Electrospun scaffolds for stem cell engineering

Advanced Drug Delivery Reviews 61 (2009) 1084–1096

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Electrospun scaffolds for stem cell engineering☆ Shawn H. Lim a, Hai-Quan Mao b,⁎ a

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 3400 North Charles Street, 102 Maryland Hall, Baltimore, MD 21218, USA Department of Materials Science and Engineering and Whitaker Biomedical Engineering Institute, Johns Hopkins University, 3400 North Charles Street, 102 Maryland Hall, Baltimore, MD 21218, USA

b

a r t i c l e

i n f o

Article history: Received 23 January 2009 Accepted 16 July 2009 Available online 30 July 2009 Keywords: Electrospinning Nanofiber Stem cell niche Topographical cue Regenerative medicine Stem cell expansion Fate specification Directed differentiation

a b s t r a c t Stem cells interact with and respond to a myriad of signals emanating from their extracellular microenvironment. The ability to harness the regenerative potential of stem cells via a synthetic matrix has promising implications for regenerative medicine. Electrospun fibrous scaffolds can be prepared with high degree of control over their structure creating highly porous meshes of ultrafine fibers that resemble the extracellular matrix topography, and are amenable to various functional modifications targeted towards enhancing stem cell survival and proliferation, directing specific stem cell fates, or promoting tissue organization. The feasibility of using such a scaffold platform to present integrated topographical and biochemical signals that are essential to stem cell manipulation has been demonstrated. Future application of this versatile scaffold platform to human embryonic and induced pluripotent stem cells for functional tissue repair and regeneration will further expand its potential for regenerative therapies. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrospun nanofibers recapitulate features of the stem cell niche . . . . 2.1. Electrospun scaffolds as stem cell culture supports . . . . . . . . . 2.2. Stem cell interactions with ECM-mimetic electrospun fibers . . . . 2.3. Nanofiber modification for presentation of biochemical cues to stem 3. Electrospun scaffolds directly influence stem cell/progenitor differentiation 3.1. Effects of fiber alignment . . . . . . . . . . . . . . . . . . . . . 3.2. Effects of fiber diameter . . . . . . . . . . . . . . . . . . . . . 3.3. Release of bioactive molecules for control of stem cell fate . . . . . 4. Rational design of stem cell constructs for tissue engineering . . . . . . . 4.1. Engineering cardiovascular tissue . . . . . . . . . . . . . . . . . 4.2. Myotube formation for skeletal muscle engineering . . . . . . . . 4.3. Nanocomposites for osteogenesis . . . . . . . . . . . . . . . . . 5. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Nanofibers in Regenerative Medicine & Drug Delivery”. ⁎ Corresponding author. Tel.: +1 410 516 8792; fax: +1 410 516 5293. E-mail addresses: [email protected] (S.H. Lim), [email protected] (H.-Q. Mao). 0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.07.011

The aim of regenerative medicine is to repair or replace damaged or diseased tissues in the human body. Progress in the field has been achieved at an accelerated pace, largely due to the improved understanding of the cell and tissue development process. Much of the focus has been on the development of cell transplantation strategies, where implanted cells either replace the loss-of-function, such as insulin-

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secreting cells for treatment of diabetes [1], or provide appropriate signals to recruit host cells to repair the damage site, or in the case of stem cell transplantation to differentiate into the desired lineages and contribute to formation of new tissue [2–4]. Cell therapy based upon stem and progenitor cells have many distinct advantages and offer tremendous potential for regenerative medicine. The multipotency and proliferative nature of stem cells makes them a more reliable cell source than terminally differentiated phenotypes. Autologous adult tissue-derived stem cells have the additional advantage of being immune-compatible, although they are lineage-restricted. On the other hand, embryonic stem (ES) cells are pluripotent, but their derivation is ethically controversial, and much work remains to be done to refine the control over ES cell differentiation prior to and after implantation before these therapies can be put to widespread use. Nevertheless, the flexibility and promise of stem cells has generated great scientific interest and they are currently a major focus in regenerative medicine. Regenerative medicine has much to benefit from rapid advances in the biomaterials engineering toolbox. For example, stem cell therapy can be complemented by combining the cells with a supportive scaffold to fill the damage site and assist in tissue repair. Rational design of cell-supportive scaffolds has led to the evolution of scaffolds beyond that of merely a passive support, towards systems that can interact with and direct stem cell fates, in addition to promoting integration with the host tissue. Much experimental evidence exists to support the notion that stem cell fate can be controlled via interactions with a synthetic scaffold, either prior to or after in vivo implantation [5–8]. A widely-cited example is the work reported by Silva et al. demonstrating that neural stem cells encapsulated within a peptide-based amphiphilic hydrogel presenting an artificially high density of the laminin-inspired signaling epitope isolucine–lysine– valine–alanine–valine (IKVAV) selectively differentiated into neurons at the expense of astrocyte differentiation [9]. Indeed, an ideal implantable scaffold would recapitulate many of the salient features of the native extracellular matrix (ECM) in the target tissue, including but not limited to presentation of adhesion molecules, topographical and biochemical cues. Aside from creating cell–scaffold constructs for implantation, another potential contribution of biomaterials science to regenerative medicine is the development of ex vivo methods for efficient expansion and differentiation of stem cells. Due to the relative scarcity of adult stem cells, access to an abundant supply of cells could easily become a therapeutic bottleneck. Current methods for stem cell manipulation have low efficiencies for either expansion or differentiation, yielding mixed populations of cells that require the additional step of separation and purification [10–13]. Uncontrolled differentiation of neural stem cells following transplantation has also been shown to generate undifferentiated mitotic neuroepithelial cells, which may be tumorigenic [14]. From both a fundamental and clinical perspective, further understanding of the interactions of stem cells with artificial scaffold platforms in vitro will provide instructive insights towards the development of new technologies for stem cell manipulation. Over the past decade, electrospinning has gained rising popularity as a means of fabricating scaffolds with micro to nanoscale features similar to the hierarchical structure of the ECM. The ability to mimic the ECM structural organization is an important consideration in rational design of a cell-responsive scaffold platform upon which additional functionalities can be incorporated. Electrospinning offers great flexibility in terms of the choice of scaffold material, as well as finer control over the scaffold geometry. Fibers with diameters ranging from tens to hundreds of nanometers can be easily produced, where the diameter is adjusted empirically via modulation of spinning parameters such as flow rate and collecting distance, and polymer solution properties such as solvent, concentration, conductivity, and surface tension [15–17]. Electrospun polymeric fibrous meshes also

