electrospun fibers as a scaffolding platform for bone tissue repair

Electrospun Fibers as a Scaffolding Platform for Bone Tissue Repair

Seungyoun Lyu,1 Chunlan Huang,2 Hong Yang,1 Xinping Zhang2

1Department of Chemical Engineering, University of Rochester, Rochester, New York, 2Center for Musculoskeletal Research, University ofRochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, 14642, New York

Received 26 August 2013; accepted 11 March 2013

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22367

ABSTRACT: The purpose of the study is to investigate the effects of electrospun fiber diameter and orientation on differentiation andECM organization of bone marrow stromal cells (BMSCs), in attempt to provide rationale for fabrication of a periosteum mimetic forbone defect repair. Cellular growth, differentiation, and ECM organization were analyzed on PLGA-based random and aligned fibersusing fluorescent microscopy, gene analyses, electron scanning microscopy (SEM), and multiphoton laser scanning microscopy(MPLSM). BMSCs on aligned fibers had a reduced number of ALPþ colony at Day 10 as compared to the random fibers of the samesize. However, the ALPþ area in the aligned fibers increased to a similar level as the random fibers at Day 21 following stimulationwith osteogenic media. Compared with the random fibers, BMSCs on the aligned fibers showed a higher expression of OSX andRUNX2. Analyses of ECM on decellularized spun fibers showed highly organized ECM arranged according to the orientation of thespun fibers, with a broad size distribution of collagen fibers in a range of 40–2.4 mm. Taken together, our data support the use ofsubmicron-sized electrospun fibers for engineering of oriented fibrous tissue mimetic, such as periosteum, for guided bone repair andreconstruction. � 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res

Keywords: electrospinning; extracellular matrix (ECM); periosteum

Tissue engineering holds enormous potential to providefunctional substitutes for damaged tissues. A key com-ponent of tissue engineering is to fabricate man-madesubstitutes (scaffolds) to guide the regenerative processof the damaged tissue. While underlying cellular mech-anisms for functions may vary greatly in different tis-sue types, all engineered materials must closely mimicthe natural tissue environment to successfully meet orperhaps surpass the original mechanical, structural,and functional properties. To this end, providing bio-logical and structural cues that mimic the complexproperties of the native tissue has become an essentialelement in the design of tissue-engineering scaffolds.

Electrospinning has emerged as a mainstay intissue engineering due to its versatility in fabricatingrandomly oriented or aligned fibers that are character-istic of the extracellular matrix (ECM).1,2 Electrospin-ning is used to draw micron- and nanometer-sizednon-woven fibers through the electrostatic interactionsof the charged polymers. Fibers produced from thistechnique exhibit high uniformity and mechanicalstrength and form porous scaffolds with a high sur-face-to-volume ratio.3 A wide range of synthetic biode-gradable polymers as well as natural macromoleculeshave been used to create fibrous scaffolds.4–7 Althoughelectrospun natural polymers show higher hydrophilic-ity, synthetic polymers are more robust and presenthigher mechanical properties.8 Scaffolds consisting ofsynthetic electrospun fibers can be further functional-ized for enhanced cellular activities by incorporating

compounds or morphogens such as hydroxyapatite,9

glycosaminoglycan,10 and recombinant human bonemorphogenetic protein-2.11 Release of these com-pounds from the scaffolds can be controlled by carefulblending of different synthetic biodegradable poly-mers.12,13 By offering both topographical and biochem-ical signals, the electrospun nanofibrous scaffolds mayprovide an optimal microenvironment mimicking na-tive ECM for the seeded cells. With the development ofincreasingly complicated techniques, electrospinninghas become not only a versatile tool for fabrication ofvarious tissues, but also a valuable approach forunderstanding the complex tissue-specific microenvi-ronment for biomimetic tissue regeneration.14

