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Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes

This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2013 Nanotechnology 24 365102 (http://iopscience.iop.org/0957-4484/24/36/365102) View the table of contents for this issue, or go to the journal homepage for more

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IOP PUBLISHING Nanotechnology 24 (2013) 365102 (10pp)

NANOTECHNOLOGY

doi:10.1088/0957-4484/24/36/365102

Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes
Benjamin Holmes1 , Nathan J Castro1 , Jian Li1 , Michael Keidar1 and Lijie Grace Zhang1,2,3
1

Department of Mechanical and Aerospace Engineering, The George Washington University, Washington, DC 20052, USA 2 Department of Medicine, The George Washington University, Washington, DC 20052, USA E-mail: lgzhang@gwu.edu

Received 20 February 2013, in nal form 14 June 2013 Published 19 August 2013 Online at stacks.iop.org/Nano/24/365102 Abstract Cartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to its extremely poor inherent regenerative capacity and complex stratied architecture. Current treatment 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 marrow mesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning to design and fabricate a series of novel 3D biomimetic nanostructured scaffolds based on hydrogen (H2 ) treated multi-walled carbon nanotubes (MWCNTs) and biocompatible poly(L-lactic acid) (PLLA) polymers. Specically, a series of electrospun brous PLLA scaffolds with controlled ber dimension were fabricated in this study. In vitro MSC studies showed that stem cells prefer to attach in the scaffolds with smaller ber diameter. More importantly, the MWCNT embedded scaffolds showed a drastic increase in mechanical strength and a compressive Youngs modulus matching to natural cartilage. Furthermore, our MSC differentiation results demonstrated that incorporation of the H2 treated carbon nanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCs than controls. After two weeks of culture, PLLA scaffolds with H2 treated MWCNTs and poly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promising for 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 be largely intractable due to the poor regenerative properties of this tissue. Cartilage, unlike other self-repairing tissues such as bone, has a low regenerative capacity and,
3 Address for correspondence: 801 22nd Street NW, 726 Phillips Hall,

Washington, DC 20052, USA. 0957-4484/13/365102+10$33.00 1

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 been performed for many years, none of them is perfect due to limited donor tissues and numerous complications associated with implantations. As an emerging interdisciplinary eld, regenerative medicine or tissue engineering aims to create living and functional tissues or even organs via use of
c 2013 IOP Publishing Ltd Printed in the UK & the USA

