.8 6 0.7 lm-diameterfibers [Fig. 2(d)]). Micropores were observed at their sur-face and they were most likely caused by solvent

FIGURE 2. Morphology of poly(L-lactic-co-e-caprolactone) electrospun microfibers. (a) and (c) aligned microfibers obtained at a rotating speed of

1500 rpm showing a high level of alignment, (b) and (d) random microfibers. SEM-observation.

ORIGINAL ARTICLE


evaporation. The aligned microfibers had a smaller diameterof 2.6 6 0.3 lm [Fig. 2(c)]). Their surface presented micro-roughness but no micropores due to solvent evaporationcould be observed.

Biological evaluation. The biocompatibility of the mem-branes was evaluated and the results are shown in Figure 3.The percentage of reduction, related to the cell activity,stayed around 5%, during the first 3 days of culture forboth the control and fibrous membranes. At day 7, a signifi-cant increase in the percentage of reduction was observedindicating that cells initiated proliferation. For the rest ofthe study, the cell activity observed for the control was sig-nificantly higher than for the fibrous membranes. Whenaligned and random microfibers were compared it wasfound that aligned microfibers promoted higher cell activity.From days 7 to 14, an increase in the cellular activity wasobserved until the percentage of reduction reached 70% forthe control and around 30% for the fibrous membranes.

The morphology of the adhered cells to the electrospunmembranes was investigated by SEM and the results areshown in Figure 4. After 3 days of culture, cell islets couldbe observed on the randomly orientated microfibers [Fig.4(a,b)]. Cells were well spread on the microfibers, and theyshowed a healthy morphology. From day 7, cells started tocolonize the membranes as shown in Figure 4(c) and after14 days of culture they covered the whole surface confirm-ing that cell proliferation occurred. Some cells were seen tomigrate under the first layers of microfiber at the surface ofthe membrane [Fig. 4(d)]. This migration could be attrib-uted to the small size of the rat b-MSC (tenth of micro-meters) compared to the large pore size of the of the micro-fibrous membrane (several tenths of micrometers).

At day 1, on the aligned microfibers, cells adopted aspindle-like morphology and were spontaneously orientatedalong the microfibers [Fig. 4(e)]. Long pseudopods, covering

the surface of some aligned microfibers, were also observed[Fig. 4(e), black oval form]. From day 1 to day 14, a sheetcomposed of aligned cells was formed [Fig. 4(f)]. Alignedcells presented a very rough membrane [Fig. 5(a,b)] charac-teristic of matrix production and secretion. Indeed, severalvesicles and extracellular fibers could be observed after sev-eral days of culture. This particular morphology was notobserved in cells cultured on random microfibers.

Composite scaffoldsMorphology. Brush-coating was performed to enhance theadhesion of the microfibers on the knitted structure. Thisresulted in a thin sheet of PLCL covering the sutures asshown in Figure 6. However, the morphology of the coatingwas different depending on the type of sutures. On thePLGA sutures (Vicryl), the PLCL coating was a thin and con-tinuous porous sheet of polymer [Fig. 6(a,b)], whereas onthe silk suture, the coating was smooth and cracks could beobserved between the filaments of the suture [Fig. 6(c,d)].

The morphology of the composite knitted scaffolds/microfibers was studied by optical microscopy since elec-tronic microscopy did not allow the simultaneous observa-tion of the fibrous mat and of the sutures. The compositescaffolds were composed of aligned PLCL-microfibers depos-ited on the surface of the knitted structure. The level ofmicrofiber alignment on the composite scaffold was lowercompared to the aligned microfibers created by the rotatingmandrel without knitted scaffolds [Fig. 2(a)]. This suggestedthat the knitted scaffold disturbed the polymer jet and thedeposition stability. However, the space between the loopsformed by the sutures was filled by the aligned microfibers[Fig. 7(a)]. Figure 7(b) shows the microfibers adhesion siteson the knitted structures, mostly located on the top of thestitches. Wet microfibers were able to adhere to the PLCL-coating as proved by the presence of the white zones on thetop of the sutures. Manual stretching of the composite

FIGURE 3. In vitro biocompatibility of rat mesenchymal stem cells on poly(L-lactic-co-e-caprolactone) electrospun membranes. þ, *, and # repre-

sent statistical difference with the previous measure. Bars show the statistical difference between the different groups.


showed that the aligned microfibers were elongated alongwith the knitted structure, indicating good adhesion to theknitted scaffold. No microfiber detachment occurred duringthis manual stretching. Thus, these composite scaffolds arepromising for ligament tissue engineering since they closelymimicked the morphology of the native tissue.