Although electrospun fibers have been explored inbone tissue engineering,15–17 the application is per-haps best suited for fabrication of a multilayeredmembranous type of tissue mimicking periosteum forbone graft repair and reconstruction.18–20 The versa-tile electrospinning technique will allow creation of amultilayered membrane simulating the highly orga-nized ECM in periosteum.21,22 Combined with ade-quate progenitor cell populations and molecularsignals, this biomimetic fibrous membrane could beused as a periosteum replacement for bone defectrepair and reconstruction. To further understand thedesign specifics for this application, electrospun fibersof different diameters and orientation were createdusing a modified electrospinning technique. The im-pact of the synthetic fiber topology, specifically thefiber diameter and orientation on osteogenic differenti-ation and ECM organization, was examined usingbone marrow stromal cells (BMSCs) isolated from aGFP transgenic mouse model.

MATERIALS AND METHODSElectrospinningElectrospun fibrous scaffold was fabricated using a modi-fied magnetic-field-assisted-electrospinning procedure as

Both authors Seungyoun Lyu and Chunlan Huang contributedequally to this work.Grant sponsor: Musculoskeletal Transplant Foundation;Grant sponsor: NYSTEM; Grant numbers: N08G495, N09G346;Grant sponsor: National Institutes of Health; Grant numbers:R21 DE021513, RC1AR058435, AR051469.Correspondence to: X. Zhang (T: 585-275-7928; F: 585-275-1121;E-mail: [email protected])

# 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2013 1

described previously.23 Poly (D, L-lactic-co-glycolic acid)(PLGA) with 75:25 monomer ratios between lactic andglycolic acids were purchased from Lactel Absorbable Poly-mers (Pelham, AL). 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol(HFP) with 99 þ % purity (Alfa Aesar, Ward Hill, MA) wasused as the solvent for all polymers. Homogenous polymersolutions in HFP were prepared by stirring overnight atroom temperature. The concentrations of the polymer from10 to 20 � 100 g/ml were used. Briefly, a 10 ml syringe witha 22G1½ needle (BD, Bedford, MA), used as a solutionreservoir and a spinneret, respectively, was attached to asyringe pump (Chemyx Incorporation, Houston, TX). The tipof the needle was connected to a high-voltage power supply(Gamma High Voltage Research, Ormond Beach, FL). Theapplied voltage and flow rate of the pump were kept constantduring an electrospinning session. To minimize possibleeffect of airflow on electrospinning, the collection areawas kept inside a custom-made polycarbonate box (35 cm �35 cm � 40 cm). Nonstick aluminum foil was used to collectdeposited fibers. Needle-to-collector distance was kept at12 cm. A pair of ceramic block magnets (4.8 cm � 2.2 cm� 0.95 cm; Hillman, Cincinnati, OH) covered in aluminumfoil was used for generating aligned fibers. The pore size ofthe scaffold, as determined by analyses using Image J,ranges from 100 to 50 nm. For all in vitro analyses, a singlelayer 2 cm � 2 cm fibrous membrane was glued to thebottom of the 6-well culture plate for cell seeding.

Isolation of BMSCsBone marrow cells were isolated from 2-month-old transgenicmice engineered to express GFP ubiquitously as previouslydescribed.24 The use of a GFP transgenic mouse model allowseasy tracking of the cells using fluorescence-based microscop-ic techniques in vitro. Briefly, femora and tibiae wereremoved aseptically and dissected free of adherent softtissue. The bone ends were cut, and bone marrow cells wereflushed from the marrow cavity by injecting alpha-MEMmedium slowly at one end of the bone using a sterile 21-gauge needle. About 5 � 106 recovered bone marrow cellswere seeded on sterile electrospun fibers in 6-well plates andcultured in alpha-MEM media containing 15% fetal bovineserum (Hyclone, Laboratories, Salt Lake City, UT) for10 days. Osteogenic differentiation media containing 50 mg/ml ascorbic acid, 5 mM b-glycerophosphate, and 10% FBS inalpha-MEM was added at Day 10 and cultured for additional21 days.

