Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age

Abstract

Skeletal aging is accompanied by decreased cancellous bone mass and increased formation of pores within cortical bone. The latter accounts for a large portion of the increase in nonvertebral fractures after age 65 years in humans. We selectively deleted Bak and Bax, two genes essential for apoptosis, in two types of terminally differentiated bone cells: the short-lived osteoblasts that elaborate the bone matrix, and the long-lived osteocytes that are immured within the mineralized matrix and choreograph the regeneration of bone. Attenuation of apoptosis in osteoblasts increased their working lifespan and thereby cancellous bone mass in the femur. In long-lived osteocytes, however, it caused dysfunction with advancing age and greatly magnified intracortical femoral porosity associated with increased production of receptor activator of nuclear factor-κB ligand and vascular endothelial growth factor. Increasing bone mass by artificial prolongation of the inherent lifespan of short-lived osteoblasts, while exaggerating the adverse effects of aging on long-lived osteocytes, highlights the seminal role of cell age in bone homeostasis. In addition, our findings suggest that distress signals produced by old and/or dysfunctional osteocytes are the culprits of the increased intracortical porosity in old age.

Keywords: AGING; APOPTOSIS; BONE FORMATION; CORTICAL POROSITY; OSTEOBLASTS; OSTEOCYTES; RANKL.

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 vs. Bak^ΔBax^f/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 femoral BMD (C) spinal BMD (D), and body weight (E) in a cohort of female mice; n=9–14/group. *p<0.05 vs. littermate controls by random coefficients model. (F–I) Experiments with the Osx1-Cre deleter strain. (A) Level of Bax genomic DNA in osteocyte-enriched femoral shafts from 2-month-old female mice; n=3 animals per group; *p<0.05 vs. Bak^Δ;Bax^f/f. Sequential determination of femoral BMD (G), spinal BMD (H) and body weight (I) in a cohort of female mice; n=6–11/group, *p<0.05 vs. Bak^Δ and BakΔOsx1-Cre littermate controls by random coefficients model.

Figure 2. Deletion of Bax with OCN-Cre reduces osteoblast and osteocyte apoptosis and increases femoral cancellous bone mass

Histomorphometric determination of cancellous osteoblast (Ob) apoptosis, and cortical osteocyte (Ot) apoptosis (A); as well as cancellous osteoblast number (B), cancellous bone area (C), and osteocyte density (D) in femoral sections from 8-month-old male mice, n=4–5/group. (E) Representative micro-CT images of femora from Bak^ΔBax^f/f and Bak^ΔBax^ΔOCN littermates; scale bar, 1 mm. Micro-CT 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. Micro-CT determination of trabecular bone volume (BV/TV) (F), trabecular number (Tb.N) (G), trabecular separation (Tb.Sp) (H), trabecular thickness (Tb.Th) (I) and connectivity density (Conn.D) (J) in the distal metaphysis of femora from Bak^ΔBax^f/f mice at 3 months (n=6), 8 months (n = 7) or 22 months (n = 8) of age; and from Bak^ΔBax^ΔOCN mice at 3 months (n = 6) , 8 months (n = 5) or 22 months (n = 9). *p <0.05 vs. Bak^ΔBax^f/f littermates.

Figure 3. Deletion of Bak and Bax with Osx1-Cre increases femoral cancellous bone mass

(A) Representative micro-CT images from Bak^ΔBax^ΔOsx1 and Bak^ΔOsx1-Cre littermates at 7 months of age in males, and at 21 months of age in females. Scale bar, 1mm. Micro-CT determination of trabecular bone volume (BV/TV) (B), trabecular number (Tb.N) (C), trabecular separation (Tb.Sp) (D), trabecular thickness (Tb.Th) (E) and connectivity density (Conn.D) (F) in the distal metaphysis of femora from Bak^ΔOsx1-Cre mice at 7 months of age (n = 9) and 21 months of age (n = 7); and Bak^ΔOsx1-Cre mice at 7 months of age (n = 8) and 21 months of age (n = 9). *p<0.05 vs. Bak^ΔOsx1-Cre littermates.

Figure 4. Lack of Bax and Bak increases cortical porosity in aged mice

(A) Representative micro-CT images of femora from 22-month-old female Bak^ΔBax^ΔOCN and Bak^ΔBax^f/f littermates (left panel), and 21-month-old female Bak^ΔBax^ΔOsx1 and Bak^ΔBax^f/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 Bak^ΔBax^ΔOsx1 mice, with the periosteal surface on the left; scale bar, 1 mm. (C) Representative femoral Trichrome-stained nondecalcified sections of femoral cortex from 22-month-old female Bak^ΔBax^ΔOCN mice, with the periosteal surface on the left.; scale bar, 50 µm. (D) Inverse micro-CT 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 micro-CT images of the distal half of femora from 3-mo-old female mice. (G) Porosity, pore number and pore volume in 3-month-old female mice, n=3/group, *p<0.05 vs. littermate controls.

Figure 5. Increased cortical porosity of aged Bak^ΔBax^ΔOCN mice is restricted to the endosteal zone

Representative BSEM images of the femoral diaphyseal cortex from (A) a 22-month-old female Bak^Δ mouse, and (B) a 22-month-old female Bak^ΔBax^ΔOCN 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.

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 (arrow heads). scale bar, 10 µm (left and middle panels) or 100 µm (right panels). (B) Cathepsin K (CatK), osteocalcin (Ocn), RANKL, OPG and VEGF mRNA levels in whole tibiae from 22-month-old female mice (n=8–10/group) *p<0.05 vs. Bak^ΔBax^f/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 vs. combined littermate controls.

Figure 7. Increased prevalence of dysmorphic osteocytes is associated with increased porosity of aged Bak^ΔBax^ΔOCN mice

(A) Representative histologic nondecalcified sections showing viable osteocytes with typical morphology (upper panel) and dysmorphic osteocytes (red arrow heads, lower panel) in the periosteal zone of the femoral cortex from 22-month-old female mice. Scale bar, 10µm. (B) The percentage of dysmorphic osteocytes in endosteal (EZ) and periosteal (PZ) zones of the cortex of 22-mo-old female mice, n=7–8/group. *p<0.05 vs. EZ; #p<0.05 vs. 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.