Biological effects of LIPUS
LIPUS的生物学效应
Passing through the tissue, the ultrasonic energy is absorbed at a rate proportional to the density of the tissue. Thus, the radical changes in density inherent in a healing tissue may well establish the gradients of mechanical strain.13 Absorption of the ultrasound signal also results in energy conversion to heat.14 While this heating effect is extremely small for low frequency ultrasonic waves, well below 18C, some enzymes, such as matrix metalloproteinase-1, or collagenase, are exquisitely sensitive to small variations in temperature.15 Therefore, ultrasound may serve to re-establish or normalize effective metabolic temperatures in tissue-healing regions; this effect, although subtle, may be biologically profound.16 Furthermore, at interfaces of distinct densities, much of the incident radiation energy will be reflected, resulting in complex gradients of acoustic pressure through the tissue.17
穿过组织，超声能量以与组织密度成比例的速率吸收。因此， 愈合组织固有的密度的根本变化可能很好地建立了机械应变 的梯度。13 超声信号的吸收也会导致能量转换为热量。14 尽管 这种加热效果对于低频而言非常小在温度低于 18°C 的超声 波中，某些酶（例如基质金属蛋白酶-1 或胶原酶）对温度的 细微变化非常敏感。15 因此，超声可用于在组织修复中重新建 立有效的代谢温度或使其正常化。这种作用虽然微妙， 但在生物学上可能很深远。16 此外，在不同密度的界面处，许 多入射辐射能量将被反射，从而导致穿过组织的声压梯度复 杂。17
Having removed the high thermal component of the higher intensities while maintaining the transmission of acoustic mechanical stress to the target tissue, LIPUS stimulates the union of non-uniting fractures, promotes the healing in a congenital pseudoarthrosis of the tibia, increase the matrix synthesis in chondrocytes, stimulates the differentiation in osteoblast precursor cells and induces the expression of growth factors, proteins and prostaglandins within osteoblasts, chondrocytes and fibroblasts.1,8 – 27 Furthermore, cartilage healing assisted with LIPUS produces fewer degenerative changes in the long term, as compared with controls.28
LIPUS 去除了较高强度的高热量成分，同时保持了声机械应 力向目标组织的传递，从而刺激了非结合性骨折的愈合，促 进了先天性胫骨假性关节炎的愈合，增加了软骨细胞的基质 合成刺激成骨细胞前体细胞的分化，并诱导成骨细胞，软骨 细胞和成纤维细胞内的生长因子，蛋白质和前列腺素的表达。 1,8 – 27 此外，与对照组相比，LIPUS 辅助的软骨愈合在长期内 产生的退行性变化更少。 28
In this review, the role of LIPUS in a soft-tissue healing will be discussed at cellular level first, giving a background of laboratory-based evidence before dwelling into various animal and human studies focusing on different soft tissues individually.
在这篇综述中，LIPUS 在软组织愈合中的作用将首先在细胞 水平上进行讨论，为基于实验室的证据提供背景知识，然后 再进行各种针对不同软组织的动物和人体研究。
Effect of LIPUS on fibroblasts, myoblasts, epithelial cells and chondrocytes
LIPUS 对成纤维细胞，成肌细胞，上皮细胞和软骨细胞的影响
In 2004, Zhou et al.29 investigated the effects of daily exposure to LIPUS at an intensity of 0.03 W/cm2 on primary human foreskin fibroblasts.LIPUS activates RhoA (a GTPase protein) which induces the formation of F-actin stress fibres which in turn cause the recruitment of paxillin, a signal transduction adaptor protein—by a Rho-associated coiled— coil-containing protein kinase (ROCK)-dependent pathway. Furthermore, LIPUS exposure promoted a 2-fold increase in extracellular signalrelated kinase (ERK) 1/2 activation, triggering DNA synthesis, and thus, cell proliferation. RhoA/ROCK is an upstream regulator of the LIPUS-induced ERK pathway. Similarly, LIPUS triggered Src, a tyrosine kinase, which further regulates the ERK cascade. ERK pathways are typically activated by mechanical stress, although, in this case, LIPUS activated a pathway, with no evidence of epidermal growth factor receptor (EGFR) involvement (EGFR is a tyrosine kinase, usually expressed in ERK pathways, triggered by direct mechanical stress). Thus, LIPUS produced results similar to mechanical stress at cellular level.18