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biomaterials, growth factor and stem cells and shows huge potential for future cartilage regeneration [1, 2]. Specically, autologous human mesenchymal stem cells (MSCs) from bone marrow, which have easy accessibility (via the iliac crest), high proliferative capacity, and multiple differentiation potentials, are one of the most popular and promising cell sources for various types of complex tissue regeneration. However, the current stem cell based cartilage regeneration is still in its infancy owing to its very low level of stem cell engraftment and survival in recipient tissue, induction of hypertrophy, and uncontrollable chondrogenic differentiation of stem cells [1, 36]. Since native stem cells are facing a 3D porous nanostructured extracellular matrix (ECM) environment and are extremely sensitive to even minute changes in their surrounding environment, creating a biomimetic environment via nanobiomaterials and scaffold fabrication techniques holds great potential in removing current challenges and improving MSC chondrogenic differentiation and cartilage regeneration. Thus, the objective of this work is to use nanomaterials and electrospinning to create novel biologically inspired tissue engineered cartilage scaffolds with biomimetic nano- and microstructure and improved mechanical properties for MSC chondrogenesis in vitro. In native cartilage tissue, the ECM forms naturally nano/microbrous structured environments that promote cell adhesion, proliferation and differentiation [7, 8]. Therefore, it is important to construct a scaffold that mimics the scale and structure of the ECM [9]. Electrospinning is such a well-established and popular scaffold fabrication method for tissue regeneration [1013]. It has been considered favorable because of the ability of researchers to create biomimetic brous polymer scaffolds on the micro- and nanoscale to improve cell differentiation and proliferation [14]. However, it is difcult to get purely electrospun polymers truly on the nanoscale, because of the limitations of electrospinning and due to the loss of structural and mechanical integrity of a scaffold with smaller ber dimensions. Because of these considerations, more and more research has been carried out to modify the surface characteristics of micro-scale and submicron scaffold bers. Methods such as co-spinning ner bers onto thicker support bers and the incorporation of inherently porous materials have been considered, but an emerging fabrication method that has begun to gain popularity is wet electrospinning [15, 16]. The process of wet electrospinning basically entails a standard electrospinning setup augmented with the collector plate submerged in a methanol bath. When the polymer bers are spun into this bath, they crosslink and form a complex block co-polymeric structure that is highly porous. Shin et al wet electrospun a 3D poly(trimethylenecarbonate-co-epsilon-caprolactone)block-co-poly(p-dioxanone) scaffold for bone regeneration that was 90% porous and exhibited interconnection among pores. This highly porous scaffold showed good cellular adhesion of osteoblasts at the center of the scaffold after only four days of in vitro cell seeding. The cells also proliferated 1.5 times faster than the control after seven days. In addition, the activity of the bone formation marker alkaline
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phosphate was four times faster than the control at twenty eight days [15]. These results show that wet electrospun scaffolds can be a promising approach to creating scaffolds that are advantageous for tissue growth. Thus, we will use this approach for our study. More importantly, nanomaterials with biomimetic features and excellent physicochemical properties are promising in cartilage regeneration, although related studies are extremely limited [11, 17, 18]. For this purpose, carbon nanotubes are emerging candidates. Carbon nanotubes mimic the dimensions of the constituent components of tissues, where cells are accustomed to interacting with nanobrous proteins. This property makes them excellent candidates for invoking positive cellular responses when employed as implants [19, 20]. In addition, the superior mechanical properties of carbon nanotubes are efcient for their usage as a secondary phase for high load bearing such as cartilage and bone applications [21]. Their electrical properties make them a potential choice in neural applications where signal transfer between growing axons necessitates electrical conductance. The unique chemical properties they possess permit them to be functionalized with different chemical groups, which further promote cell growth. In this study, by integrating wet electrospinning and nanomaterials (i.e., hydrogen treated multi-walled carbon nanotubes, MWCNTs), we will engineer and evaluate a series of novel biomimetic cartilage constructs with nano- to microstructure. We will investigate whether the mechanical and cytocompatibility properties of the electrospun cartilage scaffolds can be enhanced, with the addition of carbon nanomaterials. It was also a goal to evaluate whether the nanoscaffolds modied with a cell-favorable molecule (i.e., poly-L-lysine) can effectively control chondrogenic differentiation of MSCs in vitro.

