Robert L. Mauck et al- Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration

8/3/2019 Robert L. Mauck et al- Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-

1/24

Engineering on the Straight and Narrow:The Mechanics of Nanofibrous Assemblies

for Fiber-Reinforced Tissue Regeneration

Robert L. Mauck, Ph.D.,1,2 Brendon M. Baker, B.S.,1,2 Nandan L. Nerurkar, M.S.,1,3

Jason A. Burdick, Ph.D.,2 Wan-Ju Li, Ph.D.,4 Rocky S. Tuan, Ph.D.,5 and Dawn M. Elliott, Ph.D.1,2

Tissue engineering of fibrous tissues of the musculoskeletal system represents a considerable challenge because ofthe complex architecture and mechanical properties of the component structures. Natural healing processes inthese dense tissues are limited as a result of the mechanically challenging environment of the damaged tissue and

the hypocellularity and avascular nature of the extracellular matrix. When healing does occur, the orderedstructure of the native tissue is replaced with a disorganized fibrous scar with inferior mechanical properties,engendering sites that are prone to re-injury. To address the engineering of such tissues, we and others haveadopted a structurally motivated approach based on organized nanofibrous assemblies. These scaffolds arecomposed of ultrafine polymeric fibers that can be fabricated in such a way to recreate the structural anisotropytypical of fiber-reinforced tissues. This straight-and-narrow topography not only provides tailored mechanicalproperties, but also serves as a 3D biomimetic micropattern for directed tissue formation. This reviewdescribes theunderlying technology of nanofiber production and focuses specifically on the mechanical evaluation and theo-retical modeling of these structures as it relates to native tissue structure and function. Applying the samemechanical framework for understanding native and engineered fiber-reinforced tissues provides a functionalmethod for evaluating the utility and maturation of these unique engineered constructs. We further describeseveral case examples where these principles have been put to test, and discuss the remaining challenges andopportunities in forwarding this technology toward clinical implementation.

Introduction

Fiber-reinforced tissues: structure and function

in health and disease

Fibrous tissues of the musculoskeletal system aredense connective tissues that serve critical load-bearingroles. The fibrous architecture of these tissues is organized tooptimize this mechanical functionality. For example, tendons,ligaments, the knee menisci, and the annulus fibrosus (AF) ofthe intervertebral disc all transmit tensile loads generatedwith physiologic motion through their aligned extracellular

matrix (ECM).14

This ECM is most commonly comprised ofcollagen assembled in a hierarchical fashion into dense bun-dles (Fig. 1). In tissues that operate primarily in one direction,such as tendons and some ligaments, collagen is organizedalong the prevailing line of action, and imbues such tis-sues with mechanical properties that are highly anisotropic

(direction dependent) and highest in the prevailing fiber ori-entation. In structures that experience more complicatedloading patterns,such as the anterior cruciate ligament (ACL),the knee meniscus, and the AF, collagen alignment changes asa function of position within the tissue (either continuously orwithin discrete bundles or layers). In the AF, which experi-ences multiaxial tension, compression, and shear, collagenfibers in adjacent lamellae alternate between 30 and 30degrees with respect to the circumference, resulting in a morecomplex reinforced composite (Fig. 1, center). In the meniscus,which is exposed to a similarly complicated loading envi-

ronment, the majority of collagen fibers are circumferentiallyorganized, ranging from horn insertion site to horn insertionsite, with radial tie fibers interspersed and oriented perpen-dicular to these prevailing fiber bundles and serving to bindthe tissue together. These tissues have common develop-mental antecedents, in that this ordered structure arises very

1McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania.Departments of 2Bioengineering and 3Mechanical Engineering, University of Pennsylvania Philadelphia, Pennsylvania.4Department of Orthopaedics and Rehabilitation and Biomedical Engineering, University of WisconsinMadison, Madison, Wisconsin.5Department of Health and Human Services, Cartilage Biology and Orthopaedics Branch, National Institute of Musculoskeletal and Skin

Disorders, National Institutes of Health, Bethesda, Maryland.

TISSUE ENGINEERING: Part BVolume 15, Number 2, 2009 Mary Ann Liebert, Inc.DOI: 10.1089=ten.teb.2008.0652

171


2/24

early in gestation.57 Cell orientation and cytoskeleton polar-ity is followed by sequential deposition of ECM components(e.g., fibronectin and laminin), which in turn serves as atemplate for structural protein (collagen) deposition and as-sembly in an orderly fashion.5,8 This dense network is thenrefined and remodeled with load-bearing use to achieve theunique mechanical properties of adult tissues. In addition tothese mechanical characteristics, these tissues also share acommon feature that in the adult, the matured structures areincreasingly hypovascular and hypocellular. Shown, for ex-ample, in Figure 2 is H&E staining of fetal compared to adultmeniscus, demonstrating the early alignment of formed col-

lagen, and the decreasing cellularity that occurs with age.While critical for musculoskeletal function, the demanding

mechanical environment in which these tissues perform pre-disposes them to damage. Likewise degenerative changes thatoccur in all tissues with aging are inadequately counter- balanced by regeneration. As a consequence, tendon andligament injuries are common; ACL rupture occurs at a rate of1 in 3000 in the general population of the United States eachyear (*100,000 ACL tears per year).9 Similarly, there are morethan 750,000 operations performed each year to repair or re-move damaged or degenerate meniscus.10 Failed conservativetreatment of lumbar disc herniation leads to surgical dis-

cectomy at a rate of over 250,000 per year, making it the mostfrequently performed neurosurgical procedure in the UnitedStates.11 Notably, the rate of re-herniation is nearly 20%, oftenoccurring at the same disc location.12 In all of these instances,the limited vascularity and the hypocellularity of these tissuesin the adult engender only limited endogenous repair pro-cesses. Indeed, this repair does not restore normal tissuestructure and function, and the once highly ordered tissue isinstead replaced by a disorganized scar, that is, mechanicallyinferior and prone to re-injury.7,13,14 Thus, there exists anunmet clinical need for a mechanically functional implantabletissue or tissue substitute that can either guide repair or re-

place damaged fiber-reinforced tissues.

Approaches to fibrous tissue repair and regeneration

Engineering of functional replacements for these complexfiber-reinforced structuresis and remains a daunting challengeto the field. These tissues are dense, avascular, and hypocel-lular, have refined direction-dependent mechanical propertiesand hierarchical structures, and function in a demandingmechanical environment. All of these unique characteristicsmust be considered and enabling technologies must be de-veloped to affect repair for these challenging load-bearing

FIG. 1. Fiber organization in dense connective tissues. Most tendons and simple ligaments show fiber alignment in acommon prevailing direction over which load is transferred. Annulus fibrosus (AF) and meniscus achieve fiber reinforcementin more complicated loading environments through multilamellar planar alignment of extra-cellular matrix (ECM) or viainterspersion of fibers perpendicular to the predominant fiber direction. Image on left from Ref. 137 Scale bar100 mm, usedwith permission from Wiley. Middle image from Ref.138 Scale bar 200mm, used with permission from Elsevier. Right imagefrom Ref.139 Scale bar 500mm, used with permission from Springer.

172 MAUCK ET AL.


3/24

tissues. To this end, a number of strategies have been devel-oped for tissue repair and replacement. The most sensible andoft tried is the simple replacement of these tissues with similarstructures taken from cadaveric donor tissue or alternative

donor sites in the patients own body. It has been shown thatdevitalized (acellular) tendon grafts are re-colonized by fi-broblasts from adjacent tissues, and that these cells take ona preferential alignment coincident with the pre-existing col-lagen network.15 In practical applications, patellar tendontransfer (in bone-tendon-bone units) is commonly employedin the repair of intra-articular ligaments such as the ACL.16

Meniscus transplantation from cadaveric sources is also rela-tively common in clinical practice. With meniscus transplan-tation, it hasbeen shown that some cellular re-colonizationcanbe achieved over 6 months, though the central core remainsdevoid of cells.17 Both techniques are limited, however, by theavailability and proper size of donor tissues, the creation ofpoints of donor-site morbidity, concerns related to disease

transmission, and the observed decline in tissue properties asremodeling events transpire post-implantation.

To overcome these challenges, numerous tissue engineer-ing strategies have been developed toward the production offibrous tissues. These constructs can be matured in vitro andimplanted with fully functional properties, or be designed tomature in situ with controlled rehabilitative regimens. Ulti-mately, the construct needs to operate under loading condi-tions experienced by the tissue in vivo.18 Toward this end,some of the earliest work in this field was carried out in col-lagen gels. These studies took advantage of the fact that fi- broblast-like cell-mediated contraction around a fixedboundary (a post for example) generates ordered cellular andECM networks.19,20 These findings have been extended to

more complicated structures with different organization un-der a variety of boundary constraints.21,22 While useful forunderstanding the developmental antecedents of fiber-reinforced tissues, the resulting mechanical properties of mostgel-based constructs remain very low, potentially limitingtheir in vivo application. To address mechanics specifically,others have turned to natural and synthetic scaffolds for im-plantation. These efforts have included woven and nonwovenfibrous scaffolds composed of a diverse range of materials,from standard biodegradable polyesters to silk fibroin.23

Novel weaving technologies have further refined the me-chanical response of such constructs, and commercial prod-

ucts based on these approaches are making their way tomarket.2427

The approach that we and others have adopted for fiber-reinforced tissue engineering builds on these past efforts, but

focuses specifically on the multiscale rendering of fiber re-inforcement from the nano- and micron-scale through to thetissue level. These efforts employ electrospinning of ultrafineor nanofibrous polymer networks as a base technology. Thisreview is intended to serve as a general overview of thisunique scaffold fabrication technology, and to specificallydiscuss the mechanical characterization of such scaffolds. Insubsequent sections, we discuss the intrinsic properties ofnatural and synthetic nanofibrous assemblies, methods forinstilling anisotropy in these networks, relevant testing mo-dalities as they relate to physiologic loading, and modelingapproaches that provide additional insight into scaffoldformation and cell-mediated tissue growth. Finally, we pro-vide several case examples where these guiding principles

have been deployed toward the engineering of fiber-reinforced tissues, and discuss the challenges that must beovercome for the clinical translation of this technology forthe repair of damaged or diseased fibrous tissues.

