Article 1 - Nantechonology-1

TISSUE ENGINEERING: Part A
Volume 15, Number 1, 2009

ª Mary Ann Liebert, Inc.
DOI: 10.1089=ten.tea.2008.0014
Novel Nanofiber-Based Scaffold

for Rotator Cuff Repair and Augmentation
Kristen L. Moffat, M.S.,1 Anne S.-P. Kwei,1 Jeffrey P. Spalazzi, Ph.D.,1 Stephen B. Doty, Ph.D.,2
William N. Levine, M.D.,3 and Helen H. Lu, Ph.D.1,4
The debilitating effects of rotator cuff tears and the high incidence of failure associated with current grafts
underscore the clinical demand for functional solutions for tendon repair and augmentation. To address this
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challenge, we have designed a poly(lactide-co-glycolide) (PLGA) nanofiber-based scaffold for rotator cuff tendon
tissue engineering. In addition to scaffold design and characterization, the objective of this study was to evaluate
the attachment, alignment, gene expression, and matrix elaboration of human rotator cuff fibroblasts on aligned
and unaligned PLGA nanofiber scaffolds. Additionally, the effects of in vitro culture on scaffold mechanical
properties were determined over time. It has been hypothesized that nanofiber organization regulates cellular
response and scaffold properties. It was observed that rotator cuff fibroblasts cultured on the aligned scaffolds
attached along the nanofiber long axis, whereas the cells on the unaligned scaffold were polygonal and randomly
oriented. Moreover, distinct integrin expression profiles on these two substrates were observed. Quantitative
analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber organization and
that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds
were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically
relevant mechanical properties were maintained. These observations demonstrate the potential of the PLGA
nanofiber-based scaffold system for functional rotator cuff repair. Moreover, nanofiber organization has a pro-
found effect on cellular response and matrix properties, and it is a critical parameter for scaffold design.
Introduction repair of chronic cuff injuries,5 generally attributed to factors

such as osteoporotic bone, degenerative and poorly vascu-
T he rotator cuff consists of four muscles and tendons,

