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.

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.

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.

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.

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.

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.

Approaching time is important for assessment of endoscopic surgical skills.

This study aimed to verify whether the approaching time (the time taken to reach the target point from another point, a short distance apart, during point-to-point movement in endoscopic surgery), assessed using the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD), could distinguish the skill level of surgeons. Expert surgeons (who had performed more than 50 endoscopic surgeries) and novice surgeons (who had no experience in performing endoscopic surgery) were tested using the HUESAD. The approaching time, total time, and intermediate time (total time--approaching time) were measured and analyzed using the trajectory of the tip of the instrument. The approaching time and total time were significantly shorter in the expert group than in the novice group (p < 0.0001). The intermediate time did not significantly differ between the groups (p > 0.05). The approaching time, which is a component of the total time, is very mportant in the measurement of the total time to assess endoscopic surgical skills. Further, the approaching time was useful for skill assessment by the HUESAD for evaluating the skill of surgeons performing endoscopic surgery.

The importance of stressing the use of laparoscopic instruments in the initial training for laparoscopic surgery using box trainers: a randomized control study.

BACKGROUND: The goal of this study was to improve the efficiency of initial box training for laparoscopic surgery. MATERIALS AND METHODS: The study used the following task: suturing and knot tying task under a combination of four conditions. (1) The C (use of conventional instruments)-D (direct vision); (2) the C-V (indirect vision via a video monitor); (3) the L (use of laparoscopic instruments)-D; and (4) the L-V (the standard laparoscopic suturing and knot tying). The first study assessed 11 medical students undergoing L-V training. The time to complete each of the four assessment tasks was recorded pre- and post-training. The second study was a randomized, controlled trial involving 36 students grouped according to three types of training methods: the L-D, the C-V, and the L-V group. The improvement in the time to complete the L-V task was assessed and the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD) was used for assessment as well. RESULTS: In the first study, a significant improvement in the performance time between pre- and post-training for the L-D task was found, but not for the C-V task. The second study found that the improvement rate of the L-D trained group was significantly greater than that of the C-V and L-V groups. The HUESAD assessment also showed the similar results. CONCLUSIONS: A training program stressing the use of laparoscopic instruments and compensating for the fulcrum effect is more effective for novices using box trainers in the initial laparoscopic surgery instruction than one emphasizing performing the tasks via a video monitor.

Scientific assessment of endoscopic surgical skills.

Recently, significant attention has been focused on training and education for safe endoscopic surgery. A new assessment method, the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD), has been designed at Hiroshima University to evaluate the smoothness of the movement of endoscopic instruments from velocity. Experts (with experience in performing more than 100 laparoscopic surgeries) and novices (with no experience in performing laparoscopic surgery) were recruited for this study. The aim of task 1 was to move the tip of the endoscopic instrument on the tops of poles from A to C, and task 2 was to move it from the right pole B to the left pole D. The peak velocity (Vp) and the time when peak velocity appears (Tp) were analyzed. Both the peak velocity (Vp) and the time when peak velocity appears (Tp) to perform task 1 and task 2 were significantly faster in the expert group than in the novice group. The peak velocity (Vp) and the time when peak velocity appears (Tp) in HUESAD, which indicate the smoothnes of the endoscopic procedure, are among the most important factors for assessing endoscopic surgical skills.

Development of broad-view camera unit for laparoscopic surgery.

A disadvantage of laparoscopic surgery is the narrow operative field provided by the endoscope camera. This paper describes a newly developed broad-view camera unit for use with the Broad-View Camera System, which is capable of providing a wider view of the internal organs during laparoscopic surgery. The developed camera unit is composed of a miniature color CMOS camera, an indwelling needle, and an extra-thin connector. The specific design of the camera unit and the method for positioning it are shown. The performance of the camera unit has been confirmed through basic and animal experiments.

Objective assessment of endoscopic surgical skills by analyzing direction-dependent dexterity using the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD).

PURPOSE: We evaluated our system of objectively assessing endoscopic surgical skills. METHODS: We developed the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD), which records the movement of the tip of an endoscopic instrument precisely. The orbits of experienced surgeons (expert group) and those of medical students (novice group) were evaluated by measuring the deviation from the ideal course on horizontal and vertical planes. These data were integrated with the time taken to move the tip of an endoscopic instrument between a distal side pole (A) and a proximal side pole (C) (Task 1), and between a left side pole (D) and a right side pole (B) (Task 2). RESULTS: The integrated deviation of the expert group was significantly lower than that of the novice group on both the horizontal and vertical planes in Task 1 (P=0.0004, P=0.009) and Task 2 (P<0.0001, P=0.0002). Thus, the spatial perception of experts was significantly better than that of novices. We also found that the direction of the scope and the movement of the endoscopic instrument were related to difficulties in spatial perception for both experts and novices. HUESAD detected and resolved these differences based on the directions of the scope and movement of the endoscopic instruments. CONCLUSIONS: The HUESAD is a reliable system for assessing a surgeon's dexterity, based on direction and movement. It helps us to attain a higher degree of accuracy and to create an ideal setting for optimal endoscopic surgery.