ARTICLE IN PRESS Differentiation 79 (2010) 102–110
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Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications$ Sambit Sahoo a,1, Lay-Teng Ang a,2, James Cho-Hong Goh b,3, Siew-Lok Toh c,n a
Division of Bioengineering, National University of Singapore, Singapore-117574, Singapore Division of Bioengineering & Department of Orthopaedic Surgery, National University of Singapore, Singapore-119074, Singapore c Division of Bioengineering & Department of Mechanical Engineering, E3A-04-15, 7 Engineering Drive 1, National University of Singapore, Singapore-117574, Singapore b
a r t i c l e in f o
a b s t r a c t
Article history: Received 25 May 2009 Received in revised form 24 October 2009 Accepted 11 November 2009
Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications. & 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.
Keywords: Functional tissue engineering Biomimetic scaffolds Electrospinning Sustained release Fibroblast growth factor Bone marrow stromal cells
1. Introduction Tendon and ligament injuries account for about half of all musculoskeletal injuries and are associated with pain, suboptimal healing and permanent loss of extremity function. Grafts and prostheses, which are currently used for treatment, face problems such as donor scarcity, donor-site morbidity, tissue rejection, disease transmission and poor long-term performance. Tissue engineered tendons and ligaments could overcome these shortcomings by regenerating a tissue that is biomechanically,
$ Work performed in: Tissue Repair Lab, Division of Bioengineering, National University of Singapore, Singapore-117574, Singapore. Tel.: +65 6516 5985; fax: + 65 6872 3069. n Corresponding author. Tel.: + 65 6874 2920; fax: + 65 6872 3069. E-mail addresses:
[email protected] (S. Sahoo),
[email protected] (L.T. Ang),
[email protected] (J.C.H. Goh),
[email protected] (S.L. Toh). 1 Present address: Department of Orthopaedic Surgery, NUS Tissue Engineering Program, #04-01, DSO (Kent Ridge) Building, 27 Medical Drive, National University of Singapore, Singapore-117510, Singapore. Tel.: + 65 6516 5447, + 65 6516 5985; fax: + 65 6776 5322, +65 6872 3069. 2 Tel.: + 65 6516 5985; fax: + 65 6872 3069. 3 Tel.: + 6772 4424; fax: + 6778 0720.
biochemically and morphologically similar to the normal tissue (Butler et al., 2003, 2004; Woo et al., 2004). Though researchers have developed and tried various scaffolds, there is still the need for an ideal scaffold that could provide suitable mechanical properties along with biological signals required for tendon/ ligament regeneration, especially in stem cell-based approaches. Mesenchymal stem cells (MSCs) present an attractive way for engineering tendon/ligament tissues as not only can they be derived from replenishable sources like the bone marrow (unlike differentiated tendon/ligament fibroblasts that would require harvesting and enzymatic digestion of healthy donor tissues), but their lack of immunogenicity also makes them suitable for allogeneic implants (Gao and Caplan, 2003; Tuan et al., 2003; Jorgensen et al., 2004; Ge et al., 2005). For the stem cell approach to succeed, adequate biological signals would be required to be delivered via the tissue engineering scaffold to encourage proliferation and fibroblastic differentiation of the seeded precursor cells. Though the exact environment or cocktail of signals necessary to differentiate stem cells into tendon/ligament fibroblasts is still unknown, stimuli like cyclic mechanical stretch applied through a mechanical bioreactor, and specific growth and differentiation factors have been shown to promote tendon and ligament cell
0301-4681/$ - see front matter & 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) doi:10.1016/j.diff.2009.11.001
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proliferation and matrix formation in several in vitro and in vivo studies (Goh et al., 2003; Yamada et al., 2008; Jenner et al., 2007; Hoffmann and Gross, 2007). Among the putative growth factors such as basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-b, epidermal growth factor, platelet derived growth factor, growth and differentiation factor-5 and insulin like growth factor, bFGF is particularly relevant as it stimulates bone marrow stem cell (BMSC) proliferation, self-renewal and differentiation into fibroblastic cells specific for tendon and ligament lineages (Hankemeier et al., 2005; Petrigliano et al., 2006, 2007). However, delivering any bioactive growth factor locally at the healing site in a sustained fashion has been a challenge, as most growth factors have extremely small plasma half-lives and are rapidly inactivated (Chan et al., 2000). Incorporation of growth factors within the tissue engineering scaffold has been proposed as a means for ensuring a sustained delivery of growth factors to the repair site (Whitaker et al., 2001). Our earlier studies have shown that a fibrous ‘‘nano-microscaffold’’ combining knitted microfibers and electrospun nanofibers, when seeded with BMSCs, could be used for tendon/ ligament tissue engineering (Sahoo et al., 2006, 2007). This hybrid scaffold system combined the advantages of mechanical integrity of microfibers and the large biomimetic surface offered by nanofibers; due to their high surface area to volume ratio and resemblance to the nanostructure of natural extracellular matrix (ECM), the nanofibrous substrate facilitated cell attachment, proliferation and ECM deposition. Nanofibrous scaffolds have been demonstrated to also support MSC proliferation and differentiation along multiple lineages (Bhattarai et al., 2004; Li et al., 2005). A combination of nanofibrous substrate and sustained growth factor release can be expected to make a tissue engineering scaffold both structurally and functionally biomimetic. Protein growth factors have been incorporated into electrospun nanofibers for continued release from scaffolds (Chew et al., 2005; Liao et al., 2006; Li et al., 2006). We have recently developed, using a technique of blend-electrospinning, PLGA nanofibers that are capable of continued release of bioactive bFGF over 1–2 weeks (Sahoo et al., 2009). Since injured tendons/ ligaments have increased tissue levels of bFGF and its receptors during the 1st week of injury (Chang et al., 1998; Cool et al., 2004; Kobayashi et al., 2006; Berglund et al., 2006; Wurgler-Hauri et al., 2007), we hypothesise that an electrospun scaffold releasing bFGF over 1 week would biomimic the ECM of injured tendon/ligament in both structure and function, by providing a nanofibrous topography as well as a week-long supply of bioactive bFGF to the resident cells, and would be favourable for tendon/ligament tissue engineering. In this study, we demonstrate the feasibility of bFGF-releasing blend-electrospun nanofibers for tendon/ligament tissue engineering applications. The scaffolds are characterised morphologically, the released bFGF is evaluated for its bioactivity on BMSCs via demonstration of activation of intracellular signaling pathways, and the resulting proliferation and differentiation of seeded BMSCs into tendon/ligament fibroblasts.
