Histology and biomechanics were examined in 36 10-week-old male DT mice (72 limbs), containing the transgenes pOBCol3.6GFPtpz and pCol2-ECFP. Four treatment groups were tested in this study: (1) nonoperated control (normal), (2) surgical sham consisting of internal fixation without fracture (sham), (3) subcritical fracture (0.6 mm long) consisting of a surgically induced transverse fracture with internal fixation (subcritical), and (4) critical fracture (1.6 mm long; exceeding the local bone diameter) with internal fixation (critical). Bilateral surgeries were performed, and the groups were randomized among limbs. Histology of the defects (n = 3 for each group) was analyzed at 2 and 5 weeks postsurgery, and biomechanics (n = 12 for each group) at 5 weeks postsurgery.
All animal protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee.
Each animal was anesthetized and maintained on isoflurane. The hair over both hindlimbs was clipped, and they were prepped using chlorhexidine and alcohol. Limbs designated for the normal group were not manipulated. For sham surgery, longitudinal incision was made over the anterolateral aspect of the thigh. The surgeon then applied a novel, centrally tapered four-hole plate to the femur (Fig. d, e). Each plate was aligned and centered along the length of the femur using a custom-designed positioning clamp (Fig. a–c). The plates allowed for bicortical fixation of four screws, and provided a central taper to allow surgeons to perform an osteotomy. The undersurface of each plate includes a series of centering tabs both to provide alignment and to ensure limited contact with the bone surface. A 0.6-mm-long defect was created in limbs designated for subcritical fracture using a drill-bit after stabilization with the fixation plate (Fig. d). In limbs receiving critical-sized fracture defects, 1.6-mm-long defects were created in a band-saw-like fashion using a larger drill-bit (Fig. e). All debris was irrigated from the fracture site prior to muscle reapposition. The skin was then closed, and mice were allowed to recover from surgery. After recovery, mice were housed in individual cages and allowed unrestricted activity until sacrifice.
Fig. 1 Fracture fixation technique. a–c Top, side, and bottom views of centrally tapered fracture plate. d, e The plate is first clamped to the femur, screw holes are drilled, and screws are inserted. f, g Subcritical and critical defects, respectively (more ...)
Femurs were excised, trimmed of skin, and fixed in 10 % neutral buffered formalin (HT501320.9; Sigma) at 4 °C before shipment to the Rowe laboratory for histological imaging. The fixator device was removed after the femur was radiographed at 24 kV for 8 s with a digital X-ray (LX 60; Faxitron, USA). Subsequently, each femur was soaked overnight in 30 % sucrose/phosphate-buffered saline (PBS) solution, positioned in Neg-50 frozen-section medium (Richard-Allan Scientific, MI), frozen over chilled methylbutane, and kept at −20 °C. Longitudinal full-length 5-μm cryosections were taken either in line with or in cross-section to the screw holes (CM3050S cryostat; Leica, Germany) using a steel blade (cat# 3051835; Fisher Scientific, MA) and nonautofluorescent adhesive film (Section Lab, Co., Ltd., Toyota-gun, Hiroshima, Japan). Three sections were taken at three different depths, and each section was transferred to a glass slide, soaked for 10 min in PBS, stained in 30 mg/mL calcein blue solution (#M1255-1G; Sigma) for 30 min, and cover-slipped with 50 % glycerin in PBS prior to microscopy for the endogenous fluorescent signals.
After imaging of the endogenous signals, the cover-slip was removed and the slide was processed for additional stains. Osteoclasts were identified using fluorescent ELF-97 phosphatase substrate (E6589; Invitrogen), to detect tartrate-resistant acid phosphatase (TRAP) activity. Next, the slide was stained for alkaline phosphatase (AP) activity. After washing in PBS, the slide was re-cover-slipped for imaging using 50 % glycerol containing 10 μg/mL Hoechst 33342 (#H-3570; Molecular Probes). Hematoxylin only or hematoxylin and eosin (H&E) staining was performed on the same slides once the fluorescent staining and imaging were completed. After the cover-slip was removed, the slides were first stained in Myers modified hematoxylin solution (#S216-16 oz; Poly Scientific R&D Corp) for 1 min and then washed with tap water. Then, slides were soaked in bluing solution (#6769001; Shandon) for 2 min and washed with tap water. Slides were then cover-slipped again for imaging.
Fluorescent expression within the femoral sections was examined at 2 and 5 weeks using a Zeiss Mirax Midi scanning fluorescent microscope (Carl Zeiss, Thornwood, NY) and imaged with a high-resolution monochrome digital camera (Zeiss AxioCamHRm). A differential interference contrast (DIC) image was acquired at the same time as the endogenous fluorescence imaging. After detecting bone mineralization with three filters [4′,6-diamidino-2-phenylindole (DAPI), Chroma, #49000ET; Col3.6 blue with cyan fluorescent protein (CFP) (blue), #49001ET; Col3.6 green with yellow fluorescent protein (YFP), 49003 ET], each slide was removed and stained with ELF-97 and reimaged with a yellow filter optimized for tetracycline (Custom HQ409sp, 425dcxr, HQ555/30, set lot C-104285; Chroma Technology). Sections were then stained for: (a) AP activity [fast red substrate with tetramethylrhodamine isothiocyanate (TRITC) filter; Chroma 49005 ET], and (b) hematoxylin imaging (Zeiss AxioCamMRc 5 color camera). The Mirax software created an image stack for each filter setting that could be merged and exported.
Micro-computed tomography (μCT) imaging and biomechanical analysis
At 5 weeks postoperatively, the femora were excised, trimmed of skin and muscle, and frozen at −80 °C before shipment overnight on dry ice to the Awad laboratory for micro-CT imaging and biomechanical testing. On the day of mechanical testing, each femur was thawed and scanned by micro-CT (VivaCT 40; Scanco Medical, Bassersdorf, Switzerland) at 70 kVp and 145 μA with 300 ms integration time.
Specimens were then hydrated for 1 h in PBS. Each femur was potted in poly(methyl methacrylate) (PMMA) bone cement (DePuyOrthopaedics, Inc., IN) in sections of square aluminum tubing in a custom jig to ensure axial alignment and constant gage length. The bone cement was allowed to set for 2 h prior to rehydration in PBS at room temperature for 1–2 h. Plated specimens were centered in the gage length based on the center of the fixation plate. Nonplated specimens were centered in the gage length based on anatomical landmarks (lateral ridge on the proximal half) for consistency. Prior to testing, the titanium plates were cut perpendicularly through the center using a hand Dremel and a stainless steel, diamond coated disc (Part # 011960U0, Brasseler USA Dental Instrumentation, GA). A #11 scalpel blade was inserted in the space between the bone and plate to protect the femur from the Dremel blade during cutting. Specimens were tested in torsion using an EnduraTec TestBench system (200 N-mm torque cell; Bose Corporation, MN) at 1°/s until failure. Testing was stopped if specimens showed no torsional resistance by 30–40° of rotation.
Biomechanical properties from the normal, sham, subcritical, and critical defects were compared using two-way analysis of variance (ANOVA). Tukey’s honestly significant difference (HSD) post hoc comparisons were made in the event of statistical significance (p < 0.05). All data were analyzed using SPSS 13.0 (Chicago, IL).