nanotechnology for tissue regeneration
TRANSCRIPT
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ano Today (2009) 4, 66—80
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anotechnology and nanomaterials: Promises formproved tissue regeneration
ijie Zhang, Thomas J. Webster ∗
ivisions of Engineering and Orthopaedics, Brown University, 182 Hope Street, Providence, RI 02912, USA
eceived 23 September 2008; received in revised form 14 October 2008; accepted 15 October 2008
KEYWORDSNanomaterials;Tissue engineering;Nanotechnology;Scaffold;Biomimetic;Regenerativemedicine
Summary Tissue engineering and regenerative medicine aim to develop biological substitutesthat restore, maintain, or improve damaged tissue and organ functionality. While tissue engi-neering and regenerative medicine have hinted at much promise in the last several decades,significant research is still required to provide exciting alternative materials to finally solve thenumerous problems associated with traditional implants. Nanotechnology, or the use of nano-materials (defined as those materials with constituent dimensions less than 100 nm), may havethe answers since only these materials can mimic surface properties (including topography,energy, etc.) of natural tissues. For these reasons, over the last decade, nanomaterials havebeen highlighted as promising candidates for improving traditional tissue engineering materials.Importantly, these efforts have highlighted that nanomaterials exhibit superior cytocompatible,mechanical, electrical, optical, catalytic and magnetic properties compared to conventional (or
micron structured) materials. These unique properties of nanomaterials have helped to improvevarious tissue growth over what is achievable today. In this review paper, the promise of nano-materials for bone, cartilage, vascular, neural and bladder tissue engineering applications willbe reviewed. Moreover, as an important future area of research, the potential risk and toxicityof nanomaterial synthesis and use related to human health are emphasized.© 2008 Elsevier Ltd. All rights reserved.oc
anotechnology and nanomaterials:
iomimetic tools for tissue regenerationn 1959, Nobel award winner Richard Feynman first proposedhe seminal idea of nanotechnology by suggesting the devel-
∗ Corresponding author. Tel.: +1 401 863 2318;ax: +1 401 863 9107.
E-mail address: Thomas [email protected] (T.J. Webster).
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pment of molecular machines. Ever since, the scientificommunity has investigated the role that nanotechnologyan play in every aspect of society. The intrigue of nanotech-ology comes from the ability to control material propertiesy assembling such materials at the nanoscale. The tunableaterial properties that nanotechnology can provide were
tated in Norio Taniguchi’s paper in 1974 where the term‘nanotechnology’’ was first used in a scientific publication1,2]. Nanotechnology has achieved tremendous progress inhe past several decades. Recently, nanomaterials, whichre materials with basic structural units, grains, particles,
Nanotechnology and nanomaterials: Promises for improved tissue regeneration 67
Figure 1 (A) Scanning electron microscopy (SEM) image of poly(L-lactic acid) (PLLA) nanofibrous scaffold with interconnectedspherical macropores created by a phase-separation technique [6]. (B) Electrospun polycaprolactone/hydroxyapatite/gelatin(PCL/HA/gelatin, 1:1:2) nanofibers which significantly improved osteoblast functions for bone tissue engineering applications [7].
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(C) Densely aligned single wall carbon nanotube (SWCNT) forest[8]. (D) Transmission electron microscopy (TEM) image of monodhexane dispersion and dried at room temperature [9].
fibers or other constituent components smaller than 100 nmin at least one dimension [3], have evoked a great amount ofattention for improving disease prevention, diagnosis, andtreatment.
The intrigue in nanomaterial research for regenerativemedicine is easy to see and is wide spread. For example,from a material property point-of-view, nanomaterials canbe made of metals, ceramics, polymers, organic materialsand composites thereof, just like conventional or micronstructured materials. Nanomaterials include nanoparti-cles, nanoclusters, nanocrystals, nanotubes, nanofibers,nanowires, nanorods, nanofilms, etc. To date, numerous top-down and bottom-up nanofabrication technologies (such aselectrospinning, phase separation, self-assembly processes,thin film deposition, chemical vapor deposition, chemicaletching, nano-imprinting, photolithography, and electronbeam or nanosphere lithographies [4]) are available tosynthesize nanomaterials with ordered or random nanoto-pographies (Fig. 1, [6—9]). Nanomaterials can also be grownor self-assembled into nanotubes/nanofibers which can evenmore accurately simulate the dimensions of natural enti-
ties, such as collagen fibers. After decreasing material sizeinto the nanoscale, dramatically increased surface area, sur-face roughness and surface area to volume ratios can becreated to lead to superior physiochemical properties (i.e.,mechanical, electrical, optical, catalytic, magnetic proper-istcb
n with novel water-assisted chemical vapor deposition in 10 minrsed magnetic Fe3O4 nanoparticles (6 nm) deposited from their
ies, etc.) [5]. Therefore, nanomaterials with such excellentroperties have been extensively investigated in a wideange of biomedical applications, in particular regenerativeedicine.With the striking increase in the world’s population,
here are enormous demands each year for various biomed-cal implants to repair diseased or lost tissues. However,onventional tissue replacements (such as autografts andllografts) have a variety of problems that cannot satisfyigh performance demands necessary for today’s patient.onsequently, tissue engineering (or regenerative medicine)merged initially defined by Robert Langer and Josephacanti as ‘‘an interdisciplinary field that applies therinciples of engineering and life sciences toward the devel-pment of biological substitutes that restore, maintain, ormprove tissue function’’ [10]. However, it is clear thatoday, materials used in a wide range of tissue engineeringpplications still require improvement. Since natural tissuesr organs are nanometer in dimension and cells directlynteract with (and create) nanostructured extra-cellularatrices (ECM), the biomimetic features and excellent phys-
ochemical properties of nanomaterials play a key role intimulating cell growth as well as guide tissue regenera-ion. Even though it was a field in its infancy a decade ago,urrently, numerous researchers fabricate cytocompatibleiomimetic nanomaterial scaffolds encapsulating cells (such
68 L. Zhang, T.J. Webster
Figure 2 The biomimetic advantages of nanomaterials. (A) The nanostructured hierarchal self-assembly of bone. (B) Nanophasetitanium (top, the atomic force microscopy image) and nanocrystalline HA/HRN hydrogel scaffold (bottom, the SEM image). (C)S s maT ral be al m
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chematic illustration of the mechanism by which nanomaterialhe bioactive surfaces of nanomaterials mimic those of natufficiently stimulate more new bone formation than convention
s stem cells, chondrocytes and osteoblasts, etc.) for tissuengineering applications. In this review, we will focus onhe recent progress of the use of nanomaterials for bone,artilage, vascular, neural and bladder tissue engineeringpplications in vitro and more importantly in vivo. As theext frontier in nanotechnology research, toxicity concernsf nanomaterials and nanoparticles during manufacturingnd/or implantation will be covered as well.