including the supraspinatus, infraspinatus, teres minor
and subscapularis, which function in synchrony to stabilize
larized tendons, severe tendon weakening, muscle atrophy,
and size of the original defect.6–8 Moreover, surgical repair
often results in excessive tension on the cuff tissues at the
the glenohumeral joint and actively control shoulder kine- repair site.9,10 Although the use of synthetic tendon grafts
matics. Rotator cuff tears are among the most common in- have been reported,11,12 poor biocompatibility and insuffi-
juries afflicting the shoulder, with more than 75,000 repair cient mechanical strength have rendered these grafts sub-
procedures performed annually in the United States alone.1 optimal for rotator cuff repair.
Clinical intervention is required, since rotator cuff injuries do Recently, biological grafts based on allogeneic and xeno-
not heal due to the complex anatomy and extended range of geneic extracellular matrices have been considered for cuff
motion of the shoulder joint, as well as the relative weak- repair and augmentation.4,13 Collagen-rich dermis and small
ening and hypovascularization of the cuff tendons post- intestinal submucosa (SIS)14 have been marketed commer-
injury.2–4 cially as graft patches for reinforcing cuff repair.15,16 With its
Due to the high failure rates associated with current repair biomimetic, collagen nanofiber-based architecture and align-
procedures, coupled with the clinical need to treat large tears ment, SIS is particularly attractive because it can be readily
and chronic cuff degeneration, a significant demand exists remodeled by host cells, while encouraging angiogenesis and
for a functional rotator cuff grafting system. For example, neo-collagen production.14 Although promising results have
failure rates as high as 94% have been reported after primary been reported in animal models,4,13 augmentation with SIS
1
Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York,
New York.
2
Analytical Microscopy Laboratory, Hospital for Special Surgery, New York, New York.
3
Department of Orthopedic Surgery, Columbia University Medical Center, New York, New York.
4
College of Dental Medicine, Columbia University Medical Center, New York, New York.
115
116 MOFFAT ET AL.
did not improve the rate of tendon healing in human trials.16 grafting system for functional rotator cuff repair and aug-
Recently, using a canine model, Derwin et al.15 conducted a mentation.
systematic comparison of the biomechanical properties of
four commercially available extracellular matrices for rotator Materials and Methods
cuff augmentation. Significantly lower mechanical properties
Nanofiber scaffold fabrication
than those of the infraspinatus tendon were observed in all
grafts tested, and the mechanical properties of SIS decreased Nanofiber scaffolds (Fig. 1) based on poly(D,L-lactide-co-
due to premature resorption.15 Similar outcomes have been glycolide) 85:15 co-polymer (PLGA, Mw & 123.6 kDa; Lake-
reported for other biological grafts.17,18 Therefore, despite shore Biomaterials, Birmingham, AL) were produced using
their apparent structural advantages, the suboptimal clinical electrospinning.33–35 Briefly, a 35% (v=v) solution of PLGA
outcomes of biologically derived grafts may be attributed to was mixed with 55% N,N-dimethylformamide (Sigma-
inferior mechanical properties, the mismatch in kinetics of Aldrich, St. Louis, MO) and 10% ethyl alcohol. The polymer
graft remodeling, and the rate of neo-matrix production. solution was loaded into a 5-mL syringe with a 18.5-gauge
The debilitating effect of rotator cuff tears, coupled with stainless steel blunt-tip needle and electrospun at 8 to 10 kV
the high incidence of failure associated with existing graft using a custom electrospinning device. To fabricate un-
choices, underscore the clinical need for alternative grafting aligned nanofiber scaffolds, the collecting surface consisted
solutions. Our long-term objective is to engineer biomimetic, of a stationary plate, and a custom rotating mandrel (20 m=s)
functional, and integrative scaffold systems for rotator cuff was used to produce aligned scaffolds. The polymer solution
repair and augmentation. The ideal scaffold for rotator cuff was dispensed using a syringe pump (Harvard Apparatus,
tendon repair must be able to meet the physiologic demand Holliston, MA; 1 mL=h).
of the native tendon by matching its mechanical properties,
and it should promote host cell–mediated healing by mim- Nanofiber scaffold characterization
icking the ultrastructural organization of the native tendon. In
The structural and material properties of the nanofiber
addition, the scaffold should be biodegradable so that new
scaffolds were characterized post-fabrication. Specifically,
tissue can replace it gradually while physiologically relevant
nanofiber morphology and organization were imaged using
mechanical properties are maintained. Finally, the scaffold
scanning electron microscopy (SEM, 5 kV, FEI Quanta 600,
must be able to integrate with the host tendon and sur-
FEI Co. Hillsboro, OR). For SEM analysis, the scaffolds were
rounding bone tissue. This study, guided by these design cri-
sputter-coated with palladium to reduce charging effects. Fi-
teria, evaluated the potential of a degradable nanofiber-based
ber diameter was quantified using image analysis of SEM
scaffold system for rotator cuff repair. Nanofibers are advan-
micrographs (2000, n ¼ 5 images=group, ImageJ, National
tageous for tendon tissue engineering because of their superior
Institutes of Health, Bethesda, MD). In addition, scaffold po-
biomimetic potential and physiological relevance. They
rosity and pore diameter (n ¼ 5=group) were evaluated using
have been investigated with promising results for bone,19,20
mercury porosimetry (Micromeritics Autopore III, Norcross,
meniscus,21 intervertebral disk,22 cartilage,23 and ligament24,25
GA) following published protocols,36 in which porosity was
tissue engineering. A distinct advantage of nanofiber scaffolds
determined by measuring the volume of mercury infused
is that they can be engineered to resemble the native tendon
into the sample. Scaffold permeability (n ¼ 5=group) was de-
extracellular matrix, and designed to exhibit high aspect ratio,
termined by measuring the pressure difference across the
surface area, permeability, and porosity.26–31 Moreover, na-
scaffold with a custom device37,38 and then calculating per-
nofiber organization and alignment can be modulated during
meability according to Darcy’s Law.
fabrication,31,32 which allows for scaffold structural and ma-
The mechanical properties of the as-fabricated aligned
terial properties to be readily tailored to meet the functional
and unaligned nanofiber scaffolds were evaluated under
demands of the rotator cuff tendons.
uniaxial tensile testing.39 Briefly, the scaffolds (6 cm1 cm,
Specifically, this study focused on the design, charac-
n ¼ 5=group) were secured with custom clamps and mounted
terization, and in vitro evaluation of a poly(D,L-lactide-co-
on a mechanical testing device (Instron, Model 8841, Nor-
glycolide)-based nanofiber scaffold for rotator cuff repair. The
wood, MA) with an average gauge length of 3 cm. The sam-
effects of nanofiber organization (aligned vs unaligned) on the
ples were evaluated to failure at a strain rate of 5 mm=min,
attachment, gene expression, and matrix deposition of human
with the aligned scaffolds tested along the nanofiber long axis.
rotator cuff fibroblasts were determined over time. The three
Scaffold yield strength and ultimate tensile stress were de-
study objectives were first to characterize the structural and
termined, and elastic modulus was calculated from the linear
mechanical properties of aligned and unaligned nanofiber
region of the stress-strain curve.
scaffolds, then to evaluate the effects of fiber organization
(aligned vs unaligned) on the response of human rotator cuff
fibroblasts, and finally, to examine the effects of in vitro fi- In vitro culture of human rotator cuff fibroblasts
broblast culture on the mechanical properties of the tissue- on nanofiber scaffolds
engineered scaffolds. The nanofiber scaffold is designed to Cells and cell culture. Human rotator cuff fibroblast-like
match the structural and mechanical properties of the rotator cells were derived from explant culture of tissues obtained
cuff tendon, and it is hypothesized that the underlying na- from three patients (female, aged 65 to 70 years, institutional
nofiber organization will guide fibroblast attachment, mor- review board exempt) undergoing rotator cuff repair surgery.
phology, and matrix elaboration. This is the first reported Briefly, the tissue samples were rinsed with phosphate buff-
study on the application of tissue engineering for rotator cuff ered saline (PBS, Sigma-Aldrich), plated in tissue culture
regeneration using biomimetic, nanofiber-based scaffolds, dishes, and maintained in Dulbecco’s modified Eagle medium
and it is anticipated that this scaffold will serve as a promising supplemented with 10% fetal bovine serum, 1% non-essential
NANOFIBER FOR CUFF REPAIR 117
FIG. 1. Aligned and