Electrospinning of Nanofibrous Assemblies

Overview of electrospinning

The technique of electrospinning to create fibrous scaffoldsis becoming increasingly prevalent in the extant literature.While the basic technique was first patented in the 1930s,28 asearch for nanofiber or electrospinning in the PubMed da-tabase finds nearly 300 entries in the last 12 months alone,with applications as diverse as bone tissue engineering to

drug delivery to treatment of burns. Several recent reviewson electrospinning2934 highlight much of this literature andprovide an excellent foundation for the basic technology andits potential applications. Given the availability of these re-cent reviews, it is not the intent of this work to review all ofelectrospinning literature, but rather to focus on those fea-tures of electrospun scaffolds that have not been discussed ingreat detailnamely, the mechanical properties of nano-fibrous networks, how these properties are properly assessedand understood, and which considerations must be appre-ciated for application in load-bearing conditions, and, inparticular, the engineering of fibrous tissues.

FIG. 2. Cell and matrix characteristics of fiber-reinforced connective tissues. H&E staining of ( A) fetal ovine meniscus (130days gestation) and (B) adult ovine meniscus (1 year old). With aging, fibrous tissues decrease in cellularity while thecollagen-rich ECM grows denser and more organized. Color images available online at www.liebertonline.com=ten.

MECHANICS OF NANOFIBROUS SCAFFOLDS 173


4/24

The basic electrospinningsetup consists simply of a polymersource (or sources), a high voltage power supply, and agrounded target (Fig. 3). The solution is expressed through afine capillary or syringe (called the spinneret) by gravity orpositive pressure, and forms a pendant droplet. Applicationof a high voltage to this solution causes charge buildupand chargecharge repulsion among the individual polymerchains within the droplet, until these intermolecular forces

overcome the surface tension holding the droplet in place.Once this critical threshold is reached, the polymer emits fromthe spinneret as a fine jet, and rapidly travels to the nearestgrounded surface.35,36 As the jet is drawn from its sourcethrough the high-voltage gradient, solvent evaporation andwhipping instability produces ultrafine fibers (501000nm).37 With time, these fibers accumulate on the groundedsurface to create a mesh composed of randomly oriented fi-bers. Mesh thickness can be controlled by simply increasingthe time of deposition. Nanofiber features in the networkdepend on the polymer composition and several controllableprocessing variables37 (discussed below). Nanofibers can betuned to range from as small as 50nm up to several microns indiameter, and as such are many times smaller than most

mammalian cells; in fact, they are similar in scale to collagenfibers normally present in the ECM.38 This nano- and micron-scale topography has been shown to modulate cell signalingpathways39 and to elicit superior metabolic and matrixforming activities by seeded cells.40 Nanofibrous meshes areporous structures with a continuous distribution of pore sizesin the range of 2465mm and void volumes of 8090%.41

Electrospinning synthetic and natural polymers

Production of meshes via electrospinning has been carriedout with numerous polymers, including polyurethanes,42

biodegradable polyesters (e.g., polycaprolactone [PCL],41,4345

polyglycolic acid,46 polylactic acid,4749 and polydiaxanone50),and natural biopolymers, including collagen,44,5154 elas-tin,53,54 silk fibroin,55,56 chitosan,57,58 dextran,59 and wheatgluten.60 Additionally, liquid blends of biosynthetic and nat-ural components have been electrospun (with componentsthus mixed in every fiber) to create meshes with enhanced cellcompatibility.61,62 The most common appearance of such

blends is in the combination of two dissimilar synthetic ma-terials to result in a blend fiber that has properties of both, or anatural and a synthetic fiber combined to impart biologicfunctionality to the fibers as they form.63 Additional studieshave modified fiber surfaces to enhance cell binding and=orgrowth factor retention.6466 Further, methacrylate-based co-polymers have been electrospun to form nanofibrous coatingsthat can be crosslinked after formation.67,68 We have recentlyreported on the electrospinning of several elements of a libraryof 120 poly(b-aminoester)s that were photo polymerized afterformation69 as well as novel photocrosslinkable and hydro-lytically degradable elastomers.70 Clearly, there exists a widerange of polymers that can be processed into the nanofibrousformat.

Optimization of electrospinning parameters

For each polymer utilized, spinning parameters are opti-mized to generate a homogeneous fiber array. Common in-trinsic parameters that can be varied include the solvent typeand composition, the mass concentration of the polymer,molecular weight of the polymer, solution viscosity, appliedvoltage, electric field strength, spinneret-to-collector distance,and polymer flow rate.30,36,37,71 Additional extrinsic param-eters include atmospheric conditions such as ambient tem-perature and humidity. These individual parameters are

FIG. 3. Electrospinning ultrafine fibrous networks. (A) Schematic of a common electrospinning setup. These simple systemsconsist of a polymer source flowing (actively or passively) through a highly charged spinneret, where charge repulsion in thesolution generates a dispersion of fiber jets that travel rapidly to a grounded collecting plate or mandrel. ( B) Electrospunfibers have diameters that range from 50 nm to several microns, providing a surface topography in which cells interactsimultaneously with multiple fibers. Shown are human mesenchymal stem cells (MSCs) on a nonaligned polycaprolactone(PCL) nanofibrous sheet.

174 MAUCK ET AL.


5/24

tuned to generate fibers that meet the criteria of the intendedapplication, most commonly that the fibers minimize theirdiameter, be free of defects (beads), and show little fiberfiber welding at the interstices. Figure 4A shows examples ofsuch defects in a PCLscaffold formedwith suboptimal electro-spinning conditions. Fiber diameter and defects can usuallybe controlled by adjusting the solution mass concentration,voltage gradient, and distance over which fibers are collected.

Solvent characteristics strongly influence these features. A re-cent study by Kidoaki and co-workers showed that whenelectrospinning poly(ester urethane)urea (PEUU), increasingN,N-dimethylformamide content in the spinning solution ledto enhanced fiberfiber welding.72 In some cases, for example,when the molecular weight is low, polymers simply cannotbe electrospun on their own using traditional measures. In

such instances, alternative strategies must be adopted. In ourrecent efforts to electrospin low-molecular-weight photo-crosslinkable macromers from a poly(b-aminoester) library, acarrier molecule, poly ethylene oxide (PEO), was required toincrease the viscosity and chain length of the polymer solu-tion.69 Once formed, nanofibers of this kind could be stabi-lized by photocrosslinking, and the carrier removed. In otherapplications, synthetic polymers that are readily spun on

their own have been used as carriers for biologic proteins,such as collagen and elastin, which are generally more dif-ficult to spin and=or stabilize after production.62,63,73

Even after spinning conditions have been optimized, somevariation exists between batches, most likely caused by smallchanges in extrinsic and intrinsic conditions on the day ofproduction. Indeed, some in the field have suggested thatelectrospinning is an art rather than a science because of thisinherent variability. An example of this is shown in Figure 4B,where the mechanical properties of 10 different batches ofPCL nanofibers (aligned, tested in the fiber direction) showsome intra-scaffold variability (based mostly on position onmandrel), and extensive intra-batch variability (from smalldeviations in intrinsic and extrinsic factors). In our studies, we

address these concerns in two ways. First, when multiplescaffolds produced on different days are to be combined foruse in a large study, rigorous characterization (structuraland mechanical) is carried out on each batch, and those notmatching desired criteria are removed. Second, when usingscaffolds for a cell-based study, we commonly include acel-lular controls to be tested in the same manner and at the sametime points for the duration of the study. These steps haveincreased consistency between studies, and have aided in theinterpretation of research findings.

Order from Disorder: Methods for Inducing Anisotropy

When standard electrospinning methods are employed,

and fibers are collected onto a stationary grounded plate, theresultant mesh contains fibers organized in a random fash-ion. As indicated above, most fiber-reinforced tissues showpronounced mechanical anisotropy (different properties indifferent directions), which is based on the underlying or-ganized collagen ultrastructure. Methods to induce fiberalignment in nanofibrous scaffolds have thus been of greatinterest to the fiber-reinforced tissue engineering community,and were recently reviewed by Teo and Ramakrishna.31 Oneof the first instances of nanofiber alignment was presentedby Theron and colleagues,74 who focused fiber collection onthe thin edge of a rotating disk. Nanofiber alignment wasalso demonstrated by Xia and colleagues, using a system inwhich the collecting surface was composed of pairs of elec-

trodes that could be differentially grounded and separatedby an air gap or insulating surface. In this way, linear arraysof fibers were generated, and by varying the state of theelectrodes (grounded or not), different rosette patterns wereachieved.32,75,76 More recently, Sun and colleagues77 dem-onstrated the capabilities of near-field electrospinning, wherea point spinneret is situated only a few microns from thegrounded surface. The probe tip, which is dipped in polymersolution, becomes a stylus from which polymer is ejected,and its position relative to the ground can be controlled tofabricate ordered structures. These methods are somewhatlimited in their throughput; therefore, the most common

FIG. 4. Optimization of electrospun scaffolds. Solution,fabrication, and environmental parameters can be optimized

to generate sheets with homogenous fiber populations.Shown in (A) are several features that arise when electro-spinning conditions are not optimized; *, bead-like inclu-sions; large arrow, thick fibers; small arrowhead, thin fibers.Quality control of microstructure is required to ensure ho-mogeneity across production runs. Even when precautionsare taken, small changes in fiber properties can yield a rangeof mechanical properties in the resultant scaffolds. Shown in(B) is the modulus of aligned PCL scaffolds produced over10 separate production runs. Note the deviations in me-chanical properties within each batch, as well as the scatter ofaverage properties across production runs. Gray area indi-cates 1 standard deviation for this grouping of scaffolds.



