Scaffolds and materials
For the fabrication of the silver-containing nanofabric, clinically approved poly(lactide-co-glycolide) (PLGA) with a copolymer ratio of 85:15 (Resomer Sample MD Type RG) was purchased from Boehringer Ingelheim with a weight and number average molecular weight of 380,300 and 181,900 g mol−1, respectively. Fibers with PLGA/Ag-TCP containing 2% silver in a 70:30 ratio were fabricated by an electrospinning process [13]. Each electrospinning solution was prepared with a concentration of 5.9% (w/w) PLGA in chloroform (Riedel de Haen, Ph. Eur.) containing 6.4% (w/w) of the surfactant Tween20 (Polysorbate20, Fluka, Ph. Eur.) referred to the polymer. For the preparation of the electrospinning solution, corresponding amounts of the nanoparticles were first dispersed in a chloroform/Tween20 stock solution using an ultrasonic processor at 70 W for 5 min. PLGA was subsequently added and dissolved for 15 h by magnetic stirring. Electrospinning was performed by feeding the solutions through four capillaries (inner diameter 1.0 mm) using a syringe pump. The feeding rate was set to 8 mL h−1. A voltage of 24 kV was applied to the needle tips, which was kept in a chloroform–air stream (160 mlmin−1) by a concentrically mounted polyetheretherketone (PEEK) adapter. A positively charged jet was formed from the Taylor cone and was sprayed onto a rotating (100 rpm) collection tube covered by an aluminium foil. The distance between the needle tip and the collection tube (diameter 8 cm) was kept constant at 17 cm. The as-spun scaffolds were dried and stored in vacuum at room temperature.
As reference, a collagen scaffold with and without hyaluronan acid (Coll-HA, Coll) was used in the present study (Bio-Gide®, Geistlich, Wolhusen, Switzerland; LOT 81500056; molecular weight of collagen 1 300 KDa). The hyaluronan acid (Regedent, Zurich, Switzerland; LOT MK2-1714/1) applied on the membrane contained 2 mg sodium hyaluronate (molecular weight 2.5 MDa), 16 mg cross-linked sodium hyaluronate (molecular weight 1 MDa), 6.9 mg sodium chloride and water for injection ad 1 ml (Vol. 1.2 ml).
Cell culture
Human dental pulps of two anonymous donors below 30 years were isolated from caries-free third molars after informed consent was obtained (Ethics Committee of the Medical University Vienna; EK 631/2007). Dental pulp tissues were left in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FCS; PAA Laboratories, Linz, Austria) and antibiotics (Invitrogen) at 37 °C, 5% CO2, and 95% humidity. Dental pulp cells grown out from the pulp were cultured. Cells that had not undergone more than five passages were used and cell seeding for indicated experiments was performed in growth medium at 30,000 cells/cm2. The cell density was based on previous in vitro studies on the impact of biomaterials on cell activity [19]. Experiments on viability and proliferation were performed with two different cell donors, each in duplicates. Gene expression analyses were performed twice, each time in duplicates, using pooled dental pulp cells of the two donors.
Preparation and pretreatment of the scaffolds
For MTT tests and BrdU incorporation assays silver-containing nanofabric (PLGA/Ag-TCP) scaffolds and collagen (Coll) scaffolds of Ø 6 mm were gained from corresponding scaffolds using a biopsy punch. For gene expression, analysis scaffolds of Ø 2.2 cm were cut. One half of the collagen scaffolds was soaked in cross-linked hyaluronan acid for 1 min and thoroughly washed with PBS for 2 min (Coll-HA) immediately before stimulation of the cells.
In vitro models (Model I, Model II)
Two different in vitro models were used. Cells were precultured and the scaffold was placed thereon (Model I – scaffold on cells) and cells were cultured directly on the scaffold (Model II cells on scaffold). In both models cell viability and proliferation was assessed based on formazan formation (MTT test) and BrdU incorporation, respectively, after 24, 48, and 72 h. Gene expression of IL-6, TNF-α and AP was analyzed after 24 h.
MTT assay
For model I, where cells were cultured in the presence of the scaffolds, the latter were removed from the plates just before 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added. In model II, where the cells were cultured directly on the scaffold, MTT was added in each well and incubated for 2 h at 37 °C. Afterwards, formazan crystals, formed by the NAD(P)H-dependent oxidoreductases, were dissolved in dimethyl sulfoxide. Optical density was measured with a microplate reader (EL 808, Biotek Instruments, Winooski, VT, USA) and normalized to untreated cells.
BrdU incorporation assay
For model I similarly to the MTT assay, scaffolds were removed from the culture plates before the BrdU incorporation assay was performed. For model II growth medium was removed, dental pulp fibroblasts were washed with PBS and serum-free media containing 5-bromo-2′-deoxyuridine (BrdU) was added to the cells for 2 h. BrdU incorporation was determined following the manufacturer’s instructions (Cell Proliferation ELISA, BrdU colorimetric kit from Roche; Basel, Switzerland) and normalized to untreated cells.
Scanning electron microscopy
Surface structure topography of the scaffolds was examined by a scanning electron microscope at magnifications of × 500 at the Center of Microscopy and Image Analysis, University of Zurich (scanning electron microscopy (SEM); Carl Zeiss Supra 50 VP FESEM, Carl Zeiss). For this purpose, the samples were fixed for 24 h in 2.5% glutaraldehyde solution. Afterward, the scaffolds were rinsed with PBS and dehydrated in ascending concentrations of alcohol (50, 70, 80, and 90%) twice for 15 min. Finally, the scaffolds were immersed three times for 15 min in 94% and 60 min in 100% ethanol. Samples were then subjected to critical point drying (Bal-Tec CPD030), mounted on SEM mounts (Bal-Tec AG, Blazers, Liechtenstein), and were gold sputtered (Balzers SCD 030, Balzers Union, Balzers, Liechtenstein) for 60 s in an argon gas atmosphere at a target distance of 50 mm, at pressure of 5 Pascal (Pa) at 45 mA. SEM images were taken at a working distance of an acceleration voltage of 10 kV.
Gene expression analysis
Twenty-four hours after stimulation, the scaffolds were either removed (Model I) or retained (Model II) for gene expression analysis. Total cellular RNA was isolated with the High Pure RNA Isolation Kit (Roche F. Hoffmann-La Roche, Basel, Switzerland). Reverse transcription (RT) was performed with Transcriptor Universal cDNA Master (Roche) and PCR was performed with the FastStart Universal SYBR Green Master (Roche) on a 7500 Real-Time PCR System (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA). Primers against the pro-inflammatory cytokines human IL-6 and TNF-α representing first reaction to an inflammation, as well as against AP as an early differentiation marker [20], and GAPDH, the house-keeping gene were designed in the online Universal ProbeLibrary Assay Design Center (Roche). The mRNA levels were calculated by normalizing to GAPDH using the ΔΔCt method.
Statistical analysis
Data obtained by MTT and BrdU incorporation assays were reported as median, 25% percentile, 75% percentile, minimum and maximum of two independent experiments, each performed in duplicates. Differences between cells stimulated with the three scaffolds were tested using a non-parametric Kruskal-Wallis test followed by a post-hoc Mann-Whitney U-test. Furthermore differences between the two cell seeding methods and the three different stimulation periods were statistically analyzed (SPSS version 19.0, SPSS Inc., Chicago, IL, USA). Statistical significance was considered at p < 0.05. Data obtained by RT PCR, showing the differences in mRNA expression of target genes between cells stimulated with the different scaffolds and unstimulated cells were described by the mean and standard deviation.