2. Materials and methods

2.1. Hydrogen (H2 ) treated and non-treated multi-walled carbon nanotubes As-synthesized MWCNTs were obtained from Shanghai Xinxing Chenrong Technology Development Co., Ltd. The MWCNTs were synthesized by the oating-catalyst technique in a chemical vapor deposition process. Dimethylbenzene (C8 H10 ) and thiophene (C4 H4 S) served as carbon sources and the iron atom from ferrocene (Fe(C5 H5 )2 ) was used as catalyst for the growth of MWCNTs. The synthesis processes were carried out in a cylindrical chamber at a temperature of 1100 C in hydrogen environment. The as-synthesized MWCNTs were further treated by H2 heating. The process for H2 heating treatment is to place the as-synthesized MWCNTs in a mixture of hydrogen and nitrogen environment at a temperature of 800 C for 2 h and then turn off the hydrogen supply and cool down the samples naturally. The H2 treatment removes amorphous carbon and nanohorns encapsulating metal catalyst nanoparticles and MWCNTs, making the tubes more uniform. The morphologies of these tubes, both treated and untreated, were evaluated using scanning electron microscopy (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 polymer to be electrospun. Fibers were fabricated using an in house setup, consisting of a syringe pump, a Harvard Apparatus variable voltage supply and an aluminum collector plate (gure 1). For the scaffold preparation for MSC adhesion study, PLLA was dissolved at 18% weight by volume in a 9 to 1 solution of dichloromethane (DCM) and dimethylformaldehyde (DMF). DMF evaporates at a higher temperature than DCM, and was thus postulated to induce porosity. All adhesion study scaffolds were electrospun at 12, 14, 16, 18 and 20 cm working distance from the collector plate, at voltages varying from 14 to 18 kilovolts (kV) in order to create a series of brous scaffolds with different diameters. The proliferation and differentiation study used PLLA dissolved in pure DCM at 18% weight by volume, which was then wet electrospun into a coagulation bath of methanol. The optimized working distance (i.e., 18 cm, see sections 2 and 3) evaluated above was used here. More importantly, PLLA scaffolds were electrospun with solutions containing 0.5% w/v untreated MWCNTs, 0.5% w/v H2 treated MWCNTs and 1% w/v H2 treated MWCNTs. All samples were mixed using ultrasonication for 75 min, with the nanotubes being sonicated in the solvent rst for 45 min in order to assure uniform distribution. The scaffolds in all studies were on average 2 mm in thickness. 2.3. Scaffold characterization All of the electrospun samples were sputter-coated with a thin layer (9 nm) of goldpalladium, and were then imaged using a Zeiss SigmaVP SEM at 5 kV accelerating voltage. In addition, the compressive mechanical properties of all scaffolds were evaluated via an ATS axial tester, a 50 Newton load cell and a compression placard, under dry conditions. Circular samples of 8 mm in diameter and about 12 mm in height were taken and tested in compression, at a strain rate of 0.2 mm s1 . The forcedeformation data were then used to calculate and compare the Youngs modulus of each sample. 2.4. MSC function study in vitro 2.4.1. Primary MSC culture. Primary human bone marrow MSCs were obtained from 20 ml aspirates from
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a healthy consenting donors iliac crest (female; age 27) from the Texas A&M Health Science Center, Institute for Regenerative Medicine and thoroughly characterized [22]. Briey, the mononuclear cells were separated using density centrifugation. The cells were plated, expanded, harvested and frozen at passage 1 (P1). Two trials of the frozen P1 cells were analyzed over three passages for colony forming units, cell growth, and differentiation into fat and bone. Flow cytometry results for various cell markers and infectious agents blood screen were evaluated as well. The characterized cells were used to evaluate the cytocompatibility properties of the electrospun nanocomposite. MSCs (passage #36) were cultured in a complete medium comprised of Alpha Minimum Essential medium ( -MEM, Gibco, Grand Island, NY) supplemented with 16.5% fetal bovine serum (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 the above medium recipe, but with the addition of 100 nM dexamethasone, 40 g ml1 proline, 100 g ml1 sodium pyruvate, 50 g ml1 L-ascorbic acid 2-phosphate, ITS+ at a concentration of 1% of the total volume of prepared medium, and 10 ng ml1 TGF- 1 during the rst three days. The medium was supplemented with 100 ng ml1 IGF-I throughout the experimental period. All electrospun scaffolds were sterilized via exposure to ultraviolet light for 15 min, ipped and exposed for another 15 min, and then rinsed with PBS prior to cell experiments. 2.4.2. MSC adhesion study in vitro. In order to evaluate the diameter effects on MSC attachment, sample groups consisting of circular dry spun pure PLLA scaffold, fabricated at working distances of 12, 14, 16, 18, and 20 cm, were then seeded with MSCs at 10 000 cells per scaffold and allowed to incubate for 4 h. Cells were then lifted and counted using a hemocytometer and light microscope. 2.4.3. MSC proliferation study in vitro. Sample groups consisting of a pure PLLA control, PLLA scaffold containing 0.5% w/v untreated MWCNTs, PLLA scaffold containing 0.5% w/v H2 treated MWCNTs and PLLA scaffold containing 1.0% w/v H2 treated MWCNTs were then seeded with MSCs at 10 000 cells per scaffold. All sample groups were fabricated using wet electrospinning. Samples were then cultured for 1, 3 and 5 days. After the prescribed time periods, the scaffolds were rinsed using PBS to remove non-adherent cells. The adherent cells were lifted via trypsinization, i.e., 0.25% trypsin/1 mM EDTA solution for 3 min at room temperature. The cell proliferation was quantied via a CellTiter 96 R AQueous Non-Radioactive Cell Proliferation Assay (MTS assay) and analyzed using a Thermo Scientic Multiskan GO spectrophotometer at a setting of 490 nm wavelength light.