he promise of nanomaterials for bone andartilage tissue engineering applications
oday various bone fractures, osteoarthritis, osteoporosisr bone cancers represent common and significant clin-cal problems. The National Center for Health StatisticsNCHS) reported that bone fractures for all sites num-ered 1,039,000 in 2004 in the U.S. In addition, around18,700 patients (home health care) had osteoarthritis and
ssociated disorders in 2000. The American Academy ofrthopedic Surgeons also reported that in just a 4 yeareriod, there was an 83.72% increase in the number of hipeplacements performed from nearly 258,000 procedures in000 to 474,000 procedures in 2004 [11]. Such traumaticiboac
y be superior to conventional materials for bone regeneration.ones to promote greater amounts of protein adsorption andaterials.
one and cartilage damage happens frequently each year. Aimilar trend has been documented for other industrializedountries as well. However, traditional implant materialsnly last 10—15 years on average and implant failures orig-nating from implant loosening, inflammation, infection,steolysis and wear debris frequently occur. It is clearlyrgent to develop a new generation of cytocompatible bonend cartilage substitutes to regenerate bone/cartilage tis-ue at defect sites that will last the lifetime of the patient.
Using nanotechnology for regenerative medicineecomes obvious when examining nature. For example,one is a nanocomposite that consists of a protein basedoft hydrogel template (i.e., collagen, non-collagenousroteins (laminin, fibronectin, vitronectin) and water)nd hard inorganic components (hydroxyapatite, HA,a10(PO4)6(OH)2) [12,13] (Fig. 2A). Specifically, 70% ofhe bone matrix is composed of nanocrystalline HA whichs typically 20—80 nm long and 2—5 nm thick [14]. Otherrotein components in the bone ECM are also nanometer
n dimension. This self-assembled nanostructured ECM inone closely surrounds and affects mesenchymal stem cell,steoblast (bone-forming cell), osteoclast and fibroblastdhesion, proliferation and differentiation. Moreover,artilage is a low regenerative tissue composed of a small
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Nanotechnology and nanomaterials: Promises for improved t
percentage of chondrocytes but dense nanostructured ECMrich in collagen fibers, proteoglycans and elastin fibers.The limited regenerative properties of cartilage originatesfrom a lack of chondrocyte mobility in the dense ECMas well as an absence of progenitor cells and vascularnetworks necessary for efficient cartilage tissue repair [15].Apparently, the design of novel nanomaterials which possessnot only excellent mechanical properties but that are alsobiomimetic in terms of their nanostructure (Fig. 2B), hasbecome quite popular in order to improve bone cell andchondrocyte functions.
In addition to the dimensional similarity tobone/cartilage tissue, nanomaterials also exhibit uniquesurface properties (such as surface topography, surfacechemistry, surface wettability and surface energy) due totheir significantly increased surface area and roughnesscompared to conventional or micron structured materials.As is known, material surface properties mediate specificprotein (such as fibronectin, vitronectin and laminin)adsorption and bioactivity before cells adhere on implants,further regulating cell behavior and dictating tissue regen-eration [12]. Furthermore, an important criterion fordesigning orthopedic implant materials is the formation ofsufficient osseointegration between synthetic materials andbone tissue. Studies have demonstrated that nanostruc-tured materials with cell favorable surface properties may
promote greater amounts of specific protein interactions tomore efficiently stimulate new bone growth compared toconventional materials [16—18] (Fig. 2C). This may be one ofthe underlying mechanisms why nanomaterials are superiorwmbb
Figure 3 Histology of rat calvaria after 6 weeks of implantationnanocrystalline HA coated tantalum. Greater amounts of new bonetalline HA coated tantalum than uncoated and conventional HA cocollagen. Images are adapted from [21].
regeneration 69
o conventional materials for tissue growth. Therefore, byontrolling surface properties, various nanophase ceramic,olymer, metal and composite scaffolds have been designedor bone/cartilage tissue engineering applications.
Nanophase ceramics, especially nano-hydroxyapatiteHA, a native component of bone), are popular bone sub-titutes, coatings and other filler materials due to theirocumented ability to promote mineralization. The nanome-er grain sizes and high surface fraction of grain boundariesn nanoceramics increase osteoblast functions (such as adhe-ion, proliferation and differentiation). For example, somen vitro studies demonstrated that nanophase HA (67 nmrain size) significantly enhanced osteoblast adhesion andtrikingly inhibited competitive fibroblast adhesion com-ared to conventional, 179 nm grain size HA, after just 4 h ofulture [17]. Researchers believe they know why. They havelucidated the highest adsorption of vitronectin (a proteinell known to promote osteoblast adhesion) on nanophaseeramics, which may explain the subsequent enhancedsteoblast adhesion on these materials [17]. In addition,nhanced osteoclast-like cell functions (such as the synthe-is of tartrate-resistant acid phosphatase (TRAP) and theormation of resorption pits) have also been observed onano-HA compared to conventional HA [19]. In a recenttudy, Nukavarapu et al. fabricated a biodegradable nano-ydroxyapatite/polyphosphazene microsphere 3-D scaffold
hich had suitable mechanical properties (compressiveoduli of 46—81 MPa) and cytocompatibility properties forone tissue engineering applications [20]. It should note surprising that nanostructured composites have similarof uncoated tantalum, conventional HA coated tantalum andformation occur in the rat calvaria when implanting nanocrys-ated tantalum. Red represents new bone and blue represents
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echanical properties to bone since bone itself is a nanos-ructured composite.
