Abstract
Longitudinal in vivo micro-computed tomography (micro-CT) is of interest to non-invasively capture the healing process of individual animals in preclinical fracture healing studies. However, as adverse imaging effects associated with anesthesia, handling, and radiation have been reported in some cases, this study assessed imaging-associated effects on fracture healing in a mouse femur defect model. A scan group received weekly micro-CT measurements (week 0-6), whereas controls were only scanned post-operatively and at week 5 and 6. Registration of consecutive scans enabled assessment of bone turnover with distinct characteristics of the different healing phases. Weekly micro-CT application did not significantly change any of the assessed callus parameters in defect and periosteal volumes. This was supported by histology showing only small amounts of cartilage residuals in both groups, indicating progression towards the end of the healing period. Also, immunohistochemical staining of Sclerostin, previously associated with mediating adverse radiation effects on bone, did not reveal differences between groups.
The established longitudinal in vivo micro-CT-based approach allows monitoring of healing phases in mouse femur defect models without significant effects of anesthesia, handling and radiation on callus properties. Therefore, this study supports application of longitudinal in vivo micro-CT for healing-phase-specific monitoring of fracture repair in mice.
Introduction
Adequate monitoring and characterization of the healing process is important in preclinical fracture healing studies. One recent approach to non-invasively capture the formation and remodeling of the osseous fracture callus is the repeated application of in vivo micro-computed tomography (micro-CT) 1-3. This approach allows three-dimensional assessment of callus structures and by registering consecutive scans, dynamic parameters such as bone formation and resorption can be evaluated 4,5. As each animal can be followed individually throughout the healing process with low variance in the assessed parameters, animal numbers can be reduced compared to well-established cross-sectional studies with endpoint micro-CT and two-dimensional histological callus evaluation.
In non-fractured bone, longitudinal time-lapsed in vivo micro-CT has been increasingly used to monitor changes in bone properties associated with different diseases and external factors, e.g. estrogen-deficiency 6-8, mechanical-(un)loading 9-11, and drug application 12. However, several studies indicate that anesthesia, cumulative radiation dosage and stress due to the required handling for the CT measurements may have effects on animal well-being and bone properties 7,13-16. According to the EU Directive 2010/63, the severity of repeated isoflurane anesthesia can be categorized as mild, although repeated anesthesia was considered worse than a single session with sex-dependent differences in perceiving the severity of a procedure 13. Specifically, female mice were shown to be more susceptible to anesthesia-induced effects on well-being compared to male mice. Nevertheless, in both sexes repeated isoflurane anesthesia caused only short-term mild distress and impairment of well-being, mainly in the immediate post-anesthetic period. Radiation has also been shown to have dosage-dependent effects on bone cells in vivo and in vitro (12): Whereas high dose x-ray radiation (2.5-8Gy) was associated with reduced osteoblast and osteoclast proliferation 9,17-19, lower doses (<2Gy) had a stimulatory effect on osteoclasts. Some studies reported radiation-associated effects on structural bone parameters, whereas other studies did not see any changes 7,20. These findings indicate the importance of study-specific adaptation of micro-CT protocols, and to protect animals scanning times and radiation settings should be minimized.
Recently, several fracture healing studies have applied longitudinal in vivo micro-CT to monitor callus formation 2,3,21-23. However, as adverse effects of longitudinal imaging on the development of bone properties have been reported in the literature in some cases during normal bone remodeling, there is a need to also assess imaging-associated impact on the callus formation during the highly metabolically active process of fracture healing.
Therefore, the objectives of this study were to establish an in vivo micro-CT based approach for longitudinal monitoring of fracture healing in a mouse femur defect model and to assess the combined effect of radiation, anesthesia and handling associated with weekly time-lapsed micro-CT measurements on callus properties during the remodeling phase of fracture healing.
Results
General physical observation
All mice recovered rapidly from surgery. During the healing period, in both groups, the animals’ body weight did not significantly change compared to pre-operative values without significant differences between the two groups (see Supplementary Fig. S1 online). Social interaction between mice and nesting behaviour did not differ from pre-surgical observations and was similar for animals of the scan and control groups.
