cartilage scaffolds

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Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage

scaffolds with hydrogen treated multi-walled carbon nanotubes

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2013 Nanotechnology 24 365102

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IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 24 (2013) 365102 (10pp) doi:10.1088/0957-4484/24/36/365102

Enhanced human bone marrow

mesenchymal stem cell functions in novel3D cartilage scaffolds with hydrogentreated multi-walled carbon nanotubesBenjamin Holmes 1, Nathan J Castro 1, Jian Li 1, Michael Keidar 1 andLijie Grace Zhang 1,2,3

1 Department of Mechanical and Aerospace Engineering, The George Washington University,Washington, DC 20052, USA2 Department of Medicine, The George Washington University, Washington, DC 20052, USA

E-mail: [email protected]

Received 20 February 2013, in nal form 14 June 2013Published 19 August 2013Online at stacks.iop.org/Nano/24/365102

AbstractCartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to itsextremely poor inherent regenerative capacity and complex stratied architecture. Currenttreatment methods are highly invasive and may have many complications. Thus, the goal of this work is to use nanomaterials and nano/microfabrication methods to create novel

biologically inspired tissue engineered cartilage scaffolds to facilitate human bone marrowmesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning todesign and fabricate a series of novel 3D biomimetic nanostructured scaffolds based onhydrogen (H 2) treated multi-walled carbon nanotubes (MWCNTs) and biocompatiblepoly(L-lactic acid) (PLLA) polymers. Specically, a series of electrospun brous PLLAscaffolds with controlled ber dimension were fabricated in this study. In vitro MSC studiesshowed that stem cells prefer to attach in the scaffolds with smaller ber diameter. Moreimportantly, the MWCNT embedded scaffolds showed a drastic increase in mechanicalstrength and a compressive Youngs modulus matching to natural cartilage. Furthermore, ourMSC differentiation results demonstrated that incorporation of the H 2 treated carbonnanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCsthan controls. After two weeks of culture, PLLA scaffolds with H 2 treated MWCNTs andpoly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promisingfor further exploration for cartilage regeneration.

(Some gures may appear in colour only in the online journal)

1. Introduction

Articular cartilage repair and regeneration continue to belargely intractable due to the poor regenerative propertiesof this tissue. Cartilage, unlike other self-repairing tissuessuch as bone, has a low regenerative capacity and,

3 Address for correspondence: 801 22nd Street NW, 726 Phillips Hall,Washington, DC 20052, USA.

consequently, once injured it is much less likely to self-heal.Even tiny cartilage defects seem permanently unhealed.Although various traditional therapies such as autograft,allograft and autologous chondrocyte implantation have beenperformed for many years, none of them is perfect due tolimited donor tissues and numerous complications associatedwith implantations. As an emerging interdisciplinary eld,regenerative medicine or tissue engineering aims to createliving and functional tissues or even organs via use of

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Nanotechnology 24 (2013) 365102 B Holmes et al

biomaterials, growth factor and stem cells and shows hugepotential for future cartilage regeneration [1, 2]. Specically,autologous human mesenchymal stem cells (MSCs) frombone marrow, which have easy accessibility (via the iliaccrest), high proliferative capacity, and multiple differentiationpotentials, are one of the most popular and promising cell

sources for various types of complex tissue regeneration.However, the current stem cell based cartilage regenerationis still in its infancy owing to its very low level of stem cell engraftment and survival in recipient tissue,induction of hypertrophy, and uncontrollable chondrogenicdifferentiation of stem cells [ 1, 36]. Since native stem cellsare facing a 3D porous nanostructured extracellular matrix(ECM) environment and are extremely sensitive to evenminute changes in their surrounding environment, creatinga biomimetic environment via nanobiomaterials and scaffoldfabrication techniques holds great potential in removingcurrent challenges and improving MSC chondrogenicdifferentiation and cartilage regeneration. Thus, the objectiveof this work is to use nanomaterials and electrospinning tocreate novel biologically inspired tissue engineered cartilagescaffolds with biomimetic nano- and microstructure andimproved mechanical properties for MSC chondrogenesis invitro .

