Development of a rodent high-energy blast injury model for investigating conditions associated with traumatic amputations.

AIMS: In recent conflicts, most injuries to the limbs are due to blasts resulting in a large number of lower limb amputations. These lead to heterotopic ossification (HO), phantom limb pain (PLP), and functional deficit. The mechanism of blast loading produces a combined fracture and amputation. Therefore, to study these conditions, in vivo models that replicate this combined effect are required. The aim of this study is to develop a preclinical model of blast-induced lower limb amputation. METHODS: Cadaveric Sprague-Dawley rats' left hindlimbs were exposed to blast waves of 7 to 13 bar burst pressures and 7.76 ms to 12.68 ms positive duration using a shock tube. Radiographs and dissection were used to identify the injuries. RESULTS: Higher burst pressures of 13 and 12 bar caused multiple fractures at the hip, and the right and left limbs. Lowering the pressure to 10 bar eliminated hip fractures; however, the remaining fractures were not isolated to the left limb. Further reducing the pressure to 9 bar resulted in the desired isolated fracture of the left tibia with a dramatic reduction in the fractures to other sites. CONCLUSION: In this paper, a rodent blast injury model has been developed in the hindlimb of cadaveric rats that combines the blast and fracture in one insult, necessitating amputation. Experimental setup with 9 bar burst pressure and 9.13 ms positive duration created a fracture at the tibia with total reduction in non-targeted fractures, rendering 9 bar burst pressure suitable for translation to a survivable model to investigate blast injury-associated diseases. Cite this article: Bone Joint Res 2021;10(3):166-172.

Author Correction: Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations.

The range of the mosquito Aedes aegypti continues to expand, putting more than two billion people at risk of arboviral infection. The sterile insect technique (SIT) has been used to successfully combat agricultural pests at large scale, but not mosquitoes, mainly because of challenges with consistent production and distribution of high-quality male mosquitoes. We describe automated processes to rear and release millions of competitive, sterile male Wolbachia-infected mosquitoes, and use of these males in a large-scale suppression trial in Fresno County, California. In 2018, we released 14.4 million males across three replicate neighborhoods encompassing 293 hectares. At peak mosquito season, the number of female mosquitoes was 95.5% lower (95% CI, 93.6-96.9) in release areas compared to non-release areas, with the most geographically isolated neighborhood reaching a 99% reduction. This work demonstrates the high efficacy of mosquito SIT in an area ninefold larger than in previous similar trials, supporting the potential of this approach in public health and nuisance-mosquito eradication programs.

Tibial Osteotomy as a Mechanical Model of Primary Osteoarthritis in Rats.

This study has presented the first purely biomechanical surgical model of osteoarthritis (OA) in rats, which could be more representative of the human primary disease than intra-articular techniques published previously. A surgical tibial osteotomy (TO) was used to induce degenerative cartilage changes in the medial knee of Sprague-Dawley rats. The presence of osteoarthritic changes in the medial knee compartment of the operated animals was evaluated histologically and through analysis of serum carboxy-terminal telepeptides of type II collagen (CTX-II). In-vivo biomechanical analyses were carried out using a musculoskeletal model of the rat hindlimb to evaluate the loading conditions in the knee pre and post-surgically. Qualitative and quantitative medial cartilage degeneration consistent with OA was found in the knees of the operated animals alongside elevated CTX-II levels and increased tibial compressive loading. The potential avoidance of joint inflammation post-surgically, the maintenance of internal joint biomechanics and the ability to quantify the alterations in joint loading should make this model of OA a better candidate for modeling primary forms of the disease in humans.

A Novel In Vitro Model of Blast Traumatic Brain Injury.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

Xenon Protects against Blast-Induced Traumatic Brain Injury in an In Vitro Model.

