Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

Abstract

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitmentin vitro In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cellsin vivo Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitmentin vivoand that the primary cilium contributes to this process.-Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism.

Keywords: bone; homing; mesenchymal stem cell; primary cilium; ulna loading.

Figure 1.

Generation of chimeric animals. A) Histologic analysis of donor engraftment. GFP positive (brown) donor cells were detected in the bone marrow. B) Fluorescence activated cell sorting representative analysis in which 95.4 ± 1.2% of nucleated blood cells were positive for GFP. C–E) Cells were isolated from bone marrow, and the plastic-adherent population was analyzed for GFP. Phase contrast (C), GFP (D), and merge images (E) are shown. Nearly all of the adherent cells were positive for GFP. F) DNA was extracted from isolated bone marrow cells. PCR amplification demonstrated that the floxed allele of Kif3a was amplified in the control animal with very faint amplification of wild-type Kif3a, indicating that the marrow stromal cell population was repopulated by transplantation. G) PCR amplification of marrow stromal cells isolated from tamoxifen-treated control animals demonstrated expression of the floxed allele of Kif3a, whereas experimental animals expressed the deleted Kif3a target, indicating successful deletion of Kif3a in the experimental group.

Figure 2.

Mechanical stimulation promotes osteogenic differentiation of bone-marrow–derived cells. A) Experimental timeline. B) Sixteen-week-old nonloaded control animal. Bone marrow–derived cells were detected only within the endocortical region (arrows). C) Control animal after mechanical loading. Bone marrow–derived cells were embedded within the endocortical and periosteal bone (arrows). D) Experimental animal after mechanical loading. Bone marrow–derived cells were also embedded in endocortical bone (X, arrow), but fewer were detected in the periosteal region (Y, arrow) than in the control animals illustrated in (C).

Figure 3.

Primary cilia of progenitor cells mediate mechanically induced bone formation. A) Transverse sections with fluorochrome labels marking new bone formation (green, calcein; red, alizarin). Bone formation measures were increased in loaded vs. nonloaded ulnae of control and experimental animals with primary cilia disrupted in bone marrow cells. B–D) Active mineralizing surface (MS/BS) (B), average interlabel width MAR (C), and BFR/BS (D). E–G) Relative measures (r) were obtained by subtracting nonloaded from loaded values: rMS/BS (E), rMAR (F), rBFR/BS (G). Disruption of primary cilia resulted in a diminished response to mechanical loading.