Volumes of interest (VOI) for evaluation by time-lapsed in vivo micro-CT
One animal from the scan group could not be included in the analysis due to failure in VOI generation caused by too little cortical bone being present in the field of view. The four different VOIs (depicted in Fig. 1) encompassed the following volume for the control (n=8) and scan group (n=10): 2.51±0.34mm3 vs. 2.60±0.34mm3 for the defect center (DC), 17.26±2.21mm3 vs. 18.73±4.64mm3 for the defect periphery (DP), 1.86±0.48mm3 vs. 1.73±0.45mm3 for the cortical fragments (FC), 14.47±3.30mm3 vs. 13.10±2.66mm3 for the fragment periphery (FP). The total volume (TOT) between the inner pins of the fixator was 36.10±2.81mm3 for the control and 36.15±3.74mm3 for the scan group. No significant differences in volume were detected in any of the VOIs between groups.
Longitudinal monitoring of fracture healing by time-lapsed in vivo micro-CT
In the scan group (n=10), the repeated micro-CT scans (1×/week, Fig. 2) covered the period from the day of the defect surgery (d0) until post-operative week 6 with distinct callus characteristics indicative of the different healing phases (inflammation, repair, remodeling). From week 0-1 to week 1-2 a significant 5.7× increase in bone formation was detected (Fig. 3a) in the total VOI (TOT=DC+DF+FC+FP, p<0.0001; Fig. 1), indicating progression from the inflammation to the reparative phase. This led to a significant gain in bone volume by week 2 (BV/TVweek2: 39±7% vs. BV/TVweek0: 25±3%, p=0.0134; Fig. 3b). As bone formation triggers the onset of bone resorption, a significant 2.8x increase in resorptive activities was seen from week 1-2 to week 2-3 (p=0.0020), indicating the progression to the remodeling phase. Two weeks after surgery the highly mineralized bone fraction in the TOT VOI was significantly lower compared to the post-operative measurement (BV645/BV395 in week 2: 59±5% vs. BV645/BV395 in week 0: 84±1%, p<0.0001), indicating formation of mineralized callus of low density. From postoperative week 2 onwards, the highly mineralized bone fraction in the TOT VOI gradually increased in all subsequent weeks of the healing period reaching statistical significance by week 5 (BV645/BV395 in week 2: 59±5% vs. BV645/BV395 in week 5: 79±3%, p=0.0134; Fig. 3c).
In order to better capture the regions where bone is mainly formed and resorbed, we evaluated the different VOIs separately (Table 1): In the early postoperative phase from week 0-1 to week 1-2 a strong onset of bone formation was seen in the DC and FP sub-volumes, leading to significant 11.8× and 3.4× gain in mineralized tissue from week 0 to week 2 for DC (p=0.0090) and FP (p=0.0091), respectively. This indicates that both intra-cortical as well as periosteal callus formation takes place in this femur defect model. In both VOIs bone formation triggered the initiation of bone resorption from week 2-3. In detail, there was a significant 6.2× and 2.9× increase in bone resorption from week 1-2 to week 2-3 in the DC (p=0.0040) and the FP VOI (p=0.0079), respectively. Compared to the DC and FP VOIs, the initiation of bone formation was much less pronounced in the DP sub-volume with less deposition of mineralized tissue leading to only little peripheral callus formation in this VOI (week 2-6) and subsequently low bone resorption activities from week 2-3 to week 5-6. In all three regions (DC, DP, FP) the fraction of highly mineralized tissue considerably increased from week 2 to week 5 (+192%, +774%, +227%), indicating callus maturation. In this femur defect model, callus formation and remodeling mainly took place in the defect region (DC) with only little peripheral callus formation and remodeling (DP). Looking at the cortical fragments (FC), a significant 3.2× increase in resorptive activities was detected in week 1-2 compared to week 0-1 (p=0.0421), whereas no significant weekly change in bone formation activities was seen in this region throughout the assessed healing period. This resulted in a significant 24% reduction in bone volume from week 0 to week 4 (p=0.0091). The fraction of highly mineralized bone also gradually decreased in the remaining osseous tissue reaching statistical significance by week 3 (BV645/BV395 in week 3 reduced by 9% compared to week 0, p=0.0027).
