Low intensity ultrasound treatment increases strength in a rat femoral fracture model

Bilateral closed femoral shaft fractures were made in 22 male Long-Evans rats. In 16 animals, ultrasound was applied to one limb for 15 minutes daily 10 times within the first 14 postoperative days. The treated limbs received a 200 μsec burst of 1.5 or 0.5 MHz sine waves repeated at 1.0 KHz at a spatial average and temporal average intensity of 30 mW/cm². The contralateral limb of each animal served as a nontreated control. Six remaining animals with fractures and six additional animals without fractures received sham ultrasound treatment to control for the effects of anesthesia and handling. Fracture repair was evaluated on postoperative day 21 by radiography, mechanical testing in torsion, and histology. Five of 16 ultrasound-treated fractures showed obliteration of the fracture gap on radiographs, whereas none of the 28 controls did. The average maximum torque of fractures treated with either signal was 22% greater than that of the contralateral controls (p < 0.05). The stiffness of treated fractures was greater than that of control fractures, but the difference was significant only in animals treated with the 1.5 MHz signal (p < 0.02). Sham treatment did not affect repair in the control group. These results indicate that low-intensity pulsed ultrasound at either 0.5 or 1.5 MHz can accelerate fracture repair at 21 days in this highly controlled model.     

Bone penetrance of locally administered vancomycin powder in a rat femur fracture model

IntroductionLocally delivered, crystalline vancomycin has been suggested as a potential prophylactic measure against the development of deep and superficial surgical site infection. Clinical expectations regarding the duration and peak of drug concentration in local tissues following administration are unknown. Our goal was to develop concentration vs time curves for locally administered vancomycin powder in a high-energy, open femur fracture rat model in local tissues and to compare that data to two well performed similar, systemic administration studies.MethodsAfter approval for animal research, 24 adult Sprague-Dawley rats sustained closed, midshaft femoral fracture under anesthesia. Fractures were caused via blunt guillotine with 750 g metal rod dropped 50 cm. Injured hindlimbs were surgically opened at fracture to simulate open injury and stabilized using 0.054 Kirschner wires. Vancomycin powder was administered using weight-based protocol (goal: 25 mg/kg). Rats were sacrificed in groups of 4 at 4, 8, 24, 48, 72, 96 h. Samples harvested included rat-tail venous blood prior to sacrifice, and femoral bone and anterior thigh soft-tissue were harvested post-mortem. High Performance Liquid Chromatography (HPLC) was performed on all samples.ResultsConcentration vs. time curves demonstrated that the surrounding soft-tissues demonstrated highest maximum concentration (1.5 mg vancomycin/g muscle). Bone reached maximum average of 199 μg vancomycin/g femur: approximately 13% of maximal soft-tissue absorption. Plasma reached maximum concentration of 1.8 μg/mL plasma. All peaks at t = 4 h. Within 48 h, average muscle vancomycin concentration dropped to 3 μg/g muscle (0.2% maximum muscle concentration) and the average bone concentration dropped to 1.9 μg/g femur (0.9% maximum bone concentration). Vancomycin was undetectable on all samples at 96 h. Comparison to classical animal studies suggest local delivery to bone exceeds that of IV dosing for approximately 48 h and may peak near concentrations of 10² multiples.ConclusionsLocally administered vancomycin provides drug delivery in excess of IV dosing for approximately 48 h after intervention. Exponential decay demonstrates rapid removal of drug to near undetectable levels in bone, plasma, and local soft tissue thereafter in a rat model. Local delivery may generate concentrations exceeding that achievable by steady state systemic dosing for 48 h.     

Transosseous application of low-intensity ultrasound for the enhancement and monitoring of fracture healing process in a sheep osteotomy model

The purpose of this study is twofold: (a) to investigate the application of transosseous low-intensity pulsed ultrasound (LiUS) on the enhancement of fracture healing and (b) to demonstrate the ability of transosseous ultrasound propagation to monitor the healing process. A midshaft tibial osteotomy model was used on 40 skeletally mature sheep, and an external fixator was applied to maintain the reduction and stabilization of the osteotomy. Two ultrasound transducers were implanted into the fracture site in contact with the bone. For investigating the efficacy of LiUS, the animals were randomly divided in two equal groups: the treatment group and the control group. The LiUS-treated animals received 200-micros bursts of 1-MHz sine waves with a pulse repetition rate of 1 kHz and average intensity of 30 mW/cm2, for 20 min daily. For monitoring purposes, an ultrasound dataset was constructed consisting of serial ultrasound measurements obtained from healing bones. Animals' sacrifice took place on the 100th post-operative day. The effect of LiUS on fracture healing was evaluated using radiographs, destructive three-point bending testing and quantitative CT-based bone mineral density (BMD) measurements. Survival analysis using Kaplan-Meier curves showed significantly higher probability of radiographic healing for the animals in the treatment group (P = 0.009). Statistical significance was also observed for callus BMD (P = 0.003, Wilcoxon nonparametric test), the breaking load (P = 0.001), extrinsic stiffness (P = 0.019), Young's modulus (P = 0.043) and ultimate strength (P = 0.051) in favor of the LiUS-treated limbs. Analysis of the obtained ultrasonic measurements showed that the propagation velocity across healing bones constitutes a significant feature able to early distinguish between healed and nonhealed bones (area under ROC curve was 0.810 and 0.841 on the 80th and 100th post-operative days, respectively). Both the LiUS application and the ultrasonic measurements are supported by an integrated telemedicine system which also incorporates an ultrasound wearable device and a patient management system.     

