Mice and treatments

Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University. Experiments were performed in compliance with NIH guidelines on the use and care of laboratory and experimental animals. C57BL6 mice were provided from Charles River Laboratories (Wilmington, MA, USA) and housed under a 12-hour light, 12-hour dark cycle. Food and water were provided ad libitum. Male mice were used for the study based on our previous data showing that old male and female C57BL6 mice have identical fracture repair characteristics6,7. Nefopam hydrochloride was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and dissolved in normal saline as carrier control, then delivered by intraperitoneal injection (i.p.) (30 mg/kg). XAV939 was purchased from Selleck (Houston, TX, USA) and dissolved in 4% dimethyl sulfoxide (DMSO). Corn oil was used for vehicle or carrier control. XAV939 was subsequently delivered by i.p. injection (20 mg/kg). Mice were randomly selected to either receive treatment or carrier. Treatment was started from the day of surgery and progressed at a duration of 3, 7, or 10 days. Doses were selected from prior studies. Animals were observed for any deleterious symptoms and their weight was recorded before, during and after treatment.

Fracture generation

A tibia fracture with intramedullary stabilization was used to study bone repair8. Briefly after anesthesia was induced, the surgical area was shaved and disinfected. A 3-mm longitudinal incision was made in the patella tendon and 0.5mm hole was drilled by a 25G syringe needle above the tibial tuberosity. Intramedullary fixation was achieved using a 0.7 mm insect pin then the mid-tibial diaphysis was transected with small surgical scissors. Osteotomies were made parallel to the tibial plateau. The incision was then closed using sutures. Analgesia was provided to all mice. As in our prior work6,7,8,16,20. Slow release buprenorphine hydrochloride analgesia (0.03 mg/kg). was provided as an analgesic during the generation of a tibial injury and the initial painful period after the fracture was generated until animals returned to their usual activity level.

Radiograph and μCT analyses

The fractured tibia, as well as, and the intact, contralateral tibiae were studied after mice were sacrificed at postoperative 21 days. Tibiae were then fixed in 10% formalin for 48 h at room temperature (25 °C) and then transferred to 70% ethanol. Implants were removed from the tibiae prior to scanning. The quality of fracture repair was assessed using the Faxitron MX20 X-ray system (Faxitron, Tucson, AZ, USA). Quantitative three-dimensional evaluation of the fracture callus and healing was undertaken by μCT using a Viva CT 80 (Scanco, Brüttisellen, Switzerland) at 55 kVp and 145 μA with a resolution of 15.6 μm voxel size. A hydroxyapatite calibration phantom was used to scale values of linear attenuation for the calcified tissues to bone density values (mg/cm3). Morphometric parameters were quantified21 at the site of fracture which included 1mm proximal and distal from the fracture location. The original grayscale image datasets of the tibia region of interest were imported into the manufacturer software and the cortical and callus regions were separated using an automated contouring method. Calcified tissues were segmented from soft tissues using a global thresholding procedure with a threshold of 480 mg HA/cm3, which represents 45% of the attenuation of mature cortical bone (19, 20). Callus maturity was measured using differential thresholding in which immature bone was defined by a threshold of 169 to 400 mg HA/cm3. As an additional analysis, the unfractured tibia was also examined using μCT. Parameters such as Bone volume/total volume in the tibia were analyzed using ASBMR guidelines22.

Histology and histomorphometry

After the μCT imaging process, tibiae were decalcified in 14% EDTA (pH 7.4) for 7 days at room temperature. Decalcified samples were dehydrated in a graded ethanol series, embedded into paraffin. Sections 5 µm thick were prepared and stained using Safranin-O (to stain proteoglycans/cartilage red) and counterstained with Fast Green (to stain bone green/blue). For evaluation of osteoclastogenesis, the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was visualized by TRAP staining (Sigma-Aldrich). The sections were examined and photographed using a Leica digital imaging system (Leica, Wetzlar, Germany). Histomorphometry measurements were quantified using ImageJ software (National Institutes of Health, USA).

Protein and RNA analysis

Bone fragments were collected from the tibia from mice treated with carrier, Nefopam, or XAV939, harvested on the day 3 of treatment. After dissection, the harvested tissues were immediately frozen in liquid nitrogen, pulverized then homogenized in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). Total protein was quantified with Bradford reagent using a bovine serum albumin (BSA) standard curve as a reference. SDS-polyacrylamide gels at 8% concentration were loaded with 25 µg of protein lysate. Anti-β-catenin antibody (AB19022) was purchased from Millipore, an antibody that recognizes activated (unphosphorylated at Ser-37 and Thr-41) β-catenin (clone 8E7) was also obtained from Millipore and an anti-β-actin (13E5) antibody was purchased from Cell Signaling and was probed as a loading control. Indicated antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and ECL chemiluminescence (Bio-Rad). Protein expression was analyzed by measuring the intensity of detected bands compared to intact tibia samples and normalized to β-actin with ImageJ software. RNA was extracted from CFU cell cultures using Trizol (Invitrogen) and cDNA was generated with Superscript II (Invitrogen). For real-time reverse transcriptase PCR (RT-PCR), mouse–specific Taq-Man fluorogenic probes for alkaline phosphatase, collagen type I, osteocalcin, and osteopontin were used. Asparagine synthetase and glyceraldehyde-3-phosphate were used as internal control genes.