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offer a higher surface area for cell attachment, are easy and inexpensive to fabricate and scale-up, and are relatively reproducible. Synthetic polymer-based systems offer additional advantages with their adjustable mechanical properties, as well as ease of surface functionalization via protein coatings, or chemical conjugation of specific signaling molecules. In this review, we will provide an overview of recent efforts and current progress in manipulation and control of stem cell fates via cellular interactions with electrospun fibrous scaffolds for ex vivo stem cell expansion and differentiation, as well as applications of stem cell-fiber scaffold constructs for tissue engineering and regenerative medicine. We will first present the earlier applications of unmodified electrospun scaffolds for stem cell culture, followed by discussion of the various modifications to the basic electrospun fiber platform, such as introduction of ECM molecules and ECM-like analogs, surface presentation of biochemical cues, and controlled release of bioactive molecules for initiation of stem cell signaling. Finally, we will outline several future perspectives on engineering functional tissue constructs from stem cells seeded on electrospun scaffolds. 2. Electrospun nanofibers recapitulate features of the stem cell niche Stem cells exist in vivo within a unique tissue-specific unique microenvironment commonly termed the stem cell niche (Fig. 1). The ECM comprises the structural component of the niche; in addition to providing the physical support matrix for cell attachment, migration and division, it also presents biochemical signals to cells that are modulated through molecular interactions with ECM proteins such as heparin sulfate proteoglycans (HSPGs) or through adjacent cells [18,19]. Electrospun nanofibrous scaffolds are able to recapitulate both the structural features of the ECM, and, via various modifications to the fiber material or surface, the biochemical cues as well. This type of artificial scaffold with enhanced biofunctionality would comprise a more biomimetic microenvironment for ex vivo stem cell culture. 2.1. Electrospun scaffolds as stem cell culture supports One of the major motivating factors supporting the use of electrospun fibrous scaffolds as supports for stem cell culture is the observation that these fibrous scaffolds recapitulate the scale and three-dimensional arrangement of collagen fibrils in the ECM. Electrospun meshes generally comprise of nonwoven fibers with diameters in the hundreds of nanometers, and highly interconnected pores that are tens of micrometers in diameter [16,20]. The high surface area–volume ratio of these fibrous meshes also ensures abundant area for cell attachment, which allows for a higher density of cells to be cultured as compared with a flat, two-dimensional surface. The morphological resemblance of electrospun nanofibers to native ECM suggests their natural application as a supportive matrix for creating scaffold constructs from stem cells. Much of the earlier work on electrospun fibrous scaffolds as matrices for stem cell culture was aimed at establishing the biocompatibility of such a scaffold architecture and its suitability for the creation of tissue-like constructs in vitro. In an effort to assess bone formation from human MSCs (hMSCs), Vacanti et al. used a rotational oxygen-permeable bioreactor to provide uniform oxygen tension and mechanical stress during osteogenic induction of hMSCs seeded on polycaprolactone (PCL) nanofibrous scaffolds [21]. After 4 weeks of culture, scaffolds were found to be qualitatively stiffer, and Von Kossa staining revealed that significant calcification was present throughout the construct. Constructs fabricated from nanofibrous scaffolds maintained their original size and shape, demonstrating improved structural integrity over the culture period as compared with previously tested macroporous poly(lactic-co-glycolic acid) foams, which had a tendency to collapse or exhibit severe shrinkage. When

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Fig. 1. The concept of the stem cell niche, and role of the extracellular matrix in regulating stem cell survival and signaling. ECM: extracellular matrix; GF: growth factor; [GF]: growth factor concentration. Matrix topography may facilitate these processes. Adapted with permission from Macmillan Publishers Ltd [18].

this study was extended a further four weeks following implantation of constructs into the omenta of rats, constructs adopted a rigid and bone-like appearance, with histological evidence of extensive alkaline phosphatase and collagen I deposition both on the outer and inner portions of the scaffolds [22]. Concurrent with the efforts to engineer bone tissue using nanofibrous scaffolds, Li et al. also showed that these substrates were suitable for supporting chondrogenic induction of human bone marrow-derived MSCs in vitro [23]. Culturing MSCs on the scaffolds not only produced comparable quantities of GAGs, but also exhibited similar levels of chondrogenic gene expression as MSCs cultured as in a cell pellet, an established model of chondrogenic induction. Of note, culture on scaffolds abolished the requirement for the extremely high cell densities previously shown to be critical for MSC differentiation. The same group later demonstrated the general flexibility of this scaffold system in supporting multiphasic MSC differentiation by successfully achieving differentiation of MSCs into chondrogenic, osteogenic and adipogenic phenotypes [24] (Fig. 2). Recently, the feasibility of this technology was tested in vivo by evaluating hMSC-nanofibrous scaffold constructs in the repair of a full-thickness articular cartilage defect created in a swine model [25]. Cartilage repair is a particularly challenging problem in regenerative medicine due to the avascular nature of the tissue, with limited access to nutrients as well as a cell source for repopulating the defect. Currently explored therapies involve implantation of tissue plugs or autologous cells [26], which necessitate donor site morbidity; artificial scaffolds thus hold great promise as an alternative therapy for cartilage repair. Electrospun PCL nanofibrous scaffolds were seeded with either hMSCs or porcine chondrocytes and allowed to repopulate the scaffold for 3 weeks in vitro, following which time the constructs were sutured onto defects created on the load-bearing portion of the femoral condyle of the animals' hind knees. The most complete repair was observed in defects treated with the hMSC-seeded scaffolds, which regenerated into a smooth surface with hyaline cartilage-like tissue formation. In contrast, scaffolds seeded with chondrocytes produced tissue resembling fibrocartilage and the repaired surface was discontinuous. In both test conditions, significant remodeling of subchondral bone was observed, with the loss of the tidemark separating bone and cartilage. hMSC-seeded constructs also showed superior equilibrium compressive stress compared with chondrocyteseeded or acellular scaffolds, albeit still inferior to the mechanical properties of native cartilage. The authors proposed that hMSCs supported by an electrospun nanofibrous scaffold is a promising approach for treatment of cartilage defects, as the stem cells are able

to proliferate within the scaffold, resulting in higher cell density as well as matrix biosynthesis and deposition. 2.2. Stem cell interactions with ECM-mimetic electrospun fibers Structural proteins of the ECM such as collagen, fibronectin and laminin present cells with a myriad of recognition sites for binding cell surface integrins, and HSPGs sequester and present growth factors and cytokines. For example, fibronectin contains the peptide sequence arginine–glycine–aspartic acid (RGD) and its synergy site proline– histidine–serine–arginine–asparagine (PHSRN), which is widely implicated in integrin-mediated cell adhesion across a variety of cell types [27,28]. Interactions between stem cells and ECM molecules are implicated in supporting normal cell fates, including cell migration, proliferation, and differentiation. The incorporation of ECM molecules or ECM-inspired peptide analogs into electrospun scaffolds is thus a means of combining the structural features of the scaffolds with the biofunctionality of ECM proteins. Such biologically active nanofibers can better support stem cell attachment and growth. For example, fibers with surface laminin coating are favorable for the attachment, proliferation and differentiation of neural stem cells and progenitor cells [29]. A number of research groups have successfully fabricated electrospun scaffolds comprised of type I collagen, and evaluated their effectiveness at supporting stem cell differentiation. Shih et al. compared the growth and osteogenic induction of bone marrow-derived hMSCs on type I collagen nanofibers [30]. Nanofibrous collagen scaffolds supported greater cell proliferation over uncoated tissue culture polystyrene (TCPS) surfaces; hMSCs on collagen fibers also had elevated expression of differentiated osteogenic markers. hMSCs on collagen nanofibers showed fewer focal adhesions than on TCPS, hinting at the possible involvement of cytoskeletal-linked signaling pathways. A study by Sefcik et al. suggested that simply presenting collagen in nanofiber form better supports osteogenic differentiation than coating collagen onto 2D tissue culture surfaces [31]. Using a novel serum-free osteogenic induction medium, adipose-derived stem cells cultured on electrospun collagen nanofibers enhanced their expression of osteogenic genes compared to 2D collagen coating. Although the integrity of the collagen nanofibers did not persist beyond 9 days in culture, the initial cell–scaffold interactions were ostensibly sufficient to account for the observed differences in degree of osteogenic differentiation. However, the difficulty of achieving a relatively stable scaffold in culture underlines the fundamental difficulties in

