以下文字版
The number of cases was determined as the number of ulcers. The groups were divided according to the following criteria: when a patient had an ulcer on each leg, each ulcer received a different treatment (LIPUS or SDZ); when a patient had two or more ulcers on the same leg, all ulcers received the same treatment.
病例数确定为溃疡数。根据以下标准将组划分：当患者的每条腿都有溃疡时，每个溃疡接受不同的治疗（LIPUS或SDZ）；当患者在同一条腿上有两个或多个溃疡时，所有溃疡均接受相同的治疗。
The ulcers of the SDZ group were washed with 0.9% physiological saline and covered with SDZ cream (a standard topical treatment used by health facilities), gauze and a bandage. The ulcers of the LIPUS group were washed in the same manner, and then ultrasound was applied rapidly to different sites on the wound margins. The head of the transducer remained stationary at each site. The distance between 2 adjacent sites of treatment was 2 cm, and the time of treatment at each site was 3 min (Fig. 1a).
SDZ组的溃疡用0.9％生理盐水清洗，并用SDZ乳膏（卫生机构使用的标准外用治疗方法），纱布和绷带覆盖。以相同的方式清洗LIPUS组的溃疡，然后将超声波迅速施用于伤口边缘的不同部位。换能器的头部在每个位置均保持静止。两个相邻治疗部位之间的距离为2厘米，每个部位的治疗时间为3分钟（图1a)。
Due to the lack of consensus in the literature regarding the time of treatment and based on the fact that the fifirst studies with LIPUS (30 mW/cm2 SATA) suggested a wave exposure of at least 20 min of stationary mode [31,32], the ultrasound was applied around the ulcer in a sequence of 3 min every 2 cm for a minimum of 21 min (7 applications of 3 min each) on alternate days, with longer exposure times for ulcers with areas 614 cm2 . At the end of the treatment, the ulcers were washed with saline and covered with gauze soaked in saline and then bandaged. The duration of treatment for both experimental groups was 90 days, according to the treatment protocol for physical treatment methods used by Peschen et al. in 1997 to confifirm a healing rate of 55.4% for chronic venous ulcers after 12 weeks of treatment with ultrasound at a frequency of 30 kHz, continuous 100 mW/cm2 , 10 min, 3 times per week for 12 weeks [33].
由于有关治疗时间的文献缺乏共识，并且基于以下事实：使用LIPUS（30 mW / cm2 SATA）进行的首次研究表明，固定模式下的波暴露时间至少为20分钟[31,32]，将超声波按每2厘米3分钟的顺序在溃疡周围施加至少21分钟（每次7次，每次3分钟），隔天间隔至少≤14 cm2。在治疗结束时，用盐水清洗溃疡，并用浸在盐水中的纱布覆盖，然后包扎。根据Peschen等人使用的物理治疗方法的治疗方案，两个实验组的治疗时间均为90天。在1997年确认慢性静脉溃疡的愈合率为55.4％，超声治疗频率为30 kHz，连续100 mW / cm2，超声治疗12周，每次10分钟，每周3次[33]。
2.3. Ultrasonic power loss through a latex fifilm 通过乳胶膜的超声波功率损耗
To prevent contamination of the head of the ultrasound probe and the risk of transmitting pathogens to the patients, a latex fifilm (condom) was applied to the head of the ultrasound probe for each session. A gel layer was applied between the surface of the head of the probe and the inner surface of the latex fifilm and between the outer surface of the latex fifilm (0.1 mm thick) and the margin of the ulcer (Fig. 1b). The attenuation caused by the fifilm was determined experimentally by measuring the acoustic power with and without the latex fifilm using an ultrasonic power meter (UPM-DT1, Ohmic Instruments, Easton, MD, USA). The mean percentage loss in the ultrasonic power through the latex fifilm as measured in water was 11%. The ultrasonic energy transmitted by the transducer was adjusted to compensate for the power loss caused by the latex fifilm and to obtain a SATA intensity of 30 mW/cm2 at the site of tissue treatment.