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Table 1. Diameters of the electrospun bers, as compared to the working distance between the needle and the collector plate. Data are means standard deviation, n = 3. Working distance (cm) Average ber diameter (m) 12 3.390 0.093 14 1.715 0.005 16 1.672 0.185 18 1.332 0.114 20 1.543 0.108

2.4.4. MSC differentiation study in vitro. A two-week differentiation study was conducted, using a pure PLLA wet electrospun scaffold and four more experimental groups containing 0.5% w/v MWCNTs, 0.5% w/v H2 treated MWCNTs, 0.5% w/v MWCNTs with poly-L-lysine and 0.5% w/v H2 treated MWCNTs with poly-L-lysine. The cell favorable poly-L-lysine was used as a means to increase the hydrophilicity and change the surface chemistry of the scaffolds. The scaffolds were cultured in chondrogenic media with MSCs seeded at 250 000 cells per scaffold, and incubated for two weeks, with samples being collected at one and two weeks. The collected samples were freeze dried in a lyophilizer and treated in a Papain digestion solution for 18 h in a 60 C water bath for chondrogenic differentiation evaluations. Glycosaminoglycan (GAG): glycosaminoglycan, a key component of cartilage matrix, was measured using a standard BlyscanTM GAG assay kit according to the manufacturers instructions. Briey, sample digest solutions were centrifuged in a microcentrifuge of 10 min at 10 000 rpm, then reacted with a Blyscan dye reagent for 20 min. Samples were then centrifuged again, reacted with a Blyscan dissociation reagent and analyzed in the spectrophotometer at 656 nm. According to a standard curve run in parallel with the experimental samples, the total GAG synthesized by MSCs cultured in the samples of interest to this study was calculated. Total collagen synthesis: the collagen content of the samples was also evaluated using a Sircoll collagen assay kit according to the manufacturers instructions. Samples were prepared in the same way as for the GAG assay, except that before the addition and removal of collagen dye, the collagen was reacted with a coagulation reagent and refrigerated overnight. Samples were evaluated with the spectrophotometer at 555 nm. Total protein content: total protein content was measured using a commercial BCATM Protein Assay Reagent kit (Pierce Biotechnology) and following the manufacturers instructions. For this purpose, 150 l of aliquot supernatants of the protein-containing sample solutions were mixed with 150 l of working agent solutions (including 1:24:25 of cupric sulfate:bicinchnoninic acid:a reagent with sodium bicarbonate, sodium carbonate and sodium tartrate) and then incubated at 37 C for 2 h. Light absorbance was measured at 562 nm on the spectrophotometer. 2.5. Statistics All cellular experiments were run in triplicate and repeated three times for each substrate. Data are presented as the mean value standard error of the mean (SEM) and were analyzed with students t-test for pair-wise comparison. Statistical signicance was considered at p < 0.1.
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3. Results and discussion

3.1. Electrospinning parameter optimization and MSC adhesion on microbrous scaffolds 3D scaffold architecture and geometric cues play a major role in directing MSC behavior [23]. It has been shown that different sizes of electrospun polymer bers have different inuences on stem cell functions [2427]. Our adhesion study sought to compare the effectiveness of microber size on cellular adhesion, as well as to optimize our setup for further carbon nanotube electrospinning. As shown in table 1, smaller ber diameters were yielded by increasing the working distance, with a slight increase at 20 cm. These results can also be observed via SEM imaging (gure 2). More importantly, it is shown that the scaffolds with the smallest ber diameter promoted the greatest cellular adhesion of MSCs (gure 3), displaying that the smallest and thus more biomimetic ber dimensions promote the best stem cell adhesion. It should be noted that our larger bers also achieved good cell adhesion. This may be due to the fact that the larger bers had induced nanoscale (60 nm) surface pores. These data were observed by measuring the induced pore sizes on SEM images of bers directly. The presence of nanopores may have helped to create more surface area for MSC adhesion. Besides the surface pores, the variance of scaffold porosity may also contribute to the above result. The SEM image (gure 2(A)) showed that the scaffold with the largest bers had less densely packed bers and was more porous than the others. This suggested that both the ber dimensions and the surface topography can contribute to the creation of a stem cell-favorable environment. 3.2. MWCNT and MWCNT embedded PLLA scaffold characterization SEM and TEM images of the untreated MWCNTs compared to the H2 treated MWCNTs were taken (gure 4). These images show that H2 heating changed the morphology of the nanotubes from bundles of nanotube aggregates into a more homogenous distribution, which makes them suitable for co-electrospinning into the PLLA scaffold. In addition, the H2 treatment can facilitate the removal of impurities and metallic catalyst material in the nanotubes. SEM images were also taken of the electrospun PLLA scaffolds fabricated via dry and wet spinning (gure 5). The wet electrospun scaffolds show more 3D porous structure than the dry electrospun scaffolds. Wet spinning, in addition to increasing the three-dimensionality of scaffolds, is also known to induce nanopores and nanoroughness