6/24

method for aligning fibers for the generation of tissue engi-neering scaffolds (and the method that our lab uses) is todeposit fibers onto a rotating drum or mandrel.7884 Fromhigh-speed imaging studies, it has been shown that a singlefiber can transit from the source spinneret to the ground atspeeds >2 m=s.85 Thus, presenting a surface moving fasterthan the fiber jet will pull fibers into alignment as they aredeposited. We and others have shown that the degree of

alignment is a function of the rotation speed of the collectingsurface.81,83,84 Further, this structural anisotropy is reflectedin the measured mechanical properties, with more highlyaligned scaffolds possessing greater levels of mechanicalanisotropy.81

Mechanical Characterization of Nanofibrous Scaffolds

Mechanical properties of nanofibrous assemblies

Most load-bearing tissue engineering applications withnanofibrous scaffolds require some level of mechanical func-tionality. Therefore, the most common assay (aside fromscanning electron microscopy observation) of any nanofibrous scaffold is the assessment of mechanical properties.

These properties are typically assessed at the time of forma-tion, as well as with subsequent degradation under physio-logic conditions or with cell-mediated matrix deposition.When formed into random or nonaligned meshes, nanofiberscaffolds exhibit isotropic properties (same in all directions)that are reflective of the mechanical properties of their poly-mer composition. For example, polymers such as poly(D,L-lactide-co-glycolide) (PLGA) produce meshes that are quitestiff in tension, while scaffolds composed of PCL are 10 timesless stiff and remain elastic over a wider range (up to 8

10%)49 (Fig. 5). When biologic and synthetic molecules aremixed (in the same solution) before electrospinning, me-chanical properties vary with composition.34,73 Given thelarge number of polymers (as well as biopolymers) that have been successfully electrospun, there is a correspondinglywide range of mechanical properties that can be achieved(see Table 1). Multi-jet electrospinning systems have also been developed to increase production rates.29,8688 These

systems may be used to create composite scaffolds (with adifferent polymer in each jet) whose properties reflect theproperties and ratios of the individual components.8994

Several such multicomponent scaffolds are described in de-tail below.

In addition to properties imparted by the polymer com-position itself, several other factors may influence the mea-sured mechanical properties. As noted above, recent studieshave shown that alignment and mechanical properties in thefiber direction increase substantially as the collecting surface(rotating mandrel) increases in velocity.81,83,84 For example,for PCL scaffolds, the ratio of properties in the fiber directioncompared to transverse to the fiber direction can increase by1020-foldwithincreasingalignment81 (Fig.6).Testingaligned

scaffolds in directions that do not correspond to the prevailingfiber orientation also influences the measured mechanicalproperties in a predictable fashion.82 Random scaffolds ex-hibit a relatively linear stressstrain response in the pre-yieldregion, and extend linearly after yield. Aligned scaffold testedin the fiber direction have a sharper increase in stress withincreasing deformation, and yield and fail at similar pointsearlier in the strain regime. When these same scaffolds aretested in the transverse direction, a much lower stressstrainprofile is observed. Other factors that can change mechanical

FIG. 5. Tensile properties of common biodegradable polymers. (A) Tensile modulus and (B) yield strain for nonalignednanofibrous scaffolds fabricated from a common set of biodegradable polymers. A range of properties can be achieved, butfew polymers can be distended to greater than 2% elongation before yielding. Adapted from Ref. 49 with permission fromElsevier.

176 MAUCK ET AL.


7/24

properties include the amount of solvent remaining in a fiberwhen it reaches thecollecting plate andconsequently thenum-ber of fiberfiber welds that form. Kidoaki and co-workersshowed a fourfold increase in properties of electrospun scaf-folds with increasing degrees of fiberfiber welding.72 Somematerials show profound changes in mechanical propertieswhen they are tested in a dry versus hydrated state. For ex-ample, collagen nanofibers (which must be cross-linked to be

hydrated) decrease in tensile properties by approximately100-fold with hydration.95,96 In composite fibers formed witha temporary carrier (such as PEO), bulk scaffold propertiesmay change as the carrier material is eluted. We have shown,for example, that mechanical properties can increase in scaf-folds that are cross-linked after formation, and then subse-quently decrease as the carrier PEO is eluted.69 Given the largenumber of factors influencing these mechanical properties,and the importance of these properties for fiber-reinforcedtissue engineering applications, the followingsectionsdescribenanofiber and scaffold evaluation across multiple lengthscales and in different testing configurations.

Single-fiber mechanics

At their most basic level, nanofibrous scaffolds are the sumof many thousands of ultrafine fibers traversing a small vol-ume. While these fibers sometimes interact, via welding orfrictional interactions, bulk scaffold properties should builddirectly from these constituent fibers. Testing of a single fiberis an exacting science, requiring specialized and sensitive

equipment, and as such there are few examples in the litera-ture. Of these few, perhaps most common is the microtensiletesting of single fibers. In one study by Tan and colleagues,97

single PCL fibers were collected across a small gap, and uni-axial extension applied until failure was achieved. They re-ported that smaller fibers have a higher modulus, but are lessductile than larger fibers. Similarly, Chew and co-workersfound that small fibers were stiffer than thick fibers, and thatindividual fiber properties change when model proteins andpharmacologics were incorporated.98 Subsequent work byLim and colleagues showed that crystallinity within a fiberstrand influenced mechanical behavior; more crystalline fibers

Table 1. Tensile Properties of Common Electrospun Scaffolds

Polymer AlignmentModulus

(MPa)Yield strain

(%)Ultimate

strain (%)Yield stress

(MPa)Ultimate

stress (MPa) References

1 Thermoplastic polyurethane Both n=a n=a n=a n=a n=a 1392 Polydiaxanone-elastin blend NA 510 n=a 60200 n=a 35 633 Poly(ester urethane)urea NA 8 n=a 2.2 n=a 13 140

Poly(ester-urethane)urea-collagen

blends

NA 17 n=a 160280 n=a 211 140

4 Biodegradable polyurethane NA 5.7 61 79 61 1.3 1415 Poly(D,L-lactide-co-glycolide) (85:15) NA 323 n=a 96 n=a 23 416 Poly(p-diaxanone-co-L-lactide)-

block-poly(ethylene glycol)NA n=a n=a n=a n=a 1.31.4 142

7 Silk fibroin NA 625 n=a 4 n=a 13.6 1438 Poly(L-lactide-co-e-caprolactone) NA 156 n=a 127 n=a 5 439 Collagen-poly(ethylene oxide) blends NA 12 n=a n=a n=a 0.37 144

10 Collagen AL 52.326.1

n=a n=a n=a 1.50.7

51

11 Poly(ethylene glycol)-g-chitosan NA n=a n=a 78112 n=a 817 14512 Gelatin NA 105 n=a 64 n=a 2.5 73

Gelatin-poly(e-caprolactone) NA 5 n=a 126 n=a 2.7 73Poly(e-caprolactone) NA 31 n=a 138 n=a 1.3 73

13 Poly(vinyl alcohol)-cellulose acetateblends

NA 44 n=a 420 10 11 88

Poly(vinyl alcohol) NA 1834 n=a 214334 5.97.2 7.09.4 88Cellulose acetate NA 3 n=a 175 1.5 2 88

14 Poly(L-lactic acid) NA 8.5 1.9 2025 0.1 n=a 49Poly(D,L-lactic acid) NA 70 1.9 n=a 1.1 n=a 49Poly(D,L-lactic-co-glycolic acid)

(50:50)NA 144 2.1 38 2.8 n=a 49

Poly(D,L-lactic-co-glycolic acid)(85:15)

NA 114 1.9 n=a 1.9 n=a 49

Poly(glycolic acid) NA 138 2.1 n=a 2.7 n=a 49Poly(e-caprolactone) NA 8.5 7.5 n=a 0.7 n=a 49

15 Poly(e-caprolactone) AL 3.34.72.73.9

n=a 200231216277

0.60.70.60.4

1.42.11.22.0

146

16 Poly(glycolic acid) AL 551052055

n=a 6080105130

n=a n=a 78

Poly(glycolic acid) NA 4095 n=a 80100 n=a n=a 78

Mechanical properties of electrospun scaffolds for fibrous tissue engineering. Depending on the choice of polymer and formation method,electrospun scaffolds can achieve a range of mechanical properties. For example, the modulus can range from one to several hundred MPa.Moreover, yield strains can be quite low for many common biodegradable polyesters, but markedly higher for elastomeric polymers.

NA, nonaligned; AL, aligned; n=a, not available.