Importantly, such results have not been limited to initro studies. In vivo (specifically, rat) studies also demon-trated that nanocrystalline HA accelerated new boneormation on tantalum scaffolds when used as an osteo-onductive coating compared to uncoated or conventionalicron size HA coated tantalum [21]. Histological exami-
ation (Fig. 3) revealed that nanocrystalline HA coatingsromoted greater amounts of new bone growth in the ratalvaria than uncoated or conventional HA coated tan-alum after 6 weeks of implantation. Similar tendenciesave been reported for other nanoceramics including alu-ina, zinc oxide and titania, thus, providing strong evidence
hat, to some extent, it may not matter what implanthemistry is fabricated to have nanometer surface featureso promote bone growth. For example, osteoblast adhe-ion increased by 146% and 200% on nanophase zinc oxide23 nm) and titania (32 nm) compared to microphase zincxide (4.9 �m) and titania (4.1 �m), respectively [22]. Fur-hermore, nanophase zinc oxide, nanophase titania andanofiber alumina enhanced collagen synthesis, alkalinehosphatase activity and calcium mineral deposition bysteoblasts compared to conventional equivalents [22—23].
Because collagen in bone and cartilage is a triple helixelf-assembled into nanofibers 300 nm in length and 1.5 nmn diameter, many recent efforts have been dedicated toxploring the influence that novel biomimetic nanofibrousr nanotubular scaffolds have on regenerative mediciney following a bottom-up self-assembly process. Specifi-ally, Hartgerink et al. reported that a peptide-amphiphilePA) with the cell-adhesive ligand RGD (Arg-Gly-Asp) self-ssembled into supramolecular nanofibers (Fig. 4A and B)24]. By directly nucleating and aligning HA on the long axisf a nanofiber, a new nanofiber composite was designed withhe same self-assembly pattern as collagen and HA crystalsn bone. Moreover, Hosseinkhani et al. investigated mes-nchymal stem cell (MSC) behavior on self-assembled PAanofiber scaffolds [25]. Significantly enhanced osteogenicifferentiation of MSC occurred in the 3-D PA scaffoldompared to 2-D static tissue culture. RGD modified PAanofibers promoted the maximum amount of alkaline phos-hatase activity and osteocalcin content by osteoblasts.
Promise has also been demonstrated for other novelanostructured self-assembled chemistries. For example,steogenic helical rosette nanotubes obtained through theelf-assembly of DNA base pairs (Guanine∧Cytosine) in aque-us solutions (Fig. 4C) have been reported for bone tissuengineering applications. They have tailorable amino acidnd peptide side chains (such as lysine, RGD and KRSR (Lys-rg-Ser-Arg, which selectively promotes osteoblast adhesionnd inhibits fibroblast adhesion)) and are excellent mineral-zation templates to assemble a biomimetic nanotube/HAtructure (Fig. 4D). Furthermore, significantly improvedsteoblast adhesion has been observed on helical rosetteanotubes regardless of whether they are incorporated intoydrogels or coated on titanium (compared to untreated
ontrols [26,27]). Cartilage tissue engineering has alsoenefited from nanostructured self-assembled chemistries.isiday et al. designed a self-assembling peptide (the pep-ide KLD-12, Lys-Leu-Asp) hydrogel for cartilage repair [28].he chondrocyte encapsulated scaffold supported chon-pnttt
L. Zhang, T.J. Webster
rocyte differentiation and promoted the synthesis of aartilage-like ECM matrix (rich in proteoglycans and typeI collagen) in 3-D cell cultures after 4 weeks, thus, showingromise for cartilage tissue engineering. In summary, by thiself-assembly process, one can create a biologically inspired-D scaffold with self-assembled biomimetic features moreuitable for reconstructing 3-D bone and cartilage.
