Carbon nanotubes: Their potential and pitfalls for bone tissue regeneration and engineering

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Nanomedicine: Nanotechnology, Biology, and Medicinexx (2013) xxx–xxx

nanomedjournal.com

NANO-00779; No of Pages 20

Carbon nanotubes: Their potential and pitfalls for bone tissue regenerationand engineering

Peter Newman, BE, Honsa,b, Andrew Minett, PhDb, Rutledge Ellis-Behnke, PhDc,d,Hala Zreiqat, PhDa,⁎

aBiomaterials and Tissue Engineering Research Unit, School of Aeronautical Mechanical and Mechatronics Engineering, University of Sydney, Sydney,NSW, Australia

bLaboratory for Sustainable Technology, School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, NSW, AustraliacNanomedicine Translational Think Tank Augenklinik, Medical Faculty Mannheim of the Ruprecht-Karls-University Heidelberg, Germany

dMassachusetts Institute of Technology, Department of Brain & Cognitive Sciences, Cambridge, Massachusetts

Received 18 January 2013; accepted 2 June 2013

Abstract

The extracellular environment which supports cell life is composed of a hierarchy of maintenance, force and regulatory systems whichintegrate from the nano- through to macroscale. For this reason, strategies to recreate cell supporting environments have been investigatingthe use of nanocomposite biomaterials. Here, we review the use of carbon nanotubes as part of a bottom-up approach for use in bone tissueengineering. We evaluate the properties of carbon nanotubes in the context of synthetic tissue substrates and contrast them with the nanoscalefeatures of the extracellular environment. Key studies are evaluated with an emphasis on understanding the mechanisms through whichcarbon nanotubes interact with biological systems. This includes an examination of how the different properties of carbon nanotubes affecttissue growth, how these properties and variation to them might be leveraged in regenerative tissue therapies and how impurities orcontaminates affect their toxicity and biological interaction.© 2013 Published by Elsevier Inc.

Key words: Carbon nanotubes; Bone; Tissue engineering; Review; Toxicology; Nanobiomaterials

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Introduction

Multicellular life is composed of a hierarchy of systemsoperating from the nano- through to the macroscale.1 Throughthe combination and integration of processes at smaller lengthscales, biological systems can perform complex macroscaletasks. This is evident as the first level of cellular organization iscomposed of functioning macromolecules with lengths at the

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Sources of support for research: Commonwealth Government –Australian Postgraduate Award Scholarship. The Australian National Healthand Medical Research Council. Rebecca Cooper Foundation. AustralianResearch Council.

⁎Corresponding author: Tissue Engineering & Biomaterials ResearchUnit, School of Aerospace, Mechanical & Mechatronic Engineering, Facultyof Engineering and IT and Bosch Institute, The University of Sydney, TheUniversity of Sydney, NSW, 2006, Australia.

E-mail address: [email protected] (H. Zreiqat).

1549-9634/$ – see front matter © 2013 Published by Elsevier Inc.http://dx.doi.org/10.1016/j.nano.2013.06.001

Please cite this article as: Newman P, et al, Carbon nanotubes: Their potentialNBM 2013;xx:1-20, http://dx.doi.org/10.1016/j.nano.2013.06.001

nanoscale. This includes extracellular proteins that provide tissuestructure such as type-1 collagen which is approximately 1.5 nmin diameter and 200 nm long2; the protective cell membranewhich is 5–10 nm in thickness; integrin cell-adhesion proteinswhich have a 5 nm binding site at the end of their 20–23 nm‘head’ structure external to the cell membrane3 and biomoleculessuch as DNA in which conformational changes lead to structuraland functional effects.

Changes to this first level of cellular hierarchy can haveunprecedented effects as the summation of small nanoscalechanges becomes compounded at higher levels. Nanoscalefeatures have demonstrated regulatory effects over almostevery facet of cell behavior including adhesion, migration,proliferation, signaling, genetic expression and stem cell fate.4–6

As a consequence of these findings, many biomaterials are beingdesigned with nanoscale details that elicit favorable cellularbehavior. Such designs require that there are reliable methods tofabricate and control such details and that there is anunderstanding of the mechanisms by which nanoscale details

and pitfalls for bone tissue regeneration and engineering. Nanomedicine:

http://dx.doi.org/10.1016/j.nano.2013.06.001




mailto:[email protected]



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Figure 1. Shows transmission electron micrographs of a SWNT (A) and a MWNT (B); note the hollow center and two sides of the graphitic layers can be seen.Scale bars are 10 nm.

2 P. Newman et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2013) xxx–xxx

influence cells. Given this, biomaterials can be optimized to bestreplace damaged tissues.

Some of the most popular fabrication methods that allowcontrol over nanoscale details are electrospinning,7 nano-lithography,8 molecular self-assembly,9 chemical etching,10

vapor phase deposition11 and composite formation withnanomaterials such as carbon nanotubes12 – the subject ofthis review.

Carbon nanotubes (CNTs) are graphitic tubular structures thathave attracted a high interest due to their extraordinary physical,electrical and chemical properties. There are many ways in whichCNTs can be used within the field of tissue engineering. Theycan improve both the mechanical properties and biocompatibilityof composite biomaterials13 showing biocompatibility in vivoboth in solution14 or in composites15; interact favorably with cellbinding proteins16; modulate cell shape through a preferentialcell-binding affinity12; regulate stem cell differentiation (partic-ularly that of osteogenic17 and neuronal18 lineages); since CNTshave high conductivity and can be used to investigate theproliferative effects of electrical stimulation,19,20 the electricalactivity of cells19,21 or as electronic cell based sensors22; theycan be used to create controlled nanoscale textures23–26 as wellas nanoscale patterns over substrates12,27,28 and finally, their rod-like shape and nanoscale dimensionality may allow them to actas a morphological biomimetic of the fibrillar proteins in theextracellular matrix (ECM).

The structure of CNTs is shown in Figure 1. CNTs arecommonly divided into two subsets: single-walled carbonnanotubes (SWNTs) and multi-walled carbon nanotubes(MWNTs). While SWNTs are a single-layer of tubular graphiticcarbon, MWNTs have multiple layers (typically in the range of2–40) of concentric tubular graphitic carbon. Figure 1 showstransmission electron micrographs of SW- and MWNTs wherethe tubular graphitic lattice of the CNTs can be seen at either sideof its hollow center. There are also finer classifications such asthe number of walls, length, diameter, chirality and presence offunctional groups which can alter interactions between cells ortissue with CNTs.25

Unfortunately, despite the progress which has been madedeveloping materials with controllable nanoscale details, themechanisms by which nanoscale interactions influence cellularbehavior are still not fully understood. Efforts to achieve anunderstanding in this area fall into two distinct categories i) thestudy of cell-cell/ECM-cell interactions and ii) the attributes ofthe ECM and its structures, composition, formation, degradation,chemical and electrical conductivity. An understanding in theseareas supports the aims of tissue engineering: to recreate theextracellular environment thus restore healthy cell interactions.A brief overview of these two areas including a look at thespecific nanoscale details of bone is given below.

The structure and composition of the extracellular matrix

The typical mammalian ECM is made from a core ofapproximately 300 different proteins.29,30 ECM proteins arebuilt from repeating units which form into long fiber-like strandsof insoluble, cross-linked macromolecules with nanoscalefeatures. These proteins are loosely defined into three separatefamilies: collagens, proteoglycans and glycoproteins. The manydistinctive types of ECM are created through variations to theseprotein families, their composition and assembly.31 Collagen isthe most abundant protein in the human body and constitutesaround 30% of all proteins by mass. Proteoglycans are a type ofnegatively charged protein which bind with sulphate or uronicacid groups and are interwoven throughout the ECM. While thecollagen family provides structural support to the ECM,proteoglycans and attached sulphate groups, such as chondroitinsulphate, create biochemical boundaries and interaction sitesthroughout the ECM. These sites play roles in cell processesduring cell and ECM growth and regeneration. Similar toproteoglycans, glycoproteins adhere to chemical mediators andperform functions assisting other biochemical interactions. Theyare involved in cell adhesion, intercellular signaling and theassembly and strengthening of the ECM.32 One of the most well-

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Figure 2. Shows a generic cell (orange) with integrin clusters (red) interactingwith the ECM (dark green/light green spirals are proteoglycans/glycoproteinsand blue fibers are collagen).

3P. Newman et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2013) xxx–xxx

known glycoproteins is fibronectin; a protein involved in thebinding of integrin proteins with the ECM.

