Primer sequences of the target genes.

<p>GAPDH  =  glyceraldehide-3-phosphate dehydrogenase.</p><p>Primer sequences of the target genes.</p

Histological and immunohistochemical results.

<p>(<b>A</b>) Representative H&E and safranin-O/fast green staining showed that in UG and UAG, there were large amounts of woven bone formation in the fracture areas, with almost no chondroid tissues (stained red by safranin-O), whereas lots of chondroid and fibrous tissues still existed at the fracture site in CG at week 4. Scale bars, 50 µm. (<b>B</b>, left) Representative immunohistochemistry for GFP showed that a large number of the GFP positive cells engrafted in the fracture area in UG, whereas fewer GFP positive cells were detected in CG, and even fewer GFP positive cells were found in UAG. Scale bars at ×100, 200 µm. (<b>B</b>, right) Representative images of SDF-1 staining of callus at week 4 post-fracture for young rats. Brown color indicates positive staining. SDF-1 was located mainly in the blood vessels or sinusoid-like regions. Scale bars at ×200, 100 µm.</p

µCT measurement.

<p>(<b>A</b>) Representative 3D reconstructed micro-CT images of the three groups at week 4 post-fracture showed improved fracture healing in UG and UAG. (<b>B</b>) BVh/TV in UG was increased by 27.9% than in CG (p = 0.004). (<b>C</b>) BMD in UG was higher by 10% than in UAG (p = 0.053) and by 14.5% than in CG (p = 0.006).</p

Flowchart of the study design.

<p>MSCs were isolated from two 8-week-old female SD rats and characterized by flow cytometry assay, osteogenic induction assay and adipogenic induction assay. In the in vitro experiments (n = 5), the SDF-1 protein and mRNA expression levels in condition medium were compared between control (CG) and LIPUS treatment (UG) groups, the MSCs migration ability was compared among CG, UG and LIPUS plus AMD3100 treatment (UAG) groups. In the in vivo experiments, closed femoral fractured rats were randomly divided into CG, UG and UAG groups (n = 10). GFP-labeled MSCs were intracardiac injected to all the rats on day 3 post-fracture and recruitment effects by LIPUS were compared among groups.</p

Serum SDF-1 protein concentration.

<p>The serum SDF-1 protein level measured by ELISA assay in UG was increased by 1.55 times than in CG (p = 0.005); the serum SDF-1 level in UAG was increased by 1.55 times than in CG (p = 0.005).</p

Radiographic analysis of fracture healing in young rats.

<p>(<b>A</b>) Series of representative radiographies showed better callus bridging in UG and UAG, compared with in CG at different time points. (<b>B</b>) The measurement of callus width (CW) showed: CW in UG was larger by 26.8% at week 1 (p = 0.031), by 33.6% at week 2 (p = 0.01) and by 35.0% at week 3 (p = 0.007) post-fracture than in CG, and by 27.8% at week 2 (p = 0.035) and by 30.0% at week 3 (p = 0.022) post-fracture than in UAG. (<b>C</b>) The quantitative measurement of callus area (CA) showed: CA was significantly larger in UG by 55.1% at week 1 (p = 0.002), by 55.5% at week 2 (p = 0.002), by 64.0% at week 3 (p = 0.032) than in CG, and was significantly larger by 37.7% at week 2 than in UAG (p = 0.047).</p

Mechanical properties of the femurs of the three groups at week 4 post-fracture.

<p>UG, LIPUS treatment group; UAG, LIPUS+AMD3100 treatment group; CG, control group received sham treatment.</p>a<p>p<0.05 between UG and UAG;</p>b<p>p<0.05 between UG and CG;</p>c<p>p<0.05 between UAG and CG.</p><p>Mechanical properties of the femurs of the three groups at week 4 post-fracture.</p

MSCs migration assay.

<p>(<b>A</b>) The migrated MSCs on the exterior of the insert in UG (left) were remarkably increased compared with UAG (middle) and CG (right) at ×100 magnification. (<b>B</b>) The number of migrated cells in UG was increased 12.1 times than in CG (p = 0.002); the number of migrated cells in UAG was decreased by 87.2% as compared to UG (p = 0.003).</p

<i>Ex vivo</i> GFP intensity measurement.

<p>(<b>A</b>) On the representative image of each group, blue circles indicate the region of interest (ROI) for fluorescent imaging analysis. GFP signals in UG (left) was much higher than in UAG (middle) and CG (right). (<b>B</b>) Semi-quantitative GFP intensity of fracture callus in UG was increased 2.66 times than in UAG (p = 0.014), although no significant differences were found among other groups.</p

Target genes quantification and SDF-1 protein measurement.

<p>(<b>A</b>) LIPUS enhanced SDF-1 gene expression in MSCs significantly (p<0.0001), and (<b>B</b>) LIPUS enhanced CXCR4 gene expression in MSCs significantly (p = 0.014). (<b>C</b>) ELISA assay showed increased SDF-1 protein level in the culture medium of MSCs treated with LIPUS (p = 0.018).</p