Name: a spine robotic-assisted navigation system for pedicle screw placement
Uploaded: 2020-05-11T13:00:00
Description: Watch this Scientific Journal Video about A Spine Robotic-Assisted Navigation System for Pedicle Screw Placement at JoVE.com

This study was partially supported by Point Robotics Medtech Incorporation, which provided the robot system. The funder provided support in the form of salaries for X.Y. Xiao, C.W. Chen, H.K. Chou, and C.Y. Sung, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All procedures followed were in accordance with the ethical standards of the National Taiwan University Hospital (NTUH) Research Ethics Committee (REC) and the Helsinki Declaration of 1975 (in its most recently amended version). Informed consent must be obtained from all patients if further clinical trial is prepared.
NOTE: The anesthesia procedure can be categorized into three steps: pre-operative evaluation of the patient, intraoperative management, and postoperative management. During pre-o...

<ol>
	<li>Verma, K., Boniello, A., Rihn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+techniques+for+posterior+fixation+of+the+lumbar+spine.">Emerging techniques for posterior fixation of the lumbar spine.</a> Journal of the American Academy of Orthopaedic Surgery. 24 (6), 357-364 (2016).</li><li>Gaines, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pedicle-screw+internal+fixation+for+the+operative+treatment+of+spinal+disorders.">The use of pedicle-screw internal fixation for the operative treatment of spinal disorders.</a> The Journal of Bone and Joint Surgery-American. 82 (10), 1458-1476 (2000).</li><li>Dede, O., Ward, W., Bosch, P., Bowles, A., Roach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+freehand+pedicle+screw+placement+technique+in+adolescent+idiopathic+scoliosis+surgery:+what+is+the+incidence+of+neurological+symptoms+secondary+to+misplaced+screws.">Using the freehand pedicle screw placement technique in adolescent idiopathic scoliosis surgery: what is the incidence of neurological symptoms secondary to misplaced screws.</a> Spine. 39 (4), 286-290 (2014).</li><li>Costa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erratum:+Radiation+exposure+in+spine+surgery+using+an+image-guided+system+based+on+intraoperative+cone-beam+computed+tomography:+analysis+of+107+consecutive+cases.">Erratum: Radiation exposure in spine surgery using an image-guided system based on intraoperative cone-beam computed tomography: analysis of 107 consecutive cases.</a> Journal of Neurosurgery: Spine SPI. 26 (4), 542 (2017).</li><li>Stuer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robotic+technology+in+spine+surgery:+Current+applications+and+future+developments.">Robotic technology in spine surgery: Current applications and future developments.</a> Intraoperative Imaging. 109, 241-245 (2011).</li><li>Devito, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+acceptance+and+accuracy+assessment+of+spinal+implants+guided+with+SpineAssist+surgical+robot:+retrospective+study.">Clinical acceptance and accuracy assessment of spinal implants guided with SpineAssist surgical robot: retrospective study.</a> Spine. 35 (24), 2109-2115 (2010).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiological+and+clinical+differences+among+three+assisted+technologies+in+pedicle+screw+fixation+of+adult+degenerative+scoliosis.">Radiological and clinical differences among three assisted technologies in pedicle screw fixation of adult degenerative scoliosis.