High Osteoblastic Activity In C3H/HeJ Mice Compared to C57BL/6J Mice Is Associated with Low Apoptosis in C3H/HeJ Osteoblasts

This study sought to confirm that osteoblasts of C3H/HeJ (C3H) mice, which have higher differentiation status and bone-forming ability compared to C57BL/6J (B6) osteoblasts, also have a lower apoptosis level and to test whether the higher differentiation status and bone-forming ability of C3H osteoblasts were related to the lower apoptosis. C3H mice had 50% fewer (P < 0.01) apoptotic osteoblasts on the endocortical bone surface than B6 mice as determined by the TUNEL assay. Primary C3H osteoblasts in cultures also showed a 50% (P < 0.05) lower apoptosis level than B6 osteoblasts assayed by acridine orange/ethidium bromide staining of apoptotic osteoblasts. The lower apoptosis in C3H osteoblasts was accompanied by 22% (P < 0.05) and 56% (P < 0.001) reduction in the activity of total caspases and caspases 3/7, respectively. C3H osteoblasts also displayed greater alkaline phosphatase (ALP) activity (P < 0.001) and higher expression of Cbfa1, type-1 collagen, osteopontin, and osteocalcin genes (P < 0.05 for each). To assess if an association existed between population apoptosis and the differentiation status (ALP-specific activity) and/or bone-forming activity (insoluble collagen synthesis), C3H and B6 osteoblasts were treated with several apoptosis enhancers (tumor necrosis factor-α, dexamethasone, lipopolysaccharide, etoposide) and inhibitors (parathyroid hormone, insulin-like growth factor I, transforming growth factor β1, estradiol). Both ALP (r = −0.61, P < 0.001) and insoluble collagen synthesis (r = −0.61, P < 0.001) were inversely correlated with apoptosis, suggesting that differentiation (maturation) and/or bone-forming activity of these mouse osteoblasts were inversely associated with apoptosis. In conclusion, these studies support the premise that higher bone density and bone formation rate in C3H mice could be due in part to lower apoptosis in C3H osteoblasts.     

Regulation of bone volume is different in the metaphyses of the femur and vertebra of C3H/HeJ and C57BL/6J mice

The C3H/HeJ (C3H) mice exhibited a greater bone formation rate (BFR) and a greater mineral apposition rate (MAR) in the cortical bone of the midshafts of the femur and tibia than did C57BL/6J (B6) mice. This study sought to determine if these strain-related differences would also be observed in cancellous bone. Metaphyses of the femur and lumbar vertebra (L5-6) from C3H and B6 mice, 6 and 12 weeks of age, were analyzed by histomorphometry. Similar to cortical bone, the bone volume in the femoral metaphysis of C3H mice was greater (by 54% and 65%, respectively) than that of B6 mice at both 6 and 12 weeks of age. Higher BFR and mineral apposition rate (MAR) contributed to the higher bone volume in the C3H mice compared with the B6 mice. In contrast, bone volume (by 59% and 13%, respectively, p < 0.001) and trabecular number (by 55% and 35%, respectively, p < 0.001) in the vertebrae were lower in the C3H mice than in B6 mice at 6 and 12 weeks of age. At 6 weeks of age, MAR was higher (by 43%, p = 0.004) in C3H mice, but because of a low trabecular number, the BFR (by 37%, p = 0.026) and tetracycline-labeled bone surface (by 52%, p < 0.001) per tissue were lower in the vertebrae of C3H mice than B6 mice. The low bone volume in vertebrae of C3H mice was probably not due to a higher bone resorption, because the osteoclast number (by 55%, p < 0.001) and eroded surface (by 61%, p <0.001) per tissue area in the C3H mice were also lower in B6 mice. At 12 weeks, the trabecular thickness had increased (by 36%, p < 0.001) in the C3H mice and the difference in bone volume between strains was less than that at 6 weeks. These contrasting and apparently opposing strain-related differences in trabecular bone parameters between femur and vertebra in these two mouse strains suggest that the genetic regulation of bone volume in the metaphyses of different skeletal sites is different between C3H and B6 mice.     

