Osteoprotegerin abrogated cortical porosity and bone marrow fibrosis in a mouse model of constitutive activation of the PTH/PTHrP receptor

Abstract

Intracortical porosities and marrow fibrosis are hallmarks of hyperparathyroidism and are present in bones of transgenic mice expressing constitutively active parathyroid hormone/parathyroid hormone-related protein receptors (PPR*Tg). Cortical porosity is the result of osteoclast activity; however, the etiology of marrow fibrosis is poorly understood. While osteoclast numbers and activity are regulated by osteoprotegerin (OPG), bisphosphonates suppress osteoclast activity but not osteoclast numbers. We therefore used OPG and bisphosphonates to evaluate the extent to which osteoclasts, as opposed to bone resorption, regulate marrow fibrosis in PPR*Tg mice after treatment of animals with vehicle, OPG, alendronate, or zoledronate. All three agents similarly increased trabecular bone volume in both PPR*Tg and control mice, suggesting that trabecular bone resorption was comparably suppressed by these agents. However, the number of trabecular osteoclasts was greatly decreased by OPG but not by either alendronate or zoledronate. Furthermore, intracortical porosity and marrow fibrosis were virtually abolished by OPG treatment, whereas alendronate and zoledronate only partially reduced these two parameters. The greater reductions in cortical porosity and increments in cortical bone mineral density with OPG in PPR*Tg mice were associated with greater improvements in bone strength. The differential effect of OPG versus bisphosphonates on marrow fibrosis, despite similar effects on trabecular bone volume, suggests that marrow fibrosis was related not only to bone resorption but also to the presence of osteoclasts.

Figure 1

Micro-CT images of femurs of 6-month-old wild-type (WT) and PPR*Tg mice. Longitudinal images of entire femurs (A and B), and axial images of diaphysis (C and E) and of metaphysis (D and F) are shown.

Figure 2

Histology (A–D), TRAP staining (E and F), and immunohistochemistry (G and H) of paraffin sections of decalcified tibias. H&E staining of cortical bone (A and C) and of trabecular bone (B and D). Note the presence of fibroblastoid cells as indicated by the arrowheads in both the cortical and trabecular bone area of PPR*Tg mouse (magnification = original ×20). E and F: TRAP staining of cortical (E) and trabecular bone (F) of PPR*Tg mouse. Sections were counterstained with H&E. Arrows indicate the TRAP-positive osteoclasts (magnification = original ×40). G and H: Immunohistochemistry for CD31 of cortical (G) and trabecular bone (H) of PPR*Tg mice. Sections were incubated with CD31 antibody and the antibody binding was detected by DAB. The sections were counterstained with H&E. Double arrowheads indicate the blood vessels positive for CD31 (magnification = original ×40).

Figure 3

Effect of OPG, ALN, and ZOL treatment on long bones. A: Longitudinal micro-CT images of femurs from 6-month-old wild-type (WT) (top panels) and PPR*Tg (bottom panels) mice. B: Femur lengths of wild-type and PPR*Tg mice treated with VEH, OPG, ALN, or ZOL were measured by micro-CT. ^ZP < 0.05 vs. ZOL; error bars represent SEM. C: H&E stained tibiae of 6-month-old wild-type (top panels) and PPR*Tg (bottom panels) mice treated with VEH, OPG, ALN, or ZOL (magnification = original ×4).

Figure 4

Micro-CT analysis of femoral cortices of 6-month-old wild-type (WT) and PPR*Tg mice treated with VEH, OPG, ALN, or ZOL. A: Representative two-dimensional micro-CT axial images of the femoral diaphysis. B: Periosteal perimeter. C: Endocortical perimeter. D: Cortical area excluding the cortical porosity. E: Cortical vBMD. F: Cortical porosity. G: Regression analysis of cortical porosity and cortical vBMD. r² = R-squared value. For each measurement, central region equivalent to 10% of the height of the femur was selected distal from the midpoint. Images shown represent the median value of cortical porosity for each group. *P < 0.01 vs VEH; ^ZP < 0.05 vs. ZOL; ^{^}P < 0.05 vs. ALN. Error bars represent SEM.

Figure 5

Effect of OPG, ALN, and ZOL treatment on bone strength at the femur diaphysis. Destructive 3-point bending tests were performed at the femur midshaft. A: Peak load and (B) energy to failure, (C) ultimate strength, and (D) toughness of femur diaphysis are shown. E: Regression analysis demonstrating the correlation of ultimate strength and cortical vBMD (F) and that of toughness and cortical porosity in PPR*Tg mice. *P < 0.05 vs VEH-treated PPR*Tg mice. ^ZP < 0.05 vs ZOL-treated PPR*Tg mice. Error bars represent SEM.

Figure 6

Effect of OPG, ALN, and ZOL treatment on marrow fibrosis and osteoblast number in trabecular areas of tibias. A: Histology of trabecular bone areas of VEH-, OPG-, ALN-, and ZOL-treated wild-type (WT) (top panels) and PPR*Tg (bottom panels) mice (H&E staining). Arrows indicate areas of fibroblastoid cells; (magnification = original ×20). B: Measurement of stromal cell area per total area (SCA/T.Ar). C: Osteoblast number per bone surface (Ob/BS). D: Serum osteocalcin level. *P < 0.05 vs VEH; **P < 0.01 vs VEH; ^P < 0.05 vs ALN. Error bars represent SEM.

Figure 7

Analysis of trabecular bone in 6-month-old wild-type (WT) and PPR*Tg mice treated with VEH, OPG, ALN, or ZOL. A–E: Micro-CT analysis of distal femurs: (A) Representative three-dimensional micro-CT images of the central 0.5 mm region of trabecular bone. B: Trabecular volumetric bone mineral density (vBMD). C: Bone volume fraction (BVF). D: Trabecular spacing (Tb.Sp.) and (E) structural model index. Images shown represent median values of BVF for each group. F and G: Bone histomorphometry of trabecular bones of proximal tibiae. F: Bone Volume/Total Volume and (G) Tb.Sp. *P < 0.05 vs VEH; **P < 0.01 vs VEH; ^P < 0.05 vs ALN; ^zP < 0.05 vs ZOL. Error bars represent SEM.

Figure 8

Effect of OPG, ALN, and ZOL treatment on osteoclast number and activity. A: TRAP staining. Representative pictures of trabecular bones of wild-type (WT) and PPR*Tg tibiae. Sections were counterstained with H&E. Arrows indicate osteoclasts (magnification = original ×40). B: Osteoclast number per total area (Oc/T.Ar.). C: Serum level of TRAP5b. D: Correlation of Oc/TA and SCA/T.Ar in VEH-, OPG-, ALN-, and ZOL-treated PPR*Tg mouse tibiae. Graphs (a) and (b) indicate the regression analysis of Oc/TA and SCA/T.Ar in VEH-treated PPR*Tg group and VEH-, OPG-, ALN-, ZOL-treated PPR*Tg group respectively. (a) y = 0.0869x + 1.044, R² = 0.7715 (b) y = 0.0354x + 2.069, R² = 0.257. *P < 0.05 vs VEH; **P < 0.01 vs VEH; ^zP < 0.05 vs ZOL. Error bars represent SEM.