## Introduction

Loss-of-function mutations in the Sost gene lead to osteosclerotic phenotypes in humans (sclerosteosis, OMIM 269500 and van Buchem disease, OMIM 239100)1,2 and to high bone mass phenotypes in male and female mice3,4. Interestingly, female Sost null mice have an increased volumetric cortical bone mineral density compared to males3. In contrast, transgenic female mice overexpressing Sost exhibit low bone mass5. Sost is a negative regulator of Wnt signaling and subsequently bone formation, since canonical Wnt signaling activation increases bone mass. Pharmacological inhibition of sclerostin, the product of the Sost gene, through a sclerostin-neutralizing antibody (Scl-Ab) has shown significant bone gains in rodents6 and nonhuman primates7 and reduced fracture risk in postmenopausal women with osteoporosis8. Romosozumab (Evenity™, Amgen and UCB) was recently approved by the FDA for the treatment of postmenopausal women with osteoporosis at high risk of fracture. Other sclerostin antibodies are being tested for use in children and adults with rare diseases, such as Type I, III or IV Osteogenesis Imperfecta (Setrusumab, formerly called BPS804, Mereo BioPharma).

Since the majority of pre-clinical loading studies to date focused on females, it remains unclear if physical activity will be beneficial in older as well as young males undergoing sclerostin inhibition. Less is known in general regarding how males with or without Sost deficiency respond to mechanical loading with age. Ducher et al. showed that periosteal bone formation in male tennis players’ humeri was more pronounced in prepubertal and peripubertal boys and plateaued in postpubertal players24. Mosley et al. reported no sex-related differences in the cortical bone formation response to ulnar loading in young rats25, while other studies observed an elevated mechanoresponse in cortical bone of female wild-type mice26,27,28,29. We observed that the cortical bone formation response to loading was reduced at maturation in both female LC and Sost KO mice coincident with age-dependent expression of Wnt target genes in female Sost KO and LC mice as well as Sost gene expression in LC mice10. It remains unclear if male Sost KO and LC mice have age-related changes in mechanoresponse similar to what has been reported in female wild-type mice30,31,32,33. This knowledge is especially relevant considering that men suffer from age-related bone loss and have fragility fractures and thus would benefit from sclerostin inhibition34.

## Materials and methods

### Animals

Sperm from four male Sost -/- mice was provided by Novartis. Intracytoplasmic sperm injection with the oocytes from female C57BL/6 J mice was performed and a breeding colony was maintained. The first heterozygous generation was mated among themselves. In following generations, homozygous Sost KO and LC mice were identified using a Multiplex PCR with mice tail cuts, according to a protocol provided by Novartis. Sost KO and LC male mice at age of 10, 26 and 52 weeks were used in this study. All animal experimental procedures were approved by the local animal welfare ethics committee (LAGeSo Berlin, G0021/11). All methods were carried out in accordance with relevant guidelines and regulations of the local animal welfare ethics committee.

### Finite element analysis

Since the strain gauging experiment only provided strain measures at a very local area (gauge site) on bone surface and the gauge-measured strain does not always represent the peak or average strain at the tibial midshaft, microCT-based FE analyses were conducted to determine the load-induced strain environment across the entire tibia. The FE modeling approach used here had been developed and validated in our previous studies examining female C57BL/6 and female Sost KO mice37,38,39. Briefly, FE models of typical 10- and 26-week-old Sost KO and LC tibiae (n = 1/age/genotype) were built based on ex vivo microCT scanning of the strain-gauged whole tibiae (Amira, Thermo Fisher Scientific), at an isotropic voxel resolution of 9.91 μm (Skyscan 1172, Brukers, Belgium; 100 kVp, 100 µA, 360°, 0.3° rotation step, 3 frame averaging). Heterogeneous material properties (Young´s modulus) based on spatial distribution of tissue mineral density in the microCT scans were assigned to the FE models consisting of tetrahedral elements38,39,40. Poisson´s ratio was set to 0.35 for all models39. Loading and boundary conditions were applied to mimic the experimental in vivo axial loading of the tibia. Linear elastic FE analyses were performed (Abaqus 6.13, Dassault Systemés Simulia, MA). The predicted strain value at the gauge site for each tibia was calculated by averaging the strain in the longitudinal direction of the strain gauge at its mounting position, which was visible on the scans. The predicted strains in cortical bone were calculated in a volume of interest (VOI) centered at the tibia midshaft, containing 5% of the total tibia length, analogue to the region used for the microCT analysis. The maximum absolute value between the maximal (tensile, εMax) and minimal (compressive, εMin) principal strains was calculated for each element within the heterogeneous FE models. Thereafter, the mean values of the tensile ($$\bar{\varepsilon }_{\text{Max}}$$) and compressive ($$\bar{\varepsilon }_{\text{Min}}$$) strains in the midshaft VOI were determined. Elements for which abs (εMax) > abs (εMin ) were used to calculate $$\bar{\varepsilon }_{\text{Max}}$$ and SD (εMax), while elements for which abs (εMax) ≤ abs (εMin ) were used to calculate $$\bar{\varepsilon }_{\text{Min}}$$ and SD (εMin). The SD (standard deviation) reflects the range of εMax for tension and εMin for compression within the VOI.