Mechanical evaluation. Tensile tests were carried out toevaluate the impact of both PLCL-brush-coating and micro-fibers deposition. The tensile stress–strain curves are shownin Figure 8. Three zones could be distinguished. In the firstzone, referred as to the ‘‘toe region,’’ the sample is stretched

up to 7.5% strain without stress increase. This is character-istic of collagen-rich tissue. A second zone was observed, inwhich the stress increased linearly with the strain, knownas linear region. Finally, in the third zone, structure damagewas observed. The composite scaffolds demonstrated lowerultimate tensile strength and higher ultimate tensile strainwhen compared with untreated scaffolds. However, until7.5% strain the tensile behavior of the composite scaffoldswith and without microfibers was similar (Fig. 8). Micro-fiber detachment occurred at 12–15% strain. Below thisstrain, the fibrous mats stayed fairly well adhered to theknitted scaffolds. Table I shows the tensile properties

FIGURE 4. Morphology of rat mesenchymal stem cells seeded on poly(L-lactic-co-e-caprolactone) electrospun membranes. Random microfibers

(a) day 1 (b) day 3, (c) day 7, (d) day 14, (e) aligned microfibers day 1, (f) aligned microfibers day 14. Arrows show the cell orientation along the

microfibers and the black oval indicates the presence of long pseudopods covering the microfibers. SEM observation.

ORIGINAL ARTICLE


determined from the stress–strain curves. The ultimate strainand stress for the knitted structures was 32% and 46 MParespectively. Composite scaffolds showed significantly higherultimate strain (39%) and lower ultimate stress (42 MPa).The elastic modulus was higher for the untreated knittedscaffolds (200 MPa) than for the composite (150 MPa).

Biological evaluation. Our claim was that the deposition ofthe aligned microfibers would facilitate the cell seeding on

the highly porous knitted scaffold and improved its efficacy.When an equivalent number of cells were seeded onto a 25cm2 culture flask, the DNA content measured was 3.5 6 0.1lg compared to 2.1 6 0.02 lg for the composite scaffold.The efficacy of the seeding for our scaffold was 62%.

Figure 9(a) shows the evolution of the percentage ofreduction of the Alamar Blue related to cell activity. Thepercentage of reduction increased over the 14 days ofin vitro culture. At day 1 the percentage of reduction was

FIGURE 5. Extracellular matrix secretion induced by spontaneous cells alignment. Extracellular fibers and vesicles were found on the aligned

cells. SEM-observation. a) 3600� b) 7450�.

FIGURE 6. Morphology of the polymer 2.5% poly(L-lactic-co-e-caprolactone) coating performed on knitted scaffolds. (a) and (b) poly(L-lactic-co-

glycolic) acid scaffold, (c) and (d) silk scaffold. SEM-observation.


17% and significantly increased up to 23% at day 14, indi-cating higher metabolic activity and/or cell proliferation.Figure 9(b) shows the total DNA content measured on thescaffold at day 0 and at day 14. After 4 h after seeding,around 2.1 lg of DNA was measured on the scaffold, andsignificantly increased up to 21 6 0.5 lg at day 14, indicat-ing a high level of cell proliferation. The apparent discrep-ancy between the Alamar Blue result, showing a moderate,but still significant increase in the cell activity, and the levelof proliferation is explained by the fact that cells from dif-ferent rats were used in these separate experiments leadingto different proliferation rate. However, in these both inde-pendent experiments, cells were proliferating and the levelof cellular activity increased over time. Proliferation wasfurther confirmed by SEM-observations showing the devel-opment of a cell sheet covering the scaffold (Fig. 10). At day1, the cells adhered to the microfibers and formed a discon-tinuous cell sheet that did cover the major part of scaffoldsurface [Fig. 10(a,b)]. Most of the cells were orientated inthe direction of the microfibers [Fig. 10(b)]. At day 3, thecells sheet had grown to cover completely the scaffold sur-face [Fig. 10(c)]. At this time, the cells showed a high levelof orientation [double black arrow, Fig. 10(d)]. From day 3to day 14 no significant changes could be observed, as thescaffolds were colonized by a thick sheet of orientated cells

