中药接骨Ⅰ号治疗超声透入桡骨骨折愈合过程中骨形态发生蛋白7的表达

背景:将低强度脉冲超声波与中药相结合,即中药超声透入疗法,在动物实验和临床中用于骨折的治疗并取得了确切的疗效,然而其促进骨折愈合机制的研究却少有报道.目的:观察中药超声透入对新西兰大白兔桡骨中段骨缺损愈合过程中骨形态发生蛋白7表达的影响.方法:84只兔构建双侧桡骨中段骨缺损的骨折模型,建模成功后随机分成3组:中药组给予以接骨Ⅰ号为耦合剂的低强度脉冲超声透入治疗,超声组每日于骨折部位予以普通超声耦合剂的低强度脉冲超声治疗,对照组行假刺激.分别于术后第3,6,9,12,15天取骨痂行采用RT-PCR和免疫组织化学染色法检测骨形态发生7 mRNA表达和平均吸光度值变化,第4和6周行X射线片检查以观察不同治疗方法对骨缺损愈合的疗效.结果与结论:X射线检测结果显示中药组骨折愈合效果最好.中药组骨形态发生蛋白7 mRNA表达和平均吸光度值均高于超声组和对照组(P < 0.05),在术后第9和12天最较明显(P < 0.01).结果证实,中药低强度脉冲超声透入疗法能促进兔桡骨骨折的修复、缩短骨折修复时间,其修复作用与促进骨形态发生蛋白7表达有关.     

低强度脉冲超声波促进大鼠胫骨骨折愈合中血小板衍生生长因子和胰岛素样生长因子1的表达

背景：有研究表明低强度脉冲超声波可以促进骨折愈合。目的：观察低强度脉冲超声波对胫骨骨折大鼠血小板衍生生长因子及胰岛素样生长因子1表达的影响。方法：实验采用单侧大鼠胫骨制作闭合骨折大鼠模型,建模后每日进行低强度脉冲超声波治疗。结果与结论：建模后14,21d,低强度脉冲超声波治疗的胫骨骨折模型大鼠骨痂量及厚度明显增高（P〈0.05）,建模后7,14d,其骨痂血小板衍生生长因子和胰岛素样生长因子1表达也明显增高（P〈0.05）。说明低强度脉冲超声波促进大鼠胫骨骨折愈合中血小板衍生生长因子和胰岛素样生长因子1的表达,促进早期软骨细胞成熟和软骨内成骨,从而促进骨折的愈合。     

低强度脉冲超声波促进大鼠胫骨骨折愈合中血小板衍生生长因子和胰岛素样生长因子1的表达

背景:有研究表明低强度脉冲超声波可以促进骨折愈合。目的:观察低强度脉冲超声波对胫骨骨折大鼠血小板衍生生长因子及胰岛素样生长因子1表达的影响。方法:实验采用单侧大鼠胫骨制作闭合骨折大鼠模型,建模后每日进行低强度脉冲超声波治疗。结果与结论:建模后14,21d,低强度脉冲超声波治疗的胫骨骨折模型大鼠骨痂量及厚度明显增高(P<0.05),建模后7,14d,其骨痂血小板衍生生长因子和胰岛素样生长因子1表达也明显增高(P<0.05)。说明低强度脉冲超声波促进大鼠胫骨骨折愈合中血小板衍生生长因子和胰岛素样生长因子1的表达,促进早期软骨细胞成熟和软骨内成骨,从而促进骨折的愈合。     