In addition, due to their superior cytocompat-ble, mechanical and electrical properties, carbonanotubes/nanofibers (CNTs/CNFs) are ideal scaffoldandidates for bone tissue engineering applications [29].n a recent study by Price et al., 60 nm diameter CNFs sig-ificantly increased osteoblast adhesion and concurrentlyecreased competitive cell (fibroblast, smooth muscle cell,tc.) adhesion in order to stimulate sufficient osseointegra-ion [30]. Other research efforts have also demonstratedhat CNTs are suitable to promote osteoblast functions31]. Recently, Sitharaman and colleagues reported ann vivo study of ultra-short SWCNT polymer nanocompos-tes after implanting them into rabbit femoral condylesnd subcutaneous pockets for up to 12 weeks [32]. Theanocomposites exhibited favorable hard and soft tissueesponses after 4 and 12 weeks. They induced a 300%reater bone volume than all other experimental groups atweeks and 200% greater bone growth at defect sites than
ontrol polymers without CNTs after 12 weeks. CNT/CNFeinforced polymer nanocomposites have also demonstratedxcellent electrical conductivity for tissue regeneration.or instance, using biodegradable polylactic acid (PLA)/CNTomposites as an example, an 80%/20% (w/w) PLA/CNTomposite exhibited ideal electrical conductivity for bonerowth while PLA was an insulator and not appropriateor electrically stimulating bone growth. Specifically, theLA/CNT composite promoted a 46% increase in osteoblastroliferation and a 307% increase in calcium content afterlectrical stimulation for 2 and 21 days compared to PLAlone, respectively [33]. These studies indicated that theNTs/CNFs and their composites can serve as osteogeniccaffolds with good cytocompatibility properties, reinforcedechanical properties and improved electrical conductivity
o effectively enhance bone tissue growth.As mentioned above, synthetic and natural polymers
e.g., polyglycolic acid (PGA), poly(lactic-co-glycolic acid)PLGA), PLLA, PLA, gelatin, collagen, chitosan) are excellentandidates for bone/cartilage tissue engineering applica-ions due to their biodegradability and ease of fabrication.anoporous or nanofibrous polymer matrices can be fab-icated via electrospinning, phase separation, particulateeaching, chemical etching and 3-D printing techniques.or cartilage applications, there has been great interestn incorporating chondrocytes or progenitor cells (such astem cells) into the 3-D polymer or composite scaffoldsuring electrospinning [34—36]. For example, Li et al. inves-igated in vitro chondrogenesis of MSCs in an electrospunoly(�-caprolactone) (PCL) nanofibrous scaffold [35]. Theifferentiation of the stem cells into chondrocytes in theanofibrous scaffold was comparable to an established cell
ellet culture. However, the easily fabricated and modifiedanofibers possessed much better mechanical propertieso overcome the disadvantages of using cell pellets and,hus, were presented as ideal candidates for stem cellransplantation during clinical cartilage repair. Because the
Nanotechnology and nanomaterials: Promises for improved tissue regeneration 71
Figure 4 Self-assembled nanofibers and nanotubes for bone/cartilage tissue engineering applications. (A) Schematic illustrationof the self-assembly process of peptide-amphiphiles functionalized with RGD to form a nanofiber 7.6 ± 1 nm in diameter. Images areadapted from [24]. (B) TEM image of the above self-assembled nanofibers. (C) Schematic illustration of the self-assembly processof the Guanine∧Cytosine DNA base pairs forming helical rosette nanotubes (HRNs). (D) SEM images of biomimetic nano-HA alignedwith HRNs on a porous carbon TEM grid.
Figure 5 (A) Schematic illustrating an efficient cell seeding method into a cell—nanofiber composite for cartilage tissue engi-neering applications. (B) Image of a shiny cartilage-like tissue from the cell—nanofiber composite after 42 days of culture. (C)Low-magnification histology showing well-dispersed chondrocyte distribution throughout the nanofiber scaffold after 1 day of cellculture (the cross section). (D) High-magnification histology showing distinct cell populations among the nanofibers. Arrows pointto chondrocytes dispersed among nanofibers. Images are adapted from [36].
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nfiltration of cells is usually inhibited by small pore sizesf electrospun polymer nanofibers, leading to uneven cellistributions in the scaffold, a recent study improved chon-rocyte seeding technology and obtained a homogeneousell—PLLA nanofiber composite (Fig. 5) [36]. The resultshowed that chondrocytes were uniformly present through-ut the entire cell—nanofiber composite, and the scaffoldeveloped into a smooth cartilage-like tissue with moreotal collagen and improved mechanical properties in aynamic bioreactor relative to that obtained in static cul-ure. Moreover, Park et al. reported significantly increasedhondrocyte functions (adhesion, proliferation and matrixynthesis) on 3-D nanostructured PLGA created via chemicaltching [37].
For bone tissue engineering, there are a large number oftudies which report the promise of biomimetic 3-D nanos-ructured polymer scaffolds which encapsulate stem cellsnd/or osteoblasts. For instance, Venugopal and colleagueslectrospun a fibrous nanocomposite of PCL/HA/gelatint a ratio of 1:1:2 (Fig. 1B). The results demonstratedhat osteoblast proliferation, alkaline phosphatase activitynd mineralization were the highest on the highly flexi-le PCL/HA/gelatin nanocomposite when compared to otherCL nanofibrous scaffolds [7]. Recently, Osathanon et al.eveloped a novel polymer/calcium phosphate compositeor bone tissue engineering applications. These nanofibrousbrin-based composites promoted osteoblast alkaline phos-hatase activity as well as osteoblast marker gene (mRNA)xpression to support bone maturation both in vitro and inivo in a mouse calvarial defect model [38].
Last but not the least, nanophase metals have been
xtensively investigated for orthopedic applications due toheir higher surface roughness, energy, and presence ofore particle boundaries at the surface compared withonventional micron metals. Webster et al. provided therst evidence that nanophase Ti, Ti6Al4V and CoCrMo
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igure 6 Fluorescent microscopy images of greatly increased enonventional Ti. Scale bar is 10 �m. Images are adapted from [43].
L. Zhang, T.J. Webster
ignificantly enhanced osteoblast adhesion compared toespective conventional metals [39]. In addition, Puckettt al. created linear patterns of nano-features of Ti vialectron beam evaporation. This study revealed that theanoregion of the patterned Ti induced greater osteoblastdhesion than the micron-rough regions and also controlledsteoblast morphology and alignment [40]. Moreover, anlectrochemical method known as anodization, a well-stablished nanosurface modification technique, has beensed to fabricate highly porous TiO2 nanotube layers on Ti.hrough the anodization of Ti in dilute hydrofluoric acidHF) electrolyte solutions, nanotubes with diameters around00 nm and lengths around 500 nm can be implemented intohe TiO2 layers of Ti. Yao et al. reported greatly improvedsteoblast functions on nanotubular anodized Ti comparedo unanodized Ti in vitro [41]. Moreover, increased chondro-yte adhesion was also observed on anodized nanotubular Tiompared to unanodized Ti in a recent study, thus, suggest-ng the possibility of promoting cartilage growth on anodizedi [42].
he promise of nanomaterials for vascularissue engineering applications
ue to the increasing prevalence of vascular diseases (suchs atherosclerosis), vascular grafts of greater efficacy toeplace damaged blood vessels are needed. For example,he American Heart Association reported that coronary heartisease mostly caused by atherosclerosis had led to 451,326eaths in 2004 and is the single leading cause of death
n the U.S. today [94]. In addition, peripheral arterial dis-ase related to blood vessels outside of the heart and brainffects about 8 million Americans. Over 500,000 coronarynd periphery bypass surgeries were performed in the U.S.n 2005. Since vascular tissue is a layered structure pos-dothelial cell proliferation on nanostructured Ti compared to
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sessing numerous nanostructured features (i.e., due to thepresence of collagen and elastin in the vascular ECM), nano-materials have shown much promise to improve vascular cell(specifically, endothelial and smooth muscle cells) functionsto inhibit thrombosis and severe inflammation.