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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 cm at a lower magnication (F).

on the surface [15], which make the wet spun scaffolds surface morphology similar to that of DCM/DMF dry spun scaffolds. Figure 6 shows the SEM images of various wet electrospun MWCNT embedded PLLA scaffolds. No single nanotubes could be directly observed, implying that they were embedded in the bers. It could also be seen that the ber diameters varied in the MWCNT embedded scaffolds. The ber diameter distribution was from about 1 to 10 m. This effect, which occurs when nanomaterials are electrospun in solution with a polymer, is documented in the literature [2830]. More importantly, after the addition of MWCNTs of both species, the PLLA scaffolds Youngs modulus increased dramatically when compared to a pure PLLA control (gure 7). Moreover, all MWCNT reinforced scaffolds were within the range of native articulate cartilage (0.752 MPa depending on location) [3134]. This shows that the incorporation of just a small amount of MWCNTs can greatly increase the mechanical properties of a tissue engineering scaffold to within biomimetic regimes.
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Figure 3. Enhanced MSC attachment on the electrospun PLLA scaffold with the smallest ber diameter (1.33 m). Data are mean SEM, n = 9; p < 0.1 when compared to all other samples and p < 0.1 when compared to the scaffold with 1.67 m ber diameter.

3.3. MSC proliferation and chondrogenic differentiation in vitro The proliferation study showed an increase in cellular proliferation on all scaffolds, with the greatest cell numbers

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Figure 4. SEM images of (A) MWCNTs and (B) H2 treated MWCNTs, and TEM images of (C) MWCNTs and (D) H2 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 both species at three days (gure 8). At ve days, all of the MWCNT PLLA scaffolds showed greater cellular growth than controls, which indicated the good cytocompatibility
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properties of these nanostructured scaffolds for cell growth. It is known that mechanical factors such as scaffold stiffness can have signicant inuence on regulating stem cell activities [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% H2 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 nanoporosity of the scaffold.

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

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

due to their increase in mechanical strength, could provide a stiffer surface for better stem cell growth and proliferation. In addition, both of the nanotube species may change the nanosurface roughness and surface area of the scaffolds, thus further contributing to the improved MSC proliferation after ve days when compared to controls. Moreover, the differentiation study showed increased chondrogenic differentiation activity of MSCs in these nanostructured scaffolds in vitro (gures 9 and 10). There was a dramatic increase in GAG content at one and two weeks on the scaffolds containing poly-L-lysine coated MWCNT, and among these samples the H2 treated tubes performed the best (gure 9). The positively charged poly-L-lysine can create
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an electrostatic interaction with negatively charged GAG for improved GAG nucleation in the scaffold. This implies that the surface coating of the nanotube scaffold (to decrease hydrophobicity) had the greatest impact on GAG synthesis in vitro, greater than the nanosurface topography contribution of the MWCNTs in our study. Furthermore, gure 10 reveals that both H2 treated MWCNT and poly-L-lysine coated MWCNT PLLA scaffolds can signicantly improve total collagen synthesis after one and two weeks. The fact that the H2 treated tubes yielded better results shows that the purication of nanotubes to remove various impurities and modify the nanotube morphology is advantageous for

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Figure 9. Greatly enhanced GAG synthesis of MSCs in all MWCNT (0.5%) embedded in PLLA scaffolds, compared to controls after two weeks. Data are means SEM, n = 9. # p < 0.05 when compared to all other samples at week 1; p < 0.05 when compared to all other samples at week 2; p < 0.05 when compared to control and MWCNTs without H2 treatment; and p < 0.05 when compared to controls.