2. Materials and methods 2.1. Fabrication of bFGF-releasing nanofiber matrix 20 mg of lyophilized bFGF (Raybiotech, USA) in 333 ml of 5 mM TRIS (pH 7.6) containing 0.1% Bovine Serum Albumin (BSA) was blended with 1.5 ml of 6.1% PLGA (PLA85:PGA15; Purac Asia Pacific, Singapore) solution in hexafluoro-2-propanol (HFIP; Fluka Chemie GmBH, Germany). The resulting blend was electrospun using a high voltage power supply unit (RR 30-2P/DDPM, Gamma
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Fig. 1. Electrospinning setup used to fabricate bFGF-releasing nanofibers from a blend of bFGF and PLGA solution.
High-Voltage Research, Ormond, USA) at 10–12 kV and a flowrate of 0.45 ml/h, onto glass cover-slips placed on a grounded collector, about 15 cm from the positively charged spinneret (Fig. 1). Control scaffolds not containing bFGF were also fabricated by replacing the bFGF solution with 5 mM Tris containing 0.1% BSA. The bFGF-containing scaffolds were termed bFGF(+ ) and those without bFGF were termed as bFGF( ). 2.2. Scaffold characterization The nanofiber matrices were characterized morphologically by scanning electron microscopy (SEM) using both secondary electron (SEM; JEOL JSM-5800 LV) and backscattered electron imaging (Jeol JSM-6701 field-emission SEM). SEM images were analyzed by image analysis software (Olympus MicroImage v4.5.1, Olympus Optical Co., Germany) to determine the diameter distribution of the fibers. bFGF release from the scaffolds was studied over 2 weeks, using a release buffer comprising 1 PBS with 0.1% BSA and 0.01% sodium azide at 37 1C. bFGF concentration in the buffer was estimated using a bFGF ELISA kit (Calbiochem, Merck KGaA, Germany) on days 1, 3, 7, 10, 14 (n = 3). Protein content of the nanofibers was estimated by basesurfactant mediated hydrolysis of the PLGA nanofibers (using 50 mM Tris extraction medium containing 0.1 N NaOH, 5 M Urea and 0.08% SDS, at 37 1C for 3 h), neutralization with 0.1 N HCl and centrifugation, followed by Bradford microassay of the supernatant (n= 3) (Gupta et al., 1997). Protein encapsulation efficiency was calculated from the ratio of the estimated and the theoretically obtained total protein content of the electrospun scaffolds. 2.3. Cell culture and seeding on scaffolds Bone marrow was obtained from iliac crests of New Zealand White Rabbits under approval of the NUS Institutional Animal Care and Use Committee, National University of Singapore. BMSCs were isolated and cultured, using their property of short-term selective adherence to tissue culture polystyrene, in Dulbecco’s modified Eagle’s medium (DMEM) with low glucose, supplemented with 15% fetal bovine serum (FBS), at 37 1C with 5% humidified CO2. Semi-confluent cells of second or third passage were used for cell-seeding experiments. Nanofibrous scaffolds were sterilized by exposure to formaldehyde gas and seeded with rabbit BMSCs at a density of 104 cells/cm2 in DMEM-high glucose (HG), supplemented with 5% FBS and 1% Penicillin–Streptomycin. The constructs were cultured in a 5% CO2 incubator at 37 1C for 2 weeks, with the medium being replaced every 3 days. Cell adhesion efficiency on the nanofibrous scaffolds was estimated from the proportion of unattached cells in the culture
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medium, using a method previously described (Sahoo et al., 2006). After incubating the cell-seeded scaffolds (n= 6, for each group) for 18 h, the culture medium was collected separately and centrifuged, cell pellets re-suspended in 100 ml of medium and cell count performed. The cell adhesion efficiency was expressed as the number of cells attached to the scaffold as percentage of the number of cells seeded. The morphology and proliferation of the cells on the scaffolds was studied by live-cell staining using fluorescein diacetate (FDA, Molecular Probes, Invitrogen Corporation) and by SEM. Cellseeded scaffolds were stained with 10 mg/ml FDA in 1 PBS for 30 min and visualized using an inverted fluorescence microscope (IX71 Inverted Research Microscope, Olympus). For SEM, the samples were fixed with 3.7% formaldehyde, dehydrated in graded concentrations of ethanol, air-dried, sputter-coated with gold and observed under the SEM at an accelerating voltage of 15 kV. 2.4. Demonstration of bioactivity of released bFGF: Western blot for phosphorylated tyrosine kinases in BMSCs bFGF acts through cell surface receptors that activate several intracellular second messengers (ERK/ MAPK cascade) by phosphorylation of their tyrosine residues, before finally exerting their effects on the genes. Increased activation of this signal transduction pathway in the BMSCs cultured on bFGF( +) nanofibrous scaffolds, as compared to those cultured on bFGF( ) control scaffolds, would confirm bioactivity of the released bFGF. After 1 week of culture on the scaffolds (n =3) in DMEM-HG with 5% FBS, the BMSCs were lysed by freeze-thawing and treatment with NP-40 lysis buffer (20 mM Tris–HCl at pH 8, 100 mM NaCl, 10% Glycerol, 1% nonidet P-40, 5 mM Na2-EDTA) supplemented with several protease inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate, 0.01 protease inhibitor cocktail (Sigma Aldrich, USA)). Protein concentration in the pooled lysates of each group was estimated using Bradford method (Standard microplate assay, Bio-Rad Protein Kit). 