In native cartilage tissue, the ECM forms naturallynano/microbrous structured environments that promote celladhesion, proliferation and differentiation [ 7, 8]. Therefore,it is important to construct a scaffold that mimics the scaleand structure of the ECM [ 9]. Electrospinning is such awell-established and popular scaffold fabrication method fortissue regeneration [ 1013]. It has been considered favorable

because of the ability of researchers to create biomimeticbrous polymer scaffolds on the micro- and nanoscale toimprove cell differentiation and proliferation [ 14]. However,it is difcult to get purely electrospun polymers truly onthe nanoscale, because of the limitations of electrospinningand due to the loss of structural and mechanical integrity of a scaffold with smaller ber dimensions. Because of theseconsiderations, more and more research has been carriedout to modify the surface characteristics of micro-scaleand submicron scaffold bers. Methods such as co-spinningner bers onto thicker support bers and the incorporationof inherently porous materials have been considered, but

an emerging fabrication method that has begun to gainpopularity is wet electrospinning [ 15, 16]. The process of wetelectrospinning basically entails a standard electrospinningsetup augmented with the collector plate submerged in amethanol bath. When the polymer bers are spun into thisbath, they crosslink and form a complex block co-polymericstructure that is highly porous. Shin et al wet electrospuna 3D poly(trimethylenecarbonate-co-epsilon-caprolactone)-block-co-poly(p-dioxanone) scaffold for bone regenerationthat was 90% porous and exhibited interconnection amongpores. This highly porous scaffold showed good cellularadhesion of osteoblasts at the center of the scaffold afteronly four days of in vitro cell seeding. The cells alsoproliferated 1.5 times faster than the control after seven days.In addition, the activity of the bone formation marker alkaline

phosphate was four times faster than the control at twentyeight days [ 15]. These results show that wet electrospunscaffolds can be a promising approach to creating scaffoldsthat are advantageous for tissue growth. Thus, we will use thisapproach for our study.

More importantly, nanomaterials with biomimetic fea-

tures and excellent physicochemical properties are promis-ing in cartilage regeneration, although related studies areextremely limited [11, 17, 18]. For this purpose, carbonnanotubes are emerging candidates. Carbon nanotubes mimicthe dimensions of the constituent components of tissues,where cells are accustomed to interacting with nanobrousproteins. This property makes them excellent candidatesfor invoking positive cellular responses when employedas implants [19, 20]. In addition, the superior mechanicalproperties of carbon nanotubes are efcient for their usage asa secondary phase for high load bearing such as cartilage andbone applications [21]. Their electrical properties make thema potential choice in neural applications where signal transfer

between growing axons necessitates electrical conductance.The unique chemical properties they possess permit themto be functionalized with different chemical groups, whichfurther promote cell growth.

In this study, by integrating wet electrospinning andnanomaterials (i.e., hydrogen treated multi-walled carbonnanotubes, MWCNTs), we will engineer and evaluate aseries of novel biomimetic cartilage constructs with nano- tomicrostructure. We will investigate whether the mechanicaland cytocompatibility properties of the electrospun cartilagescaffolds can be enhanced, with the addition of carbonnanomaterials. It was also a goal to evaluate whetherthe nanoscaffolds modied with a cell-favorable molecule(i.e., poly-L-lysine) can effectively control chondrogenicdifferentiation of MSCs in vitro .

