2.2. Preparation and Characterization of Electrospun Meshes

To obtain ALD-loaded electrospun fibers, two acid-terminated PLGA polymers with 50:50 lactide:glycolide ratio, Resomer^® RG502 (0.19 dL/g, Evonik), and Resomer^® RG504 (0.4 dL/g Evonik) were chemically conjugated with ALD following the protocol described by Choi and coworkers (Choi et al., 2007). A 10% PLGA solution was prepared in 20 mL acetone to which 60 mg of N-hydroxysuccinamide (NHS, Sigma-Aldrich) and 100 mg of N, N′-dicyclohexylcarbodiimide (DCC, Sigma-Aldrich) were added and allowed to react in inert atmosphere under stirring for 24 h at room temperature. Afterwards, the reaction mixture was filtrated through 0.45 µm (Chromafil^® Xtra PET-45/25, Macherey-Nagel, Lake Forest, CA, USA) to remove dicyclohexylurea and the obtained PLGA-NHS was precipitated in cold ethyl ether (Scharlau^®, Barcelona, Spain) and dissolved in DCM. This solution was concentrated at room temperature until drying using a rotavapor.

ALD conjugation was performed by dissolving 1.9 g of the obtained PLGA-NHS in 19 mL of Acetone:DMSO (50:50) and adding an adequate amount of ALD previously dissolved in 1 mL of water. This mixture was allowed to react at room temperature for another 24 h. Then, the solution was concentrated by rotavapor at high vacuum and 37 °C. The obtained ALD conjugated polymer (PLGA-ALD) was dissolved in the minimum amount of acetone, precipitated in ethyl ether and dissolved again in DCM to avoid free ALD. Finally, this solution was dried for 24 h under high vacuum and the PLGA-ALD was precipitated with distilled water, filtrated through 0.45 μm, and freeze-dried. To ensure adequate functionalization, the obtained polymer was characterized by proton nuclear magnetic resonance. ¹H-NMR spectra were recorded at 500 MHz (Bruker Avance 500, Billerica, MA, USA), and chemical shifts were reported in ppm and calibrated on non-deuterated solvent residual peak. Elemental analysis was performed using a CHNS TruSpec Micro analyzer (LECO, St. Joseph, MI, USA).

Electrospun meshes composed by RG504-ALD: RG502-ALD: RG 855 at 1:1:1 ratio were prepared. The polymers mixture (375 mg) was dissolved in 2.5 mL of Hexafluoroisopropanol (HFIP, Sigma-Aldrich) and loaded into a syringe equipped with a 18G needle. This solution was ejected at a continuous flow of 2.75 mL/h using a syringe pump (Harvard Apparatus ^®, Holliston, MA, USA) under an electric field of 7 KV. Fibers were collected on a cylindrical metal collector that rotates at 200 rpm located at a 10 cm distance from the syringe. The process took place at room temperature and 65% of relative humidity. Blank meshes with the same polymers proportions and conditions were also obtained using unfunctionalized PLGAs. Blank and ALD loaded electrospun meshes were surface treated with plasma (O₂). The plasma oxygen treatment was carried out under vacuum during 4 min for each side of the meshes. The generator power was at 75% of the capacity (Diener electronic, Plasma-Surface-Technology, Ebhausen, Germany).

Meshes were characterized in terms of thickness, porosity, contact angle, and fiber diameter. Mean fiber diameter was obtained measuring 50 fibers for each sample after silver coating using scanning electron microscopy images (SEM, JSM 6300, JEOL) at a 1500 magnification by an image analysis software (ImageJ v1.52, National Institute of Health, Madison, WI, USA). Mesh thickness was obtained using stereomicroscope images (Leica M205 C, LAS, v3 software, Leica, Wetzlar, Germany).

Real mesh density was obtained by helium pycnometry (Micromeritics, AccuPyc 1330, Norcross, GA, USA) while apparent density was calculated from the weight and volume values. Volume was obtained from the length, width, and thickness as described in Equation (2). Mesh total porosity was calculated using real density and apparent density values as shown in Equation (3).

Mesh surface wettability was measured by contact angle assessments using a drop shape analyzer (DSA100; Krüss GmbH, Hamburg, Germany) and distilled water as the liquid media. The obtained images were analyzed with an image analyze software (ImageJ v1.52, National Institute of Health).

A proton NMR spectrum was performed on a 500 MHz equipment to determine the amount of ALD present on ALD-loaded electrospun meshes. To do so, 4.41 mg of the sheet were dissolved in 0.6 mL of CDCl₃, and 18.4 μL of a standard solution of 1,1,2,2-tetrachloroethane (Cl₂CHCHCl₂) in CDCl₃ (33.07 mM) was added. In this way, the amount of Cl₂CHCHCl₂ was four times the theoretical amount of alendronate presented in the polymer sheet. The quantity of ALD was obtained by integrating the signals corresponding to 1,1,2,2-tetrachloroethane (5.96 ppm) and the alendronate signal of the protons (2.98 ppm). These measurements were performed also at different time points on ALD-loaded meshes incubated in water at 37 °C to evaluate ALD release. To this end, samples were washed twice with water and freeze-dried previous to NMR characterization.