Direct bone formation during distraction osteogenesis does not require TNFalpha receptors and elevated serum TNFalpha fails to inhibit bone formation in TNFR1 deficient mice

Abstract

Distraction osteogenesis (DO) is a process which induces direct new bone formation as a result of mechanical distraction. Tumor necrosis factor-alpha (TNF) is a cytokine that can modulate osteoblastogenesis. The direct effects of TNF on direct bone formation in rodents are hypothetically mediated through TNF receptor 1 and/or 2 (TNFR1/2) signaling. We utilized a unique model of mouse DO to assess the effects of 1) TNFR homozygous null gene alterations on direct bone formation and 2) rmTNF on wild type (WT), TNFR1(-/-) (R1KO), and TNR2(-/-) (R2KO) mice. Radiological and histological analyses of direct bone formation in the distraction gaps demonstrated no significant differences between the WT, R1KO, R2KO, or TNFR1(-/-) and R2(-/-) (R1 and 2KO) mice. R1 and 2KO mice had elevated levels of serum TNF but demonstrated no inhibition of new bone formation. Systemic administration by osmotic pump of rmTNF during DO (10 microg/kg/day) resulted in significant inhibition of gap bone formation measures in WT and R2KO mice, but not in R1KO mice. We conclude that exogenous rmTNF and/or endogenous TNF act to inhibit new bone formation during DO by signaling primarily through TNFR1.

Figure 1

New gap bone formation after DO in rmTNF and vehicle treated mice was assessed by single beam radiography and histology. Comparison of the distracted tibial radiographs demonstrated an rmTNF–associated inhibition of in % new bone mineralization (TNF: 43.2% ± 5.6 vs control: 60.5% ± 3.7, p= 0.019). Further, analysis of histological sections supported the radiological analyses by revealing a significant decrease in new gap bone formation in rmTNF treated 41.7 ± 6.2% vs control 69.0 ± 3.0%, p< 0.001.

Figure 2

Representative H&E stained histological sections and radiographs of distracted tibial DO gaps from vehicle and rmTNF treated mice are shown. The area of new gap bone formation in the histological sections is roughly outlined with dashed lines for clarification. Notice the significant reduction in the % area of new bone formation in the rmTNF specimen in comparison to the vehicle control.

Figure 3

New gap bone formation after DO in 1) rmTNF treated versus vehicle treated TNFR1 KO mice and in 2) control mice was assessed by single beam radiography and histology. Comparison of the distracted tibial radiographs demonstrated the percent of mineralized new bone per gap was equivalent in the rmTNF treated, vehicle treated R1KO mice, and control mice (WT: 60.5% ± 6.0 vs R1KO/vehicle: 64.4% ± 8.9 vs R1KO/TNF: 54.2 ± 4.2, p=0.63). Comparison of the distracted tibial histology supported the radiographic results by demonstrating no significant differences between the three groups (WT: 58.6 ± 6.3 vs R1KO/vehicle: 71.3 ± 6.6%, vs R1KO/TNF: 61.6 ± 4.0%p=0.36).

Figure 4

Representative H&E stained histological sections and radiographs of distracted tibial DO gaps from control, vehicle treated, and rmTNF treated mice are shown. The area of new gap bone formation in the histological sections is roughly outlined with dashed lines for clarification. Notice the lack of significant changes in the % area of new bone formation among the three groups.

Figure 5

New gap bone formation after DO in WT, R2KO and R1&2 KO mice was assessed by single beam radiography and histology. Comparison of the distracted tibial radiographs demonstrated the percent of mineralized new bone per gap was equivalent in all the groups: WT, R2KO and R1&2 KO mice (WT: 53.6% ± 5.2 vs R2KO: 58.5% ± 7.0 vs R2&1KO 58.3 % ± 8.4, p=0.85). Comparison of the distracted tibial histology supported the radiographic results by demonstrating equivalent robust new gap bone formation in all groups (WT: 68.5% ± 4.6 vs R2KO: 63.5% ± 3.8, vs R2&1KO: 62.5 % ± 4.4, p=0.60).

Figure 6

Representative H&E stained histological sections and radiographs of distracted tibial DO gaps from WT, R2KO and R1&2 KO mice are shown. The area of new gap bone formation in the histological sections is roughly outlined with dashed lines for clarification. Notice the lack of significant changes in the % area of new bone formation among the three groups.

Figure 7

New gap bone formation after DO in TNF R2KO mice treated with rmTNF vs vehicle was assessed by single beam radiography and histology. Comparison of the distracted tibial radiographs demonstrated that the percent of mineralized new bone per gap was significantly decreased in the R2KO mice treated with rmTNF vs the vehicle controls (TNF: 23.3% ± 6.2 vs vehicle: 58.3% ± 5.0, p<.001). Comparison of the distracted tibial histology supported the radiographic results by demonstrating decreased new gap bone formation in the R2KO treated mice (TNF: 29.8% ± 6.3 vs vehicle: 66.9% ± 3.5, p<.001).

Figure 8

Representative H&E stained histological sections and radiographs of distracted tibial DO gaps from vehicle and rmTNF treated R2KO mice are shown. The area of new gap bone formation in the histological sections is roughly outlined with dashed lines for clarification. Notice the significant reduction in the % area of new bone formation in the rmTNF specimen in comparison to the vehicle control.