2. Materials and methods

2.1. Hydrogen (H 2) treated and non-treated multi-walled carbon nanotubes

As-synthesized MWCNTs were obtained from ShanghaiXinxing Chenrong Technology Development Co., Ltd. TheMWCNTs were synthesized by the oating-catalyst techniquein a chemical vapor deposition process. Dimethylbenzene(C8H10) and thiophene (C 4H4S) served as carbon sources

and the iron atom from ferrocene (Fe(C5H5)2) was used ascatalyst for the growth of MWCNTs. The synthesis processeswere carried out in a cylindrical chamber at a temperatureof 1100 C in hydrogen environment. The as-synthesizedMWCNTs were further treated by H 2 heating. The process forH2 heating treatment is to place the as-synthesized MWCNTsin a mixture of hydrogen and nitrogen environment at atemperature of 800 C for 2 h and then turn off the hydrogensupply and cool down the samples naturally. The H 2 treatmentremoves amorphous carbon and nanohorns encapsulatingmetal catalyst nanoparticles and MWCNTs, making thetubes more uniform. The morphologies of these tubes, bothtreated and untreated, were evaluated using scanning electronmicroscopy (SEM) and transmission electron microscopy(TEM).

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Figure 1. The laboratorys electrospinning setup.

2.2. Electrospun brous scaffold fabrication

For all experiments, biocompatible poly-L-lactic acid (PLLA)purchased from Sigma Aldrich was used as the base polymerto be electrospun. Fibers were fabricated using an in housesetup, consisting of a syringe pump, a Harvard Apparatus

variable voltage supply and an aluminum collector plate(gure 1). For the scaffold preparation for MSC adhesionstudy, PLLA was dissolved at 18% weight by volumein a 9 to 1 solution of dichloromethane (DCM) anddimethylformaldehyde (DMF). DMF evaporates at a highertemperature than DCM, and was thus postulated to induceporosity. All adhesion study scaffolds were electrospun at 12,14, 16, 18 and 20 cm working distance from the collectorplate, at voltages varying from 14 to 18 kilovolts (kV) in orderto create a series of brous scaffolds with different diameters.

The proliferation and differentiation study used PLLAdissolved in pure DCM at 18% weight by volume, which wasthen wet electrospun into a coagulation bath of methanol. Theoptimized working distance (i.e., 18 cm, see sections 2 and3) evaluated above was used here. More importantly, PLLAscaffolds were electrospun with solutions containing 0.5%w/v untreated MWCNTs, 0.5% w/v H 2 treated MWCNTs and1% w/v H2 treated MWCNTs. All samples were mixed usingultrasonication for 75 min, with the nanotubes being sonicatedin the solvent rst for 45 min in order to assure uniformdistribution. The scaffolds in all studies were on average 2 mmin thickness.

2.3. Scaffold characterization

All of the electrospun samples were sputter-coated with a thinlayer (9 nm) of goldpalladium, and were then imaged using aZeiss SigmaVP SEMat 5 kV accelerating voltage. In addition,the compressive mechanical properties of all scaffolds wereevaluated via an ATS axial tester, a 50 Newton load cell and acompression placard, under dry conditions. Circular samplesof 8 mm in diameter and about 12 mm in height were takenand tested in compression, at a strain rate of 0 .2 mm s 1.The forcedeformation data were then used to calculate andcompare the Youngs modulus of each sample.

2.4. MSC function study in vitro

2.4.1. Primary MSC culture. Primary human bonemarrow MSCs were obtained from 20 ml aspirates from

a healthy consenting donors iliac crest (female; age 27)from the Texas A&M Health Science Center, Institute forRegenerative Medicine and thoroughly characterized [22].Briey, the mononuclear cells were separated using densitycentrifugation. The cells were plated, expanded, harvestedand frozen at passage 1 (P1). Two trials of the frozen

P1 cells were analyzed over three passages for colonyforming units, cell growth, and differentiation into fat andbone. Flow cytometry results for various cell markers andinfectious agents blood screen were evaluated as well. Thecharacterized cellswere used to evaluate the cytocompatibilityproperties of the electrospun nanocomposite. MSCs (passage#36) were cultured in a complete medium comprisedof Alpha Minimum Essential medium ( -MEM, Gibco,Grand Island, NY) supplemented with 16.5% fetal bovineserum (Atlanta Biologicals, Lawrenceville, GA), 1% (v/v)L-glutamine (Invitrogen, Carlsbad, CA), and 1% penicillin:streptomycin solution (Invitrogen, Carlsbad, CA) and cultured