Influence of the longitudinal in vivo micro-CT protocol on callus properties
To assess the combined effects of radiation, anesthesia and handling associated with weekly micro-CT measurements (week 0 - week 6) on callus properties, callus parameters of the scan group were compared to control animals that were scanned only directly post-operatively (d0) and after 5 and 6 weeks (Fig. 4).
Post-operatively (d0), no significant group differences in bone volume (BV/TV) were found in any of the VOIs: 1.82±0.39% (control) vs. 1.47±0.15% (scan) in DC, 0.16±0.12% (control) vs. 0.13±0.06% (scan) in DP, 56.82±2.31% (control) vs. 57.14±6.99% (scan) in FC, 4.66±0.30% (control) vs. 4.74±0.76% (scan) in FP. Likewise, no group differences in bone volume were seen in the total volume (TOT; 26.92±4.69% in controls and 24.90±2.88% in the scan group). During the remodeling phase (post-operative weeks 5-6), no significant difference in bone turnover was seen in the total volume (TOT) between the inner pins of the fixator (BFR: 0.76±0.23% in controls, 0.64±0.15% in scan group; BRR: 0.82±0.25% in controls, 0.78±0.26% in scan group). Also, bone volume did not significantly change due to the applied micro-CT protocol with a similar fraction of highly mineralized tissue after 5 weeks (BV645/BV395: 79±3% in controls, 79±3% in scan group) and 6 weeks (BV645/BV395: 81±3% in controls, 82±4% in scan group).
Regarding the four sub-volumes (Table 2), the applied micro-CT protocol did not significantly affect bone formation and resorption activities in the callus VOIs (DC, DP and FP) from week 5-6 with similar bone volume observed in week 5 and 6 for controls and scanned animals. In both groups, bone volume remained stable from week 5 to week 6, whereas the density of the mineralized tissue increased during the same period (DC, DP, FP). Looking at the adjacent cortical fragments (FC) also no significant differences were detected in any of the assessed parameters between the control group and the scan group. Similarly to the callus VOIs, bone volume also remained stable in this region from week 5 to week 6. However, in contrast to the callus VOIs, the density of the mineralized tissue in the cortical VOI FC did not change from week 5 to week 6. In week 6, the FC VOI comprised 33% and 35% of the osseous tissue in the total VOI (TOT) for the control and scan group, respectively. In the DC VOI, 37% (control group) and 36% (scan group) of the total bone volume were seen. Less osseous tissue was detected in the two peripheral VOIs, 11% (control group) and 12% (scan group) in the DP and 19% (control group) and 17% (scan group) in the FP VOI.
As group comparisons were only performed from week 5-6, we particularly focused on the defect VOIs (DC+DP) which are most important for evaluating later healing time points during the remodeling phase of fracture healing. No significant differences in bone turnover, bone volume and mineralization were seen between the scan and control group. In addition, according to the standard clinical evaluation of X-rays, the number of bridged cortices per callus was evaluated in two perpendicular planes and animals with ≥3 bridged cortices were categorized as healed. By week 5, cortical bridging occurred in 75% of the control animals and 64% of the scanned animals (Table 3). However, non-unions only occurred in defects of ≥1.5mm length. Taking into account only these defects (≥1.5mm length), both groups had a non-union rate of 67% indicating no significant effect of the chosen micro-CT protocol on clinical fracture healing outcome.
Histology
As shown by Safranin-O staining six weeks after osteotomy no cartilage residuals were present in the defect region in the bridged defects indicating normal progression of the healing process in the final remodeling stage. In the non-union defects, only small areas of cartilage were seen in the defect region. No differences were seen by visual inspection between animals from the scan and control group (Fig. 5).
