The Effect of Material Surface Microstructure on the Enhancement of Bone-Bonding Ability of a Hydroxyapatite-Implant by Low-Intensity Pulsed Ultrasound (LIPUS)

Excellent firm bonding between the biomaterials and bone tissue (osseointegration and osteo-conductivity) has been desired for the stability in vivo of dental implants and artificial joints. Much has been learned about this concept, which has led to significant improvements in the design and surface modification of implants in the field of implant dentistry, orthopedic surgery. We have already reported that low-intensity pulsed ultrasound (LIPUS) irradiation can accelerate the bone bonding ability of the bio-conductive materials such as bioactive titanium and hydroxyapatite implant. However, it is still unclear whether the LIPUS could have same effect to different types of the bioactive-materials. Therefore, in this study, the differences of bone-like hydroxyapatite formation on some kind of hydroxyapatite surface in simulated body fluid (SBF) under the LIPUS irradiation were investigated. Two kinds of hydroxyapatite samples immersed in SBF was exposed to ultrasound waves, the bone-like apatite on the surface was analyzed by Scanning electron microscopy and X-ray diffraction. As a result, the enhancement of hydroxyapatite formation on the surface by LIPUS was confirmed, the initial epitaxial nucleation and crystal growth of apatite depended on crystal structure of the surface of matrix materials.

Effect of low intensity pulsed ultrasound on expression of TIMP-2 in serum and expression of mmp-13 in articular cartilage of rabbits with knee osteoarthritis

Objective:To study the effect of low intensity pulsed ultrasound(LIPUS) on the expression of tissue inhibitor of metalloproteinase-2(TIMP-2) in the serum and expression of matrix metallopeptidase 13(MMP-13) in the articular cartilage cells of rabbits with knee osteoarthritis(OA).Methods:Inner patellar ligament defect method was used to establish the model of knee OA.Four weeks after the modeling,the arterial blood was drawn from the ear of each rabbit,while ELISA was employed to detect the expression of TIMP-2 in the serum.The chondrocytes were separated from animals in each group and then cultured in vitro.All rabbits were divided into control group,OA model group and OA + LIPUS group.Cells in the control and OA groups were not treated,while cells in the OA+ LIPUS group were treated with LIPUS(40 mW/cnr.1 time/day).Cells were collected 7 d later and the RNA and total protein were extracted respectively.Real-time PCR and Western blotting were employed to analyze the expression of MMP-13 in chondrocytes at the mRNA and protein level,respectively.Results:The success rate of establishment of OA model was 83%.The results of ELISA showed that the content of TIMP-2 in the serum of animals with OA was 22.3%,lower than the one in the control group(P<0.05).Compared with the normal control group,the expression of TIMP-2in the OA model group was significantly increased,while the expression of MMP-13 was significantly increased(P<0.05).After the stimulation of LIPUS,the expression of TIMP-2 and MMP-13 was close to the one in the normal control group.Conclusions:The inner patellar ligament defect method is a mature method to establish the rabbit OA model,with high success rate.The expression of serum TIMP-2 in the OA model group is significantly decreased.LIPUS can up-regulate TIMP-2 and down-regulate MMP-13.

Osteogenic Effect of Low Intensity Pulsed Ultrasound on Rat Adipose-derived Stem Cells in vitro

The osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adi-pose-derived stem cells (ADSCs) was investigated.Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro,and then the alkaline phos-phatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs.To further explore the underlying mechanism,the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st,3rd,5th,7th day after exposure repectively.Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively.It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro.LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2),osteocalcin (OCN),ALP and bone sialoprotein (BSP) genes than con-trols,and the protein levels of Runx2 and BSP were also increased.The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.