[Fig. 10(d,e)]. Throughout these experiments, the cells hadrough membranes indicating high level of extracellular ma-trix production [Fig. 11(a)]. Figure 11(b) shows that cellswere secreting and forming extracellular fibers of severalmicrometers in length. Figure 12 shows the immunostainingof collagen type I and type III. Cells seeded on tissue cultureplastic produced some collagen type I, although in smallamount as revealed by the low level of fluorescence in Fig-ure 12(a). However, cells seeded on the aligned microfib-ers/knitted scaffolds, produced a higher level of collagentype I as shown by the intense fluorescence at day 7 [Fig.12(c)]. At day 14 more collagen type I was secreted andglobular and fiber-like structures were observed on thescaffolds. The production of collagen type III followed iden-tical trend, that is a gradual increase in the protein produc-tion as shown in Fig. 12(d,f). Indeed, more collagen seemedto be secreted at day 14 compared to day 7. In addition,collagen type III started to form a highly organized networkas small fibers were observed as shown in Fig. 12(f) indi-cating a high level of in vitro tissue maturation. The controldid not show any presence of collagen type III showing thatthe aligned microfibers had a morphological surface signal-ing that induced an up-regulation in the production of colla-gen type III.

DISCUSSION

Fibrous membranesExperimental set-up. The use of a rotating mandrel to alignmicrofibers was developed recently by Theron et al. whichwere among the first to use this kind of technology.35 Adecrease in the aligned microfiber diameter compared tothe microfibers electrospun on a static collector is com-monly reported. This is caused by the additional microfiber

FIGURE 7. Morphology of the composite scaffolds. (a) overview, (b) detail. Microfibers were highly aligned and they were able to adhere onto

the external side of the knitted meshes. Optical microscopy.

FIGURE 8. Tensile behavior of silk knitted scaffolds and silk compos-

ite scaffolds. Until 7.5% strain the behavior of both scaffold types

remained similar.

TABLE I. Mechanical Properties of Knitted and Composite

Scaffolds.

KnittedScaffolds

CompositeScaffolds

Ultimate strain [%] 32 6 3 39 6 2Ultimate stress [MPa] 46 6 4 42 6 3Modulus of elasticity [MPa] 202 6 12 150 6 14

All values were significantly different (p < 0.05).

ORIGINAL ARTICLE


elongation applied by the rotating mandrel producing adecrease in the microfiber diameter.39,40 In our experimentthe microfiber diameter had a twofold decrease, which isconsistent with Yang et al.39 The solvent evaporation holesencountered on the randomly orientated microfibers surfacewere stretched during the alignment giving the specific sur-face morphology that was observed on the aligned microfib-ers [Fig. 2(c)].

Biological evaluation. The biological evaluation showed anincrease in the Alamar Blue percentage of reduction, thatcan be correlated to cell proliferation proving the nontoxic-ity of the PLCL-microfibrous membranes. The biocompatibil-ity of different aliphatic polyesters is discussed in severalother publications,39,41–44 which generally highlighted thehigh cell/fibrous scaffold interaction. Different groupsshowed higher cell affinity for fibrous membrane than forsolvent cast films. When comparing a polyurethane cast filmand nanofibers from the same material, Lee et al. provedthat more cells adhered to the fibrous mats 24 h after seed-ing.36 Similar observations were reported by other researchgroups.39,42 In our experiment the percentage of reductionwas higher for the control than for the microfibrous mem-branes. This can be attributed to higher hydrophobicity ofPLCL in comparison to the plastic culture plate as men-tioned in Zhang et al. study.45

In our study the percentage of reduction was higher forthe aligned microfibers than for the random microfibers(Fig. 3), indicating higher cell metabolism and/or prolifera-tion. Lee et al. showed that cells expressed higher levels ofcollagen when adhered to aligned PU microfibers36 and Xuet al. observed an increase in cell proliferation for alignedmicrofibers when compared with random microfibers.42

These two facts could explain the results observed in ourstudy. The higher cell metabolism on aligned microfibers wasconfirmed by the observation of an important extracellularsecretion (Fig. 5). In some cases, cell developed long extracel-lular components that encapsulated the microfibers [Fig.4(e)]. Yan et al. suspected that these filament-like structures,extending from the cells and attaching them to the nanofib-ers, could be focal contacts or focal adhesions.39 Thus, thealignment of the microfibers provides the guidance and con-trol of cell morphology, inducing a spontaneous cell orienta-tion as reported by different groups.36,39,46

This preliminary biological study assessed the use ofPLCL-microfibers in the fabrication of composite scaffoldsfor ligament tissue engineering.

Composite scaffoldsAs shown in Figures 10 and 11, the composite scaffoldsshowed similar morphology to ligament or tendon architec-tures, that is, aligned microfibers where bipolar spindle cellsare inserted.1 Thus, the composite scaffolds closely mimickedthe architecture of the tissue aiming to be reconstructed.

