Nanotechnology and nanomaterials: Promises for improved tissue regeneration

Nano Today (2009) 4, 66—80

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journal homepage: www.elsevier.com/locate/nanotoday

REVIEW

Nanotechnology and nanomaterials: Promises for improved tissue regeneration Lijie Zhang, Thomas J. Webster ∗ Divisions of Engineering and Orthopaedics, Brown University, 182 Hope Street, Providence, RI 02912, USA Received 23 September 2008; received in revised form 14 October 2008; accepted 15 October 2008

KEYWORDS Nanomaterials; Tissue engineering; Nanotechnology; Scaffold; Biomimetic; Regenerative medicine

Summary Tissue engineering and regenerative medicine aim to develop biological substitutes that restore, maintain, or improve damaged tissue and organ functionality. While tissue engineering 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 the numerous problems associated with traditional implants. Nanotechnology, or the use of nanomaterials (defined as those materials with constituent dimensions less than 100 nm), may have the answers since only these materials can mimic surface properties (including topography, energy, etc.) of natural tissues. For these reasons, over the last decade, nanomaterials have been 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 improve various tissue growth over what is achievable today. In this review paper, the promise of nanomaterials for bone, cartilage, vascular, neural and bladder tissue engineering applications will be reviewed. Moreover, as an important future area of research, the potential risk and toxicity of nanomaterial synthesis and use related to human health are emphasized. © 2008 Elsevier Ltd. All rights reserved.

Nanotechnology and nanomaterials: biomimetic tools for tissue regeneration In 1959, Nobel award winner Richard Feynman first proposed the seminal idea of nanotechnology by suggesting the devel-

∗ Corresponding author. Tel.: +1 401 863 2318; fax: +1 401 863 9107. E-mail address: Thomas [email protected] (T.J. Webster).

opment of molecular machines. Ever since, the scientific community has investigated the role that nanotechnology can play in every aspect of society. The intrigue of nanotechnology comes from the ability to control material properties by assembling such materials at the nanoscale. The tunable material properties that nanotechnology can provide were stated in Norio Taniguchi’s paper in 1974 where the term ‘‘nanotechnology’’ was first used in a scientific publication [1,2]. Nanotechnology has achieved tremendous progress in the past several decades. Recently, nanomaterials, which are materials with basic structural units, grains, particles,

1748-0132/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.nantod.2008.10.014

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Figure 1 (A) Scanning electron microscopy (SEM) image of poly(L-lactic acid) (PLLA) nanofibrous scaffold with interconnected spherical 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]. (C) Densely aligned single wall carbon nanotube (SWCNT) forest grown with novel water-assisted chemical vapor deposition in 10 min [8]. (D) Transmission electron microscopy (TEM) image of monodispersed magnetic Fe3 O4 nanoparticles (6 nm) deposited from their hexane dispersion and dried at room temperature [9].

fibers or other constituent components smaller than 100 nm in at least one dimension [3], have evoked a great amount of attention for improving disease prevention, diagnosis, and treatment. The intrigue in nanomaterial research for regenerative medicine is easy to see and is wide spread. For example, from a material property point-of-view, nanomaterials can be made of metals, ceramics, polymers, organic materials and composites thereof, just like conventional or micron structured materials. Nanomaterials include nanoparticles, nanoclusters, nanocrystals, nanotubes, nanofibers, nanowires, nanorods, nanofilms, etc. To date, numerous topdown and bottom-up nanofabrication technologies (such as electrospinning, phase separation, self-assembly processes, thin film deposition, chemical vapor deposition, chemical etching, nano-imprinting, photolithography, and electron beam or nanosphere lithographies [4]) are available to synthesize nanomaterials with ordered or random nanotopographies (Fig. 1, [6—9]). Nanomaterials can also be grown or self-assembled into nanotubes/nanofibers which can even more accurately simulate the dimensions of natural entities, such as collagen fibers. After decreasing material size into the nanoscale, dramatically increased surface area, surface roughness and surface area to volume ratios can be created to lead to superior physiochemical properties (i.e., mechanical, electrical, optical, catalytic, magnetic proper-

ties, etc.) [5]. Therefore, nanomaterials with such excellent properties have been extensively investigated in a wide range of biomedical applications, in particular regenerative medicine. With the striking increase in the world’s population, there are enormous demands each year for various biomedical implants to repair diseased or lost tissues. However, conventional tissue replacements (such as autografts and allografts) have a variety of problems that cannot satisfy high performance demands necessary for today’s patient. Consequently, tissue engineering (or regenerative medicine) emerged initially defined by Robert Langer and Joseph Vacanti as ‘‘an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function’’ [10]. However, it is clear that today, materials used in a wide range of tissue engineering applications still require improvement. Since natural tissues or organs are nanometer in dimension and cells directly interact with (and create) nanostructured extra-cellular matrices (ECM), the biomimetic features and excellent physiochemical properties of nanomaterials play a key role in stimulating cell growth as well as guide tissue regeneration. Even though it was a field in its infancy a decade ago, currently, numerous researchers fabricate cytocompatible biomimetic nanomaterial scaffolds encapsulating cells (such

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Figure 2 The biomimetic advantages of nanomaterials. (A) The nanostructured hierarchal self-assembly of bone. (B) Nanophase titanium (top, the atomic force microscopy image) and nanocrystalline HA/HRN hydrogel scaffold (bottom, the SEM image). (C) Schematic illustration of the mechanism by which nanomaterials may be superior to conventional materials for bone regeneration. The bioactive surfaces of nanomaterials mimic those of natural bones to promote greater amounts of protein adsorption and efficiently stimulate more new bone formation than conventional materials.

as stem cells, chondrocytes and osteoblasts, etc.) for tissue engineering applications. In this review, we will focus on the recent progress of the use of nanomaterials for bone, cartilage, vascular, neural and bladder tissue engineering applications in vitro and more importantly in vivo. As the next frontier in nanotechnology research, toxicity concerns of nanomaterials and nanoparticles during manufacturing and/or implantation will be covered as well.

