想在这里分享一些LIPUS（低强度脉冲超声）的相关文献，我们也是出于对相关技术的兴趣，翻译不是专业的，如有不当的地方，感谢指正！
文献基本信息
文献名称：《Clinical and immunohistopathological aspects of venous ulcers treatment by Low-Intensity Pulsed Ultrasound (LIPUS)》
文献名称：低强度脉冲超声（LIPUS）治疗静脉溃疡的临床和免疫组织病理学方面
作者：Luísiane de Ávila Santana a,b,1, José Marcos Alves a,2, Thiago Antônio Moretti Andrade b,3,
João Kazuyuki Kajiwara c,4, Sérgio Britto Garcia d,5, Fernanda Guzzo Gomes a,b, Marco Andrey Cipriani Frade b,*
a Bioengineering Post-Graduate Program (EESC-IQSC-FMRP-USP), Trabalhador São-carlense Avenue, 400 Arnold Schimidt, São Carlos, São Paulo 13566-590, Brazil
a生物工程研究生课程（EESC-IQSC-FMRP-USP），特拉巴哈多São-carlenseAvenue，400 Arnold Schimidt，圣卡洛斯，圣保罗13566-590，巴西
b Faculty of Medicine of Ribeirão Preto, Division of Dermatology, Department of Internal Medicine (FMRP-USP), Bandeirantes Avenue, 3900 Monte Alegre, Ribeirão Preto,São Paulo 14049-900, Brazil
bRibeirãoPreto医学院，内科（FMRP-USP）皮肤科，Bandeirantes Avenue，3900 Monte Alegre，RibeirãoPreto，巴西圣保罗14049-900
c Faculty of Medicine of Ribeirão Preto, Department of Cell and Molecular Biology and Pathogenic Bioagents, Bandeirantes Avenue, 3900 Monte Alegre, Ribeirão Preto,São Paulo 14049-900, Brazil
cRibeirãoPreto医学院，细胞和分子生物学与致病生物制剂部门，Bandeirantes Avenue，3900 Monte Alegre，RibeirãoPreto，巴西圣保罗14049-900
d Faculty of Medicine of Ribeirão Preto, Department of Pathology and Forensic Medicine (FMRP-USP), Bandeirantes Avenue, 3900 Monte Alegre, Ribeirão Preto, São Paulo 14049-900, Brazil
dRibeirãoPreto医学院，病理和法医学系（FMRP-USP），Bandeirantes Avenue，3900 Monte Alegre，RibeirãoPreto，圣保罗14049-900，巴西
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以下正文
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Article history:
Received 4 July 2011
Received in revised form 11 November 2012 Accepted 16 December 2012
Available online 23 December 2012
文章历史：
2011年7月4日收到
2012年11月11日2012年12月16日收到修订版表格
可于2012年12月23日在线获取
Abstract
The immunological mechanisms that are triggered by Low-Intensity Pulsed Ultrasound (LIPUS) in wound healing are unknown. In the present study, experimental groups were used to assess the treatment of chronic venous ulcers with 30 mW/cm2 SATA peripheral LIPUS three times per week compared to a daily treatment of 1% silver sulfadiazine (SDZ). The ulcers of the SDZ group (n = 7) (G1) and LIPUS group (n = 9) (G2) were photographed fifive times three months, and the images were analyzed using ImageJ software to quantify the total area (S), fifibrin/sphacel area (yellow) and granulation area (red). The healing process was evaluated by the wound healing rate (WHR), granulation tissue rate (GTR) and fifibrin/sphacel tissue rate (FTR). The ulcers were biopsied on days 1 and 45 and stained for collagen fifiber quantifification (picrosirius) and CD68+ protein and VEGF (vascular endothelial growth factor) expression using HRP-streptavidin (horseradish peroxidase–streptavidin). On day 90, G2 had a mean 41% decrease in the ulcer area, while no decrease was observed in G1 (p < 0.05). An increased tendency toward positive labeling of collagen fifibers and VEGF (p > 0.05) was observed in G2 compared to G1, and the number of CD68+ cells was greater in G2 than in G1 (p < 0.05). LIPUS presents superior activity compared to SDZ in stimulating the inflflammatory and proliferative (angiogenesis and collagenesis, respectively) phases of chronic venous wound healing. 2013 Elsevier B.V. All rights reserved.