为了防止超声探头的头部受到污染以及将病原体传播给患者的风险，每次使用时都要在超声探头的头部涂一层乳胶膜（避孕套）。在探头头部的表面和乳胶膜的内表面之间以及乳胶膜的外表面（0.1毫米厚）和溃疡边缘（图。1b).通过使用超声功率计（UPM-DT1，Ohmic Instruments，Easton，MD，USA）测量有或没有乳胶膜的声功率，通过实验确定由膜引起的衰减。在水中测得的通过乳胶膜的超声功率的平均损失百分比为11％。调节由换能器传输的超声能量，以补偿由乳胶薄膜引起的功率损耗，并在组织治疗部位获得30 mW / cm2 的SATA强度。
2.4. Image capture 图像捕获
The ulcers were photographed every fififteen days using a 5.0- megapixel digital camera (Sony dsc P93, Manaus, AM, Brazil) coupled to an 80-cm aluminum arc measuring 70 cm in diameter and standing on a 50 70 cm2 acrylic base. A bulb was also coupled to the arc, indirectly lighting the ulcer. Self-adhesive labels with length marks in centimeters were placed on the ulcer margins to adjust the angulations of the ulcers during the image analysis and were used as a ruler for scale standardization during image capture, as demonstrated previously [34–36].
使用5.0兆像素数码相机（Sony dsc P93，巴西马萨诸塞州马瑙斯）每15天拍摄一次溃疡，百万像素数码相机与直径为70厘米的80厘米铝弧连接，并立于50*70cm2 丙烯酸底座上。灯泡也被固定到电弧上，间接照亮了溃疡。如前所述，将以厘米为单位的长度标记的自粘标签放置在溃疡边缘，以在图像分析过程中调整溃疡的角度，并用作图像采集过程中标尺标准化的标尺。[34–36]。
2.5. Ulcer analysis 溃疡分析
The photos of the ulcers were transferred to a computer to measure the total area and the granulation (red area) and fifibrin/sphacel (yellow area) tissues using the public domain software ImageJ [31– 33] (developed by Wayne Rasband, Research Services Branch, National Institute of Mental Health (bethesda, MD, USA) and available at [http://rsbweb.nih.gov/ij/]).The healing process was evaluated by the following parameters:
使用公共领域的软件ImageJ将溃疡的照片转移到计算机上，以测量总面积和肉芽（红色区域）以及血纤蛋白/残渣（黄色区域）组织[31– 33] （由美国国家心理卫生研究所研究服务处Wayne Rasband开发（美国马里兰州贝塞斯达），可在[http://rsbweb.nih.gov/ij/]).通过以下参数评估愈合过程：
(a) Wound healing rate (WHR): the area of the initial ulcer area minus the ulcer area measured on the day of evaluation divided by the initial ulcer area, which is interpreted as follows: unchanged area (WHR = 0), total healing (WHR = 1), decreased area (0 < WHR < 1), and increased area (WHR < 0).
(a) 伤口愈合率（WHR）：初始溃疡面积减去评估当天测量的溃疡面积除以初始溃疡面积，其解释为：面积不变（WHR = 0），总愈合率（ WHR = 1），减小的面积（0 <WHR <1）和增大的面积（WHR <0）。
(b) Granulation tissue rate (GTR): the ratio of the granulation tissue area (red area) to the ulcer area at the time of the evaluation.
(b) 肉芽组织率（GTR）：评估时肉芽组织面积（红色面积）与溃疡面积之比。
(c) Fibrin/sphacel tissue rate (FTR): the ratio of the sphacel or fifibrin tissue area (yellow area) to the ulcer area at the time of the evaluation. The rate of sphacel/fifibrin and granulation (FGR) were obtained through the area of sphacel/fifibrin tissue over the granulation tissue area during the follow-up period.
(c)纤维蛋白/纤维蛋白组织率（FTR）：评估时，纤维蛋白或纤维蛋白组织面积（黄色面积）与溃疡面积之比。
This rate indirectly shows the dynamism of tissue changes that occur during the healing process: when the area of sphacel/fifibrin tissue is equivalent to granulation area, the rate is 1 (FGR = 1); the granulation area can also be larger than the sphacel/fifibrin area (FGR < 1) or smaller than the sphacel/ fifibrin area (FGR > 1).