unaligned nanofiber scaf-
folds. Scanning electron mi-
crographs of (A) aligned and
(B) unaligned scaffolds,
2000, bar ¼ 20 mm.
amino acids, 1% penicillin=streptomycin, and 1% amphoter- Quantitative analysis of cell attachment

icin B. The cells from the first migration were subsequently on nanofiber scaffolds
discarded, and the tissue was re-plated in fresh fully supple-
The effects of nanofiber organization (aligned vs un-
mented medium. Only cells obtained from the second and
aligned) on fibroblast attachment were quantified at 1, 7, and
third migrations were used in this study, because this method
14 days following the methods of Costa et al.42 Briefly, con-
has been shown to yield a relatively homogenous fibroblast
focal images (n ¼ 3 images=group) of fibroblast-seeded na-
population.39 All medium and supplements were purchased
nofiber scaffolds and acellular scaffolds were analyzed using
from Mediatech (Herndon, VA).
circular statistics software customized for evaluating fiber
alignment (Fiber3). The circular statistics parameters deter-
Cell seeding on nanofiber scaffolds. To prevent scaffold mined included mean vector angle, which represents the
contraction,40 the nanofiber scaffolds were secured using average fiber alignment in the matrix (908 # y # 908; 08 in-
custom-made clamps. After ultraviolet sterilization, the scaf- dicates horizontal orientation); mean vector length, which
folds were pre-incubated in fully supplemented medium at ranges from zero for a randomly distributed sample to unity
378C and 5% carbon dioxide for 16 h. Human rotator cuff
for an aligned sample (0 # r # 1); and angular deviation,
fibroblasts (passage 4) were seeded on the scaffolds at a which characterizes the dispersion of the non-Gaussian angle
density of 3104 cells=cm2 and allowed to attach for 15 min distribution of the nanofibers (0-40.58). Specifically, an angu-
before the addition of fully supplemented medium. The cells lar deviation of 08 represents an aligned sample, and 40.58 is
were cultured on the aligned and unaligned nanofiber scaf- indicative of random distribution.
folds for 2 weeks, with monolayer culture of the human ten-
don fibroblasts and acellular scaffolds (aligned as well as
unaligned) serving as controls. The effects of nanofiber orga- Gene expression
nization on cell morphology, attachment, gene expression,
Gene expression (n ¼ 5=group) was measured using re-
proliferation, and matrix production were determined
verse transcriptase polymerase chain reaction (RT-PCR) at 1,
throughout the 2-week culturing period. In addition, the ef-
3, and 14 days. Total RNA was isolated using the Trizol ex-
fects of in vitro culture on scaffold mechanical properties were
traction method (Invitrogen, Carlsbad, CA). The isolated
evaluated over time.
RNA was reverse-transcribed into complementary DNA
(cDNA) using the SuperScript First-Strand Synthesis System
Cell viability and attachment morphology
(Invitrogen), and the cDNA product was then amplified using
Cell attachment morphology (n ¼ 3=group) on the nanofi- recombinant Taq DNA polymerase (Invitrogen). Expression
ber scaffolds was evaluated at 1, 7, and 14 days using SEM of integrins a2 (sense, 5’-CAGAATTTGGAACGGGACTT-3’;
(FEI Quanta 600). The samples were first rinsed with 0.1 M antisense, 5’- CAGGTAGGTCTGCTGGTTCA-3’), a5 (sense,
sodium cacodylate buffer (Sigma-Aldrich), incubated in 5’-GTGGCCTTCGGTTTACAGTC-3’; antisense, 5’-AATAGC
Karnovsky’s fixative41 for 24 hours at 48C, and subsequently ACTGCCTCAGGCTT-3’), aV (sense, 5’-GATGGACCAATG
dehydrated with an ethanol series. In addition, cell viability AACTGCAC-3’; antisense, 5’-TTGGCAGACAATCTTCAAG
(n ¼ 3=group) and attachment morphology were evaluated C-3’), and b1 (sense, 5’- GAGGAATACAGCCTGTGGGT-3’;
using Live=Dead staining (Molecular Probes, Eugene, OR). antisense, 5’-CAGAAGGTGCAGAGATGATGA-3’) were de-
The samples were rinsed twice with PBS and stained follow- termined over time. Type I collagen (sense, 5’-TGCTGGC
ing the manufacturers suggested protocol. The samples were CAACTATGCCTCT-3’; antisense, 5’ TTGCACAATGCTCT
imaged under confocal microscopy (Olympus Fluoview IX70, GATC-3’) and type III collagen (sense, 5’- CCAAACTCTAT
Center Valley, PA) at wavelengths of 488 nm and 568 nm. To CTGAAATCC-3’; antisense, 5’-GGACTCATAGAATACAAT
evaluate cell penetration into the scaffold, a z-series of con- CT-3’) expression were also examined. Glyceraldehyde-3-
focal images were collected over a depth of 10 mm, equivalent phosphate dehydrogenase (GAPDH, sense, 5’- GGCGATGC
to 15 to 20 layers of nanofibers. TGGCGCTGAGTA-3’; antisense, 5’-ATCCACAGTCTTCT
118 MOFFAT ET AL.
Table 1. Summary of the Structural Properties of Aligned and Unaligned Scaffolds
Scaffold Fiber Pore Porosity Permeability