Inbred Strain‐Specific Response to Biglycan Deficiency in the Cortical Bone of C57BL6/129 and C3H/He Mice

Inbred strain‐specific differences in mice exist in bone cross‐sectional geometry, mechanical properties, and indices of bone formation. Inbred strain‐specific responses to external stimuli also exist, but the role of background strain in response to genetic deletion is not fully understood. Biglycan (bgn) deficiency impacts bone through negative regulation of osteoblasts, resulting in extracellular matrix alterations and decreased mechanical properties. Because osteoblasts from C3H/He (C3H) mice are inherently more active versus osteoblasts from other inbred strains, and the bones of C3H mice are less responsive to other insults, it was hypothesized that C3H mice would be relatively more resistant to changes associated with bgn deficiency compared with C57BL6/129 (B6;129) mice. Changes in mRNA expression, tissue composition, mineral density, bone formation rate, cross‐sectional geometry, and mechanical properties were studied at 8 and 11 wk of age in the tibias of male wildtype and bgn‐deficient mice bred on B6;129 and C3H background strains. Bgn deficiency altered collagen cross‐linking and gene expression and the amount and composition of mineral in vivo. In bgn's absence, changes in collagen were independent of mouse strain. Bgn‐deficiency increased the amount of mineral in both strains, but changes in mineral composition, cross‐sectional geometry, and mechanical properties were dependent on genetic background. Bgn deficiency influenced the amount and composition of bone in mice from both strains at 8 wk, but C3H mice were better able to maintain properties close to wildtype (WT) levels. By 11 wk, most properties from C3H knockout (KO) bones were equal to or greater than WT levels, whereas phenotypic differences persisted in B6;129 KO mice. This is the first study into mouse strain‐specific changes in a small leucine‐rich proteoglycan gene disruption model in properties across the bone hierarchy and is also one of the first to relate these changes to mechanical competence. This study supports the importance of genetic factors in determining the response to a gene deletion and defines biglycan's importance to collagen and mineral composition in vivo.     

Exercise and Mechanical Loading Increase Periosteal Bone Formation and Whole Bone Strength in C57BL/6J Mice but Not in C3H/Hej Mice

To identify the genes, and the mechanisms that account for the 53% higher peak bone density in C3H/HeJ (C3H) mice compared with C57BL/6J (B6) mice, we are performing quantitative trait locus and phenotypic analyses. The phenotypic studies revealed differences in bone formation and resorption, and showed that hindlimb immobilization (by sciatic neurectomy) caused a greater increase in endosteal resorption in the tibiae of B6 compared with C3H mice. The current studies were intended to examine the hypothesis that the bones of C3H mice are less sensitive to mechanical loading than the bones of B6 mice. To increase mechanical loading, 9-week-old female B6 and C3H mice (n = 10–13 mice/group) were subjected to a jumping exercise (20 jumps/day, 5 days/week, to heights of 20–30 cm) for a total of 4 weeks. Control mice did not jump. Osteocalcin, alkaline phosphatase (ALP) activity, and IGF-I were measured in serum. The left tibiae were used for histomorphometry (ground cross-sections prepared at the tibio-fibular junction) and the right tibiae and femora were used for determinations of bone breaking strength (3-point bending). The results of these studies revealed (1) significant effects of both mouse strain (B6 and C3H) and the jumping exercise on tibial strength; (2) an exercise-dependent increase in serum IGF-I in C3H, but not B6 mice; and (3) no effects on serum ALP or osteocalcin. The histomorphometric analyses showed no effect of exercise on C3H tibiae, but significant exercise-dependent increases in total bone area, periosteal perimeter, periosteal mineral apposition rate (MAR), and periosteal bone formation (P < 0.02 for each) in B6 tibiae. There were no effects of exercise on periosteal resorption or any endosteal measurement in either C3H or B6 mice. Since the jumping exercise was designed to cause a two–three fold increase in muscular-skeletal loading at the tibio-fibular junction, and the calculated stress (g/mm²) at this sampling site was only 16% greater for B6 compared with C3H mice, we had anticipated that both strains of mice would show exercise-dependent increases in periosteal bone formation, with a greater response in the B6 mice. The lack of a response in the C3H tibiae demonstrates that the bones of C3H mice are less sensitive to mechanical loading (and unloading) than the bones of B6 mice. Received: 21 July 1999 / Accepted: 2 November 1999     

Lower bone cellular activities in male and female mature C3H/HeJ mice are associated with higher bone mass and different pyridinium crosslink profiles compared to C57BL/6J mice