Through the combination of these constituents the ECMprovides a complex environment rich with mechanical, chemicaland architectural diversity. Variations to these environments arecharacteristic to a specific tissue type and will regulate cellbehaviors and differentiation. Specifically, changes to theelasticity of a cell’s environment will regulate its behavior.33

More recently this effect has been shown to interplay with thedensity of adhesion sites.34 This follows logically since anincrease in the number of adhesive sites would spread the forceover a greater area thus decreasing the local stress in a mannerequivalent to an increase in elasticity. Furthermore, the type ofchemical bonding between adhesion sites and the rest of theenvironment will influence cell behavior: stronger bonds or morenumerous bonding sites acting like increases to elasticity.35 Theshape and patterning of cells over their environment also affectsinfluences behavior,36 a reflection of the observation that almostall types of tissue consist of organized patterns of orientedcells.37

Interaction with the proteins listed above occurs directly witha cell and can thus be seen as a local regulator of cell behavior.Alternatively, the control of cell behavior may occur at a globallevel where unbound chemical mediators, such as enzymes,growth factors and cytokines will float through the extracellularfluids.

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Nanoscale cell–extracellular matrix andcell–cell interactions

Interactions between a cell and its environment arebidirectional: the ECM provides the cues for cells to adhereand mature, while cells secrete the proteins that build, degradeand remodel the ECM. These processes take place throughhierarchical systems which integrate the interactions of nano-scale processes to perform those at the micro- and macroscale.Consequently, the consideration of nanoscale interactions is ofimportance when designing a biomaterial.

Cells form connections to their environment through proteinclusters known as focal adhesions, and without the formation ofadhesions most cell types won’t grow.38 One of the mostimportant protein families found in focal adhesions is known asintegrins. Integrin proteins form a chemical linkage between acell and the ECM. They cross through the cell membrane to form

links between the inside and outside of a cell. Most of an integrinprotein is found outside the cell membrane in the shape of twolarge ‘heads’. The length of these head structures is approxi-mately 20–23 nm and it is here that integrins have their ligandbinding sites; a 5 nm region at the end of each head.3 Given thenanoscale size of integrins, it is not surprising that nanoscalevariation in a cell’s environment influences adhesion and otherdownstream cell behavior in the adhesion system or ‘theadhesome’. The presence of a focal adhesion implies that a cellis attached to a substrate through an integrin-mediated structureanchored to the ends of a stress generating actin filament. Suchstructures are important in the regulation of downstreambehaviors such as growth, migration, differentiation and celldeath.39 These structures have been studied extensively andreviews are available on cellular adhesions,39 integrins,40,41 theadhesome42 and resulting downstream behaviors.38,43,44 Figure 2shows a pictorial representation of a cell adhering to the ECM.

In addition to cell-ECM interactions, tissue function dependson signaling between cells. Such cell-cell interactions also occurby small nanoscale macromolecules which have roles in cellcommunication, the transfer of biochemical matter, and thegrowth and organization of the ECM. The interference of cell-cell interaction structures, such as adherens junctions, can disruptthese processes leading to cell disorganization and both theuncontrolled remodeling and secretion of the ECM. In thismanner cell-cell interactions play critical roles in the growth andformation of new tissue.45 Such behavior is particularly relevantto angiogenesis where intercellular cytoplasmic projections formthe prerequisite constructs for the growth of endothelial andvasculature structures.46 The structure and functions of cell-cellinteractions are reviewed elsewhere.47,48

The extracellular matrix of bone and its nanoscale features

While the structure and composition of bone ECM containsthe generic features described above, it is often described as acomposite structure with just two major constituents: organiccollagen and inorganic bone mineral carbonate apatite. Therelative compositions of collagen and bone mineral varyaccording to species, anatomical location, age, sex andexercise,49 human bone varies between 0 to 43% mineralcontent by volume.50 With increasing percentages of carbonateapatite, bone becomes stiffer and breaks under smaller strains.The two constituents of bone are hierarchically organized acrossmultiple scale lengths as depicted in Figure 3. This hierarchybegins with the organization of protein sequences to form thenanoscale molecule tropocollagen and extends through to themacroscale pores and shell of cancellous and cortical bonerespectively. Despite the simplified description, it is important toremember that bone ECM is a complex structure with a great dealof chemical diversity, containing the aforementioned proteogly-cans and glycoproteins in addition to the relatively moreabundant collagen. Likewise, biochemical regulators such asgrowth factors, cytokines and enzymes are also presentthroughout bone ECM.

The description below is specific to that of the extracellularmatrix of human cancellous bone. Cancellous bone is of the

Figure 3. Depicts the hierarchical organization of human cancellous bone from the nano- to the macroscale.


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Figure 4. Scanning electron micrographs demonstrating the morphological similarities between (A) nitrogen-doped-MWNTs and (B) collagen, both structuresare long and rod-like with a periodic structure along their long axis, scale bars 200 nm – (B – reprinted with permission of FEI, courtesy of Paul Gunning ofSmith & Nephew).


highest interest to tissue engineers since the porous structureallows transport of nutrients and minerals, vascularization andfuture remodeling; all of which are critical for tissue growth.

At the nanoscale, human cancellous bone is composed ofarrays of parallel fibrillar type I collagen over which alignedplatelet-shaped crystals of carbonate apatite sit.49–52 These fibrilarray structures are aligned longitudinal to the osteons and theplane of the lamellae, this anisotropic structure reflects theanisotropic mechanical properties of bone.50,51 The base unit ofthe collagenous component of this structure is the long, rod-liketriple helix tropocollagen macromolecule (as depicted in 6thlayer of hierarchy in Figure 3). Under physiological conditions,tropocollagen macromolecules spontaneously undergo fibrillo-genesis creating the supramolecular collagen fibers.49

The dimensional characteristics of CNTs make themparticularly interesting as a biomimetic analog for the fibrillarproteins of the ECM; particularly that of collagen. Both the triplehelix collagen molecule tropocollagen and a collagen fiber – theself-assembled tropocollagen agglomerate – have comparablelength scales to CNTs. While the diameter of CNTs can be madein the range of 1–100 nm and exceeding lengths of 5 mm, thetropocollagen molecule is 1.5 nm in diameter and 300 nm inlength. Also, CNTs can be made with dimensional similarity tocollagen fibers which have diameters in the range of 20–400 nmand lengths up to hundreds of microns (determined by thenumber of axially repeating tropocollagen units2). Furthermore,since methods exist to synthesize sheets or yarns of parallel orrandomly orientated CNTs,53,54 there is a potential to fabricateCNT structures with hierarchies at multiple scales mirroring thestructure of collagen in the ECM.

Longitudinal distributions in electric charge along thetropocollagen molecule direct the axial staggering of tropocol-lagen macromolecules within a collagen fiber. This staggerforms the characteristic 67 nm periodic ‘band/gap’ structureof collagen depicted in Figure 3. Tropocollagen moleculesoverlap forming a ‘band’ region for 27 nm and leave a ‘gap’

for 40 nm.51 It is within the gap section of collagen fibers thatplatelets of carbonate apatite originate and for this reason it isfound periodically along collagen fibers.51,55,56 The overalldimensions of the platelets are reported to be between 2–5 nmthick, 10–80 nm wide and up to 200 nm long.50,51,57,58 Thelong axis of the platelets is aligned parallel to the axis of thecollagen fibers, with an average of 4 platelets touching thefiber at any given point along its axis.51 Increases in thenumber of platelets or mineral content reflects an increase tothe stiffness of the material.50 These plates are polycrystallineand composed of smaller crystals in the order of tens ofmicrons long.59,60 The potential to use CNTs as a biomimeticof collagen is inviting as the defects in the CNT structure areoften periodic, mirroring the band/gap structure of collagen(Figure 4). Furthermore, CNTs can nucleate nanoscale bone-like carbonate apatite.61–67

The use of carbon nanotubes in bone biomaterials

In the last 11 years, there have been upwards of 50 papersinvestigating the potential use of CNTs as a synthetic bone tissuereplacement. Significant studies have been compiled into Table 1where they are further broken down into 4 categories. For adescription of the Table, its categories and the criteria used tocategorize its references, see the notes included with the table.References which have significant or interesting findings fromthis table are discussed throughout the sections below. This isdone through comparison with the properties of CNTs as well asthe nanoscale features of the ECM and bone.

