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Fibronectinimmobilizationontorobotic-dispensednanobioactiveglass/polycaprolactonescaffoldsforbonetissueengineering

ARTICLEinBIOTECHNOLOGYLETTERS·DECEMBER2014

ImpactFactor:1.59·DOI:10.1007/s10529-014-1745-5

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Jong-EunWon

SeoulNationalUniversity

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MiguelAngelMateos-Timoneda

IBECInstituteforBioengineeringofCatalonia

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OscarCastano

IBECInstituteforBioengineeringofCatalonia

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Hae-WonKim

DankookUniversity

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Retrievedon:04February2016

1

Fibronectin immobilization onto robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone 1

tissue engineering 2

3

Jong-Eun Won1,2, Miguel A.Mateos4,3, Oscar Castano3,4,5, Josep A. Planell3,4,5, , Seog-Jin Seo1,2, Eun-Jung 4

Lee1,2, Cheol-Min Han1,3, Hae-Won Kim1,2,* 5

1Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, 330-714 Korea 6

2Dept. of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, 7

Dankook University, Cheonan, 330-714 Korea 8

3Biomaterials for Regenerative Therapies, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain 9

4 CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain 10

5 Materials Science and Metallurgical Engineering, Universitat Politecnica de Catalunya, Barcelona Spain 11

12

* Corresponding author: Hae-Won Kim 13

- E-mail kimhw@dku.edu ; 14

- Tel: + 82 41 550 3081; Fax: +82 41 550 3085; 15

- Mailing address: Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, 330–714, Republic of 16

Korea 17

18

2

ABSTRACT 19

Bioactive nanocomposite scaffolds with cell-adhesive surface are considered to have excellent bone regeneration 20

capacity. Here we report fibronectin (FN)-immobilized nanobioactive glass (nBG)/polycaprolactone (PCL) (FN-21

nBG/PCL) scaffolds with an open pore architecture generated by a robotic-dispensing technique. The surface 22

immobilization level of FN was significantly higher onto the nBG/PCL scaffolds than onto the PCL scaffolds, mainly 23

ascribed to the incorporated nBG that provided hydrophilic chemical-linking sites. FN-nBG/PCL scaffolds 24

significantly improved the cell responses, including initial cellular anchorage and subsequent cell proliferation. 25

Although further in-depth studies on cell differentiation and the in vivo animal responses are required, bioactive 26

nanocomposite scaffolds with cell-favoring surface are considered to provide promising three-dimensional substrate 27

for bone regeneration. 28

29

KEYWORDS: Bone scaffolds; Cell response; Fibronectin; Nanobioactive glass; Nanocomposites; Polycaprolactone30

3

INTRODUCTION 31

Three-dimensional (3D) porous architectures should be carefully designed in bone scaffolds in order to allow 32

for effective bone cell growth and tissue formation. A myriad of scaffolds with open-porous structures have been 33

actively developed to mimic the bone micro/macro-porosity (Oh et al. 2010a, Zhang et al. 2012). Open-channeled 34

networks provide highly permissive environment for cells to grow and reorganize, enabling vascularization in bone 35

scaffolds (Tampieri et al. 2009). Recent technological advancement in scaffolds has also created customized pore 36

geometries and configurations (Seo et al. 2014). Among the techniques, robotic-dispensing (RD) has shown great 37

promise to design defined pore configuration in a controllable manner (Oh et al. 2010b). 38

In addition to the pore structure design, a nanocomposite approach of using both organic matrix and inorganic 39

nanocomponent is considered to be rationale to achieve scaffolds that mimic native bone tissue composition (Chang 40

et al. 2010). Inorganic nanocomponents have been shown to improve not only bone-bioactivity but also mechanical 41

properties of polymeric matrices. Furthermore, ionic ingredients present in the inorganic phase, such as silicon (Si), 42

calcium (Ca) and phosphorous (P), are favorable for stimulating bone cell responses (Roohani-Esfahani et al. 2011). 43