The female inbred strains of C3H/HeJ (C3H) and C57BL/6J mice (B6), having high and low femoral peak bone mass, respectively, were proposed as models for studying the genetic regulation of bone mass. Here, we compared the known bone phenotype, in 4.5-month-old C3H versus B6 mice, in both genders. Femoral bone mineral content, trabecular bone mass, and thickness at the distal metaphysis were higher in C3H mice. In the long bones, deoxypyridinoline content was lower and pyridinoline/deoxypyridinoline ratios were greater in C3H. Intrafibrillar collagen packing is different not only within strains but also within sexes. Bone resorption activity, evaluated by urinary pyridinium crosslinks and active resorption surfaces in the femoral metaphysis, was lower in C3H. Bone formation activity, evaluated by serum osteocalcin and alkaline phosphatase (ALP) levels, as well as histomorphometric indices of bone formation in the femoral metaphysis and the cortical tibia, was lower in C3H. Conversely, the ALP- and Von Kossa-positive colony-forming units were more numerous in bone marrow cell cultures originating from male C3H. In both strains, resorption and formation activities were lower in males than in females. In C3H, males had lower bone mass than females whereas the opposite was seen in B6. In conclusion, we found that the lower cellular activities in C3H were associated with high cancellous bone mass and pyridinium crosslink levels, which might account for the more mineralized bone in C3H mice compared to that in B6 mice.     

Histomorphometric studies show that bone formation and bone mineral apposition rates are greater in C3H/HeJ (high-density) than C57BL/6J (low-density) mice during growth.

High-density C3H/HeJ (C3H) and low-density C57BL/6J (B6) mice, with femoral bone density differing by 50%, were chosen as a model to investigate the mechanisms controlling peak bone density and to map peak bone density genes. The present longitudinal study was undertaken to further establish the bone biologic phenotypes of these two inbred strains of mice. To evaluate phenotypic differences in bone formation parameters in C3H and B6 mice between the ages of 6 and 26 weeks, undecalcified ground sections from the diaphyses of the tibia and femur were prepared from mice receiving two injections of tetracycline. Histomorphometric analyses revealed that the cortical bone area was significantly greater (16%-56%, p < 0.001) in both the femur and tibia of the C3H mice than in the B6 mice at all timepoints. This difference in cortical bone area was due to significantly smaller medullary areas in the C3H mice than in the B6 mice. The bone formation rates (BFR) at the endosteum in both the femur and tibia were significantly greater (28%-117%,p < 0.001) in the young C3H mice (6-12 weeks old) than in B6 mice. The higher bone formation in C3H mice was associated with higher values of the bone mineral apposition rate (25%-94%, p < 0.001), and was not associated with higher values of the forming surface length as measured by tetracycline label length. Similar interstrain differences in mineral apposition and bone formation rates were observed in the periosteum of the femur and tibia. In conclusion, the greater bone area in the high-density C3H mice vs. the low-density B6 mice was, in part, due to the greater periosteal and endosteal bone formation rates during growth in the C3H mice. Because the C3H and B6 mice were maintained under identical environmental conditions (diet, lighting, etc.), the observed interstrain differences in bone parameters were the result of the action of genetic factors. Consequently, these two inbred strains of mice are suitable as a model to identify genetic factors responsible for high bone formation rates.     

Opposing changes in osteocalcin levels in bone vs serum during the acquisition of peak bone density in C3H/HeJ and C57BL/6J mice

Our knowledge of the developmental changes in the concentration of serum and bone osteocalcin (OC) is limited. To investigate the interrelationship between skeletal and circulatory OC during acquisition of peak bone density in mice, we examined the temporal changes in the concentration of serum and bone OC from 3 to 12 weeks of age between C3H/HeJ (C3H) and C57BL/6J (B6), two commonly used inbred strains of mice with a large difference in bone density. We have demonstrated an increase in bone and decrease in serum OC during the acquisition of peak bone density in C3H and B6 mice which parallels an increase in bone mineral density. These two strains exhibited differential changes in the concentration of OC. C3H mice retained more OC in bone and secreted less into serum compared with B6, which coincides with the large differences in bone density between these two strains. These opposite changes of OC levels in bone and serum between C3H and B6 stress the importance of defining the genetic mechanisms underlying the differences in OC metabolism, differences that could be relevant to the acquisition and maintenance of bone mass in mice.     