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Fig. 2. Multiphasic differentiation of hMSCs on electrospun fibrous scaffold. Scanning electron micrographs (SEM) of PCL nanofibrous scaffold at (a) low magnification, scale bar = 30 μm; (b) high magnification, scale bar = 10 μm. (c–j) SEM of scaffolds after differentiation for 21 days. (c, e, g, i) Cross sections; (d, f, h, j) top view of scaffolds. (c, d) Constructs cultured in basal medium without supplements. (e, f) constructs cultured in adipogenic medium had globular cells; (g, h) constructs cultured in chondrogenic medium exhibited round chondrocyte‐like cells with thick ECM; (i, j) constructs cultured in osteogenic medium showed nodules that were mineralized. Reprinted from [24], with permission from Elsevier.

handling of electrospun collagen fibers. In theory, scaffolds for tissue regeneration should be able to support cells post-implantation for a sufficiently long time to allow cells to proliferate and repopulate the defect site. A report by Li et al. investigating the response of chondrocytes and hMSCs to a variety of scaffolds with different degradation rates revealed that cells had higher proliferation rate on

the more stable scaffolds [32]. Rapidly degrading scaffolds quickly lost their porosity and structural integrity, to the detriment of cell adhesion and ingrowth. Other reports likewise conclude that electrospun collagen scaffolds are insufficiently stable to carry out this role without some form of post-spinning modification, such as chemical crosslinking with gluteraldehyde, which introduces

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cytotoxicity issues [33,34]. The question is also raised whether simply coating a thin layer of collagen onto electrospun polymeric fibers would elicit the same effects as observed in these previous studies. An alternative approach would be to blend ECM proteins with a second polymeric component prior to electrospinning. Blending gelatin with PCL in a 30:70 ratio produced fibers that were structurally stable in culture, and still supported significantly greater cell adhesion and proliferation of C17.2 neural stem cells compared with pure PCL fibers alone [35]. Laminin are second only to collagen in terms of its ubiquity in the basal lamina, can self-assemble into networks either independently, or in close association with other ECM components such as collagen IV and nidogen, as found in the commercial ECM substrate Matrigel. Through integrin-mediated interactions, laminin plays a critical role in cell fate specification. Indeed, the expression of laminin-1 has been established to be essential during early embryogenesis [36]. In vitro culture on laminin-coated substrates has also been shown to be highly efficacious in the adhesion and expansion of neural progenitor cells. With this in mind, Neal et al. found that electrospun laminin supported a higher degree of attachment and neurite extension of adipose tissue-derived stem cells than laminin films, although there was not a statistically significant difference in the percentage of cells that expressed β3-tubulin, an early neuronal marker [37]. More widespread adoption of ECM molecule electrospinning has generally been hindered by the cost of acquiring sufficient material. The issue of maintaining scaffold stability is not a trivial one as well. Furthermore, there is considerable resistance to using animal-derived proteins in an implantable scaffold, due to issues of immunogenicity; for example, laminin is generally purified from tumor tissue [38]. One might also argue the necessity for electrospinning ECM proteins when studies have showed that coating the fiber or conjugating the fiber surface with ECM proteins is sufficient to induce the appropriate cell response. 2.3. Nanofiber modification for presentation of biochemical cues to stem cells Synthetic electrospun polymer scaffolds possess an advantage over naturally-derived biomaterials as the polymeric platform is amenable to a variety of functional modifications. Substrate presentation of functionally relevant biochemical cues or surface chemistry has been extensively investigated and shown to influence cell response. For example, surface modification with amine, hydroxyl, and carboxylic acid groups revealed that integrin binding specificity to different functional groups correlated with differential patterns of focal adhesion and matrix deposition in immature osteoblast-like cells [39]. Especially within the context of stem cell engineering, regulating stem cell fate decision via manipulation of biochemical interactions is an area of great interest. The ability to combine topographical and biochemical cues within a single scaffold presents a valuable opportunity to evaluate their synergistic impact. Achieving efficient ex vivo expansion of hematopoietic stem/ progenitor cells (HSCs) is an attractive option for the derivation of a consistent and reliable cell supply for the treatment of a variety of hematological disorders. In an expansion culture, the combination of nanofiber topography and surface functional groups have been shown to synergistically improve the self-renewal and proliferation of human cord blood-derived HSCs [40], as shown in Fig. 3. Amination of electrospun poly(ether sulfone) nanofibers resulted in a scaffold that supported the highest expansion fold of cryopreserved HSCs compared with carboxylation and hydroxylation (195-fold vs. 40-fold and 60-fold, respectively). Expansion of HSCs on aminated nanofibers resulted in the highest expansion of CFU-GEMM cells compared with similarly modified 2-D film and other substrates. More interestingly, HSCs showed better adhesion to this nanofiber matrix and prolifer-

ated as colonies with greater preservation of the proportion of stem and progenitor cells. This result suggests a selective enrichment effect by these functional nanofibers during expansion. The clinical potential of this technology was further demonstrated by the successful engraftment of the bone marrow of NOD/SCID mice by HSCs expanded on aminated nanofiber substrates [41]. 3. Electrospun scaffolds directly influence stem cell/progenitor differentiation When attempting to replicate the structural hierarchy of the ECM in a synthetic scaffold, we should appreciate that the natural ECM contains components with different organizational length scales. Organization on the macro- and micro-scale is largely responsible for the structural integrity, porosity and other physical properties of the matrix. Such a structural organization may play an important role in dictating the cellular responses elicited by the local ECM components. It is worth noting that most cell–cell and cell–scaffold communications occur via nanoscale molecular presentation and organization, such as in the cases of receptor clustering and focal adhesion complex formation. Due to the ability to modulate structural parameters of the electrospun fibrous scaffold, the influence of topographical cues on the control of stem cells cultured on or in these scaffolds can be explored in detail. 3.1. Effects of fiber alignment One unique feature of electrospun scaffolds is the ability to manipulate the deposition of fibers so as to achieve meshes of highly aligned fibers. Instead of using a grounded stationary collector, fibers can be deposited onto a continuously rotating mandrel, or onto the edge of a spinning disc, which preferentially orients the fibers in the direction of the axis of rotation (Fig. 4). Cells cultured on such scaffolds are shown to adhere and elongate along the long axis of fiber alignment [42–44]. These fibrous meshes present a simple, scalable and straightforward strategy to induce stem cell alignment and introducing directionality of cell growth in the cell-seeded constructs. This principle of contact guidance is particularly relevant in the engineering of tissues that intrinsically possess highly anisotropic cell organization, such as skeletal muscle tissue, ligaments, articular cartilage and blood vessel walls. The anisotropic nature of these tissues is critical for their proper development of function in vivo, for example, the alignment of skeletal muscle cells permits the fusion of myoblasts into polynucleated myotubes that make up the structural building blocks of the densely packed muscle fibers that generate longitudinal muscle contraction [45]. It is generally hypothesized that using the substrate to induce an elongated, possibly more physiologically relevant cell morphology could alter, or even improve the responsiveness of stem cells to extrinsically applied cues, resulting in enhanced differentiation and improved tissue organization and function. An example of this was shown by Baker and Mauck using aligned PCL fibers to replicate the network of radially aligned collagen fibrils that comprise the fibrocartilaginous menisci of the knee [43]. They found that MSCs seeded on aligned fibers resulted in a tissue with higher stiffness and modulus after 10 weeks of culture than the MSC-random fiber construct, even though both groups yielded comparable productions of glycosaminoglycan (GAG) and total collagen. This was likely due to the more organized deposition of ECM in aligned fiber construct. A recent report by Wise et al. demonstrated that culturing hMSCs on aligned electrospun nanofibers for the purpose of engineering the superficial zone of articular cartilage enhanced the chondrogenic differentiation of hMSCs compared with culturing them on 2-dimensional films, as evidenced by significantly higher sulfatedGAG production and collagen II mRNA expression [44]. Besides complementing stem cell differentiation and tissue organization, recent evidence has emerged to suggest that interactions