在随访期间，通过在肉芽组织区域上的血脂/血纤蛋白组织的面积获得了血脂/血纤蛋白和肉芽形成的速率。该速率间接地表明在愈合过程中发生的组织变化的动态性：当虫/纤维蛋白组织的面积等于肉芽形成面积时，该速率为1（FGR = 1）；造粒面积也可以大于血脂/血纤蛋白面积（FGR <1）或小于血脂/血纤蛋白面积（FGR> 1）。
2.6. Histopathological analysis 组织病理学分析
The margins of 11 ulcers (5 ulcers of the SDZ group and 6 ulcers of the LIPUS group) of 8 patients were biopsied on days 1 and 45 of treatment with a 6-mm punch.
在治疗的第1天和第45天，用6毫米打孔器对8例患者的11个溃疡（SDZ组为5个溃疡，LIPUS组为6个溃疡）的边缘进行活检。
The biopsies were embedded in paraffifin, cut into 4-lm sections and stained with picrosirius. Collagen fifibers were quantifified under a microscope at 160 magnifification using a millimeter ruler on the eyepiece in a 10-mm fifield. The thickness of each collagen fifiber present in this fifield was measured. The quantity of collagen fifibers was determined as the sum of the fifiber thicknesses divided by 10 mm [37]. The slides were analyzed by 2 investigators, and the fifinal results are reported as a mean.
将活检组织包埋在石蜡中，切成4 lm的切片并用picrosirius染色。胶原纤维在显微镜在10毫米视场中使用目镜上的毫米尺在160放大倍率下拍摄。测量该领域中存在的每种胶原纤维的厚度，胶原纤维的数量由纤维厚度的总和除以10毫米[37]。由两名研究人员对载玻片进行了分析，最终结果报告为平均值。
2.7. Immunohistochemical analysis 免疫组织化学分析
The 4-lm skin sections were mounted on silanized 8% slides for VEGF and CD68 immunohistochemistry staining. The blocking of endogenous peroxidase and non-specifific binding was carried out with 3.0% hydrogen peroxide and bovine serum albumin (BSA), respectively. The primary antibodies used were rabbit IgG anti-human VEGF (Santa Cruz Biotechnology , Santa Cruz, CA USA) at a concentration of 1:100 and mouse IgG anti-human CD68 (Novocastra , Newcastle upon Tyne, UK) at a concentration of 1:200 for 2 h. The secondary biotinylated anti-rabbit IgG (H + L) antibodies in goat BA-1000 VECTOR and anti-mouse IgG (H + L) antibodies in horse BA-2000 VECTOR were added, respectively, both at concentrations of 1:200, for 30 min. 3,30 -diaminiobenzidine (DAB, Sigma, St. Louis, MO, USA) diluted in 15 mL 0.01 M PBS and 30% H2O2 were used to detect peroxidase activity. The slides were counterstained with Harris hematoxylin. For both markers, the sections were analyzed under a microscope at 400 magnifification, and the quantififi- cation was performed as the mean of the sum of labeled cells in each analyzed fifield divided by the number of fifields. The slides were analyzed by 2 investigators, and the fifinal results are reported as a mean.
将4-lm皮肤切片固定在8％硅烷化载玻片上，用于VEGF和CD68免疫组织化学染色。内源性过氧化物酶的封闭和非特异性结合分别用3.0％过氧化氢和牛血清白蛋白（BSA）进行。使用的主要抗体是兔IgG抗人血管内皮生长因子（Santa Cruz Biotechnology®，Santa Cruz，CA USA），在浓度为1：100和浓度为1：200的小鼠IgG抗人CD68（Novocas-tra®，泰恩河畔纽卡斯尔），浓度为1：200，持续2小时。山羊BA-1000 VECTOR®的生物素化抗兔IgG（H + L）二级抗体和抗小鼠IgG（H + L）抗体分别添加了BA-2000 VECTOR®马，1：200，持续30分钟。用15 mL 0.01 M PBS和30％H2O2 稀释的3,30-二氨基联苯胺（DAB，Sigma，St.Louis，MO，USA）用于检测过氧化物酶活性。玻片用哈里斯苏木精复染色。对于这两种标记，在显微镜下以400倍的放大倍数对切片进行分析，并以每个分析视野中标记细胞总数的平均值除以视野数进行定量。由两名研究人员对玻片进行了分析，最终结果报告为平均值。
2.8. Statistical analysis 统计分析
The statistical analysis was performed using the nonparametric Mann–Whitney test, which compares two independent groups, to evaluate the differences between groups during the follow-up period using GraphPad Prism software. A p value of less than 0.05 was considered statistically signifificant.
