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Biomaterials 30 (2009) 4309–4317

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Three-dimensional reconstituted extracellular matrix scaffoldsfor tissue engineering

Karthikeyan Narayanan 1, Kwong-Joo Leck 1, Shujun Gao, Andrew C.A. Wan*

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669

a r t i c l e i n f o

Article history:Received 24 February 2009Accepted 28 April 2009Available online 28 May 2009

Keywords:Extracellular matrixScaffoldStem cellOsteogenesis

* Corresponding author. Tel.: þ65 6824 7134; fax: þE-mail address: awan@ibn.a-star.edu.sg (A.C.A. Wa

1 Contributed equally to this work.

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.04.049

a b s t r a c t

The extracellular matrix (ECM) is a rich meshwork of proteins and proteoglycans. Besides assuming a celladhesive and structural support role, the ECM also helps to sequester and present growth factors to cells.ECM derived from tissues has been used as biological scaffolds for tissue engineering. In contrast, it hasbeen difficult to employ ECM derived from cell lines as scaffolds due to its lack of form and structure. Wehave developed a mild, aqueous-based method for incorporating cell line derived ECM into biologicalscaffolds based on polyelectrolyte complexation, using the example of ECM from MC-3T3, a mousepreosteoblast cell line. A DNase step was incorporated in the ECM isolation procedure to further purify itof genetic material. Immunohistochemistry of fibers incorporated with MC-3T3 ECM reveal the presenceof the ECM components, collagen type I, collagen type IV, fibronectin and heparan sulfate, on theirsurface. Reconstituted ECM scaffolds retained the cell-adhesion characteristics of the ECM, as demon-strated by ‘reseeding’ the ECM-secreting cell on the scaffolds. Human mesenchymal stem cells (hMSCs)were seeded onto the fibrous scaffolds incorporated with MC-3T3 ECM, and implanted subcutaneouslyinto SCID mice. After 4 weeks of implantation, histological evidence showed that the hMSC seeded ECMscaffolds had induced bone formation at the ectopic site.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

For both tissue development and regeneration, a myriad offactors are essential for the growth and differentiation of cells toform tissues. These factors must be presented on the biomaterialmatrices or scaffolds that are employed for tissue engineering [1],in a manner whereby they are accessible to the cells. Due to theprohibitive cost of recombinant factors and our incompleteknowledge of the factors involved in tissue regeneration, strategiesfor immobilizing bioactive ligands on the scaffold or deliveringbiomolecules in a sustained fashion from the scaffolds invariablyfocus on a minute fraction of the total spectrum of biologicalactivity that a scaffold can potentially be endowed with.

Extracellular matrices (ECM) are a rich source of bioactivemolecules. They can be extracted from animal tissues, and havebeen used directly as scaffolds for tissue engineering [2–8].However, the size, shape and configuration of the scaffold arelimited by the dimensions and form of the original tissue.Besides animal tissues, ECM can also be extracted from cell lines[9,10]. The use of cell lines offers several advantages compared to

65 6478 9081.n).

All rights reserved.

animal tissues. Firstly, most cell lines can be expanded indefi-nitely and relatively cheaply in the laboratory. Secondly, celllines can be screened for pathogens and viruses, and maintainedin a pathogen-free condition for ECM harvesting. Thirdly, the useof cell lines provides the flexibility of mixing ECM harvestedfrom different cell types in any desired proportion. However,there is currently no convenient way of presenting cell linederived ECM on biological scaffolds to provide it with structureand form.

In this work, ECM was isolated from cells grown in cultureand reconstructed into fibrous scaffolds by means of interfacialpolyelectrolyte complexation [11,12]. Working on the culture ofMC-3T3, the ECM isolation procedure was optimized, especiallywith respect to the use of DNase to purify it of genetic material.The availability of ECM components such as fibronectin,collagen and heparan sulfate proteoglycan on the surface of thefibers was demonstrated by immunofluorescence staining.Retention of the cell-adhesive characteristics of the ECM wasshown by culturing MC-3T3 cells on their reconstituted ECM.To demonstrate that these ECM signals could act as a source ofdifferentiation cues to differentiate stem cells into a chosenlineage (i.e. bone), we cultured hMSCs on the MC-3T3 ECMincorporated scaffolds, and implanted them subcutaneouslyinto SCID mice.