Figure 10. Improved total collagen synthesis of MSCs in H2 treated MWCNT embedded PLLA scaffolds with poly-L-lysine coating after two weeks. Data are mean SEM n = 9. p < 0.05 when compared to all other samples and p < 0.05 when compared to controls and MWCNT scaffold at week 2. # p < 0.05 and ## p < 0.1 when compared to controls at week 1. ### p < 0.05 when compared to all other samples; $ p < 0.05 when compared to poly-L-lysine coated non-treated MWCNT samples at week 1.

biological applications. It should be noted that the scaffold containing untreated nanotubes did not improve collagen synthesis when compared to the control. This is, however, not wholly unexpected, since it is known that the metal catalyst material and impurities have cytotoxic effects, and thus may have overcome the positive effects of the addition of CNTs to the construct [36, 37]. In order to further discuss the results presented, the following is a brief overview of recent research investigating the effects of poly-L-lysine and carbon nanotubes on cellular activity. Poly-L-lysine has been long-established as a benecial chemical compound for promoting cellular growth, and has been used in regenerative studies [38]. Grohmann et al investigated the secondary structure of peptide chains and their effects on proliferating osteoblasts [39]. It was found that the peptides in poly-L-lysine adopt an intermolecular
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beta sheet structure. This reveals an increased area of spread, which consequently supports osteoblast proliferation. In addition, Santana et al used poly-L-lysine coated slides to culture human chromafn progenitor cells [40]. The coated slides were able to induce the cells to differentiate into two distinct neuron-like cell types. Although the use of carbon nanotubes in tissue engineering is in its infancy, they have been considered as exciting alternatives as templates for tissue growth, drug delivery agents and in bio-sensory applications. For instance, while there have been several studies that show the ability of CNTs and their conductive properties to incite cardiac tissue development of stem cells [41, 42], Serag et al, in a rather dramatic study, studied the effects of CNTs on plant cells that were differentiating into tracheary elements [43]. It was found that these cells readily used cup-sacked CNTs to create cell structures via oxidative cross-linking of monolignols to the CNT surface. This not only demonstrates CNTs having a desirable effect on cell growth, but also highlights potential CNT fate in a living dynamic system post-application. Furthermore, there have also been experiments similar to ours, where CNTs were used in a nanocomposite material, with promising results. Ogihara et al studied the use of CNT alumina ceramic composites in vivo for bone tissue engineering [44]. It was found that there was no increased inammation at the implant site of CNT containing samples when compared to alumina controls, suggesting that constituent CNTs embedded in a matrix material have good biocompatibility properties. Although there have been cytotoxicity concerns raised about CNTs, thus far the exact mechanisms of CNTs effects on cells are still not fully known. However, all of the results presented show that nanotubes are cytotoxic only under certain conditions, e.g. certain tube lengths, high concentration, hydrophobicity of nanomaterial and dispersion of nanotubes. It has also been reported that the formation of MWCNT aggregates can signicantly contribute to their inammatory qualities [45]. In our study, we implemented a small concentration of homogenously distributed CNTs which had been H2 puried and poly-L-lysine coated for the chondrogenic differentiation of MSCs for the rst time. Our results show the signicant benecial effects of these nanostructured scaffolds in directing stem cell differentiation in vitro. As illustrated in gure 11, we believe that our scaffolds are advantageous for cellular growth because the nanotubes are modied to have cell-favorable hydrophilic poly-L-lysine coated surfaces, and are homogenously dispersed and embedded in solid microbrous structures, which creates a stable and advantageous environment for cellular activity.

4. Conclusion
Todays clinical challenges provide great opportunity for the development of highly customizable and tailorable methodologies for the treatment and repair of diseased, damaged, and injured tissue and organs. Stem cells provide a great deal of promise, but require chemical and physical cues for adequate differentiation and tissue formation.