Effect of low-intensity pulsed ultrasound on posterolateral lumbar fusion of rabbit

Objective:To observe the effect of low-intensity pulsed ultrasound in the posterolateral Lumbar fusion of rabbit.Methods:A total of 48 New Zealand white rabbits were randomly divided into the observation group and control group,including the autogenous iliac observation group.artificial bone observation group,artificial bone control group and autogenous iliac control group according to the different bone grafting.The posterolateral lumbar fusion of rabbits in each group was analyzed and compared.Results:After 4 weeks of treatment using the low-intensity pulsed ultrasound,the fusion in the bone grafting area of observation group was good.There was the relatively dense fusion area between the right transverse process and artificial bone.The left transverse process had been completely fused,with the clear bone trabecula through the fusion area.There was no significant fusion sign in the control group.According to the fusion comparison between two groups,the fusion rale of the observation group was 83.3%and it was significantly higher than the one of the control group(37.5%).Results of the comparison were statistically significant(P<0.05).The number of chondrocytes and the increase in the relative gray-scale in the fusion area of the iliac observation group were significantly higher than ones of the iliac control group(P<0.05).The number of chondrocytes and the increase in the relative gray-scale in the fusion area of the artificial bone observation group were significantly higher than ones of the artificial bone control group(P<0.05).The expression of IL-1.IL-6 and IL-17 4 weeks after the treatment of the observation group was significantly lower than the one of the control group,with the statistical significance(P<0.05).It indicated that the treatment of low-intensity pulsed ultrasound could reduce the expression level of inflammatory factors.Conclusions:The low-intensity pulsed ultrasound can significantly increase the bone grafting fusion rate of the rabbit's posterolateral lumbar fusion.The possible mechanism is that it promotes the lumbar posterolateral endochondral ossification and reduces the inflammatory reaction.

Effect of Low-Intensity Pulsed Ultrasound on Ischaemic Heart Disease Patients (Novel Technique)

Ischaemic Heart Disease (IHD) or Coronary heart disease means that the heart is not getting enough blood and oxygen supply through the coronary arteries. The most common cause of this disease is the process of atherosclerosis in the coronary arteries. Although significant progress has been made in the management of ischaemic heart disease (IHD) The number of severe IHD patients is increasing. The treatment options for IHD have not changed much over the last three decades, which is divided between medications, coronary Angioplasty and Coronary artery bypass surgery. Thus it was crucial to develop new, non-invasive therapeutic strategies in case of Failure of medical or interventional therapy or in case patient is not fit for surgery or angioplasty. In this study, we are pleased to reveal a novel technique that was carried out on a human model. We aimed to develop low-intensity pulsed ultrasound (LIPUS) therapy for the treatment of patients with Ischaemic Heart Disease. We have set up the inclusion and exclusion criteria, the treatment protocol of LIPUS on IHD patients. In this limited group of IHD patients, We found promising clinical results and improvement on myocardial functions.

1.5 MHz LIPUS无线骨折治疗仪设计

低强度脉冲超声波作为一种骨折辅助治疗手段,与传统治疗方式相比具有很多重优势。目前,市面上缺少多参数可调的低强度超声治疗设备,因此设计一款1.5 MHz低强度脉冲超声多参数可调治疗仪。该治疗仪以STM32单片机为主控芯片,可以输出超声频率为1.5 MHz、脉冲频率为1 kHz,声强、脉冲占空比及治疗时长均可调的低强度脉冲超声波,具有空载保护功能。用户可以通过蓝牙App实时查看和调节仪器的运行状态和输出参数。该App能在蓝牙意外断连后自动重连并正确显示所有信息。实验结果表明,设计的超声波治疗仪输出波形完整,超声输出频率、超声输出功率和脉冲占空比精确稳定,为临床应用奠定了基础。

LIPUS的协同作用及在口腔中的运用

低强度脉冲超声(low⁃intensity pulsed ultrasound,LIPUS)由于其安全有效和无创伤性被引入临床,最早用于骨折的治疗,随后,大量研究将LIPUS作为协同因素作用于骨、软骨、关节、癌症的治疗以及口腔的临床研究中,共同揭示了LIPUS的强大协同效应,本文总结了LIPUS在以上研究领域中的协同作用,以期拓宽LIPUS可能的临床运用范围。