100 mg of total cell-lysates were separated on a 7.5% SDS–PAGE. After semi-dry electrotransfer onto a nitrocellulose membrane, blocking was performed with Tris-buffered saline containing 1% BSA, and the membrane incubated with phosphotyrosine antibody (pY20, mouse IgG, BD Biosciences, USA). GAPDH was probed with anti-GAPDH as a loading control on a duplicate membrane. Detection was performed using horse radish peroxidase-conjugated secondary antibody (anti-mouse IgG) and TMB Substrate (Sigma ProteoQwestTM Colorimetric Western Blotting Kit). The blot was visualized using a gel documentation system (Syngene G:BOX, Synoptics Ltd, Cambridge, UK) and differences in phosphotyrosine levels in the two cell populations were measured using densitometric analysis (Quantity-One 4.4.0, BioRad), using GAPDH as the normaliser. 2.5. Effect on BMSC number: PicoGreen assay Cell number on the scaffolds was estimated by DNA quantitation using the PicoGreen assay (Molecular Probes, Invitrogen Corporation) after 7 and 14 days of culture. Cells were lysed by a cycle of freeze-thawing, freeze-drying and homogenization in a lysis buffer. 20 ml of the cell-lysate was added to 80 ml of PicoGreen dye in separate wells of a black 96-well plate, and fluorescence intensity was measured at 520 nm wavelength using a microplate reader (FLUOstar OPTIMA, BMG Labtech GmbH, Germany) after excitation at 485 nm. BMSC numbers on bFGF( ) scaffolds and tissue culture polystyrene (TCP) were used as controls (n = 3).
2.6. Effect on BMSC stemness: differentiation assays to demonstrate loss of BMSC multipotentiality It is desirable that bioactive bFGF from the nanofibrous scaffolds, besides causing increased proliferation, should also direct BMSC differentiation along a fibroblastic lineage. Since cellsurface markers for rabbit BMSC and tendon/ligament cells are currently unknown (Doroski et al., 2007), lineage-specific differentiation of BMSCs can only be demonstrated indirectly through differentiation assays, wherein the committed progeny should exhibit a loss or reduction in multipotentiality, or ability to differentiate into bone, cartilage and adipose tissues. Primary rabbit BMSCs of P2 passage were cultured on bFGF(+ ) scaffolds in DMEM-HG with 5% FBS for 2 weeks, harvested by trypsinization, replated and cultured on TCP substrates (6-well plates), and then induced to differentiate along adipogenic, osteogenic and chondrogenic lineages, using established protocols (Pittenger et al., 1999; McBeath et al., 2004). 3 105 cells were seeded per well in 6-well plates for adipogenic and osteogenic differentiation, and 6 105 cells were grown in a pellet culture for chondrogenic differentiation. Adipogenic differentiation was induced by 3 cycles of induction (with DMEM supplemented with 10% FBS, 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM dexamethasone, 10 mg/ml insulin, 0.2 mM indomethacin and antibiotics) and maintenance treatment (with DMEM supplemented with 10% FBS, 10 mg/ml insulin and antibiotics). Differentiation was then detected by Oil Red-O staining for lipid vacuoles. Chondrogenic differentiation was induced by culturing the BMSC-pellet in serum-free DMEM with TGF-b1, 50 mg/ml L-ascorbic acid 2phosphate, 1.25 mg/ml BSA, 0.1 mM dexamethasone, 1 ITS (insulin–transferrin–selenium) and antibiotics supplements. After 3 weeks, the cell-pellet was sectioned and stained with Alcian Blue for cartilage matrix-specific sulfated glycosaminoglycans and acidic sulfated mucosubstances. Osteogenic differentiation of BMSCs was induced under the influence of 10 mM b-glycerophosphate, 0.1 mM dexamethasone, 50 mg/ml L-ascorbic acid 2phosphate and 10 mg/ml insulin, and Alizarin Red staining was carried out to detect calcium accumulation after 3 weeks. All the induction reagents, except ITS (Gibco) and TGF-b1 (R&D Systems, MN, USA), were from Sigma. As a control, naı¨ve P3 BMSCs (obtained after sub-culturing P2 cells on TCP for 2 weeks) were induced to differentiate along the three lineages, following the same protocols, and various staining were performed to evaluate directed differentiation after the end of 3 weeks.
2.7. Effect on collagen production: SirCol assay and immunostaining Fibroblastic differentiation of BMSCs would be associated with increased production and deposition of collagen, the major component of tendon/ligament ECM. After 7 and 14 days of culture, soluble collagen secreted into the culture medium was determined by picrosirius red based colorimetric assay (SirCols Assay, Biocolor Ltd, Northern Ireland), using previously described methods (Sahoo et al., 2006). The total amount of soluble collagen secreted per scaffold (n =4) was estimated from the collagen concentration and the volume of culture media. Insoluble collagen deposited in the ECM surrounding the cells was detected by immunostaining for collagen type I and type III. In addition, immunostaining was also performed for tenascin-C, a tendon/ligament-specific ECM molecule. After 10 days of culture, paraformaldehyde-fixed cell-seeded scaffolds were labeled with mouse monoclonal primary antibodies (anti-collagen type I, type III and tenascin-C; ICN Biochemicals, Aurora, OH) at 1:200 dilution for 8 h at room temperature, alkaline phosphatase
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conjugated anti-mouse secondary antibodies at 1:100 dilution for 1 h, and detected using DAB substrate (IHC Select DAB Kit, Chemicon, Millipore Corporation, MA, USA). The immunostained nanofibrous scaffolds were then directly visualized under the IX71 microscope.
