Primary cilium of Prrx1+ cells mediate bone-tendon interface development by regulating cell biology

Abstract

Abstract Background Bone-tendon interface (BTI) is a typical fibrocartilaginous insertion consisting of continuous yet functionally graded structure. However, the underlying mechanism of enthesis formation is poorly understood. It was reported that primary cilia sense changes in the microenvironment of the BTI undergoing mechanical stimulation to regulate repairing. The objective of this study is to investigate how primary cilia mediate the enthesis formation. Methods Mice at different ages were used for tracing the spatiotemporal distribution of Prrx1+ cells. Cilium were knocked out at 1, 2, 4, and 6 weeks after birth, and the specimens were harvested at 12 weeks to analyze the effect of cilia deletion on the fate of Prrx1+ cells. Histological analysis and microcomputed tomography were utilized to evaluate the formation of BTI. In vitro, Prrx1+ cells were isolated by cell sorting. The expression level of IFT88 protein was knocked down by shRNA, and it was applied to explore the effect of cilia disruption on the proliferation and differentiation of Prrx1+ cells. NVP-LDE225 was used to diminish the expression levels of the Hedgehog (Hh) signaling pathway, which was performed to understand the effect of Hh signaling pathway on the differentiation capacity of Prrx1+ cells. Results Prrx1+ cells participate in the formation of BTI, and in the early postnatal development, Prrx1+ cells are abundant at the BTI. In the late development, Prrx1+ cells gradually decreased in number until disappeared. Locally at the BTI, knockout of cilia may attenuate the chondrogenic differentiation capacity of Prrx1+ cells. Conditional knockout of cilia in the early development is not conducive to BTI formation, and few are in the middle and late stages. In vitro, the knockdown of IFT88 by shRNA could diminish the proliferation and differentiation ability of Prrx1+ cells. At the same time, inhibiting the Hh signaling pathway also diminish the differentiation ability of Prrx1+ cells. The mentioned results suggested that primary cilia may regulate the biological function of Prrx1+ cells via the Hh signaling pathway localized in itself. Conclusions Primary cilia mediate the biological function of Prrx1+ cells to modulate further BTI formation via the Hh signaling pathway.