K. Narayanan et al. / Biomaterials 30 (2009) 4309–43174310

2. Materials and methods

All chemicals were obtained from Sigma–Aldrich (St. Louis, MO) unless other-wise specified. Cell lines were obtained from American Type Culture Collection(Manassas, VA).

2.1. Isolation of ECM

MC-3T3 was seeded at a density of 1.5�104 cells/cm2, and grown for 1 weekwith one change of medium in alpha Minimum Essential Medium (MEM) (supple-mented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S)). Theprocedure of ECM isolation was modified from that of Hedman et al. [9]. To isolatethe ECM, the medium was slowly aspirated from the tissue culture dish and washedtwice with phosphate buffered saline (PBS). 1 mL of Solution A (1 mM phenyl-methanesulfonyl fluoride (PMSF), 10 mM tris(hydroxymethyl)aminomethanehydrochloride (TRIS) (Merck), pH 8, 0.5% sodium deoxycholate) was applied to each100-mm dish for 1 min. Following the removal of Solution A, each dish was washedwith 1 mL of PBS. Next, 1 mL of deionized (DI) water was forcefully squirted onto thebottom of the petri dish to detach the ECM. The suspension was transferred intoseparate vials and centrifuged at 5220g at 4 �C for 5 min. The supernatant wasremoved, after which 1 mL of Solution B (10 mM magnesium chloride, 1 mM calciumchloride, 1 mM PMSF) was added. The ECM was then dispersed by vortexing, andcollected at the bottom of the vial. The vials were then placed on a Heidolph-Unimaxshaker for 30 min at an agitation rate of 250 rpm. They were centrifuged at 5220gand 4 �C for 5 min. The supernatant was removed, and the ECM pellet was washedwith DI water by dispersion and centrifugation. Alternatively, suspensions wereconsolidated and transferred to an Amicon Ultra Centrifugal Filter device (Millipore)and centrifuged at 110g at 4 �C (for 1 h for every 1.5 mL of solution). The solid ECMwas resuspended (by brief vortexing) and stored in DI water prior to use.

2.2. DNase treatment study

Cells were cultured on 24-well plates. For ECM isolation, reagent volumes werescaled down as follows: Solution A, 200 mL; PBS, 300 mL; DI water, 200 mL. After ECMextraction, the ECM suspension was centrifuged at 5220g for 5 min at 4 �C. Thesupernatant was removed and kept for determination of baseline optical density.Next, 200 mL of Solution B containing varying amounts of DNase I (bovine pancreas)was added to the ECM pellet, which was dispersed by vortexing. The suspension wasallowed to stand for 30 min at 37 �C. This was followed by another centrifugation at5220g and 4 �C for 5 min. The supernatant was again removed, and used for opticaldensity readings at 260 nm, 280 nm and 320 nm on a UV spectrophotometer, whichcorresponded to measurements at DNA, protein and the reference wavelengths,respectively.

2.3. Preparation of MC-3T3 ECM reconstituted scaffolds

To prepare the polycation precursor, tetraethylorthosilicate (TEOS) was firsthydrolyzed by mixing TEOS and 0.15 M acetic acid at a volume ratio of 1:9, andvortexed until a homogenous solution was obtained. Hydrolyzed TEOS was thenadded to a 0.5 w/v% chitosan (Aldrich, high viscosity) solution in 1 vol% acetic acid ata volume fraction of 25%. To prepare the polyanion precursor, a suspension of MC-3T3 ECM in water (from 100-mm dish) was centrifuged to obtain a pellet, which wastypically redispersed in 100 mL of a 1 w/v% sodium alginate solution (Sigma, lowmolecular weight) by tituration and vortexing.

