Materials
Polycaprolactone (PCL) (average Mn 45000 g/mol, Sigma-Aldrich, Milan, Italy) was received in pellet form and purified via dissolution in CHCl3 (15% wt/vol) and reprecipitation in a large excess of cold MeOH, in order to eliminate residual polymerization catalysts.
Poly(L-lactic acid) (PLA) (MW = 65000 g/mol, IngeoTM biopolymer PLA 4060D, Natureworks LLC, Blair, NE, USA) was received in pellet form and purified via dissolution in CHCl3 (10% wt/vol) and re-precipitated in a large excess of cold MeOH, in order to eliminate residual polymerization catalysts.
Methanol (MeOH), ethanol (EtOH, 99.8%), 1,4-dioxan (DIOX) and chloroform (CHCl3, HPLC grade) all from Sigma Aldrich (Milan, Italy) were used as received without further purification.
Dicalcium phosphate dihydrate (DCPD; CaHPO4·2H2O) powder (Sigma-Aldrich, Steinheim, Germany) and/or calcium silicate (CaSi) powders (Aalborg, Denmark), prepared by melt-quenching technique and milling procedures, as previously reported (19), and composed of dicalcium silicate, tricalcium silicate, tricalcium aluminate, and calcium sulfate, were added to PLA or PCL.
All plastic used for standard culture condition were from BD-Falcon (Corning, NY USA). Antibiotic–antimycotic, Dulbecco’s phosphate buffered saline (DPBS), DAPI staining solution were purchased from Gibco-Life Technologies (Carlsbad CA, USA). Trypsin–EDTA solution1X, MTT based assay were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pericyte Growth Medium was purchased from Promocell (Heidelberg, Germany). NucleoSpin RNA kit was purchased from Macherey-Nagel GmbH & Co. KG (Düren, Germany).
TIPS Scaffolds preparation
The porous polymer scaffolds were prepared by TIPS starting from PLA or PCL solutions in DIOX (3.5% wt/vol). CaSi and DCPD were added to the polymer solution in powder form in 10% by weight with respect to PLA or PCL.
Homogeneous dispersions were obtained by sonicating the mixtures for 3 hours using the ultrasonic processor UP50H (Hielsher, 50 watts, 30 kHz), equipped with the sonotrode MS2 (made of titanium, tip diameter 2 mm). After mixing, solutions were placed inside disposable aluminum dishes of 60 mm in diameter, and cooled at –18°C. After 18 h, the frozen samples were extracted from the holders and fully immersed in EtOH bath precooled at –18°C, where they were kept for 48 h, with solvent refresh every 3 h. At the end of the extraction procedure, the porous scaffolds were taken out of the freezer and completely dried under vacuum (24). Six disks (diameter 60 ± 1 mm, thickness 10±0.1 mm) per composition were prepared.The prepared scaffolds were: PLA, PLA–10CaSi–10DCPD, PCL, PCL–10CaSi–10DCPD.
Surface porosity evaluated by the morphometric analysis on ESEM images
The scaffolds were examined using an environmental scanning electron microscope (ESEM, Zeiss EVO 50; Carl Zeiss, Oberkochen, Germany). Specimens were placed directly onto the ESEM stub and examined in wet conditions without any previous preparation (the samples were not coated for this analysis) at low vacuum (100 Pascal) in Quadrant Back-Scattering Detector (QBSD) mode, using an accelerating voltage of 20 kV, working distance 8.5 mm, 0.5 wt% detection level, 133 eV resolution, amplification time 100 μs, measuring time 60 s.
ESEM images were analyzed through Image J program (National Institutes of Health, Bethesda, USA) to evaluate the scaffolds porosity, in terms of percentage. Surface porosity was calculated as the ratio between the most black areas (micropores) and the total examined area (24,36). For each scaffold three measurements were performed in three different areas at 500× and 1000× magnification. For each magnification, the mean value was recorded.
