Image processing steps on MRI-derived image stacks in the absence (top, images A-D) and presence (bottom, images E-H) of hMSCs (donor 1, 5 weeks of culture).

<p>A and E represent the unprocessed MR images; B and F the images after background subtraction; C and G show the results of segmentation based on a single intensity threshold within a circular ROI, comprising scaffold and surrounding water within the MRI glass tubes; D and H are the obtained 3D mesh models, representing the tissue localization, amounts and density. Scale bars represent 2 mm.</p

MRI scans of a scaffold after 14 days of culture with hMSCs (donor 1) taken with slice thicknesses of 80 µm.

<p>Slices from one scan from the top (A), middle (B) and bottom (C) of the scaffold areas are represented, respectively, as indicated in the marques in (D) the side-view of the scaffold in which cells are stained with methylene blue. (E-F) From the bottom-view of the scaffold, it can be seen that methylene blue stained hMSCs and ECM formed with the appearance of a dense string-like tissue (indicated with arrows) distributed non-homogeneously throughout the scaffold (indicated with asterisks). (G) This was also observed during culture by bright field microscopy. Scale bars represent 2 mm.</p

Histological analysis of calcification in explanted tibiae.

<p>(A) Midsagittal sections of tibiae were stained with Alizarin red after explantation or cultured seven days under hypoxic or normoxic conditions. (B) Image analysis was used to determine the length of the intensely calcified tissue, which was taken as the broken line indicated in ‘A’. (C) The area of calcification was used to determine relative calcification of the samples. (D) Higher magnification microphotographs were used to investigate the calcification of the hypertrophic cartilage that resides on top of the intensely stained bone. The dashed line represents the osteochondral interface. (N = 15). * = P<0.05 compared to freshly isolated tibiae. # = P<0.05 compared to normoxic condition of the same time point.</p

Effect of hypoxic and normoxic culture conditions on Frzb and Dkk1 protein levels.

<p>(A) Frzb and Dkk1 levels were quantified in the conditioned medium of tibiae, which were cultured for 7 days without receiving new medium in either hypoxia or normoxia (N = 5). (B) The effect of oxygen levels on Frzb and Dkk1 on protein activity over time was studied by exposing culture medium containing 10% fetal bovine serum to either hypoxia or normoxia for 7 days in 37°C. (N = 4). Frzb and Dkk1 protein levels were analyzed using ELISA. * = P<0.05 compared to normoxic condition of the same time point.</p

Explanted tibiae cultured 21 days under hypoxic or normoxic conditions.

<p>(A) Microphotographs of representative tibiae at different points in time. (B) Using image analysis the average tibiae lengths were calculated. (N = 18). * = P<0.05. ** = P<0.01.</p

Alamar blue and DNA quantification assay results to compare metabolic activity and proliferation of SCs.

<p>(A, C) PHB(50)/PHBV(50) random nanofibers (PHB/PHBV R group) and PHB(50)/PHBV(50) aligned nanofibers (PHB/PHBV A group); (B, D) PHB(50)/PHBV(50) aligned nanofibers (PHB/PHBV A group) and PHB(45)/PHBV(45)/collagen(10) aligned nanofibers (PHB/PHBV/Col group) during 14 days of culture. Asterisks represent significant difference at <i>p</i>≤0.05.</p

SEM images of SCs on PHB(50)/PHBV(50) random (R) and aligned (A) nanofibers.

<p>(A) 1 day, (B) 3 days, and (C) 7 days after cell seeding. Scale bars represent 50 µm for top and 10 µm for button pictures, respectively.</p

Raman spectra of PHB/PHBV nanofibers in different blend compositions.

<p>Raman spectra of PHB/PHBV nanofibers in different blend compositions.</p

SEM images and histograms illustrating the orientation of PHB(50)/PHBV(50) nanofibrous mats.

<p>Nanofibers collected with speed of (A) 1000 rpm, (B) 3000 rpm, (C) 5000 rpm. The voltage, flow rate and collecting conditions were fixed at 16 kV, 1.5 ml/h and 15 cm, respectively. Scale bars represent 20 µm for SEM images.</p

Image processing algorithms on SEM images of cultured SCs on PHB(50)/PHBV(50) random (PHB/PHBV R group) and PHB(50)/PHBV(50) aligned (PHB/PHBV A group) nanofibrous scaffolds.

<p>(A) Original SEM image of SCs on scaffold after 1 day of cell culture; (B) binary image showing the processing output of nonlinear diffusion filtering; (C) Radon transform results of binary image for a range of [0,180°] and with a resolution of θ = 1°; (D) dominant peak of the maximum of Radon transform matrix.</p