脉冲电磁场对山羊骨延长区骨矿化的影响

目的 研究脉冲电磁场对山羊骨延长区骨矿化的影响。方法 选用１２只山辜制成胫骨上干骺端骨痂牵伸延长模型,并随机分为对照组和脉冲电磁场治疗组,治疗组术后第一天即给予脉冲电磁场刺激,行功能指标检测、形态学分析、Ｘ线片计算机图像分析、骨痂线密度、面密度、灰密度测量及骨痂钙元素、微量元素含量测定。结果 治疗组伤肢功能恢复显优于对照组（Ｐ〈０．０１）;治疗组骨痂线密度、面密度、灰密度、骨痂钙元素及锌、铜、铁等微量元素含量显高于对照组（Ｐ〈０．０５）。结论 脉冲电磁场刺激能够加速骨延长区新骨的生成与矿化,其机制可能是脉冲电磁场刺激加速了延长区骨修复细胞的成骨活动,促进了骨痂中微量元素的聚积。     

中药接骨Ⅰ号治疗超声透入桡骨骨折愈合过程中骨形态发生蛋白7的表达

背景：将低强度脉冲超声波与中药相结合,即中药超声透入疗法,在动物实验和临床中用于骨折的治疗并取得了确切的疗效,然而其促进骨折愈合机制的研究却少有报道。目的：观察中药超声透入对新西兰大白兔桡骨中段骨缺损愈合过程中骨形态发生蛋白7表达的影响。方法：84只兔构建双侧桡骨中段骨缺损的骨折模型,建模成功后随机分成3组：中药组给予以接骨Ⅰ号为耦合剂的低强度脉冲超声透入治疗,超声组每日于骨折部位予以普通超声耦合剂的低强度脉冲超声治疗,对照组行假刺激。分别于术后第3,6,9,12,15天取骨痂行采用RT-PCR和免疫组织化学染色法检测骨形态发生7mRNA表达和平均吸光度值变化,第4和6周行X射线片检查以观察不同治疗方法对骨缺损愈合的疗效。结果与结论：X射线检测结果显示中药组骨折愈合效果最好。中药组骨形态发生蛋白7mRNA表达和平均吸光度值均高于超声组和对照组（P〈0.05）,在术后第9和12天最较明显（P〈0.01）。结果证实,中药低强度脉冲超声透入疗法能促进兔桡骨骨折的修复、缩短骨折修复时间,其修复作用与促进骨形态发生蛋白7表达有关。     

低强度超声对骨愈合和骨融合的影响

超声是以声波形式通过生物组织传递，作为一种临床诊断手段已被人们广泛接受，且技术已经较为成熟。自从1952年意大利Corradic发现持续低强度超声（Low Intensity Uhrasotmd，LIUS）治疗兔桡骨骨折可促进骨痂的形成后，国际上对HUS在促进骨愈合骨融合方面的研究在持续进行中，且取得新进展，作者综述如下。     

重组人骨形态发生蛋白2局部注射促进兔胫骨延长区的骨愈合

目的:骨形态发生蛋白与骨延长关系的报道很少。为观察其对于牵拉骨再生延长区骨愈合的作用,将重组人骨形态发生蛋白2经皮注射到兔胫骨延长区,行组织学验证。方法:实验于2004-06/2005-03在吉林大学基础医学院实验动物中心完成。①实验材料:雄性日本大耳白兔30只;重组人骨形态发生蛋白2(由美国哈佛医学院分子骨科中心Oliver博士馈赠)。②实验干预及分组:制作日本大耳白兔延长区骨再生不良动物模型24只,行快速延长,2次/d,1mm/次,2mm/d,共延长10d,共2cm。将兔子随机分为2组,每组12只。对照组,延长结束后延长区注射醋酸盐缓冲液;重组人骨形态发生蛋白2组,延长结束后延长区注射醋酸盐缓冲液溶解的重组人骨形态发生蛋白2。③实验评估:延长结束后2周、4周时2组各取4只兔,麻醉后处死,进行延长区拍摄X射线片及延长区骨标本组织学光镜检查。结果:术中兔死亡及针道处骨折6只,24只进入结果分析。①延长区X射线片评价:延长结束后2周和4周时,显示重组人骨形态发生蛋白2组新生骨痂明显多于对照组,延长结束后4周时重组人骨形态发生蛋白2组髓腔开始形成。②延长区组织学观察结果:延长结束后4周时,对照组延长区周新生软骨、骨增多,融合成片,出现编织骨。重组人骨形态发生蛋白2延长区软骨细胞吸收矿化,转化为编织骨,并逐渐向皮质骨改建塑形。结论:牵拉骨再生延长结束后,延长区经皮注射重组人骨形态发生蛋白2,经间接X射线及组织学直接验证,对延长区骨愈合有促进作用。     