The promise of nanomaterials for bone and cartilage tissue engineering applications Today various bone fractures, osteoarthritis, osteoporosis or bone cancers represent common and significant clinical problems. The National Center for Health Statistics (NCHS) reported that bone fractures for all sites numbered 1,039,000 in 2004 in the U.S. In addition, around 118,700 patients (home health care) had osteoarthritis and associated disorders in 2000. The American Academy of Orthopedic Surgeons also reported that in just a 4 year period, there was an 83.72% increase in the number of hip replacements performed from nearly 258,000 procedures in 2000 to 474,000 procedures in 2004 [11]. Such traumatic

bone and cartilage damage happens frequently each year. A similar trend has been documented for other industrialized countries as well. However, traditional implant materials only last 10—15 years on average and implant failures originating from implant loosening, inflammation, infection, osteolysis and wear debris frequently occur. It is clearly urgent to develop a new generation of cytocompatible bone and cartilage substitutes to regenerate bone/cartilage tissue at defect sites that will last the lifetime of the patient. Using nanotechnology for regenerative medicine becomes obvious when examining nature. For example, bone is a nanocomposite that consists of a protein based soft hydrogel template (i.e., collagen, non-collagenous proteins (laminin, fibronectin, vitronectin) and water) and hard inorganic components (hydroxyapatite, HA, Ca10 (PO4 )6 (OH)2 ) [12,13] (Fig. 2A). Specifically, 70% of the bone matrix is composed of nanocrystalline HA which is typically 20—80 nm long and 2—5 nm thick [14]. Other protein components in the bone ECM are also nanometer in dimension. This self-assembled nanostructured ECM in bone closely surrounds and affects mesenchymal stem cell, osteoblast (bone-forming cell), osteoclast and fibroblast adhesion, proliferation and differentiation. Moreover, cartilage is a low regenerative tissue composed of a small

Nanotechnology and nanomaterials: Promises for improved tissue regeneration percentage of chondrocytes but dense nanostructured ECM rich in collagen fibers, proteoglycans and elastin fibers. The limited regenerative properties of cartilage originates from a lack of chondrocyte mobility in the dense ECM as well as an absence of progenitor cells and vascular networks necessary for efficient cartilage tissue repair [15]. Apparently, the design of novel nanomaterials which possess not only excellent mechanical properties but that are also biomimetic in terms of their nanostructure (Fig. 2B), has become quite popular in order to improve bone cell and chondrocyte functions. In addition to the dimensional similarity to bone/cartilage tissue, nanomaterials also exhibit unique surface properties (such as surface topography, surface chemistry, surface wettability and surface energy) due to their significantly increased surface area and roughness compared to conventional or micron structured materials. As is known, material surface properties mediate specific protein (such as fibronectin, vitronectin and laminin) adsorption and bioactivity before cells adhere on implants, further regulating cell behavior and dictating tissue regeneration [12]. Furthermore, an important criterion for designing orthopedic implant materials is the formation of sufficient osseointegration between synthetic materials and bone tissue. Studies have demonstrated that nanostructured materials with cell favorable surface properties may promote greater amounts of specific protein interactions to more efficiently stimulate new bone growth compared to conventional materials [16—18] (Fig. 2C). This may be one of the underlying mechanisms why nanomaterials are superior

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to conventional materials for tissue growth. Therefore, by controlling surface properties, various nanophase ceramic, polymer, metal and composite scaffolds have been designed for bone/cartilage tissue engineering applications. Nanophase ceramics, especially nano-hydroxyapatite (HA, a native component of bone), are popular bone substitutes, coatings and other filler materials due to their documented ability to promote mineralization. The nanometer grain sizes and high surface fraction of grain boundaries in nanoceramics increase osteoblast functions (such as adhesion, proliferation and differentiation). For example, some in vitro studies demonstrated that nanophase HA (67 nm grain size) significantly enhanced osteoblast adhesion and strikingly inhibited competitive fibroblast adhesion compared to conventional, 179 nm grain size HA, after just 4 h of culture [17]. Researchers believe they know why. They have elucidated the highest adsorption of vitronectin (a protein well known to promote osteoblast adhesion) on nanophase ceramics, which may explain the subsequent enhanced osteoblast adhesion on these materials [17]. In addition, enhanced osteoclast-like cell functions (such as the synthesis of tartrate-resistant acid phosphatase (TRAP) and the formation of resorption pits) have also been observed on nano-HA compared to conventional HA [19]. In a recent study, Nukavarapu et al. fabricated a biodegradable nanohydroxyapatite/polyphosphazene microsphere 3-D scaffold which had suitable mechanical properties (compressive moduli of 46—81 MPa) and cytocompatibility properties for bone tissue engineering applications [20]. It should not be surprising that nanostructured composites have similar

Figure 3 Histology of rat calvaria after 6 weeks of implantation of uncoated tantalum, conventional HA coated tantalum and nanocrystalline HA coated tantalum. Greater amounts of new bone formation occur in the rat calvaria when implanting nanocrystalline HA coated tantalum than uncoated and conventional HA coated tantalum. Red represents new bone and blue represents collagen. Images are adapted from [21].