Analysis of the Osteogenic Differentiation of BMSCsAn inverted fluorescence microscope (Zeiss Axiovert 40 CFL)was used to examine the attachment, growth, and differenti-ation of the murine BMSCs. To determine whether thecultured BMSCs were capable of differentiating into osteo-blastic lineage on the scaffolds, alkaline phosphatase (ALP)staining was performed on day 10, 15, and 28 followingseeding.19 Stained scaffolds at Day 10, 15, and 21 werephotographed by a digital camera mounted on a microscope(Olympus SZX12). The numbers of ALPþ colonies on thefiber mesh were recorded and manually counted. The areasof the ALPþ region were quantified using Image J.24 Todetermine osteogenic gene expression; total RNA was isolat-ed from Day 10 and 21 cultures. Quantitative real-time PCRanalyses were performed to examine RUNX2 and OSXexpression as previously described.25,26 Three separateexperiments were performed to determine osteogenic differ-entiation on different scaffolds.

Scanning Electron MicroscopeField emission scanning electron microscope (FE-SEM; CarlZeiss NTS GmbH; Model: SUPRA 40VP) was used forcharacterization and analysis of fiber morphology. All sam-ples were mounted onto imaging stubs and sputter-coatedwith gold for 40 s (Desk P, Denton Vacuum). Images weretaken at an accelerating voltage of 10 kV using the InLensmode. The specimens used for imaging the cross-sections ofelectrospun fibers were made by freezing the sample in liquidnitrogen for 5 min, followed by cutting with a sharp knife.Data are expressed as a mean value plus or minus thestandard deviation of the mean.

Analyses of the Size Distribution of Collagen Fibril/Fiber in ECMAnalyses of the thickness of collagen fibril/fiber were per-formed on seeded scaffolds at Day 21. Cells on the scaffoldwere removed by incubating a mixture of 0.5% Triton X and20 mM NH4OH for 5 min at room temperature.27 Theremaining matrix and scaffolds were mounted and imaged bySEM. Measurements of collagen fiber diameter were madeon at least five images of the ECM obtained from threesamples at 5,000� magnification. The average thickness ofthe fibers was calculated via Image J (Bethesda, MD) using amethodology described by Dougherty et al.28 The fiber sizedistribution was plotted based on raw data extracted fromImage J plug-ins. In some cases, a mean was calculatedbased on the manual measurements of at least 50 collagenfibers at the different regions of the scaffold using build-insoftware in SEM.

Multiphoton Laser Scanning Microscopy (MPLSM)A multiphoton microscope (Olympus Fluoview 1000 AOM-MPM) was used to image the cells and native collagen matrixin the scaffolds. Using the excitation wavelength 780 nm togenerate multiphoton excitation signals from GFP, andsecond harmonic generation (SHG) signals from collagen at390 nm, BMSCs and ECM were detected simultaneously oncultured cellular scaffolds. Three-dimensional images werereconstructed via Amira imaging software (VSG, Burlington,MA). The diameters of the collagen fibers were directlymeasured using Image J. The means were determined basedon at least 50 fibers at different regions of the substrate.

Statistical AnalysesData were expressed as a mean value plus or minus theSEM. Statistical significance between experimental groupswas determined using one-way analysis of variance and aTukey’s post hoc test. A p value <0.05 was consideredstatistically significant. Data analysis was performed usingGraphPad Prism version 5.0 (GraphPad Software, San Diego,CA).

