Limited field radiation therapy results in decreased bone fracture toughness in a murine model

Abstract

Fragility fractures are a well-known complication following oncologic radiotherapy, and it is suspected that radiation-induced embrittlement of bone within the treatment field may contribute to fracture risk. To explore this phenomenon, a mouse model (BALB/cJ) of fractionated, limited field, bilateral hindlimb irradiation (4x5 Gy) was used. The effects of radiation on femoral (cortical) bone fracture toughness, morphology, and biochemistry—including advanced glycation end products (AGEs)—were quantified and compared to Sham group samples prior to irradiation and at 0, 4, 8, and 12 weeks post-irradiation. Additionally, alterations to bone fracture toughness mediated directly by radiation (independent of cellular mechanisms) were determined using devitalized mouse cadaver femurs. Finally, the contribution of AGEs to reduced fracture toughness was examined by artificially ribosylating mouse femurs ex vivo. These data demonstrate that in vivo irradiation results in an immediate (-42% at 0 weeks, p < 0.001) and sustained (-28% at 12 weeks, p < 0.001) decrease in fracture toughness with small changes in morphology (-5% in cortical area at 12 weeks), and minimal changes in bone composition (tissue mineral density, mineral:matrix ratio, and AGE content). Irradiation of devitalized femurs also reduced fracture toughness (-29%, p < 0.001), but to a lesser extent than was seen in vivo. While artificial ribosylation decreased fracture toughness with time, the extent of glycation needed to induce this effect exceeded the AGE accumulation that occurred in vivo. Overall, hindlimb irradiation induced a substantial and sustained decrease in bone fracture toughness. Approximately half of this decrease in fracture toughness is due to direct radiation damage, independent of cellular remodeling. Collagen glycation in vivo was not substantially altered, suggesting other matrix changes may contribute to post-radiotherapy bone embrittlement.

Altered mechanical behavior of demineralized bone following therapeutic radiation

“…There was no apparent collagen fragmentation in tibiae from the nonirradiated control group or the groups receiving therapeutic doses of radiation (0 and 12 weeks post-RTx, and 20 Gy ex vivo; Figure 6). F I G U R E 7 Instability fracture toughness of mineralized cortical bone (as we previously reported) 11 increased with greater demineralized bone energy to failure. Mean and SE of mean shown with all-time points grouped together results in loss of bone strength, 24,25,35 diminished fracture toughness, 35,36 and fragmentation of bone collagen.…”

“…Using a mouse model of unilateral hindlimb limited field radiotherapy, we have previously demonstrated that therapeutic radiation results in loss of femoral metaphyseal trabecular structures (decreased trabecular bone volume fraction, decreased trabecular number, decreased connectivity density, and increased trabecular spacing) subsequent to increased osteoclastic activity, diminished bone strength (axial compression testing of distal femur, femoral diaphyseal bending strength), and decreased femoral diaphyseal fracture toughness without loss of bone density. [11][12][13][14] Work in other labs has supported these findings. [15][16][17] Furthermore, we have shown that the bone matrix (organic and collagen phases) becomes increasingly aligned post-RTx, accompanied by an increase in trivalent:divalent collagen crosslink ratio as measured by Raman spectroscopy, with only small transient changes in pathologic crosslinks and adducts such as advanced glycation end products (AGEs).…”

“…30 We previously demonstrated that therapeutic radiation in vivo results in an immediate and sustained loss of resistance to crack propagation (a measure of bone embrittlement) in cortical bone. 11 Our complementary biochemical data suggests that this may be due to changes in the organic phase of bone matrix (increased collagen alignment and maturity via Raman spectroscopy) rather than loss of bone mineral density. However, the ways in which these biochemical changes affect the biomechanics of bone collagen are not known.…”

“…37 Postirradiation, AGEs do not accumulate at concentrations sufficient to modulate mechanical properties of bone 37 for either kGy 24,25 or therapeutic radiation doses. 11,19 While neither AGEs nor collagen fragmentation appear to play a significant role in post-radiotherapy bone embrittlement, from the literature on bone matrix-mechanics interactions we can identify several potential factors that may be worthy of future investigation.…”

“…[21][22][23] However, in the context of RTx, our data have not supported this hypothesis. 11,19 Pendleton et al 24 similarly found that AGEs do not measurably accumulate in irradiated allograft bone until extremely high doses (≥50 Gy) are reached, while Burton et al 25 found no AGE formation at 33 kGy. Data from allograft sterilization studies indicate that collagen fragmentation induced by kGy irradiation contributes to decreased fracture toughness, loss of toughening mechanisms including ligament bridging, and overall loss of bone strength.…”