Indian hedgehog signaling is in response to the blunt trauma in articuar cartilage

Abstract

Purpose: Posttraumatic osteoarthritis (PTOA) represents end-stage organ level failure of an injured joint, especially in an intra-articular fracture. The severity of the PTOA is associated with the degree of the fracture. The initial impact injury to the cartilage, in the combination with secondary pathomechanical and pathobiologic response of the cartilage, is prone to result in the final PTOA. Inhibition of Indian Hedgehog (IHH) signaling, one of a main signaling regulating the chondrocytes hypertrophy, attenuates the severity of OA in OA cartilage explants in vitro, implying that the OA development might be involved in the expression of IHH signaling. How to prevent the development of PTOA after the occurrence of a fracture in early time seems to be essential. However, none of the previous studies had focused on the response of IHH signaling expression in articular cartilage in early stage after initial severe impact. Recently, bovine cartilage plugs were proved to express most common catabolic markers of osteoarthritic degeneration after impacted by our newly designed spring-loaded impact device. Therefore, with the help of this PTOA model, it is more meaningful and clinical relevant to test the IHH signaling response in the articular cartilage to the initial impact loading. Methods: Articular cartilage explants were isolated from the distal femoral condylar of the hind-leg stifle of 2- to 3-year-old bovine within 24 hours of slaughter. The punch protocol were followed our previous study. The blunt impact to the cartilage surface was applied using a custom-designed impact device to induce trauma to cartilage plugs as described in our recently work. Briefly, the spring-loaded impact device fitted with a hemispherical tip was used to deliver traumatic, injurious impacts of calibrated magnitude and rate into the cartilage piece, resulting in increased catabolic activities and tissue degeneration. The traumatized cartilage developed lesions similar to cartilage lesions seen in PTOA. Samples were randomly distributed into 3 different impact load magnitude groups those are 0, 17 and 36 MPa. (n=3) The samples were collected in Day 1, 4 and 7. Both gene and protein of IHH signaling were extracted and tested. The immunohistology was also performed to test the IHH signaling expression. Results: HE staining showed that the 36Mpa impact injury is a very severe trauma to the articular cartilage and an apparent reduction of matrix sulfated GAG in areas below the superficial zone of the articular cartilage by Safranin-O staining. From the PCR result, comparing with the control group of 0 Mpa, gene of IHH/HHIP/GLi-1/PTCH were increased on day 4, especially after 36 Mpa impact. The differences were significant, P<0.05. On the other hand, the IHH/Gli-1 protein was tested by western blot, which validate the PCR results. The differences were significant, P<0.05. The immunohistology results showed that on day4, the IHH protein expression was apparently increased after 34Mpa impact injury. Conclusions: We firstly proved that IHH signaling is sensitively in response to the mechanical blunt trauma on the articular cartilage. Early after the blunt trauma of 36Mpa, compared with 0 Mpa, the IHH signaling significantly increased on day 4. Our results can be utilized for the future study on the mechanism of PTOA development after the intra-articular fractures.