Thickness (mm) Diameter (nm) Diameter (mm) (%) (m4=N s)
Aligned (n ¼ 5) 0.22 0.02 615 152 4.228 1.056 80.745 2.966 (7.87 2.47)1012
Unaligned (n ¼ 5) 0.19 0.02 568 147 4.914 0.777 81.760 3.929 (5.72 0.63)1012
GGGTGG-3’) served as the house-keeping gene. All genes scaffolds. Specifically, the elastic modulus, yield strength, and
were amplified for 30 cycles in a thermocycler (Eppendorf ultimate tensile stress of the samples (n ¼ 5 samples=group)
Mastercycler gradient, Brinkmann, Westbury, NY). Semi- were determined.
quantitative analysis of gene expression (ImageJ) was per-
formed, and band intensity was normalized to that of
Statistical analysis
GAPDH.
Results are presented in the form of mean standard de-
Cell proliferation viation, with n equal to the number of replicates (n ¼ 5 for all
quantitative analyses, n ¼ 3 for all qualitative analyses). One-
Cell proliferation (n ¼ 5 samples=group) was determined way analysis of variance (ANOVA) was performed to deter-
by measuring total DNA content using the PicoGreen double mine the effects of fiber organization on scaffold structural
strand DNA assay (Invitrogen) following the manufacturer’s and mechanical properties. Two-way ANOVA was used to
suggested protocol. At designated time points, each scaffold determine fiber organization and temporal effects on cell
was rinsed with PBS and homogenized in 0.1% Triton X so- alignment, proliferation, and gene expression. Similarly, the
lution (Sigma-Aldrich). Fluorescence was measured using a effects of in vitro culture and culturing time on scaffold tensile
microplate reader (Tecan, Research Triangle Park, NC) at the mechanical properties were determined. The Tukey-Kramer
excitation and emission wavelengths of 485 nm and 535 nm, post-hoc test was used for all pair-wise comparisons, and
respectively. A standard curve was generated to correlate significance was attained at p < 0.05. Statistical analyses were
DNA content with fluorescence intensity, and the number performed with JMP IN (4.0.4, SAS Institute, Inc., Cary, NC).
of cells was determined using a conversion factor of 8 pg
DNA=cell.39 Results
Cell matrix production Characterization of the aligned and unaligned