LIPUS对骨性关节炎动物模型中炎症因子、细胞凋亡及整合素信号通路的影响

目的:研究低强度脉冲超声波(LIPUS)对骨性关节炎动物模型中炎症因子、细胞凋亡及整合素信号通路的影响。方法:选择雄性新西兰大白兔作为实验动物并随机分为假手术组(Sham)组、骨关节炎模型组(OA组)、LIPUS干预组(LIPUS组),建立右后肢膝关节的骨性关节炎动物模型后给予LIPUS干预。干预后6周,分离关节软骨并检测炎症因子、细胞凋亡分子及整合素信号通路分子的表达量。结果:OA组实验动物关节软骨中骨桥蛋白(OPN)、一氧化氮(NO)、白介素(IL)-1β、肿瘤坏死因子(TNF)-α、Fas、FasL、LC3-II、Beclin-1、Integrinβ1、FAK、ERK1/2、JNK、p38MAPK、基质金属蛋白酶(MMP)-1、MMP-3的蛋白表达量显著高于Sham组,Col-I、Col-II的蛋白表达量显著低于Sham组;LIPUS组实验动物关节软骨中OPN、NO、IL-1β、TNF-α、Fas、FasL、LC3-II、Beclin-1、Integrinβ1、FAK、ERK1/2、JNK、p38MAPK、MMP-1、MMP-3的蛋白表达量显著低于OA组,Col-I、Col-II的蛋白表达量显著高于OA组,差异均有统计学意义(P<0.05)。结论:LIPUS对骨性关节炎动物模型关节软骨中的炎症反应、细胞凋亡、整合素信号通路具有抑制作用,能够促进关节软骨的修复。

有限元法仿真LIPUS照射骨细胞的局部声场分布

低强度脉冲超声(Low-Intensity Pulsed Ultrasound,LIPUS)作为一种可用于治疗骨病的潜在物理疗法,可以有效避免药物的不良反应,且具有无创伤、无电离辐射等优点,因而有着广阔的应用前景。应用有限元法(Finite Element Method,FEM)仿真研究LIPUS照射骨细胞时的局部声场分布,从微观角度探究骨细胞与声场间的相互作用以及各超声参数和照射位置对于声场分布的影响。首先建立了骨细胞模型,然后在不同位置施加超声激励并改变超声的激励声压与频率,计算不同情况下的声场分布,最后对仿真结果进行综合分析与比较。结果表明,骨细胞会在一定程度上影响声场的分布,且超声的各个参数及照射位置均对声场有一定的影响。仿真结果从声压与能量的角度阐述了LIPUS治疗骨病的潜在物理机制,另一方面也为离体细胞实验中LIPUS的参数筛选和照射方式提供理论参考。

Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery

Background A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts.Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds,enhanced production of bone marrow cells,and improved tissue-engineered bone formation.The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells （BMSCs）.Methods BMSCs/β-tricalcium phosphate （β-TCP） composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound （LIPUS） treatment group and a control group.These implants were harvested at 5,10,25,and 50 days after implantation.The samples were then biomechanically tested and analyzed for alkaline phosphate （ALP） activity and osteocalcin （OCN） content and were also observed by light microscopy.Results The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group.Histomorphometric analysis revealed a greater degree of soft tissue repair,increased blood flow,better angiogenesis,and more extensive bone formation in the LIPUS groups than in the controls.No significant difference in the compressive strength was found between the two groups.Conclusion LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/β3-TCP composites.

低强度脉冲超声波对髁突软骨细胞炎症状态的作用研究

目的 观察低强度脉冲超声波对白介素-1β制造的大鼠髁突软骨细胞炎症模型中MAPK信号通路相关因子磷酸化的表达变化。方法 二次酶消化法分离髁突软骨细胞并完成鉴定。将分离出的大鼠髁突软骨细胞随机分为3组,加入3种不同浓度的IL-1β处理并进行Ⅱ型胶原与MMP-13免疫细胞化学染色,筛选出最佳致炎浓度。再次将所提取的大鼠髁突软骨细胞随机分为3组:NC组、OA组、LIPUS组,处理后对Ⅱ型胶原、p38、p-p38、ERK1/2、pERK1/2进行免疫细胞化学染色,观察蛋白表达情况。结果(1)成功提取大鼠髁突软骨细胞。(2)IL-1β:10 ng/mL组Ⅱ型胶原表达量降低最明显,差异有统计学意义(P<0.05),MMP-13表达量升高最明显,差异有统计学意义(P<0.05)。(3)OA组与LIPUS组Ⅱ型胶原表达量均降低,差异有统计学意义(P<0.05),OA组降低更明显,差异有统计学意义(P<0.05)。3组的p38、ERK1/2表达量无明显差异(P>0.05)。OA组与LIPUS组p-p38、p-ERK1/2表达量均升高,差异有统计学意义(P<0.05),OA组升高更明显,差异有统计学意义(P<0.05)。结论(1)二次酶消化法成功提取大鼠髁突软骨细胞。(2)10 ng/mL为IL-1β最佳致炎浓度。(3)LIPUS处理可减少炎性髁突软骨细胞中Ⅱ型胶原的降解,抑制了MAPK通路相关因子p38、ERK1/2的磷酸化。

