Volume 11, Issue 2, Pages 951-964 (September 2013) Cell biological effects of mechanical stimulations generated by focused extracorporeal shock wave applications on cultured human bone marrow stromal cells  Frank Suhr, Yvonne Delhasse, Gerd Bungartz, Annette Schmidt, Kurt Pfannkuche, Wilhelm Bloch  Stem Cell Research  Volume 11, Issue 2, Pages 951-964 (September 2013) DOI: 10.1016/j.scr.2013.05.010 Copyright © 2013 Elsevier B.V. Terms and Conditions

Figure 1 Cartoon of the experimental setup. Stem Cell Research 2013 11, 951-964DOI: (10.1016/j.scr.2013.05.010) Copyright © 2013 Elsevier B.V. Terms and Conditions

Figure 2 hBMSC characterization. (A) Morphological and biochemical approaches for characterizing hBMSCs. (A1, A2) Undifferentiated hBMSCs grown in monolayer culture shown at different confluence levels. A1 shows hBMSCs at a confluence of about 40%; A2 shows hBMSCs at a confluence of about 85%. (A3, A4) Adipogenic differentiation of hMSCs. A3 depicts Red Oil staining of lipid vacuoles after three weeks of adipogenic stimulation; A4 shows control hBMSCs stained with hematoxylin/eosin. (A5, A6) Osteogenic differentiation of hBMSCs. A5 shows von Kossa staining of calcium deposits after three weeks of osteogenic stimulation; A6 shows control hBMSCs stained with nuclear fast red-aluminum sulfate solution. (A7, A8) Chondrogenic differentiation of hBMSCs. A7 shows Toluidine blue staining after three weeks of chondrogenic stimulation; A8 shows control hBMSCs. (B) Chondrogenic differentiation (D) of hBMSCs proven by RT-PCR, but not in the proliferation approach (P). H2O was used as a negative control. Actin served as an internal housekeeping gene and showed no regulation by either D or P approaches. No signal was observed in the H2O control. Stem Cell Research 2013 11, 951-964DOI: (10.1016/j.scr.2013.05.010) Copyright © 2013 Elsevier B.V. Terms and Conditions

Figure 3 hBMSC proliferation, apoptosis, and growth rate. (A) The diagram shows proliferative Ki67 positive hBMSCs in response to fESW treatment. Compared to control hBMSCs, fESW treatments increased the percentage of Ki67 positive hBMSCs 6 and 12h post-treatment, with the exception of the 0.3mJ∗mm−2 treatment after 12h. The pictures in the right panel reflect Ki67 stained hBMSCs 6 and 12h post 0.2mJ∗mm−2 treatment compared to untreated control hBMSCs. (B) The diagram shows apoptotic, caspase-3 positive hBMSCs in response to fESW treatment. Compared to control hBMSCs, fESW treatments partially induced increased percentages of caspase-3 positive hBMSCs 6 and 12h post-treatment. Importantly, 0.2mJ∗mm−2 resulted in a decreased percentage of caspase-3 positive hBMSCs compared to control hBMSCs 12h post-treatment. The pictures in the right panel show caspase-3 stained hBMSCs 6 and 12h post 0.2mJ∗mm−2 treatments compared to untreated control hBMSCs. (C) The growth rate was significantly increased after fESW (0.2mJ∗mm−2) compared to untreated hBMSCs within 24h. The pictures in the right panel show monolayer under both control and fESW (0.2mJ∗mm−2) conditions. *p<0.05, **p<0.01. Stem Cell Research 2013 11, 951-964DOI: (10.1016/j.scr.2013.05.010) Copyright © 2013 Elsevier B.V. Terms and Conditions