牵引成骨小鼠早期骨折愈合

背景:牵引成骨技术治疗四肢骨缺损和发育不良等有一定的优势,但是有关骨延长区新骨的成骨方式,目前尚存在争议.目的:观察小鼠胫骨骨折愈合过程中与成骨方式有关的骨基质蛋白组织学与基因水平的表达,论证在外界牵张力的作用下骨折愈合方式.设计、时间及地点:组织学和蛋白基因检测,于2007-08/12在中南大学第三附属医院中心实验室完成.材料:8周龄雄性CD-1小鼠36只,体质量25～30 g,由中南大学湘雅医学院实验动物中心提供.方法:接受左胫骨中上段骨干横行截骨,安置特制延长外固定架,胫骨牵引过程包括5 d静止期,12 d牵引期和70 d固塑期,牵引期的牵引速率为0.1 mm/次,共0.2 mm/d.术后于5,9,13,17,24,31 d采集左胫骨标本,分别作组织学检查和骨基质蛋白mRNA检测.主要观察指标:①小鼠胫骨牵引成骨模型骨折端组织学变化.②小鼠胫骨牵引成骨骨痂内各种基质蛋白mRNA在不同时间点的表达.结果:组织学检查显示:静止期其修复过程基本与骨折愈合相似;牵引期,被牵引骨痂显示3个典型的生物力学功能区:纤维间区,初始骨基质前沿和微骨柱形成区;固塑早期,牵引骨痂骨性愈合.mRNA检测显示:Ihh在牵引后的第4天可检测到表达,静止期及牵引后期无明显表达.Ⅱ型胶原从截骨后第5天即可检测,直到固塑期1周,但其mRNA表达逐渐减弱,到固塑期第2周即停止表达.X型胶原的表达在牵引期第4天达到高峰,然后逐步下降.骨钙素在截骨第5天尚不能被检测,其mRNA的表达在牵引后期和固塑期早期达高峰.结论:胫骨牵引静止期软骨内成骨和膜内成骨同时存在,但在机械牵张力作用下转为以膜内成骨为主的成骨过程.     

低强度超声促进骨折愈合的实验研究

目的观察低强度超声对骨折愈合的影响。方法手术造成32只新西兰大白兔双侧桡骨中段3mm标准骨缺损,随机分成两组进行研究。治疗组对其骨折部位施以低强度超声治疗,对照组不加任何治疗。术后第2,4,6,8周进行X线摄片及组织病理学对比观察。结果实验组的X线摄片、组织学观察显示骨折修复优于对照组。结论低强度超声能够刺激骨痂形成,促进骨折愈合可望成为一种非手术治疗骨不连和骨折延迟愈合的新方法。     