Among the inorganic phases that have been added to polymeric scaffolds, bioactive glasses (BGs) have gained great 44

attention (Misra et al. 2009). For example, BG incorporation into scaffolds of polycaprolactone (PCL) or poly(D,L-45

lactic acid) improved cell growth and osteoblastic functional activity (Hidalgo-Bastida and Cartmell 2010, Kim et al. 46

2010). 47

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When the surface of bone-bioactive scaffolds possesses cell-adhesive motifs, their role in cellular anchorage 48

and subsequent differentiation processes would be greatly accelerated. In fact, a variety of cell-adhesive proteins or 49

peptides have been developed onto the surface of polymeric scaffolds (Zhang et al. 2010, Lee et al. 2013). Among the 50

molecules, fibronectin (FN) has been a potent adhesive ligand that particularly stimulates the anchorage of 51

mesenchymal stem cells (MSCs) (Lee et al. 2013). Accelerating initial adhesion events of MSCs is considered to be a 52

promising tool to improve the capacity of scaffolds targeting stem cell-based bone tissue engineering. To this end, we 53

aimed to tether FN molecules onto RD-based nanocomposite scaffolds made of BG nanocomponent (nBG) and PCL 54

matrix, called FN-nBG/PCL, and further to examine effects of FN immobilization on the adhesion and proliferation 55

of MSCs. 56

57

MATERIALS AND METHODS 58

Fabrication of RD-based nBG-incorporated PCL scaffolds 59

First, sol-gel processed nBGs with a starting composition of 85SiO2·15CaO were produced using calcium 60

nitrate tetrahydrate (Sigma-Aldrich, USA) and tetraethyl orthosilicate (Sigma-Aldrich, USA) as precursors in a 61

cosolvent of ethanol-water solution (Oh et al. 2010b). The precursors dissolved in ethanol were mixed with a surfactant 62

solution of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Sigma-Aldrich, USA) 63

containing a catalyst (NH4OH, Sigma-Aldrich, USA) and stirred overnight. The obtained nBGs were dried completely 64

5

at 70oC, heated from 100 oC to 500 oC following the crush, and pulverized by a ball mill to obtain fine nanoparticles. 65

Next, six-layered nBG/PCL lattice scaffolds (13 mm x 13 mm in square) with crisscross-aligned pores (0.5 66

mm x 0.5 mm) were fabricated using the homogenized slurry composed of nBG (10 w/v%) and PCL (30 w/v%, Sigma-67

Aldrich, USA) by a computer-based RD machine (Iwashita, Ez-ROBO3) equipped with a 0.33 mm-needle (Fig. 1) 68

(Oh et al. 2010a). Briefly, the nBG/PCL slurry homogenized in the barrel equipped with a thermo-jacket to maintain 69

high temperature (50 oC) was dispensed by an air compressor through a needle into the ethanol bath. Then, the layer 70

produced on the xy-plane was sequentially stacked along the z-axis, finally generating multi-layered scaffolds. The 71

morphology, structure, and pore size of scaffolds were analyzed by scanning electron microscopy (SEM, JSM-6510, 72

JEOL, Japan) and micro-computed tomography (CT, Skyscan 1176, USA). For the measurement of the water contact 73

angle, untreated, NaOH-treated, and FN-attached film surface from the slurry was prepared using a mold by a solvent 74

evaporation method. Each sample was glued onto a slide glass set upside-down on a water bath. Air bubbles were 75

supplied to each sample through a tube connected to an air-filled syringe. Contact angle between air bubble and water 76

droplet was analyzed by a DataPhysics Oca20 program. The roughness of the scaffold surface was measured using an 77

interferometer and represented as the three roughness parameters, Ra (average roughness), Rq (root-mean-square 78

roughness), and Rz (average partial roughness). 79

80

FN immobilization 81

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For FN immobilization, scaffolds were activated with ethyl(dimethylaminopropyl carbodiimide) (EDC, 0.2 82