Characterization of a closed femur fracture model in mice

OBJECTIVES: The goal of this study was to develop and characterize a closed femur fracture model for mice that can be used for the molecular and genetic analysis of fracture healing. STUDY DESIGN: Longitudinal time study of species-specific fracture healing. METHODS: A protocol was developed for creating reproducible, closed femur fractures in mice. Impending fractures were stabilized by retrograde insertion of a 0.01-inch-diameter, stainless steel wire into the intramedullary canal. The intramedullary wire was held in place with a wedge made from the first 2 mm of a 30-gauge needle. Fractures were produced by 3-point bending. Fracture healing was assessed by radiography, histology, and torsional mechanical testing. RESULTS: The mouse femur fracture technique produced good results with minimal loss of animals. Of the 246 mice used in the study, 22 mice were excluded due to poor fracture quality (8), loss of fracture stabilization (6), or to anesthesia death (8). Radiography showed a consistent pattern of fracture healing between mice with peak fracture callus volume evident at 10 (15 mice) to 14 days (18 mice) after fracture. Fracture bridging was apparent in all 3-week postfracture radiographs (35 mice). Histologic examination of 117 specimens at 9 time points showed chondrocyte differentiation within the fracture callus by 7 days after fracture, endochondral ossification occurring by 10 days after fracture, and bone remodeling evident as early as 3 weeks after fracture. Despite radiologic and histologic evidence of fracture bridging after 3 weeks, torsional mechanical testing of 68 mice at 3, 4, 6, and 12 weeks after fracture (group size of 15 to 18 mice at each time point) indicated that significant increases in structural or material strength did not occur until 6 to 12 weeks after fracture. CONCLUSIONS: Femur fracture healing in mice follows a typical endochondral ossification pathway with fracture bridging occurring approximately 1 week faster in mice than rats. This fracture model is amenable to the molecular and genetic analysis of fracture healing using different inbred, transgenic, and knockout strains of mice.     

Effect of low-intensity ultrasound stimulation on consolidation of the regenerate zone in a rat model of distraction osteogenesis

This study was performed to explore the tissue-level changes in mineralization caused by low-intensity ultrasound stimulation after distraction osteogenesis. Unilateral femoral lengthenings (7 mm) were performed on 34 male Sprague-Dawley rats. Half of the animals received daily ultrasound stimulation for 5 weeks; the remaining animals received sham treatments. Healing was assessed with serial radiographs, quantitative micro-computed tomography, and biomechanical testing. Twenty-one animals were evaluated at the conclusion of the study (9 experimental, 12 control). Radiographically, healing of the ultrasound-treated bones preceded that of the sham-treated bones by approximately 1 week. Bone volume fraction and trabecular bone pattern factor were significantly higher in the ultrasound-treated animals, but there were no significant differences in bone mineral content or bone mineral density. The ultrasound-treated femurs were 20% stiffer and 33% stronger than the control femurs, but the differences were not statistically significant. These findings suggest that pulsed, low-intensity ultrasound matures the regenerate by altering the microarchitecture of the newly formed bone.     

Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.     

Time-dependent aggrecan gene expression of articular chondrocytes in response to hyperosmotic loading

OBJECTIVE: To investigate the effects of increasing extracellular osmolality on aggrecan gene expression and cell size in cultured chondrocytes. DESIGN: Aggrecan promoter activity and mRNA levels were measured in bovine monolayer chondrocytes subjected to hyperosmotic loading for different time periods, using transient transfection assays or RT-PCR. Cell size changes were also determined using an epifluorescence microscopy system. RESULTS: Hyperosmotic loading for 24 h suppressed aggrecan promoter activity and mRNA levels approximately two-fold. However no suppression of promoter activity was observed when exon 1 was deleted from the human aggrecan promoter construct. Osmotic regulation of aggrecan gene expression was time-dependent and found to correlate with cell shrinking and swelling. No suppression in promoter activity was observed when the hyperosmotic stimulus was applied in a cyclic manner, or when serum was present in the culture medium. CONCLUSION: Hyperosmotic loading regulates aggrecan gene expression and cell size in isolated chondrocytes. Osmotic regulation of gene expression is also affected by the time-varying nature of loading and the presence of serum.     

