Surgery
Twenty adult Swiss-Alpine female sheep with the average of 2.7 years age and 72.9 ± 6.0 kg mass were used in this study. Animals were acquired from a private source (Knüsel, Küttigen, Switzerland). All experiments were conducted according to Swiss laws for animal welfare (TSchG 455) and granted by the local veterinary authorities (license # ZH 071/17). Animals and treated limbs were randomly selected and evenly allocated to three treatment groups with six sheep each after 12.4 days of acclimatization on average. Surgeries were alternated on right and left tibiae. A 4.5/5.0 broad six-hole, locking compression plate (426.561, Synthes, Oberdorf, Switzerland) was implanted with three different combinations of screws (Fig. 2). In group VFLS3, three Variable Fixation Locking Screws (S540032S, 5.0 mm Variable fixation Locking Screw, Ti alloy, Biomech Innovations, Nidau, Switzerland) were implanted in the proximal and three standard locking screws (413.332, 5.0 mm locking screw, Ti alloy, DePuy Synthes, Oberdorf, Switzerland) were implanted in the distal segment. Both the proximal and distal segments were implanted with three locking screws in group LS and with three Variable Fixation Locking Screws in group VFLS6.
Variable fixation has been characterized in a previous biomechanical investigation on constructs featuring combinations of technologies similar to those tested here in vivo [16]. In vitro, we have shown that, at the beginning of the treatment, the stability offered by VFLS3 and VLFS6 is equivalent to that of standard LS technology. In VFLS groups, sleeve resorption led to a significant decrease in construct stiffness and to significantly larger interfragmentary displacements, with a remarkable increase at the cis cortex. All these changes nicely commensurate to the combination of technologies, with VFLS3 providing intermediate values between LS and VFLS6.
Surgery and all associated procedures strictly followed an established protocol [21] that allowed consistently fixing the tibial segments with a 3 mm parallel gap in all animals. Briefly, a 15 cm skin incision was performed on the medial aspect of the tibia and the broad 6-hole locking compression plate was slightly contoured to fit the tibial shaft. A custom cutting guide, was temporarily fixed to the intact tibia with four mono-cortical 4 mm screws (L16-18 mm, steel, DePuy Synthes 02.204.016–18). Four rubber rings allowed to position the plate at a standard distance from the periosteum. An oscillating saw (Synthes, saw blade 519.150, 70/49*14*0.6/0.4 mm) was used to perform the osteotomy through the guiding slots under constant irrigation with 0.9% saline solution. After removal of the custom cutting guide, the fragments were repositioned and fixed with the six-hole plate and either combination of screws with the 3-mm distance holder in place to further ensure a standardized parallel gap. All devices have been implanted using the instruments and torque recommended by the manufacturers and soft tissues were closed with resorbable suture material (Fig. 3). After surgery a full cast was applied protecting the fixation while allowing full weight bearing directly after surgery. Sheep were kept in small pens and a standard suspension system for 3 weeks. Starting at week three cast changes were performed in combination with weekly standardized radiographs taken in three projections (anteroposterior and mediolateral ±5°) until sacrifice. Finally, fluorescence dyes were administered subcutaneously to assess new bone deposition and remodeling during the healing period. Calcein green (5 mg/kg BW) was injected at 3 weeks, xylenol orange (90 mg/kg BW) at 6 weeks and oxytetracycline, (20 mg/kg BW) 48-72 h prior to sacrifice.
At 9 weeks all sheep were slaughtered in the hospital’s officially accredited slaughter by a professional butcher facility according to the Swiss law (VSFK, SR 817.190 and VHyS, SR 817.190.1). They were slaughtered using a cartridge driven captive bolt gun to achieve unconsciousness. Immediately thereafter, they were completely bled by cutting the main blood vessels in the neck area. All treated limbs were dissected documenting the macroscopic appearance of soft and hard tissues around implants. Local draining poplitei and inguinal lymph nodes of treated and contralateral limbs were first macroscopically examined for changes in size, color, consistency and, after harvesting, sent for histological assessment. Macroscopic assessment of the implants included overall stability of the fixation, locking of each individual screw, callus formation also over the implants, fibrosis and specifically metallosis around the implants. Finally, implants were removed and the operated and contralateral tibiae were sent for further investigations wrapped in hydrated gauzes.
Radiologic evaluation
Weekly radiographs (week 3 to 9) were semi-quantitatively scored by two independent reviewers for cortical callus formation at the cis- and trans-cortex. There, callus formation at the periosteal and endosteal site were assessed focusing on bridging of the 3 mm gap. In the slightly oblique views, the cranial and caudal callus formation was scored separately. Additionally, opacity of the callus was recorded as well as the different reaction of the cortical bone around the tip of the screws as image of local cortical bone activation due to possible micromotion. Last, the Rust score was assessed for each osteotomy. Quantitative analysis of all radiographs (week 3–9) was performed using the imaging software (OsiriX) measuring the total callus area (cm2).