2004 年，Zhou 等人 29研究了每日暴露于 0.03 W / cm2强度的 LIPUS 对原代人包皮成纤维细胞的影响。LIPUS 激活 RhoA（一种 GTP 酶蛋白），该蛋白诱导 F-肌动蛋白应激纤维的形成，进而引起桩蛋白的募集，这是一种信号转导衔接蛋白，通过与 Rho 相关的盘绕——含蛋白激酶 （ROCK）依赖性途径。此外，LIPUS 暴露可促进细胞外信号相关激酶（ERK）1/2 激活增加 2 倍，触发 DNA 合成，从而促进细胞增殖。RhoA / ROCK 是 LIPUS 诱导的 ERK 途径的上游调节剂。同样，LIPUS 触发了Src，一种酪氨酸激酶，进一步调节 ERK 级联反应。ERK 通路通常由机械应力激活，尽管在这种情况下，LIPUS 激活了一个通路，没有证据表明表皮生长因子受体（EGFR）参与（EGFR 是一种酪氨酸激酶，通常在 ERK 通路中表达，由直接机械应力触发）。因此，LIPUS 产生的结果类似于细胞水平的机械应力。18
LIPUS involvement in the ERK 1/2 system was also demonstrated by in a mice model. Here, the differentiation pathway of C2C12 (a subclone of C2 myoblasts from a C311 mouse) to the lineage of the osteoblast and chondroblast was observed.25
LIPUS 参与ERK 1/2 系统也在小鼠模型中得到证实。在这里，观察到了 C2C12（来自 C311 小鼠的 C2 成肌细胞的亚克隆）向成骨细胞和软骨细胞谱系的分化途径。25
The effects of LIPUS appear to not only be limited to the ERK pathways. Ikai et al. noted a locally increased expression of heat shock protein-70 (hsp-70) in LIPUS-exposed canine gingival epithelium. The heat shock protein family is associated with the healing process and, more specifically, with the assembly of molecules utilized for endothelial cell migration and proliferation (Table 1).30 – 32
LIPUS 的作用似乎不仅限于 ERK 途径。Ikai 等人注意到在暴露于 LIPUS 的犬齿龈上皮中热休克蛋白 70（hsp-70）的局部表达增加。热休克蛋白家族与愈合过程有关，更具体地说，与内皮细胞迁移和增殖所用分子的组装有关（表 1）。30 – 32
Inconsistent effects have been observed in various in vitro studies regarding the exact role of LIPUS in cartilage healing (Table 1). LIPUS stimulates the aggrecan m-RNA expression and proteoglycan synthesis, but it did not influence cell proliferation of rat chondrocytes.33 However, LIPUS influences the cell proliferation in an intensity-dependent manner but did not increase the expression or synthesis of aggrecan and type II collagen of chick embryo chondrocytes.34 In an in vitro atelocollagen gel-embedded culture model, LIPUS exposure promotes the synthesis of chondroitin sulfate, although it does not significantly enhance the cell number of rabbit chondrocytes.35 From these studies, it can be concluded that exposure to LIPUS could significantly affect chondrocytes proliferation, phenotype expression and matrix production. In consistencies in these studies may be attributable to many factors, including culture media, culture models or cell senescence.
关于 LIPUS 在软骨愈合中的确切作用，在各种体外研究中观察到不一致的效果（表 1）。LIPUS 刺激聚集蛋白聚糖 m-RNA 的表达和蛋白多糖的合成，但不影响大鼠软骨细胞的细胞增殖。33 但是，LIPUS 会以强度依赖性的方式影响细胞增殖，但不会增加聚集蛋白聚糖和II型胶原蛋白的表达或合成。 34 在体外经胶原蛋白凝胶包埋的培养模型中， LIPUS 暴露可促进硫酸软骨素的合成，尽管它并不能显着增加兔软骨细胞的细胞数量。35 从这些研究中，可以得出结论，暴露于 LIPUS 会显着影响软骨细胞的增殖，表型表达和基质产生。这些研究的一致性可能归因于许多因素，包括培养基，培养模型或细胞衰老。
Effects of LIPUS on soft tissues
LIPUS 对软组织的影响
(1) Inter-vertebral discs (IVDs): Results following the exposure of IVDs to LIPUS appear to feature a distinct LIPUS-activated pathway.