摘要
低强度脉冲超声（LIPUS）在伤口愈合中引发的免疫机制尚不清楚。在本研究中，与每天使用1％磺胺嘧啶银（SDZ）的每日治疗相比，实验组用于每周30次用30 mW / c㎡ SATA外围LIPUS评估慢性静脉溃疡的治疗。SDZ组（n = 7）（G1）和LIPUS组（n = 9）（G2）的溃疡三个月拍摄五次，并使用ImageJ软件分析图像以量化总面积（S），纤维蛋白/碎屑区（黄色）和制粒区（红色）。通过伤口愈合率（WHR），肉芽组织率（GTR）和纤维蛋白/残渣组织率（FTR）评估愈合过程。在第1天和第45天对溃疡进行活检，并使用HRP-链霉亲和素（辣根过氧化物酶-链霉亲和素）对胶原纤维定量（picrosirius），CD68+ 蛋白和VEGF（血管内皮生长因子）表达进行染色。在第90天，G2的溃疡面积平均减少了41％，而G1的溃疡没有减少（p <0.05）。与G1相比，G2中观察到胶原纤维和VEGF的阳性标记趋势增加（p> 0.05），G2中CD68+ 细胞的数量大于G1（p <0.05）。与SDZ相比，LIPUS在刺激慢性静脉伤口愈合的炎症和增殖（分别为血管生成和胶原形成）阶段表现出更高的活性。
©2013 Elsevier BV保留所有权利。
Keywords: Ultrasound Venous ulcer Wound healing Immunohistochemistry Image processing
关键词：超声波 静脉溃疡 伤口愈合 免疫组织化学 图像处理
1. Introduction 介绍
Venous ulcers are mainly caused by chronic venous insuffifi- ciency due to a defificiency of the superfificial and/or deep venous system [1]. The process of healing involves the replacement of normal tissue with fifibrous connective tissue at the site of trauma. The healing process is characterized by a sequence of biological, cellular and molecular events that are directed toward the restoration of a tissue that has been damaged by injury [2]. These events occur simultaneously but can be divided into the following healing phases: injury, coagulation, inflflammation, tissue formation, and tissue remodeling. The healing process is slower for chronic wounds than for acute wounds due to the poor mitogenic activity and increased levels of inflflammatory cytokines and proteases and numbers of senescent cells [3].
静脉溃疡主要是由于表面或深层系统的缺陷而导致的机能丧失而引起的静脉营养不良引起的[1]。愈合过程包括在创伤部位用纤维结缔组织代替正常组织。愈合过程的特征是一系列生物，细胞和分子事件，这些事件直接针对已被损伤破坏的组织的恢复[2]。这些事件同时发生，但可以分为以下愈合阶段：损伤，凝血，炎症，组织形成和组织重塑。由于有丝分裂活性差，炎性细胞因子和蛋白酶的水平增加以及衰老细胞的数量增加，慢性伤口的愈合过程比急性伤口的愈合过程慢[3]。
Several treatments are used to heal chronic wounds, including 1.0% silver sulfadiazine, which has bactericidal and bacteriostatic activities (destroying and preventing the proliferation of microorganisms, respectively) [4]. This product is used as a topical treatment of ulcerated injuries of different etiologies, including venous ulcers [5] and pressure ulcers [6,7]. Although the silver sulfadiazine cream has been used frequently in public wound care units, its effificacy in treating chronic venous ulcers has not been demonstrated in the literature.
几种治疗方法可用于治愈慢性伤口，包括1.0％的磺胺嘧啶银，其具有杀菌和抑菌活性（分别破坏和防止微生物繁殖）[4]。该产品用作局部治疗不同病因的溃疡性损伤，包括静脉溃疡[5] 和压疮[6,7]。尽管磺胺嘧啶银乳膏已在公共伤口护理中频繁使用，但其治疗慢性静脉溃疡的功效尚未在文献中得到证明。
Low-Intensity Pulsed Ultrasound (LIPUS) stimulation is a noninvasive technique for the treatment of fractures. In 1983, Duarte pioneered the use of this technology to stimulate bone growth in rabbits. In that study, rabbits had their fifibulas and femurs osteotomized, and the left limbs received 15 min of LIPUS daily (5 ls pulse, intensity of 47 mW/c㎡ SATA, pulse repetition frequency of 1000 Hz), while the right limbs received no treatment. The LIPUS-treated limbs underwent a rapid increase in osseous callus within the fifirst 12 days, while the control group had a slower rate of increase during this period. Signifificant regeneration of the trabecular bone was observed on the 7th day after LIPUS treatment, which was not observed in the control group [8]. This study motivated clinical studies of recent, delayed union and non-union fractures, treatment of osteoporosis [9], implant osseointegration [10], bone repair in spinal arthrodesis [11], and bone distraction [12]. Korstjens et al. in 2008 detected LIPUS-stimulated chondrocyte proliferation and matrix production in human articular implants as well as in vitro chondrocytes [13].