一种用于尿道瘢痕防治动物实验的超声探头设计

目的 尿道瘢痕是患者尿道手术后常见的并发症之一,并且难以通过手术治疗方法清除。低强度脉冲超声(low intensity pulse ultrasound,LIPUS)在抑制纤维化,抑制炎症方面有十分突出的表现,为超声防治尿道瘢痕提供了理论的可能性。尽管如此,目前并没有对超声用于尿道瘢痕的防治作用的报道,另外有关超声抑制纤维化、抑制炎症的作用机制也仍需要进一步的研究。本文针对新西兰雄兔尿道重建模型设计了一种超声探头,用于LIPUS抑制尿道瘢痕疗效和作用机制的动物实验研究。方法 根据兔阴茎的尺寸与形状设计探头结构,通过有限元仿真预测探头声场,用3D打印技术制造原型机。最后对探头声场进行测试,并且用于动物实验验证其实用性。结果 探头超声频率为2 MHz,治疗腔长度44 mm,直径8 mm。治疗腔沿长度方向上有3个紧邻的治疗区,每个治疗区的半功率长度约6 mm,治疗区内的I_(sata)(时间平均空间平均声强)可通过前端驱动器电源电压进行调节,当电源电压为30 V时,I_(sata)大于0.87 W/cm^(2)。结论 本文设计的超声探头体积小、重量轻、声场均匀,可以对动物尿道实施稳定有效的超声照射,为超声治疗尿道瘢痕动物实验提供了一种有用的工具。

低强度体外冲击波/脉冲式超声波治疗勃起功能障碍中国专家共识

勃起功能障碍(ED)是成年男性的常见病和多发病,目前国内外ED诊疗指南推荐的口服药物、阴茎海绵体内血管活性药物注射和阴茎假体植入等治疗方案均不能针对ED病因,故无法从纠正发病机制方面彻底治愈ED。低强度体外冲击波疗法(Li-ESWT)和低强度脉冲式超声波(LIPUS)治疗ED,因其独特的作用机制和生物学效应,不仅能够改善患者的勃起功能,亦能针对病因治疗,促进阴茎功能康复。然而,目前临床上存在Li-ESWT和LIPUS治疗ED适应证不明确、治疗方案不统一、疗效差异大等问题,限制了该类疗法的进一步应用。据此,中国性学会组织本领域内多位临床专家,根据现有的Li-ESWT和LIPUS治疗ED的国内外循证医学证据,经共同研究、讨论达成一致,制定了本共识,旨在为从事Li-ESWT及LIPUS治疗ED领域的临床医生、设备研发人员提供规范的治疗方法和参考。

低强度脉冲超声在骨骼肌肉及运动神经系统中的生物学效应研究进展

低强度脉冲超声(LIPUS)是一种成本低、非侵袭性且安全性高的治疗方式,主要应用于骨骼肌肉系统疾病的治疗,尤其是骨折与骨不连的治疗。本文综述了LIPUS在多种骨骼肌肉及运动神经系统疾病中的治疗作用,并分析其可能的内在机制和潜在靶点,发现除了骨折与骨不连之外,LIPUS在治疗骨质疏松、肌肉损伤及运动神经系统疾病中同样具有临床应用前景。

