Lack of Bak and Bax reduces osteoblast and osteocyte apoptosis and increases bone mass
Deletion of Bak and Bax in osteoblasts and osteocytes was accomplished by intercrossing Bak−/−;Bax+/f mice with Bak−/− mice bearing Cre recombinase under the control of osteocalcin gene regulatory elements (Bak−/−;OCN-Cre mice). The OCN-Cre transgene becomes active in replicating osteoblast progenitors just prior to their development into matrix synthesizing osteoblasts. Bak−/−;Baxf/f;OCN-Cre mice (hereafter designated BakΔBaxΔOCN mice) as well as BakΔ, BakΔBaxf/f, and BakΔOCN-Cre littermate controls, were born at the expected Mendelian ratios and did not exhibit discernible abnormalities at birth. Deletion of the Bax conditional allele was detected in cortical bone, but not in brain, liver, or spleen of BakΔBaxΔOCN mice (Fig. 1A). Osteoblastic cell cultures established from femoral bone marrow aspirates from BakΔBaxΔOCN mice exhibited reduced expression of Bax and attenuation of camptothecin-induced apoptosis, as compared to cells cultured from littermate controls (Fig. 1B).
Figure 1. Deletion of Bak and Bax increases femoral bone mass. (A–E) Experiments with the OCN-Cre deleter strain. (A) Level of Bax genomic DNA in osteocyte-enriched femoral shafts, brain, spleen or liver from 3-month-old female mice; n = 5 animals per group; *p < 0.05 versus BakΔBaxf/f. (B) Bax expression (left panel) and caspase-3 activity (right panel) in triplicate cultures of osteoblastic cells established from marrow-derived progenitors of 3-month-old female mice. Caspase-3 activity was determined 8 hours following addition of vehicle (Veh) or 0.2 µM camptothecin (Campt), *p < 0.05. Sequential determination of (C) femoral BMD, (D) spinal BMD, (E) and body weight in a cohort of female mice; n = 9–14/group. *p < 0.05 versus littermate controls by random coefficients model. (F–I) Experiments with the Osx1-Cre deleter strain. (F) Level of Bax genomic DNA in osteocyte-enriched femoral shafts from 2-month-old female mice; n = 3 animals per group; *p < 0.05 versus BakΔ;Baxf/f. Sequential determination of (G) femoral BMD, (H) spinal BMD, (I) and body weight in a cohort of female mice; n = 6–11/group, *p < 0.05 versus BakΔ and BakΔOsx1-Cre littermate controls by random coefficients model.
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Deletion of Bak and Bax caused a progressive increase in femoral BMD in a cohort of female mice (Fig. 1C). Specifically, femoral BMD of BakΔBaxΔOCN mice was greater than that of the controls beginning at 6 months of age, and remained so up to 13 months of age. However, the higher BMD of the BakΔBaxΔOCN mice declined to the control values by 18 months of age. The femoral BMD of BakΔOCN-Cre mice was indistinguishable from BakΔ and BakΔBaxf/f mice, indicating that the increased femoral BMD of the BakΔBaxΔOCN was indeed a result of the deletion of Bax rather than a nonspecific effect of Cre recombinase. Femoral bone growth was unaffected by lack of Bak and Bax as femoral length and the morphology of the growth plate of 2-month-old BakΔBaxΔOCN mice was identical to that of littermate controls (Supplementary Fig. 1A, B). Both spinal BMD and body weight of BakΔBaxΔOCN mice was indistinguishable from controls at all ages examined (Fig. 1D, E).
We next determined whether abrogation of apoptosis in osteoblast progenitors would augment the skeletal phenotype produced by Bax deletion in mature cells alone. To do this, we used transgenic mice expressing Cre recombinase under the control of Osterix 1 (Osx1) regulatory elements. In this Osx1-Cre deleter strain, the Cre recombinase is expressed in committed osteoblast progenitors. Expression of the Cre transgene is suppressed by doxycycline and was induced in our study by removal of animals from a doxycycline diet at 7 weeks of age. Bax DNA was reduced in femurs of BakΔBaxΔOsx1 mice (Fig. 1F). Moreover, a cohort of female BakΔBaxΔOsx1 mice exhibited a similar pattern of accrual, and subsequent loss, of femoral bone mass as the BakΔBaxΔOCN mice (Fig. 1G). Spinal BMD and body weight were not affected in the BakΔBaxΔOsx1 mice (Fig. 1H, I).