低强度脉冲式超声促软骨修复及其基因表达研究进展

细胞外基质中蛋白聚糖和Ⅱ型胶原合成减少、降解增加及X型胶原的出现.是血运差、自身修复能力低下的关节软骨伤痛及退变的主要原因.一定条件的低强度脉冲式超声波(LIPUS)可促进股骨骨折大鼠模型骨痂中软骨细胞聚合素与Ⅱ型胶原mRNA表达,但后期经骨膜诱导可发生骨化.LIPUS对关节软骨细胞及基质是否产生类似效应并抑制软骨细胞的成熟与退变.则成为关节软骨伤痛无创修复的研究热点.该文通过总结近年来相关文献,就LIPUS对关节软骨伤病动物在体模型的软骨组织学及基因改变.LIPUS对体外培养软骨一软骨细胞的细胞活性、增殖、表型分化及基质合成分泌功能的影响,LIPUS的临床应用研究.LIPUS促进软骨基质合成的调控机制等进行综述,并提出目前研究存在的问题及进一步研究方向.     

低强度超声波促进桡骨远端骨折愈合

目的　观察低强度脉冲超声对桡骨远端骨折愈合的促进作用。方法　 4 1例桡骨远端骨折患者 ,随机分为超声治疗组 (2 1例 )和对照组 (2 0例 )。超声组在骨折复位后以短臂石膏固定 ,每日以低强度脉冲超声治疗 15min ;对照组仅作复位和短臂石膏固定。记录两组患者骨折临床愈合时间和X线片上骨折线处灰度值的变化。结果　超声治疗组的临床愈合时间较对照组缩短 (32 0 4d± 2 5 8dvs 4 0 75d± 5 12d ,P <0 0 1)。超声治疗组骨折愈合前后X线片上骨折线处灰度值变化较对照组大。结论　低强度脉冲超声可明显加快桡骨远端骨折的愈合 ,促进骨折处局部骨的形成。     

Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.     

DNA repair gene expression in biological tissues exposed to low-intensity infrared laser

Special properties of laser light have led to its usefulness in many applications in therapy. Excitation of endogenous chromophores in biotissues and generation of free radicals could be involved in its biological effects. DNA lesions induced by free radicals are repaired by base excision repair pathway. In this work, we evaluated the expression of APE1 and OGG1 genes related to repair of DNA lesions induced by free radicals. Skin and muscle tissues of Wistar rats were exposed to low-intensity infrared laser at different fluences and frequencies. After laser exposition of 1 and 24 h, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of APE1 and OGG1 gene expression by quantitative polymerase chain reaction. Data obtained show that laser radiation alters the expression of APE1 and OGG1 mRNA differently in skin and muscle tissues of Wistar rats depending of the fluence, frequency, and time after exposure. Our study suggests that low-intensity infrared laser affects expression of genes involved in repair of DNA lesions by base excision repair pathway.     

An internal locking plate to study intramembranous bone healing in a mouse femur fracture model

In most murine fracture models, the femur is stabilized by an intramedullary implant and heals predominantly through endochondral ossification. The aim of the present study was to establish a mouse model in which fractures heal intramembranously. Femur fractures of 16 SKH‐mice were stabilized by an internal locking plate. Femur fractures of another 16 animals were stabilized by an intramedullary screw. Bone repair was analyzed by radiographic, biomechanical, and histological methods. At 2 weeks, histological analysis showed a significantly smaller callus diameter and callus area after locking plate fixation. Cartilage formation within the callus could only be observed after screw fixation, but not after fracture stabilization with the locking plate. Radiological and biomechanical analysis after 2 and 5 weeks showed a significantly improved healing and a higher bending stiffness of fractures stabilized by the locking plate. Fractures stabilized by the locking plate healed exclusively by intramembranous ossification, which is most probably a result of the anatomical reduction and stable fixation. The fractures that healed by intramembranous ossification showed an increased stiffness compared to fractures that healed by endochondral ossification. This model may be used to study molecular mechanisms of intramembranous bone healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:397–402, 2010     

Effect of low-intensity pulsed ultrasound on fracture callus mineral density and flexural strength in rabbit tibial fresh fracture

Background  

Low-intensity ultrasound is a biophysical intervention on a fracture repair process. However, the effect of low-intensity ultrasound therapy on fracture healing is controversial. The aim of the present study was to evaluate the effect of low-intensity pulsed ultrasound (LIPUS) therapy on the fracture healing process, including mineral density and strength of callus using a rabbit model.     