超声优化水凝胶支架用于促进金纳米粒子的经皮递送

背景:金纳米粒子在多功能经皮给药系统的开发中具有重要意义,较小尺寸的金纳米粒子可通过细胞间路径向真皮层渗透,然而由于易团聚和胶体形态的限制,金纳米粒子存在着难以局限发挥效应、递送效率低的问题。目的:通过结合相变纳米液滴与生物黏附性水凝胶开发一种超声优化的水凝胶支架,用于金纳米粒子的经皮递送。方法:利用乳剂溶剂挥发法制备包载金纳米粒子的超声相变纳米液滴,并将其装载到聚多巴胺修饰的甲基丙烯酰化明胶水凝胶内制备复合水凝胶支架,对超声响应性纳米金载体进行结构与化学组分的表征,对复合水凝胶支架的微观结构、孔隙率、渗透性、流变学、体外止血及抗菌性能等进行表征。通过Live/Dead染色评价复合水凝胶支架的细胞相容性,并评估低强度脉冲超声对支架材料渗透性、孔隙率和力学性能的优化效果。结果与结论:①透射电镜和紫外-可见光光谱证明纳米金载体的成功构建,粒径及电位检测结果显示超声响应性纳米金载体具有较好的稳定性;②Live/Dead染色证明,复合水凝胶支架具有一定的生物相容性;③扫描电镜下可见复合水凝胶支架具有多孔网状结构,且部分大孔内部可见纳泡受超声辐照后产生的直径约2μm的微孔,结合渗透性实验说明强度脉冲超声可以优化水凝胶支架的孔隙和渗透性;复合水凝胶支架的止血性能优于止血海绵、聚多巴胺@甲基丙烯酰化明胶水凝胶支架;在低强度脉冲超声辐照下,复合水凝胶支架具有良好的抗氧化效应与抗菌性能;④热成像结果显示金纳米粒子包载在超声响应的纳泡中,在受到超声激励下可以得到更均匀的播散;⑤力学性能测试结果表明,负载含金纳米粒子的相变纳米液滴后水凝胶支架的储能模量增加,表现出更强的力学性能,断裂伸长率为122%,延展性优于不含金纳米粒子的相变纳米液滴组(P<0.05);⑥结果表明,复合水凝胶支架具备良好的生物相容性、抗菌性、抗氧化性及止血作用。

低强度脉冲超声对脂多糖诱导炎性活化的RAW264.7巨噬细胞迁徙和吞噬的影响

目的探讨低强度脉冲超声(LIPUS)对脂多糖(LPS)活化和未经活化的RAW264.7细胞迁徙和吞噬能力的影响。方法LPS诱导RAW264.7巨噬细胞建立体外炎症活化模型;通过CCK-8法研究LIPUS对不同环境下RAW264.7细胞活力的影响,划痕实验观察LIPUS对RAW264.7巨噬细胞迁徙的影响,Transwell实验观察LPS作为趋化因素时RAW264.7细胞趋化迁徙能力的变化情况,共聚焦显微镜及流式细胞术分析细胞内FITC荧光强度以探究巨噬细胞吞噬pHrodo Green标记的大肠埃希菌生物颗粒的能力。结果成功构建活化RAW264.7炎症模型,筛选LPS浓度为100 ng/mL;LIPUS抑制未活化的RAW264.7巨噬细胞向划痕区域内迁徙和未活化巨噬细胞的趋化迁徙;但在当前参数下LIPUS不影响巨噬细胞的细胞活力和吞噬能力。结论LIPUS抑制经LPS诱导的RAW264.7巨噬细胞的迁徙,但不影响其吞噬作用。