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Fig. 3. Aminated nanofibers support the in vitro expansion of HSCs. (a). SEM images showing that human cord blood HSCs cultured on aminated nanofibers formed circular colonies after 10 days of expansion, whereas random adherent cells were found at the cracks on 2-D film (b). Functionalized PES fibers have an average diameter of 529 nm and the average surface amino group concentration was 55 nmol/cm2. (c). Aminated nanofibers promoted the expansion of CFU‐GEMM colony forming cells (CFU‐GEMM: colony‐forming unit‐ granulocyte, erythrocyte, monocyte and megakaryocyte). (d). Phenotypic comparison between adherent and suspension populations of the cells expanded on aminated PES nanofiber scaffolds, film and TCPS. Adapted from [40], with permission from Elsevier.

with the underlying substrate topography can be exploited to actively influence stem cell fate decisions. Work done by Yim et al. demonstrated that hMSCs cultured on a uniform 350 nm wide grating exhibited elongated and orientated morphology and slower proliferation; more significantly, hMSCs on nanogratings showed significant upregulation of expression of the neuronal marker MAP2 over those on unpatterned surfaces [46]. The presence of the nanotopography alone was sufficient for induction of neuronal differentiation of hMSCs even without the addition of the neuronal inducer retinoic acid. This work is of particular interest because hMSCs do not typically differentiate into a neuronal lineage, suggesting that topographical cues might present a unique paradigm of achieving neural transdifferentiation. Although the exact signaling pathways responsible for this phenomenon have yet to be elucidated, such a fate specification mechanism might be related to the observed cytoskeletal rearrangement and nuclei elongation. Work published by Chen et al. appears to support this hypothesis. They demonstrated that changes to hMSC cell shape and

cytoskeletal tension signal through the Rho-ROCK pathway to dictate the fate choice between adipogenesis and osteogenesis [47]. Despite these promising results, most of the research conducted to date has been limited to using microfabricated substrates to demonstrate the effects of micro- and nanotopography on stem cell differentiation. Although a convenient and relatively inert platform for in vitro evaluation, microfabricated substrates are time-consuming to prepare and are not easily scaled-up for the preparation of implantable cell scaffolds. Electrospinning of aligned nanofibers is a practical solution to this obstacle. Although such scaffolds possess relatively lower degree of homogeneity in presenting the aligned topography as compared with micropatterned substrates, it is reasonable to assume that similar outcomes can be expected. A recent report published by Dang and Leong highlighted a unique application of electrospinning for the creation of highly aligned stem cell sheets [48]. Thermosensitive hydroxybutyl chitosan (HBC) was electrospun into aligned nanofiber meshes, with the aim of modulating hMSC

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Fig. 4. Control over fiber alignment and geometry. Fiber alignment can be achieved by switching from a stationary collector to a rotating disk collector. Fiber diameter can be controlled via changing parameters such as polymer solution concentration or flow rate.

response through the substrate topography. Human MSCs cultured on aligned HBC nanofibers exhibited an upregulation of the myogenic genes collagen IV, desmin, Pax-3, Pax-7 and myogenin compared with hMSCs on HBC films. The most interesting advantage of this system was that after the cells reached confluent density, the entire construct could be transferred to medium cooled to 4 °C, which dissolved the fibers and released a cell sheet. Multiple such cell sheets can conceivably be stacked to produce a thicker polymer-free tissue suitable for implantation. In neurogenesis and neural regeneration, a single axon often has to extend over relatively long distances, seeking out and forming connections with other neurons that are both meaningful and functional. During normal embryonic development, axonal pathfinding in the central nervous system is facilitated by a chemoattractants such as netrins and semaphorins. Unfortunately the same complement of spatially defined biochemical signals may not be available after traumatic injury to the nervous system. Neural regeneration in the central nervous system is complicated by the formation of the glial scar that constitutes a physical barrier to regenerating axons. On the other hand, long nerve gaps are often characteristic of peripheral nervous system injury. The use of aligned nanofibers as a physical guidance cue to simulate anisotropic directionality for regenerating axons is postulated to be a strategy that likely improves the efficiency of nerve regeneration.

and nanofiber effects on stem cells. In a recent paper, Lin et al. employed a facile modification to the electrospinning technique to control fiber diameter and dimension variability [17]. The authors demonstrated that addition of a cationic amphiphile to the polymer solution helped to reduce the surface tension of the solution, resulting in a thinner yet more stable charged polymer jet that deposited narrower, more uniform electrospun fibers. This technique was used by Christopherson et al. to investigate the impact of electrospun fiber diameter on the differentiation of adult rat hippocampal-derived neural stem cells (NSCs) [29]. Rat NSCs were cultured on laminincoated electrospun poly(ether sulfone). (PES) fiber scaffolds with average diameters of 283 nm and 749 nm, and differentiated in the presence of serum and retinoic acid (Fig. 5). NSCs cultured on the smaller diameter fibers differentiated preferentially along the oligodendrocyte lineage, whereas on the larger diameter fibers, a higher degree of neuronal differentiation was observed. This difference was attributed to the effects of the fiber diameter on limiting cell spreading and migration. On the 283 nm nanofibers, neurites were guided by the underlying fiber matrix and assumed a glial-like morphology, while cells on the 749 nm fibers were restricted to spreading along single fibers and thus did not favor differentiation into glial lineages.

3.2. Effects of fiber diameter

Tissue regeneration and repair can often be complemented by targeted delivery of a specific signaling molecules that enhance tissue growth rate, promote vascularization, or serve as chemoattractive signals for stem or progenitor cells homing to the repair site. For example, fibroblast growth factors are a mitogenic signal for a variety of cell types, and members of the transforming growth factor-beta superfamily such as BMP-2 are widely implicated in the regulation of skeletal development, including proliferation and differentiation of MSCs towards chondrogenic and osteogenic fates. Encapsulation of signaling proteins within an electrospun scaffold and their subsequent release allows the scaffold to act as a vehicle for sustained delivery of growth factors to the local cell microenvironment. This approach has been explored with a particular focus on bone regeneration. BMP-2 has been shown to have osteoinductive activity for bone formation; numerous studies demonstrated that scaffolds with encapsulated BMP-2 significantly improved bone regeneration in vivo, including extensive segmental defects in long bone [50]. Li et al. electrospun silk fibroin scaffolds containing BMP-2 and evaluated their effect on in vitro bone formation from bone marrowderived hMSCs [51]. BMP-2 incorporation elicited a four-fold higher amount of calcium deposition than scaffolds without the protein.

