Effect of Low-Intensity Pulsed Ultrasound on the Graft-Bone Healing of Artificial Ligaments: An In Vitro and In Vivo Study

Background: As many researchers have focused on promoting the graft-bone healing of artificial ligaments, even with numerous chemical coatings, identifying a biosafe, effective, and immediately usable method is still important clinically. Purpose: (1) To determine whether a low-intensity pulsed ultrasound system (LIPUS) promotes in vitro cell viability and osteogenic differentiation and (2) to assess the applicability and effectiveness of LIPUS in promoting the graft-bone healing of artificial ligaments in vivo. Study Design: Controlled laboratory study. Methods: Polyethylene terephthalate (PET) sheets and grafts were randomly assigned to control and LIPUS groups. MC3T3-E1 preosteoblasts were cultured on PET sheets. Cell viability and morphology were evaluated using a live/dead viability assay and scanning electron microscopy. Alkaline phosphatase activity, calcium nodule formation, and Western blot were evaluated for osteogenic differentiation. For in vivo experiments, the effect of LIPUS was evaluated via an extra-articular graft-bone healing model in 48 rabbits: the osteointegration and new bone formation were tested by micro–computed tomography and histological staining, and the graft-bone bonding was tested by biomechanical testing. Results: Cell viability was significantly higher in the LIPUS group as compared with control (living and dead compared between control and LIPUS groups, P = .0489 vs P = .0489). Better adherence of cells and greater development of extracellular matrix were observed in the LIPUS group. Furthermore, LIPUS promoted alkaline phosphatase activity, calcium nodule formation, and the protein expression of collagen 1 ( P = .0002) and osteocalcin ( P = .0006) in vitro. Micro–computed tomography revealed higher surrounding bone mass at 4 weeks and newly formed bone mass at 8 weeks in the LIPUS group ( P = .0014 and P = .0018). Histological analysis showed a narrower interface and direct graft-bone contact in the LIPUS group; the surrounding bone area at 4 weeks and the mass of newly formed bone at 4 and 8 weeks in the LIPUS group were also significantly higher as compared with control (surrounding bone, P < .0001; newly formed bone, P = .0016 at 4 weeks and P = .005 at 8 weeks). The ultimate failure load in the LIPUS group was significantly higher than in the control group ( P < .0001 at 4 weeks; P = .0008 at 8 weeks). Conclusion: LIPUS promoted the viability and osteogenic differentiation of MC3T3-E1 preosteoblasts in vitro and enhanced the graft-bone healing of PET artificial ligament in vivo. Clinical Relevance: LIPUS is an effective physical stimulation to enhance graft-bone healing after artificial ligament implantation.