LIPUS调控FOXO1磷酸化促进氧化损伤的人牙周膜干细胞成骨分化的作用研究

该研究探讨低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)对氧化损伤的人牙周膜干细胞(human periodontal ligament stem cells,h PDLSCs)成骨分化的保护作用及机制。采用浓度为300μmol/L的H_(2)O_(2)构建细胞氧化损伤模型,将其分为对照组、H_(2)O_(2)组、LIPUS组和LIPUS+H_(2)O_(2)组。DCFH-DA探针和TBA比色法分别检测各组细胞活性氧(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)水平;碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色分别评估各组细胞早期及晚期成骨分化能力;WB(Western blot)评价抗氧化酶[过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase 2,SOD-2)]、成骨相关蛋白[矮小相关转录因子2(runt-related transcription factor-2,RUNX2)、ALP和骨桥蛋白(osteopontin,OPN)]、叉头框蛋白O1(forkhead box protein O1,FOXO1)和(phospho-FOXO1,p-FOXO1)表达水平。随后采用FOXO1抑制剂AS1842856(100 nmol/L)处理细胞,实验分为对照组、H_(2)O_(2)+二甲基亚砜(dimethyl sulfoxide,DMSO)组、LIPUS+H_(2)O_(2)+DMSO组和LIPUS+H_(2)O_(2)+AS1842856组。CCK-8(cell counting kit-8)法检测细胞活力;DCFH-DA探针检测各组细胞ROS水平,WB检测各组RUNX2、ALP和OPN的蛋白表达情况。结果发现与空白对照组相比,H_(2)O_(2)组ROS与MDA水平均升高,抗氧化酶蛋白表达下调(P<0.0001);ALP和茜素红阳性染色以及成骨相关蛋白表达水平均显著降低(P<0.0001);p-FOXO1/FOXO1蛋白值上升(P<0.0001)。与H_(2)O_(2)组比较,LIPUS+H_(2)O_(2)组的ROS与MDA水平均下降,抗氧化酶蛋白表达上调(P<0.0001);成骨染色及相关蛋白表达量显著增加(P<0.0001);p-FOXO1/FOXO1蛋白值降低(P<0.0001)。相较于LIPUS+H_(2)O_(2)+DMSO组,LIPUS+H_(2)O_(2)+AS1842856组细胞活力下降,p-FOXO1/FOXO1蛋白表达上调,ROS水平升高且成骨分化相关蛋白表达量显著降低(P<0.0001)。研究结果显示LIPUS通过调节FOXO1的磷酸化水平,发挥抗氧化防御作用并提高氧化损伤的h PDLSCs成骨分化能力。

聚焦式低强度脉冲超声联合富血小板血浆局部注射修复大鼠面神经损伤效果观察

目的观察聚焦式低强度脉冲超声(FLIPUS)联合富血小板血浆(PRP)局部注射对大鼠面神经损伤的修复效果。方法将20只健康雄性SD大鼠随机分为模型组、PRP组、FLIPUS组及PRP+FLIPUS组,每组5只。各组均建立右侧面神经损伤模型,PRP组于神经损伤处局部注射PRP 50μL,2次/周;FLIPUS组于神经损伤处进行FLIPUS治疗10 min,1次/天;PRP+FLIPUS组参照上法给予PRP局部注射及FLIPUS治疗;模型组不予特殊处理;各组疗程均为28 d。各组术后1~4周每周进行行为学评估,记录胡须运动和瞬目反射评分。各组术前及术后第28天,使用肌电诱发电位仪行右侧面神经电生理检查,记录损伤面神经的最大复合肌肉动作电位(CMAP)振幅并计算R值。各组大鼠处死后取损伤面神经节段组织,HE染色后进行组织病理学观察,同时记录单位面积的轴突数量、直径及面积。结果各组大鼠术后1~4周瞬目反射及胡须运动评分比较P均>0.05。与同组术前比较,各组大鼠术后损伤面神经的最大CMAP振幅均降低(P均<0.05)。与模型组比较,PRP组、FLIPUS组及PRP+FLIPUS组大鼠术后损伤面神经的最大CMAP振幅及R值均升高(P均<0.05),PRP组、FLIPUS组及PRP+FLIPUS组比较差异均无统计学意义(P均>0.05)。与模型组比较,PRP组、FLIPUS组及PRP+FLIPUS组神经束变性、脱髓鞘及轴突空泡化程度减轻,再生轴突增多且神经纤维排列更有序。与模型组比较,PRP组、FLIPUS组及PRP+FLIPUS组大鼠损伤面神经节段组织单位面积内的轴突数量均升高(P均<0.05);PRP组大鼠损伤面神经节段组织单位面积内的轴突直径、轴突面积均高于模型组、FLIPUS组、PRP+FLIPUS组(P均<0.05)。结论FLIPUS联合PRP局部注射对大鼠面神经损伤的修复作用不优于二者单独应用。