### Longitudinal in vivo microCT

In vivo microCT with a voxel size of 10.5 μm was performed on anesthetized mice at day 0, 5, 10, and 15 to assess the midshaft cortical bone of both the right and left tibiae (vivaCT 40, Scanco Medical, Switzerland; 55 kVp source voltage, 145 μA source current, 300 ms integration time, no frame averaging, range of 180 degrees) (Fig. 1). To prevent motion artifacts during microCT scanning, anaesthetized mice were constrained in a custom-made plastic mouse bed. Our previous study has shown the repeated radiation exposure (0, 5, 10, 15 day) from microCT at doses used in this study (approximately 0.48 Gy per scan) does not affect cortical bone parameters measured by microCT or histomorphometry in 10 and 26 week-old C57BL/6 mice35.

### In vivo microCT analysis

Similar to previous studies, midshaft cortical bone (5% of tibial length) was analyzed from microCT images at day 0, 5, 10 and 15 using the IPL (Image Processing Language) based standard evaluation scripts provided by Scanco (Scanco Medical, Switzerland) (Fig. 1). Although whole-tibia analyses from previous tibial loading studies have observed relatively robust mechanoadaptive responses along ~ 10–60% of the tibia from its proximal end44,45,46, the midshaft was chosen here because it is a common location where strain gauging is performed and load-strain relations accounting for the effect of age, gender, genotype, etc. are established. It is also a common volume of interest for examining cortical bone adaptation in the tibial loading model32,33,35,41,46,47,48. A global threshold of 4626 HU (809.6 mg HA/ccm) was used to segment cortical bone from soft tissue and water in all mice. The measured cortical bone parameters included: principal moments of inertia (Imax, Imin), cortical bone area (Ct.Ar), total cross-sectional area (T.Ar), cortical area fraction (Ct.Ar/T.Ar), cortical thickness (Ct.Th), and cortical tissue mineral density (Ct.TMD).

### Time-lapse in vivo morphometry

MicroCT images taken at day 0 and 15 were geometrically aligned and analyzed using a registration, segmentation and quantification algorithm using Amira software (Thermo Fisher Scientific) (Fig. 1). The method has previously been described in detail30,49,50. Briefly, the algorithm involves the following steps: (1) geometrical registration of images, (2) thresholding to extract the bone region, using the same global threshold mentioned above, (3) segmentation to exclude mineralized tissue present in the medullary cavity inside the VOI, (4) labeling regions of quiescent, newly formed and resorbed bone, and (5) quantification of volumetric dynamic (re)modeling parameters of formation and resorption normalized to values at the beginning of experiment (bone volume newly mineralized between day 0 and 15 divided by the bone volume present at day 0: MV/BVday0–15; bone volume eroded between day 0 and 15 divided by the bone volume present at day 0: EV/BVday0–15; mineralizing and eroded surface between day 0 and 15 normalized to the total bone surface at day 0: MS/BSday0–15 and ES/BSday0–15). The dynamic bone formation and resorption parameters were also quantified for the endocortical and periosteal surfaces, separately using previously described methods51.

### Histomorphometry

Calcein (20 mg/kg) was administered to the LC and Sost KO mice via intraperitoneal injection at day 3 and 12 during the loading experiment (Fig. 1)52. The tibiae were dehydrated in ascending grades of ethanol to absolute, cleared in xylene, infiltrated and finally embedded in polymethyl-methacrylate. The blocks were sectioned transversal to the bone’s long axis at the cortical midshaft. The slices were ground and polished to an approximately thickness of 60 μm and viewed at a magnification of 200 × under a mercury lamp microscope (KS400 3.0, Zeiss, Germany) for evidence of fluorochrome labels (Fig. 1). Images were acquired using commercially available software (Axiovision, Zeiss, Germany). The analyzed region of interest for the cortical bone included endocortical and periosteal surface. The single- and double-labeled surface per bone surface (sLS/BS, dLS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), were analyzed as recommended and using ImageJ3. MS/BS was calculated as 0.5 × sLS/BS + dLS/BS. When a specimen had no double-labeled surface (dLS/BS = 0), it was labeled as “no data” for MAR and BFR/BS53. The amount of newly mineralized bone per day was calculated using the averaged double label distances divided by the 9-day labeling interval and expressed as the MAR in units of microns per day. For determining MAR, the entire endocortical (Ec) and periosteal (Ps) surfaces were analyzed.