CR钼靶X射线对应用抗骨增生胶囊治疗骨折模型大鼠骨痂密度的评价

目的:抗骨增生胶囊中含有多种补肾壮骨中药成分,在治疗骨折方面罕见报道.钼靶X射线检查是乳腺检查和疾病诊断的常用、有效方法之一.采用钼靶X射线测量大鼠骨折模型骨痂的密度值,以探讨抗骨增生胶囊对骨折愈合的促进作用.方法:实验于2005-07/2006-03在河北医科大学第二医院实验中心完成.①实验分组:清洁级Wistar大鼠60只,雌雄各半,体质量170～200g,随机数字表法分为对照组和实验组,每组30只.②实验方法:制作胫骨骨折模型,实验组给予抗骨增生胶囊,取胶囊倒出药粉称质量配成1mL悬浊液,14.00～15.75mg/只,使用鼠灌胃器,喂药1次/d:对照组喂服等量9g/L氯化钠注射液,1次/d.③实验评估:分别于术后3,10,17,24,33d分批过量麻醉处死,游标卡尺测量骨痂直径:CR钼靶X射线片通过图像分析软件计算出骨痂的密度值.结果:纳入大鼠60只,均进入结果分析.①骨痂密度:钼靶X射线照片骨纹清晰,骨与软组织对比明显,在骨折愈合过程中实验组与对照组相比骨痂区域的密度值差异有显著性意义(P＜0.05).②骨痂直径:3d时骨折端无明显骨痂形成.10,17,24,33d实验组开始形成大量骨痂,对照组有骨痂形成但数量少.两组比较差异均有显著性意义(P＜0.01).结论:钼靶X射线照片结合计算机图像分析是测量大鼠骨痂愈合程度的有效工具.     

低强度脉冲超声波联合牙周组织翻瓣移植修复Beagle犬骨上缺损型牙周病

背景:近年来,低强度脉冲超声波联合牙周翻瓣对骨上缺损型牙周炎组织的修复效应的尚未见报道。目的:观察低强度脉冲超声波联合牙周翻瓣术对Beagle犬骨上缺损型牙周炎组织的修复效应。方法:在4只Beagle犬左下颌第二、三、四前磨牙处构建骨上缺损型牙周炎模型,经改良Widman翻瓣及根面处理后随机分为对照组(0mW/cm2)和ISATA30mW/cm2×20min/d、ISATA60mW/cm2×20min/d低强度脉冲超声波处理组。结果与结论:低强度脉冲超声波处理前后各组牙龈组织温度变化值差异无显著性意义(P>0.05);6周后对照组和低强度脉冲超声波处理组,以及处理组之间在牙周临床指标上差异无显著性意义(P>0.05);组织学切片苏木精-伊红染色显示低强度脉冲超声波处理组成骨细胞增生及骨陷窝较对照组明显,尤其是ISATA30mW/cm2×20min/d低强度脉冲超声波处理组;Masson染色显示低强度脉冲超声波处理组红染较对照组明显;低强度脉冲超声波处理组和对照组均形成长结合上皮。提示低强度脉冲超声波具有潜在的促骨上缺损型牙周炎组织修复作用。     

低强度脉冲超声波对Beagle犬Ⅱ度根分叉病变的辅助效应

背景:低强度脉冲超声波主要用于促进骨折愈合及提高新生骨组织密度,尚未见其对Ⅱ度根分叉病变组织学修复效应的报道。目的:观察低强度脉冲超声波对Ⅱ度根分叉病变的组织学修复效应。方法:5只Beagle犬各取2颗下颌第4前磨牙颊侧作为实验区,建立Ⅱ度根分叉病变模型,分别分为超声组和对照组并在颊侧缺损龈方根面制备切迹作为参照点,高糖饲养8周。超声组行根面平整术,术后1周行低强度脉冲超声波(90mW/cm21.5MHz,200μs,1kHz)处理20min/d,对照组行根面平整术和假处理。6周后取实验区域组织制备脱钙切片,苏木精伊红染色、Masson染色行根分叉区域组织测量分析。结果与结论:超声组根分叉区新生牙槽骨沿根面生长,根分叉区可见牙龈上皮;对照组根分叉区以结缔组织生长为主,可见大量牙龈上皮和少量新生骨组织。超声组新生牙槽骨胶原呈"红-蓝"相间,以红染为主;对照组呈现蓝染,即超声组的新生牙槽骨胶原较对照组成熟。超声组牙槽骨、牙骨质、牙周膜新生量均大于对照组(P<0.05)。提示90mW/cm2低强度脉冲超声波可促进根分叉病变新生牙槽骨胶原成熟和改建。     

