Effects of Ultrasound Field Distance of Low-intensity Pulsed Ultrasound (LIPUS) on Rat Fracture Healing and Osteocyte-osteoblast Paracrine Signalling.

摘要

This study aims to address the question of how ultrasound fields influence the fracture healing through testifying the following hypotheses in animal and cell culture studies: (1) Far field LIPUS bears higher biological effect in facilitating fracture healing; (2) Far field LIPUS could enhance the osteogenic activities of osteoblastic cells via paracrine factors secreted from osteocytes; (3) Far field LIPUS setup by transducer diameter modulation could facilitate fracture healing.;In part one, femoral fractured Sprague-Dawley (SD) rats were randomized into control, near-field (fractures placed at 0mm away from transducer), mid-near field (60mm away from transducer) or far-field (130mm away from transducer) groups. Rubber gel block (lengths: 0mm, 60mm and 130mm) with attenuation coefficient equivalent to soft tissue was interposed between the transducer and the fractured limb. LIPUS was given 20min/day and 5days/week. We found the callus in 130mm group was the highest in bone volume fraction and tissue mineral density at week 4. These advancements mutually contributed to its significantly stronger mechanical properties than the control group. Our results indicated that far field LIPUS could further facilitate fracture healing by promoting bone volume increase and callus mineralization, which led to enhanced mechanical properties.;In part two, LIPUS was applied to osteocyte cell line (MLO-Y4) at three distances: 0mm, 60mm and 130mm. The conditioned medium (CM) collected from different LIPUS treatment regimens were used to culture pre-osteoblast cell line (MC3T3-E1). There were 5 groups in the CM treatment: Non group (plain alpha-MEM treatment), Con group (osteocyte CM), 0mm group (Near field LIPUS treated osteocyte CM), 60mm group (Mid-near field LIPUS treated osteocyte CM) and 130mm group (Far field LIPUS treated osteocyte CM). The effect of ultrasound fields on osteocytes and the osteogenic activities of the pre-osteoblasts after different CM treatments were assessed. The immunostaining results indicated that beyond near field LIPUS (LIPUS at 130mm and 60mm) could further promote beta-catenin nuclear translocation in osteocytes. The far field LIPUS osteocyte-CM (130mm group) caused the highest biological effect on (1) pre-osteoblasts migration (reflected by wound healing assay); (2) maturation of pre-osteoblasts: transition of cell proliferation into osteogenic differentiation (BrdU cell proliferation assay and ALP activity assay); and (3) matrix calcification (Alizarin red calcium nodule staining).;In part three, the transducer diameter was reduced by half in order to draw the far field location closer to the transducer (i.e. from 130 mm to 30 mm). As the femoral shaft fractures of rats are located at around 40 mm beneath the skin, fractures were directly exposed to far field LIPUS transcutaneoulsy. It is a more clinically applicable approach than the method of physical separation (i.e. 130 mm separation between transducer and fractured limb). Femoral fractured SD rats were randomized into control, LT-Near30 (conventional transducer diameter, near field, ISATA = 30 mW/cm2), ST-Far30 (small transducer, far field, ISATA = 30 mW/cm2) and ST-Far150 (small transducer, far field, ISATA=150 mW/cm2). Our results confirmed that the far field LIPUS emitted from the transducer diameter reduction setup (ST-Far30) could further facilitate fracture healing process by enhancing callus formation and mechanical properties. Our findings also indicated that fractures exposed to far field LIPUS with relatively higher intensity (150 mW/cm2) did not heal better.;In summary, our in vivo and in vitro findings reinforce each other to confirm the positive effects of far field LIPUS on promoting fracture healing. As far field LIPUS radiates a stable ultrasound beam, osteocytes inside the callus are triggered to secrete soluble factors to promote the osteogenic activities of osteoblastic cells. This contributes to the higher therapeutic effects of far field LIPUS on fracture healing. We also translated these findings to establish a clinically applicable LIPUS device, which directly radiates far field LIPUS to subcutaneous fracture site without any distance separation needed. (Abstract shortened by UMI.).