30 mL of the polycation (chitosan) and 20 mL of the polyanion (alginate)precursors were placed close to but not touching each other in 3-mm poly(tetra-fluoroethylene) (PTFE) channels. A pair of forceps was used to bring the droplets incontact, and an upward motion was applied to form fiber. The nascent fiber wasadhered to the rotating arms of a roll-up apparatus, and the fiber was drawncontinuously until the polyelectrolyte solutions were depleted and/or fiber termi-nation occurred. Four or five of such polyelectrolyte pairs were simultaneouslydrawn to form each ECM scaffold. The dry fibers obtained from the roll-up apparatuswere transferred to microcentrifuge tubes and weighed. Approximately 1.5 mL of DIwater was then added to wash the fibers for 5 min. The washed fibers were thentransferred onto a frit in a die, and a stream of DI water was passed through the dieat a flow rate of 300–350 mL/min for 1 min to entangle the fibers. The water flowrate was then reduced to 5–35 mL/min, and the entangled fibers (scaffolds) werewashed for another 5 min. For sterilization, the scaffolds were typically immersed in70% ethanol in a 96-well plate for at least 30 min, followed by ethanol removal andultraviolet irradiation for 45–60 min.

2.4. Characterization of MC-3T3 ECM reconstituted scaffold

The morphologies of the fibers with and without ECM incorporated weredetermined using a JEOL JSM-5600 scanning electron microscope equipped with anOxford Instruments energy dispersive X-ray (EDX) analysis system. The fibers weregold-coated for imaging using a JEOL JFC-1200 Fine Coater with a sputter time of18 s, and the samples were imaged under high vacuum at an accelerating voltage of10 kV.

Immunofluorescence staining of the ECM components was performed usingprimary antibodies against heparan sulfate proteoglycan (Abcam PLC, UK), fibro-nectin, collagen Type I and collagen Type IV (Acris Antibodies, GmbH). This wasfollowed by secondary staining with the appropriate FITC-labeled secondary anti-bodies. To characterize adhesion and viability of MC-3T3 cells on the reconstitutedECM scaffolds, the LIVE/DEAD� viability and cytotoxicity kit (L-3224, MolecularProbes) was used according to the manufacturer’s instructions. This two color-assayidentifies live versus dead cells on the basis of intracellular esterase activity thatconverts non-fluorescent calcein AM to fluorescent calcein (green) and loss ofmembrane integrity leading to nuclear staining by ethidium homodimer-1 (red),respectively. Confocal microscopy was performed on an Olympus Fluoview 300confocal unit with a 488-nm laser. Green fluorescence was observed using a 510-nmlong pass filter and a 530-nm short pass filter.

2.5. Subcutaneous implantation of hMSC-seeded MC-3T3 ECM scaffoldinto SCID mice

Four scaffolds of each type were used in the in vivo experiments. Scaffolds wererinsed thoroughly in PBS prior to use. 1�106 of hMSCs (P4) were seeded on thefibrous scaffolds (with and without MC3T3 ECM incorporated), and cultured for 2days with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 2 mMglutamine and 1% penicillin–streptomycin. The scaffolds were rinsed with PBS priorto subcutaneous implantation into SCID mice. After 2 weeks and 4 weeks postimplantation, the scaffolds were harvested and fixed with 10% formalin. Paraffin-embedded sections (5 mm) were made and stained for histological evaluation. Allanimal experiments were performed at the satellite animal husbandry unit atNational University of Singapore and approved by their Institutional Care and UseCommittee.