nanofiber scaffolds
Fibroblast elaboration of type I and type III collagen (n ¼ 3
samples=group) on the aligned and unaligned scaffolds was The structural properties of the as-fabricated scaffolds are
evaluated using immunohistochemistry at 7 and 14 days. summarized in Table 1. The nanofiber diameter of the aligned
After rinsing with PBS, the samples were fixed with 10% scaffolds averaged 615 152 nm, whereas that of the un-
neutral buffered formalin for 24 h. Monoclonal antibodies for aligned group measured 568 147 nm, with no significant
type I collagen (1:20 dilution) and type III collagen (1:100) difference found between the groups. Similarly, scaffold po-
were obtained from EMD Chemicals (Calbiochem, San Diego, rosity, pore diameter, and permeability were comparable
CA) and Sigma-Aldrich, respectively. Before staining for between the aligned and unaligned scaffolds (Table 1). In
type III collagen, the samples were treated with 1% hyal- contrast, the mechanical properties of the as-fabricated
uronidase for 30 min at 378C and incubated with primary aligned and unaligned scaffolds differed significantly (Fig. 2,
antibody overnight. After a PBS wash, biotinylated secondary p < 0.05). As shown in Figure 2, distinct stress–strain profiles
antibody and streptavidin conjugate (LSAB2 System-HRP, were observed for the aligned and unaligned scaffolds, with
DakoCytomation, Carpinteria, CA) were added. The forma- significant differences detected in tensile properties. Specifi-
tion of red-brown precipitates indicated positive staining cally, the elastic modulus of the aligned scaffold was three
with the colorimetric substrate (AEC Substrate Chromogen, times as strong, while both yield strength and ultimate tensile
DakoCytomation). At day 7, organization of the type I colla- stress were nearly four times higher than those of the un-
gen produced by the human fibroblasts was evaluated using aligned scaffolds (Table 2, p < 0.05).
the circular statistics software (Fiber3), as described above.
Effects of nanofiber organization on fibroblast
Mechanical properties of the fibroblast-seeded attachment and alignment
nanofiber scaffolds
Cell attachment morphology. The attachment morphology
The effects of in vitro fibroblast culture on the mechanical and growth of human rotator cuff fibroblasts on aligned and
properties of the aligned and unaligned scaffolds were de- unaligned nanofiber scaffolds were visualized using electron
termined at 1, 7, and 14 days. The human rotator cuff fibro- and confocal microscopy. As shown in Figure 3, the fibro-
blasts were seeded at a density of 3104 cells=cm2 on the blasts attached and assumed distinct morphologies on the
scaffold. Aligned and unaligned scaffolds without cells two types of nanofiber scaffolds. Specifically, the cells grown
(acellular) served as controls. At each designated time point, on the aligned fibers adopted a phenotypic elongated mor-
the samples were tested to failure under uniaxial tension phology and were oriented in the direction of the fiber long
following the protocol described above for the as-fabricated axis. In contrast, fibroblasts seeded on the unaligned fibers
strate, focusing on mean vector angle and angular deviation

(Fig. 5A, Table 3) and mean vector length (Fig. 5B). Fibroblasts
cultured on the aligned scaffolds were oriented horizontally at
day 1, exhibiting similar mean angle distribution (cells: 4.248,
fibers: 4.328) and narrow angular deviation (cells: 19.738, fi-
bers: 17.008) to that of the underlying aligned nanofiber sub-
strate. Fibroblasts on unaligned scaffolds also conformed to
matrix organization at day 1, demonstrating a random ori-
entation (cells: 63.468, fibers: 55.698) with a wide angular
deviation approximating those of the unaligned scaffold
matrix (cells: 37.708, fibers: 35.978). At day 14, the underlying
nanofiber organization continued to dictate fibroblast growth
and morphology, with significantly greater alignment mea-
sured for fibroblasts cultured on the aligned scaffolds than for
those found on the unaligned scaffolds (Fig. 5B, p < 0.05). For
each scaffold type (aligned or unaligned), circular statistical
FIG. 2. Mechanical properties of nanofiber scaffolds.
analysis of fibroblast response revealed no significant change
A representative stress–strain curve for aligned and un-
aligned scaffolds tested in uniaxial tension is shown. Color in alignment parameters (mean angle, angular deviation,
images available online at www.liebertonline.com=ten. mean vector length) from the day 1 results.
Effects of nanofiber organization on fibroblast growth