2.8. Q-RT-PCR analysis for expression of ligament/tendon-related ECM proteins from BMSCs In addition to loss of multipotentiality, fibroblastic differentiation of BMSC would also be associated with upregulation of gene expression for tendon/ligament-specific ECM proteins like collagen type I, collagen type III, fibronectin and biglycan. After 7 and 14 days of culture on bFGF( +) scaffolds as well as bFGF( ) and TCP controls (n= 3), total RNA were extracted from the constructs using Qiagen RNeasy Kits. Quantitative Reverse Transcriptasemediated-PCR (Q-RT-PCR) was performed using SYBR-Green chemistry for collagen type I, collagen type III, fibronectin and biglycan, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and b-actin as reference genes. Primer sequences (Table 1) were either obtained from published literature (Cooper et al., 2006; Sobajima et al., 2005) or designed from rabbit gene sequences obtained from the GenBank database, using Primer-3 software, and synthesized by Research Biolabs, Singapore. cDNA synthesis and PCR expansion (using iScript and iQ SYBR Green Supermix, Bio-Rad Laboratories, CA) were performed in a iCycler iQ detection system (Bio-Rad Laboratories, CA). Data were Table 1 Real time PCR primers used in the study. Primers for collagen type I, GAPDH and bactin were designed from NZWR gene sequences obtained from GenBank (accession numbers D49399, NM_001082253 and AF309819 respectively) using Primer3 software (http://frodo.wi.mit.edu). Primer sequences for collagen type III, fibronectin and biglycan were obtained from published literature (Cooper et al., 2006; Sobajima et al., 2005). Primer
Sequence
Collagen I (a2)
F: GCA TGT CTG GTT AGG AGA AAC C R: ATG TAT GCA ATG CTG TTC TTG C F: AAG CCC CAG CAG AAA ATT G R: TGG TGG AAC AGC AAA AAT CA F: CTC ACC CGA GGC GCC ACC TA R: TCG CTC CCA CTC CTC TCC AAC G F: TGA ACA ACA AGA TCT CCA AGA T R: ATT CAG GGT CTC TGG CAG A F: GAC ATC AAG AAG GTG GTG AAG C R: CTT CAC AAA GTG GTC ATT GAG G F: CCC ATC TAC GAG GGC TAC G R: CCA CGT AGC ACA GCT TCT CC
Collagen III (a1) Fibronectin Biglycan GADPH
b-Actin
105
analyzed for relative expression using the DDCT method, and normalized against the expression profile of BMSC grown on TCP controls on day 7. 2.9. Data reduction and statistical analysis Data were analyzed by single-factor ANOVA and post-hoc Tukey tests for multiple comparisons. Results were presented as mean7standard error and po0.05 was accepted as significant.
3. Results 3.1. Scaffold characterization Scaffolds were composed of randomly oriented continuous nanofibers of 200–700 nm diameter (from analysis of SEM images, Fig. 2A). The growth factor was distributed as a random dispersion within the fibers (Backscattered electron image on SEM observation, Fig. 2B). Release kinetics results indicated that 55% of the bFGF was incorporated into the scaffolds and released over a period of 7 days (Fig. 2C); there was an initial rapid release followed by a more gradual release (average bFGF concentration: 13.5 pg/ml on day 1, 6.5 pg/ml on day 3). 3.2. Demonstration of bioactivity of released bFGF Several tyrosine-phosphorylated proteins could be observed in the Western Blots of both groups, with a higher relative density in the bFGF(+ ) group (Fig. 3). Sustained tyrosine phosphorylation and activation of ERK1 and ERK2 (p44 and p42), as well as FRS2 (p89) were observed in the cells grown on the bFGF( + ) scaffolds, indicating that the bFGF incorporated in the scaffolds was bioactive over 7 days. p145 (FGF receptor) and p52 (shc) phosphorylation levels were also slightly increased in the bFGF(+ ) scaffold. 3.3. BMSC adhesion and proliferation on scaffolds (FDA staining, SEM, PicoGreen assay) Both scaffold groups showed similar cell adhesion, with more than 90% of the seeded rabbit BMSCs adhering onto them in 18 h. SEM (Fig. 4A, B) and fluorescent microscopy (Fig. 4C, D) after livecell staining with FDA revealed better cell proliferation and spreading on bFGF( +) scaffolds. PicoGreen assay showed a significant increase in cell population on the bFGF(+ ) scaffolds between day 3 and 14 of culture (p o0.05), at the end of which,
Fig. 2. SEM images showing blend nanofibers of 200–700 nm diameter, with a random distribution of protein within. The bFGF was released over 1 week.