70 mechanical properties to bone since bone itself is a nanostructured composite. Importantly, such results have not been limited to in vitro studies. In vivo (specifically, rat) studies also demonstrated that nanocrystalline HA accelerated new bone formation on tantalum scaffolds when used as an osteoconductive coating compared to uncoated or conventional micron size HA coated tantalum [21]. Histological examination (Fig. 3) revealed that nanocrystalline HA coatings promoted greater amounts of new bone growth in the rat calvaria than uncoated or conventional HA coated tantalum after 6 weeks of implantation. Similar tendencies have been reported for other nanoceramics including alumina, zinc oxide and titania, thus, providing strong evidence that, to some extent, it may not matter what implant chemistry is fabricated to have nanometer surface features to promote bone growth. For example, osteoblast adhesion increased by 146% and 200% on nanophase zinc oxide (23 nm) and titania (32 nm) compared to microphase zinc oxide (4.9 ␮m) and titania (4.1 ␮m), respectively [22]. Furthermore, nanophase zinc oxide, nanophase titania and nanofiber alumina enhanced collagen synthesis, alkaline phosphatase activity and calcium mineral deposition by osteoblasts compared to conventional equivalents [22—23]. Because collagen in bone and cartilage is a triple helix self-assembled into nanofibers 300 nm in length and 1.5 nm in diameter, many recent efforts have been dedicated to exploring the influence that novel biomimetic nanofibrous or nanotubular scaffolds have on regenerative medicine by following a bottom-up self-assembly process. Specifically, Hartgerink et al. reported that a peptide-amphiphile (PA) with the cell-adhesive ligand RGD (Arg-Gly-Asp) selfassembled into supramolecular nanofibers (Fig. 4A and B) [24]. By directly nucleating and aligning HA on the long axis of a nanofiber, a new nanofiber composite was designed with the same self-assembly pattern as collagen and HA crystals in bone. Moreover, Hosseinkhani et al. investigated mesenchymal stem cell (MSC) behavior on self-assembled PA nanofiber scaffolds [25]. Significantly enhanced osteogenic differentiation of MSC occurred in the 3-D PA scaffold compared to 2-D static tissue culture. RGD modified PA nanofibers promoted the maximum amount of alkaline phosphatase activity and osteocalcin content by osteoblasts. Promise has also been demonstrated for other novel nanostructured self-assembled chemistries. For example, osteogenic helical rosette nanotubes obtained through the self-assembly of DNA base pairs (Guanine∧Cytosine) in aqueous solutions (Fig. 4C) have been reported for bone tissue engineering applications. They have tailorable amino acid and peptide side chains (such as lysine, RGD and KRSR (LysArg-Ser-Arg, which selectively promotes osteoblast adhesion and inhibits fibroblast adhesion)) and are excellent mineralization templates to assemble a biomimetic nanotube/HA structure (Fig. 4D). Furthermore, significantly improved osteoblast adhesion has been observed on helical rosette nanotubes regardless of whether they are incorporated into hydrogels or coated on titanium (compared to untreated controls [26,27]). Cartilage tissue engineering has also benefited from nanostructured self-assembled chemistries. Kisiday et al. designed a self-assembling peptide (the peptide KLD-12, Lys-Leu-Asp) hydrogel for cartilage repair [28]. The chondrocyte encapsulated scaffold supported chon-

L. Zhang, T.J. Webster drocyte differentiation and promoted the synthesis of a cartilage-like ECM matrix (rich in proteoglycans and type II collagen) in 3-D cell cultures after 4 weeks, thus, showing promise for cartilage tissue engineering. In summary, by this self-assembly process, one can create a biologically inspired 3-D scaffold with self-assembled biomimetic features more suitable for reconstructing 3-D bone and cartilage. In addition, due to their superior cytocompatible, mechanical and electrical properties, carbon nanotubes/nanofibers (CNTs/CNFs) are ideal scaffold candidates for bone tissue engineering applications [29]. In a recent study by Price et al., 60 nm diameter CNFs significantly increased osteoblast adhesion and concurrently decreased competitive cell (fibroblast, smooth muscle cell, etc.) adhesion in order to stimulate sufficient osseointegration [30]. Other research efforts have also demonstrated that CNTs are suitable to promote osteoblast functions [31]. Recently, Sitharaman and colleagues reported an in vivo study of ultra-short SWCNT polymer nanocomposites after implanting them into rabbit femoral condyles and subcutaneous pockets for up to 12 weeks [32]. The nanocomposites exhibited favorable hard and soft tissue responses after 4 and 12 weeks. They induced a 300% greater bone volume than all other experimental groups at 4 weeks and 200% greater bone growth at defect sites than control polymers without CNTs after 12 weeks. CNT/CNF reinforced polymer nanocomposites have also demonstrated excellent electrical conductivity for tissue regeneration. For instance, using biodegradable polylactic acid (PLA)/CNT composites as an example, an 80%/20% (w/w) PLA/CNT composite exhibited ideal electrical conductivity for bone growth while PLA was an insulator and not appropriate for electrically stimulating bone growth. Specifically, the PLA/CNT composite promoted a 46% increase in osteoblast proliferation and a 307% increase in calcium content after electrical stimulation for 2 and 21 days compared to PLA alone, respectively [33]. These studies indicated that the CNTs/CNFs and their composites can serve as osteogenic scaffolds with good cytocompatibility properties, reinforced mechanical properties and improved electrical conductivity to 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 excellent candidates for bone/cartilage tissue engineering applications due to their biodegradability and ease of fabrication. Nanoporous or nanofibrous polymer matrices can be fabricated via electrospinning, phase separation, particulate leaching, chemical etching and 3-D printing techniques. For cartilage applications, there has been great interest in incorporating chondrocytes or progenitor cells (such as stem cells) into the 3-D polymer or composite scaffolds during electrospinning [34—36]. For example, Li et al. investigated in vitro chondrogenesis of MSCs in an electrospun poly(␧-caprolactone) (PCL) nanofibrous scaffold [35]. The differentiation of the stem cells into chondrocytes in the nanofibrous scaffold was comparable to an established cell pellet culture. However, the easily fabricated and modified nanofibers possessed much better mechanical properties to overcome the disadvantages of using cell pellets and, thus, were presented as ideal candidates for stem cell transplantation during clinical cartilage repair. Because the