The aim of this study was to evaluate the neuroprotective efficacy of the inert gas xenon as a treatment for patients with blast-induced traumatic brain injury in an in vitro laboratory model. We developed a novel blast traumatic brain injury model using C57BL/6N mouse organotypic hippocampal brain-slice cultures exposed to a single shockwave, with the resulting injury quantified using propidium iodide fluorescence. A shock tube blast generator was used to simulate open field explosive blast shockwaves, modeled by the Friedlander waveform. Exposure to blast shockwave resulted in significant (p < 0.01) injury that increased with peak-overpressure and impulse of the shockwave, and which exhibited a secondary injury development up to 72 h after trauma. Blast-induced propidium iodide fluorescence overlapped with cleaved caspase-3 immunofluorescence, indicating that shock-wave-induced cell death involves apoptosis. Xenon (50% atm) applied 1 h after blast exposure reduced injury 24 h (p < 0.01), 48 h (p < 0.05), and 72 h (p < 0.001) later, compared with untreated control injury. Xenon-treated injured slices were not significantly different from uninjured sham slices at 24 h and 72 h. We demonstrate for the first time that xenon treatment after blast traumatic brain injury reduces initial injury and prevents subsequent injury development in vitro. Our findings support the idea that xenon may be a potential first-line treatment for those with blast-induced traumatic brain injury.

CD43Lo classical monocytes participate in the cellular immune response to isolated primary blast lung injury.

BACKGROUND: Understanding of the cellular immune response to primary blast lung injury (PBLI) is limited, with only the neutrophil response well documented. Moreover, its impact on the immune response in distal organs remains poorly understood. In this study, a rodent model of isolated primary blast injury was used to investigate the acute cellular immune response to isolated PBLI in the circulation and lung, including the monocyte response, and investigate distal subacute immune effects in the spleen and liver 6 hours after injury. METHODS: Rats were subjected to a shock wave (~135 kPa overpressure, 2 ms duration) inducing PBLI or sham procedure. Rat physiology was monitored, and at 1, 3, and 6 hours thereafter, blood, lung, and bronchoalveolar lavage fluid (BALF) were collected and analyzed by flow cytometry, enzyme-linked immunosorbent assay, and histologic examination. In addition, at 6 hours, spleen and liver were collected and analyzed by flow cytometry. RESULTS: Lung histology confirmed pulmonary barotrauma and inflammation. This was associated with rises in CXCL-1, interleukin 6 (IL-6), tumor necrosis factor α and albumin protein in the BALF. Significant acute increases in blood and lung neutrophils and CD43Lo/His48Hi (classical) monocytes/macrophages were detected. No significant changes were seen in blood or lung "nonclassical" monocyte and in natural killler, B, or T cells. In the BALF, significant increases were seen in neutrophils, CD43Lo monocyte-macrophages and monocyte chemoattractant protein-1. Significant increases in CD43Lo and Hi monocyte-macrophages were detected in the spleen at 6 hours. CONCLUSION: This study reveals a robust and selective response of CD43Lo/His48Hi (classical) monocytes, in addition to neutrophils, in blood and lung tissue following PBLI. An increase in monocyte-macrophages was also observed in the spleen at 6 hours. This profile of immune cells in the blood and BALF could present a new research tool for translational studies seeking to monitor, assess, or attenuate the immune response in blast-injured patients.

Prolonged but not short-duration blast waves elicit acute inflammation in a rodent model of primary blast limb trauma.

BACKGROUND: Blast injuries from conventional and improvised explosive devices account for 75% of injuries from current conflicts; over 70% of injuries involve the limbs. Variable duration and magnitude of blast wave loading occurs in real-life explosions and is hypothesised to cause different injuries. While a number of in vivo models report the inflammatory response to blast injuries, the extent of this response has not been investigated with respect to the duration of the primary blast wave. The relevance is that explosions in open air are of short duration compared to those in confined spaces. METHODS: Hindlimbs of adult Sprauge-Dawley rats were subjected to focal isolated primary blast waves of varying overpressure (1.8-3.65kPa) and duration (3.0-11.5ms), utilising a shock tube and purpose-built experimental rig. Rats were monitored during and after the blast. At 6 and 24h after exposure, blood, lungs, liver and muscle tissues were collected and prepared for histology and flow cytometry. RESULTS: At 6h, increases in circulating neutrophils and CD43Lo/His48Hi monocytes were observed in rats subjected to longer-duration blast waves. This was accompanied by increases in circulating pro-inflammatory chemo/cytokines KC and IL-6. No changes were observed with shorter-duration blast waves irrespective of overpressure. In all cases, no histological damage was observed in muscle, lung or liver. By 24h post-blast, all inflammatory parameters had normalised. CONCLUSIONS: We report the development of a rodent model of primary blast limb trauma that is the first to highlight an important role played by blast wave duration and magnitude in initiating acute inflammatory response following limb injury in the absence of limb fracture or penetrating trauma. The combined biological and mechanical method developed can be used to further understand the complex effects of blast waves in a range of different tissues and organs in vivo.