32 wk old C3H/HeJ mice actively respond to mechanical loading

Numerous studies indicate that C3H/HeJ (C3H) mice are mildly responsive to mechanical loading compared to C57BL/6J (C57) mice. Guided by data indicating high baseline periosteal osteoblast activity in 16 wk C3H mice, we speculated that simply allowing the C3H mice to age until basal periosteal bone formation was equivalent to that of 16 wk C57 mice would restore mechanoresponsiveness in C3H mice. We tested this hypothesis by subjecting the right tibiae of 32 wk old C3H mice and 16 wk old C57 mice to low magnitude rest-inserted loading (peak strain: 1235 mu epsilon) and then exposing the right tibiae of 32 wk C3H mice to low (1085 mu epsilon) or moderate (1875 mu epsilon) magnitude cyclic loading. The osteoblastic response to loading on the endocortical and periosteal surfaces was evaluated via dynamic histomorphometry. At 32 wk of age, C3H mice responded to low magnitude rest-inserted loading with significantly elevated periosteal mineralizing surface, mineral apposition rate and bone formation compared to unloaded contralateral bones. Surprisingly, the periosteal bone formation induced by low magnitude rest-inserted loading in C3H mice exceeded that induced in 16 wk C57 mice. At 32 wk of age, C3H mice also demonstrated an elevated response to increased magnitudes of cyclic loading. We conclude that a high level of basal osteoblast function in 16 wk C3H mice appears to overwhelm the ability of the tissue to respond to an otherwise anabolic mechanical loading stimulus. However, when basal surface osteoblast activity is equivalent to that of 16 wk C57 mice, C3H mice demonstrate a clear ability to respond to either rest-inserted or cyclic loading.     

Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization

Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase. Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface. These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.     

The Sox2 high mobility group transcription factor inhibits mature osteoblast function in transgenic mice

We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1α1 promoter, which extended Sox2 expression into more mature osteoblasts. In long bones, trabecular cartilage remodeling was delayed and the transition from endochondral to cortical bone was disrupted, resulting in porous and undermineralized cortical bone. Collagen deposition was disorganized, and patterns of osteoclast activity were altered. Calvarial bones were thinner and parietal bones failed to develop the diploic space. Microarray analysis showed significant up- or downregulation of a variety of genes coding for non-collagenous extracellular matrix proteins, with a number of genes typical of mature osteoblasts being downregulated. Our results position Sox2 as a negative regulator of osteoblast maturation in vivo.     

Mice lacking thrombospondin 2 show an atypical pattern of endocortical and periosteal bone formation in response to mechanical loading

Thrombospondin 2 (TSP2) is an extracellular matrix (ECM) protein localized to bone. Since mice with a targeted disruption of the TSP2 gene (TSP2-null) have increased bone formation, we hypothesized that mice lacking TSP2 would show an enhanced osteogenic response to mechanical loading. We addressed our hypothesis by subjecting wild-type (WT) and TSP2-null mice to mechanical loading using the non-invasive murine tibia loading device, and statistical comparisons were made between loaded and unloaded bones within genotype, between genotypes, and between the periosteal and endocortical surfaces within genotype. Right tibiae of WT and TSP2-null mice received 5 days of a low-magnitude loading protocol. This low-magnitude loading (inducing approximately 900 and 500 muepsilon at periosteal and endocortical surfaces of WT bones, respectively) affected neither periosteal nor endocortical bone formation rate (BFR/BS) when comparing loaded to intact bones in either WT or TSP2-null mice, nor did it result in any significant differences between WT and TSP2-null. As well, there was no difference between loaded endocortical and periosteal surfaces in WT mice; however, endocortical BFR/BS in TSP2-null loaded tibia was significantly elevated relative to the periosteal BFR/BS-despite peak periosteal strains being significantly greater than endocortical strains in TSP2-null mice (690 versus 460 muepsilon). To confirm this counterintuitive surface-specific response in TSP2-null mice and to induce significant periosteal bone formation, osteogenic potency of the loading protocol was amplified by doubling the number of loading bouts (10 loading days) and loading magnitude (1 Hz, resulting in 1400 and 900 muepsilon peak strain at the periosteal and endocortical surfaces, respectively). Under load, both WT and TSP2-null mice showed significantly increased periosteal mineralizing surface (by nearly three-fold and five-fold, respectively), but mineral apposition rate (MAR) was not statistically changed. The increased MS/BS resulted in a five-fold increase in WT periosteal BFR/BS, but the TSP2-null periosteal BFR/BS was unchanged. Furthermore, this increase in WT loaded periosteal BFR/BS was statistically greater than the WT endocortical BFR/BS. At the endocortical surface of WT mice, loading did not significantly increase bone formation parameters (versus intact). In contrast, at the endocortical surface of TSP2-null mice, loading induced a significant two-fold increase in BFR/BS (versus intact), that was also significantly greater than the endocortical BFR/BS of loaded WT mice. Thus, exogenous loading of TSP2-null mice resulted in highly variable responses that did not reflect the induced strains at the periosteal and endocortical surfaces. While in WT mice, loading resulted in increased periosteal BFR/BS that was greater than the endocortical BFR/BS, in TSP2-null mice loading resulted in endocortical (not periosteal) BFR/BS that was elevated. This reversal in envelope-specific bone formation in TSP2-null mice occurred despite periosteal strains being significantly greater than endocortical (1290 versus 775 muepsilon) and strain distributions being similar to that of WT. These results show that the disruption of a single gene can lead to a reversal in normal pattern of load induced bone formation, and more specifically, that the functional interaction of TSP2 with mechanical loading is highly contextual and specific to the cortical bone envelope examined.     