Figure 4 hBMSC migration, directed F-actin, and disorganized F-actin. (A) hBMSC migration behavior was observed by Boyden chamber assays. The diagram shows that only the fESW (0.2mJ∗mm−2) treatment resulted in an increased number of migrated hBMSCs. The pictures in the right panel show DAPI positive hBMSCs transmigrated through the 8μm pore. Control and fESW treatments (0.2mJ∗mm−2 and 0.3mJ∗mm−2) are shown. (B) The diagram shows directed F-actin fibers in response to fESW treatment. As depicted, only the 0.2mJ∗mm−2 treatment resulted in an increased percentage of directed F-actin fibers in hBMSCs compared to control hBMSCs. The pictures in the right panel show directed F-actin fibers in control hBMSCs as well as fESW-treated (0.2mJ∗mm−2) hBMSCs 0 and 30min post-treatment. (C) The diagram shows disorganized actin fibers in response to fESW treatment. As depicted, fESW treatments increased the percentage of disorganized actin fibers at 0min post-treatment. However, at 30min post-treatment, 0.2mJ∗mm−2 applications resulted to significantly reduced percentage of disorganized actin fibers, whereas 0.3mJ∗mm−2 significantly increased disorganized actin fibers compared to control condition. The pictures in the right panel show disorganized actin fibers in control as well as fESW-treated (0.2mJ∗mm−2) at 0 and 30min post-treatment. *p<0.05, **p<0.01. Stem Cell Research 2013 11, 951-964DOI: (10.1016/j.scr.2013.05.010) Copyright © 2013 Elsevier B.V. Terms and Conditions

Figure 5 Cell tracking assay and wound healing/scratch assay. (A) The diagram shows that applications of different fESW treatments result in significantly increased migration velocities of fESW-treated hBMSCs compared to untreated control. See also video data 1 and 2 (supplementary data). (B) The diagram shows the percentage of wound closure by fESW-treated (0.2mJ∗mm−2) hBMSCs 4 and 8h post-treatments. fESW treatment of hBMSCs results in significantly increased percentage of wound closure at both investigated time points compared to the respective control situations. See also video data 3 and 4 (supplementary data). (C) The diagram shows that the time needed for a complete wound closure was significantly reduced after fESW (0.2mJ∗mm−2) treatment compared to untreated control hBMSCs. *p<0.05, **p<0.01. Stem Cell Research 2013 11, 951-964DOI: (10.1016/j.scr.2013.05.010) Copyright © 2013 Elsevier B.V. Terms and Conditions

Figure 6 Maintenance of hBMSC differentiation capacities after fESW treatment. hBMSCs were either treated with fESW (0.2mJ∗mm−2/0.3mJ∗mm−2) or remained untreated (control). The differentiation capacities were proven by Red Oil staining (adipogenic cells, A–C), von Kossa staining (osteogenic cells, D–F), Alcian blue (chondrogenic cells, G–I) or by Toluidine blue staining (chondrogenic cells, J–L). As demonstrated, hBMSCs maintain their full differentiation capacities after both fESW treatments compared to control conditions. However, some minor, but notable differences between the fESW treatment conditions during osteogenic and chondrogenic differentiation were observed. Compared to control (D) and 0.2mJ∗mm−2 (E), the osteogenic differentiation potential was slightly reduced after 0.3mJ∗mm−2 application (F, dashed arrows). Furthermore, compared to control (G, arrows) and 0.3mJ∗mm−2 (I, arrows) conditions we observed an altered chondrogenic differentiation potential after 0.2mJ∗mm−2 treatment (H, arrow heads). In the 0.2mJ∗mm−2 condition the chondrogenic cells seem to be more organized and aligned compared to the control and 0.3mJ∗mm−2 conditions, indicating a more effective chondrogenic differentiation after 0.2mJ∗mm−2 application compared to controls and 0.3mJ∗mm−2. Similarly, we observed this phenomenon also by means of the second chondrogenic evidence, Toluidine blue (k, arrow heads) when compared to control (J) and 0.3mJ∗mm−2 (L). Bar in A–C: 50μm; bar in D–F: 25μm; bar in G–L: 10μm. Stem Cell Research 2013 11, 951-964DOI: (10.1016/j.scr.2013.05.010) Copyright © 2013 Elsevier B.V. Terms and Conditions