Isolation and culture of primary porcine Vascular Wall Mesenchymal Stem Cells
Primary porcine Vascular Wall Mesenchymal Stem Cells (pVW-MSCs) were isolated from the thoracic aorta of female 3-months-old pigs (Large White) euthanized for other experimental purposes, following the published methods previously described (11,37): briefly, young commercial hybrids of Sus scrofa (4 males–aged 4–5 wk, 7 ± 0.5 Kg live weight), born at the ASA Unit (DIMEVET, University of Bologna), were enrolled in the study. Piglets were bred under the lactating sow till 28 d, then weaned and kept in a multiple box for young piglets, temperature was kept at 28 ± 1 °C with adequate ventilation and humidity in relation to the young age. Surgical procedures were carried out during the morning in the surgical theatre of the DIMEVET facilities. Animal received an i.m. bolus of tiletamine-zolazepam (5 mg/kg) 10 min before induction; general anesthesia was achieved using sevoflurane with an induction mask. Animals were then sacrificed with a single bolus (0.3 mL/kg) of Tanax (embutramide/mebezonium iodide/tetracaine hydrochloride; Msd Animal Health Srl). All procedures on pigs were reviewed and approved in advance by the Ethical Committee of the University of Bologna (Bologna, Italy) and were then approved by the Italian Ministry of Health (Protocol number n.43-IX/9 all.37; 20/11/2012).. Cells were grown and expanded not beyond till passage (P) 6 in Pericyte Growth Medium (PGM—Promocell). All the experiments described in this paper were performed with cells at the third passage (P3), cultured in Pericyte Growth Medium (PGM).
Cell seeding efficiency assay
All the scaffolds were sterilized by 30 min of incubation in absolute EtOH followed by three washes in DPBS (30 min each one), then cubic-shaped sections of about 1 mm on each side were produced with the help of scalpel blades. 30 sections were suspended in 500 ul of culture medium (Pericyte Growth Medium) then pVW-MSCs were seeded on different biomaterials (PLA–10CaSi–10DCPD and PCL–10CaSi–10DCPD) following a published method (38) with some main adaptations. The biomaterials were drop-seeded with 100 µl of a concentrated cell suspension containing 4 x 105 or 8 x 105 cells. The microcentrifuge tubes were placed on a rocker oscillating at 30 rpm for 2 hours to allow for initial cell attachment then fresh cell culture medium was slowly added to each tube and cells were cultured for additional 24, 48, 72 hours in static condition at 38.5°C at 5% CO2. Cell-free biomaterials were incubated under same conditions and used as control.
Cell seeding efficiency (CSE) was estimated using an indirect method (38) after 24, 48 and 72 hours. Briefly the CSE was calculated based on the number of unattached cells that still present in the medium using the following equation CSE (%) = (1-cellsu/cellsi) x 100, where cellsi is the number of cells initially seeded and cellsu is the number of unattached cells in the residual medium and in DPBS used for rinsing cell-seeded biomaterials. Unattached cells were counted by hemocytometer in three different aliquots of medium collected from each sample.
Metabolic cell activity assay
The metabolic pVW-MSCs activity was monitored at 24, 48 and 72 hours using MTT based assay (Sigma) following the manufacturer’s instruction, with some main adaptation. Ten sections were removed at each experimental point, then scaffold sections were washed twice with DPBS and then 20 µl of MTT substrate was added and recovered for 4 hours at 38,5 °C at 5% CO2.After that, 200 µl of solubilisation solution was added and, after 30 min, the solution was vigorously mixed and the absorbance at a wavelength of 570 nm was measured spectrophotometrically, with the background substraction at 690 nm.
RNA extraction and qPCR
RNA extraction was performed using TRI Reagent (Molecular Research Center) and NucleoSpin
Briefly, culture medium was removed at 72 h and different scaffolds where washed with DPBS (twice); 500 ml of TRI Reagent (Molecular Research Center, Inc.) was added and the materials where lysed by using TH Tissue Homogenizer (Omni International GA, 30144 United States). Following, 100 µl of chloroform was added to the suspension and mixed well. After incubation at room temperature (10 min), samples were centrifuged (12000 g for 10 min) and the aqueous phase was recovered. An equal volume of absolute ethanol was added and the resulting solution was applied to the Nucleo spin RNA Column, RNA was finally purified according to the manufacturer’s instructions. After spectrophotometric quantification (Denovix, Denovix Inc., Wilmington, NC) total RNA (500 ng) was reverse-transcripted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) in a final volume of 20 μl. Swine primers were designed using Beacon Designer 2.07 (Premier Biosoft International, Palo Alto, CA, USA). Primer sequences, expected PCR product lengths and accession numbers in the NCBI database are shown in Table 1.