使用非参数Mann-Whitney检验进行统计分析，该检验比较了两个独立的组，并使用GraphPad Prism软件评估了随访期间各组之间的差异，小于0.05的p值被认为具有统计学意义。
3. Results 结果
Both groups had similar population characteristics (Table 1) and ulcer characteristics at the beginning of the treatment; there was no signifificant difference between the groups for WRT, GTR and FTR. There was also no signifificant difference for picrosirius or CD68. On the fifirst day, however, there was a difference in VEGF expression, which was positive for the LIPUS group. However, this difference did not alter the overall results because VEGF expression was not signifificantly different on day 45 of treatment.
两组的人口特征相似（表格1）和开始治疗时的溃疡特征；WRT，GTR和FTR的组之间没有显着差异。picrosirius或CD68也没有显着差异。然而，在第一天，VEGF表达存在差异，LIPUS组为阳性。但是，这种差异不会改变总体结果，因为在治疗的第45天VEGF表达没有明显差异。
The ulcer closing or re-epithelialization as measured by the WHR is described in Fig. 2. Of the 9 ulcers that were treated with LIPUS, 1 (11.1%) increased in area, 1 (11.1%) had no change in area, 5 (55.6%) decreased in area, and 2 (22.2%) healed completely; with the SDZ treatment, 1 (14.3%) ulcer healed, and 6 (85.7%) increased in area by day 90 day of treatment. Thus, 77.8% of ulcers that were treated with LIPUS had improved healing, compared to only 14.3% of SDZ-treated ulcers. The mean WHR values were positive and higher in the LIPUS group than in the SDZ group, but this difference was only signifificant on day 90 (p < 0.05).
WHR所测量的溃疡闭合或再上皮形成描述于图2.用LIPUS治疗的9个溃疡中，面积增加1个（11.1％），面积没有变化的1个（11.1％），面积缩小5例（55.6％），完全愈合例（22.2％）；与到第90天，SDZ治疗的溃疡愈合1例（14.3％），溃疡增加6例（85.7％）。因此，与仅SDZ治疗的溃疡的14.3％相比，使用LIPUS治疗的溃疡有77.8％的愈合得到改善。LIPUS组的平均WHR值是阳性且高于SDZ组，但这种差异仅在第90天时才显著（p <0.05）。
Table 1 Population data.
表格1 人口数据
Considering the dynamism of the wound healing process and based on the tissue modifification observed during the follow-up period, the LIPUS group ulcers had a larger mean granulation area (GTR) than did the SDZ group (p < 0.05) starting on day 30 until the end of the treatment period. On the same evaluation days, the mean FTR was signifificantly lower in the LIPUS group than in the SDZ group (Figs. 3 and 4).
考虑到伤口愈合过程的动态性，并根据随访期间观察到的组织改变，从第30天开始，LIPUS组溃疡的平均肉芽面积（GTR）比SDZ组更大（p <0.05），直到治疗期结束。在相同的评估日，LIPUS组的平均FTR显着低于对照组SDZ组的（图3和图4)。
A qualitative analysis of slides stained with picrosirius, which is a specifific stain for collagen fifibers, showed no signifificant increase in fifiber thickness on day 45 in the LIPUS group compared to the SDZ group (Fig. 5).
定性分析用picrosirius染色的载玻片（胶原纤维特有的染色剂），与SDZ组相比，LIPUS组在第45天的纤维厚度没有明显增加（图5)。
Immunohistochemical staining revealed a higher number of cells labeled with CD68 on day 45 in the ulcers of the LIPUS group compared to the SDZ group (p < 0.05) (Fig. 6). There was no significant difference in VEGF expression between the two groups on day 45 (p > 0.05) (Fig. 7).
免疫组织化学染色显示，与SDZ组相比，LIPUS组溃疡第45天的CD68标记的细胞数量更高（p <0.05）（图6)。两组在第45天的VEGF表达无显着差异（p> 0.05）（图7).
4. Discussion 讨论
A measurement of the area of an ulcer remains a predominantly visual (qualitative) and subjective method due to the diffificulties in the determination of wound areas and tissue modififications. Thus, monitoring the progression of healing is a serious hallenge. Photographic monitoring has been used to illustrate the course of healing and the different degrees of severity of skin lesions, and digital photographs have been the standard method of documentation in surgical dermatology practices [38,39].