</a> Scientific Reports. 8 (1), 890 (2018).</li><li>Kantelhardt, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perioperative+course+and+accuracy+of+screw+positioning+in+conventional,+open+robotic-guided+and+percutaneous+robotic-guided,+pedicle+screw+placement.">Perioperative course and accuracy of screw positioning in conventional, open robotic-guided and percutaneous robotic-guided, pedicle screw placement.</a> European Spine Joutnal. 20 (6), 860-868 (2011).</li><li>Verma, R., Krishnan, S., Haendlmayer, K., Mohsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+outcome+of+computer-assisted+spinal+pedicle+screw+placement:+a+systematic+review+and+meta-analysis+of+23+studies+including+5,992+pedicle+screws.">Functional outcome of computer-assisted spinal pedicle screw placement: a systematic review and meta-analysis of 23 studies including 5,992 pedicle screws.</a> European Spine Journal. 19 (3), 370-375 (2010).</li><li>Ghasem, A., Sharma, A., Greif, D., Alam, M., Maaieh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arrival+of+Robotics+in+Spine+Surgery:+A+Review+of+the+Literature.">The Arrival of Robotics in Spine Surgery: A Review of the Literature.</a> Spine. 43 (23), 1670-1677 (2018).</li><li>Roser, F., Tatagiba, M., Maier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+robotics:+current+applications+and+future+perspectives.">Spinal robotics: current applications and future perspectives.</a> Neurosurgery. 72 (1), 12-18 (2013).</li><li>Chen, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+using+robotic-assisted+navigational+system+in+pedicle+screw+placement.">Results of using robotic-assisted navigational system in pedicle screw placement.</a> PLoS One. 14 (8), 0220851 (2019).</li><li>. NDI Medical Available from: <a target="_blank" href="https://www.ndigital.com/medical/products/polaris-vega">https://www.ndigital.com/medical/products/polaris-vega</a> (2020)</li><li>Gertzbein, S. D., Robbins, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+pedicular+screw+placement+in+vivo.">Accuracy of pedicular screw placement in vivo.</a> Spine. 15 (1), 11-14 (1990).</li><li>Kim, T. T., Johnson, J. P., Pashman, R., Drazin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+Invasive+Spinal+Surgery+with+Intraoperative+Image-Guided+Navigation.">Minimally Invasive Spinal Surgery with Intraoperative Image-Guided Navigation.</a> Biomed Research International. 2016, 5716235 (2016).</li><li>Bailey, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BWM+spinal+fixator+system.+A+preliminary+report+of+a+2-year+prospective,+international+multicenter+study+in+a+range+of+indications+requiring+surgical+intervention+for+bone+grafting+and+pedicle+screw+fixation.">The BWM spinal fixator system. A preliminary report of a 2-year prospective, international multicenter study in a range of indications requiring surgical intervention for bone grafting and pedicle screw fixation.</a> Spine. 21 (17), 2006-2015 (1996).</li><li>Lonstein, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+associated+with+pedicle+screws.">Complications associated with pedicle screws.</a> The Journal of Bone and Joint Surgery-American Volume. 81 (11), 1519-1528 (1999).</li></ol>