低强度脉冲超声（LIPUS）刺激是一种用于治疗骨折的非侵入性技术。1983年，Duarte率先使用该技术来刺激兔子的骨骼生长。在那项研究中，兔子的腓骨和股骨被切除，左肢每天接受15分钟的LIPUS（5ls 脉冲，强度为47 mW /c㎡ SATA，脉冲重复频率1000 Hz），而右肢未接受任何治疗。在前12天内，用LIPUS治疗的肢体骨痂迅速增加，而对照组在此期间的骨痂增长速度较慢。LIPUS治疗后第7天观察到小梁骨显著再生，而对照组中未观察到[8]。这项研究推动了近期骨折、延迟愈合和不愈合骨折的临床研究以及骨质疏松症的治疗[9]、植入物骨整合[10]，脊柱关节融合术中的骨修复[11]和骨牵引[12].Korstjens等人在2008年检测到LIPUS刺激的人体关节植入物中以及体外软骨细胞中软骨细胞的增殖和基质生成[13]。
Given these effects on bone healing, a number of studies have assessed the effect of ultrasound (US) on skin ulcer healing. In a review by Khanna et al. in 2008, it was shown that mechanical waves (ultrasound) with intensities exceeding 1 W/c㎡ SATA are not adequate for tissue healing [14]. By contrast, there is considerable evidence that US intensities below 0.5 W/c㎡ SATA can increase the rate of wound healing. Dayson et al. in 1968 was a pioneer of such studies, which confifirmed the use of 0.5 W/c㎡ ultrasound in the spatial average temporal average (SATA) in wounded skin. Pulsed ultrasound (0.5 W/c㎡ ) was applied for 5 min per day on alternate days for 21 days on lesions in the ears of rabbits; this treatment had benefificial effects in accelerating the healing process [15]. Young and Dyson in 1990 conducted research with ultrasound intensities below 0.5 W/c㎡ SATA and observed that pulsed ultrasound (2 ms on, 8 ms off) at 0.1 W/c㎡ SATA (frequency of 0.75 MHz or 3.0 MHz) for fifive days after injury resulted in extensive granulation tissue, fewer polymorphonuclear leucocytes (polymorphs) and macrophages, and more fifibroblasts than shamirradiated controls [16]. Byl et al. in 1992 reported the effects of low doses of ultrasound (0.1–0.3 W/c㎡ SATA, 1 MHz) in the acceleration of wound healing in mini-pigs. Increases in tensile strength and collagen deposition, reductions in wound sizes and degranulation of mast cells were observed compared to the control group. It was suggested that in the fifirst week of healing, low-dose ultrasound intensity SATA facilitates wound healing, but more research is needed to determine the most effective dose, pulse repetition frequency, duration of treatment and intervention time for maximum healing [17].