8/24

(produced from lower concentration solutions) were stifferthan less crystalline fibers (even when fiber diameters werecomparable).99 In addition to direct tensile tests, atomic forcemicroscopy (AFM) has been used to manipulate and testsingle fibers as well. Forexample, Kimand colleagues showedthat cross-linked poly-HEMA nanofibers snap back into placeafter being distended with an AFM probe tip.68 Tan and co-

workers fixed one end of a nanofiber, and used the AFMcantilever to deform and measure force generated in PEOnanofibers.100 Similarly, the AFM probe tip has been used tocarry out three-point bending tests of individual fibers placedover a groove microetched into a silicon wafer.101 Still morerecently, specialized MEM devices have been constructed thatcouple with AFM cantilevers, and have been used to show thedependence of ultimate strain on applied strain rate forpolyacrylonitrile nanofibers.102,103

Uniaxial tensile testing of fibrous assemblies

While scaffolds are comprised of individual fibers withtheir own set of distinct mechanical features, the mechanical

function is most commonly characterized in bulk samples,where the individual fibers combine to produce an aggregateresponse. In uniaxial testing of bulk scaffolds, load is appliedalong a single specified direction, while the sample is free tocontract laterally. A rectangular or dumbbell-shaped sampleis typically loaded into a test frame and pulled at a specifiedrate of displacement (displacement controlled) or force (loadcontrolled), and both the load and displacement are mea-sured. By normalizing force to undeformed cross-sectionalarea (engineering stress) and displacement to undeformedlength (engineering strain), we can calculate the stressstrain behavior. To evaluate cross-sectional area, noncontacting

methods such as imaging or laser systems are preferable; thisavoids permanent deformation of scaffolds, particularly atearly culture periods. The typical metric for the elastic be-havior of linear elastic materials (those in which the plot ofstress vs. strain reveals a linear relationship) is the Youngsmodulus (E), or slope of the stressstrain curve. Some elec-trospun polymers,such as aligned PCL fibers (as well as many

fiber-reinforced soft tissues), exhibit a nonlinear stressstrainbehavior and so are characterized by reporting a slope of thebeginning of the curve, known as the toe-region modulus, inaddition to the elastic region modulus. The strain at which thecurve transitions from toe to linear regions is the transitionstrain, and is an additional parameter of interest in nonlinearmaterials. When electrospun scaffolds are stretched beyondtheir elastic regime, they may experience either of two pri-mary failure modes: plastic deformation (deformations thatare not reversed upon removal of load) and catastrophicfailure (load goes instantly to zero or decreases markedly).The onset of plastic deformation occurs at a strainstress levelknown as the yield point. As noted above, electrospun poly-mers have distinct failure properties, and may involve a

combination of yielding and catastrophic failure.81,104

In thiscase, an additional material property, the ultimate tensilestrength, or the maximum stress before failure, is often re-ported. However, post-yield measures, such as ultimate ten-sile strength, are less valuable for tissue engineering thanproperties of the elastic behavior, because postyield defor-mations are not recoverable upon unloading. Due to the dy-namic loading that most tissues encounter, it is critical that anengineered construct recover after deformation, and not befully expended after a single loading event. Further, nativesoft tissues often undergo more than 25% deformation beforeyielding or failure, while many common polymers in

FIG. 6. Fiber alignment influences mechanical properties of nanofibrous scaffolds. Stressstrain response of PCL nanofi-brous scaffolds produced in a nonaligned configuration (A), produced in an aligned configuration and tested in the pre-vailing fiber direction (B), and produced in an aligned configuration and tested in the transverse direction ( C). Schematicsand images illustrate fiber organization and testing direction for each scaffold.

178 MAUCK ET AL.


9/24

electrospinning are limited to 12% strain before yielding (seeTable 1).49 Therefore, the yield point is an important designcriterion to consider when selecting polymers for specifictissue applications.

Biaxial tensile testing of fibrous assemblies

Despite the value and practicality of uniaxial tensile testing,

its physiologic relevance is limited because of the absence offreely contracting boundaries for many tissues in situ. A greatmany fiber-reinforced soft tissues, such as the meniscus, AF,arteries, and myocardium, are subject to tensile loads alongmultiple directions. For this reason, biaxial tensile testing is amore valuable testing modality for electrospun scaffolds andtissue-engineered constructs. Although uniaxial tests can beperformed in multiple orientations with respect to the pre-vailing fiber direction,51,81,82 these tests must be performedeither on multiple samples or sequentially on the same sampleat small, sub-yield strains. However, biaxial testing allows forthe measurement of the material behavior simultaneouslyalong two orthogonal axes. This method is more robust forcharacterizing scaffold and tissue function, and more closely

approximates in vivo loading. Nonetheless, there are relativelyfew studies that have considered the biaxial behavior ofelectrospun scaffolds.83,105107

Grip-to-grip versus local strain

In both uniaxial and biaxial tensile testing, it is common touse grip displacement to compute strain.81,82,95,108 However,as this method only generates information regarding theboundaries of the sample, it introduces errors associatedwith inhomogeneous strain fields and sample slippage at thegrips. Therefore, measurement of material or local strainsfrom the sample mid-substance more accurately reflects thetrue strain of the material.109 This is typically performed by

placing fiducial markers on the sample, or speckle coatingthe surface with paint or other texture, and collecting imagesof the sample surface throughout the test. These images arethen postprocessed to measure the surface strains duringthe deformation. This results in two-dimensional materialstrains, which, in the case of uniaxial tests, allow for thedetermination of an important additional material property,the Poissons ratio. An example of the local strain associatedwith tensile deformation of an aligned nanofibrous scaffoldis shown in Figure 7. Average strain within a region of in-terest (ROI) compares favorably with the bulk grip-to-gripstrain, while clear heterogeneity across the ROI is observed. Itis possible, however, that with tissue deposition by residentcells, engineered constructs may produce complex, hetero-

geneous strain fields and become increasingly susceptible toslippage at the grips. Therefore, it is of value to perform thenecessary validations for studies where grip-to-grip strainsare employed. On a fiber level, even this characterization ofsurface strain likely misses fiber rearrangements and slidingmore consequential to the response of isolated cells within thenetwork. For example, recent work by Stella and co-workersshowed that nuclear orientation and deformation correlatedwith bulk scaffold deformation up to *50% strain, and thenremained constant thereafter.107 Methods such as these,where fiber and cells are monitored continuously withapplied deformation, will be required to determine the pre-

cise local deformations that control cell activity within thesescaffolds.

Dynamic and viscoelastic properties

of electrospun scaffolds

Although electrospun scaffolds are primarily elasticstructures, they are commonly applied to the engineering ofviscoelastic and cyclically loaded tissues. Despite this fact, thetesting procedure for electrospun scaffolds is typically aquasistatic (slowly applied strain rate to eliminate rate de-pendent effects), continuous ramp to failure. Based on sometheoretical models81,82 there is evidence that electrospun PCLfibers may slide relative to one another. These frictional in-

teractions may be a source of rate-dependent effects. More-over, as resident cells deposit ECM on electrospun scaffolds,there is an increase in water content and concentration ofviscoelastic ECM elements. In effect, the rate-dependent be-havior of electrospun scaffolds and engineered tissues is ofgreat importance; however, this aspect of electrospinning fortissue engineering has not been widely characterized. Further,it is important to understand how electrospun materials be-have under conditions of repeated loading. Humans take anestimated 12 million steps per year, so materials for im-plantation must be able to withstand cyclic loading withoutexperiencingfatigue damage. We have recently tested aligned

FIG. 7. Local strain in aligned scaffolds with tensile de-formation. (A) Surface image of speckle-coated scaffoldswith superimposed strain map of an aligned nanofibrousscaffold undergoing tensile deformation. Equilibrium strainranged from 4% to 6% with a grip-to-grip strain of 5% withinthe ROI. (B) Average local strain in ROI compared to grip-to-grip strain for a range of deformations. These data illustratethat bulk deformations of aligned scaffolds are relativelyuniform in the center of scaffolds. Color images availableonline at www.liebertonline.com=ten.



10/24

PCL scaffolds for up to 20,000 cycles of dynamic tensile de-formation to 3% grip-to-grip strain at a frequency of 0.5 Hz.Results of this study are shown in Figure 8. Interestingly, thescaffolds showed very little energy loss when dynamicallyloaded in this strain regime, as evidenced by the small area ofhysteresis. Further, there was no apparent change in the ten-sile modulus or maximum stress of these scaffolds as a con-sequence of the loading regimen. Conversely, Klouda et al.

carried out a similar study investigating the effect of cyclictensile loading on nonaligned electrospun PCL scaffolds for alonger duration. They found a reduction in the failure strainand ultimate tensile stress in samples that were cyclicallyloaded for 15 days, but no change in the elastic modulus. 110

Additional work by Sell and co-workers employed dynamiccompliance testing to evaluate polydiaxanone-elastin nano-fiber tubes for vascular applications.63 These results suggestthat experiments such as these, assessing dynamic and fatigueproperties, are particularly relevant for tissue engineeringstrategies that may exploit cyclic loading bioreactors or in-volve direct implantation into load-bearing sites.

Modeling of Nanofibrous Scaffold Mechanics

Although experimental measures of mechanical functionare instructive, theoretical or constitutive models provideadditional insight into the study of both acellular scaffolds

and the maturation of cell seeded constructs. A constitutivemodel is an explicit mathematical description of the me-chanical behavior of a material. Constitutive laws permit themechanical characterization of complex materials and, moreimportantly, permit one to predict how a material will behavein response to various mechanical perturbations. Despite theirutility, constitutive models have not been widely used todescribe electrospun scaffolds, and have been used even less

to describe nanofiber-based engineered tissue constructs.Constitutive models of electrospun scaffolds vary widelyin

their complexity, from simple geometrically motivated linearmodels81,82,108 to hyperelastic continuum models.83,111,112

Mathew et al. successfully predicted the dependence of elec-trospun poly(butylene terephthalate) mechanics in uniaxialtension on the degree of alignment and prevailing fiber ori-entation using a classical equation from fiber-reinforced rub-bers.108 In a similar application, Li et al. used a linear springmodel that accounted for fiber dispersion to predict the re-sponse of electrospun PCL scaffolds; the model closely ap-proximated the experimentally measured dependence ofmodulus on degree of dispersion only when it permittedunconstrained sliding between fibers.81 Nerurkar et al.82 ap-

plied a linear homogenization model to characterize the de-pendence of aligned PCL on fiber orientation in uniaxialtension, demonstrating that fiber connectivity and nonfibrillarmatrix are key determinants of this relationship. This work

FIG. 8. Dynamic testing ofnanofibrous scaffolds. (A)Setup for dynamic and fatiguetesting. (B) Dynamic stressstrain profiles of aligned na-nofibrous scaffolds through20,000 cycles of 3% deforma-tion applied at 0.5 Hz. (C)Modulus and (D) maximumstress of constructs before andafter 20,000 loading cycles.Color images available online

at www.liebertonline.com=ten.