2.6. Histological staining

For hematoxylin and eosin (H&E) staining, the sections were deparaffinized andhydrated in distilled water. The sections were stained in Harris hematoxylin solutionfor 8 min, followed by counterstaining in eosin-y solution for 1 min. The sectionswere dehydrated and mounted with xylene-based mounting medium. For Masson’strichrome staining, the sections were deparaffinized and hydrated in distilled water,mordant in Bouin’s solution for 1 h at 56 �C, and stained with phosphomolybdic–phosphotungstic acid for 10 min and aniline blue solution for 5 min. This was fol-lowed by counterstaining with hematoxylin solution. For von Kossa staining, thesections were washed with distilled water, followed by 1 wt% AgNO3 for 1 h. Theslides were washed with distilled water, and treated with 2.5 wt% sodium thiosul-fate for 5 min. The specimens were counterstained and then examined under a lightmicroscope.

2.7. Immunostaining with human nuclear antigen

Paraffin sections were deparaffinized in xylene, rehydrated, and rinsed threetimes (5 min each) in PBS (pH7.4). Antigen epitopes were retrieved using sodiumcitrate buffer, pH 6.0 using a microwave oven. Blocking of non-specific binding siteswas carried out using 10% normal goat serum for 45 min at room temperature. Thesections were again rinsed in PBS solution and incubated overnight at 4 �C withprimary antibodies against human nuclear antigen (Millipore, cat # MAB1281).Following incubation, the sections were washed thrice with PBS. An appropriatefluorescently labeled secondary antibody was incubated with the sections for60 min at room temperature. Upon washing with PBS the sections were observedunder a fluorescent microscope. Images were processed with Image J (NIH) software.

2.8. Immunostaining with STRO-1

Fibers with ECM were seeded with hMSC cells and allowed to adhere overnightat 37 �C, in a CO2 incubator. Upon washing with PBS (twice), the fibers were fixedwith 4% paraformaldehyde, the cells were permeabilized with PBS containing 0.1%Triton X-100 (PBST). Non-specific binding sites were blocked using 5% BSA in PBSTfor 60 min. The cells were incubated overnight at 4 �C with antibody against STRO-1(R&D systems). Subsequently the samples were washed and incubated withappropriate secondary antibody. The fibers were washed extensively and observedunder a fluorescent microscope. Images were processed with Image J (NIH) software.

3. Results

3.1. Extracellular matrix isolation

The procedure for ECM isolation was based on the earlier workof Hedman et al. [9]. In the current work, a modified procedure wasemployed for the isolation of ECM from MC-3T3. In particular, theoriginal procedure did not employ DNase to remove nucleic acidsfrom the ECM, whereas we introduced the additional step of DNase

0

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200 220 240 260 280 300 320 340Wavelength (nm)

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Fig. 1. Ultraviolet–visible spectra of supernatant before treatment (d). Ultravioletspectrum of the supernatant after sequential treatment with solutions of BSA (- - - -)and DNase ( ). BSA, at the same concentration as DNAse, was used as the control.

Fig. 3. Fluorescent microscope images of extracted ECM at various stages of the extractionand (c) are fluorescent micrographs while (d), (e) and (f) are the superimposition of fluore

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Fig. 2. Mass of nucleic acid extracted into 200 mL of Solution B containing differentquantities of DNase I.

K. Narayanan et al. / Biomaterials 30 (2009) 4309–4317 4311

treatment due to consideration of the potential immunogenicity ofgenetic material in tissue engineering applications. When the ECMsample was treated with solutions of bovine serum albumin (BSA)and DNase in sequence, UV spectrophotometry of the collectedsupernatants showed a significant absorbance at 260 nm whenDNase was applied, demonstrating its effectiveness in removinggenetic material (Fig. 1). The quantity of nucleic acid extracted intothe supernatant using different amounts of DNAse was determinedusing the 260-nm wavelength. From studies employing a 24-wellplate, the optimal quantity was found to be 4 mg DNase per2.27 cm2 of confluent MC-3T3 cells (Fig. 2). This optimized quantitycould be scaled up on the basis of cell number (vessel area) whenlarger plates were used for culturing.

