Name: a training and testing system for performing vascular reconstruction in vitro
Uploaded: 2019-10-26T13:00:00.000+00:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about A Training and Testing System for Performing Vascular Reconstruction In Vitro at JoVE.com

This work was supported by grants from the Ministry of Education Innovation Team Development Program of China (No. IRT1279).

The protocol was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and was approved by the Committee on the Ethics of Animal Experiments of Xi&#39;an Jiaotong University, Xi&#39;an, Shaanxi Province, China.
1. Preparation prior to the training
NOTE: The vascular reconstruction training machine is shown in Figure 1. It consists of a control panel and an operating floor.
<ol>
	<li>Click the Clean button on the control panel to clean and drain the residual liquid from the operating floor.</li>
	<li>Click the Add Liquid button on the control panel and add 0.9% saline into the machine from the operating floor until the prompt &#34;The testing liquid is adequate&#34; appears on the control panel.</li>
	<li>Prepare the magnetic tractor, which consists of a circular permanent magnet with a diameter of 20 mm and a thickness of 1 mm, an acrylonitrile butadiene styrene (ABS) plastic casing, a spiral spring, a 30 cm nylon traction wire, and a stainless-steel clamp with plastic sleeves or a vascular clamp.
	<ol>
		<li>Glue the circular magnet and the plastic casing together using an acrylate adhesive. The traction force will increase with the lengthening of the traction wire. Use a universal testing machine to test the association between the length of the traction wire and the traction force (Figure 2).</li>
		<li>Fix the clamp and the plastic casing on the upper holder and the lower holder of the universal testing machine, respectively. Gradually elevate the upper holder to stretch the traction wire between the two holders. Test the strength of the traction wire while it is being stretched. 
		NOTE: The magnetic suture tractor and magnetic vascular tractor are shown in Figure 3.</li>
	</ol>
	</li>
	<li>Prepare a magnetic suture puller.
	<ol>
		<li>Use a quasi-oval polylactic acid board with a thickness of 2 mm, a major axis diameter of 10 cm, a minor axis diameter of 2 cm, three magnetic balls with a diameter of 5 mm, and three magnetic cylinders with a diameter of 5 mm and a height of 5 mm.</li>
		<li>Punch three holes with a diameter of 3 mm and a depth of 0.5 mm on the polylactic acid board, so that the magnetic balls can cling to the board by magnetic attraction force from the magnetic cylinders under the board. 
		NOTE: The magnetic suture puller is shown in Figure 4.</li>
	</ol>
	</li>
	<li>Fix the suture under the magnetic ball after one stitch. This plays the role of a suture puller, preventing the previous suture from loosening. Extract the end with the suture needle with a force of about 0.3 N, closely in parallel to the polylactic acid board, and continue the next stitch.</li>
	<li>Ligate all the vein branches using 3-0 silk sutures to avoid leakage after anastomosis. Use tissue scissors to trim the ends of the veins and clear the excess tissue on the wall of the veins to make the veins smooth. 
	NOTE: The vasculature used in this study included the right and left iliac veins (diameter ~10 mm) harvested from Bama pigs weighing 50&#8211;60 kg. To simplify the reconstruction, only a few branches of the iliac veins were picked, and the two veins were similar in size. The vasculature was kept at -20 &#176;C. Before training, it was immersed in 0.9% saline at room temperature.</li>
</ol>
2. Fix the veins on the operating floor
<ol>
	<li>Tie the two veins on the water inlet and water outlet of the training machine with 2-0 silk sutures. 
	NOTE: This study uses the two-point vascular anastomosis5.</li>
	<li>Adjust the length of the water outlet of the training machine and ensure that the ends of the two veins are tension-free in a parallel direction.</li>
	<li>Straighten the veins and lay two 4-0 polypropylene traction sutures at the 6 o&#8217;clock and 12 o&#8217;clock positions.</li>
	<li>Insert the needle of the traction sutures from the outside of the vein and then insert from the inside of the other vein.</li>
	<li>Wet the surgical glove and sutures to avoid damaging the sutures. Gently tie at least five knots to avoid tearing the walls of the veins.</li>
	<li>Use the two stainless-steel clamps of the magnetic suture tractor to grasp the traction sutures and attract the circular magnets of the magnetic suture tractors to the ferromagnetic stainless-steel operating floor. Adjust the position of magnetic attraction and ensure that the ends of the two veins are stretched in a vertical direction.</li>
	<li>Use the two vascular clamps of the magnetic vascular tractor to clamp the anterior wall of the veins and attract the circular magnets of the magnetic vascular tractors on the operating floor. Adjust the position of attraction and ensure that the anterior walls of the veins are retracted, and the posterior walls of the veins are clearly exposed.</li>
</ol>
3. Anastomosis of posterior walls
<ol>
