2.1 Scaffold manufacturing

The scaffolds were manufactured as described previously [16, 17]. Briefly, 8 wt.% A2P (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) was melt-mixed with PLCL 70 L/30CL (PLCL 7015, Corbion Purac BV, Gorinchem, Te Netherlands) in a twin-screw extrusion process. The extruded rods were foamed with scCO2 (Waters Operating Corporation, Milford, MA, USA) using high pressure and temperature. Thereafter, the foamed rods were cut into discs with a thickness of 3–3.5 mm, resulting in scPLCLA2P porous scaffolds containing 4.7 ± 0.4 mg of A2P. Finally, the scaffolds were gamma-irradiated for sterility with a minimum dose of 25 kGy before the cell culture experiments. The porous scPLCL scaffolds were manufactured similarly, excluding melt mixing of A2P.

2.2 MicroCT (μCT)

The porosity, pore sizes, and interconnectivity of the samples were analyzed with X-ray microtomography (μCT). The samples were imaged with a Zeiss Xradia MicroXCT-400 (Carl Zeiss X-ray Microscopy Inc., Pleasanton, CA, USA) device. A total of 1601 projections were acquired with one second of exposure time. The source voltage was 80 kV, and the current was 125 µA. The reconstruction was made with XMReconstructor software, resulting in a pixel size of 5.64 µm. Image processing and visualizations were performed with Avizo 2022.2 software (Thermo Fisher Scientific, Waltham, MA, USA). The pore size analysis was based on the BoneJ plugin in Fiji software [32]. The pore network and interconnectivity calculations were performed with an in-house MATLAB (The MathWorks Inc., Natick, MA, USA) program, which is described in more detail in [33]. The scPLCLA2P scaffold particle segmentation, labeling and measurement were performed with Avizo software. Because of the imaging resolution, particles that were smaller than 500 µm3 in volume were excluded from the analysis.

2.3 Cell isolation and seeding

Human ECs and SCs for this study were obtained from 3 different donors. Cell isolation was performed as described previously [16]. Briefly, the tissue samples were cut into small pieces and digested in a solution of collagenase and dispase. The suspension was filtered, centrifuged, and plated in a CellBind T75 flask (Sigma-Aldrich, St. Louis, MO, USA) with EpiLife™ medium (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% EpiLife™ defined growth supplement (EDGS; Invitrogen), 0.1% CaCl2 (Invitrogen) and 0.35% antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin [Lonza, BioWhittaker, Verviers, Belgium]). After primary culturing, the EC and SC lines were separated, and the cells were treated with TrypLE Select (Gibco, Thermo Fisher Scientific). The loosened cells were passaged to a T75 flask (Nunc, Thermo Fisher Scientific) in DMEM/F12 (basic medium, BM; Thermo Fisher Scientific) supplemented with 5% human serum (Biowest, Nuaille´, France), 1% GlutaMAX (Life Technologies, Thermo Fisher Scientific) and 1% antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin; Lonza), resulting in the human vaginal SC line. The remaining cells, the human vaginal EC line, were further treated with TrypLE Select and passaged to T75 flasks with EpiLife™ medium. Both cell lines were passaged when confluent and frozen until experiments. ECs and SCs at passages 3–5 were used in the in vitro experiments.

Prior to cell seeding, the scPLCLA2P scaffolds were prewetted in BM for 24 h at 37 °C and placed in the wells of 48-well plates (Nunc) for cell culture experiments. The experiments were performed either by culturing ECs or SCs as monocultures in separate scaffolds or coculturing the ECs and SCs on opposite sides of the same scaffold. In monocultures, 100,000 vaginal ECs or SCs in 15 μl of medium were plated on both sides of the scPLCLA2P scaffold, and the cells were allowed to adhere for 2 h after 500 ml of EpiLife™ or BM was added, respectively. For cocultures, 100,000 SCs were plated on the scPLCLA2P scaffold in 15 μl of medium and precultured for 5 days in BM. After 100,000 ECs were plated in 15 μl of medium on the other side of the scPLCLA2P scaffold, the cocultures were cultured on EpiLife™ medium.