低强度脉冲超声对大鼠急性肌腱损伤早期血管生成的影响及机制

背景:越来越多研究表明,低强度脉冲超声可以促进急性肌腱损伤的愈合,但其具体机制尚不清楚。目的:观察低强度脉冲超声对急性肌腱损伤后早期血管生成的影响,并检测与血管内皮生长因子相关信号通路的调控关系,进而揭示其潜在作用机制。方法:8-12周龄SPF级雄性SD大鼠局部注射Ⅰ型胶原酶3 d建立急性跟腱损伤动物模型,随机分为超声组和对照组。超声组每日使用有效辐射面积为1 cm^(2)的超声小探头垂直于跟腱部位进行低强度脉冲超声治疗,对照组不干预。治疗2周后进行超声影像学检查,观察肌腱早期愈合情况;治疗1,2周后行苏木精-伊红染色和CD31免疫组织化学染色观察组织血管数量变化,Western blot和qRT-PCR检测跟腱组织中血管内皮生长因子相关信号通路分子的表达。结果与结论:①灰阶超声显示:超声组较对照组跟腱更为连续,回声强度更低且较为均匀,肌腱厚度明显降低(P<0.05);②苏木精-伊红染色和CD31免疫组织化学结果一致显示:治疗2周后,超声组的新生血管数量明显较同期对照组增多(P<0.05);③Western blot和qRT-PCR结果显示:治疗2周后超声组跟腱中血管内皮生长因子、Yes相关蛋白、血管生成素2、富含半胱氨酸的血管生成素诱导物61的蛋白和mRNA表达均显著高于对照组(P<0.05);④结果表明:低强度脉冲超声通过上调血管内皮生长因子表达显著增加了急性肌腱损伤早期血管生成数量,加速了肌腱愈合。

低强度脉冲聚焦超声对小鼠膝关节软骨细胞损伤中炎症因子和焦亡蛋白的影响

目的探讨低强度脉冲聚焦超声(FLIPUS)对脂多糖(LPS)诱导的小鼠膝关节软骨细胞损伤中炎症因子和细胞焦亡相关蛋白表达的影响及其保护软骨的作用机制。方法提取C57BL/6J小鼠膝关节原代软骨细胞进行体外实验,用1μg/mL LPS处理软骨细胞12 h模拟骨关节炎样软骨细胞损伤。细胞分为对照组(仅用含10%FBS的培养基培养,不予任何处理)、LPS组(LPS处理12 h)和LPS+FLIPUS组(LPS处理12 h后再用FLIPUS干预20 min)。采用蛋白质印迹法检测各组软骨细胞中Ⅱ型胶原蛋白α1(COL2α1)、炎症细胞因子(IL-18和IL-1β)、焦亡相关蛋白[核苷酸结合寡聚化结构域样受体3(NLRP3)、cleaved caspase 1、消皮素D N端片段(GSDMD-N)]、软骨基质降解因子[基质金属蛋白酶(MMP)13、MMP3和血小板结合蛋白基序的解聚蛋白样金属蛋白酶5(ADAMTS5)]的表达。采用钙黄绿素乙酰氧基甲酯(calcein AM)/PI荧光染色和乳酸脱氢酶(LDH)释放实验检测细胞活性,采用扫描电子显微镜观察细胞的形态变化,采用ELISA法检测细胞上清液中IL-1β、IL-18水平。结果成功提取小鼠膝关节原代软骨细胞并模拟骨关节炎样软骨细胞损伤。与对照组相比,LPS组小鼠膝关节软骨细胞中COL2α1蛋白表达降低(P<0.01),MMP13、MMP3、ADAMTS5、NLRP3、GSDMD-N、cleaved caspase 1、IL-18和IL-1β蛋白表达增加(均P<0.01),细胞上清液中IL-18、IL-1β水平升高和LDH释放增加(均P<0.01),细胞肿胀变形,细胞膜上出现较多孔隙,丧失完整性,细胞死亡率增加(P<0.01)。与LPS组相比,LPS+FLIPUS组小鼠膝关节软骨细胞中COL2α1蛋白表达上升(P<0.01),MMP13、MMP3、ADAMTS5、NLRP3、GSDMD-N、cleaved caspase 1、IL-18和IL-1β蛋白表达下降(P<0.05,P<0.01),细胞上清液中IL-18、IL-1β水平下降和LDH释放减少(P<0.05,P<0.01),细胞肿胀程度减轻,细胞膜上孔隙减少,细胞死亡率下降(P<0.05)。结论FLIPUS可能通过抑制炎症因子和细胞焦亡相关蛋白表达减轻LPS诱导的小鼠膝关节软骨细胞损伤。