Application of a Microsuction Background Device for Microanastomosis in a Rat Femoral Vessel Model.

SUMMARY: Microvascular anastomoses can be challenging to perform when edematous fluids and blood continuously flood and compromise the field of view. Intermittent irrigation and suctioning disturb workflow, require an assistant, and can increase risk of arterial thrombosis from vessels being drawn into suction drains. The authors developed and patented a novel three-dimensionally printed background device with microfluidic capabilities to provide autonomous, continuous irrigation and suction to optimize operator autonomy and efficiency. The authors tested this in a rat femoral vessel model. Twelve end-to-end anastomoses were performed by two senior microsurgeons [six conventional, six suction-assisted background (SAB)] in a rat femoral artery model. The primary outcome was time taken to complete the anastomosis. Secondary outcomes included the validated Structured Assessment of Microsurgery Skills (SAMS) score and the total number of "wiping" events to obtain field clarity. Each procedure was recorded, and videos were independently rated by two blinded experts using the SAMS score. Time taken to complete the anastomosis was greater in the conventional group compared with the SAB group (741.7 ± 203.1 seconds versus 584 ± 155.9 seconds; P = 0.007). The median SAMS score was lower in the conventional group compared with the SAB group (32.3 ± 1.4 versus 38.3 ± 1.5; P = 0.001). The median number of wiping events was significantly greater in the conventional group compared with the SAB group (13 ± 2.2 versus 1.7 ± 1.2; P < 0.001). The authors show that a novel microfluidic background device allows continuous irrigation and suctioning without the need for an assistant, optimizing the efficiency of the microvascular anastomosis. CLINICAL RELEVANCE STATEMENT: The authors have designed a novel, patented, three-dimensionally printed microsurgical background device that provides continuous irrigation and suction, reduces operative time, and provides better vessel clarity during a microsurgical anastomosis compared to standard background.

Measuring Encapsulation Efficiency in Cell-Mimicking Giant Unilamellar Vesicles.

One of the main drivers within the field of bottom-up synthetic biology is to develop artificial chemical machines, perhaps even living systems, that have programmable functionality. Numerous toolkits exist to generate giant unilamellar vesicle-based artificial cells. However, methods able to quantitatively measure their molecular constituents upon formation is an underdeveloped area. We report an artificial cell quality control (AC/QC) protocol using a microfluidic-based single-molecule approach, enabling the absolute quantification of encapsulated biomolecules. While the measured average encapsulation efficiency was 11.4 ± 6.8%, the AC/QC method allowed us to determine encapsulation efficiencies per vesicle, which varied significantly from 2.4 to 41%. We show that it is possible to achieve a desired concentration of biomolecule within each vesicle by commensurate compensation of its concentration in the seed emulsion. However, the variability in encapsulation efficiency suggests caution is necessary when using such vesicles as simplified biological models or standards.

MicroSUCI: A Microsurgical Background That Incorporates Suction Under Continuous Irrigation.

The microsurgical anastomosis is integral to the success of autologous-free tissue transfer. Successful performance of this procedure relies strongly on operator dexterity, which can be made more challenging when blood and edematous fluids obscure the field of view. Workflow is impeded by intermittent irrigation and suctioning, necessitating presence of an assistant, with risk of arterial thrombosis, from vessels being drawn into suction drains. To negate these current disadvantages and minimize the barrier of entry to microvascular operations, we designed, manufactured, and patented a novel three-dimensional printed microsurgical background device with microfluidic capabilities that allow continuous suction and irrigation as well as provide platforms that enable multiangle retraction to facilitate operator autonomy. This was validated in an ex vivo model, with the device found to be superior to the current standard. We believe that this will have major applicability to the improvement of microsurgeon.

3D Printed Chest Wall: A Tool for Advanced Microsurgical Training Simulating Depth and Limited View.

The deep inferior epigastric perforator (DIEP) flap has become the free flap of choice for autologous breast reconstruction. However, anastomoses of DIEP pedicles to internal mammary vessels in the chest wall are difficult due to restricted access and the depth of the vessels. Successful performance of such demanding procedures necessitates advanced requirements for microsurgical training models. The current chicken thigh model has been used to acquire microsurgical skills, allowing early learning curve trainees to practice repeatedly in inconsequential environments. Despite the increasing use of this model for training purposes, the resemblance to a clinical environment is tenuous. Such models should include anastomosis practice within the depth where the recipient vessels are located. To address this, we developed a three-dimensional (3D) printed chest wall as an addition to the current chicken thigh model, which reliably mimics the complexity of the anastomosis performed during DIEP breast reconstruction. This form of rapid prototyping facilitates a newfound ability for early learning curve trainees to exercise end-to-end anastomoses on vessels located with variable depths. Our enhancement of the current chicken thigh model is simple, cost-effective and offers a significantly more realistic resemblance to a clinical situation.