Lack of Bak and Bax increases femoral cancellous bone and magnifies the age-related development of cortical porosity
As expected, the prevalence of osteoblast apoptosis was reduced in cancellous bone of 8-month-old BakΔBaxΔOCN mice, compared to controls (Fig. 2A). Cortical osteocyte apoptosis was also reduced. Complete abrogation of osteoblast and osteocyte apoptosis was not achieved, probably due to retention of Bax expression in some cells and/or nonspecific ISEL staining. In any event, osteoblast number was increased by more than twofold as measured in the same femoral sections from BakΔBaxΔOCN mice (Fig. 2B), and this effect was associated with an increase in cancellous bone area (Fig. 2C). Attenuation of osteoblast apoptosis also resulted in an increase in osteocyte density (Fig. 2D), as previously observed in mice with reduced osteoblast apoptosis following daily injections of PTH.
Figure 2. Deletion of Bax with OCN-Cre reduces osteoblast and osteocyte apoptosis and increases femoral cancellous bone mass. Histomorphometric determination of (A) cancellous osteoblast (Ob) apoptosis, and cortical osteocyte (Ot) apoptosis; as well as (B) cancellous osteoblast number, (C) cancellous bone area, and (D) osteocyte density in femoral sections from 8-month-old male mice, n = 4–5/group. (E) Representative µCT images of femora from BakΔBaxf/f and BakΔBaxΔOCN littermates; scale bar, 1 mm. µCT was used to image and quantify femoral bone from 3-month-old mice comprising 2 males and 4 females of each genotype, 8-month-old male mice, and 22-month-old female mice. µCT determination of (F) trabecular bone volume (BV/TV), (G) trabecular number (Tb.N), (H) trabecular separation (Tb.Sp), (I), trabecular thickness (Tb.Th), (J) and connectivity density (Conn.D) in the distal metaphysis of femora from BakΔBaxf/f mice at 3 months (n = 6), 8 months (n = 7), or 22 months (n = 8) of age; and from BakΔBaxΔOCN mice at 3 months (n = 6), 8 months (n = 5), or 22 months (n = 9). *p < 0.05 versus BakΔBaxf/f littermates.
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3D µCT images of femurs from BakΔBaxf/f mice showed that cancellous bone was largely confined to the metaphysis in 3-month-old and 8-month-old mice, respectively; and had practically disappeared by 22 months at of age (Fig. 2E), consistent with earlier findings on the impact of aging on murine cancellous bone.[32, 41] In contrast, cancellous bone was present throughout the marrow of 3-month-old, 8-month-old, and 22-month-old BakΔBaxΔOCN mice (Fig. 2E). A similar pattern of femoral cancellous bone retention with age was also found in 7-month-old and 21-month-old BakΔBaxΔOsx1 mice (Fig. 3A). Cancellous bone architecture was determined in the distal femoral metaphysis on a group of males and females at 3 months of age, males at 7 to 8 months of age, and females at 21 to 22 months of age. Cancellous bone volume per tissue volume (BV/TV) was higher in BakΔBaxΔOCN than in controls at 3, 8, and 22 months of age (Fig. 2F). Similarly, BakΔBaxΔOsx1 mice exhibited higher BV/TV at 7 and 21 months of age (Fig. 3B). The finding of a similar increase in femoral cancellous bone mass in both BakΔBaxΔOCN and BakΔBaxΔOsx1 mice indicates that this phenomenon was indeed due to the abrogation of apoptosis in mature cells alone.
Figure 3. Deletion of Bak and Bax with Osx1-Cre increases femoral cancellous bone mass. (A) Representative µCT images from BakΔBaxΔOsx1 and BakΔOsx1-Cre littermates at 7 months of age in males, and at 21 months of age in females. Scale bar = 1 mm. µCT determination of (B) trabecular bone volume (BV/TV), (C) trabecular number (Tb.N), (D) trabecular separation (Tb.Sp), (E) trabecular thickness (Tb.Th), (F) and connectivity density (Conn.D) in the distal metaphysis of femora from BakΔOsx1-Cre mice at 7 months of age (n = 9) and 21 months of age (n = 7); and BakΔOsx1-Cre mice at 7 months of age (n = 8) and 21 months of age (n = 9). *p < 0.05 versus BakΔOsx1-Cre littermates.