Microfibers adhesion. The fabrication technique developedin this study is innovative and allows for the production ofscaffolds possessing both the mechanical properties of knit-ted structures and the topographical characteristics ofaligned microfiber membranes, that is, high surface area pervolume for promoting cell adhesion and spontaneous cellorientation induced by the morphology of the substrate.One major problem encountered during this study was theadhesion of the microfibers to the knitted structure. Whenelectrospinning was performed on noncoated knitted scaf-folds, it resulted in very poor attachment of the microfibers.To overcome this limitation, a polymer brush-coating wasperformed and wet microfibers were deposited. The fibersadhered onto the coating before the solvent could entirelyevaporate, ensuring better adhesion on the knitted struc-ture. Thus, it seems essential to pretreat the knitted struc-tures to enhance microfiber adhesion and to obtain compos-ite scaffolds that can be used in a dynamic mechanicalenvironment, that is cyclic stretching. Even if the microfiberadhesion was enhanced by the fabrication method we uti-lized, the microfiber layer could be removed by manualpeeling. However, the microfibers did not detach whenstretching was applied, indicating that the bonding with theknitted structure was strong enough to withstand thisdynamic stimulation.

The materials utilized also seemed to affect microfiberadhesion. The material used to fabricate the microfibersshould be soft enough to avoid mechanical mismatch withthe knitted structure. Sahoo et al. developed a techniquewhich coated a knitted scaffold with relatively rigid poly-

FIGURE 9. Biological evaluation of the silk composite scaffold using

rat-mesenchymal stem cells. (a) cell metabolic activity on silk com-

posite scaffolds by Alamar Blue tests. (b) DNA content. The bars

show statistical difference with the previous measurement.


lactic-co-glycolic acid (PLGA (65/35)) random nanofibers.33

The microfiber adhesion issue was not addressed in theirwork but they most likely obtained a scaffold where themicrofibers would easily detach if the scaffold is stretched(due to the rigidity of the PLGA).

In our case, the incorporation of caprolactone units inthe rigid PLLA enhances its elasticity since the copolymerPLCL is composed of both hard and soft segments. He et al.showed that PLCL (70/30) behaved in a purely elasticmanor until 5% strain, assessing its use in our application.43

Mechanical mismatch could be reduced by using highly elas-tic materials such as PLCL (50/50)41 or other rubber likebiomaterials.

This technique for producing composite scaffolds can beextended to other materials since the mechanism of micro-fibers adhesion is not substrate-dependent but is rather de-pendent on the coating applied to the knitted structures.

Indeed, in this work Vicryl and silk composite scaffoldswere produced with similar morphology.

Mechanical properties. Knitted structures provide signifi-cant advantages for ligament tissue engineering as some oftheir mechanical properties are similar to the native tissue(toe region, multiple failures etc.).27,28,31,32 The compositescaffolds developed in this study possessed an elastic modu-lus of 150 MPa, which falls within the order of magnitudeof tendon and ligament modulus (50 to 100 MPa47).Because of their higher porosity compared to braided scaf-folds, cellular infiltration and tissue growth occurs easilywithin these structures after implantation.28 However, onedrawback comes from the low ultimate tensile force (50–250 N).32,33 This could be increased by using severalsutures simultaneously or stronger materials,27 and/orsutures of higher diameter. One can, as well, manipulate the

FIGURE 10. Morphology of composite scaffolds seeded with rat mesenchymal stem cells. (a) and (b) day 1, (c) and (d) day 3, (e) and (f) day 14.

From day 1 and day 3, cells colonized the composite scaffold and started to be orientated along the aligned microfibers. After 3 days of culture

cells were highly orientated as indicated by the black double arrow. The mesh organization of the knitted scaffolds could be clearly seen in

(a) and (b).

ORIGINAL ARTICLE


geometry of the sample. Liu et al. showed that the ultimateforce of their silk knitted scaffolds was around 250 N whenthe structures were 40 stitches wide.32 In our study thescaffolds were 12 stitches wide resulting in an ultimateforce of 70 N. This value can be increased by increasing thestitch number.

An important aspect of the composite scaffolds was themicrofiber adhesion during the tensile tests. The microfibersdid not detach until 12–15% strain, proving that they canbe used in cyclic elongation in a bioreactor where the strainis generally less than 10%48. Manual stretching was per-formed on the composite structures showing that microfib-ers were stretched along with the knitted structures indicat-ing that the mechanical stimulation will be fully experiencedby the cells.