Gait compensations in rats after a temporary nerve palsy quantified using temporo-spatial and kinematic parameters.

BACKGROUND: The aim of this work was to test a method for measuring the gait of rats with sufficient sensitivity to detect subtle locomotor changes due to pathology, injury and recovery. METHOD: The gait of female Sprague-Dawley rats was assessed using an optical motion tracking system and the DigiGait™ imaging system during normal locomotion, shortly after temporary nerve block to the left hind limb and after full recovery. RESULTS: The effect of low treadmill speeds (10-30 cm/s) was initially investigated. Significant changes were detected in the spatiotemporal gait parameters, consistent with those previously reported. The overall ranges of motion in the hip, knee and ankle joints were 37.5° (±7.1°), 50.2° (±9.4°) and 61.6° (±9.1°) and did not appear to change with speed, indicating that for low speed variations, kinematic comparisons across speeds may be possible. Following the induction of a temporary sciatic nerve block, the range of motion of the left ankle and knee during swing decreased by 23° and 33°, respectively (p<0.05). A compensatory change of a greater range of motion at the hip was noted in the contralateral limb (p<0.01). 90 min post injection, most of the gait parameters had returned to normal, however, minor walking deficits were still present. COMPARISON WITH EXISTING METHOD(S): Discriminant analysis showed that a combination of dynamic and kinematic parameters provides a more robust method for the classification of gait changes. CONCLUSIONS: This more detailed method, employing both dynamic analysis and joint kinematics simultaneously, was found to be a reliable approach for the quantification of gait in rats.

microRNA miR-275 is indispensable for blood digestion and egg development in the mosquito Aedes aegypti.

The mosquito Aedes aegypti is the major vector of arboviral diseases, particularly of Dengue fever, of which there are more than 100 million cases annually. Mosquitoes, such as A. aegypti, serve as vectors for disease pathogens because they require vertebrate blood for their egg production. Pathogen transmission is tightly linked to repeated cycles of obligatory blood feeding and egg maturation. Thus, the understanding of mechanisms governing egg production is necessary to develop approaches that limit the spread of mosquito-borne diseases. Previous studies have identified critical roles of hormonal- and nutrition-based target of rapamycin (TOR) pathways in controlling blood-meal-mediated egg maturation in mosquitoes. In this work, we uncovered another essential regulator of blood-meal-activated processes, the microRNA miR-275. The depletion of this microRNA in A. aegypti females after injection of its specific antagomir resulted in severe defects in blood digestion, fluid excretion, and egg development, clearly demonstrating that miR-275 is indispensable for these physiological processes. miR-275 exhibits an expression profile that suggests its regulation by a steroid hormone, 20-hydroxyecdysone (20E). In vitro organ culture experiments demonstrated that miR-275 is induced by this hormone in the presence of amino acids, indicative of a dual regulation by 20E and TOR. This report has uncovered the critical importance of microRNAs in controlling blood-meal-activated physiological events required for completion of egg development in mosquito disease vectors.

Butterfly wings shaped by a molecular cookie cutter: evolutionary radiation of lepidopteran wing shapes associated with a derived Cut/wingless wing margin boundary system.