Since 1990, there have been rapid developments in surgical applications involving the use of robots. The available robotic technologies have been optimized, resulting in improved accuracy, overcoming the tremor in human hands, and reduced matching and registration times of navigation systems15. The benefits of surgical robot assistance include: (1) immediate standardization without lengthy learning processes; (2) surgeons can precisely follow the pre-operative plan, which is superimposed on a CT-b...

In the field of spinal surgery, spinal fusion surgery is a fundamental surgical procedure, especially posterior pedicle screw fixation, which can provide three-column support of the vertebrae and enhance the strength of biomechanics; thus, it has become one of the most commonly used surgical procedures1. In many early studies, the clinical effect of posterior pedicle screw implantation has been confirmed, and it has been widely used in surgery for many different spinal disorders, such as degenerative, traumatic, and complicated spinal conditions2.
However, although the posterior lumbar spinal fusion surgery can achieve excellent treatment effects, it is still risky due to the human body anatomy. There are many vital tissue structures close to the pedicle, such as the central nervous system, nerve roots, and main blood vessels. The damage of these tissues during the surgical procedure may cause serious complications, such as vascular injuries, neurological deficits, or screw loosening2,3. Moreover, the surgeons and staff are exposed to additional radiation, particularly in the case of minimally invasive spinal procedures4. Surgeons may experience fatigue and hand tremors after lengthy and tedious spinal surgery procedures, such as screw placements, bone osteotomy, and nerve decompression5.
The unsatisfactory rate of the pedicle screw placement procedure necessitated the proposal for a robotic-assisted navigation system to be applied in spinal surgeries to improve the surgery accuracy and patients&#8217; safety. Several studies on robotic-assisted navigation systems have demonstrated improvements in the safety, accuracy, and precision of pedicle screw placement, as well as decreased radiation exposure and operative times6,7,8,9,10. However, thorough screw trajectory planning, pre-operative planning with images, comprehensive robotic system with fixation device, and robot control software still need to be addressed to achieve this goal. This study focuses on the description of the robotic structure and the workflow of a self-developed navigation system (i.e., the Point spine navigation system (PSNS)) for robotic-assisted pedicle screw placement surgeries.
System description and surgical protocol 
The PSNS comprises a navigation workstation that includes the following. (1) There is a user interface software responsible for image reading through three-dimensional (3D) reconstruction, pre-operative planning, spatial kinematic relationship calculation, and registration. (2) The PSNS uses infrared optical guidance systems to track the spatial position of surgical robots and patients. The infrared optical guidance system contains the following components: (i) an optical tracker that actively emits infrared light and performs stereo positioning through a dual camera (Figure 1); (ii) a marker sphere whose surface has a reflective coating with reflective properties for precise tool tracking; and (iii) a tool with a dynamic reference frame (DRF) that comprises a base and four marker spheres. To avoid the identification failure of the tracking system, each device has a unique DRF design and cannot be shared with each other. The DRF used includes a base frame (BF) attached to the base of the handpiece to confirm the handpiece position, an end-effector frame (EF) attached to the end of the handpiece to confirm the handpiece position, a fiducial frame (FF) anchored on the patient&#8217;s bone to confirm the patient&#8217;s position, and a probe whose tip is used to confirm the target position in 3D space. (3) There is a handpiece comprising a six degrees of freedom (DOF) Stewart platform, with one end of the robot equipped with an operation tool used for drilling the screw path. The handpiece is a robotic-assisted navigation system that assists surgeons toward the accurate placement of implants, such as pedicle screws, or positioning of surgical tools during spinal surgery. The movement of the surgical target is tracked as the robot automatically compensates for the correct target. The robot is designed as a semi-active system that offers surgical tool guidance; however, the actual surgery is performed by surgeons. The operating principle and equipment are illustrated in Figure 2.
PSNS is indicated for procedures including but not limited to the following sample procedures: (i) open, minimally invasive, or percutaneous spinal surgery; (ii) spinal surgery site for thoracic, lumbar, or sacral vertebrae; (iii) posterior spinal fusion for trauma, degenerative stenosis disease, instability, spondylolisthesis, herniated disc, tumor, infection, or spinal deformity correction; (iv) placement of temporary or permanent devices, such as k-wires or needles, while performing vertebroplasty, or either transforaminal or interlaminar percutaneous endoscopic lumbar discectomy; and (iv) bone tumor excision, including the ablation of osteoid osteoma or tumor biopsy, in which the robot directed needles or guidewires to a given vertebral location. This procedure is contraindicated for those with an inability to tolerate anesthesia, surgical procedure, or when satisfactory navigation images have not been acquired.
Note that the operation staff, including neurosurgeons and orthopedic surgeons, must be licensed and trained in guiding courses. All procedures for operating the robot during surgery need to follow the recommended standardized procedures to avoid causing harm to the patient or surgeon. Surgeons must possess conventional surgical experience to ensure that it is possible to switch back to conventional surgical instruments and complete the surgery when it is determined that the navigation is inaccurate, based on the surgeons&#8217; anatomical knowledge.

