| Summary: | Hedgehog (Hh) signaling plays a fundamental role in the enthesis formation process and GLI-Kruppel family member GLI1 (<i>Gli1</i>) is a key downstream mediator. However, the role of <i>Gli1</i> in tendon–bone healing after anterior cruciate ligament reconstruction (ACLR) is unknown. To evaluate the tendon–bone healing after ACLR in <i>Gli1<sup>LacZ/LacZ</sup></i> (GLI1-NULL) mice, and compare <i>Gli1<sup>LacZ/WT</sup></i> (GLI1-HET) and <i>Gli1<sup>WT/WT</sup></i> wild type (WT) mice, a total of 45 mice, 15 mice each of GLI1-NULL, GLI1-HET and WT were used in this study. All mice underwent microsurgical ACLR at 12 weeks of age. Mice were euthanized at 4 weeks after surgery and were used for biomechanical testing, histological evaluation, and micro-CT analysis. The GLI1-NULL group had significantly lower biomechanical failure force, poorer histological healing, and lower BV/TV when compared with the WT and GLI1-HET groups. These significant differences were only observed at the femoral tunnel. Immunohistology staining showed positive expression of Indian hedgehog (IHH) and Patched 1(PTCH1) in all three groups, which indicated the activation of the Hh signal pathway. The GLI1 was negative in the GLI1-NULL group, validating the absence of GLI1 protein in these mice. These results proved that activation of the Hh signaling pathway occurs during ACL graft healing, and the function of <i>Gli1</i> was necessary for tendon–bone healing. Healing in the femoral tunnel is more obviously impaired by <i>Gli1</i> deficiency. Our findings provide further insight into the molecular mechanism of tendon–bone healing and suggest that <i>Gli1</i> might represent a novel therapeutic target to improve tendon–bone healing after ACLR.
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