Bone adaptation response to sham and bending stimuli in mice.

This study presents inbred-strain-related differences in tibial bone adaptation response to low-force loading in four-point bending and sham (pad pressure) arrangements in mice. Our previous work in mice has shown that at relatively high but equal bending forces (9 N or a bending moment of 16.88 N-mm), C57BL/6J mice respond with significantly greater bone formation than C3H/HeJ mice. Because of high tibial strains, the majority of the bone response in our previous study was woven bone. In this, study, we reduced the loading forces to 5 N or a bending moment of 9.38 N-mm (to decrease the woven-bone formation response) and investigated inbred-strain-related bone adaptation differences resulting from bending and sham loading (reported here for the first time in C57BL/6J) in these mice. Twenty-four female mice within each inbred mouse strain (C3H/HeJ [C3H] and C57BL/6J [B6]) were randomly divided into the two loading groups (12 per group sham and bending, total of 48 mice). All of the external loading was done for 36 cycles at 2 Hz, 3 d/wk for 3 wk. The bone adaptation response at lower forces exhibited a pattern similar to that seen for the higher forces in the previous study, suggesting that the patterns of bone adaptation were inbred strain related and independent of bending force magnitude. The bending-related periosteal mineral apposition surface (pMS) and mineral apposition rate (MAR) were respectively 40% and 45% greater in B6 than in C3H. The cortical bone adaptation response to bending was greater when compared to sham or pad pressure for each inbred strain of mice, suggesting that the majority of the bone adaptation response was the result of bending stimulus and not local pressure from pad contact. In addition, regardless of loading arrangement (sham or bending), the bone adaptation response in C57BL/6J mice was greater than C3H/HeJ.     

Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1)

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.     

Variations of microstructure, mineral density and tissue elasticity in B6/C3H mice

200-MHz scanning acoustic microscopy (SAM) and synchrotron radiation μCT (SR-μCT) were used to assess microstructural parameters, acoustic impedance Z and tissue degree of mineralization of bone (DMB) in site-matched regions of interest in femoral bone of two inbred strains. Transverse femoral sections taken from 5 C57BL/6J@Ico (B6) and 5 C3H/HeJ@Ico (C3H) mice (5.5 months old) were explored. Mass density ρ, elastic coefficient c₁₁ and Young's modulus E₁ were locally derived in the distal epiphysis, distal metaphysis for trabecular bone and mid-diaphysis for cortical bone using a rule-of-mixture model. Structural parameter estimations obtained from X-ray tomographic and acoustic images were almost identical. Both strains had the same bone diameter, but the C3H mice had greater cortical thickness and smaller cancellous diameter than did B6 mice. The average DMB and impedance values were in the range between 1.13 and 1.33 g cm^− 3 and 5.8 and 7.8 Mrayl, respectively. All tissue parameters were lower in B6 mice than in C3H mice. However, interstrain differences of DMB were much less (up to 3.8%) than differences of Z (up to 13.2%). SAM and SR-μCT fulfill the requirement for a simultaneous evaluation of cortical bone microstructure and material properties at the tissue level. However, SAM provides a quantitative estimate of elastic properties at the tissue level that cannot be captured by SR-μCT. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.     

Postnatal and pubertal skeletal changes contribute predominantly to the differences in peak bone density between C3H/HeJ and C57BL/6J mice.