RESULTSBy varying the concentration of the polymer solution,fibrous scaffolds with different fiber diameters wereobtained by electrospinning. Characterization of thefibers showed a near linear relationship between fiberdiameters and the polymer concentration at 10%, 15%,and 20%. As the concentration of the polymericsolution increased, the resulting fiber diameter in-creased for both aligned and random fibers (Fig. 1). Ata lower or higher concentration outside the range,deformation of the fiber such as beading and branch-

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JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2013

ing occurred, compromising the uniformity of the as-spun fibers. Based on the synthetic fiber diametersand orientation, four groups of fibers were selected fordetailed analyses on cellular attachment and differen-tiation: (1) aligned fibers from 10% polymer solutionwith a diameter of 404 � 107; (2) aligned fibers from20% polymer solution with a diameter of 906 � 178;(3) random fibers created from 10% polymer solutionwith a diameter of 449 � 96; and (4) random fiberfrom 20% polymer solution with a diameter of1,183 � 174. Fibers created from 15% polymer solutiondisplayed an overlapping diameter range with thosefrom both 10% to 20% solution (Fig. 1), and thereforewere excluded from the analyses.

Fluorescence imaging showed attachment of GFP-tagged BMSCs on both aligned and randomly orientedPLGA scaffolds (Fig. 2). Cells on aligned fibers showedmore elongated and spindle-shaped morphology(Fig. 2D) compared with the ones on randomly orient-ed fibers, which were more spherical (Fig. 2C). Cellson aligned fibers oriented themselves along the direc-tion of the fibers. ALP staining showed that bothaligned and randomly oriented scaffolds supportedosteogenic differentiation of BMSCs. On randomlyoriented fibrous scaffold, the ALP-positive colonieswere irregularly shaped (Fig. 2E). On aligned fibrousscaffold, ALP-positive colonies existed in strips, sug-gesting directional guidance from the electrospun

fibers (Fig. 2F). When ALP staining was performed onDay 10, more ALP-positive colonies were found on therandom fibers than the aligned fibers (Fig. 3A and B).This difference was observed regardless of the fiberdiameters. Real-time PCR analyses further showedthat at Day 10 following seeding, BMSCs exhibitedreduced expression of OSX on aligned fibers (Fig. 3D).Interestingly, after additional 11 days of culture inosteogenic medium, ALP staining was markedly in-duced in both aligned and random fiber groups(Fig. 3A). Measurements of the area of ALPþ regionsat Day 21 showed similar differentiation on all fourtypes of fibers (Fig. 3C). Quantitative real time PCRshowed marked induction of OSX and RUNX2 expres-sion in day 21 cultures as compared to day 10 (Fig. 3Dand E), with aligned fibers exhibiting higher expres-sion of OSX and RUNX2 (p < 0.05). All four types offibers within a diameter range of 300–1,300 nm sup-ported osteogenic differentiation of BMSCs.

High-resolution analyses using SEM revealed de-tailed interactions between BMSCs and the electro-spun fibers during cellular differentiation. On Day 8,BMSCs showed attachment and growth into thefibrous PLGA scaffold (Fig. 4A). By Day 21, BMSCswere found to be embedded within cell-derived ECM

Figure 1. (A–G) Representative SEM images showing random-ly oriented (A–C) and aligned (D–F) electrospun fibers fabricatedat a concentration of 10% (A, D), 15% (B, E), and 20% (C, F)75:25 PLGA. The means of the fiber diameter are directlycorrelated with the indicated concentrations of polymer (w/v) forboth aligned and random fibers (G).

Figure 2. (A–F) SEM analyses show distinctive topology ofrandom (A, 1,000�) or aligned (B, 1,000�) PLGA fibers. Fluores-cence images of GFP-tagged murine bone marrow stromal cellscultured on both types of fibers show distinctive morphology.Cells on randomly fibers adopted a rounder morphology (C, 25�)whereas cells on aligned fibers were elongated (D, 25�). ALP-positive colonies on scaffolds at day 10 displayed a similarmorphology on random (E, 4�) and aligned fibers (F, 4�).