To investigate the effectiveness of the ECM isolation protocol inremoving DNA, fluorescent micrographs of the extracted ECM wereobtained at various stages of the extraction procedure, as shown inFig. 3. Fibrous DNA was clearly evident for the ECM obtained aftertreatment with Solution A (sodium deoxycholate solution, Fig. 3aand d), and persisted even after washing with Solution B (high ionicstrength solution containing calcium and magnesium salts, Fig. 3band e) However, application of DNAse I had dramatically brokendown the fibrous structure of the DNA and removed most of it fromthe ECM. (Fig. 3c and f) While slight quantities of DNA were stillpresent, this residual DNA could be exhaustively removed byrepeating the DNAse treatment. The compositional changes of theECM were manifested in its outward appearance; while the ECMproducts obtained after treatment with Solutions A and B werefibrous in nature, a particulate ECM resulted after treatment withDNAse I.

3.2. Incorporation of ECM into polyelectrolyte complex fibers

ECM was reconstituted into the scaffolds based on the process ofinterfacial polyelectrolyte complexation between sodium alginateand chitosan. Scanning electron microscopy (SEM) of fibers spunfrom chitosan and the ECM–alginate mixture demonstrated thesuccessful incorporation of ECM. Fig. 4 illustrates ECM’s presence asa smooth, pasty material on an otherwise microgrooved fibersurface. A significant portion of the fiber surface area was covered

procedure-after treatment with: Solution A (a,d); Solution B (b,e); DNase I (c,f). (a), (b)scent and bright field images.

Fig. 4. SEM micrographs of chitosan–alginate scaffolds (a) with and (b) without MC-3T3 ECM.

K. Narayanan et al. / Biomaterials 30 (2009) 4309–43174312

by ECM. Immunofluorescence staining of the reconstituted ECMscaffold was performed by using antibodies against fibronectin,collagen and heparan sulfate proteoglycan. The three majorcomponents of the osteoblast ECM [13] were all shown to bepresent on the surface of the fiber (Fig. 5), which would render thefiber biologically active.

3.3. In vivo and in vitro studies of ECM-reconstituted scaffolds

Fig. 6 shows the confocal micrographs of MC-3T3 cells grown onscaffolds of reconstituted MC-3T3 ECM, compared to those grownon scaffolds without ECM. Cells growing on the ECM scaffolds wereable to spread out on the fibers, while cells growing on the scaffoldswithout ECM were spherical and clustered. Cell attachment onthese scaffolds was likely to be mediated by the ECM molecules,collagen and fibronectin, both of which contained cell adhesionsequences such as RGD that would mediate cell adhesion.

Fig. 5. Immunohistochemistry of fibers incorporated with ECM from MC-3T3, demonstratinand (d) collagen Type IV.

hMSCs were cultured on MC-3T3 ECM reconstituted scaffoldsfor a period of 2 days in vitro. As with the case of the MC-3T3 cells,hMSCs attached well to the fibers. The hMSCs were shown toexhibit their normal undifferentiated phenotype 24 h after seeding,as ascertained by the expression of STRO-1. (Fig. 7) The cell–scaffoldconstruct was implanted subcutaneously into SCID mice. Chitosan–alginate scaffolds without ECM were implanted as controls. Ratswere sacrificed after 2 and 4 weeks, and histological sections of theimplant site were obtained and stained with H&E, Masson’s tri-chrome and von Kossa. The results point towards progressiveectopic bone formation and mineralization for the hMSC-seeded,ECM-reconstituted scaffolds.

Histological staining of the explant cross sections for the hMSC-seeded, ECM-reconstituted scaffolds (Fig. 8e–h) showed a markeddifference from the hMSC-seeded control scaffolds (Fig. 8a–d) interms of bone mineralization. In comparing the 2-week and 4-weektime points for the hMSC-seeded, ECM-reconstituted scaffolds,Masson’s Trichrome staining showed progressive osteoid formation

g the presence of (a) collagen Type I, (b) fibronectin, (c) heparan sulfate proteoglycans,

Fig. 6. MC-3T3 cells grown on chitosan–alginate scaffolds (a) with and (b) without reconstituted MC-3T3 ECM.

