临床及动物实验的文献报告表明, 低能量脉冲超声波 (LIPUS) 能促进骨折愈合。 可是, 不同研究小组针对LIPUS的功效所提供的数据结果往往并不一致。为了找出导致数据结果不一致的原因, 以及提升LIPUS的生物功效, 科研人员正致力于测定超声波在骨折治疗中的最佳信号参数。 在临床运用上, LIPUS对骨折的治疗一般是以经皮方式应用的。 故此, 不同层次深度的骨折会暴露在不同的超声波场区里。 超声波场有两个不同的区域, 就是近场区 (接近超声波换能器的区域) 及远场区 (远离超声波换能器的区域)。 在我们早期的临床研究中, 我们曾使用超声波的近场区治疗胫骨的复杂性骨折。 我们发现, 当超声波换能器置于胫骨骨折处的前方, 骨痂集中生成于胫骨骨折处的背面。 这研究结果显示, 近场区以外的超声波场或许更具促进骨痂形成的功效。 再者, 针对LIPUS声场仿真分析的结果显示, 近场区内的声压分布是不稳定的, 而远场区内的声压则远比近场区的均及稳定。由于声压的稳定性会大大影响超声波于组织内的能量透射, 我们相信在超声波场中, 骨折的深度会影响LIPUS的生物效应。 / 本研究采用大鼠闭合性股骨骨折模型及细胞培养实验, 探究不同的超声波场区对骨折愈合的影响。本研究作出了以下三个科研假设: (1) LIPUS 的远场区在促进骨折愈合的应用上有着更高的生物效应; (2) LIPUS 的远场区能透过促使骨细胞产生旁分泌调节因子, 从而提升成骨细胞的成骨活性; (3) 通过换能器直径的调制而产生的LIPUS远场区能有效地促进骨折愈合。 / 在第一部分的实验里, 股骨骨折的SD大鼠被随机分为对照组 (control), 近场区超声波治疗组 (near field; 伤肢跟换能器相距0mm), 中近场区超声波治疗组 (mid-near field; 伤肢跟换能器相距60mm), 远场区超声波治疗组 (far field; 伤肢跟换能器相距130mm)。在伤肢及超声波换能器之间安放了跟软组织具有同一超声波衰减系数的凝胶 (长度: 0mm, 60mm, 130mm)。LIPUS每天治疗20分钟, 每周治疗5天。 我们研究结果显示, 治疗后的第四周, 远场治疗组的骨痂组织具有各组中最高的相对骨体积及组织矿密度, 这造就远场治疗组相比对照组具有更强的力学属性。我们的研究结果表明, LIPUS的远场区治疗能通过提升骨痂的骨体积及骨矿化, 进一步促进骨折的愈合。 / 在第二部分的实验里, 我们把骨细胞株(MLO-Y4) 暴露在三種不同的超声波場中: 0 mm, 60 mm 及130 mm 。 经过不同的LIPUS处理后, 我们把条件培养基(CM) 收集, 并将其用于培养成骨前趋细胞株(MC3T3-E1)。 这部分的实验共有5组: Non组(非条件培养基处理组), Con组(骨细胞条件培养基处理组), 0mm组(条件培养基处理组; 条件培养基收集自经过LIPUS近场区刺激后的骨细胞), 60mm组(条件培养基处理组; LIPUS中近场区刺激后的骨细胞), 以及 130mm组(条件培养基处理组; LIPUS远场区刺激后的骨细胞)。我们测试了各超声波场对骨细胞的直接影响, 以及成骨前趋细胞经过各类骨细胞条件培养基培养后的成骨活性。 免疫染色的结果显示近场区以后的超声波场 (130mm 及 60mm) 能进一步诱导β-catenin 于骨细胞的入核作用。 另外, 远场区的骨细胞条件培养基 (130mm CM) 的处理促进了成骨前趋细胞的: (1) 细胞迁移的能力 (反映自细胞伤口愈合测试) ; (2) 细胞分化成熟的机制 (BrdU细胞增殖检验及ALP活性分析): (3) 基质钙化 (Alizarin red 钙化结节染色)。 / 在第三部分的实验里, 我们把换能器的直径缩减致一半, 以致换能器跟LIPUS远场区之间的距离从130 mm被拉近至30 mm。 当LIPUS以经皮的方式应用, 位于皮下大约40 mm的大鼠股骨骨折因此暴露在LIPUS的远场区。 相对于在换能器及伤肢之间安置130 mm凝胶, 以调制换能器直径而直接让骨折暴露于LIPUS远场区是更具临床应用性的方法。 在这部分, 股骨骨折的SD大鼠被随机分为对照组 (control), LT-Near30 (正常的换能器直径; 近场超声处理; I[subscript SATA] = 30 mW/cm²), ST-Far30 (缩减后的换能器直径; 远场超声处理; I[subscript SATA] = 30 mW/cm²), ST-Far150 (缩减后的换能器直径; 远场超声处理; I[subscript SATA] = 150 mW/cm²)。 研究结果证实, 以调制换能器直径而产生的远场LIPUS (ST-Far30)能透过提升骨痂的生成及力学属性, 进一步促进骨折愈合。 同时, 我们的结果显示, 相对高强度 (150 mW/cm²) 的远场LIPUS治疗不能更有效地促进骨折愈合。 / 综上所述, 动物及细胞培养实验结果证明, LIPUS的远场区在促进骨折愈合上更具功效。由于LIPUS的远场区放射稳定的超声波束, 骨痂中的骨细胞受引发释放可溶性因子, 从而进一步激发成骨样细胞的成骨活性。 这些细胞的生物效应造就LIPUS的远场区在促进骨折愈合上更具治疗效果。最后, 我们亦把以上的研究发现转化成具临床应用性的LIPUS应用方法。 这应用方法能让超声波换能器以紧贴皮肤的方式直接使骨折暴露于LIPUS远场区, 从而达成促进骨折愈合的功效。 / Low-intensity Pulsed Ultrasound (LIPUS) has been confirmed to enhance fracture healing in many clinical and animal studies. However, the evidences from literatures to support the applications of LIPUS on fracture healing were inconsistent. Therefore, scientists have been studying various ultrasound parameters aiming to find out the factors resulting in the inconsistent outcomes among research groups, and to further enhance the efficacy of LIPUS. Clinically, LIPUS is usually applied onto fracture sites transcutaneously, hence, fractures at different depths are exposed to different zones of ultrasound