Previous studies have shown that 60-70% of variance in peak bone density is determined genetically. The higher the peak bone density, the less likely an individual is to eventually develop osteoporosis. Therefore, the amount of bone accrued during postnatal and pubertal growth is an important determining factor in the development of osteoporosis. We evaluated the contribution of skeletal changes before, during, and after puberty to the development of peak bone density in C3H/HeJ (C3H) and C57BL/6J (B6) mice. Volumetric bone density and geometric parameters at the middiaphysis of femora were measured by peripheral quantitative computed tomography (pQCT) from days 7 to 56. Additionally, biochemical markers of bone remodeling in serum and bone extracts were quantified. Both B6 and C3H mice showed similar body and femoral weights. B6 mice had greater middiaphyseal total bone area and thinner cortices than did C3H mice. Within strains, males had thicker cortices than did females. C3H mice accumulated more minerals throughout the study, with the most rapid accumulation occurring postnatally (days 7-23) and during pubertal maturation (days 23-31). C3H mice had higher volumetric bone density as early as day 7, compared with B6 mice. Higher serum insulin-like growth factor I (IGF-I) was present in C3H mice postnatally at day 7 and day 14. Until day 31, B6 male and female mice had significantly higher serum osteocalcin than C3H male and female mice, respectively. Alkaline phosphatase (ALP) was found to be significantly higher in the bone extract of C3H mice compared with B6 mice at day 14. These data are consistent with and support the hypothesis that the greater amount of bone accrued during postnatal and pubertal growth in C3H mice compared with B6 mice may be caused by increased cortical thickness, increased endosteal bone formation, and decreased endosteal bone resorption.     

Effects of dietary calcium depletion and repletion on dynamic determinants of tibial bone volume in two inbred strains of mice

As an adjunct to our efforts to identify the genes that determine peak bone density, we examined phenotypic differences between two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), which are of similar size but differ with respect to peak bone density (e.g., C3H mice have 53% higher femoral bone density than B6 mice). The current studies were intended to compare the skeletal responses of C3H and B6 mice to 2 weeks of dietary calcium (Ca) depletion, followed by 2 weeks of Ca repletion. Initial studies showed that: (a) femur dry weight decreased during Ca depletion in both C3H and B6 mice (by 25% and 19%, respectively, p < 0.001) and most of this loss was recovered during Ca repletion; and (b) serum alkaline phosphatase (ALP) activity increased during Ca depletion, in both strains of mice (p < 0.001), and returned to normal after Ca repletion. Histological analyses of ground cross sections prepared at the tibiofibular junction showed that Ca-depletion increased medullary area in both C3H and B6 mice (indicating endosteal bone loss, p < 0.01), with reversal during Ca repletion. There were no effects of Ca depletion or repletion on periosteal bone growth. Endosteal bone forming surface and endosteal mineral apposition decreased during Ca depletion and increased during repletion in both C3H and B6 mice (p < 0.05). Net bone formation decreased during Ca depletion in C3H mice, but not B6 mice (p < 0.01), and was normal during Ca repletion in both strains. Endosteal bone resorbing surface and net bone resorption increased during Ca depletion and decreased during repletion in both strains (p < 0.01). A supplemental study (of Ca depletion without repletion) confirmed the effects of Ca depletion on femoral dry weight and serum ALP activity (p < 0.001 for each). This supplemental study also showed that Ca deficiency increased serum parathyroid hormone (PTH) (p < 0.05) and decreased (tibial) cortical bone area and cortical mineral content (p < 0.05 to p < 0.001) in both strains of mice. Together, these data demonstrate that the skeletal responses to Ca depletion and repletion are, qualitatively, similar in C3H and B6 mice.     

A possible non-oncogenic effect of C type bone cell virus on serum calcium levels of potentially leukemic mice

In bone of C3H/Fg mice, particles structurally identical toC-type leukemia virus arise from membranes of osteocytes and osteoblasts. Although these virus apparently do not induce morphologic or neoplastic change in bone they may have other, more subtle, effects. Thus, comparison of sera form male C3H/Fg mice, a high leukemia-prone strain, with C57BL and C3H/HeJ mice, low leukemia strains which do not containC-type virus in bone, reveals that serum calcium levels are significantly lower in the former than in the latter. Further, when C3H/Fg mice develop frank leukemia there is a corresponding increase in virus particles while the serum calcium concentration levels fall to even lower values. The presence of leukemia itself appears not to be the cause as indicated by the failure of implanted lymphocytic leukemic cells in C3H/Fg mice to significantly affect serum calcium concentration. It is postulated that the effects of the virus could be due either to increased osteoblastic activity or to inhibition of osteocytic osteolytic activity or to both.     

