The effect of low-intensity pulsed ultrasound on bone-tendon junction healing: Initiating after inflammation stage

Abstract

The purpose of this study was to explore the effect of low-intensity pulsed ultrasound (LIPUS) treatment initiating after inflammation stage on the process of bone-tendon junction (BTJ) healing in a rabbit model. Thirty-six rabbits undergoing partial patellectomy were randomly divided into two groups: control and LIPUS. The period of initial inflammatory stage is 2 weeks. So LIPUS treatment was initiated at postoperative week 2 and continued until the patella-patellar tendon (PPT) complexes were harvested at postoperative weeks 4, 8, and 16. At each time point, the PPT complexes were harvested for qRT-PCR, histology, radiographs, synchroton radiation micro computed tomography (SR-µCT), and biomechanical testing. The qRT-PCR results showed that LIPUS treatment beginning at postoperative week 2 played an anti-inflammatory role in BTJ healing. Histologically, the LIPUS group showed more advanced remodeling of the lamellar bone and marrow cavity than the control group. The area and length of the new bone in the LIPUS group were significantly greater than the control group at postoperative weeks 8 and 16. SR-µCT demonstrated that new bone formation and remodeling in the LIPUS group were more advanced than the control group. Biomechanical test results demonstrated that the failure load, ultimate strength and energy at failure were significantly higher than those of the control group. In conclusion, LIPUS treatment beginning at postoperative week 2 was able to accelerate bone formation during the bone-tendon junction healing process and significantly improved the healing quality of BTJ injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1697-1706, 2016.

Keywords: bone-tendon junction (BTJ); low-intensity pulsed ultrasound (LIPUS); synchroton radiation micro computed tomography (SR-µCT).

Figure 1

A diagram of the surgical procedure to establish the BTJ injury model in rabbits. (A) A transverse osteotomy was performed at the place of the proximal 2/3 and the distal 1/3 of the patella. (B) The distal 1/3 of the patella was removed (without remaining fibrocartilage), and then sutured the patellar tendon to the proximal patella. (C) A figure‐of‐eight tension band wire was fixed to protect the PPT reconstruction.

Figure 2

The mRNA expression of proinflammatory cytokines (A, B, C) and anti‐inflammatory cytokines (D, E) in control and LIPUS groups. (*p < 0.05 the difference between the control and LIPUS groups).

Figure 3

H&E‐stained sagittal sections from the patella‐patellar tendon at postoperative weeks 4, 8, and 16. New bone formation at the proximal patella was observed in both groups at postoperative weeks 8 and 16. The LIPUS group showed more trabecular and marrow cavities than the control group at postoperative week 16. The dotted line represents the surface of osteotomy (NB, newly formed bone; RP, remaining patella; TF, tendon fiber). Scanned bar = 1,000 µm.

Figure 4

The safranin O staining showed the cartilage‐like tissue formation (arrow) at bone‐tendon junction. Proteoglycan was stained red by safranin O in the regenerated zone of cartilage‐like tissue. With healing over time, more cartilage‐like tissue was found at bone‐tendon junction in both groups. Scanned bar = 1,000 µm.

Figure 5

Measure of the size of the newly formed bone. (A) Anteroposterior digital radiographs of the patella. The dotted line represents the surface of osteotomy, and below it shows the area of the newly formed bone. (B) Lateral digital radiographs of the patella. The line between two points represents the length of the newly formed bone.

Figure 6

Comparison of the area (A) and length (B) of the newly formed bone between control and LIPUS groups at different time points. (*p < 0.05 the difference between control and LIPUS groups; #p < 0.05 the difference among different time points for control and LIPUS groups).

Figure 7

3D tomographic reconstruction images of new trabecular bone located in the region of interest with high resolution. With the healing time increased, the new trabecular bone gradually became sparse because of marrow cavity formation. New bone formation and remodeling in the LIPUS group were more advanced than in the control group. Scanned bar = 1,000 µm.

Figure 8

The morphological parameters of new bone in both groups. (A: Tb.Th, trabecular thickness; B: Tb.N, trabecular number; C: Tb.Sp, trabecular separation; D: BV/TV, bone volume fraction) (*p < 0.05, the difference between control and LIPUS group at different time points; #p < 0.05, the difference between postoperative weeks 4 and 8 or weeks 8 and 16 within the same group).