Treatment of ununited fracture of the hook of hamate by low-intensity pulsed ultrasound: a case report

A patient presented 4 months after sustaining a fracture of the hook of hamate. X-rays and computed tomography scanning of the carpal tunnel confirmed the presence of an ununited fracture. Low-intensity ultrasound was applied to the fracture site. After 4.5 months of exposure to ultrasound, union was confirmed by both x-rays and computed tomography scanning of the carpal tunnel. (J Hand Surg 2000; 25A:77-79.     

Allergy increases susceptibility to otitis media with effusion in a rat model

To investigate the possible relationship between allergy and otitis media with effusion (OME), we investigated the hypothesis that allergen presentation to the middle ear causes functional disruption of the eustachian tube predisposing to the development of OME. Thirteen of 19 Brown-Norway rats were sensitized to ovalbumin, and the remaining 6 served as nonallergic controls. To mimic subclinical exposure to allergen, we transtympanically injected ovalbumin at a dose (0.01 mg) that produced no changes detectable by otologic examination. Next, both allergic and nonallergic rats were exposed to transtympanic injection of either low-dose (10 μg/mL) or high-dose (100 μg/mL) lipopolysaccharide to simulate bacterial exposure. The allergic rats were found to have larger middle ear effusions when exposed to high-dose lipopolysaccharide as compared with the nonallergic controls. This response could be inhibited by diphenhydramine. We conclude that allergen presentation to the middle ear of allergic rats causes eustachian tube dysfunction predisposing to OME. (Otolaryngol Head Neck Surg 1999;121:687-92.)     

Low-intensity transosseous ultrasound accelerates osteotomy healing in a sheep fracture model

BACKGROUND: Low-intensity transcutaneous ultrasound can accelerate and augment the fracture-healing process. The aim of this study was to investigate the effect of transosseous application of low-intensity ultrasound on fracture-healing in an animal model. METHODS: A midshaft osteotomy of the left tibia was performed in forty sheep. An external fixator was used to stabilize the osteotomy site. A thin stainless-steel pin was inserted into the bone, 1.0 cm proximal to the osteotomy site. Ultrasound was transmitted through the free end of this pin, with a PZT-4D transducer. In twenty animals, the treated limb received a 200-microsec burst of 1-MHz sine waves repeated at 1 kHz with an average intensity of 30 mW/cm(2) for twenty minutes daily. Twenty other animals underwent the same surgery but did not receive the ultrasound (controls). Animals were killed at seventy-five and 120 days postoperatively. Radiographic evaluation was performed every fifteen days. Mechanical testing and quantitative computed tomography were performed after death. RESULTS: Fractures treated with ultrasound healed significantly more rapidly, as assessed radiographically, than did the controls (seventy-nine compared with 103 days, p = 0.027). On day 75, the mean cortical bone mineral density (and standard deviation) was 781 +/- 52 mg/mL in the treated limbs compared with 543 +/- 44 mg/mL in the control group (p = 0.014), and the average ultimate strength (as assessed with a lateral bending test) was 1928 +/- 167 N in the treated limbs compared with 1493 +/- 112 N in the control group (p = 0.012). No significant differences were noted on day 120. CONCLUSIONS: This study demonstrated that low-intensity transosseous ultrasound can significantly accelerate the fracture-healing process, increase the cortical bone mineral density, and improve lateral bending strength of the healing fracture in a sheep osteotomy model.     

The effects of low-intensity laser therapy on hepatic ischemia-reperfusion injury in a rat model

Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role and relics of low-intensity laser therapy (LILT) in many organs, the potential effects of LILT on hepatic ischemia-reperfusion have not been explored. This study was aimed to investigate the impresses of LILT applied to the skin following hepatic ischemia and reperfusion. Thirty-six healthy male Wistar rats were allocated into three groups of twelve animals each as follows: Sham, Ischemia-reperfusion (IR), and Ischemia-reperfusion with laser treatment (IR+LILT). Hepatic ischemia was induced by clamping the arterial and portal venous for 45 min. A laser diode (400 mW, 804 nm) was applied to the skin surface at the anatomical site of the liver at a dose of 3 J/cm², and the duration of irradiation was selected 120 s with 15-min interval after beginning the reperfusion. Animals were maintained under anesthesia and sacrificed 6 h subsequent reperfusion. Hepatic samples were evaluated for histological assessment and biochemistry analysis. Serum aminotransferase levels, tumor necrosis factor-alpha (TNF-α) levels, malondialdehyde (MDA), and glutathione (GSH) levels were significantly lower (P?P?相似文献