Varying the size scale of topographical cues by changing electrospun fiber diameter is another potential means of imposing a spatial restriction on stem cells. It is well-established that electrospun fiber diameter can be varied by changing the concentration of the polymer spinning solution, changing the solution flow rate, or regulating the distance between the needle and collector. It is postulated that through interactions with fibers on different length scales, one might significantly influence stem cell spreading, migration, proliferation and differentiation. In one study, Yang et al. evaluated the impact of fiber diameter on the morphology and neurite extension of C17.2 mouse neonatal cerebellum stem cells [49]. Poly(l-lactic acid) (PLLA) was electrospun into micro- and nanofiber scaffolds by changing PLLA solution concentration. The authors found that C17.2 cells cultured on nanofiber scaffolds showed increased neurofilament 200 kD staining than cells on microfiber scaffolds; furthermore, aligned nanofiber scaffolds promoted neurite extension from C17.2 cells over random nanofiber scaffolds. Controlling the uniformity of electrospun fiber diameter is essential to facilitating a rigorous comparison between microfiber

3.3. Release of bioactive molecules for control of stem cell fate

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Fig. 5. Impact of fiber diameter on the differentiation of rNSCs. Rat adult NSCs were differentiated on laminin‐coated PES fibers with different diameters, in comparison with tissue culture polystyrene. (a–b) SEM images of 749 nm (a); and 283 nm (b) fibers. (c) NSCs on 749 nm fibers immunostained for progenitor (Nestin+) and neuronal (Tuj1+) markers. (d) NSCs on 283 nm fibers immunostained for oligodendrocyte (RIP+) and astrocyte (GFAP+) markers. (e) Quantification of NSC differentiation on all substrates. Adapted from [29], with permission from Elsevier.

Furthermore, mRNA transcript analysis showed that exposure of hMSCs to BMP-2 resulted in significantly higher expression of osteogenic genes BMP-2, bone sialoprotein-II and collagen I than the fibrous scaffold alone. Although the results appeared to demonstrate that the encapsulated BMP-2 retained some of its biological activity, a direct comparison with soluble BMP-2 supplemented into the culture media would be more indicative of the relative potency of the released growth factor. It is also uncertain at this point whether the encapsulation is sufficient to protect the growth factor from degradation over extended periods of time, a condition which is necessary for an in vivo delivery system. Gene therapy offers an attractive alternative to drug delivery, especially in instances where the signaling protein of interest is highly unstable or difficult to formulate in vitro. Additionally, gene therapy provides a means of altering cellular activity at a genetic level, which cannot be achieved by application of an external drug. For example, delivering small interfering RNA can knockout or knockdown expression of a particular gene product [52,53], thereby promoting or inhibiting a particular differentiation pathway. On the other hand,

sustained release of plasmid DNA from a scaffold can provide continuous transfection of the cells cultured on the scaffold [54]. Transfected stem cells, particularly MSCs, can then be used as localized “bioreactors” for therapeutic delivery of bioactive gene products [55]. Particularly, in the case of protein therapeutics with short half life or requiring supraphysiological levels to elicit desired bioactivity in vivo, secretion by genetically transduced cells can result in higher concentrations locally over a prolonged time period. Work by Nie and Wang have demonstrated that it is possible to electrospin plasmid DNA in the form of chitosan nanoparticles with PLGA into nanofibers as a means of achieving scaffold-based gene delivery [56] (Fig. 6). Chitosan-DNA nanoparticles were incorporated into nanofibers with high encapsulation efficiency (N65%). Gradual release of nanoparticles occurred over a 60-day time period. Formulation of plasmid DNA into nanoparticles protected the DNA from degradation during the electrospinning process; nanoparticles released from the fibers successfully transfected bone marrow MSCs seeded on the scaffold as measured by protein secretion. Further investigation is merited to determine whether transfected hMSCs are indeed more

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Fig. 6. Incorporation of chitosan–DNA nanoparticles into electrospun PLGA for scaffoldbased gene delivery of BMP‐2. (a) SEM micrographs of fibrous scaffolds encapsulating nanoparticles, 3 types of scaffolds were spun containing different proportions of hydroxyapatite; (b) cross section of a single fiber, with encapsulated nanoparticles indicated by arrows; (c) continuous release of nanoparticles was achieved over a 66‐day time period. Increasing the amount of hydroxyapatite in the scaffolds quickened the rate of release. Reprinted from [56], with permission from Elsevier.

effective at promoting bone regeneration in vivo, and if so, whether such an effect is exerted through osteogenic differentiation or by chemotactic recruitment of host stem cells. 4. Rational design of stem cell constructs for tissue engineering The ultimate goal of tissue engineering is to design and fabricate a cell–scaffold construct that, post-implantation, facilitates regeneration of neo-tissue that is both functional and well-integrated with the host. Electrospun scaffolds have been demonstrated to possess suitable biocompatibility and mechanical strength to act as temporary scaffolding; furthermore they are easily processed into templated shapes such as tubes and patches to suit the dimensions of the defect. Continuing efforts that combine electrospinning technology with stem cells for tissue regeneration are currently under way. Here we will summarize several case studies that exemplify such efforts. 4.1. Engineering cardiovascular tissue Currently, engineering of smaller diameter grafts remains a serious challenge, primarily because the acellular grafts are prone to occlusion and thrombosis following implantation. The presence of the endothelial intimal layer in vascular grafts is believed to be antithrombogenic. It was shown that grafts seeded with endothelial progenitor cells prior to implantation in an ovine model maintained graft patency over 130 days [57]. However, reproducing the endothelial layer is only part of the solution, as graft strength and contractility are provided by the medial layer of aligned smooth muscle cells and connective tissue. Due to their ability to differentiate into vascular phenotypes, bone

marrow-derived MSCs were explored as a strategy for repopulation of a decellularized matrix in a vascular graft [57]. Although there was successful formation of both the endothelial and smooth muscle cell layers, it was unclear whether MSCs differentiated into these cell types following implantation, or had recruited host cells to repopulate the graft. Nevertheless, this study established the feasibility of MSCs as a cell source for regenerating bioartificial vascular grafts. Decellularized tissue matrices pose several attractive features as scaffolds because they already possess the essential complement of relative tissue ECM. However, donor tissue is likely to elicit immunogenic responses, and contain dramatic batch-to-batch variation. Several elements of electrospinning technology can overcome these challenges, while still fulfilling the same desirable properties of a vascular scaffold. The vascular tissue macrostructure can be recapitulated by electrospinning fibers onto a thin mandrel to fabricate a tubular structure with suitable mechanical strength and flexibility. Selection of an elastomeric polymer will provide the scaffold with compliance comparable to native tissues. The porosity of the scaffold can be adjusted to facilitate cell infiltration and attachment. Using a combined electrospinning/electrospraying technique, Wagner and colleagues have taken a unique approach to rapid fabrication of a cellintegrated vascular graft with retention of high levels of cell viability [58,59]. This technique has the advantage of homogenous distribution of cells, but the rather harsh processing conditions during electrospraying might hinder its application to stem cells. Zhang et al. investigated small diameter vascular tissue engineering by seeding bone marrow MSCs on an electrospun poly(propylene carbonate) scaffold [60]. In light of the fact that nitric oxide (NO) production is correlated with vessel patency following bypass surgery,

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Fig. 7. Remodeling and cellularization of aligned electrospun vascular grafts without MSCs (a, c, e, g) and with pre‐seeded MSCs (b, d, f, h) after 60 days in vivo. (a, b) Hematoxylin/ eosin staining of graft cross‐section; (c, d) CD31+ staining indicates endothelial cell monolayer; (e, f) MHC staining for SMCs; (g,h) Verhoeff’s staining indicates collagen in pink and elastin in black. Scale bars: (a–b), 100 μm; (c–h), 100 μm. (i) Cell‐seeded aligned fibrous sheet is rolled around a mandrel into a tube, followed by (j) implantation as a common cartid artery bypass in a rat. Reproduced from reference [62].