Cell culture

Unfractured tibia of 20-month old mice was isolated and contents of medullary cavity (bone marrow) were flushed using αMEM culture medium (Gibco) on POD 21 days. Cell suspensions were passed through an 18G needle to dissociate cell aggregates, and cells were plated in 24-well plates with a density of 5 × 105 cells/ml in plating medium (αMEM, 10% FBS, 1% Penicillin/Streptomycin) or 1 × 106 cells/ml for seven days. To determine the colony forming units-fibroblastic after14 days with plating media; wells were then washed with PBS, fixed with 10% formalin, and stained with 0.05% crystal violet. Cells were cultured in osteogenic differentiation media (αMEM, 10% FBS, 1% Penicillin/Streptomycin, 30 uM ascorbic acid, 10–8 M dexamethasone, 8 mM sodium phosphate) for next 14 days. Cultures were incubated for 21 days at 37 °C with 5% CO 2 and media were replenished every 2 days. Wells were washed with PBS, fixed using 10% formalin, and stained for alkaline phosphatase (ALP) using NBT/BCIP and mineralization using 2.5% silver nitrate solution (Von Kossa) on a light box and 2% Alizarin Red S (pH 4.3). Colonies were defined by containing at least 25 cells.

Mechanical testing to determine work to failure

Tibiae for mechanical testing were harvested four weeks following the generation of a Fracture. Intact tibiae were harvested, soft tissue removed, and immediately wrapped in PBS soaked gauze and then frozen at −20 °C. The limbs were thawed prior to mechanical testing. This process has been shown to be the best method of preservation of mechanical properties in bone. The inherent variability in mechanical testing was found to be greater than any loss in bone properties due to the processing23,24,25. Tibia were tested to failure in four-point bending on a Precision Materials testing machine (Acumen 3 A/T, MTS, Eden Prairie, MN) with a 500N force transducer (model 661.11H.02, MTS, Eden Prairie, MN). The supports of the four-point bend fixture were cylindrical and 2 mm in diameter. The distance between the midpoints of the lower load supports was set to 8.9 mm, and the distance between the midpoints of the upper load supports was set to 4 mm, spanning the fracture callus and centered over the lower supports. Tibiae were loaded by a flexion moment with the flat anteromedial surface down26 on the lower supports at a constant displacement rate of 0.03 mm/s27 to failure. The data acquisition rate for load, displacement, and time was 20 Hz. Failure loads were calculated using a peak-prominence calculation in MatLab. A prominence of 0.25 was used for intact contralateral tibia and 0.4 for fractured tibia. Failure loads were then confirmed visually as the primary departure of a linear trend of the load-displacement data. Bending stiffness was calculated as the slope of the load v. displacement data between 30–70% of the load at failure to exclude the nonlinear toe-regions. Energy to failure (mJ) was calculated using a trapezoidal algorithm of the area under the load-displacement curves.

Statistical analysis and data sharing

A priori power analysis to obtain statistical significance (p = 0.05, power 80%) between the two groups. Sample sizes were determined using a power calculation based on variances from prior investigations: Micro-computed-tomography (μCT) imaging, n ≥ 7 per group on day 21; histology, n ≥ 7 per group on day 21; colony forming unit (CFU), n ≥ 3 per group on days 21; western blot, n ≥ 3 per group on days 3. μCT and histology data were analyzed in a blinded manner. Data are expressed as the means ± 95% confidence intervals. The Bartlett test was used to determine if the comparison groups followed a normal distribution. If the Bartlett test showed that the variances equal statistical significance was evaluated through a Student’s t-test for all comparisons in which there were two groups; or a one-way analysis of variance (ANOVA) followed by a Turkey post hoc testing for analyses in which there were three or more comparisons being made. If Bartlett test showed that the variances were not parametric, the Kruskal-Wallis test to test for differences in population means instead of ANOVA and the Steel-Dwass test was used instead of Turkey28,29. The data shown in Figs 1D and 4B was analyzed using a non-parametric approach. The data differences were considered significant at p < 0.05. GraphPad software was used for analysis (GraphPad Software, San Diego, CA). The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.