鉴于这些对骨骼愈合的影响，许多研究已经评估了超声对皮肤溃疡愈合的影响。2008年，Khanna等人的研究表明，强度超过1 W /c㎡ SATA的机械波（超声波）不足以治愈组织[14]。相比之下，有大量证据表明，低于0.5 W / c㎡ SATA的超声强度可以提高伤口愈合率。Dayson等人在1968年是此类研究的先驱，该研究证实了0.5 W / cm2 的超声波在受伤皮肤的空间平均时间平均值（SATA）。隔天在兔耳的病变处连续每天施加脉冲超声（0.5 W / c㎡）5分钟，共21天，这种治疗对加速愈合过程具有有益的作用[15]。Young和Dyson在1990年用低于0.5 W / c㎡ SATA的超声强度进行了研究，并观察到脉冲超声（2 ms开启，8 ms关闭）在0.1 W / c㎡ SATA（频率为0.75 MHz或3.0 MHz），损伤后五天，导致肉芽组织扩张，较少的多形核白细胞（多形体）和巨噬细胞，以及比经放射线照射的对照组少的成纤维细胞[16]。Byl等人1992年报道了低剂量超声波（0.1–0.3 W/c㎡ SATA，1 MHz）对小型猪伤口愈合的加速作用。与对照组相比，观察到抗张强度和胶原蛋白沉积增加，伤口尺寸减小和肥大细胞脱粒。有人建议，在康复的第一周，低剂量的超声强度SATA可以促进伤口愈合，但是还需要更多的研究来确定最有效的剂量，脉冲重复频率，治疗时间和干预时间，以最大程度地康复[17]。
A recent series of studies investigated the effect of lower doses of ultrasound (30 mW/c㎡ ) on the proliferation of epithelial cells [18] in bone regeneration [19,20] and in the healing tendon [21]. However, venous ulcers currently represent a serious public health problem that is associated with chronic diseases and high life expectancy; effective treatments and low-cost treatments have been diffificult to fifind [22], and LIPUS might represent such a treatment. Given the confifirmed effificacy of LIPUS in bone regeneration, it is important to assess its effectiveness as a treatment for chronic ulcers.
最近的一系列研究调查了较低剂量的超声波（30 mW/c㎡）对上皮细胞增殖[18] 在骨再生中[19,20] 在愈合肌腱上[21]的影响。但是，静脉溃疡目前是一个严重的公共卫生问题，与慢性病和较高的预期寿命有关。有效的治疗方法和低成本的治疗方法很难找到[22]，而LIPUS可能代表了这种治疗方法。鉴于LIPUS在骨再生中已证实的功效，是评估其作为慢性溃疡治疗的有效性很重要。
LIPUS was developed and patented by Professor Duarte Romariz from the School of Electric Engineering – Interunits of Bioengineering – University of São Paulo, Brazil, in 1979 and was fifirst used for bone regeneration [8,23]. Walsh et al. in 2008 observed an increase in the levels of vascular endothelium growth factor (VEGF) in animals that were treated with LIPUS for bone regeneration [24]. Lu et al. in 2008 demonstrated an increase in VEGF in rabbits that were treated for palectomy by LIPUS and concluded that the primitive regulation of VEGF expression is important for the remodeling and chondrogenesis phases [25]. Low-intensity ultrasound (0.05 W/c㎡ SATA) was used to treat moderate and severe lung ischemia in mice. Increases in vessel numbers, cellular proliferation and VEGF mRNA levels were observed by histology in the moderate ischemia group [26].
LIPUS由巴西圣保罗大学电气工程学院-生物工程学单元-Duarte Romariz教授于1979年开发并获得专利的，最早用于骨再生[8,23]。Walsh等人在2008年观察到用LIPUS治疗骨骼再生的动物中血管内皮生长因子（VEGF）的水平有所增加[24]。Lu等人在2008年研究表明，LIPUS治疗腭切除术的兔子的VEGF升高，并得出结论：VEGF表达的原始调节对于重塑和软骨形成阶段很重要[25]。低强度超声波（0.05 W/c㎡ SATA）用于治疗小鼠的中度和重度肺缺血，组织学观察到中度缺血组血管数目，细胞增殖和VEGF mRNA水平增加[26]。
Few studies of healing induced by LIPUS have analyzed VEGF expression in vitro, and no study has shown its expression level in chronic venous wounds, similar to studies of the inflfluence of CD68+ cells on the healing process and in vitro. However, countless cells, growth factors, cytokines and proteins are involved in the healing process, including macrophages, which are important during the inflflammatory phase. CD68 levels indicate macrophage activation during phagocytosis of cell remnants [27,28] and VEGF plays an important role in the tissue formation phase of healing (angiogenesis).
LIPUS诱导的愈合的研究很少在体外分析VEGF的表达，没有研究表明其在慢性静脉伤口中的表达水平，类似于CD68+ 细胞对愈合过程和体外影响的研究。但是，愈合过程涉及无数的细胞，生长因子，细胞因子和蛋白质，包括巨噬细胞，这在炎症期很重要。CD68水平表明吞噬细胞残余物中的巨噬细胞活化[27,28] VEGF在愈合的组织形成阶段（血管生成）中起重要作用。
The objective of the present study was to assess and to compare the effificacy of LIPUS with a standard treatment of 1.0% silver sulfadiazine (SDZ) in the healing of chronic venous ulcers. The wound healing process was evaluated by measuring the ulcer area and its respective tissues (granulation and fifibrin/sphacel) using ImageJ image analysis software, in addition to assessing the number of collagen fifibers and expression of VEGF and CD68 proteins by immunohistochemistry in ulcer biopsies at the beginning of the study and after 45 days of treatment. The loss of ultrasound intensity due to the use of a latex fifilm (condom) over the head of the ultrasound probe was also investigated as an asepsis technique.