180 MAUCK ET AL.


11/24

also confirmed the observation by Mathew et al. that fiberorientation plays a minimal role in the uniaxial tensile be-havior of aligned electrospun fiber mats when the direction ofloading deviates by more than 408 from the prevailing fiberdirection.

Hyperelastic models have the additional advantage of de-scribing materials with nonlinear mechanical behaviors overlarge deformations. Because nonlinearity and finite defor-

mations are functional signatures of many fiber-reinforcedsoft tissues, hyperelastic models are of great value not only forcharacterizing acellular scaffolds and engineered constructs,but also to yield comparisons of these materials with nativetissue benchmarks. De Vita et al.111 employed a hyperelasticmodel to describe the elastic and failure behavior of alignedelectrospun poly(butylene terephthalate), whereby failurewas modeled as a statistical distribution of fiber failure strainsat which fibers ceased to carry load. Although the model wasable to fit both elastic and postyield behavior with relativeaccuracy, interpretation of these types of models is limited byan inability to distinguish between the catastrophic and se-quential failure of fibers and the plastic deformations that arelikely superimposed upon them. Courtney et al.83 formulated

a hyperelastic model that incorporated fiber distributions,and applied it to ES-PEUU scaffolds with varied degrees ofalignment. One key feature that distinguishes this study fromthose discussed thus far is the application of the model to bi-axialtensiletests.UnlikeNerurkarandcolleaguesandMathewand co-workers, who investigated multidirectional propertiesby uniaxial tension, modeling biaxial tension is a more robustmethod because the model must simultaneously describebehavior along two orthogonal loading directions.

By fully characterizing the mechanics of an acellular scaf-fold, constitutive models can also be used to characterizechanges in mechanics subsequent to the deposition of ECM bya resident cell population. In effect, constitutive models canserve as quantitative measures of functional maturation for

cell-seeded constructs. This was proposed in Nerurkar et al.82

for a linear homogenization model, and later extended to anonlinear hyperelastic model by Nerurkar et al.112 (describedbelow). In this study, the authors used the material parame-ters of a fiber-reinforced constitutive model as metrics forfunctional growth of engineered AF constructs, and corre-lated these metrics with biochemical composition. Addition-ally, upon validation, the model was used to investigate (bysimulation) the consequences of construct maturation oncomplex, physiologically motivated loading scenarios such asbiaxial tension and simple shear. At their basic level, modelsof these systems may provide insight into the complex mo-lecular interactions of native tissue, and be used to guidemodel development for these more complicated assemblies.

For tissue engineering, modeling of complex dynamic scaf-folds of varying composition can be used to tune constructproperties to match the necessary mechanical requirementsexpected over the lifetime of its intended use.

Fibrous Tissue Engineering on the Straight

and Narrow

Many polymers, many choices

Given the large and growing number of polymers availablefor electrospinning, their unique mechanical and degradationcharacteristics, and the ability to precisely control their orga-

nization, the question arises: How does one choose a startingpoint for tissue engineering applications? The answer to thisquestion must be in part defined by the cell population andmatrix organization of the tissue of interest as well as theoperating conditions experienced by that tissue. Cellular in-teraction is of course important, both in the case of acellularscaffolds to be colonized after implantation, as well as cellu-larized constructs grown in vitro. While fibrous tissues are

unified in their hierarchical matrix organization, some aremore organized than others (compare for example the su-praspinatus tendon with the flexor tendon), and scaffoldsshould reflect this complexity. The approach to tissue re-placement matters as well; a construct intended for immedi-ate implantationwill require different properties than one thatis matured in vitro before implantation. For example, if thetissue of interest is one that commonly experiences in vivostrain levels of>5% strain, then polymers that yield belowthis level may be unsuitable for immediate implantation.However, these same polymers, combined with the appro-priate cell source in vitro, may be perfectly acceptable if thecell-mediated deposition of new ECM allows for a greaterrange of distensibility after some culture duration. Alterna-

tively, controlled rehabilitation regimes after surgery mightbe employed to protect the implant from loading while in vivomaturation takes place. At the end of the production and=ormaturation process, the implanted material on functional day0 should duplicate as many of the natural features of thenative tissue as possible. For example, while the linear mod-ulus of tendons and ligaments is important, the toe region isjust as important, if not more so. As measured by Butler andcolleagues, the normal operating range of a tendon does nottypically engage its failure state.113 Indeed the toe region iscritical for ensuring low-force mobility of joints and othermoving structures to enable smooth motion, while the failureproperties are operative only under extreme conditions.

Case example: knee meniscus

One tissue for which we have extensively investigated theapplication of nanofibrous scaffolds is the knee meniscus. Asnoted above, this fibrous tissue of the knee functions totransmit load from the femur to the tibia, and enhances jointcongruency. A distinguishing feature of the meniscus is thehighly organized collagen fiber structure, which imparts tothe tissue anisotropic tensile properties. An example of thestressstrain response of meniscus (tested in the fiber direc-tion) is shown in Figure 9. From this plot, we can appreciatethat the tissue shows significant nonlinearity in its stressstrain response, with clear toe and linear regions. Also shownon this plot are the stressstrain profiles of several aligned

nanofibrous constructs of differing polymer compositions (alltested in the aligned fiber direction). As can be readily ob-served, some of these polymers result in scaffolds that arestiffer than the native tissue and fail at much lower strainlevels, while others produce scaffolds that are considerablyless stiff, matching at best the toe region modulus of thenativetissue.

Of the many possible polymers, we work primarily withPCL, a slow degrading polyester that matches the native tis-sue in its toe region, shows some nonlinearity in its stressstrain response, and is distensible to greater than 10% strain.In most of our studies PCL is formed into nanofibers using the



12/24

electrospinning process, and scaffold fiber organizationis tuned by collecting on a static surface (for nonalignedscaffolds) or on a rotating mandrel (when alignment is de-sired).8082 In one recent study, we evaluated the long-termin vitro maturation of scaffolds after seeding with eithermesenchymal stem cells (MSCs) or meniscus fibrochon-drocytes (MFCs). MSCs are multipotent cells derived from bone marrow that can undergo fibro-cartilaginous differen-tiation, while MFCs are cells derived from the native tissuethat are normally responsible for the production and main-tenance of the fibro-cartilaginous ECM. Cell infiltration andconstruct maturation were evaluated as a function of topog-

raphy with culture on both aligned (AL) and nonaligned (NA)scaffolds.80 Seeded scaffolds were cultured in a defined pro-chondrogenic medium114 for 10 weeks and at bi-weeklyintervals, and were tensile tested to failure; their biochemicaland histological characteristics were assessed.

The results of this study showed that aligned nanofibrousscaffolds direct cell polarity in the scaffold (Fig. 10). At theoutset of the study, AL scaffolds had a higher linear regiontensile modulus (*12MPa) than NA scaffolds (*4 MPa).Notably, PCL degrades slowly, and so mechanical propertiesdid not change over 70 days in the absence of cells (notshown). When seeded with MSCs or MFCs, both AL and NAscaffolds increased in tensile properties with time (Fig. 11).ALconstructs increased by *10 MPa, while NA constructs in-

creased by only *1 MPa when seeded with either cell type.Conversely, biochemical composition (proteoglycan and col-lagen content) increased steadily with culture duration, andwas not dependent on the scaffold architecture. Histology oftransverse sections showed cell infiltration into the outer two-third of the scaffold and increasing matrix deposition withtime (not shown). The most interesting finding in this studywas that while cells on NA and AL scaffolds produce similaramounts of ECM, marked increases in tensile properties wereobserved only on AL scaffolds. This finding may be explainedby the organization of newly deposited ECM. On AL scaf-folds, polarized light microscopy showed collagen deposition

occurring in an ordered fashion, whereas deposited collagenwas disorganized on NA scaffolds (Fig. 11). In related studiesusing MFCs derived from surgical waste tissue from 10 hu-man donors,104 similar results were observed, with tensilemechanical property increases correlating strongly with theamount of collagen deposition. Importantly, the stressstrainresponse of these MFC-seeded scaffolds from several donorsmatched that of the native meniscus in its normal operatingrange (06% strain). However, the long time required for cellcolonization (4270 days) and the need for space for robustmatrix accumulation motivates our continued developmentof these novel scaffolds and culture systems (as described

below).

Case example: annulus fibrosus

Another tissue of focus for nanofiber-based tissue engi-neering is the AF of the intervertebral disc. Like the meniscus,this tissue relies on an ordered collagen ultrastructure toimpart defined mechanical function. Unlike the meniscus,the annulus is a laminate structure with alternating planesof organized fibers oriented at *308 relative to the cross-sectional plane. Toward engineering a single annulus layer,we have tested rectangular samples removed from alignedfibrous meshes at angles that are not coincident to the fiberdirection (Fig. 12A).82 The mechanicsof these acellularmeshes

were further characterized using a homogenization modelthat we have previously applied to native AF tissue, and wefound good agreement between modeled and measured lin-ear-region moduli.82 In subsequent studies, AF cells isolatedfrom bovine caudal discs were seeded onto scaffolds withfiber orientations of 08, 308, and 908 with respect to the lon-gitudinal axis of the sample. These cell-laden constructs werecultured in a chemically defined serum-free growth media (asabove) and mechanically tested at regular intervals. Histo-logical and biochemical analyses were also carried out.

Results of this study showed that when seeded with AFcells, scaffolds increase in tensile moduli with time for each

FIG. 9. Benchmarks for theengineering of fibrous tissues.Healthy meniscus tissue tes-

ted in the fiber direction pro-duces a nonlinear stressstrainresponse (circles indicate av-erage response; boundariesindicate high and low range ofresponses from 10 separatesamples). Superimposed onthis physiologic range are thestressstrain responses of na-nofibrous scaffolds of varyingcomposition.