<table>
	<tbody>
		<tr>
			<td>Dynamic reference frames</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FF tool kit: 
			1.Connecting Rod 
			2.Combination clamps 
			3.Multi-pin clamps 
			4.Schanz screw 
			5.Spinous process clamp 
			6.Open wrench 
			7.Hexagonal wrench</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Handpiece</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Handpiece holder</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Handpiece stand</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K-pin</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical tracker</td>
			<td>NDI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Passive spheres</td>
			<td>NDI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile box</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile drape</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trocar</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Workstation cart</td>
			<td>POINT</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a spine robotic-assisted navigation system for pedicle screw placement

Pedicle screw implantation has excellent treatment effects and is often used by surgeons in spinal fusion surgery. However, due to the complexity of human body anatomy, this surgical procedure is difficult and challenging, especially in minimally invasive surgery or patients with congenital anomalies and kyphoscoliosis deformity. In addition to the abovementioned factors, the surgical experience and technique of the surgeon also affect the recovery rates and complications of the patients after the surgical operation. Therefore, accurately performing pedicle screw implantation has is a constant topic of common concern for surgeons and patients. In recent years, with the technological development, robot-assisted navigation systems have gradually become adopted. These robot-assisted navigation systems provide surgeons with complete preoperative planning before surgery. The system provides 3D reconstructed images of each vertebra, allowing surgeons to understand the patient&#39;s physiological characteristics more quickly. It also provides 2D images of sagittal, coronal, axial and oblique planes so that surgeons can accurately perform pedicle screw placement plan.
Previous studies have demonstrated the effectiveness of robot-assisted navigation systems for pedicle screw implantation procedures, including accuracy and safety assessments. This step-by-step protocol aims to outline a standardized surgical technique note for robotic-assisted pedicle screw placement.

The safety and accuracy of robotic-assisted pedicle screw placements have been addressed in several studies6,11. We match the vertebrae with pre-operative planning images under an optical tracking system in the proposed method. After determining the planned surgical path, this information was transferred to the handpiece through the handpiece control unit. The navigation system integrates the tracking information and displays it on the monitor during the surgery....

Watch this Scientific Journal Video about A Spine Robotic-Assisted Navigation System for Pedicle Screw Placement at JoVE.com

A Spine Robotic-Assisted Navigation System for Pedicle Screw Placement

This article presents a standardized surgical technique for robotic-assisted pedicle screw placement by using robotic-assisted navigational systems. We present a step-by-step protocol and describe the workflow and precautions of this procedure.

Pedicle screw implantation has excellent treatment effects and is often used by surgeons in spinal fusion surgery. However, due to the complexity of human ...