Butterflies and moths show a remarkable diversity of specialized wing shapes, yet little is known about the molecular basis of wing shape determination. To learn more about this process we examined the expression of dorsoventral (DV) boundary candidate genes in developing wings of several species of Lepidoptera. We found that the transcription factor Cut and mRNA for the signaling molecule wingless (wg) are strongly co-expressed in a discrete zone around the larval wing disc margin. Surprisingly, the expression boundary of Cut and wg clearly presages complex future adult wing shapes, including the hindwing tails of swallowtail butterflies, very early in final-instar wing disc development. During pupal wing development the cells in this zone undergo apoptosis, thereby defining the actual margin of the adult wing. Comparison with gene expression in beetle and fly wings suggests that this delineation of a topologically independent boundary running parallel to the DV boundary is a derived feature of Lepidoptera. We propose that the developmental decoupling of wing margin determination and DV boundary formation was a major developmental innovation that facilitated the radiation of specialized wing shapes in moths and butterflies.

Stepwise introduction of a bone-conserving osseointegrated hip arthroplasty using RSA and a randomized study: I. Preliminary investigations--52 patients followed for 3 years.

BACKGROUND: We developed a total hip system using osseointegration guidelines, a metaphyseal-loading proximal femoral replacement in the retained neck and a dual-geometry titanium shell in the acetabulum. PATIENTS AND METHODS: A randomized controlled clinical trial was undertaken in 52 patients (53 hips), using the cemented Spectron stem and cementless Harris-Galante II cup as control implants (24 patients in experimental group, 29 control patients). Clinical measures of Harris Hip Score (HHS), pain score and radiostereometric analysis (RSA) at regular intervals for up to three years were used to monitor progress. RESULTS: No statistically significant differences were found in HHS and pain score; the stability of the cementless experimental implant was also comparable to that of the cemented controls by RSA. 3 revisions were required for migration in the experimental group and 1 was required for component dislocation in the control group. INTERPRETATION: Our findings indicate the practicality of osseointegration of titanium implants, but suggest that current performance is inadequate for clinical introduction. However, the stable fixation achieved in the retained neck in the majority of patients is indicative of osseointegration. This finding will encourage technical and design improvements for enhancement of clinical osseointegration and should also encourage further study. Periprosthetic osteolysis might be avoided by the establishment and maintenance of direct implant-bone connection: "osseointegration".

Stepwise introduction of a bone-conserving osseointegrated hip arthroplasty using RSA and a randomized study: II. Clinical proof of concept--40 patients followed for 2 years.

BACKGROUND: We have developed a bone-conserving commercially pure titanium hip replacement system using osseointegration principles: a metaphyseal loading proximal femoral component affixing into the retained neck and metaphysis only, leaving the femoral canal untouched. The acetabular cup closely fits a dual-geometry cavity, avoiding stress protection at the dome. PATIENTS AND METHODS: After extensive laboratory and clinical pilot trial investigations, the surface-engineered implants were submitted to a prospective randomized controlled clinical trial involving 40 patients (40 hips), in which they were compared to the cemented Spectron femoral component and cementless Trilogy cup as control implant. The following clinical measures were used to monitor progress at regular intervals for the first 2 postoperative years: radiostereometric analysis (RSA), Harris Hip Score, pain score, WOMAC, and SF-36. RESULTS: After 2 years of follow-up, no statistically significant differences were seen between the groups concerning rotation or translation along the cardinal axes. The patients receiving the Gothenburg osseointegrated titanium (GOT) system had significantly higher Harris Hip Score at 6 months, suggesting more rapid recovery. WOMAC, SF-36 and pain analysis were similar for the first 2 postoperative years. INTERPRETATION: Our RSA data suggest that osseointegration was achieved for all patients receiving the GOT hip system. This bone-conserving prosthesis may provide a good alternative, especially for young and active patients.

Improved tibial cutting accuracy in knee arthroplasty.

