Fig 1.
Fracture osteosynthesis of the tibia.
Completed osteosynthesis of the tibia with a 5-hole mini Y-plate and 4 screws, with the plate spanning the fracture gap located in the metaphyseal area of the tibia.
Table 1.
Extracorporeal shock wave therapy application scheme.
Fig 2.
Exemplary sections of a proximal tibia from the 3 x 0.15 mJ/mm2 group labeled with the investigated fluorescent dyes administered previously in vivo.
A) Shown is the schematic representation of the evaluated callus domains: dorsal, endosteal, and ventral. Only the callus and none of the cortical area was considered for data acquisition. B) Shown are the areas of the callus stained through the complexation of the fluorescent color with Calcium by Calcein green (green) and Alizarin red (red). The proximal, empty area represents the former location of one of the proximal osteosynthesis screws.
Table 2.
Specific primer sequences used for the quantitative real-time polymerase chain reaction (qRT-PCR) amplification.
Fig 3.
Micro-CT analyses of the number of trabecular nodes (n.Nd) in the tibia metaphysis above the fracture and exemplary images.
A) Mean number of trabecular nodes in the proximal metaphyseal area of the tibia proximal to the fracture site according to the respective treatment group. Treatment groups are shown as number of ESWT-application(s) and energy flux intensity (mJ/mm2). The number of mean trabecular nodes are shown as means ± standard error of the mean (SEM) according to the respective treatment group. Adjusted p-values were considered statistically significant at p < 0.05 (Two-way analysis of variance (SPSS Statistics Version 23; IBM Corp., New York, NY, USA) and Bonferroni post hoc test). * p < 0.05 vs. all OVX groups with and without ESWT treatment. B and C) Micro-computed tomography imaging (micro-CT) of the transverse cross sections of the proximal tibia metaphysis, proximal to the fracture, and exemplary demonstration of the trabecular network with the visible difference in number of trabecular nodes (n.Nd). (B: SHAM C: OVX with ESWT treatment).
Fig 4.
Biomechanical analysis results.
Biomechanical analysis results regarding the average A) stiffness in N/mm and B) yield load in N. Both are shown against the different treatment groups (number of treatment(s) and applied energy flux intensity (mJ/mm2)). Values are shown as means ± standard error of the mean (SEM). Adjusted p-values were considered statistically significant at p < 0.05 (One-way analysis of variance (SPSS Statistics Version 23; IBM Corp., New York, NY, USA), Welch-Test adjusted, and Bonferroni post hoc test for stiffness; Two- way analysis of variance ((SPSS Statistics Version 23; IBM Corp., New York, NY, USA) and Bonferroni post hoc test for the other parameters). * p < 0.05 vs. 3 x 0.55 mJ/mm2. N: Newton, mm: milli-meter, mJ: milli-Joule.
Fig 5.
Average total callus formed including simultaneous depiction of the average callus formed individually by Calcein green (white part of bar graph) and Alizarin red (grey part of bar graph). The amount of callus was measured in mm2 and is shown according to respective treatment group (number of treatment(s) and applied energy flux intensity (mJ/mm2)). Portrayed is the average total callus of the dorsal, the endosteal and the ventral aspect of the fracture callus. Values are shown as means ± standard error of the mean (SEM) of the total callus. Adjusted p-values were considered statistically significant at p < 0.05 (One-way analysis of variance (SPSS Statistics Version 23; IBM Corp., New York, NY, USA), Welch-Test adjusted, and Bonferroni post hoc test). * p < 0.05 vs. SHAM, Δ p < 0.05 vs. 1 x 0.35 mJ/mm2. mm: milli-meter, mJ: milli-Joule.
Fig 6.
Gene expression analysis of osteoblast and osteoclast specific transcripts.
Gene expression analysis through qRT-PCR of the osteoblast and osteoclast markers with regards to the respective treatment group (number of treatment(s) and energy flux intensity (mJ/mm2)) and shown in relative expression units. Portrayed values are shown as means ± standard error of the mean (SEM). Adjusted p-values were considered statistically significant at p < 0.05 (One-way analysis of variance (SPSS Statistics Version 23; IBM Corp., New York, NY, USA), Welch-Test adjusted, and Bonferroni post hoc test). Please see Supplement 2 for details on the numerous significances. qRT-PCR: Quantitative real-time Polymerase Chain Reaction, Coll1α1: Collagen 1-alpha-1, ERα: Estrogen Receptor-α, IGF-1: Insulin-like Growth Factor 1, OC: Osteocalcin, TRAP: Tartrate-resistant Acid Phosphatase, mJ: milli-Joule, mm: millimeter.