K. Narayanan et al. / Biomaterials 30 (2009) 4309–4317 4313

(blue stain for collagen of the osteoid), while the von Kossa stainingfor calcium indicated the process of bone mineralization. From thehigher magnification image, cells could be seen lining the fibers atthe 2-week time point. (Fig. 9a) Human nuclear antigen stainingrevealed that virtually all these cells were derived from hMSCs andnot recruited from the host. (Fig. 9b) At 4 weeks, the fibers hadresorbed completely, and annular deposits could be observed in thefiber cavity. (Fig. 9c) These deposits were most likely hydroxypatite[14]. The process of ECM scaffold resorption was evident from theH&E stained and autofluorescence image of the explanted hMSC-seeded, ECM-reconstituted scaffolds at the 4-week time point(Fig. 10). While fibers remained intact for the control scaffold(Fig. 10a and b), they have virtually disappeared in the case of theECM scaffold (Fig. 10c and d).

4. Discussion

Decellularization of tissue has been widely shown to be anattractive method of obtaining scaffolds for tissue engineering. Theadvantage of this method relies on the provision of signals bythe decellularized matrix (ECM), to differentiate, or maintain the

Fig. 7. Mesenchymal stem cells seeded on MC-3T3 ECM scaffold and stained with fluorescebright field images.

differentiated phenotype of seeded cells. In addition, the scaffoldserves as the template structure for the tissue or organ to beregenerated. Thus, neonatal cardiac cells, seeded onto decellular-ized rat cadaveric hearts developed substantial pump function [8],while a tissue engineered blood vessel obtained by culturingendothelial and smooth muscle cells on a decellularized porcinevascular scaffold remained patent after 6 months [15]. The otheruntapped source of ECM would be cells that are grown in tissueculture. Two aspects of reconstituting ECM from cell lines war-ranted our attention, namely, the isolation of the ECM and thereconstitution of the isolated ECM into structured scaffolds.

While protocols to isolate ECM from cell culture have beenreported in literature, to our best knowledge, cell culture-derivedECM has only been used for in vitro applications [9,10,16]. As ourscaffolds are meant for in vivo tissue engineering, our isolationprocedure called for a balance between complete removal of cellularand genetic material, and the preservation of protein activity in thereconstituted scaffold. Therefore, our selected procedure for decel-lularization involved the simple and relatively mild treatment ofa confluent cell monolayer with an ionic solution of sodiumdeoxycholate in the presence of a protease inhibitor. In contrast,

nt STRO-1 antibody: (a) co-staining with DAPI; (b) superimposition of fluorescent and

Fig. 8. (a,c,e,g) Masson’s trichrome and (b,d,f,h) von Kossa staining of hMSC-seeded chitosan–alginate scaffolds (a–d) without and (e–h) with reconstituted MC-3T3 ECM, (a,b,e,f) 2weeks and (c,d,g,h) 4 weeks after subcutaneous implantation in SCID mice.

K. Narayanan et al. / Biomaterials 30 (2009) 4309–43174314

Fig. 9. Mineralization of hMSC-seeded, MC-3T3 ECM-reconstituted scaffold. After 2 weeks of subcutaneous implantation, (a) Von Kossa staining, and (b) anti-human nuclearantigen staining of explants. (c) After 4 weeks in vivo, numerous annular deposits are present in the cavities due to the resorbed fibers (white arrows).

K. Narayanan et al. / Biomaterials 30 (2009) 4309–4317 4315

methods of obtaining decellularized scaffolds from naturallyderived tissue often consisted of a lengthy incubation with enzymes(e.g. trypsin, dispase), which would result in a disruption of collagenstructure to some extent [17]. Furthermore, the need to removeinorganic components from hard tissues such as bone would requireexposure of the ECM to acidic conditions, which might be detri-mental to the proteinaceous component of the ECM [18]. Theprotocol in this paper had been optimized with respect tothe duration of exposure to sodium deoxycholate solution and thesolution volume, since these parameters affected the removal of thecellular fraction. Over-exposure to the deoxycholate solution