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Figure 4 Self-assembled nanofibers and nanotubes for bone/cartilage tissue engineering applications. (A) Schematic illustration of the self-assembly process of peptide-amphiphiles functionalized with RGD to form a nanofiber 7.6 ± 1 nm in diameter. Images are adapted from [24]. (B) TEM image of the above self-assembled nanofibers. (C) Schematic illustration of the self-assembly process of the Guanine∧Cytosine DNA base pairs forming helical rosette nanotubes (HRNs). (D) SEM images of biomimetic nano-HA aligned with 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 engineering 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 cell culture (the cross section). (D) High-magnification histology showing distinct cell populations among the nanofibers. Arrows point to chondrocytes dispersed among nanofibers. Images are adapted from [36].

72 infiltration of cells is usually inhibited by small pore sizes of electrospun polymer nanofibers, leading to uneven cell distributions in the scaffold, a recent study improved chondrocyte seeding technology and obtained a homogeneous cell—PLLA nanofiber composite (Fig. 5) [36]. The results showed that chondrocytes were uniformly present throughout the entire cell—nanofiber composite, and the scaffold developed into a smooth cartilage-like tissue with more total collagen and improved mechanical properties in a dynamic bioreactor relative to that obtained in static culture. Moreover, Park et al. reported significantly increased chondrocyte functions (adhesion, proliferation and matrix synthesis) on 3-D nanostructured PLGA created via chemical etching [37]. For bone tissue engineering, there are a large number of studies which report the promise of biomimetic 3-D nanostructured polymer scaffolds which encapsulate stem cells and/or osteoblasts. For instance, Venugopal and colleagues electrospun a fibrous nanocomposite of PCL/HA/gelatin at a ratio of 1:1:2 (Fig. 1B). The results demonstrated that osteoblast proliferation, alkaline phosphatase activity and mineralization were the highest on the highly flexible PCL/HA/gelatin nanocomposite when compared to other PCL nanofibrous scaffolds [7]. Recently, Osathanon et al. developed a novel polymer/calcium phosphate composite for bone tissue engineering applications. These nanofibrous fibrin-based composites promoted osteoblast alkaline phosphatase activity as well as osteoblast marker gene (mRNA) expression to support bone maturation both in vitro and in vivo in a mouse calvarial defect model [38]. Last but not the least, nanophase metals have been extensively investigated for orthopedic applications due to their higher surface roughness, energy, and presence of more particle boundaries at the surface compared with conventional micron metals. Webster et al. provided the first evidence that nanophase Ti, Ti6Al4V and CoCrMo

L. Zhang, T.J. Webster significantly enhanced osteoblast adhesion compared to respective conventional metals [39]. In addition, Puckett et al. created linear patterns of nano-features of Ti via electron beam evaporation. This study revealed that the nanoregion of the patterned Ti induced greater osteoblast adhesion than the micron-rough regions and also controlled osteoblast morphology and alignment [40]. Moreover, an electrochemical method known as anodization, a wellestablished nanosurface modification technique, has been used to fabricate highly porous TiO2 nanotube layers on Ti. Through the anodization of Ti in dilute hydrofluoric acid (HF) electrolyte solutions, nanotubes with diameters around 100 nm and lengths around 500 nm can be implemented into the TiO2 layers of Ti. Yao et al. reported greatly improved osteoblast functions on nanotubular anodized Ti compared to unanodized Ti in vitro [41]. Moreover, increased chondrocyte adhesion was also observed on anodized nanotubular Ti compared to unanodized Ti in a recent study, thus, suggesting the possibility of promoting cartilage growth on anodized Ti [42].

The promise of nanomaterials for vascular tissue engineering applications Due to the increasing prevalence of vascular diseases (such as atherosclerosis), vascular grafts of greater efficacy to replace damaged blood vessels are needed. For example, the American Heart Association reported that coronary heart disease mostly caused by atherosclerosis had led to 451,326 deaths in 2004 and is the single leading cause of death in the U.S. today [94]. In addition, peripheral arterial disease related to blood vessels outside of the heart and brain affects about 8 million Americans. Over 500,000 coronary and periphery bypass surgeries were performed in the U.S. in 2005. Since vascular tissue is a layered structure pos-

Figure 6 Fluorescent microscopy images of greatly increased endothelial cell proliferation on nanostructured Ti compared to conventional Ti. Scale bar is 10 ␮m. Images are adapted from [43].

