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SUPPORTING INFORMATION

Hierarchically Engineered Fibrous Scaffolds for Bone Regeneration

Nadège Sachot,

†,‡,§

Oscar Casta

ño,*

,

†,‡,§

Miguel A. Mateos-Timoneda,

‡,†

Elisabeth Engel

†,‡,§ and Josep A. Planell

†,‡,§

Biomaterials for Regenerative Therapies, Institute for Bioengineering of Catalonia (IBEC),

Baldiri Reixac 15-21, 08028 Barcelona, Spain

‡ CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain

§

Technical University of Catalonia (UPC), Diagonal 647, 08028 Barcelona, Spain

Materials and Methods:

Fibers deposition

:

A 3% w/w polymer solution was prepared by the dissolution of polylactic acid (PLDLA

95L/5DL PURAC) in 2,2,2-trifluoroethanol (TFE, Sigma-

Aldrich ≥99%) and electrospun by a conventional horizontal electrospinning set-up under the following conditions: 7.5 kV voltage,

13 cm distance tip-collector and 0.5 mL/h flow rate.

Glass preparation

:

Calcium 2-methoxyethoxide (CaO) and phosphorous alkoxide precursors were prepared in our laboratory by refluxing metallic Ca (Sigma-Aldrich 98%) in 2-methoxyethanol (Sigma-

Aldrich, anhydrous, 99.8%)

1

and solid P

2

O

5

(Sigma-Aldrich, 99.99%) in absolute ethanol

(Panreac, 99.98%)

2

. The ormoglass solution was obtained by mixing commercial tetraethylorthosilicate (TEOS, Aldrich, 98%), CaO and P

2

O

5

alkoxide precursors under inert atmosphere (55% Si, 10% P

2

and 35% Ca molar ratio).

Coating treatment

:

Fibers were hydrolyzed with a 0.1 M NaOH (Panreac, 98%) solution for 150 seconds and immersed in a 0.1/0.2 M EDC/NHS (Aldrich 97% / Aldrich, 98%) ethanolic solution for 1 hour.

Afterwards, the APTES solution (Acros Organics, 99%, 10% v/v in ethanol) was added to react with the activated carboxyl groups for 2 hours. In parallel, the ormoglass was hydrolyzed with water (Si: H

2

O molar ratio) for 150 seconds and functionalization was performed by immersing the fibers for 2 hours in this hydrolyzed ormoglass, previously diluted with absolute ethanol (1:1.5 v:v). Between each treatment, the reagent was removed and fibers were washed with absolute ethanol. Once coated with the glass, fibers were finally placed in an ultrasonic bath during 5 minutes to remove the non reacted-residual compounds.

Material Characterization:

FESEM imaging

:

After drying and carbon coating, fibers were loaded to a field emission scanning electron microscope (FESEM, Nova™-Nano SEM-230; FEI Co., the Netherlands) to observe their topography. The pictures were taken at an accelerating voltage of 5 kV and a working distance of 5 mm. Pictures with cells fixed on the scaffolds were obtained in the same conditions. Fibers thickness average was determined by measuring a minimum of 15 fibers diameters (before and after coating) using ImageJ Software.

3

EDS elemental quantification

:

The same sample used for FESEM was loaded to an electron scanning microscope with an

EDX detector (Quanta 200 XTE 325/D8395; FEI Co., the Netherlands) to determine the exact coating composition. Three different places were arbitrarily selected on the scaffold to perform the measurements (30s acquisition time for each points - Si, Ca and P quantification).

FIB tubular structure imaging

:

The sample was coated with carbon as for FESEM imaging and loaded to a focus ion beam apparatus (Strata DB235; FEI Co., the Netherlands). Thanks to ions ablation, a square hole was created in the fiber mat in order to obtain a cross section of the tubular structure.

Pictures were taken using an accelerating voltage of 5 kV, a working distance of 5 mm and a tilted angle of 45º. Inner fiber diameter was approached by considering 10 fiber cross sections. Trigonometry was used to apply the proper correction of scale associated to the tilt of the sample.

AFM stiffness and roughness determination

:

Fibers stiffness and roughness (Root Mean Square roughness Rq) were investigated using

AFM (MultiMode 8 Atomic Force Microscope, Bruker and NanoScope Analysis, v1.2) in

PeakForce tapping mode in air. Thin layer of fibers was deposited on an adhesive substrate and scanned along the fibers length (individual fiber measurements). Stiffness was assessed by evaluating the DMT modulus which is calculated by taking into account the load force and adhesion between the tip and the sample.

4

Figure A.

AFM 3D images showing the roughness and nanostructured topography of pure

PLA and coated fibers. (P

LA fibers Rq= 3.3 ± 0.6 nm, coated fibers Rq= 15.0 ± 6.1 nm).

Tensile assays

:

Fibers strips of 50x10 mm were used to evaluate the Young’s Modulus and yield strength of the scaffolds by tensile tests (Adamel Lhomargy DY-34, France). Strips were clamped between the grips of the machine and then subjected to elongation (setting parameters previously defined). The thickness of the strips, needed for the calculation of the desired mechanical values, was determined by FESEM and measured thanks to the ImageJ

Software.

3

Figure B.

Typical example of FESEM picture used for the determination of the fibrous mat thickness.

PLA

Fibers

Coated

Fibers

Young’s

Modulus

[MPa]

Yield

Strength

[MPa]

7.6

± 2.7 0.3 ± 0.1

48.7

±

9.0

1.3

± 0.4

Table C.

Mechanical characteristics of the coated fibers obtained by tensile assays, attesting of the higher mechanical resistance of the produced scaffold (whole structure) compared to

PLA one.

Figure D.

Picture of the fibrous layer obtained after coating showing the remarkable flexibility of the scaffold despite the increase in the DMT and Young’s moduli.

Water contact angle measurements

:

Characterization of fibers wettability was performed via contact-angle measurements using an OCA 20 system (Dataphysics, GmbH) and the sessile drop technique. This method consists in the measurement of the equilibrium angle formed between a 3 µL of an ultra high pure water drop and the surface of the fibrous sheet. Measurements were performed on three different points for each sample.

In vitro

assays:

Mesenchymal stem cells obtained from rat bone marrow were used for this study. Squares were cut from the fibers layer to fit in a 24 well-plate, sterilized by immersion in ethanol for 15 min under UV radiation, pre-incubated for 2 h in complete medium at 37°C and rinsed with

PBS before seeding. Cells were seeded at a density of 10000 cells/disc in 24 well-plates.

After 24 hours, cells were fixed using a paraformaldehyde-sacarose-H

2

O-PBS solution for the fluorescence staining. 4,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei and green phalloidin for the actin cytoskeleton. Fluorescent images were collected using a confocal microscope with a x63 objective lens (oil immersion). Cells observed by FESEM were fixed using a glutaraldehyde-PBS solution and then lyophilized before carbon coating.

References:

1

Castano, O.; Navarro, M.; Planell, J. A.; Engel, E.; Aguirre, A.

European Patent

EP2386525

2012

.

2

Ali, A. F.; Mustarelli, P.; Magistris, A. Mater. Res. Bull. 1998 ,

33

, 697.

3

Rasband, W. S., J. Image, US National Institute of Health, Bethesda, USA, http://imagej.nih.gov/ij/,

1997

–2011

.

4

Derjaguin, B. V.; Muller, V. M.; Toporov, Y. P. J. Colloid Inter. Sci., 1975 ,

53

, 314.

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