Fig. 10. (a,c) H&E staining and (b,d) autofluorescence of hMSC-seeded chitosan–alginatedegradation of the ECM scaffold 4 weeks after subcutaneous implantation in SCID mice.

resulted in poor yield, whereas under-exposure led to cellularresidue in the isolated material. Once the cell culture had beendecellularized, we implemented an additional step of DNase treat-ment to remove undesirable DNA residue in the isolated ECM.Removal of genetic material was demonstrated by both spectro-photometry (extraction of DNA into the supernatant) as well as DAPIstaining of the ECM extract.

Further changes to the ultrastructure of the ECM are expected tohave occurred during the reconstitution process, namely changes inmatrix stiffness and nanotopography, factors that have been shownto influence stem cell differentiation [19–21]. Matrix stiffness has

scaffolds (a,b) without and (c,d) with reconstituted MC-3T3 ECM, demonstrating

K. Narayanan et al. / Biomaterials 30 (2009) 4309–43174316

been shown to direct lineage specification of mesenchymal stemcells [19], while Rowlands et al. have shown an interplay of thestiffness factor and the specific matrix protein in the determinationof stem cell fate [20]. Stem cells are thus differentiated followingboth mechanical and biochemical cues. By isolating the ECM,dispersing and reconstituting it into a 3D fibrous scaffold, the matrixstiffness of the resulting scaffold would be a composite of thestiffness of the original ECM and that of the polyelectrolyte complex(chitosan–alginate) and that would be expected to somewhatmodify the lineage-specific differentiation of the stem cells. Apossible method to ensure that the mechanical signal remainsunchanged would be to modify the fiber to achieve a stiffnessmatching that of the ECM concerned.

While reconstitution of the ECM would also affect the surfaceligand density and other nanotopographical aspects that would inturn affect cell phenotype, these effects remain unknown as yet. Inpart, this is because the microstructure of the native ECM itself islargely undetermined. Clearly, more studies should be focusedtowards elucidating differences in the surface architecture of nativeversus reconstituted ECM and the corresponding influence on stemcell differentiation.

Interfacial polyelectrolyte complexation (IPC) has been estab-lished as a good method for the encapsulation of biological mate-rials. In this process, a polyanion is combined with a polycation toobtain a fiber by a self-assembly phenomenon [11,12]. Due to themild, aqueous-based nature of the process, proteins, cells and otherbiomolecules can be incorporated into the fibrous scaffolds whilemaintaining their activity and viability. For example, PDGF-bb,encapsulated and released from an IPC fiber over 3 weeks, showednegligible loss in activity based on a fibroblast proliferation assay[22]. In the present work, the ECM that had been isolated from cellculture was subsequently reconstituted into ECM fibers by the IPCprocess. As an ECM suspension rather than a solution was involved,it was important to ensure good dispersion of the particulates inthe suspension, in order to ensure a smooth fiber drawing process.The isolated ECM was stored in DI water rather than the polyanionprecursor, sodium alginate, due to the greater stability of theaqueous suspension. The calcium content of the ECM would grad-ually result in the crosslinking of alginate and its subsequentprecipitation out of solution together with other ECM components.Thus, ECM was stored in DI, and was pelletized and redispersed inalginate only at the point of usage.

According to the reported mechanism of interfacial poly-electrolyte complex fiber formation [23], particulates were encap-sulated by the formation of thinner nuclear fibers that coalescedand went ‘around’ the particles, facilitating effective encapsulationwithout unduly compromising the physical properties of the fibers.Thus, while a portion of the ECM particles was encapsulated withinthe bulk of the fiber, the other portion would be exposed on thefiber surface for interaction directly with the biological environ-ment, including cells. The availability of the ECM for interactionwith cells was demonstrated by the cell adhesion experiment(Fig. 6) Good adhesion would be a pre-requisite for cell signalling bythe insoluble factors present in the ECM.

