Animal breeding, fracture model, and animal groups

Animal breeding

All animal procedures were approved by the Institutional Animal Care and Use Committee of Daping Hospital, Third Military Medical University, P. R. China. All methods were performed in accordance with approved guidelines of the Institute. Catnbtm2Kem mice, Catnblox(ex3) mice and 3.2-kb mouse pro-collagen 1 promoter mice (3.2-kb Col1-Cre-ERTM) were obtained from Jianquan Feng (Department of Biomedical Sciences, Baylor College of Dentistry) and maintained on a C57BL/6 background. Catnbtm2Kem mice possess loxP sites in introns 1 and 6 of the gene encoding β-catenin, resulting in a null allele when treated with Cre recombinase28. Catnblox(ex3) mice contain loxP sequences flanking exon 3, and Cre recombinase treatment of these animals results in the expression of a fully functional but stabilized β-catenin protein16, 29. Mice expressing tamoxifen-inducible Cre fusion protein, Cre-ERTM, under the control of the 3.2-kb mouse pro-collagen 1 promoter, which is active in osteoblasts, odontoblasts and tendon fibroblasts maintained on a C57BL/6 background were used to stabilize or delete β-catenin29,30,31. Tamoxifen (TM; Sigma-Aldrich, St. Louis, MO, USA) was injected to activate the function of the promoter29. Briefly, TM was dissolved in a small volume of ethanol and diluted with corn oil to a concentration of 10 mg/ml. The mice were intraperitoneally injected with TM at a dose of 75 mg/kg29. All of the animals used in the current study were male mice aged two months.

Animal groups and TM injection procedure

To constitutively activate β-catenin in the remodeling phase, TM was injected into Catnblox(ex3)/3.2-kb Col1-Cre-ERTM mice once every three days from weeks 3 to 8 post-fracture. To slightly activate β-catenin in the remodeling phase, TM was injected twice into Catnblox(ex3)/3.2-kb Col1-Cre-ERTM mice starting at week 3 post-fracture, and saline was then injected until week 8 post-fracture. To delete the gene encoding β-catenin in the remodeling phase, TM was injected into Catnbtm2Kem/3.2-kb Col1-Cre-ERTM mice from week 3 to 8 post-fracture. Wildtype C57BL/6 mice were obtained from the animal center of Daping Hospital and used as control and were subjected to the same TM injection procedure as the CA-β-catenin mice. The efficiency of the TM dosing regimen to represent the β-catenin and TCF/LEF1 expression level was determined by real time-PCR, and the method is described in the following section.

Fracture model

Bone fractures were generated in eight-week-old male mice as described by Huang et al.32. Briefly, the mice were anesthetized through an intramuscular injection of pentobarbital sodium (0.05 mg/g, Chuangdong Co., Chongqing, China). Under rigorously aseptic conditions, the fur of the right tibia was shaved, and the skin sterilized with betadine solution prior to surgery. A small incision was made lateral to the patella, the patellar tendon was exposed, and a 27-gauge needle was used to ream a hole through the proximal tibial plateau and into the medullary cavity. The tibia was stabilized by inserting a sterile 0.25-mm-diameter stainless-steel pin through the reamed hole and down the tibia shaft. Following pin placement, a mid-diaphyseal fracture was created in the tibia using an electric saw. After fracture injury, the wound was irrigated with sterile saline and closed with sutures. Radiographs of the tibia were taken immediately post-operatively to ensure proper alignment of the fracture at the start of the healing period. Buprenorphine (Abbot Laboratories, Abbott Park, IL, USA) was administered in the drinking water for pain relief for the first three days after surgery. The animals were housed with free access to food and water and allowed unrestricted weight bearing after recovery from anesthesia.