As radiation-mediated decreased bone formation by osteoblasts has previously been associated with the inhibition of osteoanabolic Wnt-signaling 24, we assessed Sclerostin expression in the defect region by immunohistochemistry. Visual inspection did not reveal any differences in staining intensity and amount of Sclerostin between the scan and control group (Fig. 5).
Discussion
In this study, longitudinal in vivo micro-CT was applied for monitoring the process of fracture healing in mice. In addition, the combined effect of radiation, anesthesia and handling associated with the established imaging approach on callus parameters was assessed. Until now, preclinical fracture healing studies predominantly have a cross-sectional study design with end-point callus evaluation in sub-groups of animals at different time points during the healing period. Only recently, several studies in murine diaphyseal femur defect models stabilized by external fixation 2,3,21,22 or intramedullary nails 23, have applied longitudinal in vivo micro-CT to monitor the healing progression in each animal over time. These studies were able to consecutively assess callus parameters (e.g. callus volume and density) at specific time points during the healing process for up to 12 weeks 2,3,21-23. By registering consecutive scans, we were now able to include dynamic parameters such as bone formation and resorption in our micro-CT based monitoring approach for fracture healing. This allows for characterization of the different healing phases seen by changes in formation and resorption in the osseous callus volume.
Specifically, we saw that the initiation of bone formation (maximum in week 2-3), indicative of the onset of the reparative phase, triggered bone resorption (maximum in week 3-4) with maximum osseous callus volumes in week 3. From week 4 to week 6, bone formation and resorption continuously decreased. Whereas these remodeling activities also led to a decrease in callus volume from week 4 to week 6, the density of the mineralized tissue in the callus increased during the same period indicating advanced callus maturation. These observations were further supported by histology with no remaining cartilage in the defect region of unions and only a small amount of cartilage residuals in some non-unions. The findings are also in accordance with previous longitudinal studies of up to 4 weeks in mice 2,3,22 and 8 weeks in rats 21 mainly focusing on the inflammation and reparative phase. Similar to our study, the bone volume continuously increased during the 4/8 week healing period, although no remodeling to the original bone geometry was seen due to the shorter observation period. One study in ovariectomized rats with a 12-week post-operative monitoring period detected maximum osseous callus volumes by week 6, which diminished thereafter, whereas bone mineral density continuously increased until week 12, similar to our findings. This indicates that during the reparative phase first low-density bone is formed, which is then further mineralized during the remodeling phase of fracture healing. Looking at the functional fracture healing outcome, complete cortical bridging was seen in 64% of the animals, which is similar to other studies (60-62%) using similar femur defect models with relatively stiff external fixation (fixator stiffness measured in our study 24N/mm) in mice 2,25. However, differences in defect sizes have to be considered: 0.7mm 2, 1.19±0.25 mm 25, 1.47±0.16mm (this study). In contrast to the other studies, we saw the manifestation of non-unions only in defects ≥1.5mm. This might be partially due to the fact that in some studies the exact defect length might have differed from the reported value (saw diameter used for osteotomy) and that non-union formation was assessed irrespective of defect length. Overall, all longitudinal fracture healing studies using in vivo micro-CT were able to follow the healing progression in single animals, thereby reducing variance of outcome parameters compared to cross-sectional studies, also allowing for reduction of animals per group considering the 3R principles of animal welfare 26,27. However, when applying longitudinal in vivo micro-CT, the effect of repeated anesthesia, handling and radiation associated with the scans on the study’s main outcome parameters and the general well-being of the animals have to be considered.
In this study, isoflurane was used as anesthetic agent for the in vivo micro-CT scans, which is the most commonly used inhalation anesthetic in longitudinal imaging studies with a fast on- and offset of anesthesia and low metabolism rate 13. Despite its general use, isoflurane has been associated with several adverse effects, such as hepatic degenerative changes 28, immunomodulation 29, oxidative DNA damage 30 as well as alterations in expression profiles of oncogene and tumor suppressor genes in the bone marrow 31, which might potentially also affect fracture healing and the general well-being of the animals. A recent study by Hohlbaum et al. (2017) 13, which assessed the impact of repeated isoflurane anesthesia (6 times for 45 min at an interval of 3–4 days) and the associated handling on the well-being of adult C57BL/6JRj mice, categorized the degree of distress as mild according to the EU Directive 2010/63 with only short-term impairment of well-being, mainly in the immediate post-anesthetic period. Therefore, isoflurane-based inhalation anesthesia and the associated handling of the animal is suggested for longitudinal studies with multiple anesthetic sessions per animal.
In this study, the applied micro-CT settings (55 kVp, 145 µA, 350 ms integration time, 500 projections per 180°, 21 mm field of view (FOV), scan duration ca. 15 min) were adapted from well-established protocols used for longitudinal monitoring of bone adaptation and implant integration in tail vertebrae in mice 10,32. These settings are similar to protocols used in 2 of the 5 longitudinal fracture healing studies published so far 3,21, while the 3 other studies did not specify the CT settings 2,22,23. The radiation dosage (CT dose index, CTDI) associated with each scan in the current study was previously estimated to be 0.67 Gy per scan 32,33. A radiation control experiment using similar settings did not show detrimental effects of 5 weekly in vivo micro-CT scans on bone development in tail vertebrae of mice 33.
However, some studies in non-fractured bone indicate cumulative radiation-associated effects on bone properties particularly when using high radiation doses (>2G; 15). The radiation effects also depend on animal age 15,34 and ovariectomy (OVX) 7, with younger and ovariectomized animals being more susceptible to radiation. So far, only one study assessed radiation-associated effects of longitudinal in vivo micro-CT on bone healing in a uni-cortical tibia burr-hole defect model, and did not find significant radiation effects on bone properties 1. In this study, similar CT settings (45kVp, 133 µA, 200 ms integration time, 1000 projections per 180°) were used; however, due to the smaller dimensions of the defect and resulting shorter scanning time, the radiation dosage (0.36 Gy) was lower compared to our protocol (0.64 Gy).
In the current study, we assessed the combined imaging-associated impact (anesthesia, handling, radiation; 1×/week for 6 weeks) on callus formation and remodeling in a diaphyseal femur defect model in adult female C57BL/6J mice. We did not see any significant imaging-associated changes in bone volume and turnover in the fracture callus during the remodeling phase (post-operative week 5 and 6). Furthermore, no significant differences in callus mineralization between scanned (d0, week1-6) and control animals (d0, week 5+6) in any of the assessed callus volumes (TOT, DC, DP, FP) were observed. The distribution of the osseous callus volume into the three sub-volumes was also not significantly different between groups, indicating similar healing patterns. In addition, when separately assessing the cortical fragments between the inner pins (FC), also no significant differences in any of the assessed parameters were detected between groups. In respect to clinical healing outcome, the percentage of unions in animals above the sub-critical defect length was 67% in both groups. Defects without cortical bridging only showed small cartilage residuals as visualized by Safranin-O staining, indicating manifestation of non-union formation. In the defect regions of unions, no cartilage was seen, indicating progression towards the end of the healing period. As adverse effects of radiation on bone properties have been shown to be mediated via Sclerostin inhibition of osteoanabolic Wnt-signaling 24, we also visualized Sclerostin expression in the fracture callus by immunohistochemistry, but did not see differences in number of stained osteocytes, staining intensity and pattern between groups in any of the assessed callus regions.
In summary, we did not see significant differences in a total of 7 assessed CT and histological parameters in the total volume (TOT) between the inner pins of the fixator as well as in the different callus sub-volumes (DC, DP, FP) and the adjacent cortical fragments (FC) between the scan and the control group. The sub-volume specific analysis also allowed capturing of distinct features only present in one region (e.g. significant post-operative increase in bone resorption in FC), that showed contrary progression compared to the overall picture (significant post-operative increase in bone formation in DC, DP and FP). This also shows the importance of VOI selection. The other longitudinal fracture healing studies either assessed a VOI consisting of the defect as well as adjacent bone fragments, or then only the callus in the defect region including the last slice of intact cortex 2,3, or the callus in the defect and periosteal region combined 21 was evaluated. An overall VOI including the defect region and cortical fragments can give an overview of callus characteristics, but sub-volume-specific differences might be missed. Therefore, analysis of different endosteal and periosteal callus regions as well as the adjacent cortical volume is favorable.
The longitudinal in vivo micro-CT based approach established in this study allows monitoring of the different healing phases in mouse femur defect models without significant anesthesia-, handling- and radiation-associated effects on callus properties. By registering consecutive scans of the defect region for each animal, data on bone turnover can be obtained with distinct characteristics of the different fracture healing phases. Importantly, repeated anesthesia, handling and radiation associated with the scans did not impair callus formation and remodeling. Therefore, this study supports the application of longitudinal in vivo micro-CT for healing-phase specific monitoring of fracture repair in mice. Further studies should evaluate the potential of this micro-CT-based monitoring approach for healing phase-specific discrimination of normal and impaired healing conditions.
Methods
Study design
A micro-CT based approach for longitudinal in vivo monitoring of fracture healing was established for a mouse femur defect model. All mice received a femur defect and post-operative micro-CT scans (vivaCT 40, ScancoMedical, Brüttisellen, Switzerland) were performed. The scan group then received weekly scans of the defect area (weeks 1-6). To assess the combined effect of radiation, anesthesia and handling associated with weekly micro-CT measurements, controls were only scanned after 5 weeks. Control animals then received another scan in post-operative week 6 to enable the assessment of both, static and dynamic bone parameters during the final remodeling phase of fracture healing.
Animals
All animal procedures were approved by the authorities (licence number: 36/2014; Kantonales Veterinäramt Zürich, Zurich, Switzerland). We confirm that all methods were carried out in accordance with relevant guidelines and regulations (ARRIVE guidelines and Swiss Animal Welfare Act and Ordinance (TSchG, TSchV)). Female 12 week-old C57BL/6J mice were purchased from Janvier (Saint Berthevin Cedex, France) and housed in the animal facility of the ETH Phenomics Center (EPIC; 12h:12h light-dark cycle, maintenance feed (3437, KLIBA NAFAG, Kaiseraugst, Switzerland), 5 animals/cage). At an age of 20 weeks, all animals received a femur defect by performing an osteotomy (group 1: control group, defect length - 1.45mm ± 0.16, n=8; group 2: scan group, defect length −1.47mm ± 0.16, n=11; housing after surgery: 2-3 animals/cage). Perioperative analgesia (25 mg/L, Tramal®, Gruenenthal GmbH, Aachen, Germany) was provided via the drinking water two days before surgery until the third postoperative day. For surgery and micro-CT scans, animals were anesthetized with isoflurane (induction/maintenance: 5%/1-2% isoflurane/oxygen).
Femur osteotomy
An external fixator (Mouse ExFix, RISystem, Davos, Switzerland; stiffness: 24N/mm 35) was positioned at the craniolateral aspect of the right femur and attached using four mounting pins. First, the most distal pin was inserted approximately 2mm proximal to the growth plate, followed by placement of the most proximal and the inner pins. Subsequently, a femur defect was created using 2 Gigli wire saws.
Time-lapsed in vivo micro-CT
Immediate post-surgery correct positioning of the fixator and the defect was visualized using a vivaCT 40 (Scanco Medical AG, Brüttisellen, Switzerland) (isotropic nominal resolution: 10.5 µm; 2 stacks of 211 slices; 55 kVp, 145 µA, 350 ms integration time, 500 projections per 180°, 21 mm field of view (FOV), scan duration ca. 15 min). Subsequently, the fracture callus and the adjacent bone between the inner pins of the fixator were scanned weekly using the same settings. Scans were registered consecutively and morphometric indices (bone volume - BV, bone volume/total volume – BV/TV, bone formation rate – BFR, bone resorption rate - BRR) were computed (threshold: 395mg HA/cm3; 4,11). To assess mineralization progression, a second threshold (645mg HA/cm3) was applied and the ratio between highly and lowly mineralized tissue (BV645/BV395) was calculated. According to the standard clinical evaluation of X-rays, the number of bridged cortices per callus was evaluated in two perpendicular planes (UCT Evaluation V6.5-1, Scanco Medical AG, Brüttisellen, Switzerland). A ‘‘healed fracture’’ was considered as having a minimum of at least three bridged cortices per callus. For evaluation, four volumes of interest (VOIs) were defined, which were created automatically from the post-operative measurement (Fig. 1): defect center (DC), defect periphery (DP), cortical fragment center (FC), and fragment periphery (FP). Data were normalised to the central VOIs: DC/DC, DP/DC, FC/FC, FP/FC.
