Supermicrosurgical Anastomosis Training Using Chick Embryos within the Egg-in-Cube System.

SUMMARY: Although supermicrosurgery techniques are essential skills for lymphatic surgery or perforator-flap surgery, an ideal training model is yet to be introduced. Living animal models, such as rodents, are considered to be ideal microsurgical training models. However, the use of living animal models is costly and involves bioethical considerations. Hence, the authors developed a novel, cost-effective, highly reproducible, and easy-to-handle supermicrosurgical anastomosis training system using the chicken embryo within the egg-in-cube system. Chick embryos were fertilized in the artificial cubic eggshell, which was fabricated by integrating a polycarbonate frame structure and five polydimethylsiloxane membranes. Seven days later, the trainees underwent supermicrosurgical training using the vitelline artery of the chick embryo. The trainees were able to perform supermicrosurgical training using all 11 surviving chicken embryos. The average diameter of the vitelline artery was 0.43 mm. Patency and pulsation were observed after the anastomosis in four of the 11 cases. The supermicrosurgical training system using the chicken embryo within the egg-in-cube system has several advantages. This system is ethically acceptable, less costly and easier to manage than other animal models, and suitable as a supermicrosurgical training model, such as for lymphovenular anastomosis, because the diameter of the vitelline artery was similar to that of the lymphatic vessels in patients with lymphedema. Moreover, the trainee can confirm patency and leakage after the anastomosis because this model has a circulation system. The trainee can practice the supermicrosurgical technique efficiently with simultaneous feedback on anastomosis results. CLINICAL RELEVANCE STATEMENT: This study introduces a novel, cost-effective supermicrosurgical training system using chicken embryos within an egg-in-cube, offering a practical and ethical alternative. Its close simulation to human lymphatic vessels supports skill enhancement for practicing surgeons.

Akkermansia biwaensis sp. nov., an anaerobic mucin-degrading bacterium isolated from human faeces.

A bacterial strain, WON2089T, was isolated from the faeces of healthy Japanese adults and is able to use mucin as the sole carbon and nitrogen source. Sequencing of its 16S rRNA gene showed that WON2089T has 98.0 and 94.4% similarity to Akkermansia muciniphila MucT and Akkermansia glycaniphila PytT, respectively, while phylogenetic tree analysis confirmed that it belongs to the genus Akkermansia. The whole genome of WON2089T was sequenced, which showed that it shares 84.5 % average nucleotide identity (ANI) and 24.9 % digital DNA-DNA hybridization (dDDH) with its closest relative, A. muciniphila MucT. Cells of WON2089T are non-motile, anaerobic and oval-shaped (0.4-0.5×0.5-1.0 µm). The strain is Gram-stain-negative and grows in the temperature range of 25-45 °C (optimum, 30-37 °C) and pH range of pH 5.5-9.5 (optimum, pH 6.5-8.0). WON2089T can utilize d-glucose, d-mannitol, lactose and d-mannose, as assessed by API20A strips. The major cellular fatty acids are C15 : 0 anteiso, C15 : 0 3OH and C18 : 1 ω9c (55.5, 7.5 and 5.8 % of total fatty acids, respectively). Based on 16S rRNA sequencing, ANI, dDDH and acid formation from d-mannitol, WON2089T is distinct from previously reported species of the genus Akkermansia. Based on phenotypic, phylogenetic and genetic characteristics, WON2089T represents a novel species of the genus Akkermansia and the name Akkermansia biwaensis sp. nov. is proposed. The type strain is WON2089T (= NBRC 115679T= DSM 114407T).

A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development.

The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA "in cubo", followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the "in cubo" electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.

Identification of Faecalibacterium prausnitzii strains for gut microbiome-based intervention in Alzheimer's-type dementia.

Evidence linking the gut-brain axis to Alzheimer's disease (AD) is accumulating, but the characteristics of causally important microbes are poorly understood. We perform a fecal microbiome analysis in healthy subjects and those with mild cognitive impairment (MCI) and AD. We find that Faecalibacterium prausnitzii (F. prausnitzii) correlates with cognitive scores and decreases in the MCI group compared with the healthy group. Two isolated strains from the healthy group, live Fp360 and pasteurized Fp14, improve cognitive impairment in an AD mouse model. Whole-genome comparison of isolated strains reveals specific orthologs that are found only in the effective strains and are more abundant in the healthy group compared with the MCI group. Metabolome and RNA sequencing analyses of mouse brains provides mechanistic insights into the relationship between the efficacy of pasteurized Fp14, oxidative stress, and mitochondrial function. We conclude that F. prausnitzii strains with these specific orthologs are candidates for gut microbiome-based intervention in Alzheimer's-type dementia.

