1. Glucocorticoid-induced Osteonecrosis of the Femoral Head in Mice
Temporal Sequence of Molecular, Cellular, Vascular and Anatomical Changes Leading to
Glucocorticoid-induced Osteonecrosis of the Femoral Head in Mice
Robert S. Weinstein1, Erin A. Hogan1, Marilina Piemontese1, Michael J. Borrelli2, Serguei Liachenko3, Charles A. O’Brien1 and Stavros C. Manolagas1
1Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, Department of Internal Medicine, Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, AR 2Department of Radiology, Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, AR 3National Center for Toxicological Research/Food and Drug Administration, Pine Bluff, AR
Abstract
Osteonecrosis of the hip develops in up to 40% of patients receiving long-term systemic therapy with glucocorticoids and may occur without widespread osteoporosis. However, the pathological changes responsible remain obscure. We have examined the consecutive molecular, cellular, vascular and anatomical changes that occur during the development of glucocorticoid-induced osteonecrosis. To do this, we administered pulsed doses of glucocorticoids, a regimen associated with a greater risk of osteonecrosis than steady-state treatment. Prednisolone pellets (2.1 mg/kg/d) were implanted in 7-month-old mice on day 0, 14 and 28 and necropsies were performed on day 14, 28 and 42, respectively. We found that fourteen days after the first pellet was implanted, the femoral head expression of osteocalcin, OPG, RANKL, and VEGF was decreased. Similarly, femoral head strength and bone formation rate decreased prior to any detectable changes in femoral head density or microarchitecture as determined by µCT; and prior to equivalent changes in distal femoral or vertebral bone. By day 42, femoral head density and trabecular thickness as well as vertebral compression strength decreased but there were no changes in vertebral mineral density, distal femoral µCT, or femoral 3-point bending. The loss of femoral head density and cancellous architecture was accompanied by a 2.5-fold and 6.3-fold increase in extraordinarily large, intensely TRAP stained cancellous and endocortical osteoclasts at day 14 lasting until day 28, after which the osteoclast numbers returned to control values by day 42. Unexpectedly, this pronounced osteoclastosis was accompanied by a decrease in RANKL, and particularly in OPG. Lastly, we imaged the femoral head vascularity using µCT of decalcified bone after perfusion with lead chromate. Glucocorticoids converted the normal dendritic vasculature to pools and lakes. These changes were accompanied by a striking increase in femoral head edema as detected by magnetic resonance imaging (MRI) - robust diagnostic evidence of osteonecrosis. Collectively, these findings reveal a pathophysiological sequence during which an initial decrease in femoral head OPG, RANKL, and VEGF by glucocorticoids caused increased osteoclastogenesis and reduced vascularity and mechanical strength well before the loss of bone mass and microarchitectural deterioration occurred; thus rendering the femoral head vulnerable to the development of osteonecrosis.
Cancellous Tissue
Femoral head
Distal femur
Vertebrae
1 mm
µm2/µm/d
BFR/BPM
placebo
prednisolone *
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
OCN
RANKL
OPG
VEGF
Wnt10b
Wnt5a
1 pellet
2 pellets
3 pellets
placebo
prednisolone * *
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days * *
Figure 1. In 3-month-old C57Bl/6 control mice, tetracycline labels in the cancellous bone of the femoral head (FH) were more intensely fluorescent than those in the distal femur and the bone formation rate (BFR) of the FH was more than three-fold greater than that of the distal femur. Micro-CT (µCT) showed that the arterial foramen of the FH was far smaller than the nutrient foramen of the femoral shaft. * * *
placebo
prednisolone
Figure 6. Using a 7 Tesla MRI, custom-made 20 mm surface coil, and 3D fast low angle short imaging to enhance T1 contrast, there was a striking increase in the FH water signal after prednisolone.
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
Endocortical perimeter
BFR/BPM
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days *
µm2/µm/d *
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
Summary
Glucocorticoid administration decreases FH strength and BFR before cancellous bone area and before equivalent changes in other bones.
Decreases in the FH expression of OPG, RANKL, VEGF, and Wnt10b with increases in Wnt5a were accompanied by decreased BFR, increased osteoclasts and disrupted vasculature.
The FH is more sensitive to glucocorticoids than other skeletal sites due to the load it carries and inadequate blood supply. * *
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
Figure 4. Bone histomorphometry showed that by day 14, the BFR in the FH decreased before changes in cancellous bone area. The number of cancellous osteoblasts decreased and osteoclasts increased at both the cancellous and endocortical perimeter. The purple arrow points to large, intensely TRAP+ osteoclasts in the FH. *
prednisolone
placebo *
t-test
p = 0.03
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
1 pellet
14 days
2 pellet
28 days
3 pellets
42 days
Acknowledgements
These studies were supported by VA Merit Review Grants (I01BX000436, I01BX000294, I01BX001405) from the Office of Research and Development Department of Veteran Affairs, the National Institutes of Health (P01-AG13918 and R01-AR49794) and Tobacco Settlement Funds provided by the UAMS College of Medicine. The authors have nothing to disclose.
Figure 2. In 7-month-old C57Bl/6 mice, receiving repeated implantation of pellets delivering placebo or 2.1 mg/kg/d of prednisolone on day 0, 14 and 28, FH strength decreased by 44% while vertebral strength decreased by 24%, half of the loss at the FH. These decreases occurred before changes in FH or µCT, distal or midshaft femoral bone or vertebral BMD.
Figure 3. Prednisolone administration reduced OCN expression in the FH, distal femur and vertebrae 14 days after implantation of the first pellet. However, at this time point, OPG, RANKL, and VEGF were decreased in the FH but no significant changes were found in the distal femur or vertebrae. Early decreases in Wnt10b occurred in the FH and vert with increases in Wnt5a at all 3 sites.
Figure 5. The vascular pattern of the FH was imaged using µCT of decalcified bone after perfusion with lead chromate. Prednisolone converted the normal dendritic pattern to pools of edema.