Loss of intraflagellar transport 140 in osteoblasts cripples bone fracture healing

Abstract

The indispensability of primary cilia in skeletal development has been widely recognized. We have previously shown that intraflagellar transport 140 (IFT140), a protein component of a bidirectional intraflagellar transport system required for ciliary function, controls bone development and dentinogenesis. However, it remains unknown whether IFT140 functionally contributes to bone fracture rehabilitation. Here an osteotomy-induced femoral fracture model was generated in Ift140-transgenic (Ift140-TG) and osteoblast-specific Ift140-conditional knockout (Ift140-cKO) mice. Micro-computed tomography, osteogenic induction, qualitative polymerase chain reaction, and toluidine blue and safranin O/fast green staining assays were used to characterize the dynamics of bone fracture healing from various perspectives. We found that IFT140 was relatively enriched in the bone callus and decreased in fracture-susceptible aged, or diabetic bones. Ift140-cKO mice had impaired osteogenic differentiation from bone mesenchymal stem cells, lower bone mass, and delayed fracture closure, whereas Ift140-TG mice had promising healing outcomes. Overall, our findings demonstrated for the first time that IFT140 has a beneficial role in fracture repair. Future investigation of the primary cilium in the context of aging and osteoporosis would certainly benefit patients at high risk of bone fractures.

Keywords: Bone Mesenchymal Stem Cells; Bone fracture; Intraflagellar Transport 140; Osteoblasts; Primary Cilia.

Graphical abstract

Fig. 1

Spatial-temporal IFT140 expression post-bone fracture healing. (a) Relative mRNA level of common ciliary protein in the bone callus at different times post-fracture (n = 6); (b) IFT140 immunofluorescence signal location at 1, 2, and 3 weeks post-fracture; IF-stained image magnified from the boxed area of the H&E result; the bone contour is depicted as a dotted line (scale bar, H&E 500 μm, IF 100 μm); (c-d) H&E, immunofluorescence, and quantification of changes in IFT140 expression in the femur of young and aged mice (scale bar, H&E 500 μm, c1–c4 100 μm, n = 6). (e) Ift140 expression in the femur of WT and types of diabetic mouse models (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Fig. 2

Defective bone fracture healing in Ift140 cKO mice. (a) Micro-CT and radiographic analysis of the femurs in cKO and control mice at 1–4 weeks post-fracture (scale bar 1 mm, n = 6). (b) Parameters of the bone callus from micro-CT scanning at 3 weeks in cKO and control mice (n = 6, * p < 0.05, ns, not significant).

Fig. 3

Direct histological evidence of delayed bone fracture healing. (a) HE, (b) Toluidine blue, and (c) Safranin O/fast green staining of the femurs of cKO and control mice at 1, 2, and 3 weeks. The bone contour is depicted as a dotted line, noting the almost healed fracture line in the control group at 3 weeks (scale bar of 500 μm).

Fig. 4

Improved fracture healing in Ift140-TG mice. (a) Micro-CT and radiographic analysis on the femurs in Ift140-TG and WT mice at 1–4 weeks post-fracture (scale bar 1 mm, n = 6). (b) Parameters of the bone callus from micro-CT scanning at 3w after bone fracture in Ift140-TG and WT mice (n = 6, * p < 0.05, ** p < 0.01, ns, not significant). (c) H&E and toluidine blue staining of the femurs of Ift140-TG and WT mice 3 weeks after bone fracture. The bone contour is noted as a dotted line, scale bar of 500 μm.

Fig. 5

Reduced osteogenic and chondrogenic capacity in Ift140-cKO mice. (a) mRNA expression level of chondrogenic genes in BMSCs from control and cKO mice (n = 3, * p < 0.05, **p < 0.01, ns, not significant). (b) mRNA expression level of chondrogenic genes in the bone callus at 1-week post-fracture from control and cKO mice (n = 6, * p < 0.05, ** p < 0.01, ns, not significant). (c) mRNA expression level of osteogenic genes in BMSCs from control and cKO mice (n = 3, * p < 0.05, *** p < 0.001). (d). mRNA expression level of osteogenic genes in the bone callus at 3 weeks post-fracture from control and cKO mice (n = 6, * p < 0.05, ** p < 0.01).