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rat MSCs were virally transfected with a gene encoding for endothelial nitric oxide synthase (eNOS), with the hypothesis that eNOS gene delivery would be a suitable means of inhibiting neointimal formation. The authors demonstrated that the electrospun scaffold facilitated survival and proliferation of transfected MSCs, and MSCs produced NO at a level comparable to endothelial cells from a freshly harvested artery. It remains to be verified whether MSCs differentiate into the smooth muscle cell layer and NO production induces endothelialization following in vivo implantation. In a more comprehensive study, Hashi et al. seeded bone marrow MSCs on an aligned PLLA nanofibrous mesh, allowed the cells to grow to confluence, and then rolled the mesh into a tubular structure reminiscent of a vascular graft [61]. The grafts were implanted as a common carotid artery bypass in rats. After 60 days in vivo, the MSCseeded tubes stained positively for a CD31+ endothelial layer as well as myosin heavy chain (MHC+) smooth muscle cell layer (Fig. 7). In contrast to acellular tubes, SMCs in the grafts were arranged in a tight band, with collagen and elastin deposition patterns mimicking the structure of native arteries. More interestingly, there was very little neointimal formation in the cell-seeded grafts. Further experiments showed that MSCs seeded on nanofibrous scaffolds did not support platelet aggregation in the short term. The authors attributed the antithrombogenic property of MSCs to the presence of heparin sulfate proteoglycans on the cell surface, rather than endogenous production of nitric oxide. Contrary to their initial hypothesis, most of the cells within the graft at 60 days post-implantation were not originated from the seeded MSCs, indicating their role of recruiting host cells to the implantation site likely through MSC-released trophic factors. The contribution of MSCs to maintaining patency of the graft was thus postulated to be short-term prevention of thrombus formation. 4.2. Myotube formation for skeletal muscle engineering Mature skeletal muscle consists of bundles of terminally differentiated and multinucleated muscle fibers that have formed via myoblast fusion. Damaged muscle has a limited capacity for regeneration; however, there exists a small population of quiescent muscle progenitor cells, known as satellite cells, that are activated in response to muscle damage and can fuse with damaged fibers or form new myotubes themselves [62]. Satellite cells are committed to the myogenic lineage, but still retain proliferative capabilities, and are thus considered the prototypical muscle stem cells. One paradigm for muscle reconstruction and repair is ex vivo expansion of autologously derived satellite cells, followed by scaffold-supported differentiation into muscle tissue. Riboldi et al. electrospun DegraPol, a commercially available and degradable block copolymer polyesterurethane, into aligned and random fibrous meshes and compared the differentiation and myotube formation from C2C12 and L6 myoblast cell lines [63]. These scaffolds were pre-coated with Matrigel, an animal-derived purified ECM, to facilitate cell adhesion. Cultured myoblasts exhibited a steady proliferation on both aligned and random meshes over 1 week; however, rate of proliferation on aligned meshes was significantly slower than on random meshes. The authors took this to be indicative of increased differentiation induced by aligned morphology. Both myoblast lines on aligned fibers had upregulated gene expression of myogenin and myosin heavy chain (MHC), and differentiation was further confirmed by anti-MHC immunofluorescence staining. Putative multinucleated myotubes were highly oriented in parallel arrays on aligned fibers, which was not the case on random fibers. In an earlier study, Huang et al. evaluated the differentiation of C2C12 myoblasts on aligned PLLA nanofibers and showed similar results in myoblast gene expression [64]. The authors also quantified the average length of differentiated myotubes and found that aligned fibrous scaffolds induced myotube formation that was at least twice the length of myotubes generated on random fibers. Striated sarcomeres assembled in the myotubes, although the relative

proportions of striated myotubes were not significantly different on the two types of fiber meshes. These studies demonstrated that aligned electrospun scaffolds have the potential to direct myoblast organization and assembly via simple contact guidance. As a follow-up work, satellite cells cultured on such substrates may be subjected to mechanical or electrical stimulation reminiscent of in vivo conditions that might potentially enhance myogenic differentiation. In order to realize the potential of this strategy, the next challenges to overcome would be to increase the differentiation efficiency of the myoblasts, as well as assembly of myotubes into compact, elongated bundles capable of contracting in unison. 4.3. Nanocomposites for osteogenesis A tested strategy in developing artificial scaffolds for bone regeneration is the incorporation of bioceramics in order to better mimic the mineral composition of native bone as well as promote osteointegration and osteoinduction [65]. Mixing a more ductile and flexible polymer with ceramics such as calcium phosphate or hydroxyapatite (HAp) helps to reduce the brittleness of ceramic structures and also facilitates shaping the scaffold to match the defect size. The versatility of the electrospinning technique allows for incorporation of bioactive ceramics into the polymeric nanofibers. Schneider et al. electrospun fibers of PLGA containing up to 40% nanoparticles of aerosolized amorphous tricalcium phosphate (ATCP), and found that the meshes formed had a texture similar to cotton wool [66]. Incubation of this mesh in simulated body fluid promoted deposition of a dense layer of hydroxyapatite in an amount correlated with the doping level of ATCP. This finding demonstrated that incorporation of ATCP into the scaffold promotes mineralization. The mineralized scaffolds did not have an adverse effect on biocompatibility, as indicated by the fact that the nanocomposite scaffolds supported hMSC proliferation and osteogenic differentiation to the same extent as the polymer scaffold without ATCP. A variation of ATCP encapsulation is to load scaffolds with HAp [51,56,67]. Similarly, the encapsulation of HAp particles had no deleterious effects on hMSC viability and differentiation in culture. When both HAp and BMP-2 were encapsulated into electrospun fibers, HAp particles acted as an adjuvant to modulate the release rate of growth factor from the fibers. Fibers with higher doping level of HAp had a more rapid release rate of BMP-2 over a 60-day test period (Fig. 6). As an alternative to using bone mineral composite scaffolds, Ko et al. created a novel type of nanofibrous composite scaffold by electrospinning a mixture of demineralized bone powder (DBP) with poly (l-lactide) (PLA) [68]. Although there was no significant difference in the expression of osteogenic markers in mandible bone-derived hMSCs on PLA scaffolds versus PLA/DBP scaffolds, calcium deposition onto PLA/DBP scaffolds was significantly higher after 14 days and 21 days of culture. PLA/DBP scaffolds also showed enhanced healing in a rat cranial defect model, with almost complete bone healing achieved in 12 weeks. 5. Conclusions and future perspectives Over the past decade, the body of work encompassing electrospun scaffolds for regenerative medicine has expanded exponentially. The versatility of this platform is in no small part responsible for this surge in scientific interest. Electrospun nanofibers recapitulate several key features of the stem cell niche, are relatively easy to fabricate, and most importantly are amenable to various functional modifications targeted towards enhancing stem cell survival and proliferation, directing fate decisions, or promoting tissue organization. Although much work has been done to establish the biocompatibility of scaffolds for stem cell culture, there remains a