PolliRS: A 3D-printed Pollicization Retractor System that Improves Access and Autonomy during the Surgical Procedure.

We demonstrate the design, manufacture, and deployment of the first custom-made 3-dimensional (3D)-printed hand retractor for the pollicization procedure. Radiological images of the patient's hand were taken preoperatively to measure anatomical dimensions and guide the design of the device in a patient-precise manner. The 3D-printed, sterilizable, device was autoclaved and successfully used on a patient that underwent a pollicization procedure in our unit. The radiolucency of the device and the fluency enabled by the ability to exchange between different positions demonstrated the potential of this device in increasing the overall autonomy afforded to the lead-surgeon during the operation and demonstrated the potential of rapid-prototyping techniques such as 3D printing for producing patient-precise tools on-the-fly that taken account the specific needs of the patient.

Three-dimensional Printed Customized Adjustable Mallet Finger Splint: A Cheap, Effective, and Comfortable Alternative.

Mallet finger deformity is a common and debilitating injury of the fingertip, accounting for 10% of all tendon and ligament injuries. It involves a disruption of the terminal extensor mechanism of the distal phalanx. Patients can experience significant pain and swelling of the fingertip and have significant morbidity without treatment. Nonoperative treatment using joint immobilization with splints is the mainstay of management. Traditionally, prefabricated and thermoplastic splints have been utilized; however, issues with comfort and skin complications such as maceration can lead to patient noncompliance and eventually, poor outcomes. To address this, we demonstrate our experience with the design, manufacture, and application of individualized 3D printed mallet finger splints. The splints were found to provide advantages akin to traditional thermoplastic splints, with the addition of being low cost, easy to manufacture, and environmentally friendly.

Absolute Quantification of Protein Copy Number in Single Cells With Immunofluorescence Microscopy Calibrated Using Single-Molecule Microarrays.

Great strides toward routine single-cell analyses have been made over the last decade, particularly in the field of transcriptomics. For proteomics, amplification is not currently possible and has necessitated the development of ultrasensitive platforms capable of performing such analyses on single cells. These platforms are improving in terms of throughput and multiplexability but still fall short in relation to more established methods such as fluorescence microscopy. However, microscopy methods rely on fluorescence intensity as a proxy for protein abundance and are not currently capable of reporting this in terms of an absolute copy number. Here, a microfluidic implementation of single-molecule microarrays for single-cell analysis is assessed in its ability to calibrate fluorescence microscopy data. We show that the equivalence of measurements of the steady-state distribution of protein abundance to single-molecule microarray data can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy. The methods presented have been developed using GFP but are extendable to other proteins and other biomolecules of interest.

A Standardized Hand Fracture Fixation Training Framework using Novel 3D Printed Ex Vivo Hand Models: Our Experience as a Unit.

Surgery for hand trauma accounts for a significant proportion of the plastic surgery training curriculum. The aim of this study was to create a standardized simulation training module for hand fracture fixation with Kirschner wire (K-wire) techniques for residents to create a standardized hand training framework that universally hones their skill and prepares them for their first encounter in a clinical setting. METHODS: A step-ladder approach training with 6 levels of difficulty on 3-dimensional (3D) printed ex vivo hand biomimetics was employed on a cohort of 20 plastic surgery residents (n = 20). Assessment of skills using a score system (global rating scale) was performed in the beginning and at the end of the module by hand experts of our unit. RESULTS: The overall average scores of the cohort before and after assessment were 23.75/40 (59.4%) and 34.7/40 (86.8%), respectively. Significant (P < 0.01) difference of improvement of skills was noted on all trainees. All trainees confirmed that the simulated models provided in this module were akin to the patient scenario and noted that it helped them improve their skills with regard to K-wire fixation techniques, including improvement of their understanding of the 3D bone topography. CONCLUSIONS: We demonstrate a standardized simulation training framework that employs 3D printed ex vivo hand biomimetics proved to improve the skills of residents and that paves the way to more universal, standardized and validated training across hand surgery. This is, to our knowledge, the first standardized method of simulated training on such hand surgical cases.

Hand-portable HPLC with broadband spectral detection enables analysis of complex polycyclic aromatic hydrocarbon mixtures.