Cell seeding. One of the key points for a successful tissueengineering strategy is the cell seeding and homogeneousdistribution. Hutmacher at al. reported difficulties encoun-tered when seeding highly 3D macroporous scaffolds49 asthe cells were not confined to the inside of the structurebut preferentially adhered to the plastic culture plate. It isalso a challenge to seed knitted structures for the same rea-son. Hutmacher et al. reported a seeding efficacy of only30% when seeding their scaffold. The use of microfibers tocover the void space between the knitted stitch has demon-strated its ability to improve the seeding efficacy. However,only 60% of cells were effectively seeded onto the microfib-ers. The seeding efficacy could be increased by reducing thevolume of cellular solution utilized in this experiment.Indeed, the solution was leaking from the scaffold resultingin a loss of cells. Several groups developed several techni-ques to bring cells inside the scaffold. Ouyang et al. used afibrin gel to encapsulate cells in the scaffolds.28 However,gas and nutrient diffusion, compulsory phenomena for via-ble implants, were strongly limited in these kinds of con-structs. Chen et al. developed a collagen fibrous net-work29,30 and Sahoo et al. deposited nanofibrous mats ontothe knitted structures and scaffolds could be seeded by sed-imentation technique (pipetting cell solution on the

FIGURE 11. Extracellular matrix production of aligned cells on the composite scaffolds. (a) arrows represent the cells alignment direction, (b)

presence of extracellular secretion and extracellular fibers. SEM-observation.

FIGURE 12. Immunostaining of the scaffold. (a), (c) and (e) collagen

type I, (a) representative image of cell seeded on tissue culture plas-

tic, (c) cell on scaffold at day 7, (e) scaffold at day 14. (b) (d), and (f)

collagen type III, (b) representative image of cell seeded on tissue cul-

ture plastic, (d) cell on scaffold at day 7, (f) scaffold at day 14. The

production of both collagen types increased over time. Collagen type

I formed a fibrous network after 14 days of in vitro culture. [Color fig-

ure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]


scaffold).33 We have also recently developed an alginate/knitted composite scaffold enabling easy and controlled cellseeding by cell encapsulation which could not be used in abioreactor due to the poor tensile strength of the alginategel.50 Ouyang et al. set up a technique, in which a cell sheetwas cultured until it could be mechanically detached fromthe culture plate and placed on the knitted structure.31 Theadvantage of this technique came from the development ofa natural extracellular matrix that could be further spatiallyorientated by mechanical stimulations. However, the seedingdensity was nearly impossible to control. In our study, cellsare easily and homogenously seeded by sedimentation withgood control over cell distribution as shown in Figure 10(a).Cells are indeed well distributed over a surface of severalmm2. Because of the specific topography of the composite,cells were spontaneously orientated in the direction of themicrofiber. Thus, two advantages are cumulated with theuse of composite scaffolds, easy seeding and cell alignmentwithout mechanical stimulation.

Tissue engineering is based on the ability of the cells tobuild their extracellular environment. In our study an im-portant extracellular matrix was observed when cells wereseeded on the composite scaffold. This was probably due tothe cell alignment. Lee et al. showed that cells onto alignedpolyurethane nanofibers, secreted more collagen than con-trol and random nanofibers.36 We observed similar trend,that is an increased production of collagen type I and typeIII on our scaffolds. Thus, cells started to develop their ownextracellular matrix without any external stimulation (i.e.,stretching) which is a positive phenomenon for a future im-plantation. As the aligned microfibers are adhered to theknitted structure, they could directly transmit the mechani-cal stimulus to the cell further enhancing the extracellularmatrix synthesis.48 These composite scaffolds will be usedin a bioreactor where a dynamic mechanical environmentwill be applied to enhance the production of extracellularmatrix

CONCLUSION

A composite scaffold composed by knitted structure andaligned PLCL microfibers was developed for potential use inligament and tendon tissue engineering. The innovative fab-rication technique involved electrospinning of aligned micro-fibers on knitted scaffolds. The void space between thestitches was covered by the microfibers allowing for easycell seeding by pipetting. We proved that the deposition ofthe microfibrous mat did not affect the shape of the tensilecurve and that some of the mechanical properties wereadequate for ligament tissue engineering. The biologicalevaluation showed that the composite scaffolds were non-toxic and cells were spontaneously orientated along themicrofibers making the seeded scaffolds very similar inmorphology to ligaments. Thus, an innovative scaffold fabri-cation technique was developed in our group, producinghighly organized structures possessing both the advantagesof knitted scaffolds and those of aligned microfiber mem-branes. This study discussed also the potential of these

composite scaffolds for a future use in a bioreactor for thedevelopment of a neo-ligament.

ACKNOWLEDGMENTS

The authors thank Mr. L. Marchal for the SEM imaging.

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DOI:10.1163/092050609X12560455246676.


Aligned poly(L-lactic-co-e-caprolactone) electrospun microfibers and knitted structure: A novel...

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