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References
[1] Zhang L, Hu J and Athanasiou K A 2009 The role of tissue engineering in articular cartilage repair and regeneration Crit. Rev. Biomed. Eng. 37 157 [2] Castro N J, Hacking S A and Zhang L G 2012 Recent progress in interfacial tissue engineering approaches for osteochondral defects Ann. Biomed. Eng. 40 162840 [3] Mathur D, Pereira W C and Anand A 2012 Emergence of chondrogenic progenitor stem cells in transplantation biology-prospects and drawbacks J. Cell. Biochem. 113 397403 [4] Pelttari K, Winter A, Steck E, Goetzke K, Hennig T, Ochs B G, Aigner T and Richter W 2006 Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcication and vascular invasion after ectopic transplantation in SCID mice Arthritis Rheum. 54 325466 [5] Seo S and Na K 2011 Mesenchymal stem cell-based tissue engineering for chondrogenesis J. Biomed. Biotechnol. 2011 806891 [6] Mauck R L, Yuan X and Tuan R S 2006 Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture Osteoarthritis Cartilage 14 17989 [7] Hutmacher D W 2000 Scaffolds in tissue engineering bone and cartilage Biomaterials 21 252943 [8] Yaszemski M J, Payne R G, Hayes W C, Langer R S, Aufdemorte T B and Mikos A G 1995 The ingrowth of new bone tissue and initial mechanical properties of a degrading polymeric composite scaffold Tissue Eng. 1 4152 [9] Phipps M C, Clem W C, Grunda J M, Clines G A and Bellis S L 2012 Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell inltration Biomaterials 33 52434 [10] Li W-J, Laurencin C T, Caterson E J, Tuan R S and Ko F K 2002 Electrospun nanobrous structure: a novel scaffold for tissue engineering J. Biomed. Mater. Res. 60 61321 [11] Holmes B, Castro N J, Zhang L G and Zussman E 2012 Electrospun brous scaffolds for cartilage and bone regeneration: recent progress and future developments Tissue Eng. B Rev. 18 47886 [12] Li W J and Tuan R S 2009 Fabrication and application of nanobrous scaffolds in tissue engineering Curr. Protoc. Cell Biol. 42 25.2.125.2.12 [13] Li W J, Mauck R L, Cooper J A, Yuan X and Tuan R S 2007 Engineering controllable anisotropy in electrospun biodegradable nanobrous scaffolds for musculoskeletal tissue engineering J. Biomech. 40 168693 [14] Smith L A and Ma P X 2004 Nano-brous scaffolds for tissue engineering Colloids Surf. B 39 12531 [15] Shin T J, Park S Y, Kim H J, Lee H J and Youk J H 2010 Development of 3-D poly(trimethylenecarbonate-coepsilon-caprolactone)-block-poly(p-dioxanone) scaffold for bone regeneration with high porosity using a wet electrospinning method Biotechnol. Lett. 32 87782 [16] Miao J, Miyauchi M, Dordick J S and Linhardt R J 2012 Preparation and characterization of electrospun core sheath nanobers from multi-walled carbon nanotubes and poly(vinyl pyrrolidone) J. Nanosci. Nanotechnol. 12 238793 [17] Reddi A H, Becerra J and Andrades J A 2011 Nanomaterials and hydrogel scaffolds for articular cartilage regeneration Tissue Eng B 17 3015 [18] Zhang L and Webster T J 2009 Nanotechnology and nanomaterials: promises for improved tissue regeneration Nano Today 4 6680
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Figure 11. Schematic illustration of MSC differentiation and cartilage formation in three electrospun PLLA scaffolds. Darker blue represents more cartilage matrix formation. Not drawn to scale.

In this study, a series of biomimetic electrospun brous nanoscaffolds with controlled ber dimension and surface nanoporosity were fabricated. When compared to a pure PLLA control, scaffolds with MWCNTs showed no adverse effect on MSC proliferation, and more importantly displayed enhanced MSC chondrogenesis when modied with a chemical factor. The puried H2 heated nanotubes also showed enhanced differentiation over scaffolds containing untreated tubes. In addition scaffolds with incorporated nanotubes showed signicantly improved mechanical properties when compared to a PLLA control. This demonstrates that MWCNTs can be used to greatly improve the strength of electrospun polymer bers, without having an adverse effect on cellular activity. Overall, the data presented display that electrospun PLLA scaffolds with incorporated, surface modied MWCNTs and poly-L-lyisne coating can be suitable scaffolds for cartilage regeneration.

Acknowledgments
The authors are grateful for a Research Award from the Clinical and Translational Science Institute at Childrens National (CTSI-CN) and support from the George Washington University Institute for Nanotechnology (GWIN). We are also grateful for the imaging support from the George Washington University Center for Microscopy and Image Analysis and the National Institute of Standards and Technology.

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