Low-Intensity Pulsed Ultrasound Attenuates Periodontal Ligament Cells Apoptosis by Activating Yes-Associated Protein-Regulated Autophagy

ObjectiveThe goal of the work described here was to determine if low-intensity pulsed ultrasound (LIPUS) has an anti-inflammatory effect on lipopolysaccharide (LPS)–induced inflammation in periodontal ligament cells (PDLCs). The mechanism underlying this effect remains to be explored and is likely related to PDLC apoptosis regulated by Yes-associated protein (YAP) and autophagy.MethodsTo verify this hypothesis, we used a rat model of periodontitis and primary human PDLCs. We examined alveolar bone resorption in rats and apoptosis, autophagy and YAP activity in LPS-treated PDLCs with and without application of LIPUS by cellular immunofluorescence, transmission electron microscopy and Western blotting. Then, siRNA transfection was used to decrease YAP expression to confirm the regulatory role of YAP in the anti-apoptotic effect of LIPUS on PDLCs.DiscussionWe found that LIPUS attenuated alveolar bone resorption in rats and this was accompanied by YAP activation. LIPUS inhibited hPDLC apoptosis by YAP activation, and promoted autophagic degradation to help autophagy completion. These effects were reversed after YAP expression was blocked.ConclusionLIPUS attenuates PDLC apoptosis by activating Yes-associated protein-regulated autophagy.     

Low-Intensity Pulsed Ultrasound Promotes Osteogenic Differentiation of Reamer-Irrigator-Aspirator Graft-Derived Cells in Vitro

Recently, reamer–irrigator–aspirator (RIA) systems have been increasingly used to harvest autologous bone grafts. RIA graft materials contain bone marrow, which provides a viable source to derive large numbers of mesenchymal stem cells. Low-intensity pulsed ultrasound (LIPUS) significantly accelerates the differentiation of stem cells derived from bone marrow. This in vitro study investigated the effect of LIPUS on the osteogenic activity and differentiation of RIA graft-derived cells. A small amount of RIA graft was obtained from seven patients. After the cells derived from RIA grafts were cultured, they were divided into two groups: the LIPUS and control groups. LIPUS was applied once daily for 20 min (1.5 MHz, pulse duration: 200 µs, pulse repetition rate: 1 kHz, spatial average-temporal average intensity: 30 mW/cm²). Alkaline phosphatase activity (113.4% and 130.1% on days 7 and 14), expression of osteoblast-related genes (ALP, Runx2) and mineralization (135.2% on day 21) of the RIA graft-derived cells were significantly higher in the LIPUS group than in the control group. However, LIPUS did not affect the cell proliferation of RIA graft-derived cells. This study indicates that LIPUS may enhance the healing of non-union and critical bone defects treated by autologous bone grafting using the RIA system.     

低强度脉冲超声波对Beagle犬Ⅱ度根分叉病变的辅助效应

背景：低强度脉冲超声波主要用于促进骨折愈合及提高新生骨组织密度,尚未见其对Ⅱ度根分叉病变组织学修复效应的报道。目的：观察低强度脉冲超声波对Ⅱ度根分叉病变的组织学修复效应。方法：5只Beagle犬各取2颗下颌第4前磨牙颊侧作为实验区,建立Ⅱ度根分叉病变模型,分别分为超声组和对照组并在颊侧缺损龈方根面制备切迹作为参照点,高糖饲养8周。超声组行根面平整术,术后1周行低强度脉冲超声波（90mW/cm21.5MHz,200μs,1kHz）处理20min/d,对照组行根面平整术和假处理。6周后取实验区域组织制备脱钙切片,苏木精伊红染色、Masson染色行根分叉区域组织测量分析。结果与结论：超声组根分叉区新生牙槽骨沿根面生长,根分叉区可见牙龈上皮;对照组根分叉区以结缔组织生长为主,可见大量牙龈上皮和少量新生骨组织。超声组新生牙槽骨胶原呈＂红-蓝＂相间,以红染为主;对照组呈现蓝染,即超声组的新生牙槽骨胶原较对照组成熟。超声组牙槽骨、牙骨质、牙周膜新生量均大于对照组（P〈0.05）。提示90mW/cm2低强度脉冲超声波可促进根分叉病变新生牙槽骨胶原成熟和改建。     

