INTRODUCTION: The process of ligamentization includes the histological and structural remodelling of the tendons graft to ligamentous tissue. There is little information documenting the mechanism of ligamentization process in molecular level. A number of essential genes are involved in this process and their expression can be regulated through complex biochemical pathways. Animal studies have shown that transcutaneous application of low intensity pulsed ultrasound (LiUS) accelerate the tendon and ligament healing process and recent reports have proven the efficacy of the transosseous application of LiUS for both enhancement and monitoring of the bone healing. The purpose of this study is to investigate the effect of transosseous low-intensity pulsed ultrasound (LiUS) during lingamentization process on the healing at tendon graft-bone interface in rabbits, by examining the expression levels of TGF-β1, biglycan and collagen I using semi-quantitive RT-PCR.
MATERIALS AND METHODS: Twenty-eight New Zealand rabbits were used in this study. The anterior cruciate ligament was excised and replaced with the long digital extensor. Custom-made ultrasound transducers were implanted onto the bone fragment and along the surface of the bone tunnel at the right knees of the rabbits (study group). The LiUS-treated animals received 200-μsec bursts of 1 MHz sine waves with pulse repetition rates of 1 KHz and average intensity of 30 mW/cm2, for 20 minutes daily, while the left knee received no LiUS (control group). Semi-quantitative RT-PCR was performed from RNA samples representing both study and control groups at 1, 2, 3, 5, 7, 8, 9, 12, 14 and 21 days, using specific primers.
RESULTS: Analysis of the RT-PCR products showed that there is significant up-regulation of biglycan and collagen-encoding genes in the study group compared to the control group. In addition, TGFb1-encoding gene exhibits a bimodal profile. In the study group, it represses its mRNA levels from day 1 until day 9 and then the initial expression levels are restored. The control group showed no essential alteration of expression levels for TGFb1.
DISCUSSION: Transosseous LiUS treatment affects the expression levels of significant genes like TGF-β1, big-lycan and collagen type I. All the above studied genes mediate important biochemical pathways in lingamentization process and possibly enhance the healing rate of the tendon graft-bone interface in a bone tunnel in rabbits. The present report is supportive of the hypothesis that transosseous application of LiUS enhances tendon graft healing to bone through effects on molecular level. These present findings suggest that indeed ultrasound treatment after joint ligament reconstruction may facilitate earlier rehabilitation.