M, Sigma-Aldrich, USA) and N-Hydroxysuccinimide (NHS, 0.4 M, Sigma-Aldrich, USA) after treatment with 0.5 M 83

of sodium hydroxide (NaOH, Sigma-Aldrich, USA) for 3 hr, and washed with distilled water three times to remove 84

non-adhesive FN molecules (Sigma-Aldrich, USA). The amount of FN immobilized onto the scaffold surface was 85

indirectly quantified using a micro-BCA assay (Thermo Scientific, USA) by calculating FN measurements remaining 86

in the media subtracted from a total FN amount. 87

88

Rat MSC (rMSC) isolation 89

rMSCs were obtained from bone marrow of five-week-old male Sprague–Dawley rats (Lee et al. 2011). For 90

rMSC isolation, bone marrow extracted from the tibia and femur of sacrificed rats was treated with 0.1 % of 91

collagenase type I (Sigma-Aldrich, USA) and 0.2 % of dispase II (Sigma-Aldrich, USA) for 30min. The harvested 92

cells were cultured in advanced-DMEM media (Gibco, USA) supplemented with 10 % of heat inactive fetal bovine 93

serum (Gibco, USA), 1mmol of L-glutamine (Sigma-Aldrich, US), and 1 % of penicillin (40000 U/ml)/ streptomycin 94

(40000 g/ml) (Gibco, USA) under humidified atmosphere of 5 % of CO2 at 37 oC. Media were refreshed every three 95

days for and cells were sub-cultured upon 80% of cell confluence. 96

97

Cell adhesion and proliferation 98

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rMSCs seeded at a density of 2.5x104 per scaffold were cultured for 14 days, and initial adhesion and 99

proliferation of cells were tested by a WST-1 (mitochondrial dehydrogenase activity, Roche, USA) assay. At the 100

determined time, cell-populated scaffolds were transferred to new culture wells to measure cell growth at 450 nm, 101

after replenished with a media containing 10 v/v% of WST reagent for 4hr. The absorbance was translated into cell 102

numbers using a calibration curve which was obtained by plotting the absorbance of a series of known cell 103

concentrations in culture dish. In order to further observe initial adhesion and growth, cells cultured on scaffolds were 104

investigated by SEM and confocal laser scanning microscopy (CLSM 510, Zeiss). For SEM investigation, samples 105

were dehydrated with a graded series of ethanol (70, 90, 95, and 100 %) after fixation with 2.5 % of glutaraldehyde 106

(Sigma-Aldrich, USA), and treated with a hexamethyldisilazane (Sigma-Aldrich, USA) for coating on platinum by 107

sputtering. For CLSM investigation, samples were treated with 4 % of paraformaldehyde (Dae-Jung, South Korea) 108

for 10min, with 0.2 % of triton X-100 (Sigma-Aldrich, USA) for penetration, and with 1 % bovine serum albumin for 109

blocking nonspecific binding. DAPI (Invitrogen, USA) for nucleus staining and Alexa Flour 546-conjugated 110

phalloidin (Invitrogen, USA) for F-actin staining were used and each cell dimension from the cell images was 111

measured using a ZEN 2009 program. 112

113

Statistical analysis 114

The data were expressed as the mean ± standard deviation (SD). Statistical comparisons were made by one-115

8

way ANOVA and the significances were considered at P < 0.05 and P < 0.01. 116

117

RESULTS AND DISCUSSION 118

Fabrication of RD nanocomposite scaffolds 119

3D bone scaffolds with open pore channels were fabricated by an RD technique, and their pore structure and 120

morphology were observed by μCT (Fig. 2(a)) and SEM (Fig. 2 (b)). The μCT images revealed square-shaped 121

scaffolds with a multi-layered lattice structure. The SEM micrographs showed a highly-interconnected pore structure 122

with open pores of approximately 210 μm in size, suggesting the generation of pores appropriate for cell ingrowth and 123

the associated vascularization. For the optimal cell ingrowth to the scaffolds, pore sizes of more than 150 μm were 124

generally proposed (Seo et al. 2014). In addition, the surface of nBG/PCL scaffolds appeared to be much rougher than 125

that of PCL scaffolds, due to the added nBG. The surface topography that could affect the cell adhesion and growth 126