	<li>Use the two stainless-steel clamps of the magnetic suture tractor to grasp the traction sutures and attract the circular magnets of the magnetic suture tractors on the ferromagnetic stainless-steel operating floor. Leave the tail segment of the polypropylene suture at the 12 o&#8217;clock position for the traction suture and use the segment with the needle for continuous suture.</li>
	<li>Ensure intima-to-intima contact between the two veins.</li>
	<li>Insert the first suture from the outside of the vein to the inside.</li>
	<li>In subsequent sutures, insert the needle from the inside of the vein and then insert from the outside of the other vein.</li>
	<li>Check that the sutures are not loose.</li>
	<li>After one suture, ensure that the polypropylene suture is hung on the magnetic suture puller and pull the polypropylene gently until the magnetic ball presses the polypropylene.</li>
	<li>Extract the end with the needle of the suture with a force of about 0.3 N, closely in parallel to the polylactic acid board, and continue the next stitch. 
	NOTE: By using this technique, the tail of the polypropylene suture will be sufficiently tight. As the sutures continue, the polypropylene suture will become shorter. According to the length of the suture, select the most suitable of the three magnetic balls, and then manually press the suture under it.</li>
	<li>Insert the last suture from the inside of the vein to the outside to ensure intima-to-intima contact between the two veins.</li>
	<li>Avoid stenosis after anastomosis by two means: Keep same, proper margin and needle spacing when stitching, and keep the &#34;growth factor&#34;15 when knotting. 
	NOTE: The &#34;growth factor&#34; is the reserved space away from the vessel wall when tying the first knot after the anastomosis so that vessels can remain flexible rather than stenose.
	<ol>
		<li>Maintain the same suture margin and needle spacing. 
		NOTE: In this study, iliac veins with a diameter of approximately 10 mm were used, so the suture margin and needle spacing was about 1 mm.</li>
		<li>Keep the &#34;growth factor&#34;15 when tying the knots. After anastomosis of the posterior walls, tie the end of the suture and the tail segment of the suture at the 6 o&#8217;clock position together away from the vein wall in order to prevent suture stenosis. Use the standard method for tying knots.</li>
	</ol>
	</li>
</ol>
4. Anastomosis of anterior walls
<ol>
	<li>After anastomosis of the posterior walls, remove the magnetic vascular tractor, leave the tail as a traction suture, and use the segment with the needle at the 6 o&#8217;clock position for the anastomosis of the anterior walls.</li>
	<li>Insert the needle from the outside of the vein and then insert from the inside of the other vein. 
	NOTE: The methods used in the anastomosis of the posterior walls to ensure the intima-to-intima contact between the two veins (the suture not being loose and avoiding stenosis after anastomosis) were followed in the anastomosis of the anterior walls5,15.</li>
	<li>After anastomosis of the anterior walls, cut off two traction sutures using suture scissors.</li>
</ol>
5. Test the effect of anastomosis
<ol>
	<li>Set the test parameters.
	<ol>
		<li>Set the perfusion pressure as 2.0 kPa on the control panel. 
		NOTE: The normal vein pressure will not exceed 2.0 kPa.</li>
		<li>Set the duration of peak pressure as 5 s on the control panel.</li>
		<li>Set the temperature as 25 &#176;C on the control panel.</li>
		<li>Set the pressure deviation as 0.1 kPa on the control panel.</li>
	</ol>
	</li>
	<li>Click the Test button and observe the time and pressure on the control panel and whether the reconstructed vein leaks. 
	NOTE: If the vein does not leak during the peak pressure, the anastomosis is successful. If leaks are found, the leakage position should be located and sutured, and then the test should be performed again. The results of the test in this video are shown in Figure 5.</li>
</ol>
6. Checking the safety of the magnetic suture puller
NOTE: To test whether the magnetic suture puller damaged the polypropylene suture, perform the breaking strength and light microscopy tests. In this experiment, three groups with six segments of 4-0 polypropylene suture in each were tested: a control group with no intervention on the polypropylene suture, a manual group in which the polypropylene suture was manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x.
<ol>
	<li>Test the breaking strength of the polypropylene suture on the universal testing machine. Fix the two ends of the polypropylene suture on the upper holder and the lower holder of the universal testing machine. Gradually elevate the upper holder. Test the strength of the polypropylene suture while it is being stretched. Set the breaking strength as the tension force when the suture snaps. Compare the breaking strength among the three groups and perform pairwise comparisons.</li>
	<li>Observe the damage of the polypropylene suture under a light microscope. Define the number of damage points as the number of fibrous or coarse fracture points visible at 200x magnification. Compare the number of damage points among the three groups and perform pairwise comparisons.</li>
</ol>

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The authors have nothing to disclose.