Nanotechnology and nanomaterials: Promises for improved tissue regeneration sessing numerous nanostructured features (i.e., due to the presence of collagen and elastin in the vascular ECM), nanomaterials have shown much promise to improve vascular cell (specifically, endothelial and smooth muscle cells) functions to inhibit thrombosis and severe inflammation. Choudhary et al. reported that vascular cell adhesion and proliferation were greatly improved on nanostructured Ti compared to conventional Ti (Fig. 6) [43]. Interestingly, greater competitive endothelial cell adhesion, total elastin and collagen synthesis were observed than respective vascular smooth muscle cell functions on nanostructured Ti after 5 days in culture. Since one of the current problems with vascular stents is the overgrowth of smooth muscle cells compared to endothelial cells, these results suggest that endothelial cell functions were enhanced over that of vascular smooth muscle cells, thus, increasing the probability of endothelialization on nanostructured stents. It was speculated that the increased nano-roughness and particle boundaries on nanostructured Ti contributed to the observed favorable endothelial cell functions. In addition, Miller et al. created biodegradable PLGA vascular grafts with nanometer surface features through chemical etching in NaOH and through a cast-mold technique [44—46]. Results demonstrated that both those polymers created through chemical etching and a polymer cast-mold technique possessed random nanometer structures which promoted endothelial and vascular smooth muscle cell proliferation compared to the conventional PLGA [44]. A further study provided evidence that nanostructured PLGA promoted more fibronectin and vitronectin adsorption from serum than conventional PLGA, thus, leading to the greater vascular cell responses on the nanostructured PLGA [45]. In order to elucidate specific nanometer surface features which promoted vascular cell responses, 500, 200, and 100 nm polystyrene spheres were used to cast PLGA [46]. Results demonstrated that the PLGA with 200 nm structures promoted vascular cell responses and greater fibronectin interconnectivity compared to smooth PLGA and PLGA with 500 nm surface features (Fig. 7). Such results have been translated into the design of 3-D polymer scaffolds as several random and aligned 3D nanofiber scaffolds have been fabricated for vascular applications. For example, Lee and colleagues fabricated

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and evaluated a variety of electrospun collagen, elastin and synthetic polymer (such as PLLA, PLGA and PCL) nanofiber scaffolds for vascular graft applications [47]. These scaffolds have tailorable mechanical properties and exceptional cytocompatibility properties for vascular applications. Specifically, extensive smooth muscle cell infiltration was observed in the collagen/elastin/PLLA scaffold after 21 days of culture. By electrospinning on a rotating disk collector, Xu et al. fabricated an aligned PLLA-CL (75:25) nanofibrous scaffold which mimicked the oriented fibril structure in the medial layer of an artery [48]. Not only did coronary artery smooth muscle cells favorably interact with that scaffold, but cells also oriented along the fiber, further emulating the natural environment. In addition to the electrospinning method, self-assembled peptides have been formulated into scaffolds to mimic the vascular basement membrane showing excellent cytocompatibility properties for vascular tissue repair. Genove et al. functionalized three peptide sequences from two basement membrane proteins (specifically, laminin and collagen IV) onto a self-assembled peptide scaffold [49]. These tailorable self-assembled scaffolds enhanced endothelialization and improved nitric oxide release and laminin as well as collagen IV deposition by the endothelial cell monolayer. These results indicate the promise of biomimetic nanoscaffolds for improving vascular tissue engineering applications and when coupled with the aforementioned promise of nanomaterials for orthopedic applications, suggests a possible wide spread use of nanomaterials for numerous tissue engineering applications.

The promise of nanomaterials for neural tissue engineering applications In addition to aiding in orthopedic and vascular tissue regeneration, nanomaterials are also helping to heal damaged nerves. In particular, nervous system injuries, diseases, and disorders occur far too frequently. In the U.S., there are about 250,000—400,000 patients suffering from a spinal cord injury each year [50]. Although various cell therapies and implants have been investigated, repairing damaged nerves and achieving full functional recovery are still challeng-

Figure 7 Atomic force microscopy images of fibronectin (5 ␮g/mL) coated PLGA cast nanosphere surfaces. (A) Phase images of fibronectin adsorbed on PLGA with 500 nm surface features showed no interconnectivity between proteins. (B) Phase images of fibronectin adsorbed on PLGA with 200 nm surface features showed significant interconnectivity between fibronectin. (C) PLGA with 200 nm surface features only. Images are adapted from [46].

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Figure 8 Schematic graphs of injured nerve regeneration in the central and peripheral nervous systems. (A) Central nervous system recovery process with glial scar tissue formation and (B) peripheral nervous system recovery process involving the activity of Schwann cells, macrophages, and monocytes. Images are redrawn and adapted from [52,29].

ing considering the complexity of the nervous system. For example, nearly 50,000 patients die among the average 1.4 million Americans that sustain traumatic brain injuries each year [51]. Generally, the nervous system can be divided into two main parts: the central nervous system (CNS) (including the brain and the spinal cord) and the peripheral nervous system (PNS) (including the spinal and autonomic nerves). These two systems have two different repair procedures after injury (Fig. 8) [52—54]. For the PNS, the damaged axons usually regenerate and recover via proliferating Schwann cells, phagocytosing myelin by macrophages or monocytes, forming bands of Bünger by the bundling of Schwann cells and sprouting axons in the distal segment [55]. However, it is difficult to re-extend and re-innervate axons to recover functions in the CNS due to the absence of Schwann cells. More importantly, due to the influence of astrocytes, meningeal cells and oligodendrocytes, the thick glial scar tissue typically formed around today’s neural biomaterials will prevent proximal axon growth and inhibit neuron regeneration [53]. For these reasons, CNS injuries may cause severe functional damages and are much more difficult to repair than PNS injuries.