Micro computed tomography
Bone samples wrapped with hydrated gauze were scanned with a cone-beam microCT (XtremeCT II, SCANCO Medical AG, Brüttisellen, Switzerland) with the x-ray tube operated at 68 kVp, 1470 μA; 900 projections/180° were acquired with 43 ms shutter time. The slices were reconstructed across an image matrix size of 1654 × 1654 voxels, with a nominal voxel size of 60.7 μm. A Gaussian filter was used to minimize noise and a thresholding algorithm was used for segmentation of bone (>1000mgHA/ccm) relative to background and for segmentation of callus (250-1000mgHA/ccm) relative to adjacent native bone. Bone and callus masks were refined using a custom series of opening and closing transformations. The image processing algorithms were developed with EasyIPL, a high-level library of macros using the scanner software (SCANCO Image Processing Language, IPL and HP OpenVMS Digital Command Language, DCL). Parameters of interest were bone and callus volume and density, the profile of the cross-sectional polar moment of inertia (pMOI) along the bone major axis, the callus volume at the cis and trans-cortex. Parameters were calculated in two regions of interest: the whole bone, defined as the sample volume under the plate, and the gap, defined as the sample volume between the proximal and distal screw adjacent to the osteotomy gap (Fig. 2).
Biomechanical testing
Non-destructive torsion tests were performed. Operated and contralateral tibiae were tested in torsion on an Instron® E10000 testing machine. Axial load and torque were measured with a calibrated Instron® Load Cell ±10 kN / 100 Nm and recorded through the Instron® Console 8.4 software. Each tibia was fixed to the test frame using standard polymethylmethacrylate embedding and constantly kept moist using soaked tissues. Samples were tested in angular displacement control at 5 degree/min and under 20 N axial load. The machine stopped as a 3 Nm drop in recorded torque was detected in order to allow further histological investigations. Data were recorded at 20 Hz. Torque (Nm) and angular displacement (degree) at failure, apparent stiffness (Nm/degree) in the linear region of the loading curve, yield point (Nm) and energy to failure (Nm*degree) were determined. Variables of interest for each operated tibia were recorded as absolute values as well as normalized to the values measured for the contralateral limb.
After testing, all samples were cut in smaller pieces including the osteotomy site and the first proximal and distal screw holes and were brought to the histology laboratory fixed in 50% ethanol.
Histology
Four investigations were performed: 1) analysis of the draining lymph nodes, 2) biocompatibility analysis of the local tissue effects according to ISO10993-6:2016, 3) histomorphometric measurements of callus formation in ground sections and 4) evaluation of bone deposition and remodeling using the fluorescent sections.
The inguinal and popliteal lymph nodes of treated and non-treated limbs of the VFLS6 and LS group were fixed in 4% formalin, routinely embedded in paraffin and stained with H&E for qualitative evaluation of structural changes and non-local cell content. Particular attention was paid to inflammatory cells and the presence of foreign material from the polymer sleeve and/or local metallosis. The Ziehl-Neelsen staining was used to determine the presence of acidophilic bacteria. Lymph nodes of VFLS3 were not evaluated due to the combined usage of different screws in the same animal.
After fixation of the non-decalcified bone samples in 50% ethanol, they were further fixed in an ascending series of ethanol under light protection. Thereafter, samples were defatted in xylene and subsequently infiltrated in liquid polymethylmetacrylate (PMMA) until blocks were hardened. The polymerized samples were then cut lengthwise in the axis and midline of the screws using an Exact 310 saw (EXAKT Technologies, Inc. Oklahoma City, US). Ground sections were polished and then surface stained with toluidine blue, thin sections (5 μm) were cut with a microtome (Leica RM 2155, Leica Instruments GmbH, Nussloch, Germany) and after mounting in glass slides stained with toluidine blue or von Kossa/McNeill. Toluidine blue staining allows to distinguish old from newly built bone due to its color intensity.
The evaluations of local biocompatibility (inflammation and tissue response) were performed on hematoxylin-eosin, toluidine blue and von Kossa stained thin sections (N = 138) using a light microscope (microscope Leica DMR system). The evaluation was performed by two independent observers. Assessment of biocompatibility parameters of VFLS screws in comparison to the standard locking screw was evaluated in the area of the cis- and trans-cortex screw holes including bone marrow cavity. The evaluation was performed comparing VFLS6 group screws to LS group screws as well as the VFLS screws and locking screws implanted in the same animals (VFLS3 group).
Software based quantitative histomorphometry was conducted on digital images of interactively color-highlighted toluidine blue stained ground sections captured with a microscope (Leica Z6 APOA, Leica DFC 420C, Glattbrugg, Switzerland). Measurements quantified the percentage of old and new bone, and non-bone (non-bone containing tissue like fibrous tissue, fat, bone marrow tissue) on the total section (between the proximal and distal screw adjacent to the osteotomy gap) and, in the gap, on the cis-, on the trans-cortex and on the endosteal area.
Fluorescent sections of the gap area were recorded and quantitatively evaluated with an image software (Fiji, ImageJ) for differences of dye integration between groups and at different time points (calcein green at 3 weeks, xylenol orange at 6 weeks and oxytetracycline at 9 weeks post-surgery).
Statistical analysis
Sample size has been chosen on a power analysis of previous data derived from similar experimental measurements with this same model [21]. The a priori exclusion criteria were: implants not loaded due to incorrect positioning, loss of screw to plate rigid connection or not weight bearing animals. Statistical analysis was performed using parametric or non-parametric test methods depending on the normal distribution (ANOVA or Signed rank test) and appropriate post-hoc testing (in e.g. Tukey). All statistical analyses were performed using the software SPSS (Version 2.5) or R.