under standard cell culture conditions (37

C, a humidied,5% CO2 /95% air environment). For the differentiation study,chondrogenic medium was prepared, which consisted of theabove medium recipe, but with the addition of 100 nMdexamethasone, 40 g ml 1 proline, 100 g ml 1 sodiumpyruvate, 50 g ml 1 L-ascorbic acid 2-phosphate, ITS + ata concentration of 1% of the total volume of preparedmedium, and 10 ng ml 1 TGF- 1 during the rst three days.The medium was supplemented with 100 ng ml 1 IGF-Ithroughout the experimental period. All electrospun scaffoldswere sterilized via exposure to ultraviolet light for 15 min,ipped and exposed for another 15 min, and then rinsed withPBS prior to cell experiments.

2.4.2. MSC adhesion study in vitro. In order to evaluatethe diameter effects on MSC attachment, sample groupsconsisting of circular dry spun pure PLLA scaffold, fabricatedat working distances of 12, 14, 16, 18, and 20 cm, were thenseeded with MSCs at 10 000 cells per scaffold and allowed toincubate for 4 h. Cells were then lifted and counted using ahemocytometer and light microscope.

2.4.3. MSC proliferation study in vitro. Sample groupsconsisting of a pure PLLA control, PLLA scaffold containing0.5% w/v untreated MWCNTs, PLLA scaffold containing0.5% w/v H 2 treated MWCNTsand PLLA scaffold containing1.0% w/v H 2 treated MWCNTs were then seeded with MSCsat 10 000 cells per scaffold. All sample groups were fabricatedusing wet electrospinning. Samples were then cultured for1, 3 and 5 days. After the prescribed time periods, thescaffolds were rinsed using PBS to remove non-adherentcells. The adherent cells were lifted via trypsinization, i.e.,0.25% trypsin/1 mM EDTA solution for 3 min at roomtemperature. The cell proliferation was quantied via aCellTiter 96 R AQueous Non-Radioactive Cell ProliferationAssay (MTS assay) and analyzed using a Thermo ScienticMultiskan GO spectrophotometer at a setting of 490 nmwavelength light.

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Figure 2. SEM images of varying electrospun brous PLLA scaffolds at 12, 14, 16, 18 and 20 cm working distance ((A)(E)), and 18 cmat a lower magnication (F).

on the surface [15], which make the wet spun scaffoldssurface morphology similar to that of DCM/DMF dry spunscaffolds. Figure 6 shows the SEM images of variouswet electrospun MWCNT embedded PLLA scaffolds. Nosingle nanotubes could be directly observed, implying thatthey were embedded in the bers. It could also be seen

that the ber diameters varied in the MWCNT embeddedscaffolds. The ber diameter distribution was from about 1to 10 m. This effect, which occurs when nanomaterials areelectrospun in solution with a polymer, is documented in theliterature [ 2830].

More importantly, after the addition of MWCNTs of both species, the PLLA scaffolds Youngs modulus increaseddramatically when compared to a pure PLLA control(gure 7). Moreover, all MWCNT reinforced scaffolds werewithin the range of native articulate cartilage ( 0.752 MPadepending on location) [ 3134]. This shows that theincorporation of just a small amount of MWCNTs can greatly

increase the mechanical properties of a tissue engineeringscaffold to within biomimetic regimes.

Figure 3. Enhanced MSC attachment on the electrospun PLLAscaffold with the smallest ber diameter (1 .33 m). Data aremean SEM, n = 9; p < 0.1 when compared to all other samplesand p < 0.1 when compared to the scaffold with 1 .67 m berdiameter.

3.3. MSC proliferation and chondrogenic differentiation invitro

The proliferation study showed an increase in cellularproliferation on all scaffolds, with the greatest cell numbers

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Figure 4. SEM images of (A) MWCNTs and (B) H 2 treated MWCNTs, and TEM images of (C) MWCNTs and (D) H 2 treated MWCNTs.