### Statistical analysis

The within-subject effect of loading (loaded and control limbs) and between-subject effects of age (10- and 26-week-old) and genotype (LC and Sost KO) as well as interactions between these terms were assessed using a repeated measure ANOVA (SAS 9.3, Cary, USA). A separate ANOVA was used to assess between-subject age (10- and 26-week-old) and genotype (LC and Sost KO) and interaction effects for relative values, the interlimb differences (∆interlimb = loaded limb − control limb). Paired t-tests were used to compare control and loaded limbs. Unpaired t-tests were performed to compare control limbs between age or genotype. The percent difference was presented as percentual increment [%Δ = ((loaded limb − control limb)/control limb) × 100%)]. Statistical analyses of the qPCR data were performed on the ΔCt values. Unpaired or paired t-tests were performed to determine the differences between Sost KO and LC or between loaded and control limbs, respectively. A significant difference was set for all analyses as p < 0.05.

## Results

### Tissue strains in cortical bone of Sost KO and age- and gauge strain-matched LC mice

The strain values at the strain gauge site predicted by the FE models were similar to those measured experimentally (~ 900 µε), supporting the validation of our FE models. Further, the FE models predicted that the mean compressive strains were 16% higher in 10-week-old than in 26-week-old Sost KO mice (Fig. 2). In contrast, the mean tensile strains were almost identical in the 10- and 26-week-old Sost KO mice. Both mean tensile and compressive strains were lower in the Sost KO mice than in their age-matched LC whereas this difference was much more pronounced in the 10-week-old (37% for tension and 70% for compression) than in the 26-week-old mice (7% for tension and 9% for compression) (Fig. 2).

### Sost deficiency led to an increased bone formation in male mice

Compared to LC mice, male Sost KO mice had a greater surface area of newly mineralized tissues relative to the total surface area at the midshaft cortical bone, as indicated by time-lapse in vivo morphometry (main effect of genotype for MS/BSday0–15; Fig. 3). Further, both in vivo morphometry and histomorphometry showed that Sost deficiency-induced increase in MS/BS occurred primarily at the endocortical surfaces (main effect of genotype for Ec.MS/BS and Ec. MS/BSday0–15; Fig. 4, Supplementary Tables 3 and 4). Consistent with the enhancement of bone formation due to Sost deficiency, 10- and 26-week-old Sost KO mice had greater morphological and mechanical parameters (Ct.Ar, T.Ar, Ct.Th, Imax and Imin) relative to their littermate controls (main effects of genotype; Table 1). Additionally, those microCT-measured parameters were enhanced in 26-week-old Sost KO mice compared to 10-week-old mice (interaction of age and genotype; Table 1), likely due to an accumulation of formed bone associated with long-term Sost deficiency. Sost deficiency had little effect on bone resorption (no main effect of genotype; Fig. 3B).

### Skeletal maturation led to decreased bone formation and increased bone resorption in male Sost KO mice

Skeletal maturation led to decreased bone formation apparent in time-lapse in vivo morphometry, and histomorphometry (main effect of age by ANOVA; Figs. 3 and 4). Time-lapse in vivo morphometry showed decreased MV/BVday0–15 and MS/BSday0–15 in 26-week-old compared to 10-week-old Sost KO and LC mice (Fig. 3B). Histomorphometry measures also revealed a decrease in bone formation with skeletal maturation (Ec.dLS/BS, Ec.MS/BS, Ec.MAR, Ec.BFR/BS, Ps.sLS/BS, Ps.dLS/BS, Ps. MAR) (Fig. 4 and Supplementary Table 4). Interestingly, this reduction in bone formation with skeletal maturation was independent of Sost deficiency except in the Ec.MAR (ANOVA, interaction of genotype and age; Fig. 4B). Skeletal maturation also affected bone resorption, with 26-week-old mice having larger EV/BVday0–15 and ES/BSday0–15 than 10-week-old mice (Fig. 3B).

## Discussion

Motivated by our earlier work on females, the goal of this study was to examine, in male mice, the effect of Sost deficiency on the cortical bone (re)modeling and adaptive response to a moderate level of applied loading. In vivo loading was applied to the tibiae of young and older male Sost KO and LC mice to engender a gauge strain of ~ 900 με at the midshaft, the same target strain as our previous female loading study10. This more moderate target strain level was chosen in the present study in males and previous study in female Sost KO mice10, since our pilot studies showed that 1200 με (requiring 17 N) determined through in vivo strain gauging of 10-week-old female Sost KO mice at the mid-diaphysis led to ankle swelling and limping in the mice during the first several days of loading.