The attractiveness of being able to reconstitute ECM from a widevariety of cell lines (including both human and animal cell lines)lies in the potentially unlimited selection of ECM for three-dimensional (3D) cell culture and tissue engineering. Currentmodels in cell biology and strategies in tissue engineering employproteins whose functions and usefulness have been well estab-lished, e.g. collagen, fibronectin and fibrin. Matrigel, a solubilizedbasement membrane preparation derived from Engelbreth-Holm-Swarm Sarcoma is also a popular choice. However, unlike ‘whole’ECM, these naturally derived materials pose a limit to the repertoireof biological signals that are required for cellular processes such as

differentiation. In all probability, the ideal ECM for regeneratinga specific cell/tissue type would be the ECM native to the cells inquestion. It is also conceivable that signals derived from theparticular ECM type would influence the differentiation of stemcells into the corresponding lineage; in fact, by culturing hMSCs onthe ECM produced by decellularizing MC3T3 cell cultures, weshowed that the pre-osteoblast ECM was capable of differentiatinghMSCs into the osteogenic phenotype. (Supplementary Informa-tion, S1.)

Next, to demonstrate that the reconstituted ECM scaffoldsretained the bioactivity of the intact ECM in terms of providingsignals for cell differentiation, hMSCs were seeded on the scaffolds,which were then implanted subcutaneously into SCID mice. Ectopicbone formation was observed within a period of 4 weeks, allowingus to postulate that the ECM scaffold had differentiated the seededhMSCs into bone cells with the ability to ‘remodel’ the cell–scaffoldconstruct into a bone phenotype.

Matrix degradation is well known to be an essential step of thebone remodeling process, and is a function of osteoclasts thatsecrete proteases to degrade type I collagen and other non-collagenous proteins present in the ECM. Osteogenic differentiationof preosteoblasts has also been shown to lead to an increase in thesecretion of specific metalloproteinases [24]; hMSC differentiationto bone cells in this case is also likely to result in a release of similarmatrix-degrading enzymes, which we postulate had led to subse-quent resorption of the ECM component of the fiber within 4weeks. Lysozyme is also known to be secreted into the extracellularcompartment by osteoclasts [25], and being a b-glucosidase, webelieve it to be responsible for the degradation of the chitosan–alginate component of the fibers.

Our experiments have underlined the potential of a recon-stituted ECM scaffold as a vehicle for stem cell transplantation, withstem cell differentiation capacity. The complexity of the ECM hasdictated other work in 3D cell culture, where a set of semi-syntheticECMs has been used to rebuild and replicate a given tissue [26]. Yetother studies have underlined the importance of having a mix ofECM components in order to realize the optimal differentiatedfunction of cells [27]. These observations reinforces the advantageof using ‘whole’ ECM, rather than isolated ECM components ina scaffold, the exact interplay of the different components andfactors in the natural environment being as yet, unknown.

5. Conclusion

The incorporation of cell-secreted ECM into fibers formed byinterfacial polyelectrolyte complexation provides the basis bywhich ECM can be reconstituted to form 3D scaffolds. Suchmatrices are useful as a source of ECM signals for stem cell trans-plantation and differentiation, and represent a promising approachtowards the engineering of functional tissue.

Acknowledgements

The authors would like to acknowledge Xingfang Su, BenjaminTai and Jessica Oon for helpful discussions and technical assistance,as well as Dr. Yuangang Zheng for his help with confocal micros-copy. This work was supported by the Institute of Bioengineeringand Nanotechnology (Biomedical Research Council, Agency forScience, Technology and Research, Singapore).

Appendix

Figures with essential colour discrimination. Certain figures inthis article cannot be interpreted in black and white. The full colour

K. Narayanan et al. / Biomaterials 30 (2009) 4309–4317 4317

version can be found in the online version at doi:10.1016/j.biomaterials.2009.04.049.

Appendix. Supplementary information

Supplementary data associated with this article can be found inthe online version at doi:10.1016/j.biomaterials.2009.04.049.

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