Quick and Abnormal Ossification Results in No Growth Plate Formation and Skeletal Dysplasia in Postnatal Mice Induced by Conditional Knockout of Indian Hedgehog

Abstract

Objective: To observe the dynamic effect of conditional knockout of Indian hedgehog (Ihh) on ossification of postnatal growth plate, skeletal abnormalities and the related signaling factors in mice.Methods: P0 Col2a1-Cre ERT2; Ihhfl/fl mice were randomly divided into tamoxifen (TM) injection to delete Ihh and oil injection as control. Real-time PCR (RT-PCR) was used to detect Ihh knockout rate. X-ray and micro-CT were used to evaluate gross and knee joint morphology of 8-week-old mice. The hind knee joints were harvested at P2-P14 after the animals were euthanized to observe the dynamic evolution of the growth plate. Safranin-O and Von Kossa staining were performed to assess growth plate development and the mineralization of bone respectively. The costal cartilage cells of mice were cultured in vitro on postnatal day 6. Changes in the expression of parathyroid hormone-related protein (PTHrP) and bone morphogenetic protein 6 (BMP-6) were detected by immunohistochemistry and RT-PCR.Results: RT-PCR results confirmed that the knockout rate of Ihh was 76.83%. X-ray and micro-CT scans showed that the short-limb deformity of the experimental group was associated with abnormal development of the epiphysis. Saffron O staining showed a disorder of cell arrange in the growth-plate area of Ihhd/d mice; Von Kossa staining showed an early and premature growth-plate ossification that prevents the growth plate formation in Ihhd/d mice starting at P8. Immunohistochemistry and RT-PCR showed significantly decreased PTHrP expression (P<0.05) and significantly increased BMP-6 expression (P<0.05). Conclusion: Postnatal conditional knockout of Ihh inhibited the expression of PTHrP and promotes the expression of BMP-6, which leads to early and abnormal ossification of the growth plate, no growth plate formation, and skeletal dysplasia in mice.