(1) 椎间盘（IVD）：IVD 暴露于 LIPUS 后的结果似乎表现出独特的 LIPUS 激活通路。
In nucleus pulposus (NP) cells, Hiyama et al. reported an increased expression of transforming growth factor-beta type 1 (TGF-BR1) receptor gene on exposure to LIPUS. Transforming growth factor-beta 1 (TGF-B1) promotes IVD cell proliferation and matrix synthesis, as well as playing a part in the regulation of chondrocytes.31 This is attributed to the structural similarities between chondrocytes and the NP and annulous fibrosus cells of the IVDs.36 Exposure of these cells to recombinant TGF-B1 mimicked the effects produced by LIPUS, whereas the exposure of LIPUS-treated cells to anti-TGF-B1 would reverse all the beneficial effects induced by LIPUS.31 Also, a marked increase in proteoglycan synthesis within the NP cells encouraging the uptake of water was observed, resulting in the potential regression of disc herniation.
在髓核（NP）细胞中。Hiyama等人报道了暴露于 LIPUS 后转化生长因子β1 型（TGF-BR1）受体基因表达增加。转化生长因子β1（TGF-B1）促进 IVD 细胞增殖和基质合成，并参与软骨细胞的调节。31 这归因于软骨细胞与 NP 和环状纤维细胞之间的结构相似性。 36 这些细胞暴露于重组 TGF-B1 可以模仿 LIPUS 产生的作用，而将 LIPUS 处理的细胞暴露于抗 TGF-B1 则可以逆转 LIPUS 诱导的所有有益作用。31 另外，观察到 NP 细胞内蛋白多糖合成的显着增加，促进了水的吸收，导致椎间盘突出症的潜在消退。
Also, investigating the effect of LIPUS on NP cells, Omi et al.37 noted not only a 2.06-fold up-regulation of tissue inhibitor of metallo-proteinase-1, but also a 2.3-fold up-regulation in local macrophage monocyte chemotaxis protein-1 compared with controls. Similar findings have been presented by other studies,38 suggesting that LIPUS-induced macrophage migration may play an important role in IVD remodelling and possibly IVD hernia regression, proposing that macrophages could enter IVDs following the increased capillary blood flow and cell-membrane permeability caused by LIPUS stimulation of fine vibrations in local tissue.37 Studies by Miyamoto et al.39 demonstrated increased collagen synthesis by cells isolated from the intervertebral disc by the application of LIPUS (16 –19% increase, P , 0.05–0.0001), suggesting that LIPUS may prove useful for tissue engineering of intervertebral disc tissue in future (Table 1).
此外，Omi 等人在研究 LIPUS 对 NP 细胞的作用时，不仅注意到金属蛋白酶-1 的组织抑制剂上调了 2.06 倍，而且在局部巨噬细胞也上调了 2.3 倍。与对照组相比，单核细胞趋化蛋白白-1。相似的其他研究也提出了类似的发现，暗示 LIPUS 诱导的巨噬细胞迁移可能在 IVD 重塑和可能的 IVD 疝消退中起重要作用，这表明巨噬细胞可以在LIPUS刺激局部组织中的精细振动一起的毛细血管血流量和细胞膜通透性增加后进入 IVD。 Miyamoto 等人 的研究表明，使用 LIPUS 可以从椎间盘分离的细胞中增加胶原蛋白的合成 （增加 16 – 19％，P，0.05 – 0.0001），表明 LIPUS 可能在将来对椎间盘组织的组织工程化有用（表 1）。
(2) Ligaments: Several studies investigated the effect of LIPUS in comparison to non-steroidal anti-inflammatory drugs (NSAIDs) in relation to ligamentous healing. They concluded that LIPUS accelerated but did not improve ligament healing, whereas NSAIDs delayed but did not impair healing. When used in combination, the beneficial LIPUS effect was not impaired by the detrimental NSAID effect,suggesting that LIPUS does not influence the cyclo-oxygenase pathway which is normally inhibited by NSAIDs.40,41 Other studies found LIPUS effective in accelerating rat medial collateral ligament injury healing at an intensity of 0.03 W/cm2. 42,43 (Table 2).