182 MAUCK ET AL.


13/24

fiber orientation (Fig. 12CE). Notably, constructs tested inthe fiber direction (j 08) increased by *25 MPa (140%),while oblique (j 308) and transverse (j 908) constructsincreased by *2 MPa (33100%). These cell-seeded scaffoldswith a fiber orientation similar to the AF (j 308) matchedthe circumferential linear region modulus of native inner AF(5.6 MPa) after just 8 weeks of culture (7.3 MPa). However,the j 08 fiber direction modulus (43.3 MPa) remains belowthe single lamella of the native AF (77.6 MPa).115 The toe-region modulus in the fiber direction (9.1 MPa), however,was slightly higher than native AF (5.9 MPa). Therefore,in the range of small deformations, constructs achieved

comparable tensile properties to native tissue by 8 weeks.As with meniscus constructs above, the orientation of de-posited collagen coincided with the scaffold fiber direction(Fig. 12B).

Additionally, a fiber-reinforced constitutive model wasapplied to tensile stressstrain curves of these samples toyield four scalar material parameters with physically discretemeanings:m, nonfibrillar (matrix) stiffness;n, matrix compress-ibility; g, fiber stiffness; x, fiber nonlinearity. When the hy-perelastic model was applied, data from j 908 and j 08samples fit well (Fig. 12F; R2> 0.98) and yielded excel-lent predictions for j 308 samples (not shown, R2> 0.98),

FIG. 10. Nanofibrous topography controls cell morphology. (Top row) Scanning electron micrographs of NA and ALelectrospun PCL meshes. (Middle row) Scanning electron microscopy images of MSCs seeded on these meshes after 7 days ofculture. Note that cell polarity is dictated by the underlying scaffold topography. (Bottom row) Actin staining (green, withDAPI [blue] nuclear counter stain) shows that cytoskeletal elements within cells are similarly organized to reflect theunderlying topography. Scale bar 50mm. Color images available online at www.liebertonline.com=ten.



14/24

thereby validating the model for this application. The materialparameters that represent the matrix (Fig. 12G) and fibers(Fig. 12H) increased with time in culture. Of these, the largestincrease was for fiber stiffness g, and these model parameterscorrelated well with biochemical content. These results sug-gest that thefunctional improvement of theconstructs is basedon the increasing contribution of fibrillar and matrix ECM.This work additionally confirmed the ability of AF cells tocolonize and deposit organized, functional ECM, with similarcomposition and architecture as the native tissue. Further, thisstudy demonstrates the utility of an approach that couplesexperimental measurement with constitutive modeling tocharacterize the full nonlinear behavior of constructs. Thisapproach will help to elucidate the physical and biochemicalmechanisms of functional growth and may help guide ourtissue engineering efforts for creating functional AF tissue forimplantation.

Other Considerations

While the results above clearly indicate the potential ofnanofibrous scaffolds for soft tissue engineering, there re-

main a number of challenges and opportunities for ad-vancement of the technology that should be addressed. Someof these issues are detailed below.

Enhancing cell infiltration

While cell-mediated matrix deposition can generate func-tional constructs, one significant limitation in engineeringfibrous constructs for implantation is the long durations re-quired for scaffold colonization of slow degrading aligned

polyester scaffolds. This limits both the rate of matrix accu-mulation, as well as potentially limiting the degree to whichintegrationwithnativestructurescanoccur.Asdetailedabove,using juvenile bovine and adult human MFCs, *1-mm-thickscaffolds were only colonized in the peripheral two-thirdsthickness after 10 weeks in culture.80,104 It thus appears thatthese dense fibrous arrays, while providing a suitable mi-cropattern for directing growth, can also be a physical im-pediment to cell infiltration. When formed into an alignedconfiguration, this issue is exacerbated as the apparent den-sity in scaffolds is significantly increased compared to non-aligned or random scaffolds.81

FIG. 11. Case example: meniscus tissue engineering. MSCs and MFCs were grown on either NA or AL PCL scaffolds for upto 10 weeks of in vitro culture. (A) On AL scaffolds, both MSC- and MFC-seeded constructs increased by *10 MPa over thistime course, while the same cells on NA scaffolds only increased by *1MPa. (B) Differential growth was not a function ofECM deposition, as similar amounts of collagen (as well as proteoglycan, not shown) were deposited in each scaffold.

Producing en face sections (in the scaffold plane, (C) shown schematically) and viewing under polarized light (insets, D and E)show that organized collagen is present only in aligned scaffolds (E). Scale bar 100mm. Data adapted from Ref.80 Colorimages available online at www.liebertonline.com=ten.

184 MAUCK ET AL.


15/24

While numerous potential solutions could be posited forovercoming this hurdle, ranging from fluid pressure116 to

galvanotactic mechanisms,117

few reports exist on expeditedinfiltration. The most direct method for overcoming this bar-rier would be to place cells directly into the scaffold as it isformed. This has recently been accomplished by Stankus andco-workers, who electrosprayed cells in onto a commonmandrel onto which PEUU fibers were simultaneously beingcollected.118 This is an exciting new process, though scale-upand sterility may limit its wide-spread application. Anothermethod for increasing cell infiltration may be to form ECMproteins directly into nanofiber form. Biologic elements (in-cluding collagen) provide a biomimetic environment for celladhesion and thus are more readily colonized. Telemeco and

colleagues reported enhanced cell infiltration into pure col-lagen scaffolds compared to synthetic scaffolds with subcu-

taneous implantation.119

In these biologic scaffolds, cellsmay colonize by one of two routes, either through direct in-teraction in which they pull themselves through the protein-aceous milieu or they may degrade the ECM by secretion ofmatrix metalloproteinases. One drawback of this strategy,however, is that scaffolds mechanical properties using bio-logic polymers are considerably lower than that of commonsynthetic nanofibrous scaffolds,51,96 and that pretreatmentwith crosslinking agents (such as gluteraldehyde) are re-quired for their stabilization.

An alternative to these direct approaches can be found inthe extensive literature on porous foams and sponges. From

FIG. 12. Case example: AF tissue engineering. (A) Mechanical response and properties of acellular aligned scaffolds testedin directions that are not coincident (see inset) with the prevailing fiber direction can be captured with simple compositemodels. (B) When seeded with AF cells, matrix deposition (collagen staining) is coincident with the direction of the un-derlying fibers. Mechanical properties of cell-seeded scaffolds of different orientations (C: 08, in fiber direction; D: 308, off axisto the scaffold direction; E: 908, perpendicular to the fiber direction) increase with time in culture. (F) Hyperelastic modelscapture the temporal evolution of functional matrix. Representative curves for engineered AF (j 08) demonstrate a non-linear response in uniaxial tension. With time in culture, ECM deposition increases the nonlinearity and modulus from 1 dayto 8 weeks. The model (solid lines) successfully fit experimental tensile behavior (open circles) at each time point, as shown.Model parameters for matrix (G) and fiber (H) moduli increase with culture duration. Data adapted from Ref.82,112 Colorimages available online at www.liebertonline.com=ten.



16/24

that body of work, it appears that there exists an optimal poresize to promote cell infiltration.120 For nanofibrous scaffolds,this approach hasbeen addressed by mixingfibers of differentdiameters to limit fiber packing.116 Alternatively, pores maybe introduced by including salt particles at the time of pro-duction and subsequently leaching the salt out.121 In the for-mer case, some increase in cell infiltration was observed,particularly when coupled with fluid flow through the scaf-

fold thickness. In the latter case, salt crystals created planes ofseparation within the scaffold into which cells could migrate,though this compromised the overall structural integrityof thescaffold. In place of salt, still others have induced the forma-tion of ice crystals from relative humidity with collection on asuper-cooled collecting surface to provide solid inclusionsaroundwhichfibersform.122Still,othershavesimplyincreasedthe fiber size itself, transitioning from sub-micron-sized fibersto fibers on the order of tens of microns.123 This approachincreases cellular infiltration, but sacrifices the biomimetic

characteristics of the nanofibrous topography. Such size in-creases in fiber size adversely impact cellular behavior.40

An alternativeapproach to theseaforementionedtechniquesis to increase the size of the pores themselves, while not alter-ing the nano-scale morphology of the fibers or the preinstilledfiber alignment. To achieve such a goal, Baker and co-workershave recently reported on a novel dual-electrospinning pro-cess in which two fiber populations are deposited onto a

common rotating mandrel93

(Fig. 13). By carefully selectingfabrication parameters and degree of overlap between spin-nerets, one can control the relative fraction of each polymer inthe resulting composite mesh. Inclusion of fiber populationsthat are rapidly eroding and=or soluble in aqueous solutions(such as PEO) dramatically enhances porosity as the sacrificialcomponent elutes from the composite. Figure 13 shows anexample of this process, where the interspersion of discretefibers of differing composition is achieved and organized fiberdirectionality is maintained within the scaffold. As might be

FIG. 13. Enhancing cell infiltration via the inclusion of sacrificial fibers. A dual electrospinning system ( A) delivers twointerspersed fiber populations to a common grounded rotating mandrel (red, PCL; green, PEO), and removal of the sacrificialcomponent fibers (PEO) upon submersion in an aqueous environment (B). Mechanical properties of slowly degradingmeshes, such as PCL, do not change with hydration, while pure sacrificial meshes degrade completely. Mixtures of PEO andPCL fibers decrease in mechanical properties after submersion ( C). Increased porosity enhances infiltration of MSCs into thescaffold by week 3 of in vitro culture (D). Quantification shows increased fractions of sacrificial components (60%) led toincreasing numbers of cells within the central region (50100% infiltration depth) of the scaffold ( E). Data adapted from Ref.93

Color images available online at www.liebertonline.com=ten.

