Quantitative expression of Indian hedgehog gene using real-time polymerase chain reaction

Abstract

AIM: Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) technology is an invaluable new tool to measure messenger RNA expression in vivo and in vitro to screen for tumour markers, diabetic glomerulosclerosis and other cytotoxicology tests. Indian hedgehog (Ihh) acts as a mechanotransduction mediator that converts mechanical signals resulting from anterior mandibular displacement to stimulate cellular proliferation on cartilage formation. In this study to help in understanding tissue responses to functional appliance therapy, quantitative RT-PCR measurements were carried out to: 1) investigate the temporal pattern of gene expression of Ihh and type II collagen in the condyle during mandibular advancement, and 2) quantify and compare Ihh and type II collagen mRNA expression during natural growth, single-step and stepwise advancement of mandible. MATERIALS AND METHOD: Sprague-Dawley rats were fitted with bite jumping appliances for either single advancement or stepwise advancement of the mandible. Animals of the experimental groups, together with their matched controls were sacrificed 3, 7, 14, 21, 30, 33, 37, 44, 51 and 60 days after appliance insertion. Total RNA was extracted from condylar cartilage immediately after dissection under a microscope. Ihh and type II collagen mRNA was quantified using RT-time PCR. RESULTS: In the single step group, Ihh mRNA expression reached a peak on experimental day 7 and gradually decreased to the control level. In the stepwise advancement group, Ihh mRNA significantly increased on day 7. However, the second advancement resulted in another peak on experimental day 37. CONCLUSIONS: Mandibular advancement triggers and enhances the expression of the Ihh gene, the mechanotransduction mediator that leads to more cartilage formation and acts as a template for new bone formation. Stepwise advancement of the mandible delivers mechanical stimuli that produce a series of tissue responses and lead to more chondrocyte proliferation, greater volume of cartilage and eventually more bone formation.