To evaluate gene expression profiles, quantitative real-time PCR (qPCR) was performed in CFX96 (Bio-Rad) thermal cycler using a multiplex real time reaction for reference genes (GAPDH, glyceraldehyde–3-phosphate dehydrogenase; HPRT, hypoxanthine guanine phosphoribosyl transferase; β-Actin), and using Taq-Man probes and SYBR green detection for target genes (CD90, α-SMA, NG2 and PDGFR-b). All amplification reactions were performed in 20 μL and analyzed in duplicates (10 μl/well). Multiplex PCR contained: 10 μl of iTaqMan Probes Supermix (Bio-RAD), 1 μL of forward and reverse primers (5 μM each) of each reference gene, 0.8 μl of iTaq-Man Probes (5 μM) of each reference gene, 2 μl cDNA and 2.6 μL of water. The following temperature profile was used: initial denaturation at 95°C for 30 seconds followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds.
The SYBR Green reaction contained: 10 μl of IQSYBR Green Supermix (Bio-RAD), 0.8 μl of forward and reverse primers (5 μM each) of each target gene, 2 μl cDNA and 7.2 μl of water. The real-time program included an initial denaturation period of 1.5 min at 95°C, 40 cycles at 95°C for 15 s, and 60°C for 30 s, followed by a melting step with ramping from 55°C to 95°C at a rate of 0.5°C/10 s.
The specificity of the amplified PCR products was confirmed by agarose gel electrophoresis and melting curve analysis.
The relative expressions of the studied genes were normalized based on the geometric mean of the three reference genes. The relative mRNA expression of tested genes was evaluated as fold of increase using the 2-ΔΔCT method (39) referred to control (pVW-MSCs cultured in flask under standard 2D culture condition).
DAPI staining and immunofluorescence
To test cellular distribution in the different scaffolds, pVW-MSCs cultured for 72 hours were processed for labeling DNA in fluorescence with DAPI staining and alpha-tubulin immunofluorescence. In particular biomaterials were washed twice in DPBS then were fixed ON in cold 4% Formaldehyde Solution in PBS, pH 7,4. Each sample was transferred into a 25% sucrose (Sigma-Aldrich, MO, USA) solution in PBS at 4°C for 24 hours to get cryoprotection. Finally, samples were embedded and freezed in OCT (Sakura Finetek, CA, USA). Ten micrometers sections were cut at a Leica CM1950 cryostat (Leica, Wetzlar, Germany) mounted on microscope’s slides and stainined with DAPI Staining Solution (Thermo Fischer). For immunofluorescence staining slides were completely dried at room temperature (RT), washed three times in PBS 1X for 5 minutes, permeabilized with Triton X–100 0,1% in PBS 1X for 1 hour then washed three times in PBS 1X for 5 minutes. For aspecific sites blocking slides were treated with 10% Normal Goat Serum in PBS 1X for 1 hour at RT then incubated ON in a humidified chamber with an anti alpha-tubulin antibody (Clone TU–01, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:250 in PBS 1X. In negative controls the primary antibody was omitted. At this point samples were washed three times in PBS 1X, incubated with anti-mouse FITC conjugate antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:100 in PBS 1X for 1 hour at RT in a humidified chamber. After two washes for 5 min in PBS 1X and one wash in distilled water for 5 minutes, coverslips were mounted on slides with Fluoreshield with PI (Sigma-Aldrich, St. Louis, MO, USA). Photomicrographs were obtained using a Nikon digital camera installed on a Nikon epifluorescence microscope (Nikon Inc., Melville, NY, USA).