ELECTROSPUN FIBERS FOR BONE REPAIR AND RECONSTRUCTION 3


that covered the entire scaffold (Fig. 4B). On randomlyreoriented scaffold, collagen fibers were seen extend-ing from all directions from the polygonal cells(Fig. 4B–C). These fibers were formed through bun-dling of the thin collagen fibrils. Examination of thesethin collagen fibrils at a higher magnification showedcharacteristic banded repeats with typical uniformity(Fig. 4D).29 Measurement of the collagen fibrilsshowed a narrow diameter distribution with a meanof 41 and a standard deviation of 8 nm. Followingdecellularization to remove cells, SEM showed thatthe scaffold was covered with native collagen matrixproduced by BMSCs (Fig. 4E–H). The orientation ofthe large collagen fibers or collagen bundles wereorganized according to the alignment of the electro-spun polymeric fibers. The collagen matrix adopted aswirling morphology on randomly oriented polymericfibers (Fig. 4E–F). On the aligned polymeric fibers, thecollagen fibers were organized along the longitudinaldirection of the polymeric fibers (Fig. 4G–H). Thincollagen fibrils appeared to be woven in between thelarge collagen fibers/bundles. Analyses of the thicknessof collagen fibers in BMSC cultures showed a broadrange of distribution from 35 to 1,000 nm (Fig. 4I).

Image analyses from three samples revealed anaverage diameter of 143 nm.

MPLSM analyses in the living cultures of BMSCs(Fig. 5) showed that cells (shown as green in Fig. 5A,B, E, and F) occupied the synthetic fiber network andconstructed their own collagen matrix in the surround-ings (shown as cyan in Fig. 5C, D, G, and H). Themajority of the cells were spread and differentiated onthe surface of the fibrous scaffold (Fig. 5A and B).Infiltration of cells into the deeper region of thepolymeric fibers can be found in both random andaligned fibrous meshes at 50 mm depth (Fig. 5E andF). Consistently with the previous finding in SEM,BMSCs and collagen matrix were arranged accordingto the orientation and alignment of the electrospunfibers (Fig. 5A, C, and E, G vs. B, D, F, and H). On therandom fibers, BMSCs formed “honeycomb”-like ma-trix with cells dwelling inside (Fig. 5C and G). Incultures on the aligned fibers, cells as well as thematrix were organized into strips (Fig. 5D and H).Analyses of the collagen fiber diameter using image Jshowed the coexistence of both high-contrast nano-and micro-scaled collagen fibers produced fromBMSCs. Measurements of the distinctive thicker fibers

Figure 3. (A–E) Alkaline phosphatase staining were performed on random and aligned fibers on days 10, 15, and 21 as indicated (A).Photographs of the colony formation on fibrous meshes were taken at a magnification of 1� via a dissection microscope. Numbers of theALPþ colony were quantified at day 10 (B) and areas of the ALPþ region were quantified at day 21 (C). Real time PCR analyses showosteogenic marker gene OSX (D) and RUNX2 (E) expressions at Day 10 and Day 21. � Indicates p < 0.05.

4 LYU ET AL.


showed a mean diameter of 1.786 � 0.6 mm (n ¼ 85).These thicker fibers formed an extended nest sur-rounding the cells. The decellularization procedureallowed removal of the cells without disruption of theoverall morphology of the ECM produced from BMSCsin both random and aligned fibers (Fig. 5G and H).

DISCUSSIONWe have previously demonstrated a tissue-engineeringapproach that combines structural bone graft, genes,and BMSCs in a murine model to repair a largesegmental defect.18,19,30 A major limitation to thetranslation of this approach is the lack of a cellularosteoinductive scaffold that could function as perioste-um to fit around a bone of any size or shape. Electro-spun nanofibers have offered a versatile technique forfabrication of a biomimetic scaffold best suited forsuch an application. To provide further rationales forthe use of electrospun fibers as a scaffolding platformfor fabrication of a periosteum replacement, we per-formed a series of in vitro experiments to determinethe impact of fiber diameter and orientation on cellu-lar differentiation and ECM organization of BMSCs.Our studies showed that the orientation of the fibers

had a significant impact on the differentiation ofBMSCs and the organization of ECM. Using orientedelectrospun fibers could provide significant advantagesfor guided bone tissue repair and reconstruction.