should be fundamentally considered for the rational design of cell-adhesive scaffolds (Gentile et al. 2010). Thus, the 127

surface roughness parameters (Ra, Rq, and Rz) were obtained by interferometry (Table 1). All the parameters were 128

much higher for the nBG/PCL scaffolds. Recently, dental pulp-derived stem cells showed stimulated osteogenic 129

differentiation on the rougher laser-sintered titanium surface with respect to that on the conventional acid-etched 130

titanium surface (Mangano et al. 2010). Similarly, Kumar et al. reported that a rough topography on freeform 131

fabricated scaffolds improved the cell growth and osteogenic differentiation (Kumar et al. 2012). In line with these 132

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previous studies, our results on roughness suggest possible stimulation in cellular responses to the rougher 133

nanocomposite scaffolds. 134

135

Characteristics of FN-tethered scaffolds 136

Tethering the surface with cell-adhesive proteins by a simple coupling chemistry is well known to improve 137

cellular responses. Several tethering techniques of the proteins on the materials surface have been reported, such as 138

direct crosslinking (Zhang et al. 2010) and the biotinylation (Gorbahn et al. 2012). Here, we used an EDC-mediated 139

crosslinking method under aqueous conditions for FN immobilization, as depicted in Fig. 3(a). For EDC-mediated 140

crosslinking, the hydrophobic surface of PCL scaffolds was tailored to be hydrophilic through NaOH treatment. For 141

the case of nBG/PCL scaffolds, silanol groups (Si-OH) present on nBG should also be an effective site for chemical 142

crosslinking. FN immobilized to the surface was measured with varying initial FN concentration used (Fig. 3(b)). 143

With increasing initial FN concentration, FN immobilization increased quite linearly on nBG/PCL scaffolds; however, 144

on PCL scaffolds FN immobilization level reached a plateau above 20 g/ml. Particularly with the FN treatment of 145

40 g/ml, immobilized FN on nBG/PCL scaffolds became almost twice that on PCL scaffolds. 146

Next, water contact angles were measured at each surface modification step (untreated, NaOH-treated, and 147

FN-immobilized) using film-typed samples with the same compositions as the scaffolds, as presented in Table 2. The 148

contact angles were slightly lower on nBG/PCL than on PCL, indicating the incorporated nBG enhanced the 149

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hydrophilicity. Taken together, nBG incorporation improved the hydrophilic properties of the scaffolds that ultimately 150

increased the FN immobilization level. 151

152

In vitro cell tests 153

The initial adhesion of rMSCs to the scaffolds was assessed at 4 hr of culture, as shown in Fig. 4(a). Cell 154

adhesion levels on the FN-immobilized scaffolds were significantly higher than those on the untreated surfaces. The 155

highest adhesion level was found on the FN-nBG/PCL scaffold, which showed ~4 times higher than that on pure PCL 156

scaffold. Fig. 4(b) shows the cell morphology on the scaffolds after 4 hr of culture. Cell spreading behaviors were 157

observed to be better on FN-immobilized scaffolds than on untreated scaffolds. The cell spreading was then quantified 158

based on the cell images using ZEN 2009 software. The cell dimensional parameters, including perimeter, area, and 159

length of major axis could be obtained, as summarized in Table 3. All the measured values were substantially higher 160

on FN-immobilized scaffolds than on untreated scaffolds, demonstrating an effective role of FN molecules tethered to 161

the scaffolds in the initial cell adhesion and spreading processes of rMSCs. 162

Next, FN-tethering effects on cell growth were evaluated by means of WST-1, CLSM and SEM, as shown in 163

Fig. 5. Cell growth levels were measured for up to 14 days (Fig. 5(a)). Cells grew continuously on all the scaffolds 164

for 14 days with slight difference in their growth kinetics. The presence of FN significantly improved the cell growth 165

level; approximately twice increase in FN-treated scaffolds with respect to non-treated scaffolds, particularly at day 7. 166

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The cell morphology on FN-nBG/PCL scaffolds at day 14 was further analyzed by CLSM (Fig. 5 (b)) and SEM (Fig. 167