本研究的目的是评估和比较LIPUS与1.0％磺胺二嗪银（SDZ）的标准治疗在慢性静脉溃疡愈合中的疗效。通过使用ImageJ图像分析软件测量溃疡区域及其各个组织（肉芽和纤维蛋白/残渣），评估伤口的愈合过程，此外还通过免疫组织化学评估胶原纤维的数量以及溃疡活检组织中VEGF和CD68蛋白的表达。研究开始和治疗45天后，作为无菌技术，还研究了由于在超声探头的头部使用乳胶膜（避孕套）而导致的超声强度损失。
2. Methods 方法
2.1. Patient selection 患者选择
Ten patients with multiple chronic venous ulcers were selected at the Outpatient Dermatologic Clinic of Neurovascular Ulcers (Clinical Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo) and at the School Health Center of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, from February 2005 to January 2006. The study was approved by the Research Ethics Committee of HCFMRP-USP (Process number 12488/2004), and all patients were informed and signed the consent terms to participate in the study.
在神经血管性溃疡的门诊皮肤病诊所（圣保罗里贝朗·普雷图大学医学院，临床医院）和里贝雷朗·普雷图医学院的学校卫生中心，选择了10名患有多发性慢性静脉溃疡的患者，圣保罗大学，2005年2月至2006年1月。该研究得到HCFMRP-USP研究伦理委员会的批准（程序编号12488/2004），并告知所有患者并签署同意书以参与该研究。
Each patient was accompanied by a medical team that determined the ulcer etiology. The CEAP scale was used to classify the level of chronic lower extremity venous disease [29].
每位患者均由确定溃疡病因的医疗团队陪同。使用CEAP量表对慢性下肢静脉疾病的水平进行分类[29]。
The inclusion criteria were the presence of venous ulcers, characterized by signs of venous insuffificiency such as varicosity, edema, eczema, lipodermatosclerosis and elephantiasis; lack of infection; brachial index larger than 0.9; and, when associated with systemic arterial hypertension, a diastolic arterial pressure below 95 mmHg.
纳入标准为是否存在静脉溃疡，以静脉功能不全为特征，如静脉曲张，水肿，湿疹，脂肪性皮肤硬化和象皮病。缺乏感染肱指数大于0.9；当与全身性动脉高压相关时，舒张压低于95 mmHg。
2.2. Experimental groups 实验组
Ten patients with multiple chronic venous ulcers were followed, and their wounds were randomly included in one of two experimental groups: (a) the SDZ group, with 7 ulcers treated with daily SDZ cream dressings, and (b) the LIPUS group, with 9 ulcers treated with LIPUS three times per week. The ultrasound device is a custom instrument that was made by the School of Electrical Engineering of São Carlos – USP for research use. It is designed to generate a burst sinusoidal signal of 1.5 MHz frequency, 200 ls pulse width, 1.0 kHz repetition frequency and 30 mW/c㎡ SATA intensity, according to the guidelines proposed for authors and reviewers of journals of ultrasound in medicine that were published by Edmonds et al. in 2005 [30]. This same LIPUS was produced by Exogen in 2000 (Smith and Nephew Inc., Memphis, TN, USA)
对10例多发性慢性静脉溃疡的患者进行了随访，并将他们的伤口随机分为两个实验组：（a）SDZ组，每天治疗7个溃疡，用SDZ乳膏敷料；（b）LIPUS组，用LIPUS治疗9次溃疡，每周3次。超声仪是由美国电气工程学院制造，圣卡洛斯– USP供研究使用的定制仪器。根据为医学超声杂志的作者和审阅者建议的准则，它旨在产生1.5 MHz频率，200 ms脉冲宽度，1.0 kHz重复频率和30 mW/ c㎡ SATA强度的突发正弦信号，由Edmonds等人在2005年出版 [30]。同样的LIPUS由Exogen在2000年生产（美国田纳西州孟菲斯市的史密斯和内弗公司）。
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