Chemical properties of carbon nanotubes

Carbon atoms in CNTs form bonds in the hexagonal sp2

hybridization state. Consequently these atoms have strong non-

Table 1t1:1

Summary of different studies that used CNTs as an osteogenic substrate.t1:2

t1:3 Osteogenic Outcome Proliferative Outcome

t1:4 Positive Negative Neutral NR Positive Negative NR

t1:5 CNT Type and Diameter (nm)t1:6 SWNT .5-2 143, 12, *148, *148, 130 21, 96, 28, 117,

91, 16, 93, *72,107

130, 117, 143,*72, 12, 93,107

21, *96,130, *148

28, 91, 16

t1:7 MWNT 10-19 130 21, 25, 28, 93,*20, *116, 109

25, 130, 93,*20, *116, 109

21, 130, *20,*116, 109

28

t1:8 20-39 *149, 149, *104, 25, *72 25, *149, 149,*72, *104,

t1:9 40-79 25, 150 25 150t1:10 80+ 14, 73, 97, 97,

68, 151, *13, 77*13 14, 73, 97, 97,

68, 68, *13,13, 77

97, 68,151, *13

t1:11 NR 19, 145, 152 145 153 87 19, 145 87, 145, 152 153t1:12 COOH-SWNT .5-2 *154, 92 *26, 74,

75, 92130, *90 21, 91 130, 154, 92 21, *26, 130,

74, *90, 75,*154, 92

91

t1:13 COOH-MWNT 2-9 108 *26, 75 108 *26, 75t1:14 10-19 118 *26, 118 118, 118, *26, 118t1:15 20-39 *106 89, *106 89, *106t1:16 40-79 155 105 155, 105 155, 105t1:17 80+ *77, 77 77 *77t1:18 NR 15, 87 15, 87 87 15t1:19 CNF 23, 147, 24, 23, 27, 85 23, 147, 24, 27 23 85t1:20 Func-CNT *148, 17 *148 21, 156, *88, 87 156, *88, 92,

87, 1721, 87,*148, 17

t1:21 Biological Modelt1:22 None 153 85 85, 153t1:23 Osteoblastic 19, 23, 147, 24,

155, *149, 73,97, 143, *154,118, 68, 151,*13, *104,

23, *26, 89,118, *13

21, 27, 25, 96,150, 105, *88,*72, 91, 93, 87,*20, *116, 107,109

19, 23, 147, 24,27, 25, 155, *149,105, 73, *88, 97,89, 143, *72, 93,87, 118, 68, *13,*20, *104,*116, 107, 109

21, *96, 155,*26, 105, 97,*154, 87, 118,68, 151, *13,23, *20, *116,109

150, 91,

t1:24 Multipotent *106, 12, 92,145, *77, *148,152, 17, 77, 108

74, *106,92, 145,*148, 75

130, *90, 156, 28, 15, 16 156, 130, *106,12, 92, 145, 77,108, 17,

130, 74, *90,75, 92, 145,*77, *148,152, 17

28, 15, 16

t1:25 in vivo 149, 14, 97 117 15, 149, 117, 14,97, 154, 118, 68,13, 77

t1:26 Substrate Morphologyt1:27 aq. 68, *26, 74, 75 130, 130, 68, 68 *26, 130, 74,

75, 68t1:28 2D 19, 23, 147, 24,

*149, 73, 143,*154, *106, 12,92, *77, 152,*13, 17, 77

23,*106,92, *13

*90, 21, 27, 25, 96,85, 150, 28, *88,*72, 91, 16, 93,87, *116, 107,109

19, 23, 147, 24,27, 25, *149, 73,*88, 143, 154,*106, *72, 12,93, 92, 87, 77,*13, 13, 17,*116, 107,77, 109

21, *96, *90,*154, 92, 87,*77, 152, *13,23, 17, *116,109

85, 150, 28,91, 16

t1:29 3D 155, 149, 14, 97,97, 118, 145,151, *148, *104,108

89, 118,145, *148

153 156, 105, 15,117, *20

156, 155, 15,149, 105,117, 14, 97,97, 89, 118,118, 145, *20,*104, 108

155, 105, 118,145, 151, *148,97, *20

15, 153


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t1:30 Table 1 (continued)

t1:31 Osteogenic Outcome Proliferative Outcome

t1:32 Positive Negative Neutral NR Positive Negative NR

t1:33 Time of Biological Modelt1:34 NR 153, *90 21, 15, 27, 25,

96, 156, 85, 150,28, 105, 117,*88, *72, 91, 16,93, 87, *20,*116, 107, 109

85, 150, 153,91, 16

t1:35 1 hrs 147, 24, 97 147, 24, 97, *13,*116,

97

t1:36 2-12 hrs 74 *13, *116, 107,108

74, *116,

t1:37 13-24 hrs 73, 97, 118,*77, *13

*26, 74 130 23, 25, 156,*149, 130, 73,97, 143, 118,*13, *116, 107,77, 108

*26, 130, 74, 97,*90, *77, 152,23, 17, *116,

28, 15

t1:38 25-48 hrs *26, 74 19, 27, 155,17, 108

*96, 155, *26,105, 74, 75

15

t1:39 49-72 hrs 143, *104, *26, *106 23, 25, *149,143, *106, *13,*20, *104, 107,108, 109

*26, *90, 75, 13,23, *20, 17, 109

15

t1:40 4-7 days 23, 73, 92, 145,151, *77, *148,*13, *104, 77,108

75, *106,92, 118,*148, *13

23, 25, 155,*149, 73, *88,*106, 93, 92, 87,145, 68, *13,*20, *104, 107,77

105, *90, *154,92, 87, 118, 68,151, *77, *148,13, 21, *20

t1:41 8-14 days 23, 14, 143,*106, 12, 92,151, *148, 17,*104, 108

23, 89, 75,92, *148

155, 105, 14, 89,143, *106, *72,12, 68, 68, *104,

155, *90, *148

t1:42 15-21 days 19, 23, 14, 143,*106, 68, 152,*104,

23, 89 15, 14, 97, 89,143, *106, 68,77, *104

t1:43 22-28 days 155, 143, *104, 145 105, 117, 118, *104 155, 145t1:44 N28 days 149, 97, *154, 68 149, 117, 97,

154, 13

The studies have been categorized according to four characteristics: the type of CNT used, the biological model used, the substrate morphology and the durationof the cell culture. In each category the substrates performance is reported as ‘positive’, ‘negative’, ‘neutral’ or ‘not reported’. Bold references show outcomesthat showed statistical significance: P b .05; italics denote use of a multipotent cell culture; underlined references denote in vivo testing, and asterisks denote useof tissue culture plates as control medium. The positive osteogenic response for references24,147 are determined to have significant increase in osteogenicoutcome through comparison of osteogenic and non-osteogenic cell lines, while the other osteogenic outcomes are determined by increases to osteogenicmarkers. Proliferative outcome is determined from viability or proliferation assays, cell counts or increases to cell/protein mass. If a reference has bothstatistically significant and non-significant results for the one table cell entry only the statistically significant results are shown.t1:45


polar covalent bonds and a hydrophobic surface making themdifficult to disperse in solution. Such surface interactionscombined with Van de Waals forces and the high aspect ratioof CNTs, results in the agglomeration of CNTs. Preventing theformation of CNT aggregates remains one of the main challengesfacing the widespread use of CNTs. Aggregates prohibit theformation of a reinforcing or conducting network which couldotherwise toughen the material, increase its conductivity and/orchange its chemical properties. Accordingly, most applicationsof CNTs begin with the preparation of a homogeneousdispersion. Tissue-friendly dispersion mediums that have been

proposed for this purpose include carboxymethycellulose,68 aform of hydrolyzed collagen, gelatine69 and the tissue cultureprotein supplement bovine serum albumin.70 It should be notedthat for cells cultures which use aqueous CNTs (that is, CNTs notwithin a composite or bound to a substrate), the state of CNTdispersion affects their interactions with proteins71 and growthfactors.70

Graphitic carbon such as that in CNTs is able to chemicallybind to a wide range of functional groups. These chemical bondsare sufficiently weak that a diverse range of reactions will easilyproceed in a biochemical environment, but are also strong

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enough as to avoid chemical decay. When placed within in vitroenvironments CNTs adsorb extracellular and serum proteinswhich enhance interactions with cell cultures.72,73

The chemical interactions of CNTs can be modified throughthe addition of functional groups. Common functionalisationsinclude the addition of carboxyl or alcohol groups which arenormally used to assist the dispersion of CNTs in water. In 2006,Zanello et al. varied CNT functionalization to investigate the invitro behavior of osteosarcoma cells over CNT coated glasscover slips with a varying surface energy.21 The study looked atthe proliferative capability of osteosarcoma cells over function-alized and ‘pristine’ CNTs coated glass coverslips (CNTs with ahighly graphitic structure, lacking chemical or structural defectsas well as functionalisations are described as ‘pristine’). All CNTcontaining groups showed a reduction to overall proliferationrates when compared to the uncoated glass cover slip control.Unmodified SWNTs showed the best proliferative capabilities(59% of control cell count) closely followed by the chemicallyneutral group SWNT-(poly ethylene glycol) (57%), withMWNTs showing the worst performance (b15%).