Initial stability and development of long-term fixation for cementless tibial components at the knee both depend on the accuracy of fit between implanted components and prepared bone surfaces. Tibial surfaces prepared for total knee replacement with conventional saw-blades and guides were shown by Toksvig-Larsen to vary by over 2 mm, from a flat surface at the point of maximum variation, and all points varied with a standard deviation of up to 0.4 mm. Surface cutting errors are caused by flexion of the saw-blade and blade angulation from the ideal alignment, due to poor guidance or control by the saw-block or guide. Most conventional knee instrumentation relies on flat surface or slotted cutting blocks, constraining the moving saw-blade against one or two guide surfaces. Improved cutting action was achieved by constraining the saw from the pivot point of the blade, and controlling motion of this constraint with parallel action slides. Using this saw-guide and an improved saw-blade, tibial cuts were made in mock arthroplasty procedures on twenty four cadaveric tibiae in mortuo. Analysis of Variance and Tukey's HSD test showed that the improved saw technique yielded significantly better flatness (p < 0.03) and greatly improved roughness (p < 0.0005).

Suturing technique and the integrity of dural closures: an in vitro study.

OBJECTIVE: The watertight closure of the dura mater is fundamental to intracranial procedures in neurosurgery. Nevertheless, for any given operator and type of suture, it is still not certain which suturing technique affords the most watertight dural closure. We have developed a laboratory model that allows us to compare the pressures at which dural closures leak when different suturing techniques are used. METHODS: Human cadaveric dura was secured to a glass cylinder filled with colored saline. By application of force to a bag of saline attached to the cylinder, the pressure at which sutured dural incisions leak can be recorded. Using this method, we have compared the closure of 2-cm dural incisions with 3-0 silk using the following techniques (10 per group): 1) interrupted simple, 2) running simple, 3) running locked, and 4) interrupted vertical mattress. We have also compared the closure of 1- x 3-cm dural windows with cadaveric dura and 3-0 silk using the same suturing techniques (10 per group). RESULTS: The pressure at which 2-cm linear dural incisions leaked was significantly higher when they were closed with the interrupted simple suturing technique (P < 0.05). There was no significant difference among the different suturing techniques when they were used to close a 1- x 3-cm dural window with a duraplasty. Overall, the pressures at which sutured linear dural incisions leaked were higher than the pressures at which sutured dural windows closed with duraplasties leaked. CONCLUSION: In the experimental model described, an interrupted simple suturing technique affords the most watertight dural closure for linear incisions, whereas no suturing technique proved advantageous for the closure of a duraplasty.

Variation in surface texture measurements.

Surface texture influences cellular response to implants, implant wear, and fixation, yet measurement and reporting of surface texture can be confusing and ambiguous. Seven specimens of widely different surface textures were submitted to three internationally renowned laboratories for surface texture characterization. The specimens were from dental implants, orthopedic implants, and femoral heads. Areas to be measured were clearly marked; simplified instructions were supplied but specific measurement parameters were not requested. Techniques used included contact profilometry, two- and three-dimensional laser profilometry, and atomic force microscopy. Four to thirteen parameters were reported, 2D or 3D, including R(a) or S(a); only three were common to all centers. The results varied by as much as +/-300-1000%, depending on technique and surface type. Some surfaces were not measurable by some techniques. One dental implant surface was reported with R(a) of 0.17, 0.85, 1.9, and 4.4 microm. The CoCr femoral head ranged from an R(a) of 0.011 to 0.10 microm; the zirconia head from 0.006 to 0.05 microm. Similar variability was reported for the other parameters. Useful surface texture characterization requires reporting of all measurement parameters. Comparisons between studies may be compromised if differences in technique are not considered.

A novel liner locking mechanism enhances retention stability.

Acetabular liner retention of a novel design of liner locking was evaluated in static and cyclic endurance modes. The locking mechanism combines geometric form and accurate machining to give high conformity to the acetabular shell and minimise relative motion against the metal shell, minimising debris generation and escape or ingress. Using amended test liners with integral coupling, mean static pullout strength was determined to be 399+/-53 N and lever-out strength 28.03+/-2.8 N m. Cyclic loading of 5 N m for up to 10 million cycles caused no significant reduction in strength, no detectable fretting wear, and the sealing mechanism prevented particle access between the cup interior and the "effective joint space". The stability measured ensures secure and reliable in vivo retention of the liner, comparable with extant component designs using other liner locking mechanisms.