186 MAUCK ET AL.


17/24

predicted, elution of sacrificial fibers from the composite has apronounced effect on scaffold properties. Combining indi-vidual fiber components with dissimilar mechanical proper-ties influences the composite scaffold mechanics, and does soas a function of the fiber fractions employed. Fiber mixingalsoalters several key features of the stressstrain profile, includ-ing the toe region and the plastic deformation response of thescaffolds after reaching their yield point. Both visual inspec-

tion and mechanical testing of scaffolds before and after re-moval of PEO fibers show that mechanical anisotropy waspreserved. Further, by offsetting the source spinnerets withrespect to one another, a graded fibrous mesh was producedwith varying PEO contents. The tensile properties (maximumstress, stiffness, and modulus) reflected the amount of sacri-ficial component removed from the scaffold. When seededwith MSCs, scaffolds with greater amounts of removed sac-rificial fibers showed pronounced increases in their cell infil-tration rates.93

To further this area of inquiry, and to potentially offset theloss in properties with sacrificial fiber removal, we have re-cently adapted our techniques to allow for as many as three

individual polymers to be spun simultaneously onto the samecollecting mandrel94 (Fig. 14). The mechanical properties ofthese multipolymer composites can be tuned to compensatefor losses in properties from sacrificial fiber removal, such thatcell ingress can be promoted despite the removal of a signif-icant sacrificial fiber fraction.

Reconstituting anatomic form

Fabrication and maturation of a simple planar structurethat recapitulates the primary mechanical features of the na-tive tissue is a considerable challenge. Added to this is the factthat most tissues possess a specific anatomic form, and insome tissues, such as the intervertebral disc and meniscus,this form is a critical component of tissue function. Most na-nofibrous scaffolds begin as flat sheets or hollow tubes (whencollected on a mandrel). This latter form is particularly usefulfor small blood vessel tissue engineering, and unique multi-layered tissues have been formed using this technique.90

For other more complicated structures, few construction al-gorithms exist. Recent reports show that an MSC-seeded

FIG. 14. Multipolymer nanofibrous composites. Scaffold mechanics can be tuned with the integration of different polymersinto nanofibrous composites. Here three polymers [PEO, PCL, and poly(D,L-lactide-co-glycolide)] were collected simulta-neously to produce a fully interspersed fiber mixture (A). Changing component ratios affects the resulting stressstrainresponse (B). Mechanical properties of the composite are dictated by the relative fraction of each constituent (C). Using thisapproach, and based on the starting properties of each individual polymer, a wide range of scaffold properties can begenerated (D). Data adapted from Ref.94 Color images available online at www.liebertonline.com=ten.



18/24

nanofibrous scaffold can be press-fit into a molding appara-tus, and maintain their form for up to 6 weeks of in vitrorotating wall bioreactor culture.124 Others have investigatedmulticomponent scaffolds, where a gelatinous core is estab-lished (by injection) in the mid-substance of a nanofibroussheet for intervertebral disc applications.125 Still others havebegun to investigate how layers of nanofibers can be seededwith cells,and then stacked to achieve anatomic forms such as

periodontal ligament126

as well as more complicated struc-tures recreating the anatomic form of the IVD and the kneemeniscus127,128 (Fig. 15).

Conclusions and Future Directions

The large body of literature that has recently emergedconcerning nanofibrous scaffolds is a testament to the degreeof excitement in the field regarding their potential for tissueengineering applications. As the field advances, several ex-citing new possibilities arise, and what first emerged as asimple straight-and-narrow template for organizing cells will

develop into multifunctional scaffolds that enhance tissuerepair and regeneration. Emerging work focused on thecombination of multiple polymers into composite arrays will

FIG. 15. Engineering anatomic form with nanofibrous scaffolds. Nanofibrous scaffolds are typically formed as planarsamples, while most fiber-reinforced tissues have specialized anatomic form. For complex structures, such as the kneemeniscus (AC) and the intervertebral disc (DH), construction algorithms must be developed for in vivo application. Customwrapping can produce cell-laden wedge-shaped structures for meniscus applications ( B) that can be colonized by seeded cells(C). For IVD, annular composites with multiple layers can be formed ( D, E). Cell-seeded IVD composites localize cells tolamellar interfaces (F), and direct cell (G) and matrix (H) organization in each layer. Color images available online atwww.liebertonline.com=ten.

188 MAUCK ET AL.


19/24

allow for the tuning of scaffold properties to match those ofnumerous native tissues.34,94 New drug and gene deliverymethods are emerging that will impart additional function-ality,129131 potentially improving in vivo maturation rates andexpanding the potential clinical application. Bioreactor sys-tems that promote tissue growth via nutrient provision132

and=or mechanical stimulation133 will increasingly be utilizedto foster nanofiber-based tissue development. Further, con-

sideration of softhard interfaces, such as tendon insertionsites,134 may be addressed via selective induction of miner-alization and bone formation with inclusion of hydroxyapa-tite crystals.135 As thesedevelopments occurand, in particular,as advances are made in the area of load-bearing tissue en-gineering, special attention must be given to the appropriatecharacterization methods and analysis of mechanical prop-erties of these unique scaffolds. Moreover, production meth-ods must be optimized, such that nanofibrous scaffoldproduction is less of an art, and more of a science. For this, wemust endeavor toward higher throughput production meth-ods, and the establishment of good manufacturing protocolsand rigorous analyses. Finally, we must ultimately test thesescaffolds in realistic and challenging load-bearing situations

to evaluate their in vivo efficacy.136

These include the tensileloading methods described in this review, as well as com-pressive testing modalities (including equilibrium and dy-namic testing) when implants are to be used in compressiveand or mixed load-bearing situations (e.g., in the knee me-niscus). Taken together, this unique approach for the multi-scale rendering of fiber reinforcement from the nano- andmicron-levels holds tremendous potential for the repair orreplacement of fiber-reinforced tissues.

Acknowledgments

Funding for this work was provided by the National In-stitutes of Health (RO1 EB02425), the Aircast Foundation,The NFL charities, and the Penn Center of Musculoskeletal

Disorders (P30 AR050950). The authors would like to ac-knowledge Ms. Lara Ionescu for provision of the fetal andadult meniscus images and Mr. Ashwin Nathan for strainmaps of deformed scaffolds.

Disclosure Statement

No competing financial interests exist.

References

1. Lynch, H.A., Johannessen, W., Wu, J.P., Jawa, A., and El-liott, D.M. Effect of fiber orientation and strain rate onthe nonlinear uniaxial tensile material properties of tendon.

J Biomech Eng 125, 726, 2003.

2. Setton, L.A., Guilak, F., Hsu, E.W., and Vail, T.P. Bio-mechanical factors in tissue engineered meniscal repair.Clin Orthop 367 Suppl, S254, 1999.

3. Holzapfel, G.A., Schulze-Bauer, C.A., Feigl, G., and Re-gitnig, P. Single lamellar mechanics of the human lumbaranulus fibrosus. Biomech Model Mechanobiol 3, 125, 2005.

4. Proctor, C.S., Schmidt, M.B., Whipple, R.R., Kelly, M.A.,and Mow, V.C. Material properties of the normal medial

bovine meniscus. J Orthop Res 7, 771, 1989.5. Hayes, A.J., Benjamin, M., and Ralphs, J.R. Extracellular

matrix in development of the intervertebral disc. MatrixBiol 20, 107, 2001.

6. Clark, C.R., and Ogden, J.A. Development of the menisci ofthe human knee joint. Morphological changes and theirpotential role in childhood meniscal injury. J Bone JointSurg Am 65, 538, 1983.

7. Beredjiklian, P.K., Favata, M., Cartmell, J.S., Flanagan, C.L.,Crombleholme, T.M., and Soslowsky, L.J. Regenerativeversus reparative healing in tendon: a study of biome-chanical and histological properties in fetal sheep. AnnBiomed Eng 31, 1143, 2003.

8. Wang, J.H., Jia, F., Gilbert, T.W., and Woo, S.L. Cell ori-entation determines the alignment of cell-produced col-lagenous matrix. J Biomech 36, 97, 2003.

9. Frank, C.B., and Jackson, D.W. The science of reconstruc-tion of the anterior cruciate ligament. J Bone Joint Surg Am79, 1556, 1997.

10. Greis, P.E., Bardana, D.D., Holmstrom, M.C., and Burks,R.T. Meniscal injury: I. Basic science and evaluation. J AmAcad Orthop Surg 10, 168, 2002.

11. Asch, H.L., Lewis, P.J., Moreland, D.B., Egnatchik, J.G., Yu,Y.J., Clabeaux, D.E., and Hyland, A.H. Prospective multi-ple outcomes study of outpatient lumbar microdiscectomy:should 75 to 80% success rates be the norm? J Neurosurg96, 34, 2002.

12. Koebbe, C.J., Maroon, J.C., Abla, A., El-Kadi, H., and Bost, J.Lumbar microdiscectomy: a historical perspective and cur-rent technical considerations. Neurosurg Focus 13, E3, 2002.

13. Newman, A.P., Anderson, D.R., Daniels, A.U., and Dales,M.C. Mechanics of the healed meniscus in a canine model.Am J Sports Med 17, 164, 1989.

14. Provenzano, P.P., Hurschler, C., and Vanderby, R., Jr. Mi-crostructural morphology in the transition region betweenscar and intact residual segments of a healing rat medialcollateral ligament. Connect Tissue Res 42, 123, 2001.

15. Matsumoto, N., Horibe, S., Nakamura, N., Senda, T., Shino,K., and Ochi, T. Effect of alignment of the transplanted graftextracellular matrix on cellular repopulation and newlysynthesized collagen. Arch Orthop Trauma Surg 117, 215,1998.

16. Schoderbek, R.J., Jr., Treme, G.P., and Miller, M.D. Bone-patella tendon-bone autograft anterior cruciate ligamentreconstruction. Clin Sports Med 26, 525, 2007.