Similar to the study by Zanello et al., a comparative studyinvestigating the effects of pristine SWNTs against carboxylfunctionalized SWNTs was completed by Mu et al. in 2009.74 Itwas shown that carboxyl groups inhibited cell proliferation by anonapoptotic mechanism: the cessation of the cell cycle by thesuppression of proliferation and differentiation associated Smad-dependant human bone morphogenic proteins. This mechanismis supported by Liu et al. who observed the inhibition of Smad,and hence BMP, when MSC were cultured in cell culturemedium containing carboxylated CNTs.75

The poor proliferative performance of MWNTs observed byZanello et al. may be a result of the cell culture medium that theyused. In 2010, Akasaka investigated the effects of changing theconcentration of fetal bovine serum (FBS) in CNT culturemediums.72 This study showed that cell proliferation on differenttypes of CNT films is significantly decreased relative to apolystyrene control when using culture mediums with lowprotein concentrations. At low concentrations of FBS (1%),SWNTs showed significantly higher proliferation when com-pared to MWNTs. Comparable proliferation for MWNTs wasn’tachieved until FBS concentrations were between 10–15%. Here,the relative increase in surface area and hence reactivity ofsmaller diameter CNTs may allow for a greater concentration ofproteins to bind with the CNT substrate. Strong interactionsbetween nanoparticles and proteins, or the formation of a protein‘corona’ is a typical behavior of nanoparticles since they have arelatively high reactivity as well as large surface areas.76 Theformation of a protein corona may then indirectly facilitatefavorable interaction between cells and CNTs. This may includecell behaviors such as adhesion or internalization. Such a processis analogous to heterogeneous catalysis wherein, reactions arecatalyzed over a surface by the immobilization of one or both ofthe reactants. This process facilitates cell-material interaction asit provides a surface over which reactions can occur, increasingthe local concentration of the reactants and in turn facilitatingcell-material interaction.

In heterogeneous catalysis theory, interactions can be furtherpromoted by high surface areas (such as those of CNTs)77 since

the surface area determines the availability of catalytic sites. Li etal. demonstrated increased rates of proliferation, osteogenicexpression and ectopic bone formation on the carbon isomorphsCNTs, when compared to graphite microparticles with arelatively smaller surface area.

Other studies have looked to define the protein corona ofCNTs in cell culture conditions.78 In this work, SWNTs orMWNT were carboxylated, coated with polyviynlpyrodine (acommon dispersant for CNTs) or used as purchased. They werethen incubated in cell culture conditions (DMEM with glutamaxand 10% FBS), washed, resuspended in PBS and their proteincorona was assessed using liquid chromatography with massspectroscopy. All nanotubes were found to associate with highaffinity to a common subset of proteins including albumin, titin,and apolipoproteins. However, there was a changed associatedwith the presence of functional groups. Unique proteins werefound on such CNTs suggesting that hydrogen bonding andelectrostatic interactions were important in the formation of theCNT protein corona. The authors were unable to demonstrate arelationship between the isoelectric point, hydropathy andaliphatic index further suggesting marginal contributions of pi-stacking and hydrophobic chemical interactions.

It is important to note that the protein corona is highlydependent on not only intrinsic properties of the corona, but also,the extrinsic properties of the environment. Furthermore, inbiological systems, additional and more complex actions byadsorbed proteins have been suggested. The adsorption processmay induce conformational changes to proteins that give accessto hidden protein sites responsible that may be responsible forcell adhesion or other downstream cell behaviors such asmigration or differentiation.79 Additional mechanisms in whichcells modify protein conformation through the application ofmechanical strains have also been proposed.80

In the study conducted by Zanello et al., the culture mediumcontained 5% FBS along with 5% Serum Plus (contains lowlevels of FBS along with other proteins for cell nutrition andgrowth). The study by Akasaka shows that such low concentra-tions of FBS are insufficient to support high levels ofproliferation, especially on MWNT containing substrates.

Zanello et al. also investigated the effects of CNTs on thefunction of the cell membrane; a regulator of protein secretionand thus the formation of bone matrix. This study showed thatthe electrical function of the cell membrane was maintained andthat calcium ion channel functions were enhanced in thepresence of neutral CNTs. Furthermore, the morphology andformation of hydroxyapatite was found to mirror that observedwhen the same cell type is cultured on woven bone.

As mentioned above, the carboxylation of CNTs is commonas it aids in the creation of a homogeneous dispersions. Whatremains unclear are the effects that carboxylation has upon CNT-cell interactions. This ambiguity is further convoluted since thisprocess can also have the effect of purifying the CNTs, changingthe size of the CNTs used for experimentation (as CNTs can bebroken by sonication and/or stirring, or are selectively used afterfiltration/centrifugation) and further changing the chemicalcomposition of CNTs with the addition of oxygen or alcoholgroups. When the new CNTs are placed onto a substrate they willbehave differently as a result of all of these parameters. What is

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unclear is the extent to which the various changes to the CNTschange their interaction with cells: is it a more homogeneousdispersion, a higher purification, the presence of differentfunction groups, a different surface energy or any combinationof these parameters that is affecting changes to cellularproliferation?

One possible mechanism by which carboxylated CNTs maysupport favorable interaction with osteogenic cells is through arelative increase in affinity for calcium and the resultingformation of bone mineral. In particular, the preferentialnucleation of bone-like carbonate apatite crystals from solutionswith carboxylated CNTs.62,64,81 Such an interaction may havefurther-reaching consequences. For example, in 2012 Shimizu etal. hypothesized the presence of a positive feedback cycleexisting through the bidirectional interactions of CNTs withcells.68 Here, CNTs attract calcium for bone mineralizationwhich then promote osteoblast differentiation and the release ofalkaline phosphatase. This in turn attracts more calcium and thusbegins the cycle again.

CNTs can also be functionalized with organic chemicalgroups including carbohydrates,82 nucleic acids83 and fats.71,84

Of greater interest to tissue engineers is the ability of CNTs toreadily form bonds with cell binding epitopes such asfibronectin.16 This demonstrates their potential as a biomimeticof the fibrillar proteins in the ECM and provides an opportunityto spatially control ECM proteins through changes to thedispersion and concentration of protein-bound-CNTs. In addi-tion, since chemical interactions between CNTs and organicmolecules can occur by non-covalent interactions such as π-stacking, the structure of both the CNTs and proteins will bepreserved.83

In 2006, Khang et al. hypothesized that protein interactionsbetween proteins and carbon nanofibers or CNFs (which differfrom CNTs in that they are stacked cones or horizontal layers ofgraphitic carbon rather than concentric cylinders) could be usedto enhance cell adhesion and proliferation.27 This was testedthrough the use of a polycarbonate urethane (PCU) substratepatterned with 30 μm stripes of CNFs. When cells were grownon this substrate, they preferentially adhered to the CNF stripedsections which, with use of atomic force microscopy, showed arelatively stronger interaction with the protein fibronectin whencompared to fibronectin interactions over the bare PCU sections.This again suggests that the biocompatibility of CNTs may bedue to the indirect interaction of cells with the adsorbed proteincorona rather than direct interaction with the material itself.85 Ina follow up study of this work in 2007, Khang et al. used CNTsto show that the adsorption of fibronectin is mediated by theindependent contributions of surface chemistry (70%) and thesurface roughness (30%).85 This again suggests that thebiocompatibility of CNTs is due to the indirect interactions ofan adsorbed proteins corona.

In 2007 Park et al. published work which further supports thehypothesis that the high biocompatibility of CNTs is due toindirect interaction with an adsorbed protein corona.28 Osteo-blasts were cultured over a patterned CNT substrate withalternating 3 μm thick stripes of randomly tangled CNTs and 1-octadecanethoil. Fluorescent tagging and imaging of fibronectinshowed an increase to fibronectin concentration over CNT

coated areas. The preferential cellular adhesion and growth overthe CNT patterned sections was then observed. Furthermore,increases to the concentration of fibronectin demonstrated furtherimprovement to cell adhesion. Such improvements wereobserved through an increase to the coincidence of vinculin(present at integrin adhesive sites) and actin (a protein in theintegrin linkage to cytoskeletal machinery). This demonstratesthe presence of a holistic integrin mediated adhesion betweencells and CNT substrates. Using this technique, the investigatorswere then able to control the morphology of mesenchymal stemcells (MSCs), thus demonstrating that CNT substrates could beused to as an effective means to control cell morphology – aregulator of stem cell fate.86 Namgung et al. also investigated theeffects of fibronectin interactions with CNT substrates wheresimilar increases to proliferation and integrin-mediated adhesionwere observed.16 Additionally, through attachment with an AFMtip, Namgung et al. showed that the unfolding force offibronectin bound to CNT substrates was unchanged whencompared to fibronectin its natural form. This indicates thatCNTs do not induce conformational changes to fibronectin andwe can therefore expect cellular interaction with fibronectinbound with CNT substrates to be similar to interactions withfibronectin in its natural form.

Vidal et al.87 reported similar effects to the study by Park etal. This study investigated the use of polymer and CNTsubstrates with both fibronectin and the short amino acidsequence RGD, a peptide epitope found in extracellular proteinssuch as fibronectin and thought to play roles in cell adhesion. Byfunctionalizing CNTs, Vidal et al. investigated the effectsbetween adhesion proteins, surface charge and cell behavior.Cell adhesion, spreading and proliferation were all enhanced inthe presence of fibronectin on both hydrophobic and hydrophilicCNT substrates, while RGD peptides only showed enhancementthese behaviors on hydrophilic (i.e. functionalized) CNTs. Theabsence of enhanced cell behavior when cells were cultured onhydrophobic CNTs with RGD peptides may be due to aninability for the RGD peptide to bind with the hydrophobicCNTs, thus indirectly inhibiting the potential for enhancedadhesion. This hypothesis is supported by the greater weightpercentage of carbon (measured by XPS) when comparing RGDpeptides on hydrophobic CNTs to those on hydrophilic CNTs(greater amounts of RGD peptide should decrease the carbonweight percentage since RGD peptides contain relatively lesscarbon than CNTs). Moreover, since cell behavior was enhancedon both hydrophilic and hydrophobic substrates that were treatedwith fibronectin, we expect to see similar amounts of fibronectin(i.e. weight percentage decrease in carbon) on the substrates: aresult supported by the XPS data in this study. This behaviorprovides additional support to the hypothesis that CNTs promotecell adhesion and maturation in directly through the adsorptionof a protein corona (as shown in Figure 5).

Many papers report conflicting evidence and, in fact, it ismore commonly observed that CNTs facilitate a high level of cellspreading, elongation and a flat morphologies.12,16,28,72,75,85,88–93

Many of these papers attributed this to the high affinity of CNTsto cell-adhesion stimulating proteins such as FBS or fibronectin,which increase integrin mediated adhesion,16,28,77,85,92–94 whileothers suggest the high aspect ratios and thus contact guidance

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Figure 5. Shows the adhesion of cells to CNTs. The adhesive properties ofsuch substrates may have an indirect mechanism, where binding betweenCNTs first adhere to cell adhesion mediators which in turn adhere to cells.The similarities of such a substrate and that of the natural ECM arenoteworthy (i.e. comparison with Figure 2); both contain fibrillarmacromolecules in meshes and bind with macromolecule proteins.


effects of CNTs12 enhance spreading. In addition, it is believedthat increased cell spreading can result in high intercellularstress, which is correlated to increased proliferation andosteogenic differentiation.36,86,95

Contrary results have been demonstrated by Mwenifumbo etal. who was unable to demonstrate high levels of cell spreading,elongation and flat morphologies with the use of a non-patterned2D MWNT substrate.25 Using vinculin and actin stainsMwenifumbo showed diffuse actin fibers with the non-coincidence of vinculin and actin. Mwenifumbo et al hypothe-sized this to be a result of the high tensile strength of CNTssuggesting that actin stress fibers were unable to organize, whichundermined the cells ability to spread. These results arecomparable to Kalbacova et al. who explain this behavior inanother manner.96 They attribute the relatively disorganizedcytoskeleton to the increase in nanoroughness of the CNTsubstrate, stating that increased nanoroughness corresponds toincreased nanodisorganisation and an inability to spread.

Another chemical interaction through which CNTs have beenfound to enhance osteogenic potential was demonstrated byNarita et al. with the use of pellets containing CNTs, BMP andcollagen in vitro with osteoblast and osteoclast cells.97 It washypothesized that CNTs increase bone mineralization by adisruption to osteoclastogenesis which ultimately preventsosteoclastic bone resorption. This hypothesis was supported bythe selective presence of CNTs inside osteoclast progenitor cellsand attenuation of genetic transcription necessary for osteoclas-togenesis. Furthermore, this study included an in vivo studywhere pellets were implanted into the dorsal musculature of ddYmice demonstrating a decrease to osteoclastic bone resorption asa result of CNTs.

One problematic aspect of CNT use is their chemicalinteraction with indicator dyes used to assess cell viability incell culture studies. In 2006 Wörle-Knirsch et al. showed thatMTT cell viability assays can show false negatives due to MTT-formazan crystal formation over CNT substrates.94 In the samestudy, the negative viability result from the MTT assays wascompared to other tetrazolium-based assays (WST, INT XTT)which did not interact with the CNTs. Using these and othermeasures of cell viability (LDH assays, FACS assistedmitochondrial membrane potential determination and AnnexinV/PI staining) they demonstrated a varying degrees of neutral orpositive effect results between CNTs and cell viability further

demonstrating the inconsistent nature of using such measures onCNTs. Another study in 2007 by Casey et al. showed thatCommassie Blue, Alamar Blue, Neutral Red, MTT and WSTassays all indicate varying degrees of cell viability.98 For thesereasons, Casey et al. suggested that the use of cytotoxic assaysthat rely on light absorbance should be avoided, insteadsuggesting an approach using cell counting or time consumingclonogenic assays. An alternative suggestion has been made byAli-Boucetta et al. who avoided interference between the LDHassay and CNTs by artificially lysing the cells with Triton X-100,centrifuging the cell lysate sample to precipitate the CNTs andthen incubating the assay as per normal.99 The above studieshighlight the need to carefully consider the possible interactionsbetween CNTs and cytotoxic assays.99 Alternative (such as thecell counting or clonogenic assays suggested by Casey et al.) ormodified techniques (see techniques of Ali-Boucetta et al.)should be used to avoid these complications.

Physical properties of carbon nanotubes

Another appealing property of CNTs is their high mechanicalstrength. They are among the strongest materials known withreported tensile strengths higher than 60 GPa,100 a valueapproximately three times that of bone.50 This makes themideal candidates for use as reinforcement in composite materials.Such a property may allow material scientists to address one ofthe more pertinent limitations of synthetic tissues: an inability tocreate materials with comparable mechanical properties to that ofnatural tissues. Unfortunately, the application of CNTs incomposite materials has been hampered by two problems: poorCNT dispersions and poor interfacial adhesion between CNTsand the composite matrix. Poor dispersions of CNTs result inagglomerates which act to increase local stress and seed failurewithin the composite100,101 while poor interfacial adhesion limitsthe amount of failure energy that can be shared between thedifferent constituents of a composite.101 Despite this, CNT-polymer composites have demonstrated increases to stiffness ofnearly 700%100 as well as increases to strength and toughness of1200%.102 With improvements this significant it may bepossible to fabricate CNT-polymer biomaterials with mechanicalproperties that rival the healthy tissue which they aim to replace(non-composite polymers typically have toughness’s a tenth thatof biologically created materials103).

The creation of CNT composite materials can improve boththe mechanical properties and proliferative capabilities of abiomaterial.13,104–109 In 2012, Ogihara et al. used CNTs toreinforce an alumina composite for bone tissue replacement.Mechanical testing demonstrated a 120% increase to fracturetoughness.13 The in vitro response of the substrate showedsuperior proliferative ability for osteoblasts, chondrocytes,smooth muscle cells and fibroblasts when compared to non-composite alumina. In an in vivo study, the composite substrateswere implanted into 15 week-old Japanese white rabbits wherethey demonstrated direct integration into the bone and acomparable or lessened inflammatory response to that of thealumina control. A similar result was shown by Jell et al., whereincreasing concentrations of CNTs in a CNT/PU composite both

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increased the compressive strength and proliferative ability of thesubstrate.105 Lin et al. is another such example where theinclusion of CNTs into PLGA substrate yielded a 500% increasein the stiffness and a 270% increase to the tensile strength.106

The composite was also able to demonstrate superior prolifer-ative potential when compared to either a substrate withoutCNTs or tissue culture plates.

As mentioned above, one appealing physical property ofCNTs is their dimensionality: CNTs can be synthesized withdimensions comparable to the proteins of the ECM. In 2007,Mwenifumbo investigated the in vitro behavior of osteosarcomacells over substrates with varying CNT diameter. This includedthree different diameter distributions centered at 14, 22 and53 nm. While all of the CNT groups showed increases toproliferation when compared to the highly ordered pyrolyticgraphite control group, cell proliferation was shown to benegatively correlated to CNT diameter. This trend may be aresult of the interaction between CNTs and proteins as describedabove: smaller diameter CNTs have relatively greater surfaceareas and hence a greater potential to bind with growthpromoting and adhesive proteins.

The reverse of this trend is revealed by regrouping the data ofTable 1. Figure 6, A and B show that CNTs with larger diametersbetter promote both proliferation and osteogenic differentiation.Such differences could be the result of a balance between thepositive and negative effects of smaller CNTs. While smallCNTs may have a greater interaction with growth promotingproteins, small particle sizes are also associated with anincreasing potential for toxicity by the disruption of biochemicalprocesses, the creation of reactive oxygen species (ROS),increased reactivity110 and the rupture or penetration of thecell membrane.94 Furthermore, on average, smaller diameterCNTs contain a larger percentage of the toxic transmission-metalcatalyst particles required for CNT growth.

Another physical feature of significance in tissues is thepresence of patterns. The use of patterns in synthetic substratesmay help to orientate cells, as cell orientation is present in almostall natural tissues.37 Different cell types favorably interact withpatterns which have micro and nanoscale features.4,36 Twoexamples of favorable cell interactions due to substratepatterning have already been given above when discussing thechemical properties of CNTs.27,28 These studies demonstratedthat microscale rows of CNTs increased osteogenic cellproliferation.

A study in 2011 by Namgung et al. was able to provide moreinsight into the mechanisms which may be responsible forincreased proliferation on CNT substrates.12 By spin coatingglass substrates with CNTs Namgung et al. were able to alignSWNTs using centrifugal forces. When compared to unalignedor randomly-orientated CNTs the patterned substrate showed a120% increase in proliferation as well as an increase in theexpression of osteogenic markers osteocalcin, osteopontin,alkaline phosphatase and Runx2. This also included the down-regulation of genes DKK1 and sFRP3 which are known to inhibitosteogenesis. Namgung et al. hypothesized that increases toproliferation and osteogenic differentiation were achieved by thecontact guidance effects of the CNTs. This was achieved byusing a CNT density that was sufficiently low (~1 CNT/μm),

such that adjacent CNTs were further apart than their adhesions(~0.25 μm). Under these conditions cells would preferentiallyspread along the axis of CNT alignment. Such spreadingincreases cytoskeletal tension activating mechanotransductivepathways (measured as increases to adhesion mediators RhoA,ROCK2 and FAK) and promoting osteogenic differentiation.This result has also been demonstrated with the use ofelectrospun CNT/polymer composite fibers, where alignmentincreased spreading and proliferation,20 or with sheets of alignedCNTS that have been drawn from the side of vertically alignednanotube ‘forests’.111

Electrical properties of carbon nanotubes

Carbon nanotubes can display either metallic or semicon-ducting electronic properties according to the chirality of thenanotube. While methods to synthesize chiral specific nanotubesdo exist,112 the aforementioned variations within any given CNTproduct as well as non-orthogonal relationships between CNTsynthesis variables makes the fabrication of CNTs with specificelectronic problems problematic. Instead, methods to obtain aspecific electronic properties favor the use of post-processingsuch as ultra-high speed centrifugation. For the above reasons,any sample of CNTs typically displays a wide range of electronicproperties, even though the CNTs may superficially appear thesame in length, diameter or otherwise. Despite these problems,bulk samples of CNTs display high conductivity – a propertywhich may be leveraged in bone tissue regeneration.

It has been shown that conducting substrates can be used toelectrically stimulate tissue thereby accelerating bone formationand repair113 as well as increasing the rate of ECM proteinsynthesis, expression of osteogenic markers114 and production ofrelevant growth factors.115 The aforementioned benefits provideCNTs with the potential to fulfill the stringent criteria requiredfor an electrically active biomaterial; a criteria matched only bygraphene. While metal electrodes have the correct conductiveproperties, they have insufficient biocompatibility and thusrequire removal. In addition, metals cannot be dispersed as anetwork within a composite because this worsens theirtoxicological potential and the difficulties associated with theirremoval. Unable to form networks, metals cannot achievegrowth throughout a substrate as electrical activity is confined tothe tips of the electrode. Likewise the use of polymers orceramics has not yielded a material that has simultaneouslyshown sufficient conductivity and biocompatibility.19 Thus,CNTs and CNT composites show promise for use as electricallyactive biomaterials with high conductivity, biocompatibility anda potential to be distributed in a network throughout a compositethus inducing uniform bone growth throughout.

The first reported use of CNTs as an osteogenic biomaterialwas in 2002 by Supronowicz et al.19 In this study, CNTs weredispersed through polylactic acid in order to enhance itsconductive properties and thus induce cell growth throughelectrical stimulation. This involved the preparation of a 1 mmthick, 40 mm diameter disk for in vitro study with osteoblasts.This substrate showed that CNTs can effectively expose cells toelectrical stimulation, leading to an increase in cell proliferation

Figure 6. (A, B) Each respectively show the proliferative and osteogenic outcome for studies using CNT as an osteogenic biomaterial broken down by CNTdiameter and type. Diameter is measured in nanometers and shown at left along with the CNT type. SWNT andMWNT are CNTs as prepared, annealed, purifiedor pristine while COOH-SWNTs and COOH-MWNTs are those functionalized with carboxyl groups. (C) Proliferative outcome data from the same studies but isbroken down according to the substrate morphology: CNTs in dispersion mediums are ‘aqueous’ and CNTs bound are 2D or 3D substrates are ‘2D’ and ‘3D’accordingly. These figures all show the percentage of study data points which have positive, positive P b 0.05 (stat.sig.pos.), negative, negative P b 0.05(stat.sig.neg.), neutral or not reported (NR) proliferative or osteogenic outcomes against the CNT diameter and type (A, B) or the substrate morphology (C). Atthe center of each bar the number of data points is shown. These figures are a reinterpretation of the data included in Table 1.A andB show that CNTs with largerdiameters better promote both proliferation and osteogenic differentiation. C shows CNTs in an aqueous dispersion have lower proliferative potential whencompared to 2D and 3D substrates to which CNTs are bound.


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of 46% and the increase in osteogenic expression markersincluding extracellular calcium, collagen, osteocalcin, osteonec-tin and osteoprotegerin.

In 2011, Shao et al. showed that nanotopology and electricalsimulation had synergistic effects on cell proliferation.20 Thiswas achieved with the use of a composite material made fromelectrospun PLA/MWNT nanofibers. Samples with eitherrandomly orientated or aligned CNTs were created. Thematerials were then cultured with osteoblasts with or withoutthe electric stimulation. Without electrical stimulation, the cellsshowed greater extensions, directional outgrowth and prolifer-ation over fibers containing aligned CNTs. With electricalstimulation, there were further increases to proliferation and cellalignment along the direction of electrical stimulation for bothrandomly orientated and aligned fiber types. While alignmentalone improved cell elongation and proliferation, electricalstimulation was shown to have much more significant effects,which became more pronounced from 3 to 5 and then 7 days.Electrical stimulation was applied at 0 (control group) 50, 100and 200 μA, with statistically significant increases to prolifer-ation (as indicated by Alamar blue assay) for 50 and 100 μA, buta decrease to proliferation for 200 μA. The researchershypothesized that 200 μA exceeded the osteoblasts tolerancefor electrical stimulation and hence led to cell death. Similarresults were shown Vila et al. in 2013 where it was shown thatelectrical stimulation can have positive synergistic effects to cellproliferation when combined with the drug Zoledronic acid fortreating osteoporosis.116

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The in vivo response of carbon nanotubes

Abarrategi et al. evaluated a freeze cast nanocomposite madefrom CNTs (N80 weight %) and chitosan for use as a syntheticbone tissue scaffold.15 It included two groups: one with and onewithout BMP in the scaffold. These scaffolds were implantedinto subcutaneous tissue made in the back tissue of Balc typemice for 3 weeks. They observed the degradation of the scaffold,no indication of chronic inflammation and a high expression ofcollagen concluding good tissue biocompatibility and potentialfor future applications.

Later in 2008, Sitharaman et al. evaluated CNT composites invivo for use as a synthetic bone substrate.117 This involved thepreparation of a porous biodegradable polymer composite withthe inclusion of short (20–80 nm) SWNTs. When the in vivoresponse of CNT containing substrates was compared to acontrol without CNTs, the CNT composite showed 300%increase in bone area as observed in micro-CT analysis andbone area under histological analysis. The histological scoring at12 weeks was also significantly higher, indicating higher tissuequality, greater tissue ingrowth, reduced inflammatory celldensity, increased connective tissue organization and a greaterbone volume. Similar results were shown by Hirata et al. in2011.118 The in vivo subcutaneous implantation of a CNT/collagen tissue substrate showed minimal inflammation at 1 and4 weeks with a significant increase to bone formation around thesubstrate at both 4 and 8 weeks.

The favorable interaction of CNTs in vivo was furthersupported by Usui et al. in 2008.14 In this study, an aqueousinjection of MWNTs was found to induce little local inflamma-tion and high osteoinductivity, integrating directly into the bonematrix. This result was also demonstrated by Li et al.77 andOgihara et al.13 who observed the direct integration of CNTcontaining substrates into the newly formed bone matrix.

Toxicological effects of carbon nanotubes

As the particle size of a material decreases, there is acorresponding elevation in reactivity and hence toxicological andinflammatory potential.110 This is because nanomaterials such asCNTs have relatively high surface area to volume ratios overwhich more chemical interactions can occur. Furthermore,nanomaterials share similar dimensional scales to many largebiological molecules rendering them inherently more likely todisrupt natural interactions and cause toxicity.119 For thesereasons, the toxicological response of CNTs and all nanomater-ials in general is a pertinent issue regarding their use in medicalapplications. This issue is further complicated by the fact thatCNTs have demonstrated both positive117 and negative120

toxicological effects and that the mechanisms regulating theirtoxicity are still to be understood.121

While this can be partly attributed to experimental differ-ences, such as the different cytotoxic assays mentionedabove,94,98 most of the variability is suspected to be reflectiveof the wide range of properties that CNTs may have. Asdiscussed above, CNTs may have functional groups or defects intheir side walls; they can be conducting, or semi conducting; theycan be short, long, thick, thin, multiwalled or singlewalled; andare often still bound to the catalytic metal particle from whichthey grow. The many different properties of CNTs reflect themany different potential toxicological interactions and pathwaysthat these may have, and indeed, their toxicity is dependent onnot only their chemical and dimensional properties but also,factors such as dosage and method of uptake.71

In bone tissue applications, CNTs in bulk, within a compositeor attached to substrate have been shown to promote cellproliferation, tissue formation and have been evidencedharmlessly integrating into the bone matrix.13,15,77,117 For thisreason, CNTs in such applications are only thought to posehealth risks when migrating from the desired site of implantation.Such behavior is problematic since CNTs in dispersion/detachedfrom a substrate show a different toxicological profile to those incomposites or attached to a surface. Dispersions show toxicity asa result of cell lysis, membrane rupture and/or interaction withbiomolecules. For this reason, the following section is primarilyconcerned with the toxicological effects of CNTs is dispersion.

Toxicological Mechanisms – Reactive Oxidative Species

The most widely researched toxicological mechanism is thecreation of ROS as a result of chemical interactions withCNTs.110 Such interaction can induce in vitro apoptosis by theformation of free radicals, accumulation of peroxidative productsand the depletion of cell antioxidants.110 While ROS aresuspected to have many pathways of action, Mu et al.

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demonstrated that ROS disrupted the in vitro cellular behavior bychanges to DNA transcription regulators.74

Several studies suggest that most of the toxicologicaleffects of CNTs are in response to the catalytic metal particlesused for CNT growth.69,122,123 These metal particles oftenremain attached to CNTs and their presence results in theproduction of ROS and thus acute toxicity. In order to avoidsuch effects, the purity of CNTs used in medical devices mustbe very high. Furthermore, high purity CNTs are essential torealizing their unique theoretical properties which are dimin-ished due to metal and graphitic impurity. The purification ofCNTs is commonly achieved by chemical routes such asoxidation in aqueous solution, physical methods such ascentrifugation and thermal methods such as annealing or anycombination of these. It should be noted that these techniquescan have undesirable affects. For example, depending on thestrength and time for which an oxidant is used, chemicalpurification can introduce different amounts of alcohol,carboxyl and ketone groups.124 This means that purifiedCNTs will have different chemical and electrical properties tothat of the CNTs containing impurities. Such changes can alsooccur due to physical methods of purification. Centrifugationand sonication can both have the effect of shortening CNTs asa result of fracture which in turn changes CNT electrical,chemical and mechanical properties. For a review on CNTpurification see references.125,126

Although it is true that CNTs in suspension can cause cellapoptosis,75 CNTs within composites, bulk or bound to a surfaceseem to minimize many of the toxic pathways.22,127 In thesecontexts, CNTs have demonstrated favorable biological re-sponses, particularly with osteogenic14 and neuronal celllineages.128 Such positive behavior indicates a strong potentialfor CNT use as substrates for tissue growth.

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Toxicological mechanisms – membrane rupture

Another proposed mechanism of CNT/cell toxicity is throughdamage to the cell membrane. The ability of CNTs to rupture thecell membrane has been demonstrated in vitro.121 Specifically,CNTs that rupture the cell membrane can migrate and depositwithin cells. CNT have been observed in cellular cytoplasm,129

within the mitochondria of lung epithelial cells,94 or depositedalong the edge of a the nucleus in mesenchymal stem cells.130

Such needle-like structures are poorly dealt with by theimmunological system and evoke comparison with asbestos.131

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Toxicological mechanisms – biochemical disruption

CNTs within an organism may induce a cytotoxic response bya different mechanism: the disruption of biochemical processes.This follows from the potential for CNTs to easily bond withvarious life supporting organic chemicals. Such disruptions mayoccur to inter- or intracellular signaling, metabolic processes andprotein synthesis.132 This may be further exaggerated by the factthat CNTs can deposit along the nuclear membrane, possiblyinteracting with cell metabolites, chromatins or other geneticmaterials.74

Toxicological effects – skin toxicity

At present, little is known about the uptake mechanism ofCNTs.122 Due to their scale, it is suspected that nanomaterialsmay penetrate, be absorbed by or find their way into an organismthrough virtually any means.110 Since skin forms our outermostlayer of protection, it is also the most likely to interact withforeign substances. Huczko and Lange reported no toxicologicaleffects of CNTs when routinely applied to a skin patch of 40human volunteers over 96 hours. This included no presentationof allergenic symptoms, irritation or abnormalities otherwise.They also found this result for the application of CNTs to the eyeof a rabbit model. After 72 hours from the time of application,there was no allergenic or irritation observed. Since these studiesonly investigated short-term health effects of CNTs, cautionwhile handling CNTs is still advised.

Toxicological effects – respiratory toxicity

CNTs are believed to present the highest risk of adversehealth effects to the respiratory system due to their light weightand propensity to be aerosolized and inhaled. In 2004, Lam et al.showed CNTs to cause granulomas in the lung tissue of mice as aresult of intra-tracheal instillation.120 Increasing doses corre-sponded to higher rates of inflammation and prominence oflesions. When compared to the quartz positive control, a knowncause of granulomas and lung lesions, CNTs showed a higherincidence of negative health effects. This included increasedinflammation, fibrosis, granulomas, necrosis, and lethality.Similar results have been demonstrated repeatedly.133–135

Comparing more directly to asbestos, in which the mesotheliumof the lungs is affected, Poland et al. exposed the mesothelialtissue lining the lung/body cavity of mice to CNTs with a medianlength of approximately 20 μm.136 Toxic behavior wasevidenced including inflammation and the formation ofgranulomas. Such effects were not present for the controlgroup, which were exposed to carbon black, nor for the groupsexposed to short CNTs (median length less than 5 μm) or tangledmats of short CNTs. Contrary to this, Mutlu et al. demonstratedthat the toxic effects of CNTs were linked to particle aggregationrather than CNT length.137 Following intratracheal administra-tion of poorly dispersed and well dispersed CNTs to mice, onlymice treated with poorly dispersed CNTs showed the develop-ment of granuloma-like structures. Furthermore, the uptake andclearance of CNTs was observed by macrophage cells for thegroups administered with well-dispersed CNTs. While thissuggests that CNTs can be processed to minimize negative healtheffects, the relevance of this result is questionable. CNTs aredifficult to disperse and studies that make concerted efforts tocreate homogeneous dispersions which are then injected orinhaled directly are unrealistic. Consequently, concerns regard-ing the negative health efforts of exposure to CNTs arewarranted.

Toxicity effects – concentration

The toxicity of CNT suspensions has been demonstrated byZhang et al. who prepared CNT suspensions of SWNTs, double-walled-CNTs and MWNTs that, when cultured with osteoblast

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Figure 7. Differences in the morphology of hMSC grown over (A) a glasssubstrate coated with aligned CNTs and (B) a glass substrate with unalignedor randomly orientated CNTs (reprinted with permission from Namgung etal. (2011), Copyright 2011, American Chemical Society).


cells, showed cell proliferation and an inverse proportionalitybetween proliferation and CNT concentration.26 An inhibitedosteogenic response was also measured by decreases to calciummineralization as well as type-1-Collagen and the expression ofthe osteogenic marker Runx2. Similar results were observed byMooney et al.,130 Mu et al.,74 and Liu et al.,75 where theconcentration of CNT in suspension had a negative correlationwith cell proliferation. Both Liu et al. and Mu et al. were alsoable to demonstrate a reduction in osteogenic potential for cellsin CNT mediums. This can also be shown through therepresentation of the data from Table 1 shown in Figure 6, Cdemonstrating that CNTs in an aqueous dispersion have a lowerproliferative potential when compared to 2D and 3D substrates towhich CNTs are bound or form a composite.