While the electrospun fibrous scaffolds have beenshown to support cellular attachment, proliferation,and differentiation in a variety of cell types, theimpact of fiber diameter and fiber orientation onosteogenic differentiation of BMSCs has not been wellcharacterized. Careful examination of the literatureshows contradictory results or ambiguous descriptionsin regards to the effects of alignment and fiberdiameter on the differentiation of BMSCs.31–36 Anearlier report using MC3T3-E1 osteoblast cell lineshows that the fiber diameter affects cellular densityat the early stage of osteoblastic differentiation.35

Following addition of osteogenic differentiation media,cell density and differentiation become comparable infibers ranging from 0.5–2 mm. Other reports usingvarious polymeric materials show conflicting results incultures.31–35 In our current study, in which freshlyisolated bone marrow stromal cell cultures were used,we found that fiber diameter at a range of 300–1,200 nm had a minimal effect on differentiation of

Figure 4. (A–H) Representative SEM images (1,000�) of bone marrow stromal cells cultured on randomly oriented electrospun fiberson Day 8 (A) and Day 21 (B). Higher magnification image (10,000�) shows detailed morphology of and nano and micron-sized collagenmatrix with polygonal cells (C). Characteristic collagen fibrils and collagen bundles (arrows) are shown at 30,000� (D). Followingremoval of the cells (decellularization), the native collagen matrix produced by BMSCs cultured on randomly oriented (E, F) or analigned fibers (G, H) is shown at 1,000� and 5,000�.



BMSCs. However, at the same diameter range, fiberorientation significantly impacted the early behaviorand differentiation of BMSCs. Cells cultured onaligned fibers had a reduced number of ALPþ colonyat Day 10, indicating a slight delay in early osteoblas-tic differentiation. However, this negative effect ondifferentiation could be overcome by the addition ofosteogenic differentiation media containing ascorbicacid and b-glycerophosphate, such that by Day 21 thecells on aligned fibers exhibited higher levels ofRUNX2 and OSX gene expression. These results showthat the aligned fibers of nano- and sub-micron sizecould be used as a suitable scaffolding platform tosupport the osteogenic differentiation of BMSCs.

Two complementary imaging technologies wereused to analyze ECM deposition on the electrospunfibers. While both SEM and MPLSM allow analyses athigh resolution, SEM only permits high resolutionimaging of the surfaces. In comparison, MPLSM offersa significant advantage in analyzing fluorescence

labeled cells throughout the fiber mesh in livingcultures. MPLSM further allows visualization of colla-gen-rich ECM via Second Harmonic Generation(SHG). SHG is produced in the noncentrosymmetricbiomolecules such as collagen fibrils or collagen fiberin ECM. SHG is enhanced when disordered collagentriple helices are converted into ordered fibrils andfurther compacted into collagen fibers.37–40 SHG mi-croscopy has been widely used for visualization andassessment of tissue structure in wound healing,malignancy, and development.41–43 As an intrinsicsignal from the collagen, measurement of SHG over-comes variability and uncertainty that are oftenassociated with immunohistochemical staining.39,40

The analyses of the native ECM showed that theECM deposited on the fibrous scaffolds was organizedlargely according to the orientation of the polymericfibers.44–51 This important feature could be used toproduce highly organized ECM of the native tissues,such as periosteum during healing. Although thedetailed microstructure of periosteum remains elusive,early study of periosteum in chick calvaria shows thatperiosteum explant forms densely packed, highly ori-ented, crossbanded collagen fibrils prior to mineraliza-tion,22 suggesting highly organized collagen matrixformation prior to bone formation. A recent study fromFoolen et al.21 further shows that collagen orientationin periosteum and perichondrium is aligned withpreferential directions of tissue growth. Our unpub-lished data indicated that the collagen fiber arrange-ment at the cortical bone gap consisted ofmultilayered, crisscrossing, and highly oriented colla-gen bundles. The unique arrangement of the collagenfibers at the site of repair suggests the necessaryguidance cues for directional growth and migration ofprogenitor cells during repair and reconstruction. Insupport of this notion, several recent studies indicatethat the spatially oriented electrospun fibers couldserve as a guidance substrate for wound repair. MSCsmigrate faster along oriented fibers than non-orientedcounterparts.52,53 Thus, from a biomimetic standpoint,electrospun fiber with its versatility in producingpatterned ECM could be used as a bonding matrixthat bridges damaged bone tissues, providing neces-sary cues for guided bone tissue repair and reconstruc-tion.