exhibited a polygonal morphology without preferential ori- and matrix deposition
entation. Moreover, as fibroblasts proliferated on both types Tendon fibroblasts proliferated on the nanofiber scaffolds
of substrates, these morphological differences were main- over the 2-week culturing period, with no significant differ-
tained over the 2-week culturing period (Fig. 3). ence in cell growth found between the aligned and unaligned
groups (Fig. 6A). Matrix deposition by human fibroblasts on
Gene expression. The expression of integrins and type I and the nanofiber scaffolds was also evaluated over time, and
type III collagen were compared over time on the aligned and the cells produced a collagen-rich matrix containing type
unaligned scaffolds (Fig. 4). a5 expression did not vary sig- I and type III collagen (Fig. 6B). Additionally, circular statis-
nificantly between the nanofiber scaffolds, and no appar- tical analysis of the type I collagen immunohistochemistry
ent change was observed over time (Fig. 4A). a2 expression images revealed an oriented collagen matrix on the aligned
(Fig. 4B) differed significantly between the two types of na- nanofiber scaffold (Fig. 6C), with a mean angle angular
nofiber scaffolds, with 15, 9, and 10 times greater expression deviation of 9.088 30.048 and a mean vector length of 0.45.
detected in the aligned than in the unaligned group at 1, 7, and In contrast, a randomly oriented collagen matrix with a mean
14 days, respectively. After 2 weeks of culture, significantly angle angular deviation of 12.048 34.378 and a mean vec-
higher aV expression was seen on the unaligned scaffold (Fig. tor length of 0.28 was observed on the unaligned scaffolds.
4C). Although the expression of b1 (Fig. 4D) was similar be-
tween the two scaffold groups, it increased significantly over
Effects of in vitro culture on scaffold
time, with higher expression ( p < 0.05) detected at day 14 than
on day 1 and day 3. Expression of type I collagen was evident mechanical properties
by day 3, whereas type III collagen expression was main- The mechanical properties of fibroblast-seeded PLGA
tained on aligned and unaligned scaffolds at all time points nanofiber scaffolds were determined over time and com-
examined (Fig. 4A). All genes evaluated were also consis- pared as a function of cell culture (cellular vs acellular),
tently detected in the monolayer control (data not shown). culturing time, and nanofiber organization (aligned vs un-
aligned). As expected for PLGA, in vitro culture decreased
Quantitative analysis of cell attachment and alignment. The scaffold mechanical properties, with significantly lower ulti-
attachment response of human rotator cuff fibroblasts to the mate tensile stress and yield strength over time for aligned
inherent organization of the nanofiber scaffolds (aligned vs and unaligned scaffolds than for the as-fabricated scaffolds
unaligned) was quantified using circular statistical analy- (Figs. 7A, C; p < 0.05). In contrast, in vitro culture had no sig-
sis.42,43 Specifically, cell alignment and orientation over time nificant effect on scaffold elastic modulus (Fig. 7B). The ulti-
were compared with those of the underlying nanofiber sub- mate tensile stress, elastic modulus, and yield strength of the
Table 2. Summary of the Mechanical Properties of Aligned and Unaligned Scaffolds
Elastic Modulus Yield Strength Ultimate Stress

(MPa) (MPa) (MPa)
Aligned (n ¼ 5) 341 30* 9.8 1.1* 12.0 1.5*

Unaligned (n ¼ 5) 107 23 2.5 0.4 3.7 0.2
*p < 0.05.
120 MOFFAT ET AL.
FIG. 3. Effects of nanofiber organization on cell morphology. Top panel: Confocal microscopy of human rotator cuff
fibroblasts at day 1 and day 14 (green: live, red: dead), 20, bar ¼ 100 mm. Bottom panel: Scanning electron micrographs of
cells on aligned and unaligned scaffolds at day 1 and day 14, 1000, bar ¼ 50 mm. The fibroblasts remained viable and grew on
both types of substrate over time, with the rotator cuff cells exhibiting phenotypic elongated morphology on the aligned
nanofiber scaffolds. Color images available online at www.liebertonline.com/ten.
FIG. 4. Gene expression on aligned and unaligned scaffolds over time. (A) Fibroblast expression of glyceraldehyde-
3-phosphate dehydrogenase (GAPDH), integrins, and type I and type III collagen. (B) Expression of the a2 integrin differed
significantly on the aligned and unaligned scaffolds at all time points examined (*p < 0.05). (C) Expression of aV was signifi-
cantly higher on the unaligned scaffolds at day 14 (*p < 0.05). (D) Although b1 expression did not vary significantly between the
two scaffold types, it increased significantly over time (*p < 0.05). Color images available online at www.liebertonline.com/ten.
FIG. 5. Quantitative Analysis of fibroblast response on