Radiographic imaging and tissue preparation

Eight weeks after surgery, the mice were deeply anesthetized, and radiographic images of their entire skeletons and the fractured tibia were obtained using a Faxitron MX-20 system (Faxitron, Wheeling, IL, USA). Subsequently, whole blood was extracted from the mice via a heart puncture, incubated at room temperature for 30 min and then centrifuged for 10 min at 5000 rpm. The serum was collected for the examination of bone biomarkers. The mice were then sacrificed by an overdose of the anesthesia, and the tibia were removed and fixed overnight. Some of the samples were either used for mechanical tests or directly embedded in plastic, and 8-μm undecalcified sections were cut for von Kossa staining. The remaining samples were decalcified in 10% ethylenediaminetetraacetic acid. Once they were adequately decalcified, the samples were tissue-processed, embedded in paraffin, and sectioned coronally at a thickness of 5 μm. The sections were de-paraffinized, rehydrated and used for hematoxylin and eosin (H&E), Fast Green/Safranin O, Sirius Red and Masson staining.

Examination of serum bone biomarkers

The serum was used to determine the concentrations of CTX through an ELISA according to the instructions provided by the manufacturer. The ELISA kits were procured from Immunodiagnostic Systems, Ltd. (Boldon, UK). The concentrations of serum ALP were determined by routine methods using an automatic biochemical analyzer (UniCel DxC 800 Synchron Clinical Systems; Beckman Coulter, Fullerton, CA, USA).

MicroCT examination

At week 8 post-fracture, the tibia were dissected and subjected to three-dimensional microCT analysis using a Viva CT 40 (Scanco Medical, Bassersdorf, Switzerland) following the procedural recommendations provided by the American Society for Bone and Mineral Research33. Ethanol was used as the scanning medium, the X-ray tube potential was 45 kVp, and the voxel size was 10 μm3. Images were reconstructed and analyzed with EVS Beam software using a global threshold of 1400 Hounsfield units. For analysis, the volume of interest (VOI) was defined as the region between the first and last slices with callus formation, resulting in an approximately 800-slice segment of bone. The total volume (TV), maximum moment of inertia (Imax), and bone volume fraction (BV/TV) were measured. Global thresholding was performed to distinguish between mineralized and non-mineralized tissue. Additionally, transverse sections of the central 1-mm region of the fracture were used to observe the bone remodeling process.

Real-time polymerase chain reaction (real time-PCR)

Three mice from each group were sacrificed eight weeks after the operation. The whole tibia was dissected, and the whole fracture site was obtained. The samples were immediately shock-frozen in liquid nitrogen and were homogenized, and the total RNA was then isolated using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Real-time PCR using the SYBR green detection method was performed to examine the expression levels of β-catenin, TCF, LEF1, ALP, Osterix, Collagen1, Runx2, RANKL, and OPG. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control, and the expression levels of specific genes are expressed as proportions relative to the mean GAPDH value. The primers that were used are presented in Table 1.

Table 1 Primers used for real-time PCR. Full size table

H&E staining

H&E staining was performed as described in a previous report16, 17, 29. Briefly, the sections were stained in Harris hematoxylin solution for 5 min, differentiated in 1% acid alcohol for 30 s, and bluing in 0.2% ammonia water for 30 s. Between each step, the sections were washed fully with tap water. The sections were subsequently rinsed in 95% alcohol, counterstained in eosin-phloxine solution for 30 s, rinsed in 95% alcohol for 2 min, in 100% alcohol for 3 min (twice), and in xylene for 2 min (twice), and coverslipped with Permount.

Fast Green/Safranin O staining

The rehydrated sections were stained with Weigert’s iron hematoxylin for 1 min and rinsed in distilled water until clear. Subsequently, the sections were stained sequentially with 0.02% Fast Green for 5 min, 1% acetic acid for 30 s, and 0.1% Safranin O for 20 min. The slides were not rinsed between steps, and subsequently, the slides were rinsed with 95% alcohol for 2 min, 100% alcohol for 3 min (twice), and xylene for 2 min (twice) and coverslipped with Permount29. All chemicals were purchased from Chuandong Corporation (Chongqing, China). The purpose of these stains was to visualize stromal (hematoxylin in connective tissue), cartilaginous (Safranin O), and bony (Fast Green) tissues, allowing the reviewers to differentiate between these types of tissues for appropriate quantification.