While submicron to micro-sized polymeric fiberswere shown to effectively support BMSC growth anddifferentiation, analyses of the native ECM/collagenmatrix indicated that the majority of collagen fibers(74%) deposited by BMSCs had a diameter range of35–200 nm, significantly smaller than polymeric fibersgenerated via electrospinning. Spun fibers in nanome-ter scale could be produced using our current method.However, as previously reported the uniformity offibers in the order of 50–200 nm were often compro-mised due to beading and branching.54 To overcomethis, we demonstrated an ECM-coated spun fiber-based scaffold created by removal of the cells from the

Figure 5. (A–D) Fluorescent excitation and SHG of live GFP-positive cells cultured on random or aligned fibers were imagedvia MPLSM at Day 21. Cells are shown as green and collagenmatrix produced by these cells shown as cyan. Distinctivecellular morphology and collagen matrix organization are shownin randomly oriented (A, C, E, and G) and aligned (B, D, F, andH) fibers. Multiphoton microscopy further allows optical section-ing of the live specimens at a depth of 5 mm (A–D, surface layer)and 50 mm (E and F, deeper layer). G and H show collagenmatrix in random or aligned decellularized electrospun fibers.

6 LYU ET AL.


seeded fiber meshes (Fig. 4E–H). These decellularizedmatrices maintained the spatial orientation of thesynthetic fibers with newly deposited collagen fibersorganized according to the instructive cues of thesynthetic fibers. These ECM coated polymeric fiberscombine the benefits of ECM, such as the rich contentof growth factors for cell proliferation and differentia-tion,27,55,56 and the direction guidance of alignedsynthetic fibers, therefore could provide advantagesfor progenitor cell migration, proliferation, and differ-entiation in a more suitable bone healing microenvi-ronment. The efficacy of these native ECM-coatedelectrospun fibrous scaffolds for repair and reconstruc-tion is currently under investigation.

Although electrospinning still faces issues such ascontrol of porous structure, cell infiltration, and fiberdegradation, its versatility and simplicity in producingpatterned and oriented nano and submicron-sizedfibers has provided distinctive advantages in scaffolddesign and fabrication. Many recent advances suchas co-axial or core-shell electrospinning, two-phaseelectrospinning, simultaneous cell-spraying, and elec-trospinning of nanoparticle-incorporated polymerssuggest that there is a tremendous potential for thistechnique in tissue engineering.57–62 With such meth-ods as blowing-assisted or multi-jet electrospinning, alarge industry-scale production of electrospun fibersfor clinical use is also conceivable.63

In summary, using a versatile electrospinning tech-nique that allows fabrication of polymeric fibers withcontrolled uniformity and alignment, in this study, weexamined the impact of fiber diameter and orientationon BMSC differentiation and ECM deposition in vitro.Our data demonstrated an advantage of using orientedand organized nano- and submicron-fibers as a scaf-folding platform for engineering periosteum mimeticsfor bone tissue repair and reconstruction.

ACKNOWLEDGMENTSThis study is supported by grants from the MusculoskeletalTransplant Foundation (XPZ), NYSTEM N08G-495 (XPZ)and N09G346 (XPZ), and the National Institutes of Health(R21 DE021513 to XPZ, RC1AR058435 to XPZ, AR051469 toXPZ). We thank Ming Xue for her assistance in isolation ofbone marrow stromal cells from GFP transgenic mice.

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8 LYU ET AL.

electrospun fibers as a scaffolding platform for bone tissue repair

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