The cells were incubated in a humified atmosphere of 5% CO2 in air at 37 °C until analysis, and the specific medium was changed three times per week. The analyses were performed after 1, 7 or 14 d of cell culture. For cocultures, the time points followed the implantation of ECs; therefore, the corresponding culturing days for SCs in cocultures were 6, 12 or 19 days. The experiments and time points in mono- and/or cocultures are represented in the Table 1.

Table 1 Overview of the analyses, samples and timepoints2.4 Scanning electron microscopy (SEM)

To evaluate the attachment and morphology of the ECs and SCs in monocultures, scanning electron microscopy (SEM) imaging was performed at d 1, d 7, and d 14 [16]. Briefly, the cells were fixed with 5% glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.4, Sigma-Aldrich) at room temperature (RT) for 48 h. Then, the samples were dehydrated through a sequence of increasing concentrations (30, 50, 70, 80, 90. 95 and 100%) ethanol for 5 min and finally in a solution of 1:2 hexamethyldisilazane (HMDS, Sigma-Aldrich) and 100% ethanol (Altia Oyj, Helsinki, Finland) for 20 min following incubation in 2:1 HMDS and ethanol for 20 min. The samples were allowed to evaporate overnight in a fume, carbon sputtered and imaged with SEM (Zeiss ULTRAplus, Oberkochen, Germany).

2.5 Viability staining and cell proliferation

The viability of ECs and SCs in both mono- and cocultures was studied at d 1, d 7, and d 14 time points with qualitative live/dead viability staining as described previously [13, 16]. The samples were incubated at 0.5 mM CalceinAM (green fluorescence; Molecular Probes) and 0.25 mM EthD-1 (green fluorescence; Molecular Probes) in DBPS (Sigma-Aldrich) for 45 min at RT and imaged with a fluorescence microscope (Olympus IX51S8F-2; camera DP71, Tokyo, Japan). The viable cells were stained green, and the dead cells were stained red. The background fluorescence caused by the scaffold without cells was used as a negative control.

The CyQUANT™ cell proliferation assay kit (Invitrogen) measuring the total DNA amount was used to evaluate the proliferation of ECs and SCs in monocultures at d 1, d 7, and d 14 as previously described[16]. Briefly, the cells were lysed with 0.1% Triton-X-100 buffer (Sigma-Aldrich) and stored at − 70 °C until analysis. After the freeze–thaw cycle, the working solution containing CyQUANT™ GR dye and cell lysis buffer was added, and the fluorescence at 480/520 nm was measured with a Victor 1420 Multilabel Counter microplate reader (Wallac, Turku, Finland).

2.6 Total collagen content

The total soluble collagen content of ECs and SCs in monocultures was evaluated using the Sircol™ Soluble Collagen Assay (Biocolor, Carrickfergus, United Kingdom) measuring mammalian type I-V collagen at the d 14 time point, as described previously [18, 34]. Briefly, the samples were incubated in 0.5 M acetic acid (Merck, Darmstadt, Germany) with 0.1 mg/ml pepsin (Sigma-Aldrich) for 4 h at + 4 °C to extract the acid-soluble collagen from samples. Sircol Dye reagent (Biocolor) was added to liquid samples and incubated with gentle shaking for 30 min at RT. The samples were centrifuged for 10 min at 12 000 rpm, and collagen pellets were washed with ice-cold Acid-Salt Wash Reagent (Biocolor). The samples were centrifuged again, and the dyed collagen was further dissolved in 0.5 M sodium hydroxide solution (Biocolor), and the intensity of the red dye was measured with a Victor 1420 microplate reader (Wallac) at 540 nm.