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cell lysis and DNA extraction from the 2D substrate of culture flasks as compared to 3D nanofibrous scaffolds (Ng et al., 2005). 3.4. BMSC differentiation assay Naı¨ve P3 BMSCs could be successfully differentiated into adipocytic, osteocytic and chondrocytic lineages after 3 weeks of specific induction. However, P2 BMSCs, after being passaged on bFGF(+ ) nanoscaffolds for 2 weeks, failed to show any adipocytic differentiation while osteocytic and chondrocytic differentiation was markedly reduced (Fig. 5). 3.5. Collagen production: SirCol assay and immunostaining
Fig. 3. Western blot showing increased tyrosine phosphorlyation events (marked by arrows in the blot) in BMSCs cultured for 7 days on bFGF( +) scaffolds, indicating activation of bFGF signal transduction pathways. The relative increase in band density, normalized against GAPDH intensities, is indicated next to each band.
While Sircol assays showed that BMSCs on bFGF(+ ) and bFGF( ) scaffolds produced similar amounts of soluble collagen (36.673.56 mg/ bFGF( +) scaffold, 32.8 71.99 mg/ bFGF( ) scaffold, and 31.070.84 mg/TCP well, on day 14), immunostaining revealed a denser deposition of collagen type I and III in the ECM on bFGF(+ ) scaffolds as compared to control bFGF( ) scaffolds after ten days of culture. In addition, a denser deposition of tenascin-C, another tendon/ligament-specific ECM protein, was also observed on bFGF(+ ) scaffolds (Fig. 6). Cellular outlines were only faintly visible under the imaging conditions due of the thickness and interference from the irregular surface of the nanofibrous scaffolds. 3.6. Q-RT-PCR analysis for gene expression of ligament/tendonspecific ECM proteins Gene expression analyses by single-factor ANOVA demonstrated upregulation of gene expression for various tendon/ ligament ECM proteins in the BMSCs cultured on nanofibrous scaffolds compared to BMSCs on TCP, at the end of 1 week. By the end of 2 weeks, bFGF(+ ) scaffolds showed significantly higher gene upregulation compared to bFGF( ) scaffolds and TCP controls (Fig. 7). Collagen type I expression was lower on the bFGF(+ ) scaffolds than on the bFGF( ) control on day 7; but the scenario was reversed by day 14. While the expression of collagen type I and biglycan on the bFGF( +) scaffolds increased significantly between day 7 and 14, that of fibronectin dropped significantly; the decrease in collagen type III expression was not statistically significant.
4. Discussion A polymeric nanoscaffold was developed with the capability of releasing an encapsulated bioactive growth factor over 1 week. The scaffold facilitated BMSC attachment and subsequent proliferation, production and deposition of collagen as well as tenascin-C, and upregulation of gene expression for tendon/ ligament-specific ECM proteins, suggesting BMSC differentiation into a tendon/ligament phenotype. Fig. 4. SEM and live cell imaging showing better cell proliferation and spreading on bFGF( +) scaffolds (A, C). PicoGreen assay (E) confirmed significantly higher cell population on bFGF(+ ) scaffolds compared to bFGF( ) scaffolds after two weeks.
4.1. Electrospun nanofibers as carriers for sustained release of bioactive molecules
bFGF(+ ) scaffolds had significantly higher cell population than bFGF( ) scaffolds (Fig. 4E). Cells cultured on TCP gave prominently higher PicoGreen readings compared to the nanofibrous scaffolds (significant difference based on Tukey test on day 3 and day 14); this may be the result of higher efficiency of
Several recent studies have also reported growth factorreleasing electrospun nanofibers for tissue engineering scaffolds (Chew et al., 2005; Liao et al., 2006; Li et al., 2006). It has been hypothesised that a combination of passive diffusion across nanopores on the nanofiber surface and material degradation of the nanofibers causes protein release (Jiang et al., 2005, 2006; Ramakrishna et al., 2006). Chain scission of PLGA molecules
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Fig. 5. Oil red O (for fat deposits, indicated by black arrows), alizarin red (for calcium deposits, indicated by white arrows) and alcian blue staining (for sulfated mucosubstances), showing adipogenic, osteogenic and chondrogenic differentiation of BMSCs. Data demonstrate a reduction of multilineage differentiation potential of BMSCs after culture on bFGF(+ ) nanoscaffolds (bottom) as compared with untreated naı¨ve BMSCs (top). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Immunostaining for collagen type I, collagen type III and tenascin-C showing a denser deposition (indicated by arrows) of the ECM proteins in the BMSC-seeded bFGF(+ )scaffolds, compared to bFGF( )scaffolds, on day 10. Cell outlines were not clearly visible in the stained samples compared to unstained controls under the imaging conditions.
during electrospinning increases the rate of hydrolytic degradation of PLGA nanofibers, particularly in the amorphous regions, allowing small proteins to diffuse through (Zong et al., 2003; Kim et al., 2009). Protein release from the nanofibers developed in this study is also expected to have the underlying mechanisms of diffusion and degradation. Growth factor bioactivity, in previous studies, has only been indirectly demonstrated by increased proliferation of cells when cultured in media supplemented with supernatant containing the released growth factor (Chew et al., 2005; Liao et al., 2006), or by enhanced differentiation of MSCs when cultured on the scaffolds (Li et al., 2006). In this study, bioactivity of the released bFGF was directly demonstrated though specific signal activation in BMSCs. In vitro studies have shown that optimal differentiation of BMSCs into tendon/ ligament fibroblasts requires the simultaneous and sequential administration of multiple growth factors (Moreau et al., 2005a, 2005b). It would therefore be desirable to fabricate nanofibrous scaffolds incorporated with several relevant growth factors; the initiation, duration and concentration of release of the different growth factors could be controlled by modifying the amount of growth factor loaded into the nanofibers, by choosing biomaterials of different degradation rates and by addition of porogens such as PEG in the biomaterials used for the different nanofibers,
and by using techniques like coaxial electrospinning (Liao et al., 2006; Jiang et al., 2006).