(2) 韧 带：数项研究调查了 LIPUS 与非甾体类抗炎药 （NSAIDs）在韧带愈合方面的效果。他们得出结论，LIPUS 可以加速但不会改善韧带的愈合，而 NSAID 可以延迟但不会损害愈合。联合使用时，有害的 LIPUS 效果不会受到有害的 NSAID 效果的损害，这表明 LIPUS 不会影响通常被 NSAID 抑制的环加氧酶途径。40,41 其他研究发现 LIPUS 可有效促进大鼠内侧副韧带强度为 0.03 W / cm 的伤口愈合 2。42,43（表 2）。
(3) Tendons: Therapeutic ultrasound has been found to have no effect over and above placebo on tendon healing.44 Research focused on the role of LIPUS on tendon healing use BTJ as the main area of investigation. A closer look into the finding of these studies, however,reveals that LIPUS improves BTJ healing mainly by enhancing bony in-growth and osteo-integration.44 – 48 The study by Warden et al.49 demonstrates no benefit of LIPUS therapy over and above placebo in the management of symptoms associated with patellar tendinopathy.Thus, LIPUS does not seem to have direct effect on healing of tendon tissue itself.
(3) 肌腱：已发现超声治疗对肌腱愈合的作用没有超过安慰剂的影响。44 研究重点是使用 BTJ 作为主要研究领域，研究 LIPUS 在肌腱愈合中的作用。然而，仔细研究这些研究发现，发现 LIPUS 主要通过增强骨向内生长和骨整合来改善 BTJ 愈合。Warden 等人的研究表明，LIPUS 疗法没有优于 BIP 及以上安慰剂骨肌腱病相关症状的管理。因此，LIPUS 似乎对腱组织本身的愈合没有直接影响。
The mechanism of action pertaining LIPUS at the BTJ revolves around the acoustic mechanical stimulation of osteoblasts and fibrocytes, acoustic vibrational aligning of collagen fibres, promoting local influx of calcium and the increased expression of angiogenic factors [i.e. vascular endothelial growth factor (VEGF)].45 – 47 The increase in VEGF expression of 7% from LIPUS-treated rabbit patellar-tendon samples over control samples (P , 0.05) suggests that a reason for the acceleration in BTJ healing was increased early vascularity and osteoblastic differentiation45 (Table 2).
LIPUS 在 BTJ 的作用机制围绕成骨细胞和纤维细胞的声学机械刺激，胶原纤维的声学振动排列，促进钙的局部流入以及血管生成因子[即血管内皮生长因子（VEGF）]的表达增加。 45 – 47 与对照组相比，LIPUS 处理的兔髌腱样品的 VEGF 表达增加了 7％（P，0.05），这表明 BTJ 愈合加速的原因是早期血管形成和成骨细胞分化（表 2）。
(4) Cartilage: In vivo studies50,51 in New Zealand rabbit and canine models with full-thickness osteochondral defects have demonstrated that exposure to LIPUS significantly improves the morphologic features and histologic characteristics of repaired cartilage. Jia et al.52 studied the effect of LIPUS on 10 adult New Zealand rabbits with bilateral full-thickness osteochondral defects. The scores of the gross appearance grades, histological grades and the optical density of toluidine blue of the tissues in the experimental group were significantly higher than those of the controls at 8 weeks after injury (P , 0.05), suggesting that LIPUS can accelerate the repair of injured articular cartilage.
(4) 软骨：对具有全厚度骨软骨缺损的新西兰兔和犬模型进行的体内研究表明，暴露于 LIPUS 可以显着改善修复后软骨的形态学特征和组织学特征。Jia 等人研究了 LIPUS 对10只双侧全厚度骨软骨缺损的成年新西兰兔的作用。实验组在损伤后 8 周的总外观等级，组织学等级和甲苯胺蓝的光密度得分明显高于对照组（P，0.05），表明 LIPUS 可以加速修复关节软骨损伤。
Chondrocytes isolated from young children’s articular cartilage of ablated polydactylia, showed that LIPUS was found to increase aggrecan synthesis in a time-dependent manner,53 with a maximal response observed at an intensity of 48 mW/cm2 . After 14 days of exposure at this intensity, the aggrecan and type II collagen synthesis were significantly higher than control group. However, LIPUS treatment revealed no significant influence on cell proliferation, confirming that the stimulation of aggrecan and type II collagen synthesis by LIPUS was not the result of an increase in chondrocyte cell proliferation. In addition, human chondrocytes harvested from older donors were less responsive to LIPUS.53 Similar studies on human articular chondrocytes isolated from osteoarthritis patients demonstrated