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The increased cancellous bone volume could be accounted for by increased trabecular number, decreased trabecular separation, and increased trabecular connectivity in 3-month-old and 22-month old BakΔBaxΔOCN mice (Fig. 2G, H, J), and in 21-month-old female BakΔBaxΔOsx1 mice (Fig. 3C, D, F). In male mice examined at 8 months of age, there was only a small increase in trabecular number and a small decrease in trabecular separation. These changes were statistically significant in BakΔBaxΔOsx1 mice (Fig. 3C, D) but not BakΔBaxΔOCN mice (Fig. 2G, H). The reason for the discrepancy between the large change in BV/TV as compared to the small changes in Tb.N and Tb.Sp in 7-month-old to 8-month-old males lacking Bak and Bax is unclear, and requires further investigation. Trabecular thickness was unaffected by lack of Bak and Bax at all ages examined in both the OCN-Cre (Fig. 2I), and the Osx1-Cre (Fig. 3E) models.
Vertebral cancellous bone volume was unaffected in BakΔBaxΔOCN mice at 3 and 8 months of age, and in 7-month-old BakΔBaxΔOsx1 mice (Supplementary Fig. 2). Although there was an increase in mean BV/TV in 21-month-old to 22-month-old Bak/Bax-deficient mice as compared to controls, the change was not statistically significant. These findings are consistent with the lack of an effect of the deletion of Bak and Bax on spinal BMD in both strains (Fig. 1D, H).
In difference to the pronounced cancellous bone phenotype of femurs of Bak/Bax-deficient mice, earlier attempts to abrogate apoptosis by overexpression of the antiapoptotic protein Bcl-2 in osteoblasts caused either a transient increase or no change in femoral cancellous bone mass, probably due to an inhibitory effect of high Bcl-2 levels on osteoblast differentiation.[42, 43] However, deletion of Bak and Bax had no such effect (Supplementary Fig. 3). As expected for osteoblasts with a prolonged lifespan, mineral deposition and expression of alkaline phosphatase and osteocalcin was significantly higher in cultures of bone marrow–derived osteoblastic cells from BakΔBaxΔOCN mice, compared to cells cultured from control mice.
Strikingly, µCT images revealed far more intracortical pores, or voids, in femurs of 22-month-old female BakΔBaxΔOCN mice (Fig. 2E, and enlarged images in Fig. 4A) and 21-month-old BakΔBaxΔOsx1 mice (Figs. 3A and 4A), as compared to age-matched control mice. The presence of intracortical voids was confirmed in both the femur and tibia of aged female BakΔBaxΔOsx1 mice by histology (Fig. 4B). In some areas, trabecularization of the endocortical bone had occurred, whereas in others the interior of the cortex contained voids of various size (Fig. 4C). Very large voids were sometimes associated with cortical expansion into the marrow space, along with the formation of new endosteal boundary (Fig. 4A, C). As a result, large porous areas (eg, right panel of Fig. 4C) appeared to be located in the middle of the cortex. Lack of a well-defined endosteal boundary prevented accurate determination of cortical thickness in aged Bak/Bax-deficient mice. However, in 3-month-old and 7-month-old to 8-month-old mice lacking Bak and Bax, which had an intact endosteal boundary, femoral cortical thickness was indistinguishable from controls (Supplementary Fig. 1C).
Figure 4. Lack of Bax and Bak increases cortical porosity in aged mice. (A) Representative µCT images of femora from 22-month-old female BakΔBaxΔOCN and BakΔBaxf/f littermates (left panel), and 21-month-old female BakΔBaxΔOsx1 and BakΔBaxf/f littermates (right panel). Scale bar = 1 mm. White arrowheads mark location of pores in cortical bone. (B) Representative femoral and tibial H&E-stained decalcified sections from 21-month-old female BakΔBaxΔOsx1 mice, with the periosteal surface on the left. Scale bar = 1 mm. (C) Representative Trichrome-stained nondecalcified sections of the femoral cortex from 22-month-old female BakΔBaxΔOCN mice, with the periosteal surface on the left. Scale bar = 50 µm. (D) Inverse µCT images of the distal half of femora from 21-month-old female mice. Void areas are depicted in grey within a transparent bone matrix. (E) Cortical porosity (Ct.Po), pore number (Po.N), and pore volume (Po.V) in the cortex of the distal half of femora from 21-month-old female mice, n = 3–4/group. (F) Inverse µCT images of the distal half of femora from 3-month-old female mice. (G) Porosity, pore number and pore volume in 3-month-old female mice, n = 3/group, *p < 0.05 versus littermate controls.