Choudhary et al. reported that vascular cell adhesionand proliferation were greatly improved on nanostructuredTi compared to conventional Ti (Fig. 6) [43]. Interestingly,greater competitive endothelial cell adhesion, total elastinand collagen synthesis were observed than respective vas-cular smooth muscle cell functions on nanostructured Tiafter 5 days in culture. Since one of the current problemswith vascular stents is the overgrowth of smooth musclecells compared to endothelial cells, these results suggestthat endothelial cell functions were enhanced over that ofvascular smooth muscle cells, thus, increasing the proba-bility of endothelialization on nanostructured stents. It wasspeculated that the increased nano-roughness and particleboundaries on nanostructured Ti contributed to the observedfavorable endothelial cell functions. In addition, Miller et al.created biodegradable PLGA vascular grafts with nanome-ter surface features through chemical etching in NaOH andthrough a cast-mold technique [44—46]. Results demon-strated that both those polymers created through chemicaletching and a polymer cast-mold technique possessed ran-dom nanometer structures which promoted endothelial andvascular smooth muscle cell proliferation compared to theconventional PLGA [44]. A further study provided evidencethat nanostructured PLGA promoted more fibronectin andvitronectin adsorption from serum than conventional PLGA,thus, leading to the greater vascular cell responses on thenanostructured PLGA [45]. In order to elucidate specificnanometer surface features which promoted vascular cellresponses, 500, 200, and 100 nm polystyrene spheres wereused to cast PLGA [46]. Results demonstrated that the PLGAwith 200 nm structures promoted vascular cell responses andgreater fibronectin interconnectivity compared to smooth
PLGA and PLGA with 500 nm surface features (Fig. 7).Such results have been translated into the design of3-D polymer scaffolds as several random and aligned 3-D nanofiber scaffolds have been fabricated for vascularapplications. For example, Lee and colleagues fabricated
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Figure 7 Atomic force microscopy images of fibronectin (5 �g/mLfibronectin adsorbed on PLGA with 500 nm surface features showedfibronectin adsorbed on PLGA with 200 nm surface features showed s200 nm surface features only. Images are adapted from [46].
regeneration 73
nd evaluated a variety of electrospun collagen, elastinnd synthetic polymer (such as PLLA, PLGA and PCL)anofiber scaffolds for vascular graft applications [47].hese scaffolds have tailorable mechanical propertiesnd exceptional cytocompatibility properties for vascularpplications. Specifically, extensive smooth muscle cell infil-ration was observed in the collagen/elastin/PLLA scaffoldfter 21 days of culture. By electrospinning on a rotat-ng disk collector, Xu et al. fabricated an aligned PLLA-CL75:25) nanofibrous scaffold which mimicked the orientedbril structure in the medial layer of an artery [48]. Not onlyid coronary artery smooth muscle cells favorably interactith that scaffold, but cells also oriented along the fiber, fur-
her emulating the natural environment. In addition to thelectrospinning method, self-assembled peptides have beenormulated into scaffolds to mimic the vascular basementembrane showing excellent cytocompatibility properties
or vascular tissue repair. Genove et al. functionalized threeeptide sequences from two basement membrane proteinsspecifically, laminin and collagen IV) onto a self-assembledeptide scaffold [49]. These tailorable self-assembled scaf-olds enhanced endothelialization and improved nitric oxideelease and laminin as well as collagen IV deposition byhe endothelial cell monolayer. These results indicate theromise of biomimetic nanoscaffolds for improving vascu-ar tissue engineering applications and when coupled withhe aforementioned promise of nanomaterials for ortho-edic applications, suggests a possible wide spread use ofanomaterials for numerous tissue engineering applications.
he promise of nanomaterials for neuralissue engineering applications
n addition to aiding in orthopedic and vascular tissue regen-ration, nanomaterials are also helping to heal damagederves. In particular, nervous system injuries, diseases, and
isorders occur far too frequently. In the U.S., there arebout 250,000—400,000 patients suffering from a spinal cordnjury each year [50]. Although various cell therapies andmplants have been investigated, repairing damaged nervesnd achieving full functional recovery are still challeng-) coated PLGA cast nanosphere surfaces. (A) Phase images ofno interconnectivity between proteins. (B) Phase images of
ignificant interconnectivity between fibronectin. (C) PLGA with
74 L. Zhang, T.J. Webster
Figure 8 Schematic graphs of injured nerve regeneration in the central and peripheral nervous systems. (A) Central nervouss ) pero rawn
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ystem recovery process with glial scar tissue formation and (Bf Schwann cells, macrophages, and monocytes. Images are red
ng considering the complexity of the nervous system. Forxample, nearly 50,000 patients die among the average 1.4illion Americans that sustain traumatic brain injuries each
ear [51]. Generally, the nervous system can be dividednto two main parts: the central nervous system (CNS)including the brain and the spinal cord) and the peripheralervous system (PNS) (including the spinal and autonomicerves). These two systems have two different repair pro-edures after injury (Fig. 8) [52—54]. For the PNS, theamaged axons usually regenerate and recover via prolifer-ting Schwann cells, phagocytosing myelin by macrophagesr monocytes, forming bands of Bünger by the bundling ofchwann cells and sprouting axons in the distal segment55]. However, it is difficult to re-extend and re-innervatexons to recover functions in the CNS due to the absencef Schwann cells. More importantly, due to the influencef astrocytes, meningeal cells and oligodendrocytes, the
hick glial scar tissue typically formed around today’s neuraliomaterials will prevent proximal axon growth and inhibiteuron regeneration [53]. For these reasons, CNS injuriesay cause severe functional damages and are much moreifficult to repair than PNS injuries.niftr
ipheral nervous system recovery process involving the activityand adapted from [52,29].