17. Arnoczky, S.P., DiCarlo, E.F., OBrien, S.J., and Warren, R.F.Cellular repopulation of deep-frozen meniscal autografts:an experimental study in the dog. Arthroscopy 8, 428, 1992.

18. Butler, D.L., Shearn, J.T., Juncosa, N., Dressler, M.R., andHunter, S.A. Functional tissue engineering parameters to-ward designing repair and replacement strategies. ClinOrthop Relat Res 427 Suppl, S190, 2004.

19. Awad, H.A., Butler, D.L., Harris, M.T., Ibrahim, R.E., Wu,Y., Young, R.G., Kadiyala, S., and Boivin, G.P. In vitrocharacterization of mesenchymal stem cell-seeded colla-gen scaffolds for tendon repair: effects of initial seeding

density on contraction kinetics. J Biomed Mater Res 51, 233,2000.20. Garvin, J., Qi, J., Maloney, M., and Banes, A.J. Novel system

for engineering bioartificial tendons and application ofmechanical load. Tissue Eng 9, 967, 2003.

21. Costa, K.D., Lee, E.J., and Holmes, J.W. Creating alignmentand anisotropy in engineered heart tissue: role of boundaryconditions in a model three-dimensional culture system.Tissue Eng 9, 567, 2003.

22. Barocas, V.H., Girton, T.S., and Tranquillo, R.T. Engineeredalignment in media equivalents: magnetic prealignmentand mandrel compaction. J Biomech Eng 120, 660, 1998.



20/24

23. Altman, G.H., Horan, R.L., Lu, H.H., Moreau, J., Martin, I.,Richmond, J.C., and Kaplan, D.L. Silk matrix for tissue en-gineered anterior cruciate ligaments. Biomaterials 23, 4131,2002.

24. Cooper, J.A., Lu, H.H., Ko, F.K., Freeman, J.W., and Laur-encin, C.T. Fiber-based tissue-engineered scaffold for liga-ment replacement: design considerations and in vitroevaluation. Biomaterials 26, 1523, 2005.

25. Cooper, J.A., Jr., Sahota, J.S., Gorum, W.J., 2nd, Carter, J.,Doty, S.B., and Laurencin, C.T. Biomimetic tissue-engineered anterior cruciate ligament replacement. ProcNatl Acad Sci USA 104, 3049, 2007.

26. Horan, R.L., Collette, A.L., Lee, C., Antle, K., Chen, J., andAltman, G.H. Yarn design for functional tissue engineering.

J Biomech 39, 2232, 2006.27. Moutos, F.T., Freed, L.E., and Guilak, F. A biomimetic

three-dimensional woven composite scaffold for functionaltissue engineering of cartilage. Nat Mater 6, 162, 2007.

28. Formhals, A. Process and apparatus for preparing artificialthreads. 1934, United States Patent Office. Patent no.1,975,504.

29. Burger, C., Hsiao, B.S., and Chu, B. Nanofibrous materialsand their applications. Annu Rev Mater Res 36, 333, 2006.

30. Li, W.J., Mauck, R.L., and Tuan, R.S. Electrospun nanofi-brous scaffolds: production, characterization, and applica-tions for tissue engineering and drug delivery. J BiomedNanotechnol 1, 259, 2005.

31. Teo, W.E., and Ramakrishna, S. A review on electrospin-ning design and nanofibre assemblies. Nanotechnology 17,R89, 2006.

32. Li, D., and Xia, Y.N. Electrospinning of nanofibers: re-inventing the wheel? Adv Mater 16, 1151, 2004.

33. Pham, Q.P., Sharma, U., and Mikos, A.G. Electrospinningof polymeric nanofibers for tissue engineering applications:a review. Tissue Eng 12, 1197, 2006.

34. Barnes, C.P., Sell, S.A., Boland, E.D., Simpson, D.G., andBowlin, G.L. Nanofiber technology: designing the nextgeneration of tissue engineering scaffolds. Adv Drug Deliv

Rev 59, 1413, 2007.35. Shin, Y.M., Hohman, M.M., Brenner, M.P., and Rutledge,

G.C. Experimental characterization of electrospinning: theelectrically forced jet and instabilities. Polymer 42, 9955, 2001.

36. Reneker, D.H., and Chun, I. Nanometre diameter fibres ofpolymer, produced by electrospinning. Nanotechnology 7,216, 1996.

37. Deitzel, J.M., Kleinmeyer, J., Harris, D., and Beck Tan, N.C.The effect of processing variables on the morphology ofelectrospun nanofibers and textiles. Polymer 42, 261, 2001.

38. Riesle, J., Hollander, A.P., Langer, R., Freed, L.E., andVunjak-Novakovic, G. Collagen in tissue-engineered carti-lage: types, structure, and crosslinks. J Cell Biochem 71, 313,1998.

39. Nur, E.K.A., Ahmed, I., Kamal, J., Schindler, M., and Mei-ners, S. Three dimensional nanofibrillar surfaces induce ac-tivation of Rac. BiochemBiophys ResCommun331, 428,2005.

40. Li, W.J., Jiang, Y.J., and Tuan, R.S. Chondrocyte phenotypein engineered fibrous matrix is regulated by fiber size.Tissue Eng 12, 1775, 2006.

41. Li, W.J., Laurencin, C.T., Caterson, E.J., Tuan, R.S., and Ko,F.K. Electrospun nanofibrous structure: a novel scaffold fortissue engineering. J Biomed Mater Res 60, 613, 2002.

42. Khil, M.S., Cha, D.I., Kim, H.Y., Kim, I.S., and Bhattarai, N.Electrospun nanofibrouspolyurethane membraneas wounddressing. J Biomed Mater Res B Appl Biomater 67, 675, 2003.

43. Xu, C., Inai, R., Kotaki, M., and Ramakrishna, S. Electro-spun nanofiber fabrication as synthetic extracellular matrixand its potential for vascular tissue engineering. Tissue Eng10, 1160, 2004.

44. Venugopal, J., Ma, L.L., Yong, T., and Ramakrishna, S. In vitrostudy of smooth muscle cells on polycaprolactone and col-lagen nanofibrous matrices. Cell Biol Int 29, 861, 2005.

45. Yoshimoto, H., Shin, Y.M., Terai, H., and Vacanti, J.P. Abiodegradable nanofiber scaffold by electrospinning and itspotential for bone tissue engineering. Biomaterials 24, 2077,2003.

46. Boland, E.D., Telemeco, T.A., Simpson, D.G., Wnek, G.E.,and Bowlin, G.L. Utilizing acid pretreatment and electro-spinning to improve biocompatibility of poly(glycolic acid)for tissue engineering. J Biomed Mater Res B Appl Biomater71, 144, 2004.

47. Yang, F., Murugan, R., Wang, S., and Ramakrishna, S.Electrospinning of nano=micro scale poly(L-lactic acid)aligned fibers and their potential in neural tissue engi-neering. Biomaterials 26, 2603, 2005.

48. Yang, F., Xu, C.Y., Kotaki, M., Wang, S., and Ramakrishna,S. Characterization of neural stem cells on electrospunpoly(L-lactic acid) nanofibrous scaffold. J Biomater Sci

Polym Ed 15, 1483, 2004.49. Li, W.J., Cooper, J.A., Jr., Mauck, R.L., and Tuan, R.S.

Fabrication and characterization of six electrospunpoly(alpha-hydroxy ester)-based fibrous scaffolds for tissueengineering applications. Acta Biomater 2, 377, 2006.

50. Boland, E.D., Coleman, B.D., Barnes, C.P., Simpson, D.G.,Wnek, G.E., and Bowlin, G.L. Electrospinning polydioxan-one for biomedical applications. Acta Biomater 1, 115, 2005.

51. Matthews, J.A., Wnek, G.E., Simpson, D.G., and Bowlin,G.L. Electrospinning of collagen nanofibers. Biomacromo-lecules 3, 232, 2002.

52. Rho, K.S., Jeong, L., Lee, G., Seo, B.M., Park, Y.J., Hong,S.D., Roh, S., Cho, J.J., Park, W.H., and Min, B.M. Electro-spinning of collagen nanofibers: effects on the behavior ofnormal human keratinocytes and early-stage wound heal-

ing. Biomaterials 27, 1452, 2006.53. Buttafoco, L., Kolkman, N.G., Engbers-Buijtenhuijs, P.,

Poot, A.A., Dijkstra, P.J., Vermes, I., and Feijen, J. Electro-spinning of collagen and elastin for tissue engineering ap-plications. Biomaterials 27, 724, 2006.

54. Li, M., Mondrinos, M.J., Gandhi, M.R., Ko, F.K., Weiss,A.S., and Lelkes, P.I. Electrospun protein fibers as matricesfor tissue engineering. Biomaterials 26, 5999, 2005.

55. Min, B.M., Jeong, L., Nam, Y.S., Kim, J.M., Kim, J.Y., andPark, W.H. Formation of silk fibroin matrices with differenttexture and its cellular response to normal human kerati-nocytes. Int J Biol Macromol 34, 281, 2004.

56. Min, B.M., Lee, G., Kim, S.H., Nam, Y.S., Lee, T.S., and Park,W.H. Electrospinning of silk fibroin nanofibers and its ef-

fect on the adhesion and spreading of normal humankeratinocytes and fibroblasts in vitro. Biomaterials 25, 1289,2004.

57. Geng, X., Kwon, O.H., and Jang, J. Electrospinning ofchitosan dissolved in concentrated acetic acid solution.Biomaterials 26, 5427, 2005.

58. Bhattarai, N., Edmondson, D., Veiseh, O., Matsen, F.A., andZhang, M. Electrospun chitosan-based nanofibers

Documents

Robert L. Mauck et al- Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration