PTH(1–34) treatment and/or mechanical loading have different osteogenic effects on the trabecular and cortical bone in the ovariectomized C57BL/6 mouse

In preclinical mouse models, a synergistic anabolic response to PTH(1–34) and tibia loading was shown. Whether combined treatment improves bone properties with oestrogen deficiency, a cardinal feature of osteoporosis, remains unknown. This study quantified the individual and combined longitudinal effects of PTH(1–34) and loading on the bone morphometric and densitometric properties in ovariectomised mice. C57BL/6 mice were ovariectomised at 14-weeks-old and treated either with injections of PTH(1–34); compressive loading of the right tibia; both interventions concurrently; or both interventions on alternating weeks. Right tibiae were microCT-scanned from 14 until 24-weeks-old. Trabecular metaphyseal and cortical midshaft morphometric properties, and bone mineral content (BMC) in 40 different regions of the tibia were measured. Mice treated only with loading showed the highest trabecular bone volume fraction at week 22. Cortical thickness was higher with co-treatment than in the mice treated with PTH alone. In the mid-diaphysis, increases in BMC were significantly higher with loading than PTH. In ovariectomised mice, the osteogenic benefits of co-treatment on the trabecular bone were lower than loading alone. However, combined interventions had increased, albeit regionally-dependent, benefits to cortical bone. Increased benefits were largest in the mid-diaphysis and postero-laterally, regions subjected to higher strains under compressive loads.


Methods
Animals and treatment. Twenty-four virgin female C57BL/6 mice were purchased at 13-weeks old (Charles River UK Ltd., Margate, UK). Mice were housed, four per cage, in The University of Sheffield's Biological Services Unit at 22 °C, with a twelve-hour dark/light cycle and ad libitum access to 2918 Teklad Global 18% protein rodent diet (Envigo RMS Ltd., UK) and water. All the procedures were performed under a British Home Office licence (PF61050A3) and in compliance with the Animal (Scientific Procedures) Act 1986. This study was reviewed and approved by the local Research Ethics Committee of The University of Sheffield (Sheffield, UK). The findings and experiments in this paper were designed and reported in accordance with the ARRIVE guidelines 35 . C57BL/6 female mice were chosen due to documented skeletal responsiveness to mechanical loading, PTH  or OVX 27,29,31,36 . Peak cortical bone mass was reported in the appendicular skeleton of female C57BL/6 mice at 3-4 months of age 37 , thus the mice herein were considered to be skeletally mature at the onset of this study (14 weeks of age). An a priori estimate of sample size based on large loading effects on the trabecular bone volume fraction and cortical thickness after six weeks of loading in PTH-treated mice 27 , indicated that six mice per group was necessary to achieve 80% statistical power and assuming Cohen's d = 2, α = 0.05.
At age 14 weeks, and following one week acclimatization, all mice underwent OVX and remained untreated for 4 weeks following surgery to allow oestrogen-deficiency related bone loss 36 . OVX mice were randomly assigned into 4 treatment groups (n = 6 mice/group) and then treated, per schedule in Fig. 1(A), with either (1) PTH  between weeks 18 and 22, subgroup "PTH", (2) mechanical loading during weeks 19 and 21, "ML", (3) concurrent treatment with PTH(1-34) and mechanical loading, "ML + PTH", (4) weekly alternating treatment with PTH(1-34) during weeks 18, 20 and 22 of age and mechanical loading during weeks 19 and 21, "ML + PTH alt ". All mice were withdrawn from treatment for the final two weeks of the study (weeks 23 and 24 of age). We confirmed treatment effects of PTH  and mechanical loading by comparing bone properties within the same animals before the treatment started (i.e. relative to week 18 values) and with a group of age-matched C57BL/6 ovariectomized mice from a previous study in our laboratory 36 .  (Bachem, Bubendorf, Switzerland) at 100 µg/kg/day 29,31 , 5 days/week (groups: PTH, ML + PTH) or vehicle (group: ML). PTH was prepared in 1% acetic acid and 2% heat inactivated mouse serum in HBSS 31 . Mechanical loading. A minimally invasive method was used for uniaxial compressive loading of the right tibia per a previously published protocol 38 . Briefly, the flexed knee and ankle were fixed between two soft cups and the tibia loaded along the superior-inferior axes to a peak load of 12 N. Tibiae were loaded to 12 N peak by superimposing a dynamic load of 10.0 N upon a static 2.0 N preload at a rate of 160,000 N/second. Forty trapezoidal waveform load cycles were applied (held for 0.2 seconds at 12 N) with a 10 second interval between each cycle. A 12 N load was previously shown to promote significant bone apposition in female C57BL/6 mice without impairing mobility following treatment 38 . Mechanical loading was applied to all mice in groups ML, ML + PTH and ML + PTH alt , three days per week (Mon, Wed, Fri) at weeks 19 and 21. In-vivo microCT imaging. The whole right tibia of each mouse was imaged in vivo with microCT (VivaCT80, Scanco Medical, Bruettisellen, Switzerland). A baseline scan (before OVX surgery) was performed at 14 weeks of age, then follow up in vivo scans performed every two weeks until week 22 (Fig. 1). At week 24 mice were euthanized by cervical dislocation and both the left and right tibia were imaged ex vivo using the in Image alignment and preprocessing. From each reconstructed microCT image two analyses were performed ( Fig. 1(B)): standard 3D morphometric analysis as defined in the guidelines of the American Society of Bone and Mineral Research (ASBMR) 39,42 and a spatial densitometric analysis 31 . The image of one tibia from one mouse at week 14 of age was randomly chosen and used as a reference. The longitudinal axis of each reference tibia was approximately aligned with the z-axis of the global reference system 43 . All remaining images (from different mice and different time points) were rigidly registered to the reference images prior to the below image analyses. The rigid registrations were performed by using a Quasi-Newton optimizer and the Normalised Mutual Information as the similarity measure (Amira 5.4.3, Thermo Fisher Scientific, France) 44 . The registered grayscale image datasets were smoothed with a Gaussian filter (convolution kernel [3 3 3], standard deviation = 0.65) in order to reduce the high frequency noise and bone voxels were defined using a global threshold, which was calculated as the average of the grey levels corresponding to the bone and background peaks in each image histogram (frequency plot) 39 .

Intraperitoneal PTH(1-34) injections. Mice received either intraperitoneal injection of PTH
Standard 3D morphometric analysis. For trabecular bone analysis a region of interest (ROI) of 1 mm height was selected, 0.3 mm below a reference line defined as the most distal image slice that included the growth plate and adapted from previous research 42, 45 . This was necessary to minimize analysis of the newly formed (modelled) trabeculae emerging from the growth plate due to continuous longitudinal growth in rodents 46 . For cortical bone analysis a region of 1 mm height was selected in the tibia diaphysis and centred at 50% of the tibia bone length 43 . ROIs in the trabecular and cortical bone were manually marked and the following 3D bone parameters were computed (CT Analyser v1.18.4.0, Skyscan-Bruker, Kontich, Belgium): trabecular bone volume fraction (Tb.BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N); cortical total cross-sectional area (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar) and cortical thickness (Ct.Th). In the midshaft cortical ROI, minimum (I min ) and maximum (I max ) principal moments of inertia, polar moment of inertia (J) and eccentricity (Ecc) were computed.

Spatiotemporal densitometric analysis. Densitometric properties were estimated in multiple regions
within the tibia adapting a previously described procedure 31 . Briefly, the length of each tibia (L) was measured at each time point, computed as the distance between the most proximal and distal bone voxels in the registered image stack, and a region 80% of L was cropped starting from the section below the growth plate (MatLab, 2018a, The MathWorks, Inc. USA). The tibia was divided longitudinally into ten transverse sections (from most proximal, section C01 to most distal, section C10) with the same thicknesses (i.e. 8% of L) and each section was then divided into quadrants (anterior, medial, posterior and lateral sectors) for a total of 40 ROIs across the length of the tibia. Anterior, medial, posterior and lateral compartments were defined by two perpendicular lines passing through the centre of mass of each slice. Bone mineral content (BMC) and tissue mineral density (TMD, mg HA/cm³) were measured in each of the 10 sections and in each of the 40 quadrants. This approach provides a reasonable compromise between the measurement spatial resolution along the tibia length (number of sections) and the densitometric measurement reproducibility, while accounting for small but still present growth of the tibia between weeks 14 and 24 of age 43,47 .
TMD in each voxel was obtained from its grey level by using the calibration curve provided by the manufacturer of the microCT scanner. The BMC was calculated in each voxel as TMD multiplied by the volume of the voxel. The BMC in each compartment was calculated as the sum of BMC in each bone voxel, while TMD in each compartment was defined as the ratio between BMC and the bone volume (BV) 36 .

Statistics.
All morphometric and densitometric properties were tested for assumptions of normality (Shapiro-Wilks test), homogeneity of variance (Levene's Test) and sphericity (Mauchly's Test). To determine whether anabolic treatments reverse OVX-induced trabecular bone loss and cortical bone adaptations, data were analysed by two-way mixed Analysis of Variance (ANOVA). Where for a given bone property the F values were significant for a 'time by intervention' interaction, the simple "time effect" was investigated using paired t-tests between (1) treatment baseline (week 18) and proceeding time-points (week 20-24) and (2) between sequential time-points (e.g. week 20-22, 22-24 comparisons) 36,48 . Between-group differences in bone properties due to treatment and treatment withdrawal (i.e. at weeks 20-24) were analysed using Analysis of Covariance (ANCOVA), adjusted for values at 18-weeks-old (treatment onset) and with post hoc pairwise comparisons (Bonferroni-adjusted for six comparisons among treatment groups). Adjustment for week 18 values mitigates bias due to potential differences in the bone properties at the onset of treatment. Statistical significance was set at α = 0.05. All analyses were performed using SPSS Statistics 25 (IBM Corp., Armonk, NY, USA).
Data are presented as mean ± standard deviation (SD) unless otherwise specified. The percentage change in morphometric properties were computed per Eq. (1), where "BP" is the mean bone property value (e.g. of Tb.BV/ TV, Ct.Th) and "i" defines a subsequent time point (weeks 20-24): Changes of tibia densitometric properties are presented as the mean relative percentage difference between the two treatment groups, normalized for the baseline values of the second group (week 14). See the Supplementary Materials for computation of mean relative percentage difference as per Lu et al. 31 .

Results
All mice completed this study without complications. One mouse in the ML + PTH alt group was removed from densitometric analysis as reconstruction of the image data in the distal tibia failed at baseline, but this did not affect morphometric analysis. Data collected in this study are accessible at https://doi.org/10.15131/shef. data.12292787.  36 ). In PTH, a small and transient, albeit non-statistically significant, increase in Tb.BV/TV was observed at week 20 (12% increase relative to week 18) corresponding with a significant increase in Tb.Th (+27%, p=0.002). Tb.BV/TV returned to baseline at week 22 (2% reduction relative to week 18). In ML, ML + PTH and ML + PTH alt , Tb.BV/TV values were significantly higher at week 22 than week 18 (+66-89%, p < 0.01), attributed to a significant increase in Tb.Th (+57-62%, p < 0.02). Individual or combined treatments with mechanical loading did not improve Tb.Sp nor Tb.N, relative to week 18 values. In cortical bone, a detectable (significant) change in morphometric properties was observed within two weeks from treatment onset (week 20). With loading (individually or combined with PTH), a significant and persistent increase in Ct.Ar and Ct.Th was observed from weeks 18 to 20 to 22 (6-20% increase/week, p < 0.01), but only from weeks 18 to 20 with PTH alone (+17-18%, p < 0.001).

Effects of treatments and withdrawal on the trabecular and cortical bone morphometry.
Significant intervention effects were observed among treatment groups ( Fig. 2). At week 22, Tb.BV/TV, adjusted for week 18 values, significantly differed among all treatment groups and was higher in ML, ML + PTH and ML + PTH alt than PTH (76-148% higher, p < 0.05), and higher in ML than ML + PTH and ML + PTH alt (14-60%, p < 0.05). Tb.Th and Tb.N were significantly lower in PTH than all other groups (−20%, −22%, and −24% for Tb.Th, and −58%, −50%, and −50% for Tb.N compared to ML, ML + PTH, and ML + PTH alt , respectively); and Tb.N was 17% lower with combined treatments than ML. In cortical bone, Ct.Th was 14-16% higher with combined treatment than PTH. Ct.Ar was 12% higher in ML + PTH than PTH. www.nature.com/scientificreports www.nature.com/scientificreports/ Significant changes in trabecular bone morphometry were observed following treatment withdrawal (Table 1 and Figs. 2 and 3). In PTH, a significant reduction in Tb.BV/TV (31%) and Tb.Th (17%), with corresponding increase in Tb.Sp (8%) was observed between weeks 22 and 24; Tb.BV/TV reductions being similar to change in untreated OVX mice. With combined treatment, significant thinning of the trabecular bone was observed, corresponding with a significant reduction in Tb.BV/TV in ML + PTH alt . In ML, changes in trabecular morphometry were not significant following treatment withdrawal. No significant change in cortical bone morphometry ( Table 2) was evident between weeks 22 and 24 in PTH, nor ML. In ML + PTH and ML + PTH alt , a significant increase in the Tt.Ar persisted following treatment withdrawal.
With PTH and/or loading, a persistent increase in the cortical midshaft moments of inertia (I max , I min and J) and eccentricity were observed (10-49% increase at week 22 compared with week 18 values, p < 0.05), which were retained two-week following treatment withdrawal (Supplementary Table S1). Whereas, in untreated mice, no change in bone properties were observed with ovariectomy over time. At week 22, I max and J were significantly higher in ML + PTH than PTH alone (Fig. S4). www.nature.com/scientificreports www.nature.com/scientificreports/ Effects of treatment on the bone densitometric properties. At week 20, BMC significantly increased between 7% and 26% along the tibia length in all treatment groups (Fig. 5). With PTH, a significant and persistent increase in BMC, from weeks 18-20-22, were observed in the most proximal (C01, 25% increase from week 18 to 22), the mid (C03, + 17%) and in the distal tibia (C06-C10, + 23 to +25%). In ML and co-treated mice, persistent increments in BMC (from 18 to 20 to 22) were observed in the proximal to mid-tibia (C01-C08, + 17 to +45%). BMC remained above week 18 values following treatment withdrawal. Sub-regionally, the greatest osteogenic benefits of loading and co-treatment were observed posteriorly and laterally, particularly in the most proximal (C01, up to +71%, Fig. 6) and mid-tibia (C03-C06; up to +63%), whereas in PTH a more homogeneous response among quadrants was observed. Every treatment increased slightly the TMD (up to 7% in the most proximal region of the PTH treated mice at week 22 of age) in most regions of the tibia (Fig. 5). While PTH increased the TMD homogeneously across the tibia and among all quadrants, for all loaded mice the central portion of the tibia was less affected particular in the anterior and medial regions (Fig. S6). In most cases small effects on TMD were maintained after treatment withdrawal.
Treatment effects differed among groups (Fig. 7). In ML, the increase in BMC was significantly higher proximally (C01; 10% difference) than PTH at week 22. With PTH, the increase in BMC was significantly higher in the distal tibia (C08-C10; 5-13% difference) than ML at weeks 20 and 22, with differences persisting following treatment withdrawal (week 24; C08-C10; 9 to 14%). By subregional analysis, greater osteogenic benefits of ML than PTH were observed in the mid-tibia postero-laterally, whereas PTH had greater benefit to the medial and more distal portions of the bone (Fig. S5, Supplementary Materials).
For TMD, the differences were small (less than 5%) and significantly differed only between PTH and combined treatment groups in the most proximal tibia region (C01; 4% lower in the combined treatments compared to PTH, Fig. 8).

Discussion
In this study we quantified for the first time the longitudinal effects of PTH(1-34) and mechanical loading on bone morphometric and densitometric properties in an ovariectomised mouse model of osteoporosis. The results herein suggest a dominant effect of mechanical loading compared to injections of PTH, with increased and regionally-dependent benefits of combined treatments to the tibia cortical bone, but limited benefits for the trabecular bone.
PTH monotherapy had no significant anabolic benefit to metaphyseal trabecular bone consistent with neutral effects in intact mice 29,31,49 , but in contrast to increasing bone mass shown in OVX rodents elsewhere 17,19,20 . PTH www.nature.com/scientificreports www.nature.com/scientificreports/ inhibited further OVX-induced bone loss by trabecular thickening, a characteristic adaptive response 17,19,20 , in the presence of declining trabecular number. Interestingly, in 50% of the mice examined the trabecular changes were characterised by a small increase in Tb.BV/TV to week 20 and then bone loss thereafter (Supplementary Materials, Fig. S1), supporting a transient response to ongoing treatment described cross-sectionally 17 . Anabolic effects of PTH depend on its ability to stimulate osteoblast, osteocyte and osteoclast activities 50 . While typically favourable to osteoblastic activity, benefits may be compromised in mice with very low (<5%) baseline Tb.BV/TV as in C57BL/6 mice herein and reported elsewhere 17 . This is in line with the limited efficacy of PTH in the less trabecular rich femoral neck, relative to benefits in the lumbar vertebra shown clinically 51 Fig. 1(a), at the beginning of week 18 and was withdrawn at the end of week 22. +Tb.Sp was significantly higher in PTH than in ML (p=0.013) and ML + PTHalt (p=0.036) at the onset of treatment. Remaining morphometric parameters did not significantly differ among the four treatment groups at week 18 following randomisation. ‡ 3D morphometry of untreated ovariectomized mice (group "OVX") and intact controls ("CTRL") from Roberts et al. 36 are reported for comparison of trends in bone adaptation.
Scientific RepoRtS | (2020) 10:8889 | https://doi.org/10.1038/s41598-020-65921-1 www.nature.com/scientificreports www.nature.com/scientificreports/ et al. 19 report, in OVX rats, strong positive interrelationships between baseline Tb.BV/TV and bony adaptations to PTH, though relationships were not confirmed by our current data (Tb.BV/TV 18 vs. ∆Tb.BV/TV [18][19][20][21][22] ; Spearman's ρ=−0.657, p=0.156, see Supplementary Materials). In the midshaft, PTH treatment lead to an immediate increase (from week [18][19][20] in cortical thickness and bone area consistent with cross-sectional findings on OVX mice 17,20 , although osteogenic benefits, except in Tt.Ar, desisted thereafter. This finding in Ct.Th is contrary to the constant linear increase observed in OVX rats 19 , and in 19-months-old intact mice where PTH exacerbated age-related thinning of the cortical bone over time 29 . PTH had relatively homogeneous benefits along the bone length, increasing BMC with constant benefits to the mid-to distal tibia, and contrary to intact mice, where benefits propagated proximal to distally and in postero-medial sectors 31 . *treatment commenced, as per Fig. 1(A), at the beginning of week 18 and was withdrawn at the end of week 22. Morphometric parameters did not significantly differ among the four groups at onset of treatment (p > 0.05). ‡ 3D morphometry of untreated ovariectomized mice (group "OVX") and intact controls ("CTRL") from Roberts et al. 36 are reported for comparison of trends in bone adaptation.
Scientific RepoRtS | (2020) 10:8889 | https://doi.org/10.1038/s41598-020-65921-1 www.nature.com/scientificreports www.nature.com/scientificreports/ Tibia loading had anabolic benefits to both the secondary trabecular and cortical bone shown previously in intact and orchidectomised mice 23,24,27,29 . In age-matched C57BL/6, OVX and age-related changes in trabecular metaphyseal bone are characterised by a significant decline in Tb.BV/TV (12-31% bone loss from 18-to 22-weeks-old) 36 . Whereas, bone adaptations after loading were characterised by a persistent increase in Tb.BV/ TV (89% increase from week 18 to 22) due to trabecular thickening particularly in posterior regions (Fig. 3). In the mid-tibia, loading increased the cortical thickness and total cross-sectional area, and led to a constant increase in BMC in the proximal to mid-tibia, agreeable with cross-sectional 23,24,27,52 and recent longitudinal findings 53 in intact mice. Notably, the largest loading induced increase occurred posterior to laterally at the mid-shaft (Figs. 4 and 6), consistent with higher bone formation and decreased resorption processes at the periosteal surface of these sites documented elsewhere, and where the compressive strains are greatest under uniaxial load 54 . Compared with PTH, loading showed greater benefits to trabecular, but not cortical bone morphometry, except a higher cortical thickness two weeks after treatment withdrawal. PTH had greater benefit to distal BMC, whereas loading was more beneficial to the proximal and mid-tibia, consistent with heterogeneous strain distribution in this loading model 55 .
Both concurrent and alternating co-treatment had anabolic benefits to morphometric and densitometric properties of the mouse tibia. In general, owing to dominant loading effects as discussed above, combined treatment induced longitudinal adaptations in morphometric properties similar to loading alone, e.g. increased Tb.BV/TV with trabecular and cortical thickening. Interestingly, with co-treatment, PTH appeared to limit the osteogenic benefits of loading on the trabecular bone, confirming a possible antagonistic interaction on metaphyseal trabeculae observed cross-sectionally in intact 19-months-old C57BL/6 29 , though contrary to additive benefits on both appendicular or axial trabecular bone observed in 3-4 months-old intact 27 and OVX C57BL/6 mice 15 . Combined treatments had increased benefits to cortical thickness than PTH alone, but not loading monotherapy which is consistent with short (2 weeks), but not with prolonged (3-6 weeks) tibia loading previously shown 27,29 . In BMC, PTH(1-34) enhanced loading effects in the proximal tibia, particularly in posterolateral regions that are subjected to higher compressive strain under controlled mechanical load, while conferring osteogenic benefits to the distal portion where mechanical effects were low. Compared with PTH, loading had increased benefits only to the proximal tibia and confirmed by site-specific analysis of cortical morphometry elsewhere 29 . Meanwhile, alternating PTH lead to a lower anabolic response at 22 weeks of age in the most proximal part of the tibia (significantly lower Tb.BV/TV and in C01-C04 BMC compared with ML + PTH). An appropriate animal model of the human disease is recommended for preclinical testing of novel anti-osteoporotic treatment strategies. In C57BL/6, OVX-induced changes are characterized by rapid and www.nature.com/scientificreports www.nature.com/scientificreports/ persistent bone loss with concomitant reductions in circulating