5(c)). The surface of the scaffolds was almost completely covered with cells, indicating that cells reached a confluence. 168

Particularly, some cells were found deep in the pore channels indicating their possible ingrowth into the pores; 169

furthermore, they interconnected the scaffold stems actively (indicated by white arrows), which however, was not 170

readily found in non-treated scaffold (not shown images). Collectively, FN molecules linked effectively to the surface 171

of nanocomposite scaffolds demonstrated their biological roles in accelerating initial cell adhesion and spreading, and 172

subsequent cell proliferation. 173

174

CONCLUSIONS 175

Cell-adhesive FN protein was effectively linked to bone-bioactive nanocomposite porous scaffolds. The 176

presence of nBG within scaffolds significantly enhanced the FN immobilization level. FN molecules linked to 177

scaffolds demonstrated significant improvements in adhesion, spreading and proliferation of rMSCs. Although further 178

in-depth studies on cell differentiation and in vivo findings still remain, the results support the promising uses of FN-179

nBG/PCL scaffolds for bone tissue engineering. 180

181

Acknowledgments 182

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This work was supported by a grant of Priority Research Centers Program (grant #2009-0093829), through the 183

National Research Foundation of Korea (NRF), Republic of Korea. This works has been also supported by the 184

Spanish Ministry of Economy and Competitiveness-MINECO (grant #MAT2011-29778-C02-01). 185

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Tables 232

Table 1. Surface roughness parameters of scaffolds (coefficient unit: m, Ra: average roughness, Rq: root mean square 233

of the values, Rz: average partial roughness). 234

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Coefficient PCL nBG/PCL

Ra 0.11 0.17

Rq 0.14 0.22

Rz 0.29 0.50

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Table 2. Water contact angles measured on the samples at each treatment process (n=8). 237

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Chemical processes PCL nBG/PCL

Untreated 80° 43°

NaOH-treated 43° 31°

FN-immobilized 31° 26°

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Table 3. Quantitative measurement of surface perimeter, area, and axis length of cells spread on the scaffolds with 240

four different characteristics. Results represent mean±SD (n=10). 241

Perimeter (m) Area (m2) Length of major axis (m)

PCL 102.9 ± 36.8 436.3 ± 180.7 39.4 ± 14.9

nBG/PCL 114.4 ± 42.1 419.6 ± 186.7 34.5 ± 13.8

FN-PCL 233.7 ± 67.8 1701.3 ± 606.9 63.5 ± 11.2

FN-nBG/PCL 248.2 ± 53.5 1612.3 ± 510.3 64.4 ± 17.9

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Figure captions 244

Fig. 1. Schematic drawing of a computer-based RD machine for the fabrication of open-macrochanneled scaffolds. 245

Fig. 2. (a) μ-CT image and (b) SEM images of scaffolds. 246

Fig. 3. (a) Chemical processes for FN immobilization onto scaffolds via EDC-mediated crosslinking, and (b) 247

quantitative analysis of FN immobilization as a function of initial FN treatment by a micro BCA assay. Results are 248

represented as mean ± SD (n=8). 249

Fig. 4. (a) Initial adhesion of rMSCs measured by a WST-1 assay (**P < 0.01 from comparison between FN-treated 250

and non-treated groups; #P<0.05 from comparison between nBG-added and non-added groups, by ANOVA for n=3). 251

(b) Morphology of rMSCs observed by CLSM. Actin cytoskeletons and nuclei were stained with Alexa Fluor 546-252

conjugated phalloidin (red) and DAPI (blue), respectively. All measurements and observation were performed on the 253

scaffolds after a 4-hr culture. 254

Fig. 5. (a) rMSC growth assessed by a WST-1 assay after culture for 1, 3, 7 and 14 days. (**P < 0.01 from comparison 255

between FN-treated and non-treated groups; #P<0.05 from comparison between nBG-added and non-added groups, 256

by ANOVA for n=3). (b) CLSM and (c) SEM images of rMSCs grown on FN-nBG/PCL scaffolds at day 14. The 257

white arrows indicate cells inside of the scaffold. 258

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