This robot-assisted navigation system is very effective, accurate, and safe. It provides a friendly user interface and automatically compensated handpiece for pedicle screw replacement. The point handpiece of this system has a parallel platform architecture, which has the advantages of a strong load capacity, fast positioning speed, portability, small footprint, and high curvability.This system can be used to perform minimally invasive surgeries, complicated kyphoid scoliosis deformities and other challenging cases. We plan to use the Robotics system in other surgical procedures such as nerve decompression. In contrast to a traditional surgical methods, this system uses a unique technology that allows visualization of the surgical goals and operation steps within the application interface.Demonstrating the procedure with me will be Chin-Wei Chen and Xiu-Yun Xiao, engineers from Point Robotics. After anesthesia and draping, place one percutaneous wire with a ply of 1.5 millimeters on the posterior iliac crest to anchor the fiducial frame to the iliac bone of the saw bone and check the entry point under fluoroscopy. Mark the entry point with ink.Use a power drill set to 1, 000 revolutions per minute to insert the first percutaneous pin into the patient's posterior iliac crest. Use the pin to anchor fiducial frame and adjust the frame until it is recognized by the optical tracking camera. When the frame is in position, use a screwdriver to fix the frame to the pin and use the drill to insert the second percutaneous pin, further securing the frame with the second pin.Mark the surgical field with ink and use a surgical scalpel to make a skin incision along the mark. Then, use a screwdriver to fix the frame to the spinal process of interest and have the surgeon confirm that the frame has been firmly anchored. For spatial labeling and registration, enter the dynamic reference frame monitoring interface to the Point Spine Navigation System software with multiple planar views.All of the dynamic reference frames should be inside the vision area of the optical tracking system. When the dynamic reference frame vector arrow is displayed, the tracker has been stably recognized by the tracking system. To ensure a correct registration, select at least four non-coplanar feature points on the patient's pre-operative 3D reconstruction CT images.Use the tip of the probe to keep in contact with the first feature point within the actual surgical area. Then press Probe Selection'to confirm the access point. When all of the feature points have been confirmed, press Calculation'The system will calculate the result of the landmark registration and present it in the software interface.When the acceptance criteria for the registration accuracy meets the needs of the clinical indications, use the probe tip. Continuously contact any point on the bone surface in the actual surgical area and press the Probe Selection'button to confirm the access points. When at least 50 pic points have been confirmed, press Calculation'The system will calculate the surface matching result and present it on the software interface.Once the registration result has been accepted, use the probe to select the obvious anatomical landmarks of the actual surgical area for confirmation. To assemble the robot, angle the handpiece in space so that the two circles representing the angle of the handpiece coincide in the software interface. Then, horizontally and vertically adjust the position of the handpiece, so that the dots representing the position of the handpiece in the software interface are aligned with the entry points of the planned path.We recommend becoming familiar with the first person orientation of the user interface and the movement we have here of the handheld robot before attempting a surgery. To prepare the pedicle, activate the drill function of the handpiece and drill the instruments mounted on the front end along the planned path. Use the C-arm to confirm the position of the K-pin and the trocar and use the anterior/posterior view to determine whether the instrument is located in the oval area formed by the pedicle in the perspective image.Then, under the lateral view, determine whether the instrument is within the range of the pedicle and the vertebrae. When the K-pin and trocar positions are appropriate for the procedure, replace them with guide wires and insert the pedicle screws through the guide wires. In a previous study, a low-overall screw malposition rate of 1.7%from a total of 59 screws placed on 34 seen vertebrae through the Point Spine Navigation System was demonstrated.No spinal canal perforations or injuries to any other major vessels were observed and all of the pedicle screws were positioned within the safe zone. The robot can, not only drill, but also perform even soft tissue manipulations because of the high accuracy of robot, many surgical procedures can be performed safely. Our team is currently focusing on making the user learning curve easier and providing a supporting arm to reduce the weight of the handpiece.

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A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Fast Pyrolysis of Biomass Residues in a Twin-screw Mixing Reactor

Fast pyrolysis is being increasingly applied in commercial plants worldwide. They run exclusively on woody biomass, which has favorable properties for conversion with fast pyrolysis. In order to increase the synergies of food production and the energetic and/or material use of biomass, it is desirable to utilize residues from agricultural production, e.g., straw. The presented method is suitable for converting such a material on an industrial scale. The main features are presented and an example of mass balances from the conversion of several biomass residues is given. After conversion, fractionated condensation is applied in order to retrieve two condensates &#8212; an organic-rich and an aqueous-rich one. This design prevents the production of fast pyrolysis bio-oil that exhibits phase separation. A two phase bio-oil is to be expected because of the typically high ash content of straw biomass, which promotes the production of water of reaction during conversion.
Both fractionated condensation and the use of biomass with high ash content demand a careful approach for establishing balances. Not all kind of balances are both meaningful and comparable to other results from the literature. Different balancing methods are presented, and the information that can be derived from them is discussed.

A procedure for thermochemical conversion of biomass residues is presented that aims at maximizing the yield of liquid products (fast pyrolysis). It is based on a technology proven on an industrial scale and especially suitable for treating a straw type of biomass.

Fast pyrolysis is being increasingly applied in commercial plants worldwide. They run exclusively on woody biomass, which has favorable properties for ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...