The ideal materials for neural tissue engineering applications should have excellent cytocompatible, mechanical and electrical properties. Without good cytocompatibility properties, materials may fail to improve neuron growth and at the same time may elicit severe inflammation or infection. Without sufficient mechanical properties, the scaffold may not last long enough to physically support neural tissue regeneration. In addition, superior electrical properties of scaffolds are required to help stimulate and control neuron behavior under electrical stimulation, thus, more effectively guiding neural tissue repair. To date, various natural and synthetic materials have been adopted as nerve grafts to repair severely damaged nerves by bridging nerve gaps and guiding neuron outgrowth. However, there are still many shortcomings for these neural biomaterials including: for autografts, it is usually difficult to collect sufficient donor nerves from patients and it is possible donor site nerve functions may be impaired [56], and for allografts, inflammation, rejection and transmission of diseases may frequently occur leading to implant failures [57]. Other traditional biomaterials (such as silicon probes used in neuroprosthetic devices and polymers used as nerve conduits)

Nanotechnology and nanomaterials: Promises for improved tissue regeneration used for neural tissue repair have been limited by the extensive formation of glial scar tissue around the material as well as non-optimal mechanical and electrical properties for nerve regrowth. Nanotechnology provides a wide platform to develop novel and improved neural tissue engineering materials and therapy including designing nanofiber/nanotube scaffolds with exceptional cytocompatibility and conductivity properties to boost neuron activities. Nanomaterials have also been used to encapsulate various neural stem cells and Schwann cells into biomimetic nanoscaffolds to enhance nerve repair. For example, work by Ramakrishna et al. has led to the fabrication of various nanofibrous PLLA or PCL scaffolds via electrospinning and phase separation; such scaffolds have demonstrated excellent cytocompatibility properties for neural tissue engineering applications [58—60]. Recently this research group incorporated laminin (a neurite promoting ECM protein) into electrospun PLLA nanofibers in order to create a biomimetic scaffold for peripheral nerve repair [58]. The results showed that neurite outgrowth improved on laminin-PLLA scaffolds produced by facile blended electrospinning. In another recent report, electrospun PCL/chitosan nanofiber scaffolds exhibited improved mechanical properties compared to chitosan [59]. Schwann cells also proliferated well on this PCL/chitosan nanofiber scaffold. In addition, Zhang and colleagues also reported favorable neural cell responses on the self-assembled peptide nanofiber scaffold (called SAPNS). Holmes et al. reported that the self-assembled peptide scaffold supported neuronal cell functions, neurite outgrowth and functional synapse formation among neurons [61]. Furthermore, Ellis-Behnke et al. investigated SAPNS for in vivo axon regeneration in the CNS [62]. The SAPNS aided in CNS regeneration to help axonal growth, even ‘‘knitting’’ the brain tissue together and successfully improving functional recovery. Due to the fact that carbon nanotubes/fibers have excellent electrical conductivity, strong mechanical properties, and have similar nanoscale dimensions to neurites, they have been used to guide axon regeneration and improve neural activity as biomimetic scaffolds at neural tissue injury sites. In particular, Mattson et al. found for the

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first time that neurons grew on multiwalled carbon nanotubes (MWCNTs) [63]. They observed over a 200% increase in total neurite length and nearly a 300% increase in the number of branches and neurites on MWCNTs coated with 4-hydroxynonenal compared to uncoated MWCNTs. Hu et al. revealed that different surface charges of MWCNTs, obtained through chemical functionalization, resulted in different neurite outgrowth patterns (such as neurite length, branching and the number of growth cones) [64]. They demonstrated that positively charged MWCNTs significantly increased the number of growth cones and neurite branches compared to negatively charged MWCNTs, thus, controlling neural growth. Lovat et al. demonstrated that purified MWCNTs potentially boosted electrical signal transfer of neuronal networks (Fig. 9A and B) [65]. Moreover, highly ordered CNT/CNF matrices or free standing nanotube films have been fabricated for neural tissue engineering applications [66—67]. For instance, Gheith et al. investigated the biocompatibility of a freestanding positively charged SWCNT/polymer thin-film membrane prepared by layer-by-layer assembly [66]. They observed that 94—98% of neurons were viable on the SWCNT/polymer films after a 10 day incubation. The SWCNT/polymer films favorably induced neuronal cell differentiation, guided neuron extension and directed more elaborate branches than controls. In order to inhibit activated astrocyte functions which result in the formation of glial scar tissue, McKenzie et al. incorporated different weight ratios of high surface energy CNFs into polymers and demonstrated for the first time that astrocyte adhesion can be effectively inhibited by using CNF/polymer composites [68]. In addition, decreased astrocyte proliferation was observed on nanostructured CNFs, thus, leading to decreased glial scar tissue formation on such materials. On the other hand, NguyenVu et al. fabricated a vertically aligned CNF nanoelectrode array by creating a thin conductive polymer film coating (such as polypyrrole) for neural implants [69]. The vertical CNF arrays had more open and mechanically robust 3-D structures as well as better electrical conductivity which contributed to forming an intimate neural-electrical interface between cells and nanofibers (Fig. 9C—E). Gabay et