an increase in the number and size of glycosaminoglycan-positive lacunae and cellular organelles, appearing as rough endoplasmic reticulum and mitochondria in LIPUS exposed group, suggesting that viability and metabolism of human articular chondrocytes in alginate culture were induced by LIPUS treatment.54 Studies on both animal and human tissues have shown that LIPUS enhance the healing of cartilage defects (Table 2). Whether this acceleration of chondrogenesis in vitro will lead to the maturation of tissue-engineered neo cartilage in a clinical setting has been questioned.55
从幼儿的消融多乳突软骨中分离出的软骨细胞表明，LIPUS 被发现以时间依赖性的方式增加了聚集蛋白聚糖的合成 ，在 48 mW / cm 的强度下观察到了最大的反应 。在此强度下暴露 14 天后，聚集蛋白聚糖和 II 型胶原蛋白的合成显着高于对照组。但是，LIPUS 治疗对细胞增殖没有显着影响，证实 LIPUS 对软骨聚集蛋白聚糖和 II 型胶原合成的刺激不是软骨细胞增殖增加的结果。此外，从较年长的供体中收集的人软骨细胞对 LIPUS 的反应较弱。对从骨关节炎患者分离的人关节软骨细胞的类似研究表明，糖胺聚糖阳性腔和细胞器的数量和大小均增加，在 LIPUS 暴露组中表现为粗面内质网的线粒体和线粒体，表明通过 LIPUS 处理可诱导藻酸盐培养物中人关节软骨细胞的活力和代谢。 对动物和人体组织的研究表明，LIPUS 增强了软骨缺损的愈合（表 2）。 在体外，这种软骨形成的加速是否会导致组织工程化的新软骨在临床环境中的成熟受到质疑。
Economical aspects of LIPUS therapy
LIPUS 治疗的经济方面
Every fracture or soft-tissue injury is accompanied by direct and indirect implication on the economy of an individual and the establishment for which the individual works. The goal of modern orthopaedic surgery is to minimize this financial strain while maximizing the functional recovery after injury.56,57 Study on potential economical benefit of LIPUS therapy in tibial fractures demonstrated substantial cost savings for third party payers, employers and government agencies after the use of LIPUS therapy, as there was lesser need for secondary procedures after LIPUS treatment thereby substantial reduction of the amount of Workers’ Compensation payments.58 The literature, however, is deficient on the economical benefits of LIPUS on soft-tissue healing.
每种骨折或软组织损伤都伴随着对个人经济和个人工作场所的直接和间接影响。现代骨科手术的目标是最大程度地减少这种财务负担，同时使损伤后的功能恢复最大化。对 LIPUS 治疗胫骨骨折的潜在经济利益的研究表明，使用后，第三方付款人，雇主和政府机构可以节省大量成本 。LIPUS 治疗的原因在于 LIPUS 治疗后无需进行二次手术，从而大大减少了工人的赔偿金。 然而，有关 LIPUS 在软组织愈合方面的经济效益却缺乏文献报道。
Conclusions 结论
Research, mainly pre-clinical, so far have shown encouraging result with LIPUS in promoting healing in various soft tissues such as cartilage, inter vertebral disc, etc. The effect on BTJ is primarily on osseous tissue. The role of LIPUS in treating tendinopathies is questionable . These preclinical studies that support the positive effect of LIPUS therapy on soft-tissue healing could be translated into human use. However, poor methodological standards in animal studies mean that positive results rarely translate to the clinical domain59 suggesting that sufficient supporting evidence in the ‘clinically justifiable’ role of LIPUS in soft-tissue healing is lacking. Adequately, powered human studies together with the standardization of intensities and dosages for each target tissue are needed to build a stronger clinical database for routine clinical use. Furthermore, if the aforementioned results with LIPUS concerning IVD’s, tendon and cartilage healing are repeatable and consistent in humans, they could change the future of the management of soft-tissue injuries.
迄今为止，主要是在临床前的研究表明，LIPUS 在促进各种软组织 （例如软骨，椎间盘等）的愈合方面取得了令人鼓舞的结果。对 BTJ 的作用主要是在骨组织上。LIPUS 在治疗肌腱病变中的作用值得怀疑。这些支持 LIPUS 治疗对软组织愈合产生积极作用的临床前研究可以转化为人类用途。然而，动物研究方法学标准差，意味着阳性结果很少会转化为临床领域，这表明缺乏足够的支持证据证明 LIPUS 在软组织愈合中具有“临床上可证明的”作用。适当地，需要进行有力的人体研究以及每个靶组织的强度和剂量的标准化，才能为常规临床应用建立更强大的临床数据库。此外，如果上述关于 LIPUS 的有关 IVD，肌腱和软骨愈合的结果在人类中是可重复且一致的，它们可能会改变软组织损伤管理的未来。
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