Histology
Histological analyses were performed in a sub-set of animals (n=6/group). On day 42, femora were excised, the femoral head was removed and the samples were placed in 4% neutrally buffered formalin for 24 hours and subsequently decalcified in 12.5% EDTA for 10-14 days. The samples were embedded in paraffin and 4.5 µm longitudinal sections were stained with Safranin-O/Fast Green: Weigert’s iron haematoxylin solution (HT1079, Sigma-Aldrich, St. Louis, MO) - 4min, 1:10 HCl-acidified 70% ethanol - 10s, tap water - 5min, 0.02% Fast Green (F7258, Sigma-Aldrich, St. Louis, MO) - 3min, 1% acetic acid - 10s, 0.1% Safranin-O (84120, Fluka, St. Louis, MO) - 5min. Images were taken with Slide Scanner Pannoramic 250 (3D Histech, Budapest, Hungary) at 20× magnification.
For immunohistochemical staining of Sclerostin, nonspecific sites were blocked (1% BSA/PBS + 1% rabbit serum) for 60 min at room temperature. Subsequently, the sections were incubated with the primary antibody against Sclerostin (AF1589, R&D Systems, Minneapolis, MN; 1:150 in 1%BSA/PBS + 0.2% rabbit serum) overnight at 4°C. To detect the primary antibody, a secondary biotinylated rabbit anti-goat-IgG antibody (BAF017, R&D Systems, Minneapolis, MN) was added for 1 h at room temperature. For signal amplification, the slides were incubated with avidin-biotin complex (PK-6100 Vector Laboratories, Burlingame, CA) for 30 min. Diaminobenzidine (Metal Enhanced DAB Substrate Kit, 34065 ThermoFisher Scientific, Waltham, MA) was used as detection substrate. Counterstaining was performed with FastGreen (F7258, Sigma-Aldrich, St. Louis, MO). Species-specific IgG was used as isotype control. Images were taken with Slide Scanner Pannoramic 250 (3D Histech, Budapest, Hungary) at 40× magnification.
Statistics
CT analysis: Data were tested for normal distribution (Shapiro-Wilk-Test) and homogeneity of variance (Levene-Test). Depending on the test outcome, group comparisons (scan versus control group) of data derived at single time points were performed by two-tailed Student’s t-test or Mann-Whitney U-test (IBM SPSS Statistics Version 23). For statistical evaluation of repeated measurements (scan group) dependent on results from normality and variance tests, either one-way analysis of variance (ANOVA) with Bonferroni correction or Friedman test with Dunn correction for multiple comparisons (GraphPad Prism 7) were performed. Two-way ANOVA was used for longitudinal comparison of the body weight between the control and the scan group. The level of significance was set at p < 0.05.
Author Contributions Statement
The study was designed by E.W., G.A.K., S.H. and R.M.. The experiments were performed by E.W., G.A.K. and A.C.S.. Data analyses were performed by E.W. and D.C.B.. The manuscript was written by E.W. and reviewed and approved by all authors.
Data availability
All necessary data generated or analyzed during the present study are included in this published article and its Supplementary Information files (preprint available on BioRxiv (BIORXIV/2019/692343). Additional information related to this paper may be requested from the authors.
Competing Interests
The authors declare no competing interests.
Acknowledgements
The authors gratefully acknowledge support from the EU (BIODESIGN FP7-NMP-2012-262948 and ERC Advanced MechAGE ERC-2016-ADG-741883). E. Wehrle received funding from the ETH Postdoctoral Fellowship Program (MSCA-COFUND, FEL-25_15-1).