Figure 9 SEM images of neural cell adhesion on carbon nanotube/fiber substrates. (A) Neonatal hippocampal neurons adherent on purified MWCNT glass substrates with extended neurites after 8 days; inset image (B) shows a single neurite in close contact to CNTs. Images are adapted from [65]. (C), (D) and (E) PC12 neural cells grown freestanding on vertically aligned CNFs coated with polypyrrole at different magnifications. Images are adapted from [69].

76 al. developed a novel method to fabricate islands of CNT on substrates. Neurons preferably attached on the CNT islands and further extended their neurites to form interconnected neural networks according to pre-designed patterns [70]. In this manner, the CNTs/CNFs and their composites are promising scaffold candidates for injured neural tissue repair. Studies have also provided evidence that individual CNTs/CNFs may be useful in treating neurological damage when combined with stem cells. Stem cells have the potential to differentiate and self-renew into controllable, desirable cell types: i.e., neural stem cells in the CNS can differentiate into neurons and astrocytes [71]. Therefore, many efforts have focused on impregnating multi-potential stem cells into CNTs/CNFs and other nanoscaffolds, which can be directly transplanted into injury sites and assist neural tissue recovery. However, a challenging problem has been to determine how to effectively deliver and selectively differentiate stem cells into favorable neuronal cell types at injury sites in order to regenerate desirable tissue. Although the underlying mechanisms triggering differentiation of stem cells are not entirely clear, accumulated evidence has indicated that novel biomimetic nanomaterials may contribute to selective stem cell differentiation (without the use of growth factors) [72,73]. For example, Lee et al. injected CNFs impregnated with stem cells into stroke damaged neural tissue in rat brains and found

L. Zhang, T.J. Webster extensive neural stem cell differentiation with little glial scar tissue formation in vivo [72]. After 1 and 3 weeks of animal implantation, histological sections showed that neural stem cells favorably differentiated into neurons (Fig. 10A and B) and little to no glial scar tissue (Fig. 10C and D) formed around CNFs compared to controls (only implanting stem cells without CNFs or implanting CNFs without cells). Furthermore, Jan et al. successfully differentiated mouse embryonic neural stem cells including neurospheres and single cells into neurons on layer-bylayer assembled SWCNT/polyelectrolyte composites [73]. The layer-by-layer SWCNT composites promoted slightly more neurons and fewer astrocytes on substrates during a 7 day culture period than poly-L-ornithine (a common substrate for neural stem cell studies). Clearly, CNTs/CNFs played an important role in effectively delivering stem cells into injured sites and promoted stem cells to differentiate into favorable neurons to repair damaged neural tissues.

The promise of nanomaterials for bladder tissue engineering applications Nanomaterials have also been used in soft tissues, such as the bladder. As the 6th most common cancer in the U.S., urinary bladder cancer affects over 53,200 Americans and

Figure 10 Histology of CNFs impregnated with stem cells into stroke damaged rat neural tissue after 3 weeks. (A) and (B), numerous active neuroprogenitor cells and fully differentiated neurons (brown stained cells, marked by nestin and MAP2, respectively) were found around CNFs. (C) and (D), few glial cells interacting with CNFs led to little or no glial scar tissue formation. GFAP is a marker for astrocytes; CD11b is a marker for activated microglia cells. Black areas in the images are CNFs. Scale bar is 25 ␮m. Images are adapted from [72].

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Figure 11 Schematic illustration of the bilayer smooth muscle cell/urothelial cell (SMC-UC) encapsulation in a PA/PGA gel. Image is adapted from [76].

leads to 12,200 deaths annually [74]. Although standard treatments such as surgery to remove bladder tumors followed by radiation, chemotherapy and immunotherapy have improved, various complications (such as systemic infections, flu-like symptoms and cancer recurrence, etc.) with these procedures are still too commonly reported. Sometimes, radical cystectomy by removing parts of and even the entire bladder is needed. However, such a drastic approach requires the implantation of a bladder tissue replacement to quickly recover bladder functions. As emerging bladder tissue engineering materials, nanomaterials provide a promising approach to more efficiently improve bladder tissue regeneration for the same reasons mentioned earlier for other tissue systems (biologically inspired roughness, increased surface energy, selective protein adsorption, etc.). In particular, Harrington and colleagues have coated a series of branched or linear self-assembling peptideamphiphile nanofibers containing cell-adhesive RGDS on traditional PGA scaffolds [75]. Human bladder smooth muscle cell densities on the branched PA/PGA nanocomposite were greater than on the uncoated PGA after 17 days of culture. In a recent review, they encapsulated bladder smooth muscle cells and urothelial cells into a PA/PGA nanofibrous gel containing specific growth factors (Fig. 11) [76]. Due to their ability to mimic the oriented nanostructured bladder ECM, electrospun polymer nanofibers have been used in bladder tissue engineering. Baker et al. showed that bladder smooth muscle cells were aligned on oriented electrospun polystyrene scaffolds similar to the native bladder tissue [77]. This study also demonstrated that argon plasma treated electrospun polystyrene nanofibers significantly improved smooth muscle cell attachment. Fibrinogen has also been electrospun into a scaffold for urinary tract tissue regeneration [78]. This study demonstrated that human bladder smooth muscle cells rapidly migrated into, proliferated onto and remodeled the 3-D fibrinogen scaffold. Other nanostructured polymers with superior biocompatibility properties have been widely investigated by Haberstroh and colleagues for bladder tissue regeneration applications [79—81]. For instance, this research