aligned and unaligned scaffolds. Nanofiber organization
guided cell attachment based on analysis of (A) mean vector
angle (0 ¼ horizontally aligned) and (B) mean vector length
(0 ¼ random, 1 ¼ aligned, *p < 0.05). Insert: Confocal images
of cells on aligned (top) and unaligned (bottom) nanofibers,
60, bar ¼ 50 mm. Color images available online at www
.liebertonline.com=ten.
fibroblast-seeded scaffolds did not change significantly over not found until 1 week later in the aligned group (Fig. 7A). The
time (Fig. 7). yield strength of the aligned scaffolds decreased significantly
At all time points examined, the tensile properties of the at day 1 (Fig. 7C) and continued to drop over time, with a
aligned scaffolds were significantly greater than those of the significantly lower yield strength found at day 14 ( p < 0.05).
unaligned scaffolds (Fig. 7, p < 0.05), and this trend was In contrast, no significant change in yield strength was seen in
consistently observed in the cellular and acellular groups. For the unaligned scaffolds over time.
the unaligned group, a significant decrease in ultimate tensile
strength was detected at day 1, whereas such a decrease was Discussion
Our long-term goal is to engineer biomimetic, functional,
and integrative scaffold systems for rotator cuff repair and
Table 3. Summary of the Alignment Analysis Results augmentation. This study focuses on the design, character-
Day 1 (n ¼ 3) Mean Angle Angular Deviation (8) ization, and in vitro evaluation of a degradable polymer-based
nanofiber scaffold with pre-engineered mechanical properties
Aligned Cells 4.24 19.73 matching those of native tissue. In this study, the effects of
Aligned Fibers 4.32 17.00 nanofiber organization on the scaffold structural and me-
Unaligned Cells 63.46 37.70 chanical properties and the response of primary cells derived
Unaligned Fibers 55.69 35.97 from the human rotator cuff tendons were systematically in-
vestigated. Additionally, the effects of fibroblast culture on
Day 14 (n ¼ 3) Mean Angle Angular Deviation (8)
the ability of the nanofiber scaffold to maintain stable me-
Aligned Cells 6.19 18.74 chanical properties were evaluated over time. It is observed
Aligned Fibers 4.62 17.78 that nanofiber organization controls scaffold mechanical
Unaligned Cells 19.45 35.93 properties and is the primary factor guiding the attachment
Unaligned Fibers 20.84 38.14 morphology, alignment, integrin expression, and matrix de-
position by human rotator cuff fibroblasts. Moreover, al-
Cell and fiber alignment parameters evaluated at 1 and 14 days
of culture, represented as mean vector angle (MA: 908 # y # 908)
though in vitro culture resulted in an expected decrease in
angular deviation (AD: 0–40.58). scaffold mechanical properties with polymer degradation,
08 indicates horizontal orientation. nanofiber organization modulated these changes. Based on
122 MOFFAT ET AL.
these observations, it is apparent that nanofiber organization

exerts significant control over cell response as well as scaffold
properties and is a critical graft design parameter for func-
tional rotator cuff repair.
The scaffolds used in this study were pre-designed with
similar structural properties (nanofiber diameter, porosity,
pore size, permeability); thus, these observed differences in
cell response can be attributed to the differences in nanofiber
organization (aligned vs unaligned). Qualitative and quanti-
tative analyses revealed that human rotator cuff fibroblasts
exhibited a phenotypic morphology and attached preferen-
tially along the nanofiber axis of the aligned scaffolds,
whereas only random cell attachment was observed on the
unaligned scaffold. These differences were maintained over
time, and more importantly, subsequent cell-mediated pro-
duction of type I and type III collagen also conformed to na-
nofiber organization.
To further explore this contact guidance phenomena,44–46
where topography of the biomaterial substrate regulates
cell adhesion, expression of key integrins on aligned and

