Cisplatin inhibits bone healing during distraction osteogenesis

Abstract

Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents. Many patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. Surgical reconstructions after tumor resection include structural allografts, non-cemented endoprostheses, and distraction osteogenesis (DO), which require direct bone formation. Although cisplatin (CDP) is extensively used for OS chemotherapy, the effects on bone regeneration are not well studied. The effects of CDP on direct bone formation in DO were compared using two dosing regimens and both C57BL/6 (B6) and tumor necrosis factor receptor 1 knockout (TNFR1KO) mice, as CDP toxicity is associated with elevated TNF levels. Detailed evaluation of the five-dose CDP regimen (2 mg/kg/day), demonstrated significant decreases in new bone formation in the DO gaps of CDP treated versus vehicle treated mice (p < 0.001). Further, no significant inhibitory effects from the five-dose CDP regimen were observed in TNFR1KO mice. The two-dose regimen significantly inhibited new bone formation in B6 mice. These results demonstrate that CDP has profound short term negative effects on the process of bone repair in DO. These data provide the mechanistic basis for modeling peri-operative chemotherapy doses and schedules and may provide new opportunities to identify molecules that spare normal cells from the inhibitory effects of CDP.

Keywords: chemotherapy; cisplatin; distraction osteogenesis; limb salvage; mouse.

Figure 1

Five day CDP Regimen 1: Left: Comparison of the distracted tibial radiographs. A significant decrease in the mineralized area of distraction gaps of CDP treated mice (3.6% ± 2.1) versus vehicle treated mice (63.3% ± 5.8) (p<0.001). Right: Histology of the DO gaps. A significant decrease in cellular parameters of bone formation between the CDP treated mice (7.6% ± 7.0) versus vehicle treated mice (75.4% ± 5.4) (p<0.001).

Figure 2

Representative radiographs (A) and H&E histology (B) for B6 mice in Regimen 1 are shown. In 2B new bone is outlined by dashes.

Figure 3

(A) Representative micro CT images of tibial DO gaps from vehicle and CDP-treated mice. (B) New mineralized endosteal bone formation is significantly inhibited by CDP (3% ± 3) vs. vehicle control (38% ± 10) (p<0.01) (C) New mineralized periosteal bone formation is significantly inhibited by CDP (8%±3) vs. vehicle control (50% ± 8) (p<0.01).

Figure 4

Five day CDP Regimen 1. Left: Comparison of the distracted tibial radiographs. No significant decrease in mineralized area of CDP treated (20.6% ± 8.7) vs. vehicle-treated TNFR1 KO mice (44.1% ± 7.5) (p=0.09). Right: Comparison of the distracted tibial H&E histology. No significant decrease in cellular bone formation of CDP treated mice (40.5% ± 12.7) versus vehicle treated mice (64.1% ± 4) (p=0.07).

Figure 5

Two day CDP Regimen 2. Left: Comparison of the distracted tibial radiographs. No significant decrease in mineralized area in the distraction gaps of CDP treated (15.5% ± 9.0) versus vehicle treated B6 mice (42.8% ± 12.67) (p= 0.1). Right: Comparison of the distracted tibial H&E histology. A significant decrease in the area of new bone formation in distraction gaps of CDP treated mice (26.1% ± 13.4) versus vehicle treated mice (67% ± 5.3) (p<0.02)..

Figure 6

(A) Representative microCT images of tibial DO gaps from vehicle and CDP-treated B6 mice in Regimen 2. (B) Total new mineralized endosteal bone formation is significantly inhibited by CDP (19% ± 3) vs. vehicle control (33% ± 7) (p<0.05).