group used nanotextured PLGA and poly(ether urethane) (PU) films to successfully enhance bladder smooth muscle cell functions [79]. Through chemical etching technologies, PLGA and PU were transformed from their native nano-smooth surface features into those possessing a high degree of nano-roughness. This study revealed that nanoroughness played a critical role in promoting bladder smooth muscle cell proliferation once the influence of surface chemistry change was eliminated (through castmold techniques using the chemical treated polymer as the cast). Recently, Pattison et al. also demonstrated that nanostructured PLGA and PU 3-D scaffolds prepared by a solvent casting and salt leaching methods significantly enhanced bladder smooth muscle cell functions and ECM protein synthesis compared to conventional nanosmooth polymers in vitro [80]. Furthermore, preliminary in vivo studies have provided evidence that nanostructured polymer scaffolds form little to no calcium oxalate stones (stone formation is a common problem during bladder replacements) in augmented rat bladders. Although there are many unknowns for the use of nanomaterials in bladder tissue engineering applications, utilizing these biomimetic nanomaterials with progenitor cells is undoubtedly a promising future research direction to regenerate bladder tissue in resected bladder cancerous tissue locations.

Potential risks of nanomaterials towards human health As described, nanotechnology has achieved tremendous progress in a relatively short time period in medical applications. As a result, nanomaterials have begun to enter wide spread industrial production. For instance, nanoceramics are commercially available as new bone grafts or as implant coating materials (i.e., nano-HA paste—–Ostim® from Obernburg, Germany; nano-beta-tricalcium phosphate-Vitoss from Orthovita, USA) [82]. However, it is important to note that the research on nanomaterials for tissue engineering applications is still at its infancy and, most importantly, the

78 influence of nanomaterials on human health and the environment is not well understood. In particular, toxic responses to nanoparticles generated from the degradation of implanted nanomaterials, via wear debris from artificial joints with nanofeatures, and heavy metals (iron, nickel and cobalt catalysts) remaining in CNTs, have all been reported. Many reports on the cellular uptake of nanoparticles in the lungs, immune system, as well as other organs have been published [83—85]. Nanoparticle uptake by endothelial cells, alveolar macrophages, pulmonary or intestinal epithelium, nerve cells etc. has been reviewed and, thus, may possess a problem for this field if not thoroughly understood before being applied widely [83]. Gutwein et al. investigated the viability of osteoblasts in vitro when cultured in the presence of nanoalumina and titania particles for 6 h [84]. This study demonstrated that ceramic nanoparticles were safer to osteoblasts than conventional, micron-sized, ceramics particles. In contrast, in an in vivo study, Lam et al. showed that CNTs were more toxic than carbon black in the lungs, which may be a serious occupational health hazard in chronic inhalation exposures [85]. Sometimes nanoparticle interactions with biomolecules in vivo or their aggregation states may change their toxicity to humans. But the often contradictory results of current studies are clearly not enough to provide the final answer concerning nanomaterial toxicity. In depth investigations of nanomaterials on human health and the environment are necessary to fully elucidate whether nanoparticles should be used in biomedical applications.

Conclusions To date, there has been an exponential increase in studies using nanotechnology for tissue engineering applications. To be concise, this paper only covered the recent progress using nanomaterials for bone, cartilage, vascular, neural and bladder tissue regeneration. Other reviews of nanotechnology applications for the specific regeneration of tissues can be found [15,76,86—92]. Nanotechnology approaches for the regeneration of other types of tissues (such as the muscle, skin, kidneys, liver, pancreas, and the immune system) have also been reviewed [92,93]. Although many challenges may lie ahead, synthetic nanomaterials can mimic properties of the natural ECM and thus, show great potential for numerous tissue engineering applications. Particularly, due to their excellent cytocompatibility properties, research interest has been evoked to use nanomaterials as the next generation of tissue repair materials. In the future, the underlying mechanisms of the in vivo interactions between nanomaterials and cells at the molecular level will significantly advance the development of this field.

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L. Zhang, T.J. Webster Thomas J. Webster is an associate professor of Engineering and Orthopedics at Brown University. His degrees are in chemical engineering from the University of Pittsburgh (BS, 1995) and in biomedical engineering from Rensselaer (MS, 1997; PhD, 2000). His research addresses the design, synthesis, and evaluation of nanophase materials for various regenerative medicine applications. His lab group has generated 4 books, 33 book chapters, 85 invited presentations, 215 literature

articles, and 245 conference presentations. He is the founding editor-in-chief of the International Journal of Nanomedicine and is on the editorial board for 10 other journals. Among other awards, Dr. Webster received the 2002 Biomedical Engineering Society Rita Schaffer Young Investigator Award, 2004 Outstanding Young Investigator Award from Purdue University, 2005 Wallace Coulter Foundation Early Career Award, and in 2007 was elected as a Fellow of the American Academy of Nanomedicine. His research has led to the formation of two nanotech companies.