Figure 5. SEM images of (A) and (B) pure PLLA scaffold prepared by normal dry electrospinning at low and high magnications, and (C)and (D) pure PLLA scaffold prepared by wet electrospinning with increased porosity at low and high magnications.

on the scaffolds with incorporated 0.5% MWCNTs of bothspecies at three days (gure 8). At ve days, all of theMWCNT PLLA scaffolds showed greater cellular growththan controls, which indicated the good cytocompatibility

properties of these nanostructured scaffolds for cell growth.It is known that mechanical factors such as scaffold stiffnesscan have signicant inuence on regulating stem cellactivities [35]. It is possible that our 0.5% MWCNT scaffolds,

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Figure 6. SEM images of varying electrospun brous PLLA scaffolds with (A) 0.5% MWCNTs, (B) 0.5% H 2 treated MWCNTs, and (C)1.0% H2 treated MWCNTs. (D) High magnication of the PLLA scaffold with 0.5% MWCNTs (image A), which shows the nanoporosityof the scaffold.

Figure 7. Signicantly enhanced compressive Youngs modulus of electrospun PLLA scaffolds with MWCNTs when compared toPLLA control ( p < 0.05 and p < 0.05). The modulus of thenanocomposites matches to natural cartilage.

due to their increase in mechanical strength, could provide astiffer surface for better stem cell growth and proliferation.In addition, both of the nanotube species may change thenanosurface roughness and surface area of the scaffolds, thusfurther contributing to the improved MSC proliferation afterve days when compared to controls.

Moreover, the differentiation study showed increasedchondrogenic differentiation activity of MSCs in thesenanostructured scaffolds in vitro (gures 9 and 10). There wasa dramatic increase in GAG content at one and two weeks onthe scaffolds containing poly-L-lysine coated MWCNT, andamong these samples the H 2 treated tubes performed the best(gure 9). The positively charged poly-L-lysine can create

Figure 8. MSC proliferation on various electrospunPLLA/MWCNT scaffolds. Data are mean SEM; n = 9. p < 0.05 when compared to controls at day 5; p < 0.05 whencompared to all other scaffolds at day 3.

an electrostatic interaction with negatively charged GAG forimproved GAG nucleation in the scaffold. This implies thatthe surface coating of the nanotube scaffold (to decreasehydrophobicity) had the greatest impact on GAG synthesisin vitro , greater than the nanosurface topography contributionof the MWCNTs in our study. Furthermore, gure 10reveals that both H 2 treated MWCNT and poly-L-lysinecoated MWCNT PLLA scaffolds can signicantly improvetotal collagen synthesis after one and two weeks. The factthat the H2 treated tubes yielded better results shows thatthe purication of nanotubes to remove various impuritiesand modify the nanotube morphology is advantageous for

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Figure 11. Schematic illustration of MSC differentiation andcartilage formation in three electrospun PLLA scaffolds. Darkerblue represents more cartilage matrix formation. Not drawn to scale.

In this study, a series of biomimetic electrospun brousnanoscaffolds with controlled ber dimension and surfacenanoporosity were fabricated. When compared to a pure

PLLA control, scaffolds with MWCNTs showed no adverseeffect on MSC proliferation, and more importantly displayedenhanced MSC chondrogenesis when modied with achemical factor. The puried H 2 heated nanotubes alsoshowed enhanced differentiation over scaffolds containinguntreated tubes. In addition scaffolds with incorporatednanotubes showed signicantly improved mechanical prop-erties when compared to a PLLA control. This demon-strates that MWCNTs can be used to greatly improvethe strength of electrospun polymer bers, without havingan adverse effect on cellular activity. Overall, the datapresented display that electrospun PLLA scaffolds withincorporated, surface modied MWCNTs and poly-L-lyisnecoating can be suitable scaffolds for cartilage regenera-tion.

Acknowledgments

The authors are grateful for a Research Award from theClinical and Translational Science Institute at Childrens Na-tional (CTSI-CN) and support from the George WashingtonUniversity Institute for Nanotechnology (GWIN). We are alsograteful for the imaging support from the George Washington

University Center for Microscopy and Image Analysis and theNational Institute of Standards and Technology.

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