2.7 Quantitative real-time polymerase chain reaction (qRT–PCR)

The quantitative relative expression of the genes cytokeratin (CK) 7 and CK19 for ECs and alpha smooth muscle actin (αSMA), elastin, collagen I (Col I) and collagen III (Col III) for SCs was studied using real-time reverse transcription-polymerase chain reaction (qRT–PCR). For qRT–PCR analysis, ECs and SCs were monocultured on scPLCL and scPLCLA2P for 14 days, and polystyrene cell culture plastic (PS) without A2P served as a control as previously described [16, 34]. Briefly, the cells were lysed, and total RNA was isolated with Nucleospin kit reagent (Macherey–Nagel GmbH & Co. KG, Düren, Germany). Next, the mRNA was reverse transcribed to cDNA with a high-capacity cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific). The qRT–PCR mixture contained cDNA, forward and reverse primers (Table 2, OligomerOy, Helsinki, Finland), and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA).

Table 2 The qRT–PCR primer sequences used in this study

The reaction was conducted with an AbiPrism 7000 Sequence Detection System (Applied Biosystems), and the initial enzyme activation was performed at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 60 s. The gene expression levels of CK7, CK8, CK19, αSMA, elastin, Col I and Col III were normalized to the expression of the housekeeping gene large ribosomal protein P0 (RPLP0), and the relative expression was calculated using a previously described mathematical model [35]. Furthermore, the Col I/Col III ratio was calculated by using average qRT–PCR cycle threshold (Ct) values for Col I and Col III mRNA.

2.8 Immunostaining

The staining of Col I (anti-collagen I antibody, 1:2000, Abcam, Cambridge, UK) and αSMA (anti-actin α-smooth muscle, 1:400, Sigma) was evaluated after 7 and 14 d in SC monocultures on scPLCL and scPLCLA2P. Additionally, in cocultures of ECs and SCs on scPLCLA2P, the staining of pancytokeratin (AE1/AE3, 1:250, Cytokeratin Pan Ab, Thermo Fisher Scientific) and actin cytoskeleton organization (phalloidin-tetramethylrhodamine B isothiocyanate, 1:500, Sigma-Aldrich) was evaluated after 7 and 14 d of cell culturing.

The samples were fixed with 0.2% Triton X-100 (Sigma-Aldrich) in 4% PFA (Sigma-Aldrich) and incubated overnight in the abovementioned primary antibody dilutions. The following day, the SC monocultures were incubated in secondary antibody dilutions (1:400, goat anti-mouse IgG1 or 1:300, goat anti-mouse IgG (H + L), Alexa-fluor 488, green fluorescence, Invitrogen). The EC and SC cocultures were incubated in a mixture of secondary antibody (1:400, goat anti-mouse IgG (H + L), Alexa-fluor 488, green fluorescence, Invitrogen) and phalloidin. Finally, the cell nuclei were stained with DAPI (1:200, blue fluorescence, Sigma-Aldrich), and the samples were imaged with a fluorescence microscope (Olympus).

2.9 Statistical analysis

Statistical analyses were performed with SPSS v 23 (IBM SPSS Statistics for Windows, Armonk, NY, USA). The CyQUANT™ cell proliferation assay was repeated with cells from three different donors using three parallel cell samples and three technical replicates (n = 27). The change in cell amount in relation to culturing time points was analyzed. The total collagen content measured with the Sircol™ collagen assay was repeated using cells from three different donors with three parallel cell samples and two technical replicates (n = 18). qRT–PCR was repeated with cells from three different donors using two parallel samples; however, some expression values were required to be removed from the analyses (n = 5–6). The differences in gene expression between the materials were evaluated. The data were nonnormally distributed; therefore, nonparametric Kruskal–Wallis (CyQuant and qRT–PCR) or Mann–Whitney (Sircol) tests were used to analyze the data in both analyses and are reported as medians and quartiles (p < 0.05 considered significant).