Low-Intensity Pulsed Ultrasound for the Treatment of Bone Delayed Union or Nonunion: A Review

The goal of this review is to present the most updated knowledge derived from basic science, animal studies and clinical trials, concerning biophysical stimulation of bone repair through low-intensity pulsed ultrasound (LIPUS), with particular reference to the management of delayed unions and nonunions. Low-intensity pulsed ultrasound LIPUS has been proved to significantly stimulate and accelerate fresh fracture healing in animal studies and in randomized controlled clinical trials. LIPUS also appears as an effective and safe home treatment of aseptic and septic delayed-unions and nonunions, with a healing rate ranging from 70% to 93% in different, nonrandomized, studies. Advantages of the use of this technology that may avoid the need for additional complex operations for the treatment of nonunions, include efficacy, safety, ease of use and favourable cost/benefit ratio. Outcomes depend on the site of nonunion, time elapsed from trauma, stability at the site of nonunion and host type. The detailed biophysical process by which low-intensity pulsed ultrasound LIPUS stimulates bone regeneration still remains unknown, even if various effects on bone cells in vitro and in vivo have been described. (E-mail: carlo.romano@grupposandonato.it)     

Low-intensity pulsed ultrasound produced an increase of osteogenic genes expression during the process of bone healing in rats

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery.     

Ultrasound Enhances Dentoalveolar Remodeling in an Ex Vivo Orthodontic,Ovariectomy-Induced Osteoporotic Model

The aim of the study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on dentoalveolar structures during application of force to a cultured mandible slice taken from an ovariectomized rat model of osteoporosis. Rats were divided based on whether they had ovariectomy and/or LIPUS application into four groups: control osteoporosis group, control normal group, ultrasound-treated osteoporosis group and ultrasound-treated normal group. The mandibles were dissected, sliced and cultured before application of a 0.5-N force. Tissue specimens from five rats per group received LIPUS; the remaining rats served as untreated controls. Tissue sections were evaluated histologically and histomorphometrically. Osteoporosis significantly affected the alveolar bone without any effect on the dentin–pulp complex. LIPUS enhanced osteoporotic alveolar bone remodeling and increased cementum and predentin thickness. Furthermore, LIPUS application significantly increased odontoblast and periodontal ligament cell counts (p < 0.05) in both groups. Therefore, LIPUS enhances alveolar bone remolding and increases cementum and predentin formation in osteoporotic rat mandible slice organ cultures.     

Acceleration of Bone Defect Healing and Regeneration by Low-Intensity Ultrasound Radiation Force in a Rat Tibial Model

The objective was to evaluate the effect of low-intensity pulsed ultrasound (LIPUS)-induced acoustic radiation force on trabecular bone defect repair and healing in a rat tibial model. A uniform surgical defect, 3.5 mm in diameter, was generated in the proximal bilateral tibial region of rats (N?=?20). LIPUS was applied to the defects in the left tibia for 20 min every day for 2 wk. Contralateral defects in the right tibia served as a control without active LIPUS treatment. The micro-computed tomography data revealed that LIPUS-treated tibia exhibited higher bone volume/total volume, connectivity density, trabecular number, and bone mineral density and significantly lower trabecular separation. Histomorphometry analysis indicated a similar trend. Mechanical testing data revealed that LIPUS treatment significantly increased bone stiffness relative to that of the control group. Short-term (2-wk) LIPUS therapy initiated trabecular bone repair and regeneration in large trabecular bone defects, whereas cortical bone remained in the initial non-mineralization stage.