4.2. Biomimiking the ECM in structure and function The ECM in a tissue is structurally and functionally integrated with resident cells, providing them support and anchorage, as well as regulating their survival, differentiation and function. In addition, the ECM sequesters several cellular growth factors, thereby stabilizing and protecting them and acting as their local depot. Tissue engineering scaffolds that present a combination of nanofibrous substrate and sustained growth factor release would thus be both structurally and functionally biomimetic. Nanofiber coating on scaffolds can provide a large biomimetic surface aiding in BMSC differentiation into a tendon/ligament lineage, even without any supplementation of growth or differentiation factors (Sahoo et al., 2006, 2007). Aligned nanofibers have been demonstrated to induce fibroblast alignment and collagen production (Lee et al., 2005), suggesting that nanotopographic cues from electrospun scaffolds affect cell behavior and fate. The current study also demonstrated gene upregulation of tendon/ ligament matrix proteins in BMSCs after culture on control
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Fig. 7. Q-RT-PCR analysis showing a significant gene upregulation of tendon/ligament ECM proteins on bFGF(+ ) scaffolds after 2 weeks.
nanofibrous scaffolds without any bFGF, at the end of 1 week. A significantly higher gene upregulation was observed on bFGF(+ ) nanoscaffolds suggesting a synergistic effect of nanotopography and growth factor release on the cells.
4.3. BMSC proliferation and differentiation on nanofibrous scaffolds Compared to the control scaffolds, enhanced cell proliferation and fibroblastic differentiation of BMSCs (indicated by loss of multipotency, gene upregulation of tendon/ligament ECM proteins, and increased collagen production and deposition) were observed on the bFGF(+ ) nanoscaffolds. The temporal pattern of collagen type I gene expression on the bFGF(+ ) scaffolds (lower expression on day 7, but higher expression by day 14) suggests that while high levels of bFGF initially maintained BMSCs in an actively proliferating undifferentiated phenotype, sustained bFGF levels resulted in BMSC differentiation by day 14. Collagen type III and fibronectin showed earlier gene upregulation (day 7) compared to collagen type I and biglycan (day 14). Collagen type III is an embryonic form of collagen, characterized by short disorganized fibrils, that is typically over-expressed during the early inflammatory and proliferative stages of tendon healing. It is gradually replaced by longitudinally aligned long collagen type I fibrils with remodeling and maturation of the healing tissue (Lin et al., 2004). A similar relationship between the expression of collagen type I and III was observed in this study. Fibronectin is known to be a marker for active reparative connective tissue processes and regulates initial cell attachment and survival (Venugopal et al., 2006). Down-regulation of fibronectin transcript levels in the second week, in this study, is
consistent with other studies where cells were observed to synthesize fibronectin during proliferation and early differentiation; once the cells reached maturation and accumulated collagenous ECMs, they sharply reduced the production of fibronectin (Venugopal et al., 2006; Chen et al., 2006). Biglycan and tenascin-C are known to modulate growth factor activity during tendon development and repair; biglycan also controls the diameter and assembly of ECM collagen fibrils (Berglund et al., 2006) and tenascin-C modulates cell–ECM interactions like cell adhesion and migration (Doroski et al., 2007). They are generally over-expressed during the reorganization phases of tissue development and repair, and coincide with collagen type I overexpression in this study. In contrast to 2-dimensional cultures on tissue culture polystyrene, where bFGF helps in BMSC self-renewal and maintains their multipotency (Tsutsumi et al., 2001), 2 weeks’ culture on bFGF-releasing nanoscaffolds resulted in their differentiation, further suggesting that the nanofibrous substrate plays a crucial role in determining cell behavior and fate. While previous in vitro studies have demonstrated significant effects of bFGF on BMSC proliferation, self-renewal and differentiation using twice-weekly replenished culture media supplemented with 0.1–10 ng/ml of bFGF (Hankemeier et al., 2005; Tsutsumi et al., 2001), in this study, sustained picogram concentrations of bFGF over 1 week was shown to be sufficient to induce the same effects. These results are supportive of our hypothesis that a biomimetic nanofibrous scaffold allowing a biomimetic sustained release of bFGF over 1 week would be suitable for tendon/ligament tissue engineering. The bFGF( +) scaffold developed in this study biomimics the ECM of injured tendons in both structure and function by providing a nanofibrous topography as well as a
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week-long supply of bioactive bFGF to the resident cells. Some of the cellular response to bFGF stimulation were observed on the bFGF(+ ) scaffolds in the second week, when the incorporated bFGF had already been released from the underlying scaffolds. This suggests that the released bFGF may have resulted in persistent, long-term signaling events in the BMSCs resulting in their proliferation and differentiation in the second week. A similar long-term response has been reported with TGF-b elsewhere (Wormstone et al., 2006). There were several limitations in this study that could be addressed in future studies. The current study used primary rabbit BMSCs, for which surface antigenic and gene markers are currently unknown. Therefore, demonstration of multipotency (and its loss) could only be demonstrated through differentiation assays. Also the relative contributions of the nanotopographic substrate and the released growth factor on cell fate has not been studied. Since multiple growth factors coordinate the healing of tendon/ligament injuries (Moreau et al., 2005a, 2005b), nanofibrous scaffolds incorporated with several relevant growth factors could be a more effective model than the single growth factor model used in this study. As nanofibers alone cannot provide sufficient mechanical support required for healing tendons/ ligaments, hybrid scaffolds have been fabricated by coating nanofibers on microfibrous scaffolds (Sahoo et al., 2006, 2007) growth factor releasing nanofibers developed in this study could be suitable candidates for such coating.