Sirius Red and Masson staining for collagen

Sirius Red staining was used to demonstrate differences in collagen structure as previously reported17. The prepared sections were stained with Weigert’s hematoxylin for 8 min, and the slides were then washed for 10 min in running tap water. Subsequently, the slides were stained with PicroSirius Red for 1 h and washed in acidified water. The water was removed, and the slides were dehydrated, rinsed in 95% alcohol for 2 min, 100% alcohol for 3 min (twice), xylene for 2 min (twice), and coverslipped with Permount. The collagen morphology (structure) was then observed under a polarized light microscope.

Masson staining was used to demonstrate the collagen content as previously reported16. Prepared sections were stained with Weigert’s iron hematoxylin working solution for 10 min, followed by Biebrich scarlet-acid fuchsin solution for 10 min. The sections were then differentiated in phosphomolybdic–phosphotungstic acid solution for 10 min and transferred directly (without rinsing) to aniline blue solution and stained for 5 min. After rinsing briefly in distilled water, they were differentiated in 1% acetic acid solution for 2 min, then rinsed in 95% alcohol for 2 min, 100% alcohol for 3 min (twice), xylene for 2 min (twice), and coverslipped with Permount. Collagen was stained blue in color. The content of collagen were assessed by analyzing the optical density with Image-Pro Plus 4.5 (Media Cybertics, The Netherlands).

Von Kossa staining

Von Kossa staining was employed to monitor the mineralization ability of bone. After the sections were degreased and rehydrated, 100 µl of 2% silver nitrate solution was applied to each section, and the slides were exposed to strong light for 30 min. After the silver nitrate was removed, 5% sodium thiosulfate was added to the section for 10 s prior to rinsing with distilled water. The sections were then incubated with Van Gieson working solution for 5 min and prepared for observation16, 17. After von Kossa staining, the mineralized bone appeared black, with osteoid seams appearing bright pink and non-mineralized bone appearing pink. The optical density for the color of black was analyzed by using Image-Pro Plus 4.5 (Media Cybertics, The Netherlands).

TRAP staining

TRAP staining was performed as described previously16, 29. Briefly, two Coplin jars (A and B) with 50 ml of stock basic incubation medium (9.2 g of sodium acetate anhydrous, 11.4 g of sodium tartrate dibasic dehydrate, and 2.8 ml of glacial acetic acid dissolved in 1000 ml of distilled water; the pH was adjusted to 4.7–5.0 with 5 M sodium hydroxide) were pre-heated to 37 °C. Fifty microliters of 2% naphthol AS-BI phosphate substrate in ethylene glycol monoethyl ether followed by the slides were added to jar A, and the jar was then incubated at 37 °C for 45 min. A few minutes prior to completion of the 45-min incubation, 1 ml of 5% pararosaniline chloride and 1 ml of 4% sodium nitrite were mixed for 30 s, and the resulting mixture was incubated at room temperature for 2 min without mixing, transferred into jar B and mixed well. The slides from jar A were then transferred to jar B without rinsing. Incubation at room temperature was performed for 1–3 min until color development, and the slides were then rinsed, counterstained with methyl green for 5 min, dehydrated, and covered with Permount. All chemicals were purchased from the Chuandong Corporation (Chongqing, China).

Pictures of each section were taken under a magnification of 400×, and the numbers of TRAP-positive cells corrected by bone area were counted in five random fields. The average numbers and standard deviations of TRAP-positive cells were calculated and subjected to statistical analyses.

Mechanical testing

The fractured tibias, which had previously been measured by microCT, were tested for mechanical strength through a three-point bending test using a BOSE ElectroForce ELF 3200 computer-controlled testing machine, which has a force resolution of 0.05 N. During the test, each tibia was placed horizontally on two lower supports located 6.5 mm apart, with the anterior surface facing upward. The pressing force was applied vertically to the midshaft of the bone. Each bone was compressed at a speed of 0.05 mm/s until failure, and force-displacement data were collected every 0.01 s. Based on the data, a force-displacement curve was created, and the ultimate force (UF; N) was defined as the bending force at failure. The stiffness was calculated as the slope of the linear portion of the curve.

Statistical analysis

All of the data are expressed as the means ± standard deviations. Statistical significance was evaluated through one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. The data were considered significant at P < 0.05.