Polycyclic aromatic hydrocarbons (PAHs) are considered priority hazardous substances due to their carcinogenic activity and risk to public health. Strict regulations are in place limiting their release into the environment, but enforcement is hampered by a lack of adequate field-testing procedure, instead relying on sending samples to centralised analytical facilities. Reliably monitoring levels of PAHs in the field is a challenge, owing to the lack of field-deployable analytical methods able to separate, identify, and quantify the complex mixtures in which PAHs are typically observed. Here, we report the development of a hand-portable system based on high-performance liquid chromatography incorporating a spectrally wide absorption detector, capable of fingerprinting PAHs based on their characteristic spectral absorption profiles: identifying 100% of the 24 PAHs tested, including full coverage of the United States Environmental Protection Agency priority pollutant list. We report unsupervised methods to exploit these new capabilities for feature detection and identification, robust enough to detect and classify co-eluting and hidden peaks. Identification is fully independent of their characteristic retention times, mitigating matrix effects which can preclude reliable determination of these analytes in challenging samples. We anticipate the platform to enable more sophisticated analytical measurements, supporting real-time decision making in the field.

A Novel Customized 3D Printed Arm Stand Improving Skin Preparation Efficiency in Hand Surgery.

Patient preparation for hand surgery often necessitates skin preparation via the use of an assistant to hold the arm to be operated on in mid-air while disinfectant is applied. This study introduces a three-dimensional printed arm stand that decreases dead time during skin preparation, while also enabling the more efficient use of an assistant. The arm stand devices were customized on the anatomy of the patients and then successfully used on patients having general or regional anesthesia. A practical, reusable, and effective three-dimensional printed arm stand has been developed and applied on both adult and pediatric patients. We have found the bespoke device to be beneficial in terms of reducing theater dead time and overall costs, while increasing the efficiency of an upper limb operating theater list. The rapid prototyping cycle afforded by 3D printing renders this technology a valuable tool for developing medical devices with patient-precise dimensions.

Three-dimensional Printed Microvascular Clamps: A Safe, Cheap, and Effective Instrumentation for Microsurgery Training.

Microsurgical training involves practice in ex vivo models during the early learning curve, and poor instrument handling by the inexperienced microsurgeons can cause damage to microsurgical instrumentation or clamps, which is particularly costly. To address this, we demonstrate the development, design, manufacturing, and application of 3 different types of 3-dimensional printed microvascular clamps in an ex vivo simulation training model. This report provides evidence of a low-cost and easily accessible device that facilitates the process of microsurgical training. The clamps were found to provide advantages akin to normal stainless-steel microvascular clamps in training settings.

Zero electrical power pump for portable high-performance liquid chromatography.

A major trend in analytical chemistry is the miniaturization of laboratory instrumentation. We report a pump requiring no power to operate based on the controlled expansion of a pre-pressurised gas for use in portable applications of high-performance liquid chromatography. The performance of the gas pump is characterised and integrated into a compact liquid chromatography system capable of isocratic separations integrating an LED-based UV-absorption detector. The system weighed 6.7 kg when the mobile phase reservoir was fully charged with 150 mL solvent and included an on-board computer to control the system and analyse data. We characterise the flow-rate through chromatography columns with a variety of geometries and packing materials for a range of pressures up to 150 bar. The maximum variation in flow rate was measured to be 6.5 nL min-1, limited by the resolution of the flow detector. All tests were made on battery power and results are a mixture of those made in the laboratory and in the field. Additionally, we performed a series of 1 m drop tests on the device and show the system's high tolerance to mechanical shocks during operation in the field.

Micropatterning of planar metal electrodes by vacuum filling microfluidic channel geometries.

We present a simple, facile method to micropattern planar metal electrodes defined by the geometry of a microfluidic channel network template. By introducing aqueous solutions of metal into reversibly adhered PDMS devices by desiccation instead of flow, we are able to produce difficult to pattern "dead end" or discontinuous features with ease. We characterize electrodes fabricated using this method and perform electrical lysis of mammalian cancer cells and demonstrate their use as part of an antibody capture assay for GFP. Cell lysis in microwell arrays is achieved using the electrodes and the protein released is detected using an antibody microarray. We show how the template channels used as part of the workflow for patterning the electrodes may be produced using photolithography-free methods, such as laser micromachining and PDMS master moulding, and demonstrate how the use of an immiscible phase may be employed to create electrode spacings on the order of 25-50 µm, that overcome the current resolution limits of such methods. This work demonstrates how the rapid prototyping of electrodes for use in total analysis systems can be achieved on the bench with little or no need for centralized facilities.

Counting Proteins in Single Cells with Addressable Droplet Microarrays.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.