由于难以确定伤口面积和组织改变，溃疡面积的测量仍然是主要的视觉（定性）和主观方法。因此，监测愈合的进展是一个严峻的挑战。使用图像监控来说明治疗的过程以及皮肤损伤的不同程度，数码照片已成为外科皮肤病学实践中记录的标准方法[38,39]。
ImageJ software has been used extensively for image measurements [34–36,40]. According to the dynamics of the healing process, ulcer evolution can be monitored on the basis of modififications in the fifibrin/sphacel (yellow tissues) and granulation tissues (red tissues) [41].ImageJ software, which is a free, easily accessible, non-invasive option for ulcer area quantitative analysis, has been used effectively to quantify wound areas.
ImageJ软件已被广泛用于图像测量[34–36,40].根据愈合过程的动力学，可以根据血纤蛋白/血脂（黄色组织）和肉芽组织（红色组织）的变化监测溃疡的发展[41].ImageJ软件是一个免费的、方便的、可用于溃疡面积定量分析的无创易用选项，已被有效地用于量化伤口面积。
On the fifirst day of treatment, there was no signifificant difference in the leg ulcer area, fifibrin/sphacel area or granulation area between the SDZ group and the LIPUS group, which highlights the homogeneity of the experimental groups at the beginning of the treatment period.
在治疗的第一天，SDZ组和LIPUS组之间的下肢溃疡区域，纤维蛋白/散布区域或肉芽区域没有显着差异，这突出了实验组在治疗开始时的同质性治疗期。
The majority of the ulcers in the SDZ group had negative WHR, i.e., they presented an ulcer area at the end of the treatment that was larger than the ulcer area on the fifirst day, as shown in Fig. 2. Some of the ulcers in the LIPUS group showed a negative WHR until the 30th day of follow-up. However, starting on day 45, ulcers of the LIPUS group showed an increase in the WHR, which was evident in 8 of the 9 evaluated ulcers by day 90, in contrast to the SDZ group in which a decrease occurred in 6 out of 7 ulcers. The necrotic tissue on the borders of chronic wounds gives the impression that the wound area is smaller than its actual area. This necrotic tissue must be removed during the pro-inflflammatory phase to encourage better healing. The results showed that treatment with both SDZ and LIPUS initially led to an increase in the ulcerated areas, suggesting activation of the inflflammatory phase and consequent ulcer debridement. Young and Dyson in 1990 suggested a pro-inflflammatory activity of ultrasound, which is important for the acceleration of the proliferative phase of tissue repair [16]. Sulfadiazine contributes to the removal of necrotic tissue but does not act in the formation of granulation tissue or neovascularization, which might explain the unchanged state of the ulcer and even its increase in area. Silver sulfadiazine is known to have important antimicrobial activities against gram-positive and gram-negative bacteria, fungi, dermatophytes, viruses and protozoa, but its action in other phases of healing is unclear [42].
SDZ组中大多数溃疡的WHR均为负值，即治疗结束时出现的溃疡面积大于第一天的溃疡面积，如图2所示。LIPUS组中的某些溃疡在随访的第30天之前显示出WHR阴性，但是，从第45天开始，LIPUS组的溃疡表现出WHR升高，到90天时，这9个评估过的溃疡中有8个是明显的。与SDZ组相反，SDZ组的7个溃疡中有6个发生了减少溃疡，慢性伤口边界上的坏死组织给人的印象是伤口面积小于其实际面积。在促炎阶段必须切除这种坏死组织以促进更好的愈合。结果表明，同时使用SDZ和LIPUS进行治疗可导致溃疡区域的增加，这表明炎症期已激活，继而溃疡清创。Young和Dyson在1990年提出超声的促炎活性，这对于加速组织修复的增殖期很重要[16]。磺胺嘧啶有助于去除坏死组织，但不参与肉芽组织的形成或新血管形成，这可能解释了溃疡的状态不变，甚至面积增大。已知磺胺嘧啶银对革兰氏阳性和革兰氏阴性细菌，真菌，皮肤真菌，病毒和原生动物具有重要的抗菌活性，但尚不清楚其在其他愈合阶段的作用[42].