The ideal materials for neural tissue engineering appli-ations should have excellent cytocompatible, mechanicalnd electrical properties. Without good cytocompatibilityroperties, materials may fail to improve neuron growth andt the same time may elicit severe inflammation or infec-ion. Without sufficient mechanical properties, the scaffolday not last long enough to physically support neural tis-
ue regeneration. In addition, superior electrical propertiesf scaffolds are required to help stimulate and controleuron behavior under electrical stimulation, thus, moreffectively guiding neural tissue repair. To date, various nat-ral and synthetic materials have been adopted as nerverafts to repair severely damaged nerves by bridging nerveaps and guiding neuron outgrowth. However, there are stillany shortcomings for these neural biomaterials including:
or autografts, it is usually difficult to collect sufficientonor nerves from patients and it is possible donor site
erve functions may be impaired [56], and for allografts,nflammation, rejection and transmission of diseases mayrequently occur leading to implant failures [57]. Otherraditional biomaterials (such as silicon probes used in neu-oprosthetic devices and polymers used as nerve conduits)
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Nanotechnology and nanomaterials: Promises for improved t
used for neural tissue repair have been limited by the exten-sive formation of glial scar tissue around the material aswell as non-optimal mechanical and electrical properties fornerve regrowth. Nanotechnology provides a wide platform todevelop novel and improved neural tissue engineering mate-rials and therapy including designing nanofiber/nanotubescaffolds with exceptional cytocompatibility and conduc-tivity properties to boost neuron activities. Nanomaterialshave also been used to encapsulate various neural stem cellsand Schwann cells into biomimetic nanoscaffolds to enhancenerve repair.
For example, work by Ramakrishna et al. has led to thefabrication of various nanofibrous PLLA or PCL scaffoldsvia electrospinning and phase separation; such scaffoldshave demonstrated excellent cytocompatibility propertiesfor neural tissue engineering applications [58—60]. Recentlythis research group incorporated laminin (a neurite pro-moting ECM protein) into electrospun PLLA nanofibers inorder to create a biomimetic scaffold for peripheral nerverepair [58]. The results showed that neurite outgrowthimproved on laminin-PLLA scaffolds produced by facileblended electrospinning. In another recent report, electro-spun PCL/chitosan nanofiber scaffolds exhibited improvedmechanical properties compared to chitosan [59]. Schwanncells also proliferated well on this PCL/chitosan nanofiberscaffold. In addition, Zhang and colleagues also reportedfavorable neural cell responses on the self-assembledpeptide nanofiber scaffold (called SAPNS). Holmes et al.reported that the self-assembled peptide scaffold sup-ported neuronal cell functions, neurite outgrowth andfunctional synapse formation among neurons [61]. Further-more, Ellis-Behnke et al. investigated SAPNS for in vivo axonregeneration in the CNS [62]. The SAPNS aided in CNS regen-eration to help axonal growth, even ‘‘knitting’’ the braintissue together and successfully improving functional recov-ery.
Due to the fact that carbon nanotubes/fibers have excel-
lent electrical conductivity, strong mechanical properties,and have similar nanoscale dimensions to neurites, theyhave been used to guide axon regeneration and improveneural activity as biomimetic scaffolds at neural tissueinjury sites. In particular, Mattson et al. found for the(cscf
Figure 9 SEM images of neural cell adhesion on carbon nanotubeon purified MWCNT glass substrates with extended neurites after 8 dCNTs. Images are adapted from [65]. (C), (D) and (E) PC12 neural cepolypyrrole at different magnifications. Images are adapted from [6
regeneration 75
rst time that neurons grew on multiwalled carbon nan-tubes (MWCNTs) [63]. They observed over a 200% increasen total neurite length and nearly a 300% increase inhe number of branches and neurites on MWCNTs coatedith 4-hydroxynonenal compared to uncoated MWCNTs. Hut al. revealed that different surface charges of MWC-Ts, obtained through chemical functionalization, resulted
n different neurite outgrowth patterns (such as neuriteength, branching and the number of growth cones) [64].hey demonstrated that positively charged MWCNTs signifi-antly increased the number of growth cones and neuriteranches compared to negatively charged MWCNTs, thus,ontrolling neural growth. Lovat et al. demonstrated thaturified MWCNTs potentially boosted electrical signal trans-er of neuronal networks (Fig. 9A and B) [65]. Moreover,ighly ordered CNT/CNF matrices or free standing nanotubelms have been fabricated for neural tissue engineeringpplications [66—67]. For instance, Gheith et al. inves-igated the biocompatibility of a freestanding positivelyharged SWCNT/polymer thin-film membrane prepared byayer-by-layer assembly [66]. They observed that 94—98%f neurons were viable on the SWCNT/polymer films after
10 day incubation. The SWCNT/polymer films favor-bly induced neuronal cell differentiation, guided neuronxtension and directed more elaborate branches than con-rols.
