Irradiation induces bone injury by damaging bone marrow microenvironment for stem cells

Abstract

Radiation therapy can result in bone injury with the development of fractures and often can lead to delayed and nonunion of bone. There is no prevention or treatment for irradiation-induced bone injury. We irradiated the distal half of the mouse left femur to study the mechanism of irradiation-induced bone injury and found that no mesenchymal stem cells (MSCs) were detected in irradiated distal femora or nonirradiated proximal femora. The MSCs in the circulation doubled at 1 week and increased fourfold after 4 wk of irradiation. The number of MSCs in the proximal femur quickly recovered, but no recovery was observed in the distal femur. The levels of free radicals were increased threefold at 1 wk and remained at this high level for 4 wk in distal femora, whereas the levels were increased at 1 wk and returned to the basal level at 4 wk in nonirradiated proximal femur. Free radicals diffuse ipsilaterally to the proximal femur through bone medullary canal. The blood vessels in the distal femora were destroyed in angiographic images, but not in the proximal femora. The osteoclasts and osteoblasts were decreased in the distal femora after irradiation, but no changes were observed in the proximal femora. The total bone volumes were not affected in proximal and distal femora. Our data indicate that irradiation produces free radicals that adversely affect the survival of MSCs in both distal and proximal femora. Irradiation injury to the vasculatures and the microenvironment affect the niches for stem cells during the recovery period.

Fig. 1.

Local Irradiation on left distal femur. (A) SARRP system. (B) Radiograph of left lower limb of a mouse that received local irradiation of the distal femur. Light rectangle indicates the beam of X-ray. (C) Femora collected from the mice 1 wk after irradiation. (D) Light micrographs of H&E staining performed on trabecular bone sections from proximal and distal femora of mice 1 wk and 4 wk after irradiation. Yellow cross indicates left proximal femur, green cross indicates irradiated area of left distal femur, yellow triangle indicates right proximal femur, and green triangle indicates right distal femur. L, left. R, right. (Scale bar: 100 μm.)

Fig. 2.

Bone histomorphometric analysis of trabecular bone from the mice 1 wk and 4 wk after irradiation. Bone volume fraction (BV/TV) (A), trabecular thickness (Tb.Th) (B), trabecular number (Tb.N) (C), trabecular separation (Tb. Sp) (D), number of osteoblasts per bone perimeter (N.Ob⁺/B.Pm) (E), and number of osteoclasts per bone perimeter (N.Oc⁺/B.Pm) (F) were measured (LP, left proximal femur; LD, left distal femur; RP, right proximal femur; RD, right distal femur). Data represent the mean ± SEM; n = 10 (*P < 0.05).

Fig. 3.

Effects of local irradiation on potential of MSCs. (A) Colonies formed from harvested bone marrow of femora of the mice as indicated in CFU-F and CFU-Ob assays (1 × 10⁵ bone marrow nucleated cells were plated into six-well plates). (B and C) The colony-forming efficiency was determined by number of colonies per 10⁵ marrow cells plated. Data represent the mean ± SEM of triplicate cultures of bone marrow nucleated cells from five individual mice (*P < 0.05). (D) Colonies formed from harvested bone marrow of left tibia of the mice as indicated in CFU-F assay. (E and F) FACS analysis of the sorted Sca-1, CD29, CD45, and CD11b cells from the peripheral blood plasma (E) and the bone marrow cell suspension (F) obtained from the mice. Data represent the mean ± SEM; n = 5.

Fig. 4.

Effects of local irradiation on the production of free radicals and angiogenesis. Free radicals were assessed in both bone marrow (A) and peripheral blood (B) before irradiation and 1 wk and 4 wk after irradiation detected by TBARS assay. (C) Representative 3D micro-CT images of femora from the mice after local irradiation. (Scale bar: 1 mm.). (D–F) Quantitative micro-CT angiography analysis. Vascular volume fraction (VV/TV) (D), vascular thickness (V.Th) (E), and vascular number (V.N) (F) were measured. Data represent the mean ± SEM; n = 10 (*P < 0.05).