Strain differences in bone density and calcium metabolism between C3H/HeJ and C57BL/6J mice.

Previous reports indicate that peak bone density is significantly higher in C3H/HeJ (C3H) than in C57BL/6J (C57BL) mice, making these two inbred strains useful models for studying the genetic basis for peak bone density. The following study was undertaken to examine whether strain differences in the bone density of C3H and C57BL mice are associated with differences in intestinal calcium (Ca) absorption. Calcium absorption was measured by the balance technique and animals received two injections of fluorochromes 5 days apart before killing. Subsequently, the femurs were removed and, following measurement of volumetric density, the left femur was divided into three equal parts and the middle third served as the femoral cortical diaphysis. Femur diaphyseal volumetric bone density, ash, and Ca content were 10%, 29%, and 29% higher in C3H than in C57BL mice (p < 0.001), respectively. Bone length, periosteal mineral apposition rate, and periosteal bone formation rate of femoral diaphyseal cortical bone were not significantly different between the two strains of mice, but the marrow area of C57BL mice was almost twofold that of C3H mice (p < 0.0001). Intestinal Ca absorption and 1,25-dihydroxyvitamin D [1,25(OH)2D]-stimulated Ca2+ uptake by intestinal mucosal cells were 38% and 51% higher in C3H than in C57BL mice p < 0.001), respectively. Serum Ca and 1,25(OH)2D levels were 6% and 32% higher in C3H than in C57BL mice (p < 0.001), respectively, and the number of intestinal-occupied vitamin D receptors was 51% higher in C3H than in C57BL mice (p < 0.01). In a second experiment, three groups of C3H mice and three groups of C57BL mice were fed diets that contained 0.4%, 0.1%, or 0.02% Ca, and serum Ca, 1,25(OH)2D, parathyroid hormone (PTH), and intestinal Ca absorption measured. At all dietary Ca levels, C3H mice maintained positive Ca absorption and absorbed significantly more Ca than C57BL mice. In contrast, at low dietary Ca levels (0.1% and 0.02% Ca), C57BL mice maintained negative Ca absorption. Low dietary Ca increased serum PTH significantly in C57BL but not in C3H mice, and decreased serum 1,25(OH)2D and Ca levels in both strains of mice. Our findings indicate that the C57BL mice relied more on the mobilization of Ca from bone to maintain extracellular Ca homeostasis than the C3H mice. We conclude that strain differences in bone mass and density between C3H and C57BL mice is expressed, in part, through the vitamin D and PTH endocrine systems and their effects on the maintenance of extracellular Ca homeostasis.     

Bisphosphonates suppress periosteal osteoblast activity independently of resorption in rat femur and tibia

Recent studies demonstrate that bisphosphonates suppress bone resorption by leading to apoptosis of the osteoclast and inhibiting the differentiation to mature osteoclasts. The influence of bisphosphonates on bone formation is unknown, although it has been hypothesized that bisphosphonates inhibit osteoblast apoptosis and stimulate osteoblast proliferation and differentiation in vitro, leading to increased bone formation. The purpose of this study was to investigate the effect of bisphosphonates on bone formation. We administered risedronate at 0.05, 0.5 or 5.0 microg/kg/day or alendronate at 0.1, 1.0 or 10 microg/kg/day subcutaneously for 17 days to 6-month-old female Sprague-Dawley rats. Control rats were given a daily subcutaneous injection of saline. Following sacrifice, the femoral and tibial mid-diaphyses were harvested and mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/BS) were measured on periosteal and endocortical surfaces. In the femur, periosteal MAR was significantly lower in all treatment groups (22-29% for risedronate, 26-36% for alendronate) than in control. In the tibia, periosteal MAR and BFR of all treatment groups were significantly lower (41-50% for risedronate, 43-52% for alendronate) than in the control group. Because the periosteal surfaces of these bones are only undergoing bone formation in modeling mode, our results show that bisphosphonates suppress bone formation independently of bone resorption. Because this effect is seen on periosteal MAR rather than on periosteal MS/BS, we hypothesize that bisphosphonates affect the activity of individual osteoblasts at the cell level. This may help to explain the reason that the anabolic effects of teriparatide are blunted when administered concurrently with or following a course of bisphosphonates in humans.