In order to inhibit activated astrocyte functions whichesult in the formation of glial scar tissue, McKenzie etl. incorporated different weight ratios of high surfacenergy CNFs into polymers and demonstrated for the firstime that astrocyte adhesion can be effectively inhib-ted by using CNF/polymer composites [68]. In addition,ecreased astrocyte proliferation was observed on nanos-ructured CNFs, thus, leading to decreased glial scar tissueormation on such materials. On the other hand, Nguyen-u et al. fabricated a vertically aligned CNF nanoelectroderray by creating a thin conductive polymer film coating
such as polypyrrole) for neural implants [69]. The verti-al CNF arrays had more open and mechanically robust 3-Dtructures as well as better electrical conductivity whichontributed to forming an intimate neural-electrical inter-ace between cells and nanofibers (Fig. 9C—E). Gabay et/fiber substrates. (A) Neonatal hippocampal neurons adherentays; inset image (B) shows a single neurite in close contact tolls grown freestanding on vertically aligned CNFs coated with
9].
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l. developed a novel method to fabricate islands of CNTn substrates. Neurons preferably attached on the CNTslands and further extended their neurites to form intercon-ected neural networks according to pre-designed patterns70]. In this manner, the CNTs/CNFs and their compositesre promising scaffold candidates for injured neural tissueepair.
Studies have also provided evidence that individualNTs/CNFs may be useful in treating neurological dam-ge when combined with stem cells. Stem cells have theotential to differentiate and self-renew into controllable,esirable cell types: i.e., neural stem cells in the CNS canifferentiate into neurons and astrocytes [71]. Therefore,any efforts have focused on impregnating multi-potential
tem cells into CNTs/CNFs and other nanoscaffolds, whichan be directly transplanted into injury sites and assist neu-al tissue recovery. However, a challenging problem haseen to determine how to effectively deliver and selec-ively differentiate stem cells into favorable neuronal cellypes at injury sites in order to regenerate desirable tissue.lthough the underlying mechanisms triggering differen-iation of stem cells are not entirely clear, accumulated
vidence has indicated that novel biomimetic nanomate-ials may contribute to selective stem cell differentiationwithout the use of growth factors) [72,73]. For exam-le, Lee et al. injected CNFs impregnated with stem cellsnto stroke damaged neural tissue in rat brains and foundt
Ntu
igure 10 Histology of CNFs impregnated with stem cells into strokective neuroprogenitor cells and fully differentiated neurons (brownound around CNFs. (C) and (D), few glial cells interacting with CNFsor astrocytes; CD11b is a marker for activated microglia cells. Blacdapted from [72].
L. Zhang, T.J. Webster
xtensive neural stem cell differentiation with little glialcar tissue formation in vivo [72]. After 1 and 3 weeksf animal implantation, histological sections showed thateural stem cells favorably differentiated into neuronsFig. 10A and B) and little to no glial scar tissue (Fig. 10Cnd D) formed around CNFs compared to controls (onlymplanting stem cells without CNFs or implanting CNFsithout cells). Furthermore, Jan et al. successfully dif-
erentiated mouse embryonic neural stem cells includingeurospheres and single cells into neurons on layer-by-ayer assembled SWCNT/polyelectrolyte composites [73].he layer-by-layer SWCNT composites promoted slightlyore neurons and fewer astrocytes on substrates during7 day culture period than poly-L-ornithine (a common
ubstrate for neural stem cell studies). Clearly, CNTs/CNFslayed an important role in effectively delivering stemells into injured sites and promoted stem cells to differ-ntiate into favorable neurons to repair damaged neuralissues.
he promise of nanomaterials for bladder
issue engineering applicationsanomaterials have also been used in soft tissues, such ashe bladder. As the 6th most common cancer in the U.S.,rinary bladder cancer affects over 53,200 Americans and
damaged rat neural tissue after 3 weeks. (A) and (B), numerousstained cells, marked by nestin and MAP2, respectively) wereled to little or no glial scar tissue formation. GFAP is a marker
k areas in the images are CNFs. Scale bar is 25 �m. Images are
Nanotechnology and nanomaterials: Promises for improved tissue regeneration 77
cell
g(cgndrssmttbcEsitsdtibebt
Ph
Aptsa
Figure 11 Schematic illustration of the bilayer smooth muscleis adapted from [76].
leads to 12,200 deaths annually [74]. Although standardtreatments such as surgery to remove bladder tumors fol-lowed by radiation, chemotherapy and immunotherapy haveimproved, various complications (such as systemic infec-tions, flu-like symptoms and cancer recurrence, etc.) withthese procedures are still too commonly reported. Some-times, radical cystectomy by removing parts of and even theentire bladder is needed. However, such a drastic approachrequires the implantation of a bladder tissue replacementto quickly recover bladder functions. As emerging blad-der tissue engineering materials, nanomaterials providea promising approach to more efficiently improve blad-der tissue regeneration for the same reasons mentionedearlier for other tissue systems (biologically inspired rough-ness, increased surface energy, selective protein adsorption,etc.). In particular, Harrington and colleagues have coateda series of branched or linear self-assembling peptide-amphiphile nanofibers containing cell-adhesive RGDS ontraditional PGA scaffolds [75]. Human bladder smooth mus-cle cell densities on the branched PA/PGA nanocompositewere greater than on the uncoated PGA after 17 days of cul-ture. In a recent review, they encapsulated bladder smoothmuscle cells and urothelial cells into a PA/PGA nanofi-brous gel containing specific growth factors (Fig. 11) [76].Due to their ability to mimic the oriented nanostructuredbladder ECM, electrospun polymer nanofibers have beenused in bladder tissue engineering. Baker et al. showedthat bladder smooth muscle cells were aligned on orientedelectrospun polystyrene scaffolds similar to the native blad-der tissue [77]. This study also demonstrated that argonplasma treated electrospun polystyrene nanofibers signifi-cantly improved smooth muscle cell attachment. Fibrinogenhas also been electrospun into a scaffold for urinary tracttissue regeneration [78]. This study demonstrated thathuman bladder smooth muscle cells rapidly migrated into,proliferated onto and remodeled the 3-D fibrinogen scaf-
fold.Other nanostructured polymers with superior biocom-patibility properties have been widely investigated byHaberstroh and colleagues for bladder tissue regener-ation applications [79—81]. For instance, this research
cbOtc
/urothelial cell (SMC-UC) encapsulation in a PA/PGA gel. Image
roup used nanotextured PLGA and poly(ether urethane)PU) films to successfully enhance bladder smooth muscleell functions [79]. Through chemical etching technolo-ies, PLGA and PU were transformed from their nativeano-smooth surface features into those possessing a highegree of nano-roughness. This study revealed that nano-oughness played a critical role in promoting bladdermooth muscle cell proliferation once the influence ofurface chemistry change was eliminated (through cast-old techniques using the chemical treated polymer as
he cast). Recently, Pattison et al. also demonstratedhat nanostructured PLGA and PU 3-D scaffolds preparedy a solvent casting and salt leaching methods signifi-antly enhanced bladder smooth muscle cell functions andCM protein synthesis compared to conventional nano-mooth polymers in vitro [80]. Furthermore, preliminaryn vivo studies have provided evidence that nanostruc-ured polymer scaffolds form little to no calcium oxalatetones (stone formation is a common problem during blad-er replacements) in augmented rat bladders. Althoughhere are many unknowns for the use of nanomaterialsn bladder tissue engineering applications, utilizing theseiomimetic nanomaterials with progenitor cells is undoubt-dly a promising future research direction to regenerateladder tissue in resected bladder cancerous tissue loca-ions.