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The percentage of cortical bone occupied by voids (porosity), measured in histologic sections, was fourfold to sevenfold higher in femurs and tibias from BakΔBaxΔOCN and BakΔBaxΔOsx1 mice as compared to controls (Table 1). Femoral porosity of BakΔOCN-Cre mice varied more than all the other controls, but this phenomenon was not seen in tibias, excluding the possibility of a Cre effect. Because the cortical porosity phenotype was practically identical in BakΔBaxΔOCN and BakΔBaxΔOsx1 mice, bones from either genotype were used interchangeably in the subsequent work.
Table 1. Deletion of Bak and Bax Increases Cortical Porosity in the Femur and Tibia in Aged Female Mice
|(A) OCN-Cre model (22 months old)|
| ||Porosity, % (n)|
|Femur||4.7 ± 1.4 (2)||3.6 ± 4.7 (3)||11.4 ± 8.3 (4)||23.3 ± 11.5 (7)a|
|Tibia||4.1 ± 4.1 (7)||6.4 ± 3.1 (5)||6.2 ± 5.2 (9)||27.2 ± 16.5 (4)b|
|(B) Osx1-Cre model (21 months old)|
| ||Porosity, % (n)|
|Femur||2.1 ± 0.9 (4)||NA||4.7 ± 1.2 (3)||19.2 ± 6.8 (4)b|
|Tibia||4.1 ± 2.2 (6)||3.2 ± 1.6 (3)||3.3 ± 2.9 (7)||16.6 ± 7.1 (6)b|
Inverse 3D µCT images revealed that the intracortical pores were distributed throughout the distal portion of the femur of 21-month-old BakΔBaxΔOsx1 mice, whereas pores were largely located in the metaphyseal cortex of BakΔBaxf/f controls (Fig. 4D). Quantification of the inverse 3D images gives a more accurate measure of porosity than that obtained using a single histologic section. Using this approach, we found that porosity of femoral bone of aged BakΔBaxΔOsx1 mice was sevenfold greater than that of controls due to an increase in pore size (Fig. 4E). The number of pores, however, was reduced due to coalescence of small pores into larger ones. Indeed, in a series of 8-µm-thick cross-sectional µCT images taken at the femoral mid-diaphysis of a 22-month-old BakΔBaxΔOCN mouse, small pores were frequently contiguous with larger pores (Supplementary Video 1).
Pores were rare in femurs from 3-month-old Bak-deficient and Bax-deficient mice or controls, whether viewed in longitudinal sections (not shown), 3D µCT images (Supplementary Fig. 4), or in inverse 3D µCT images (Fig. 4F). The femoral cortical porosity of 3-month-old control mice was approximately 1.5% and increased to 6% by 21 months of age (Fig. 4G, E, respectively), consistent with earlier evidence that cortical porosity is a feature of old age in rodents.[44-46] Nevertheless, even at 3 months of age, cortical porosity was 17% higher in BakΔBaxΔOCN mice than in control mice, due to a small increase in pore size (Fig. 4G).
In contrast to the situation in femoral bone, porosity was not observed in histologic sections or µCT images of vertebral cortical bone of aged Bak/Bax-deficient mice or controls (data not shown).
The age-related increase in cortical porosity is a result of increased intracortical bone remodeling
Backscattered scanning electron microscopy (BSEM) of the femoral diaphyseal cortex of 22-month-old control mice revealed two distinct zones separated by a mineral-rich boundary (Fig. 5A). The endosteal zone contained highly mineralized scalloped tidemarks that mark previous episodes of bone remodeling.[47, 48] In contrast, the periosteal zone had a lamellar structure, and lacked markings of remodeling activity. Cortical voids in femurs from 22-month-old BakΔBaxΔOCN mice were largely restricted to the endosteal zone, and rarely invaded the periosteal zone (Fig. 5B). Moreover, the porous endosteal zone of these mice exhibited areas of bright mineralized matrix, reflecting older bone, juxtaposed to areas of dimmer newly formed bone. These features are diagnostic of recent remodeling activity.
