Additional file 4: Figure S3. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Surface marker expression (in percentage) of the acoustic stimulated cells represented as a bar plot. Each bar represents the average expression obtained from three independent donors. Represented are only the surface markers that were expressed in the obtained populations. Negative markers are not shown. No statistically significant differences were found between the two conditions. (PDF 184 kb

Additional file 3: Figure S2. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Biological characterization of isolated hMSCs from acoustically stimulated BM at 300 Hz for 5 min at different volumes, 11.5, 10, 8, 6 and 5 ml. The results are presented as the fold change over the non-stimulated bone marrow (baseline). (A) Graphic representation of the bone marrow volumes, donor dependent. (B) Proliferation of hMSCs calculated as PD/day from P1 to P2, donor and volume dependent. (C) CFU potential of hMSCs, donor and volume dependent. (D) ECM production, quantification of nodule size area in mm2, donor and volume dependent. (E) Osteogenic potential calculated as percentage of ALP positive colonies within the CFUs, donor and volume dependent. (F) Adipogenic potential, quantification of Oil red O staining relative to 100% Oil red O staining solution, donor and volume dependent. Values are represented as mean ± standard deviation of at least three independent experiments (n ≥ 3). Statistically significant differences were found with ***p < 0.001, **p < 0.01 and *p < 0.05. (PDF 694 kb

Additional file 2: Figure S1. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Macroscopic appearance of bone marrow aspirated from different locations: ilium, proximal femur, distal femur and proximal tibia. (PDF 311 kb

Additional file 5: Figure S4. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Alizarin red staining of calcium nodules after osteogenic induction of hMSC isolated under varying culture condition from different donors. No differences were observed between the culture conditions, though differences between the donors were identified. Donor 2 and 11 showed less calcium nodules formation than the rest of the donors. All the controls stained negative for calcium nodules formation. Values are represented as mean ± standard deviation of at least three independent experiments (n = 3). (PDF 2096 kb

In vivo vascularization of polymeric construct combined with Matrigel and cells.

<p>Vessel-like network formation in vitro in 3D constructs. Tissue construct sections were stained with eosin (orange for tissue, red for erythrocytes) and counterstained with hematoxylin (brown) or with Masson’s trichrome staining (blue or green for collagen, red for erythrocytes) and counterstained with hematoxylin (brown).</p

Wound healing assay.

<p>Pictures were taken directly after making the wound, 12 and 24 hours later (A). Quantification of wound recovery was performed in TScratch program and presented on the graph (B). Error bars represent standard deviation, * denotes statistical significance (P<0.05).</p

Primers used for qPCR.

<p>Primers used for qPCR.</p

Quantitative analysis of vessels in polymeric constructs.

<p>Number of vessels per sample was quantified by four people blinded for the conditions (A). ** denotes statistical significance (P<0.01), *** denotes statistical significance (P<0.001). Tissue construct sections were stained with anti-human CD31 antibody (brown, indicated by black arrows) and counterstained with hematoxylin (blue) (B).</p

Image segmentation steps.

<p>Original image (A), quality enhancement (B), intensity thresholding (C), edge detection (D), edge connection/filling (E), final segmentation result (F). Print Screen of the analysis of tube topology (G).</p

In vitro vascularization of polymeric construct combined with Matrigel and cells.

<p>PLLA/PLGL scaffold before cell seeding (A). Scanning electron microscope (SEM) pictures of scaffold taken after gold coating (B). Eosin/hematoxylin staining of tissue sections taken from constructs after 10 days of in vitro culture without cells (C), with C2C12 (D), with C2C12 and MSCs (E), with C2C12 and HUVECs (F), with C2C12 and EL-MSCs (G).</p