5. Conclusion bFGF could be successfully incorporated, randomly dispersed within the electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF could activate tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, production and deposition of collagen and tenascin-C, and induced tendon/ligament-like fibroblastic differentiation, suggesting their potential in tendon/ligament tissue engineering applications. References Berglund, M., Reno, C., Hart, D.A., Wiig, M., 2006. Patterns of mRNA expression for matrix molecules and growth factors in flexor tendon injury: differences in the regulation between tendon and tendon sheath. J. Hand Surg. [Am.] 31, 1279– 1287. Bhattarai, S.R., Bhattarai, N., Yi, H.K., Hwang, P.H., Cha, D.I., Kim, H.Y., 2004. Novel biodegradable electrospun membrane: scaffold for tissue engineering. Biomaterials 25, 2595–2602. Butler, D.L., Dressler, M.R., Awad, H.A., 2003. Functional tissue engineering: assessment of function in tendon and ligament repair. In: Guilak, F., Butler, D.L., Goldstein, S.A., Mooney, D. (Eds.), Functional Tissue Engineering. Springer-Verlag, New York, pp. 213–226. Butler, D.L., Shearn, J.T., Juncosa, N., Dressler, M.R., Hunter, S.A., 2004. Functional tissue engineering parameters toward designing repair and replacement strategies. Clin. Orthop. Relat. Res., S190–199. Chan, B.P., Fu, S., Qin, L., Lee, K., Rolf, C.G., Chan, K., 2000. Effects of basic fibroblast growth factor (bFGF) on early stages of tendon healing: a rat patellar tendon model. Acta Orthop. Scand. 71, 513–518. Chang, J., Most, D., Thunder, R., Mehrara, B., Longaker, M.T., Lineaweaver, W.C., 1998. Molecular studies in flexor tendon wound healing: the role of basic fibroblast growth factor gene expression. J. Hand Surg. [Am.] 23, 1052–1058. Chen, J., Horan, R.L., Bramono, D., Moreau, J.E., Wang, Y., Geuss, L.R., Collette, A.L., Volloch, V., Altman, G.H., 2006. Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering. Tissue Eng. 12, 3085–3095. Chew, S.Y., Wen, J., Yim, E.K., Leong, K.W., 2005. Sustained release of proteins from electrospun biodegradable fibers. Biomacromolecules 6, 2017–2024. Cool, S.M., Snyman, C.P., Nurcombe, V., Forwood, M., 2004. Temporal expression of fibroblast growth factor receptors during primary ligament repair. Knee Surg. Sports Traumatol. Arthrosc. 12, 490–496. Cooper Jr., J.A., Bailey, L.O., Carter, J.N., Castiglioni, C.E., Kofron, M.D., Ko, F.K., Laurencin, C.T., 2006. Evaluation of the anterior cruciate ligament, medial
109
collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament. Biomaterials 27, 2747–2754. Doroski, D.M., Brink, K.S., Temenoff, J.S., 2007. Techniques for biological characterization of tissue-engineered tendon and ligament. Biomaterials 28, 187–202. Gao, J., Caplan, A.I., 2003. Mesenchymal stem cells and tissue engineering for orthopaedic surgery. Chir. Organi. Mov. 88, 305–316. Ge, Z., Goh, J.C., Lee, E.H., 2005. Selection of cell source for ligament tissue engineering. Cell Transplant. 14, 573–583. Goh, J.C., Ouyang, H.W., Teoh, S.H., Chan, C.K., Lee, E.H., 2003. Tissue-engineering approach to the repair and regeneration of tendons and ligaments. Tissue Eng. 9 (Suppl 1), S31–44. Gupta, R.K., Chang, A.C., Griffin, P., Rivera, R., Guo, Y.Y., Siber, G.R., 1997. Determination of protein loading in biodegradable polymer microspheres containing tetanus toxoid. Vaccine 15, 672–678. Hankemeier, S., Keus, M., Zeichen, J., Jagodzinski, M., Barkhausen, T., Bosch, U., Krettek, C., Van Griensven, M., 2005. Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng. 11, 41–49. Hoffmann, A., Gross, G., 2007. Tendon and ligament engineering in the adult organism: mesenchymal stem cells and gene-therapeutic approaches. Int. Orthop. 31, 791–797. Jenner, J.M., van Eijk, F., Saris, D.B., Willems, W.J., Dhert, W.J., Creemers, L.B., 2007. Effect of transforming growth factor-beta and growth differentiation factor-5 on proliferation and matrix production by human bone marrow stromal cells cultured on braided poly lactic-co-glycolic acid scaffolds for ligament tissue engineering. Tissue Eng. 13, 1573–1582. Jiang, H., Hu, Y., Li, Y., Zhao, P., Zhu, K., Chen, W., 2005. A facile technique to prepare biodegradable coaxial electrospun nanofibers for controlled release of bioactive agents. J. Control. Release 108, 237–243. Jiang, H., Hu, Y., Zhao, P., Li, Y., Zhu, K., 2006. Modulation of protein release from biodegradable core–shell structured fibers prepared by coaxial electrospinning. J. Biomed. Mater. Res. B Appl. Biomater. 79, 50–57. Jorgensen, C., Gordeladze, J., Noel, D., 2004. Tissue engineering through autologous mesenchymal stem cells. Curr. Opin. Biotechnol. 