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critical need to demonstrate in a rigorous manner that such an electrospun fiber platform can augment stem cell-based regeneration in vivo. More significantly, the vast majority of existing work has focused primarily on influencing differentiation of MSCs. Research into human embryonic stem cells (hESCs) for regenerative medicine has progressed at a rapid pace over the last ten years, ever since the establishment of the first hESC lines [69,70]. hESCs offer a significant advantage over the lineage-restricted adult-derived stem cell lines due to their pluripotency as well as superior expansion capability. The goal of bringing human pluripotent stem cells into the clinic is now brought closer to realization by the successful derivation of human induced pluripotent stem cells (iPSCs) from terminally differentiated adult somatic cells. True validation of the electrospinning platform will come when it is successfully used to support development of hESCs and iPSCs into functional tissue in vivo, paving the way for widespread adoption in a variety of biomedical applications. Acknowledgements This work is partially supported by National Science Foundation Faculty Early Career Award (DMR-0748340) and Maryland Stem Cell Research Commission (2007-MSCRFE-018). References [1] M.E. Furth, A. Atala, Stem cell sources to treat diabetes, J. Cell Biochem. 106 (2009) 507–511. [2] I.L. Weissman, J.A. Shizuru, The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases, Blood 112 (2008) 3543–3553. [3] H. Taylor, S.L. Minger, Regenerative medicine in Parkinson's disease: generation of mesencephalic dopaminergic cells from embryonic stem cells, Curr. Opin. Biotechnol. 16 (2005) 487–492. [4] K. Fukuda, Progress in myocardial regeneration and cell transplantation, Circ. J. 69 (2005) 1431–1446. [5] S.M. Dellatore, A.S. Garcia, W.M. Miller, Mimicking stem cell niches to increase stem cell expansion, Curr. Opin. Biotechnol. 19 (2008) 534–540. [6] W. Potter, R.E. Kalil, W.J. Kao, Biomimetic material systems for neural progenitor cell-based therapy, Front. Biosci. 13 (2008) 806–821. [7] K. Ghosh, D.E. Ingber, Micromechanical control of cell and tissue development: implications for tissue engineering, Adv. Drug Deliv. Rev. 59 (2007) 1306–1318. [8] C. Chai, K.W. Leong, Biomaterials approach to expand and direct differentiation of stem cells, Mol. Ther. 15 (2007) 467–480. [9] G.A. Silva, C. Czeisler, K.L. Niece, E. Beniash, D.A. Harrington, J.A. Kessler, S.I. Stupp, Selective differentiation of neural progenitor cells by high-epitope density nanofibers, Science 303 (2004) 1352–1355. [10] T.C. McDevitt, S.P. Palecek, Innovation in the culture and derivation of pluripotent human stem cells, Curr. Opin. Biotechnol. 19 (2008) 527–533. [11] B.M. Abdallah, M. Kassem, Human mesenchymal stem cells: from basic biology to clinical applications, Gene. Ther. 15 (2008) 109–116. [12] P. Herve, Donor-derived hematopoietic stem cells in organ transplantation: technical aspects and hurdles yet to be cleared, Transplantation 75 (2003) 55S–57S. [13] A. Alhadlaq, J.J. Mao, Mesenchymal stem cells: isolation and therapeutics, Stem Cells. Dev. 13 (2004) 436–448. [14] N.S. Roy, C. Cleren, S.K. Singh, L. Yang, M.F. Beal, S.A. Goldman, Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes, Nat. Med. 12 (2006) 1259–1268. [15] S.G. Kumbar, R. James, S.P. Nukavarapu, C.T. Laurencin, Electrospun nanofiber scaffolds: engineering soft tissues, Biomed. Mater. 3 (2008) 034002. [16] T.J. Sill, H.A. von Recum, Electrospinning: applications in drug delivery and tissue engineering, Biomaterials 29 (2008) 1989–2006. [17] K. Lin, K.N. Chua, G.T. Christopherson, S. Lim, H.Q. Mao, Reducing electrospun nanofiber diameter and variability using cationic amphiphiles, Polymer 48 (2007) 6384–6394. [18] M.P. Lutolf, J.A. Hubbell, Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering, Nat. Biotechnol. 23 (2005) 47–55. [19] A. Spradling, D. Drummond-Barbosa, T. Kai, Stem cells find their niche, Nature 414 (2001) 98–104. [20] D. Liang, B.S. Hsiao, B. Chu, Functional electrospun nanofibrous scaffolds for biomedical applications, Adv. Drug Deliv. Rev. 59 (2007) 1392–1412. [21] H. Yoshimoto, Y.M. Shin, H. Terai, J.P. Vacanti, A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering, Biomaterials 24 (2003) 2077–2082. [22] M. Shin, H. Yoshimoto, J.P. Vacanti, In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold, Tissue Eng. 10 (2004) 33–41.

1095

[23] W.J. Li, R. Tuli, C. Okafor, A. Derfoul, K.G. Danielson, D.J. Hall, R.S. Tuan, A threedimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells, Biomaterials 26 (2005) 599–609. [24] W.J. Li, R. Tuli, X. Huang, P. Laquerriere, R.S. Tuan, Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold, Biomaterials 26 (2005) 5158–5166. [25] W.J. Li, H. Chiang, T.F. Kuo, H.S. Lee, C.C. Jiang, R.S. Tuan, Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study, J. Tissue Eng. Regen. Med. 3 (2009) 1–10. [26] S.N. Redman, S.F. Oldfield, C.W. Archer, Current strategies for articular cartilage repair, Eur. Cell Mater. 9 (2005) 23–32 discussion 23–32. [27] S. Aota, M. Nomizu, K.M. Yamada, The short amino acid sequence Pro–His–Ser– Arg–Asn in human fibronectin enhances cell-adhesive function, J. Biol. Chem. 269 (1994) 24,756–24,761. [28] S.K. Akiyama, Integrins in cell adhesion and signaling, Hum. Cell 9 (1996) 181–186. [29] G.T. Christopherson, H. Song, H.Q. Mao, The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation, Biomaterials 30 (2009) 556–564. [30] Y.R. Shih, C.N. Chen, S.W. Tsai, Y.J. Wang, O.K. Lee, Growth of mesenchymal stem cells on electrospun type I collagen nanofibers, Stem Cells 24 (2006) 2391–2397. [31] L.S. Sefcik, R.A. Neal, S.N. Kaszuba, A.M. Parker, A.J. Katz, R.C. Ogle, E.A. Botchwey, Collagen nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human adipose stem cells, J. Tissue Eng. Regen. Med. 2 (2008) 210–220. [32] W.J. Li, J.A. Cooper Jr., R.L. Mauck, R.S. Tuan, Fabrication and characterization of six electrospun poly(alpha-hydroxy ester)-based fibrous scaffolds for tissue engineering applications, Acta Biomater. 2 (2006) 377–385. [33] K.J. Shields, M.J. Beckman, G.L. Bowlin, J.S. Wayne, Mechanical properties and cellular proliferation of electrospun collagen type II, Tissue Eng. 10 (2004) 1510–1517. [34] C.P. Barnes, C.W. Pemble, D.D. Brand, D.G. Simpson, G.L. Bowlin, Cross-linking electrospun type II collagen tissue engineering scaffolds with carbodiimide in ethanol, Tissue Eng. 13 (2007) 1593–1605. [35] L. Ghasemi-Mobarakeh, M.P. Prabhakaran, M. Morshed, M.H. Nasr-Esfahani, S. Ramakrishna, Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering, Biomaterials 29 (2008) 4532–4539. [36] H. Colognato, P.D. Yurchenco, Form and function: the laminin family of heterotrimers, Dev. Dyn. 218 (2000) 213–234. [37] R.A. Neal, S.G. McClugage, M.C. Link, L.S. Sefcik, R.C. Ogle, E.A. Botchwey, Laminin nanofiber meshes that mimic morphological properties and bioactivity of basement membranes, Tissue Eng. Part C 15 (2008) 11–21. [38] H.K. Kleinman, Preparation of basement membrane components from EHS tumors, Curr. Protoc. Cell Biol. (1998) 10.2.1–10.2.10. [39] B.G. Keselowsky, D.M. Collard, A.J. Garcia, Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding, Biomaterials 25 (2004) 5947–5954. [40] K.N. Chua, C. Chai, P.C. Lee, Y.N. Tang, S. Ramakrishna, K.W. Leong, H.Q. Mao, Surface-aminated electrospun nanofibers enhance adhesion and expansion of human umbilical cord blood hematopoietic stem/progenitor cells, Biomaterials 27 (2006) 6043–6051. [41] K.N. Chua, C. Chai, P.C. Lee, S. Ramakrishna, K.W. Leong, H.Q. Mao, Functional nanofiber scaffolds with different spacers modulate adhesion and expansion of cryopreserved umbilical cord blood hematopoietic stem/progenitor cells, Exp. Hematol. 35 (2007) 771–781. [42] W.J. Li, R.L. Mauck, J.A. Cooper, X. Yuan, R.S. Tuan, Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering, J. Biomech. 40 (2007) 1686–1693. [43] B.M. Baker, R.L. Mauck, The effect of nanofiber alignment on the maturation of engineered meniscus constructs, Biomaterials 28 (2007) 1967–1977. [44] J.K. Wise, A.L. Yarin, C.M. Megaridis, M. Cho, Chondrogenic differentiation of human mesenchymal stem cells on oriented nanofibrous scaffolds: engineering the superficial zone of articular cartilage, Tissue Eng. Part A 15 (2009) 913–921. [45] J.C. Chen, D.J. Goldhamer, Skeletal muscle stem cells, Reprod. Biol. Endocrinol. 1 (2003) 101. [46] E.K. Yim, S.W. Pang, K.W. Leong, Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage, Exp. Cell. Res. 313 (2007) 1820–1829. [47] R. McBeath, D.M. Pirone, C.M. Nelson, K. Bhadriraju, C.S. Chen, Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment, Dev. Cell 6 (2004) 483–495. [48] J.M. Dang, K.W. Leong, Myogenic induction of aligned mesenchymal stem cell sheets by culture on thermally responsive electrospun nanofibers, Adv. Mater. 19 (2007) 2775–2779. [49] F. Yang, R. Murugan, S. Wang, S. Ramakrishna, Electrospinning of nano/micro scale poly(l-lactic acid) aligned fibers and their potential in neural tissue engineering, Biomaterials 26 (2005) 2603–2610. [50] C.M. Cowan, C. Soo, K. Ting, B. Wu, Evolving concepts in bone tissue engineering, Curr. Top. Dev. Biol. 66 (2005) 239–285. [51] C. Li, C. Vepari, H.J. Jin, H.J. Kim, D.L. Kaplan, Electrospun silk-BMP-2 scaffolds for bone tissue engineering, Biomaterials 27 (2006) 3115–3124. [52] A. Aigner, Nonviral in vivo delivery of therapeutic small interfering RNAs, Curr. Opin. Mol. Ther. 9 (2007) 345–352. [53] J.N. Moreira, A. Santos, V. Moura, M.C. Pedroso de Lima, S. Simoes, Non-viral lipidbased nanoparticles for targeted cancer systemic gene silencing, J. Nanosci. Nanotechnol. 8 (2008) 2187–2204.