Toxicological effects – clearance and biopersistence

The biopersistence of CNTs warrants additional concernswhen considering them for biological uses, particularly since thisvalidates their comparison with the carcinogen asbestos. Whilethis is concerning, the in vivo study of mice has demonstratedthat ‘pristine’ CNTs have a relatively low half-life of 3 hours inthe bloodstream.138 Through the use of various imagingtechniques, the in vivo study of mice has also shown thatCNTs pose risks specifically to the lungs, liver and spleen,71,138–141 persisting in these locations at up to 10% of the initial dose atthe studies completion time of 28 days.71 Furthermore it ispossible that the toxicological behavior of CNTs activates at acertain threshold meaning that this activation barrier would needto be identified and further dosage of CNTs limited according tothe biopersistence (Figure 7).69

An example of CNT biopersistence is the observation byMooney et al. who showed that CNTs dispersed in cell culturemedium can migrate and remain at the edge of the cell’snucleus.130 Usui et al. also observed the migration of CNTs intothe extracellular location of the bone matrix.14 While evidenceexists to support the benign nature of such integration intobone,117,118 CNTs in different locations such as the mesotheliumand lung tissues can form granulomas, as discussedabove.120,133,136 Consequently, the approval for CNTs to beused clinically will be greatly accelerated by demonstrating theircomplete removal from the body.

Removal through biodegradation has been demonstrated fromfunctionalized carbon nanotubes. Although pristine, non-func-tionalized nanotubes have been shown to resist biodegradation,the biodegradation of functionalized SW- and MWNTs has beendemonstrated by neutrophils and macrophages as well as bysimulated biodegrading fluids: horseradish peroxidase andphagolysosomal fluid.142 The oxidizing actions of enzymaticfluids are believed to slowly exfoliate the CNTs, degrading themover time into CO2. In these studies, the rate of degradation isshown to be proportional to degree of functionalization. Thissuggests that the appropriate functionalization can minimize thehealth concerns of CNTs.

Toxicological effects – time based in vitro behavior

Tutak et al. observed another toxicological behavior of CNTsubstrates when comparing cell proliferation at different time

points.143 When compared to the control, no significant changesin proliferation were present at 1 through to 8 days of cellculture, however, as time periods increased past 8 days, so didrelative increases to cell proliferation over the CNT substrates.Furthermore, it was found that the proliferation of osteoblasticcells on CNT substrates required a minimal seeding density.Cells cultured below densities of 9000 cells/cm2 resulted in notonly initial decrease, but also the death of the entire cellpopulation. Tutak et al. hypothesized that this is a result of theinteraction of loose CNTs which are, as mentioned above,believed to have a greater toxicity when compared to thosebound on substrates. It was also observed that for cell culturesseeded with densities above 9000 cells/cm,2 there was an initialplateau in cell proliferation followed by exponential increase.Tutak et al. describe this as the ‘rebound effect’. In this situation,it is hypothesized that loose nanotubes are responsible for theinitial cell toxicity. When this initial period of cell death islimited, long term cell proliferation is increased by the subsidingeffects of loose CNTs and the release of growth factors fromdead cells. The ‘rebound effect’ is an explanation for therelatively low proliferation of osteogenic cells observed bystudies which use low cell-seeding densities or durationsinsufficient to observe the recovery of cell proliferation.

The delayed increase in cell proliferation on CNT substrates isan effect that has been observed in a number of studies.Observation of the ‘# of days in culture’ category in Table 1shows that references23,92,105,106,130,144 all initially have arelatively decreased proliferative or osteogenic ability whencompared to a control, while as time increases the CNT substratesoutperform the non-CNT substrates. This trend can also beobserved through the representation of this data shown inFigure 8, where, the total percentage of studies showing a positiveeffect on proliferation is greater for trials lasting a longer time.

While evidence of the rebound effect does exist, itsprominence and the extent of its action are disputable. The

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Figure 8. Shows the proliferative outcome for studies using CNT as an osteogenic biomaterial broken down by the time at which the proliferative outcome wasmeasured. Specifically it shows the percentage of study data points which have positive, positive p b 0.05 (stat.sig.pos.), negative, negative P b .05(stat.sig.neg.), neutral or not reported (NR) outcomes. At the center of each bar the number of data points is shown. This data is a reinterpretation of the dataincluded in Table 1.


presence of loose CNTs is not inevitable, nor is the subsidence oftheir negative interaction. Many studies investigating CNT-cellinteractions show a long-term worsening in proliferativeability.26,90,118,145 Furthermore, while toxicology can vary dueto the properties of CNTs, it may also vary due to extrinsicproperties such as cell type, for example, Narita et al. showednegative interaction specific to osteoclastic cells.146 In otherwords, while some CNT substrates or cell types may becharacterized by an initial stage of cell death, and later, througha subsiding concentration of loose CNTs, a stage of proliferation;other substrates and cell types may maintain toxicity through thesustained CNT detachment, biomagnification or creation ofROS. The study in which Tutak et al. proposes the ‘reboundeffect’ uses carboxylated CNTs which may have been respon-sible for complex effects such as those described by Mu el at.Such effects may have gone unnoticed since, as previouslynoted, the use of carboxylated CNTs is commonly used to aidCNT dispersion.147,148

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Conclusion

The dependency of cellular behaviors on nanoscale details is areflection of the fact that tissue is a complex and highly organizedstructure which integrates details at every scale to performfunctions. Consequently, successfully engineering functionalsynthetic tissues will rely of our ability to manufacture andcontrol environments with comparable complexity and detail.While this is yet to be achieved, approaches which make use ofnanomaterials such as CNTs have demonstrated an ability topositively influence cell behavior and move us closer to reachingthis goal. Specifically, CNTs have shown positive in vivobehavior,13,14,77,117 while in vitro experimentation has demon-strated that CNTs can be used in a variety of different ways toincrease both cell proliferation and osteogenic differentiation.These methods include using CNTs as a conducting material toexpose cells to electrical stimulation19,20,116; adding CNTs with

specified morphologies to control nanoscale architecture23–26 oradd specified patterns to a substrate12,27,28; creating CNTcomposite materials to improve the mechanical properties and/or biocompatibility of a material13,104–106; and changing thechemical behavior of a material through the addition of CNTswith simple functional or biochemical groups.21,62,74 Further-more, the use of CNTs in synthetic bone substrates may allowtissue engineers to take advantage of the fibrillar/ECM-protein-mimicking morphology of CNTs; their affinity to adsorb a coronacomposed of proteins that stimulate cell-adhesion16,28,85 andpreferential adhesion and strengthening of integrin mediatedadhesions between CNTs and cells.12

Through discussion of the studies above we have proposed anumber of mechanistic explanations for the interactions betweenCNTs and biological environments. In particular, we suggest thatthe positive proliferative ability of CNTs may occur through anindirect route. Here, proteins involved in enhancing cell behaviorsuch as adhesion and proliferation may preferentially bind withCNTs which in turn enhance cell behaviors.149,150 We alsosuggest that CNTs may have time dependent effects on cellproliferation characterized by an initial period of cell death.Following this, the toxic potential of CNTs is removed and cellsthen increase proliferation. This may require that cells are seededat a minimum density to overcome the toxicological effects andrecover. We also propose that aqueous dispersions of CNTsmaximize toxicity since the toxicological pathways of CNTssuch as cell lysis, interaction with ROS and disruption ofbiochemical pathways are maximized while in an aqueousdispersion. Additionally, because toxicological pathways areminimized when CNTs are entrapped or bound to substrates,bulk CNT or CNT composite biomaterials will maximize thepositive biological effects of CNTs when compared to materialswithout CNTs.151,152

From the many different applications and successes, it is clearthat the use of CNTs in bone tissue engineering has a greatpotential. However, in order to reach this potential, there are stilla number of issues needing to be addressed. While the individual

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effects of diameter, various functionalisations, patterning andsubstrate architecture are discussed above, the mechanismsthrough which these properties (or combinations thereof) affectcell behaviors are still unclear.153,154 This is because there is stilla great deal of inconsistency between studies investigating theeffects of CNTs on cell behavior. Research which controls forthese properties and elucidates the mechanisms of interactionwill allow further progress for the future use of these materials intissue engineering. Furthermore, by realizing this goal, it will bepossible for tissue engineers to take advantage of the variety ofproperties discussed above and thus tailor make CNT substratesoptimized for the most favorable tissue engineeringoutcomes.155,156

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Acknowledgments

The authors acknowledge the facilities, and the scientific andtechnical assistance of Victor Lo with SEM and TEM analysis,and of the Australian Microscopy and Microanalysis ResearchFacility at the Australians Centre for Microscopy and Micro-analysis, The University of Sydney. The authors also thankTamara Church, Kieran Mackenzie and Alice King for theirassistance editing this work.

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Carbon nanotubes: Their potential and pitfalls for bone tissue regeneration and engineering

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