beam. There are two characteristic zones of ultrasound beam: the near field (close to the transducer) and far field (farther from the transducer). In our previous clinical study, direct transcutaneously applied LIPUS (near field LIPUS exposure) was used to treat human tibial complex fractures. We found that callus usually formed on the posterior side when the transducer was placed on the anterior side. This finding implied that ultrasound beam beyond near field bears higher potential in promoting callus formation. Moreover, beam mapping measurement of LIPUS shows a variable spatial pressure in near field; while a more uniform pressure profile was found beyond it (far field). As the stability of pressure profile influences the ultrasound energy transmission in tissue, we postulate that the biological effects of LIPUS are affected by the fracture depths within the ultrasound field. / This study aims to address the research 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 study, 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 study, 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 α-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 β-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 study, 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, I[subscript SATA] = 30 mW/cm²), ST-Far30 (small transducer, far field, I[subscript SATA] = 30 mW/cm²) and ST-Far150 (small transducer, far field, I[subscript SATA]=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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fung, Chak Hei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 186-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 中 文 摘 要 --- p.v / Publications --- p.ix / Acknowledgements --- p.xii / List of Abbreviations --- p.xiii / Index for Figures --- p.xvi / Index for Tables --- p.xviii / Chapter Chapter 1. --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Long Bone Fracture - A Growing Global Challenge --- p.2 / Chapter 1.2 --- Long-Bone Fracture - Current Management and Limitations --- p.5 / Chapter 1.3 --- Cellular Biology of Fracture Healing --- p.7 / Chapter 1.3.1 --- Stage 1: Inflammation --- p.7 / Chapter 1.3.2 --- Stage 2: Soft callus formation --- p.8 / Chapter 1.3.3 --- Stage 3: Hard callus formation --- p.9 / Chapter 1.3.4 --- Bone Remodeling --- p.10 / Chapter 1.4 --- Biophysical Stimulation to Bone --- p.13 / Chapter 1.5 --- Low-intensity Pulsed Ultrasound --- p.14 / Chapter 1.5.1 --- Application of LIPUS on Fracture Healing --- p.14 / Chapter 1.5.2 --- Physics of Ultrasound --- p.16 / Chapter 1.5.3 --- Ultrasound Parameters --- p.20 / Chapter 1.5.3.1 --- Ultrasound Frequency --- p.20 / Chapter 1.5.3.2 --- Duty Cycle --- p.22 / Chapter 1.5.3.3 --- Intensity --- p.22 / Chapter 1.5.3.4 --- Angle of Incidence --- p.24 / Chapter 1.5.3.5 --- Ultrasound Field --- p.25 / Chapter 1.5.4 --- Possible Mechanism of LIPUS on Tissue --- p.31 / Chapter 1.5.4.1 --- Thermal Effect --- p.31 / Chapter 1.5.4.2 --- Cavitation --- p.31 / Chapter 1.5.4.2 --- Acoustic Streaming --- p.32 / Chapter 1.5.4.3 --- Frequency Resonance Hypothesis --- p.32 / Chapter 1.5.4.4 --- Micromotion --- p.33 / Chapter 1.6 --- Possible Cellular and Molecular Mechanotransduction Mechanism of LIPUS --- p.34 / Chapter 1.6.1 --- Osteocyte: Potential Mechanosensor --- p.34 / Chapter 1.6.3 --- Osteocyte-osteoblast mechanotransduction --- p.39 / Chapter 1.7 --- Hypothesis --- p.39 / Chapter 1.8 --- Study Plan and Objectives --- p.40 / Chapter 1.8.1 --- Study Plan --- p.40 / Chapter 1.8.2 --- Objectives --- p.42 / Chapter Chapter 2. --- Characterization of Ultrasound Field Distances on Rat Fracture Model --- p.43 / Chapter 2.1 --- Introduction --- p.44 / Chapter 2.2 --- Material & Methods --- p.47 / Chapter 2.2.1 --- Closed Femoral Shaft Fracture Model in Rat --- p.47 / Chapter 2.2.2 --- Ultrasound Field Distances Setup --- p.51 / Chapter 2.2.3 --- Animal Grouping & LIPUS Treatment Protocol --- p.53 / Chapter 2.2.4 --- Assessments --- p.56 / Chapter 2.2.4.1 --- Radiological Analysis --- p.56 / Chapter 2.2.4.2 --- Micro-computed Tomography --- p.61 / Chapter 2.2.4.3 --- Histomorphometry --- p.64 / Chapter 2.2.4.4 --- Mechanical Testing --- p.66 / Chapter 2.2.4.5 --- Statistical Analysis --- p.66 / Chapter 2.3 --- Results --- p.68 / Chapter 2.3.1 --- Radiological Analysis --- p.71 / Chapter 2.3.2 --- MicroCT --- p.77 / Chapter 2.3.3 --- Histomorphometry --- p.82 / Chapter 2.3.4 --- Mechanical Testing --- p.85 / Chapter 2.4 --- Discussion --- p.87 / Chapter 2.4.1 --- Far Field LIPUS Enhances Mechanical Properties of Healing Callus --- p.88 / Chapter 2.4.2 --- Mid-near field and Near field LIPUS Enhances Woven Bone Formation --- p.90 / Chapter 2.4.3 --- The Biological Effects of LIPUS with Different Ultrasound Field Exposure --- p.94 / Chapter 2.5 --- Conclusion --- p.97 / Chapter Chapter 3. --- The Effect of Ultrasound Field Distances on Bone Cells --- p.100 / Chapter 3.1 --- Introduction --- p.101 / Chapter 3.2 --- Material & Methods --- p.102 / Chapter 3.2.1 --- Cell Culture --- p.102 / Chapter 3.2.2 --- Ultrasound Field Distances Setup & Treatment Protocol --- p.102 / Chapter 3.2.2 --- Immunostaining of β-catenin --- p.106 / Chapter 3.2.3 --- Wound Healing Assay --- p.109 / Chapter 3.2.4 --- BrdU Cell proliferation Assay --- p.111 / Chapter 3.2.5 --- Alkaline phosphatase activity assay --- p.112 / Chapter 3.2.6 --- Alizarin calcium nodule staining --- p.113 / Chapter 3.2.7 --- CM characterization - PGE₂ ELISA assay --- p.114 / Chapter 3.2.8 --- CM characterization - nitrite assay --- p.114 / Chapter 3.2.9 --- Statistical Analysis --- p.115 / Chapter 3.3 --- Results --- p.116 / Chapter 3.3.1 --- Immunostaining of β-catenin --- p.116 / Chapter 3.3.2 --- Wound healing assay --- p.119 / Chapter 3.3.3 --- BrdU Cell proliferation Assay --- p.119 / Chapter 3.3.4. --- Alkaline phosphatase activity assay --- p.122 / Chapter 3.3.5 --- Alizarin calcium nodule staining --- p.122 / Chapter 3.3.6. --- CM characterization - PGE₂ ELISA assay --- p.125 / Chapter 3.3.7 --- CM characterization - nitrite assay --- p.125 / Chapter 3.4 --- Discussion --- p.128 / Chapter 3.4.1 --- The Osteogenic Effect of Far Field LIPUS-Osteocyte Conditioned Medium --- p.128 / Chapter 3.4.2. --- Mechanisms of Mechanotransduction between Osteocyte and Osteoblast --- p.131 / Chapter 3.5 --- Conclusion --- p.136 / Chapter Chapter 4. --- Rat Fracture Exposed to Far Field LIPUS by Modulating Ultrasound Transducer Diameter --- p.139 / Chapter 4.1 --- Introduction --- p.140 / Chapter 4.2 --- Material & Methods --- p.143 / Chapter 4.2.1 --- Closed Femoral Shaft Fracture Model in Rat --- p.143 / Chapter 4.2.2 --- Ultrasound Field Distances Setup & Treatment Protocol --- p.143 / Chapter 4.2.3 --- Assessments --- p.148 / Chapter 4.2.3.1 --- Radiological Analysis --- p.148 / Chapter 4.2.3.2 --- Micro-computed Tomography --- p.148 / Chapter 4.2.3.3 --- Histomorphometry --- p.149 / Chapter 4.2.3.4 --- Mechanical Testing --- p.151 / Chapter 4.2.3.5 --- ex vivo Temperature Measurements --- p.151 / Chapter 4.2.3.6 --- Statistical Analysis --- p.151 / Chapter 4.3 --- Results --- p.152 / Chapter 4.3.1 --- Radiological Analysis --- p.154 / Chapter 4.3.2 --- MicroCT --- p.157 / Chapter 4.3.3 --- Histomorphometry --- p.160 / Chapter 4.3.4 --- Mechanical Testing --- p.166 / Chapter 4.3.5 --- ex vivo Temperature Measurement --- p.168 / Chapter 4.4 --- Discussion --- p.170 / Chapter 4.4.1 --- Far field LIPUS Setup by Transducer Diameter Modulation Enhanced Fracture Healing --- p.170 / Chapter 4.4.2 --- Fractures Exposed to Far Field LIPUS with Higher Intensity Did Not Heal Better --- p.174 / Chapter 4.4.3 --- Biphasic Effect of LIPUS Intensities on Fracture Healing --- p.176 / Chapter 4.5 --- Conclusion --- p.178 / Chapter Chapter 5. --- Conclusion --- p.179 / Chapter 5.1 --- Differential Biological Effects of Ultrasound Fields --- p.180 / Chapter 5.2 --- Far Field LIPUS exposure can be achieved by transducer diameter modulation --- p.181 / Chapter 5.3 --- Biphasic Effect of Ultrasound Intensities on Fracture Healing --- p.181 / Chapter 5.4 --- Mechanotransduction between Osteocyte and Osteoblastic cells --- p.182 / Chapter 5.5 --- Clinical Implications --- p.183 / Chapter 5.6 --- Future Investigations --- p.184 / Chapter 5.7 --- Limitations --- p.184 / Bibliography --- p.186 / Appendix --- p.208
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328186 |
| Date | January 2012 |
| Contributors | Fung, Chak Hei., Chinese University of Hong Kong Graduate School. Division of Orthopaedics & Traumatology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xxii, 208, [8] leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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