otential risks of nanomaterials towardsuman health
s described, nanotechnology has achieved tremendousrogress in a relatively short time period in medical applica-ions. As a result, nanomaterials have begun to enter widepread industrial production. For instance, nanoceramicsre commercially available as new bone grafts or as implant
oating materials (i.e., nano-HA paste—–Ostim® from Obern-urg, Germany; nano-beta-tricalcium phosphate-Vitoss fromrthovita, USA) [82]. However, it is important to note thathe research on nanomaterials for tissue engineering appli-ations is still at its infancy and, most importantly, the
7
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nfluence of nanomaterials on human health and the environ-ent is not well understood. In particular, toxic responses to
anoparticles generated from the degradation of implantedanomaterials, via wear debris from artificial joints withanofeatures, and heavy metals (iron, nickel and cobaltatalysts) remaining in CNTs, have all been reported. Manyeports on the cellular uptake of nanoparticles in the lungs,mmune system, as well as other organs have been pub-ished [83—85]. Nanoparticle uptake by endothelial cells,lveolar macrophages, pulmonary or intestinal epithelium,erve cells etc. has been reviewed and, thus, may possessproblem for this field if not thoroughly understood beforeeing applied widely [83]. Gutwein et al. investigated theiability of osteoblasts in vitro when cultured in the pres-nce of nanoalumina and titania particles for 6 h [84]. Thistudy demonstrated that ceramic nanoparticles were safero osteoblasts than conventional, micron-sized, ceramicsarticles. In contrast, in an in vivo study, Lam et al. showedhat CNTs were more toxic than carbon black in the lungs,hich may be a serious occupational health hazard in chronic
nhalation exposures [85]. Sometimes nanoparticle interac-ions with biomolecules in vivo or their aggregation statesay change their toxicity to humans. But the often con-
radictory results of current studies are clearly not enougho provide the final answer concerning nanomaterial tox-city. In depth investigations of nanomaterials on humanealth and the environment are necessary to fully eluci-ate whether nanoparticles should be used in biomedicalpplications.
onclusions
o date, there has been an exponential increase in stud-es using nanotechnology for tissue engineering applications.o be concise, this paper only covered the recent progresssing nanomaterials for bone, cartilage, vascular, neuralnd bladder tissue regeneration. Other reviews of nanotech-ology applications for the specific regeneration of tissuesan be found [15,76,86—92]. Nanotechnology approachesor the regeneration of other types of tissues (such ashe muscle, skin, kidneys, liver, pancreas, and the immuneystem) have also been reviewed [92,93]. Although manyhallenges may lie ahead, synthetic nanomaterials canimic properties of the natural ECM and thus, show greatotential for numerous tissue engineering applications.articularly, due to their excellent cytocompatibility proper-ies, research interest has been evoked to use nanomaterialss the next generation of tissue repair materials. In theuture, the underlying mechanisms of the in vivo interac-ions between nanomaterials and cells at the molecularevel will significantly advance the development of thiseld.
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Lijie Zhang received a BS degree in chemicalengineering from Tianjin University, China,in 2001 and a MS degree in chemical engi-neering from Brown University in 2007. She iscurrently pursuing her PhD in biomedical engi-neering at Brown University. She has interestsin developing novel nanobiomaterials forbone tissue engineering, orthopedic implantand vascular stent applications. She has pub-
lished over fourteen journal papers as well asconference proceedings, three book chaptersnd has presented her work at over eighteen conferences. Sheas also the recipient of the Society for Biomaterials STAR Award
2007).
8
aiaonOutstanding Young Investigator Award from Purdue University, 2005
0
Thomas J. Webster is an associate profes-sor of Engineering and Orthopedics at BrownUniversity. His degrees are in chemical engi-neering from the University of Pittsburgh(BS, 1995) and in biomedical engineeringfrom Rensselaer (MS, 1997; PhD, 2000). His
research addresses the design, synthesis, andevaluation of nanophase materials for variousregenerative medicine applications. His labgroup has generated 4 books, 33 book chap-ters, 85 invited presentations, 215 literatureWeHc
L. Zhang, T.J. Webster
rticles, and 245 conference presentations. He is the found-ng editor-in-chief of the International Journal of Nanomedicinend is on the editorial board for 10 other journals. Amongther awards, Dr. Webster received the 2002 Biomedical Engi-eering Society Rita Schaffer Young Investigator Award, 2004
allace Coulter Foundation Early Career Award, and in 2007 waslected as a Fellow of the American Academy of Nanomedicine.is research has led to the formation of two nanotechompanies.