Figure 5. Increased cortical porosity of aged BakΔBaxΔOCN mice is restricted to the endosteal zone. Representative BSEM images of the femoral diaphyseal cortex from (A) a 22-month-old female BakΔ mouse, and (B) a 22-month-old female BakΔBaxΔOCN mouse. Endosteal (“E”) and periosteal (“P”) zones are separated by a highly mineralized boundary, indicated by green arrowheads. Red arrowheads mark highly mineralized cement lines that reflect previous remodeling activity. Red arrows denote areas of recently remodeled bone that have not yet achieved full mineralization.
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Cortical voids in both the aged BakΔBaxΔOCN mice and the control mice contained osteoclasts as well as osteoblasts adjacent to osteoid matrix, blood vessels, and cells indistinguishable from those inhabiting the bone marrow (Fig. 6A). In addition, surfaces outlining the voids were labeled by tetracycline, confirming active intracortical remodeling. Expression of cathepsin K and osteocalcin—markers of osteoclasts and osteoblasts, respectively—was increased in tibias of BakΔBaxΔOCN mice, as compared to control mice (Fig. 6B). Moreover, expression of RANKL was increased, whereas expression of the RANKL antagonist osteoprotegerin (OPG) was unchanged, compared to controls. In line with the presence of blood vessels in the voids and a higher porosity in BakΔBaxΔOCN mice, the expression of VEGF was also increased in these mice. The increase in RANKL and VEGF transcripts was confirmed in osteocyte-enriched humeri from BakΔBaxΔOsx1 mice (Fig. 6C), indicating that osteocytes are the principal source of the increased RANKL and VEGF.
Figure 6. The age-related increase in porosity in Bak/Bax-deficiency is associated with intracortical bone remodeling and increased expression of RANKL and VEGF by osteocytes. (A) Femoral bone sections from 22-month-old female mice were stained for TRAPase to visualize osteoclasts, stained red (left panels); with toluidine blue (middle panels) to view osteoblasts (red asterisk) and blood vessels (bv); or viewed with fluorescence microscopy (right panels) to observe tetracycline labeling (arrowheads). Scale bar = 10 µm (left and middle panels), 100 µm (right panels). (B) Cathepsin K (CatK), osteocalcin (Ocn), RANKL, OPG and VEGF mRNA levels in whole tibias from 22-month-old female mice, n = 8–10/group; *p < 0.05 versus BakΔBaxf/f littermates. (C) RANKL and VEGF mRNA levels in collagenase-digested humeri, enriched in osteocytes, from 21-month-old female mice, n = 6–7/group; *p < 0.05 versus combined littermate controls.
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Histomorphometric measurements showed that void area, intracortical bone perimeter, and osteoclast perimeter were increased in the endocortical zone of the femoral cortex of aged BakΔBaxΔOCN mice compared to controls (Table 2). These measurements reflect the increase in cortical porosity, and indicate that osteoclastic erosion of the cortex is responsible. However, when expressed per millimeter of intracortical bone surface, osteoclast number was not affected by Bak/Bax deficiency; nor were dynamic indices of bone remodeling, including mineralizing surface, mineral apposition rate, and bone formation rate. Taken together, these findings demonstrate an increase in the amount of intracortical bone engaged in remodeling in aged Bak/Bax-deficient mice. The cancellous bone perimeter and area were also increased in aged BakΔBaxΔOCN mice, consistent with the µCT findings. The paucity of cancellous bone prevented precise determination of the number of osteoclasts at this site. Nevertheless, there was sufficient tetracycline labeling in the limited amount of bone available to show that, as in the intracortical pores, dynamic indices of cancellous bone remodeling were unaffected by Bak/Bax deficiency in these aged mice.
