Prevalence of glucocorticoid induced osteonecrosis in the mouse is not affected by treatments that maintain bone vascularity

Abstract

Objective: Determine if LLP2A-Ale or PTH (1-34) affects the prevalence of glucocorticoid-induced osteonecrosis (ON) in a mouse model.

Methods: Eight-week-old young adult male BALB/cJ mice were weight-randomized into Control (Con), glucocorticoid (GC)-only, or concurrent treatments with GC and LLP2A-Ale (250 μg/kg or 500 μg/kg, IV, Days 1, 14, 28) or parathyroid hormone hPTH (1-34) (40 μg/kg, 5×/week). Mice were necropsied after 45 days for qualitative evaluation of prevalent ON and quantitative evaluation of vascularity in the distal femoral epiphysis (DFE); and quantitative evaluation of bone mass, microarchitecture, and strength in the distal femoral metaphysis and lumbar vertebral body.

Results: The prevalence of ON was 14% in the Con group and 36% in the GC-only group (P = 0.07). The prevalence of ON did not differ among GC-only, GC + LLP2A-Ale, and GC + PTH groups. GC-only mice had significantly lower trabecular and cortical bone strength than Con, while GC + LLP2A-Ale (500 μg/kg) and GC + PTH (1-34) groups had significantly greater trabecular bone strength than the GC-only group. GC + LLP2A-Ale (250 μg/kg and 500 μg/kg) and GC + PTH had significantly higher trabecular bone volume than GC-only mice at the vertebrae, distal femoral epiphyses and distal femoral metaphyses. DFE vascularity was lower in GC-only mice than in all other groups.

Conclusion: Neither LLP2A-Ale nor hPTH (1-34) reduced the prevalence of GC-induced ON, compared to GC-only mice. However, GC-treated mice given LLP2A-Ale or hPTH (1-34) had better bone mass, microarchitecture, and strength in trabecular-rich regions, and higher levels of vascularity than GC-only mice.

Keywords: Dexamethasone; Distal femoral epiphysis; LLP2A-Ale; Prevention; hPTH (1–34).

Fig. 1

Photomicrographs of Osteonecrosis-Free and Osteonecrosis-Positive Distal Femoral Epiphyses A&B- Representative 5 μm thick H&E-stained parasagittal section of an ON-free distal femoral epiphysis. B is higher magnification photomicrograph of box in A. Note abundant hematopoietic marrow with occasional adipocytes and trabeculae with most osteocyte lacunae showing healthy nuclei. Occasional, isolated empty lacunae exist randomly in normal bone due to the relative size of lacunae, 5 μm section thickness, and positioning of nuclei in neighboring sections. Scale bars = 1 mm (A) and 200 μm (B). C&D- Representative section from an ON-positive distal femoral epiphysis. D is higher magnification photomicrograph of box in C. A positive diagnosis of ON in a DFE required the presence of both fatty marrow or necrotic bone marrow stroma that surrounded trabeculae, and multiple confluent empty osteocyte lacunae (Kawedia et al., 2012; Janke et al., 2013; Liu et al., 2016; Yang et al., 2009). Most ON-positive DFE's had only a portion, occasionally as little as 10%, of the epiphysis involved with ON. Nonetheless, note copious amount of fatty marrow in D, with trabeculae containing multiple confluent empty lacunae. Fields with fatty marrow or stromal necrosis surrounding trabeculae that contained only lacunae with healthy osteocyte nuclei were not uncommon. Only when fat or marrow necrosis-enveloped trabeculae also had multiple confluent empty osteocyte lacunae was the specimen designated as ON-positive. Multiple confluent empty osteocyte lacunae in trabeculae were never observed in the midst of healthy, hematopoietic marrow. Scale bars as for A&B = 1 mm (C) and 200 μm (D).

Fig. 2

Immunofluorescence Labeled Percent Area for CD31/PECAM1 and Endomucin in the Distal Femoral Epiphysis A) % of CD31+ Cells; Mean ± SEM; *Compared to GC-only (p < 0.05). B) % of Endomucin+ Cells; Mean ± SEM; *Compared to GC-only (p < 0.05).

Fig. 3

Immunofluorescence Labeling for CD31/PECAM1 and Endomucin of same Region in the Distal Femoral Epiphysis A) Representative photomicrograph of immunofluorescence labeling for CD31/PECAM1. Scale bar (100 μm) in lower right corner of Fig. 3C applies. B) Representative photomicrograph of immunofluorescence labeling for Endomucin. Scale bar (100 μm) in lower right corner of Fig. 3C applies. C) Representative photomicrograph of immunofluorescence labeling for merged CD31/PECAM1 and Endomucin. Scale bar (100 μm) in lower right corner.