unaligned scaffolds were compared over time. Significantly
greater a2 expression was consistently detected on the aligned
nanofiber scaffolds, whereas aV and b1 expression levels were
upregulated on the unaligned scaffolds over time. These ob-
servations suggest that human rotator cuff fibroblasts can
detect differences in nanofiber alignment during initial at-
tachment, as well as post-adhesion matrix synthesis. Because
elevated expression and production of aV and b1 has been
associated with healing tendons47 and ligaments,48 the dif-
ferential integrin expression found in this study suggests that
cells exposed to the unaligned nanofiber matrix may be pri-
marily undergoing a repair response. In contrast, the physi-
ologically relevant aligned fibers circumvent such a healing
response, which may result in disorganized scar tissue for-
mation. Thus the aligned nanofibers directly promote the
deposition of a biomimetic collagen matrix instead. The sup-
pression of a2 integrin expression observed on the unaligned
scaffolds is also significant. Li et al.23 evaluated fetal bovine
chondrocyte response on unaligned nanofiber scaffolds of
poly(e-caprolactone) and also observed that a2 expression
was down-regulated when compared with monolayer con-
trol. Because a2 complexes with b1 to form the key integrin
heterodimer that mediates cell attachment to collagenous
matrices,49–51 its upregulation, only when fibroblasts are
cultured on the aligned nanofiber, suggests that this matrix
may better mimic the collagen matrix native to the rotator cuff
fibroblasts. Although these results are promising, only gene
expression was determined in this study. Future work will
focus on evaluation of integrin production and the down-
stream effects of these observed cell responses as a function of
nanofiber organization.
Because the nanofiber scaffold is based on degradable
polymers, it is important to characterize whether scaffold
mechanical properties will be maintained during in vitro
FIG. 6. Cell proliferation and matrix elaboration on aligned culture and, moreover, whether cell-mediated matrix pro-
and unaligned scaffolds. (A) Cells proliferated on both types
duction will compensate for the reduction in mechanical
of scaffolds independent of fiber alignment. (B) Im-
munohistochemical staining for types I and III collagen (day properties due to nanofiber degradation. It was observed
7, 20, bar ¼ 100 mm). (C) Nanofiber organization also guided that, although tensile properties decreased because of hy-
matrix production on the nanofiber scaffolds, with an aligned drolytic degradation of the PLGA nanofibers,39 its material
collagen I matrix found on the aligned scaffolds (day 7, mean properties were still within range of those reported for hu-
angle analysis). Color images available online at www man rotator cuff tendons.52 This indicates that physiological
.liebertonline.com=ten. loading can be sustained while scaffold degradation occurs
FIG. 7. Effects of in vitro culture on scaffold mechanical properties. Mechanical properties of the cell-seeded and acellular
scaffolds decreased because of polymer degradation, and aligned scaffolds remained significantly stronger than unaligned
scaffolds, (A) ultimate tensile strength, *p < 0.05; (B) elastic modulus, *p < 0.05 vs unaligned, and #p < 0.05 vs unaligned cellular
scaffolds; and (C) yield strength, *p < 0.05 for aligned vs as-fabricated aligned scaffolds, and #p < 0.05 day 1 vs day 14. Color
images available online at www.liebertonline.com=ten.
and the cell-mediated response is underway. Additionally, enchymal stem cells and meniscal fibrochondrocytes cultured
the aligned nanofiber scaffolds maintained the as-fabricated on aligned and unaligned poly(e-caprolactone) nanofiber
tensile mechanical properties longer than the unaligned scaffolds.21 Furthermore, collagen synthesis by human liga-
group. These observations demonstrate that degradation ment fibroblasts grown on aligned polyurethane nanofiber
kinetics of the nanofiber scaffolds and associated changes in scaffolds was significantly greater after being subjected to
mechanical properties are dependent on fiber organization. tensile loading.24 It is likely that mechanical stimulation and
No significant increase in tensile properties with fibroblast fiber organization may be coupled to promote overall colla-
culture was observed, which is probably due to the relatively gen production over time and in turn improve cuff healing
short culturing time evaluated in this study. Cell-mediated and long-term clinical outcome.
increases in mechanical properties were reported only after The PLGA nanofiber-based scaffold represents a promis-
more than 2 months of in vitro culture.21 The effects of long- ing system for functional repair and augmentation of rota-
term culture on scaffold mechanical properties will be in- tor cuff tendons. In this study, the tensile modulus of the
vestigated in future studies. unaligned and aligned scaffolds averaged 107 MPa and
In this study, the human rotator cuff fibroblasts prolifer- 341 MPa, respectively, with mean ultimate tensile strength
ated over time on both types of nanofiber scaffolds, with no ranging from 3.7 to 12.0 MPa. In addition to possessing
significant differences observed between the scaffold groups. physiologically relevant mechanical properties and fiber di-
These results corroborate previous studies that also reported ameter approximating that of native collagen matrix, the
minimal effect on cell proliferation due to nanofiber organi- nanofiber scaffold reported here exhibits structural proper-
zation.21,24 Although fiber organization differed, types I and ties that are optimal for soft tissue repair, including porosity
III collagen deposition were observed on the aligned and un- greater than 80% and high permeability, which can facilitate
aligned scaffolds. While total collagen content was not quan- nutrient transport and promote cell viability and tissue
tified in this study, fiber architecture has been reported to growth in vitro and in vivo. The permeability of the scaffold is
have minimal effect on collagen production for bovine mes- also within range of those reported for musculoskeletal
124 MOFFAT ET AL.
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TISSUE ENGINEERING: Part A

Volume 15, Number 1, 2009

Novel Nanofiber-Based Scaffold

Introduction repair of chronic cuff injuries,5 generally attributed to factors

T he rotator cuff consists of four muscles and tendons,

FIG. 1. Aligned and

amino acids, 1% penicillin=streptomycin, and 1% amphoter- Quantitative analysis of cell attachment

Table 1. Summary of the Structural Properties of Aligned and Unaligned Scaffolds

Scaffold Fiber Pore Porosity Permeability

Cell matrix production Characterization of the aligned and unaligned

strate, focusing on mean vector angle and angular deviation

Effects of nanofiber organization on fibroblast growth

Table 2. Summary of the Mechanical Properties of Aligned and Unaligned Scaffolds

Elastic Modulus Yield Strength Ultimate Stress

Aligned (n ¼ 5) 341 30* 9.8 1.1* 12.0 1.5*

FIG. 5. Quantitative Analysis of fibroblast response on

these observations, it is apparent that nanofiber organization

cell adhesion, expression of key integrins on aligned and

tissues.37,53–56 Moreover, the observed differences in cell re- References

Cellulose Derivatives 506-546. [Crossref]

repair. Journal of Materials Science: Materials in Medicine 24:6, 1605-1614. [Crossref]

Bulletin 94:1, 165-188. [Crossref]

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