15, 406–410. Kim, B.S., Oh, J.M., Kim, K.S., Seo, K.S., Cho, J.S., Khang, G., Lee, H.B., Park, K., Kim, M.S., 2009. BSA-FITC-loaded microcapsules for in vivo delivery. Biomaterials 30, 902–909. Kobayashi, M., Itoi, E., Minagawa, H., Miyakoshi, N., Takahashi, S., Tuoheti, Y., Okada, K., Shimada, Y., 2006. Expression of growth factors in the early phase of supraspinatus tendon healing in rabbits. J. Shoulder Elbow. Surg. 15, 371–377. Lee, C.H., Shin, H.J., Cho, I.H., Kang, Y.M., Kim, I.A., Park, K.D., Shin, J.W., 2005. Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast. Biomaterials 26, 1261–1270. Li, W.J., Tuli, R., Huang, X., Laquerriere, P., Tuan, R.S., 2005. Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 26, 5158–5166. Li, C., Vepari, C., Jin, H.J., Kim, H.J., Kaplan, D.L., 2006. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 27, 3115–3124. Liao, I.C., Chew, S.Y., Leong, K.W., 2006. Aligned core-shell nanofibers delivering bioactive proteins. Nanomedicine 1, 465–471. Lin, T.W., Cardenas, L., Soslowsky, L.J., 2004. Biomechanics of tendon injury and repair. J. Biomech. 37, 865–877. McBeath, R., Pirone, D.M., Nelson, C.M., Bhadriraju, K., Chen, C.S., 2004. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 6, 483–495. Moreau, J.E., Chen, J., Bramono, D.S., Volloch, V., Chernoff, H., Vunjak-Novakovic, G., Richmond, J.C., Kaplan, D.L., Altman, G.H., 2005. Growth factor induced fibroblast differentiation from human bone marrow stromal cells in vitro. J. Orthop. Res. 23, 164–174. Moreau, J.E., Chen, J., Horan, R.L., Kaplan, D.L., Altman, G.H., 2005. Sequential growth factor application in bone marrow stromal cell ligament engineering. Tissue Eng. 11, 1887–1897. Ng, K.W., Leong, D.T., Hutmacher, D.W., 2005. The challenge to measure cell proliferation in two and three dimensions. Tissue Eng. 11, 182–191. Petrigliano, F.A., McAllister, D.R., Wu, B.M., 2006. Tissue engineering for anterior cruciate ligament reconstruction: a review of current strategies. Arthroscopy 22, 441–451. Petrigliano, F.A., English, C.S., Barba, D., Esmende, S., Wu, B.M., McAllister, D.R., 2007. The effects of local bFGF release and uniaxial strain on cellular adaptation and gene expression in a 3D environment: implications for ligament tissue engineering. Tissue. Eng. 13, 2721–2731. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. Ramakrishna, S., Fujihara, K., Teo, W.-E., Yong, T., Ma, Z., Ramaseshan, R., 2006. Electrospun nanofibers: solving global issues. Mater. Today 9, 40–50. Sahoo, S., Ouyang, H., Goh, J.C., Tay, T.E., Toh, S.L., 2006. Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering. Tissue Eng. 12, 91–99. Sahoo, S., Goh, J.C.H., Toh, S.L., 2007. Development of hybrid polymer scaffolds for potential applications in ligament and tendon tissue engineering. Biomed. Mater. 2, 169–173. Sahoo, S., Ang, L.T., Goh, J.C.H., Toh, S.L., 2009. Growth factor delivery through electrospun nanofibers in scaffolds for tissue engineering applications. J. Biomed. Mater. Res. A. (in press).
ARTICLE IN PRESS 110
S. Sahoo et al. / Differentiation 79 (2010) 102–110
Sobajima, S., Shimer, A.L., Chadderdon, R.C., Kompel, J.F., Kim, J.S., Gilbertson, L.G., Kang, J.D., 2005. Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction. Spine J. 5, 14–23. Tsutsumi, S., Shimazu, A., Miyazaki, K., Pan, H., Koike, C., Yoshida, E., Takagishi, K., Kato, Y., 2001. Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF. Biochem. Biophys. Res. Commun. 288, 413–419. Tuan, R.S., Boland, G., Tuli, R., 2003. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res. Ther. 5, 32–45. Venugopal, J.R., Zhang, Y., Ramakrishna, S., 2006. In vitro culture of human dermal fibroblasts on electrospun polycaprolactone collagen nanofibrous membrane. Artif. Organs 30, 440–446. Whitaker, M.J., Quirk, R.A., Howdle, S.M., Shakesheff, K.M., 2001. Growth factor release from tissue engineering scaffolds. J. Pharm. Pharmacol. 53, 1427–1437.
Wurgler-Hauri, C.C., Dourte, L.M., Baradet, T.C., Williams, G.R., Soslowsky, L.J., 2007. Temporal expression of 8 growth factors in tendon-to-bone healing in a rat supraspinatus model. J. Shoulder Elbow Surg. 16, S198–203. Woo, S.L., Abramowitch, S.D., Loh, J.C., Musahl, V., Wang, J.H., 2004. Ligament healing: present status and the future of functional tissue engineering. In: Guilak, F., Butler, D.L., Goldstein, S.A., Mooney, D. (Eds.), Functional Tissue Engineering. Springer, pp. 17–29. Wormstone, I.M., Anderson, I.K., Eldred, J.A., Dawes, L.J., Duncan, G., 2006. Shortterm exposure to transforming growth factor beta induces long-term fibrotic responses. Exp. Eye Res. 83, 1238–1245. Yamada, M., Akeda, K., Asanuma, K., Thonar, E.J., An, H.S., Uchida, A., Masuda, K., 2008. Effect of osteogenic protein-1 on the matrix metabolism of bovine tendon cells. J. Orthop. Res. 26, 42-48. Zong, X., Ran, S., Kim, K.S., Fang, D., Hsiao, B.S., Chu, B., 2003. Structure and morphology changes during in vitro degradation of electrospun poly (glycolide-co-lactide) nanofiber membrane. Biomacromolecules 4, 416–423.