Mechanosensitive myosin II but not cofilin primarily contributes to cyclic cell stretch-induced selective disassembly of actin stress fibers.

Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major cross-linker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism.

Identification of Aquatic Organisms Using a Magneto-Optical Element.

In recent advanced information society, it is important to individually identify products or living organisms automatically and quickly. However, with the current identifying technology such as RFID tag or biometrics, it is difficult to apply to amphibians such as frogs or newts because of its size, stability, weakness under a wet environment and so on. Thus, this research aims to establish a system that can trace small amphibians easily even in a wet environment and keep stable sensing for a long time. The magnetism was employed for identification because it was less influenced by water for a long time. Here, a novel magnetization-free micro-magnetic tag is proposed and fabricated with low cost for installation to a living target sensed by Magneto-Optical sensor for high throughput sensing. The sensing ability of the proposed method, which was evaluated by image analysis, indicated that it was less than half of the target value (1 mm) both in the water and air. The FEM analysis showed that it is approximately twice the actual identification ability under ideal conditions, which suggests that the actual sensing ability can be extended by further improvement of the sensing system. The developed magnetization-free micro-magnetic tag can contribute to keep up the increasing demand to identify a number of samples under a wet environment especially with the development of gene technology.

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.

High repeatability from 3D experimental platform for quantitative analysis of cellular branch pattern formations.

Three-dimensional (3D) cell and tissue cultures more closely mimic biological environments than two-dimensional (2D) cultures and are therefore highly desirable in culture experiments. However, 3D cultures often fail to yield repeatable experimental results because of variation in the initial culture conditions, such as cell density and distribution in the extracellular matrix, and therefore reducing such variation is a paramount concern. Here, we present a 3D culture platform that demonstrates highly repeatable experimental results, obtained by controlling the initial cell cluster shape in the gel cube culture device. A micro-mould with the desired shape was fabricated by photolithography or machining, creating a 3D pocket in the extracellular matrix contained in the device. Highly concentrated human bronchial epithelial cells were then injected in the pocket so that the cell cluster shape matched the fabricated mould shape. Subsequently, the cubic device supplied multi-directional scanning, enabling high-resolution capture of the whole tissue structure with only a low-magnification lens. The proposed device significantly improved the repeatability of the developed branch pattern, and multi-directional scanning enabled quantitative analysis of the developed branch pattern formations. A mathematical simulation was also conducted to reveal the mechanisms of branch pattern formation. The proposed platform offers the potential to accelerate any research field that conducts 3D culture experiments, including tissue regeneration and drug development.

New noncontact sensor for detecting pulmonary tumors during video-assisted thoracic surgery.

BACKGROUND: Small pulmonary tumors are difficult to localize during video-assisted thoracic surgery (VATS) because of lack of direct tissue contact. However, in partial lung resection, tumor localization is quite important. The aim of this study was to evaluate the safety and feasibility of a new noncontact sensor for detecting pulmonary nodules during VATS using human and porcine models. METHODS: The sensor, based on the principle of phase differences, comprises an air nozzle for producing air pulse jets and an optical fiber sensor to measure phase differences and visualize object stiffness. For in vivo assessment, we developed a porcine model by inserting plastic balls mimicking tumors into the pig lungs after thoracotomy and then scanned the lungs. The sensor sensitivity was evaluated by measuring the ratio of the depth of the ball from the lung surface to the ball diameter (D/S). For the ex vivo human model, partially resected lung tissue with tumors was obtained from six patients and then scanned. RESULTS: In the porcine model, 32 of 37 (86.5%), 70 of 94 (74.5%), and 60 of 100 (60.0%) tumors were detected in the categories D/S ≤ 1, 1 < D/S ≤ 2, and D/S > 2, respectively. Sensor safety was confirmed with an air jet at pressures between 0.05 and 0.15 MPa directed onto the lung surface; all the examined lungs including the pleura remained intact microscopically. In six patients, all nodules were successfully detected. CONCLUSIONS: Our noncontact sensor is a safe and feasible tool for detecting small pulmonary tumors during VATS.