oestrogen 36 , the latter of which is not typical in aging rodents, though are cardinal features of human OP 56 . We selected skeletally mature, yet relatively young, mice to quantify adaptive response in absence of aging and related comorbidities that could confound the findings, though aging can affect the bone mechano-adaptation and responsiveness to co-therapy 29 , thus should be considered in future studies. With ovariectomy, our data highlights generally positive effects of combined bone anabolics to the cortical bone, but potentially antagonistic effects to trabecular bone in this mouse model. This www.nature.com/scientificreports www.nature.com/scientificreports/ variable response along the tibia, and often contrary to outcomes elsewhere, highlights the need for caution when extrapolating findings from young or old intact animals or otherwise positive anabolic benefits reported in axial bones 15 . Using comprehensive subregional assessment with longitudinal study design our results also provide meaningful additional information on bone's dynamic response to treatments that can be underrepresented by standard morphometric analyses. For example, characterization of mid-shaft cortical morphometry failed to capture the increased and highly region-dependent benefits of combined PTH and loading that we demonstrate by BMC partitioning in quadrants along the bone length. This spatial analysis, applied to high-resolution in vivo microCT images, represents important methodological refinements by contributing to a substantial reduction in the number of animals used for preclinical assessment of novel anti-osteoporotic treatment strategies 32 . Further, the longitudinal data could provide invaluable information for mechanistic models of bone remodeling with anabolic therapies, e.g. references [57][58][59] .
There were limitations to this study. First, PTH(1-34) was administered approximately 2-3 hours following loading. While clinically, timing of the PTH(1-34) dose, e.g. morning than in evening, can enhance its efficacy 51 , the timing to optimise treatment synergies is yet to be resolved. Regardless, increased benefits were still shown and post-loading administration may be clinically relevant given drug side-effects, e.g. cramping and nausea/dizziness, which may be contraindications to exercise 8 . Second, the applied (12 N) load was matched across time-points and the intervention groups. Due to the PTH effects on cortical morphology, given at week 18 and one week before the first application of mechanical load, potential differences in local strains among the treatment groups may occur. Nevertheless, injections of PTH have shown not to significantly affect the cortical bone and to induce only small differences (7-9%) in BMC change in the proximal medial and posterior sectors 31 . Thus, the difference in local strain under the same axial load for the different groups of mice in week 19 should be minimal. Third, the in vivo study design precludes microCT scanning at smaller voxel size without increasing the radiation dose. Thus, we could not reasonably evaluate the effects of treatment on intra-cortical remodeling given that the mean cortical pore diameter in C57BL/6 mice is often less than the voxel size (i.e. <10 µm) 60 . Fourth, although sufficiently powered per our a priori sample size estimation, the heterogeneous response of the mice (see Supplementary Materials) may confound group trends and limit our ability to detect further significant intervention effects. However, the longitudinal design is advantageous to reduce risk of study bias while improving statistical power 32 . Finally, C57BL/6 mice, particularly following OVX have very low trabecular bone mass   and mechanical loading on tissue mineral density in 10 sections along the tibia length in ovariectomized C57BL/6 mice. Sections are C01, most proximal to C10, most distal. Values are reported as the relative percentage difference between two treatment groups (g1 vs. g2), normalised for week 18 values of the latter group (g2). *p < 0.05, indicates statistically significant differences between groups (ANCOVA, adjusted for baseline values at week 18). Positive and negative values indicate greater increases in BMC in g1 and g2, respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ at treatment onset. Thus, in the tibia metaphysis there is often very few trabeculae on which to reliably assess treatment efficacy.
In conclusion, combining PTH(1-34) and tibia loading has increased, albeit highly regionally-dependent, benefits to the tibia cortical bone properties in ovariectomized mice, whereas co-treatment had lower osteogenic benefits on the trabecular bone than loading alone. While PTH(1-34) has relatively homogeneous benefits along the tibia length, loading increased BMC more focally in the mid-diaphysis and postero-laterally, which is subjected to higher stresses and strains under compressive loads. This data reinforces the need for comprehensive spatial analysis along the bone length when testing effects of novel treatment strategies.