Table 2. Increased Intracortical Bone Engaged in Remodeling in Aged BakΔBaxΔOCN Mice
|B.Pm (mm)a||11.4 ± 7.2||42.3 ± 13.4*||3.13 ± 1.55||17.6 ± 8.4*|
|Vo.Ar/EZ.Ar||0.10 ± 0.10||0.33 ± 0.22*||–||–|
|B.Ar/T.Ar||–||–||0.012 ± 0.006||0.061 ± 0.037*|
|Oc.Pm (mm)a||0.47 ± 0.38||1.48 ± 0.80*||0.19 ± 0.26||0.89 ± 1.21|
|Oc.Pm/B.Pm||0.036 ± 0.024||0.034 ± 0.012||0.055 ± 0.050||0.037 ± 0.038|
|Oc.N/B.Pm (1/mm)||1.15 ± 0.75||1.10 ± 0.49||2.25 ± 2.13||1.65 ± 1.85|
|M.Pm/B.Pm||0.24 ± 0.08||0.21 ± 0.02||0.22 ± 0.06||0.17 ± 0.06|
|MAR (µm/d)||1.64 ± 0.20||1.28 ± 0.22||1.34 ± 0.24||1.23 ± 0.09|
|BFR/BS (µm2/µm/d)||0.34 ± 0.08||0.25 ± 0.06||0.30 ± 0.12||0.20 ± 0.08|
Serum levels of Ca, Pi, and PTH, as well as 25-hydroxyvitamin D3 1α-hydroxylase transcripts in the kidney (a PTH target gene), were similar in BakΔBaxΔOsx1 mice and littermate controls (Table 3). Moreover, there was no evidence of the marrow fibrosis often seen in conditions of excess PTH (Fig. 4B, C). These findings suggest that a local, rather than a systemic, stimulus was responsible for the increased porosity in aged BakΔBaxΔOCN mice.
Table 3. Calcium Homeostasis in 21-Month-Old Female Mice Is Unaffected by Deletion of Bak and Bax
|Calcium (mg/mL)||7.64 ± 0.53 (8)||8.22 ± 0.72 (3)||8.17 ± 0.43 (7)||7.86 ± 0.55 (8)|
|Phosphate (mg/mL)||12.5 ± 2.3 (8)||16.5 ± 3.5 (3)||13.3 ± 5.0 (7)||14.3 ± 1.8 (8)|
|PTH (pg/mL)||216 ± 95 (8)||218 ± 164 (3)||219 ± 116 (7)||241 ± 189 (8)|
|Cyp27b1 mRNA (relative to GAPDH)||0.023 ± 0.019 (4)||0.025 ± 0.006 (3)||0.018 ± 0.008 (5)||0.024 ± 0.012 (5)|
The increased cortical porosity in aged BakΔBaxΔOCN mice is associated with dysmorphic osteocytes
The majority of cortical osteocytes in 22-month-old BakΔBaxΔOCN or littermate control mice occupied the entire lacunar space and had prominent nuclear profiles containing nucleoli (Fig. 7A). Nonetheless, some osteocytes displayed dysmorphic features, including condensed cytoplasm and pyknotic nucleus without nucleoli. The prevalence of dysmorphic osteocytes was higher in the periosteal zone of both control and BakΔBaxΔOCN mice, as compared to the endosteal zone (Fig. 7B). Strikingly, dysmorphic osteocytes in the periosteal zone were more numerous in BakΔBaxΔOCN mice compared to the periosteal zone of controls. Moreover, there was a correlation between the prevalence of dysmorphic periosteal zone osteocytes and the extent of cortical porosity (r = 0.6, p < 0.02), suggesting a cause and effect relationship.
Figure 7. Increased prevalence of dysmorphic osteocytes is associated with increased porosity of aged BakΔBaxΔOCN mice. (A) Representative histologic nondecalcified sections showing viable osteocytes with typical morphology (upper panel) and dysmorphic osteocytes (arrowheads, lower panel) in the periosteal zone of the femoral cortex from 22-month-old female mice. Scale bar = 10 µm. (B) The percentage of dysmorphic osteocytes in endosteal (EZ) and periosteal (PZ) zones of the cortex of 22-month-old female mice, n = 7–8/group; *p < 0.05 versus EZ, #p < 0.05 versus PZ of combined controls. (C) Lacunar and canalicular morphology of cortical osteocytes of 22-month-old mice in the endosteal (“E”) and periosteal (“P”) zones, visualized by acid-etch SEM. Scale bar = 50 µm. Inset: high magnification image of typical lacuna (arrowhead) and canaliculi in the periosteal zone. Scale bar = 5 µm; *cortical void.
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Attenuation of apoptosis by overexpression of Bcl-2 decreases osteocyte viability, most likely by inhibiting canalicular development. Thus, the dysmorphic osteocytes in Bak/Bax-deficient mice could be due to an increase in free Bcl-2 protein, which would normally bind to Bax and Bak and prevent apoptosis. However, the morphology of the lacunae and canaliculi of cortical osteocytes, visualized by acid-etch SEM, of 22-month-old BakΔBaxΔOCN mice was indistinguishable from that of controls, thus excluding this possibility (Fig. 7C).