An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

The chorioallantoic membrane (CAM) containing tiny blood vessels is an alternative to large animals for studies involving angiogenesis and tissue engineering. However, there is no technique to design the direction of growing blood vessels on the CAM at the microscale level for tissue engineering experiments. Here, a methodology is provided to direct blood vessel formation on the surface of a three-dimensional egg yolk using a cubic artificial eggshell with six functionalized membranes. A structure on the lateral side of the eggshell containing a straight channel and an interlinked chamber was designed, and the direction and formation area of blood vessels with blood flow was artfully defined by channels with widths of 70-2000 µm, without sharply reducing embryo viability. The relationship between the size of interlinked chamber and the induction of blood vessels was investigated to establish a theory of design. Role of negative and positive pressure in the induction of CAM with blood vessels was investigated, and air pressure change in the culture chamber was measured to demonstrate the mechanism for blood vessel induction. Histological evaluation showed that components of CAM including chorionic membrane and blood vessels were induced into the channels. Based on our design theory, blood vessels were induced into arrayed channels, and channel-specific injection and screening were realized, which demonstrated proposed applications. The platform with position- and space-controlled blood vessels is therefore a powerful tool for biomedical research, which may afford exciting applications in studies involved in local stimulation of blood vessel networks and those necessary to establish a living system with blood flow from a beating heart.

Oral administration of Bifidobacterium bifidum G9-1 alleviates rotavirus gastroenteritis through regulation of intestinal homeostasis by inducing mucosal protective factors.

Human rotavirus (RV) infection is a leading cause of dehydrating diarrhea in infants and young children worldwide. Since therapeutic approaches to RV gastroenteritis are limited to alleviation of dehydration with oral rehydration solutions, more direct approaches to palliate symptoms of RV gastroenteritis are required. Treatments with probiotics have been increasingly recognized as alternative safe and low cost treatments for moderate infectious diarrhea. In this study, Bifidobacterium bifidum G9-1 (BBG9-1), which has been used as an intestinal drug for several decades, was shown to have a remarkable protective effect against RV gastroenteritis in a suckling mice model. As well as prophylactic oral administration of BBG9-1 from 2 days before RV infection, therapeutic oral administration of BBG9-1 from 1 day after RV infection significantly alleviated RV-induced diarrhea. Therapeutic administration of BBG9-1 reduced various types of damage in the small intestine, such as epithelial vacuolization and villous shortening, and significantly diminished the infectious RV titer in mixtures of cecal contents and feces. It was also shown that therapeutic administration of BBG9-1 significantly increased the number of acidic mucin-positive goblet cells and the gene expression of mucosal protective factors including MUC2, MUC3, MUC4, TGFß1 and TFF3 in the small intestine. This led to alleviation of low gut permeability shown as decreased gene expression levels of occludin, claudin-1 and villin-1 after RV infection. Furthermore, in the small intestine, therapeutic administration of BBG9-1 significantly palliated the decreased gene expression of SGLT-1, which plays an important role in water absorption. In the large intestine, administered BBG9-1 was shown to replicate to assimilate undigested nutrients, resulting in normalization of the abnormally high osmotic pressure. These results suggested that water malabsorption caused by RV infection was alleviated in mice administered BBG9-1. Thus, the present study showed that oral administration of BBG9-1 palliated diarrhea partly through protection against RV-induced lesions by inducing mucosal protective factors. Oral administration of BBG9-1 is thought to be an efficient method for management of an RV epidemic for both prophylactic and therapeutic purposes.

Microrobotic Platform for Single Motile Microorganism Investigation.

We propose a microrobotic platform for single motile microorganism observation and investigation. The platform utilizes a high-speed online vision sensor to realize real-time observation of a microorganism under a microscopic environment with a relatively high magnification ratio. A microfluidic chip was used to limit the vertical movement of the microorganism and reduce the tracking system complexity. We introduce a simple image processing method, which utilizes high-speed online vision characteristics and shows robustness against image noise to increase the overall tracking performance with low computational time consumption. The design also considers the future integration of a stimulation system using microtools. Successful long-time tracking of a freely swimming microorganism inside of a microfluidic chip for more than 30 min was achieved notwithstanding the presence of noises in the environment of the cell. The specific design of the platform, particularly the tracking system, is described, and the performance is evaluated and confirmed through basic experiments. The potential of the platform to apply mechanical stimulation to a freely swimming microorganism is demonstrated by using a 50-µm-thick microtool. The proposed platform can be used for long-term observation and to achieve different kinds of stimulations, which can induce new behavior of the cells and lead to unprecedented discoveries in biological fields.

Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations.

An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented. The 3D structure of biosamples can be recognized without fluorescence. The cubic platform employs two types of hydrogels that are compatible with conventional culture dishes or well plates, facilitating growth in culture, ease of handling, and viewing at multiple angles.

Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research.

On-line tracking of living cell subjected to cyclic stretch.