1096

S.H. Lim, H.-Q. Mao / Advanced Drug Delivery Reviews 61 (2009) 1084–1096

[54] L. De Laporte, L.D. Shea, Matrices and scaffolds for DNA delivery in tissue engineering, Adv. Drug Deliv. Rev. 59 (2007) 292–307. [55] C. Conrad, R. Gupta, H. Mohan, H. Niess, C.J. Bruns, R. Kopp, I. von Luettichau, M. Guba, C. Heeschen, K.W. Jauch, R. Huss, P.J. Nelson, Genetically engineered stem cells for therapeutic gene delivery, Curr. Gene Ther. 7 (2007) 249–260. [56] H. Nie, C.H. Wang, Fabrication and characterization of PLGA/HAp composite scaffolds for delivery of BMP-2 plasmid DNA, J. Control. Release 120 (2007) 111–121. [57] S.W. Cho, H.J. Park, J.H. Ryu, S.H. Kim, Y.H. Kim, C.Y. Choi, M.J. Lee, J.S. Kim, I.S. Jang, D.I. Kim, B.S. Kim, Vascular patches tissue-engineered with autologous bone marrowderived cells and decellularized tissue matrices, Biomaterials 26 (2005) 1915–1924. [58] J.J. Stankus, J. Guan, K. Fujimoto, W.R. Wagner, Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix, Biomaterials 27 (2006) 735–744. [59] J.J. Stankus, L. Soletti, K. Fujimoto, Y. Hong, D.A. Vorp, W.R. Wagner, Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization, Biomaterials 28 (2007) 2738–2746. [60] J. Zhang, H. Qi, H. Wang, P. Hu, L. Ou, S. Guo, J. Li, Y. Che, Y. Yu, D. Kong, Engineering of vascular grafts with genetically modified bone marrow mesenchymal stem cells on poly (propylene carbonate) graft, Artif. Organs 30 (2006) 898–905. [61] C.K. Hashi, Y. Zhu, G.Y. Yang, W.L. Young, B.S. Hsiao, K. Wang, B. Chu, S. Li, Antithrombogenic property of bone marrow mesenchymal stem cells in nanofibrous vascular grafts, Proc. Natl. Acad. Sci. U S A 104 (2007) 11,915–11,920. [62] P.M. Wigmore, G.F. Dunglison, The generation of fiber diversity during myogenesis, Int. J. Dev. Biol. 42 (1998) 117–125.

[63] S.A. Riboldi, N. Sadr, L. Pigini, P. Neuenschwander, M. Simonet, P. Mognol, M. Sampaolesi, G. Cossu, S. Mantero, Skeletal myogenesis on highly orientated microfibrous polyesterurethane scaffolds, J. Biomed. Mater. Res. A 84 (2008) 1094–1101. [64] N.F. Huang, S. Patel, R.G. Thakar, J. Wu, B.S. Hsiao, B. Chu, R.J. Lee, S. Li, Myotube assembly on nanofibrous and micropatterned polymers, Nano. Lett. 6 (2006) 537–542. [65] M. Wang, Developing bioactive composite materials for tissue replacement, Biomaterials 24 (2003) 2133–2151. [66] O.D. Schneider, S. Loher, T.J. Brunner, L. Uebersax, M. Simonet, R.N. Grass, H.P. Merkle, W.J. Stark, Cotton wool-like nanocomposite biomaterials prepared by electrospinning: in vitro bioactivity and osteogenic differentiation of human mesenchymal stem cells, J. Biomed. Mater. Res. B Appl. Biomater. 84 (2008) 350–362. [67] H. Nie, B.W. Soh, Y.C. Fu, C.H. Wang, Three-dimensional fibrous PLGA/HAp composite scaffold for BMP-2 delivery, Biotechnol. Bioeng. 99 (2008) 223–234. [68] E.K. Ko, S.I. Jeong, N.G. Rim, Y.M. Lee, H. Shin, B.K. Lee, In vitro osteogenic differentiation of human mesenchymal stem cells and in vivo bone formation in composite nanofiber meshes, Tissue Eng. Part A 14 (2008) 2105–2119. [69] J.A. Thomson, J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, J.M. Jones, Embryonic stem cell lines derived from human blastocysts, Science 282 (1998) 1145–1147. [70] M.J. Shamblott, J. Axelman, S. Wang, E.M. Bugg, J.W. Littlefield, P.J. Donovan, P.D. Blumenthal, G.R. Huggins, J.D. Gearhart, Derivation of pluripotent stem cells from cultured human primordial germ cells, Proc. Natl. Acad. Sci. U S A 95 (1998) 13,726–13,731.