Pathway 435

Fluorescence imaging of mounted DRG samples was performed with a BD Pathway 435 using a 10× objective. Automated Sholl Analysis was performed using ImageJ and the Sholl Analysis Plugin (ver.4.0.0) with a 20 μm step size between concentric shell measurements. The resulting output was used to determine the 10 longest neurites, from which the average maximum length per sample was calculated.
Additional methodology information is available in supplementary material section.

After 5 weeks of mineralization in osteogenic medium, 1% tetracycline was added to the medium for 24 h before fixation in 10% formalin (Sigma). 5 images from each sample, with 3 samples for each topography using a BD Pathway 435 automated fluorescence microscope. Integrated intensities were quantified using ImageJ software [41] .

Cell morphology assessment was performed after 3 days of culture in BM (n = 3, except for flat L-PLA and pits-patterned H-PLA, where n = 2 was used, because the third sample was damaged during processing). The samples were washed with PBS and fixed with 4% paraformaldehyde for 20 min. To perform immunofluorescence staining and analysis, the samples were permeabilized with 0.1% Triton X-100 for 5 min and blocked with 1% bovine serum albumin (BSA) in PBS. Cell cytoskeleton was stained by phalloidin conjugated to Alexa Fluor 488 (1:60 dilution in 1% BSA in PBS; Invitrogen), and DAPI (in 1% BSA in PBS) was used to stain cell nuclei. Cell imaging on eight randomly selected areas of each sample was performed using fluorescence microscopy (BD Pathway 435, BD Biosciences). The analysis of the cell shape parameters was performed using CellProfiler (Broad Institute, USA) with built-in modules (MeasureObjectSizeShape), according to the detailed manual published previously [50] . This module measures several areaand shape features of identified objects and for this study, cell area, cell perimeter and minor axis length were selected. The number of cells used for the analysis varied between approximately 400 and 1400 for all the materials, except for flat H-PLA where the total number of cells was 64.

Cell attachment and morphology (n=3) were evaluated by a four-channel fluorescence imaging of nucleus, F-actin, vinculin and vimentin. Upon culture for 1 or 3 d, the medium was removed, and the samples were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. For fluorescent staining, samples were washed twice with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and washed twice with 0.1% Tween-20 in PBS (PBST). They were first incubated in blocking buffer (2% BSA in PBST) for 30 min, and subsequently with anti-Vinculin-FITC (1:750 in blocking buffer) in combination with primary rabbit antibody for Vimentin (1:450 in blocking buffer) overnight. On the next day the samples were washed twice with PBST, incubated with F-actin probe CF™594 phalloidin (1:40 in PBST) combined with secondary antibody Alexa Fluor® 647 anti-rabbit (1:400 in PBST) for 1 h, washed twice with PBS and finally incubated with DAPI (1:100 in PBS) for 15 min to stain the nuclei. The samples were then washed again and left in PBS. Incubation with fluorescent dyes was performed at room temperature in the dark. Cells were observed with a fluorescence microscope equipped with a mercury lamp, BD Pathway™ 435 (BD Biosciences, USA), at a magnification of 20× with multiple frame acquisitions.

Cells (3 × 10 4 cells per well) were seeded in 96-well clear-bottom black plates (Matrix Technologies) and transfected with purified reporter linear constructs (PCR products). Transfections were performed in a serum-free medium using Lipofectamine 2000 (Invitrogen). All transfections were performed in several replicates. The efficiency and extent of transfection were assessed by monitoring the fluorescence from the EGFP constructs (optimum excitation wavelength, 488 nm; emission wavelength, 503 nm). Images were captured using an automated laser-focus image BD high-throughput imaging system, the BD Pathway 435 (BD Biosciences). The variance in EGFP fluorescence among replicate microwells was usually <6%; thus, with this minimum variance, experiments did not require transfection normalization (47) . Image processing, segmentation, and fluorescence quantification were facilitated by ProXcell software, as previously described (48) . Data are Downloaded from https://www.science.org on September 08, 2024 means ± SEM of the total fluorescence intensity in each well, with replicate readings. For the comparison of two groups, a two-tailed unpaired t test was used. Statistical significance was reported with two-tailed P values, unless otherwise described.