We propose a novel system for the observation of living cell exposed to cyclic stretch under dynamic conditions. The developed system is mainly composed of a laptop PC, a stretching unit with three motorized stages, and a microscope with a CCD camera. The design of the cell tracking system is based on the deformation characteristics of the elastic chamber and its performance was confirmed through the basic experiments. Finally, we succeeded in on-line imaging of living single cells under the microscope with a high magnification ratio. We believe that the developed system is a promising platform for studying the immediate responses of cells exposed to cyclic stretch.

Consecutive oral administration of Bifidobacterium longum MM-2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model.

Bifidobacterium, one of the major components of intestinal microflora, shows anti-influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM-2 (MM-2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM-2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro-inflammatory cytokines such as IL-6 and TNF-α in bronchoalveolar lavage fluid. The anti-IFV mechanism of MM-2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN-γ, IL-2, IL-12 and IL-18. Even in non-infected mice, MM-2 administration also induced significant enhancement of both IFN-γ production by Peyer's patch cells (PPs) and splenetic NK cell activity. Oral administration of MM-2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti-IFV effect. MM-2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.

Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis.

Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid-polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.

Improving performance under mirror-image conditions during laparoscopic surgery using the Broadview camera system.

INTRODUCTION: Under mirror-image conditions, surgeons often become confused and their task performance may deteriorate. This study aimed to quantitatively investigate the difficulty of performing laparoscopic surgery based on a mirror image and to find methods to improve performance under these conditions. METHODS: Twelve medical students with no prior endoscopic surgical experience and 10 surgical residents, each with over 50 laparoscopic surgery experiences, participated in this study. Three measures were assessed using the Hiroshima University Endoscopic Surgical Assessment Device: the deviation with integrated time, the approaching time, and the peak velocity. The scope was placed at 0° (coaxial position), 180° (mirror-image condition), and 180° using the Broadview camera system. Each participant performed the Hiroshima University Endoscopic Surgical Assessment Device task 10 times. RESULTS: The deviation with integrated time, the approaching time, and the peak velocity were better for surgical residents than for novices at 0° (P < 0.001, P = 0.001, P = 0.011). The deviation with integrated time, the approaching time, and the peak velocity at 180° were significantly worse than the corresponding values at 0° (P < 0.0001) for both surgical residents and novices. All three types of Hiroshima University Endoscopic Surgical Assessment Device assessment at 180° were significantly better when the Broadview camera system was used than when it was not (P < 0.0001). CONCLUSION: This study showed quantitatively the difficulty in manipulating laparoscopic instruments in mirror-image conditions. Using the Broadview camera system improved surgeons' performance under mirror-image conditions during laparoscopic surgery.

On-chip microrobot for investigating the response of aquatic microorganisms to mechanical stimulation.

In this paper, we propose a novel, magnetically driven microrobot equipped with a frame structure to measure the effects of stimulating aquatic microorganisms. The design and fabrication of the force-sensing structure with a displacement magnification mechanism based on beam deformation are described. The microrobot is composed of a Si-Ni hybrid structure constructed using micro-electro-mechanical system (MEMS) technologies. The microrobots with 5 µm-wide force sensors are actuated in a microfluidic chip by permanent magnets so that they can locally stimulate the microorganisms with the desired force within the stable environment of the closed microchip. They afford centimetre-order mobility (untethered drive) and millinewton-order forces (high power) as well as force-sensing. Finally, we apply the developed microrobots for the quantitative evaluation of the stimuation of Pleurosira laevis (P. laevis) and determine the relationship between the applied force and the response of a single cell.

Virtual reality training followed by box training improves the laparoscopic skills of novice surgeons.

BACKGROUND: The detailed influence of virtual reality training (VRT) and box training (BT) on laparoscopic performance is unknown; we aimed to determine the optimal order of imparting these training programs. MATERIAL AND METHODS: This randomized controlled trial involved two groups, each with 20 participants without prior laparoscopic surgical experience: A BT-VRT group (60 min BT followed by 60 min VRT) and a VRT-BT group (60 min VRT followed by 60 min BT). We objectively assessed the laparoscopic skills with a motion-analysis system (Hiroshima University Endoscopic Surgical Assessment Device: HUESAD), which reliably assesses surgical dexterity. Skill assessment was performed before and after the training session. RESULTS: No inter-group differences were identified in the study measures at the pre-training assessment. In both groups, the performance on all tasks was significantly better at the post-training assessment than at the pre-training assessment. However, the outcome of the tests using the HUESAD was significantly better in the VRT-BT group than in the BT-VRT group at the post-training assessment. CONCLUSIONS: VRT followed by BT effectively improves the dexterity of novice surgeons during initial laparoscopic (combination) training.