With the help of magnetic tractors and a magnetic suture puller, a trainee can complete vein anastomosis individually and precisely. Magnetic tractors pull the tissue that blocks the anastomosis field and provide suitable strength for stretching the veins in a vertical direction, thus achieving clear exposure for vein anastomosis. In traditional manual anastomosis, at least one assistant is required for surgical exposure. The use of magnetic tractors could achieve the required exposure and substitute for assistants. In addition, the traction force of the magnetic tractor was dependent on the length of the traction wire, so we could adjust the site that the magnetic tractor was adsorbed onto in order to change the length of the traction wire to obtain a suitable traction force. In contrast to traditional manual anastomosis, the traction force in this study was quantifiable by the length of the traction wire. This allowed us to avoid some issues resulting from too heavy or too light a traction force, such as tearing of the vasculature and unclear exposure.
The magnetic suture puller was another novel invention in this study. It replaced the requirement for an assistant to pull the suture to prevent the previous suture from loosening, resulting in anastomotic leakage. Because it pressed the polypropylene suture, we tested the degree of damage caused by the magnetic suture puller and compared it to intact and manual pulling. Although the number of damage points in the magnetic puller group was more than in the control group (intact polypropylene sutures), it was similar to that observed in the manual pull that is widely used in clinical practice. Moreover, the breaking strength test showed a similar breaking strength among the three groups. With the microscope, we found that the changes caused by the magnetic puller were too small to damage the strength of the polypropylene suture.
It must be emphasized that the tension on the vertical and parallel directions of the vasculature during anastomosis is significant. Therefore, it is vital to adjust the length of the water outlet of the training machine as well as the position of the magnetic tractors. In addition, as we add sutures, we pick the most suitable magnetic ball to press the suture so that the tension on the suture is moderate. Moreover, to avoid stenosis after anastomosis, it is essential to keep the same suture margin, needle spacing, and &#34;growth factor&#34;.
If the trainee wishes to practice anastomosis using vasculature with a greater or smaller diameter, the magnetic balls and cylinders of the magnetic suture puller should be replaced by bigger or smaller ones, so that the pulling force changes accordingly. Simultaneously, the test parameters after anastomosis should be adjusted. In the current version of the vascular reconstruction training machine, the diameter of the inlet and outlet is only 5 mm, making it difficult to use on smaller diameter vessels. Fortunately, the inlet and outlets are detachable, so the current inlet and outlet can be replaced by smaller ones that allow changes in vessel size.
Beside the sizes of the inlet and outlet, there are still some limitations to this training system. Because there is only one water inlet and one water outlet, this training and testing system is only applicable to end-to-end anastomosis, and trainees cannot practice end-to-side or side-to-side anastomosis using this system. Additionally, the surgical instruments used in this video (e.g., the needle holder and scissors) are ferromagnetic stainless steel. They were absorbed by the magnetic tools occasionally, which could interfere with the training progress. If conditions permit, the surgical instruments can be replaced with non-ferromagnetic titanium instruments.
Open surgical vascular simulators are generally divided into two types: in vivo and in vitro. Tang6 &#65279;developed a novel technique for vascular reconstruction in vivo using &#65279;ewes as animal models. Although this technique provided a more realistic operation scene, the use of in vivo animal models is both inconvenient for training and costly. Shimizu12 and Maluf13&#65279; invented in vitro &#65279;training devices for cerebrovascular anastomosis, while Bismuth14 introduced a vascular surgery course named Cardiovascular Fellows Bootcamp for cardiovascular surgery education. Although the rationale of our training system is similar to those outlined in these studies, no previous study has recommended the use of a device to assist in exposing surgical field and maintaining the tension of the suture. Thus, the training previously described must be completed by at least two trainees. Also, previous researchers did not introduce a way to precisely check the quality of anastomosis. Therefore, compared to these open vascular simulators, our technique is economical, convenient to practice individually, and effective in terms of feedback training quality.
We intend to add smaller water inlets and water outlets to the vascular reconstruction training instrument so that trainees can practice other types of anastomosis. We expect that magnetic tractors and suture pullers will be used to help surgeons expose the surgical field in routine clinical operations in the future.
In summary, we introduce a training and testing system where the trainee can complete manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller

Vascular reconstruction is a basic skill required for surgeons. Although Obora1 and Holt2 invented several mechanical reconstruction methods to simplify the reconstruction of small vessels (diameters &#60;10 mm), these methods are not commonly applied in macrovascular anastomosis. Manual vascular anastomosis is still performed in many operations, including vascular surgery3, emergency surgery4, and solid organ transplantation5. Thus, it is essential for surgeons to practice manual vascular anastomosis. However, an optimal training system for vascular reconstruction in vitro is uncommon, and inexperienced surgeons must undergo considerable training in vivo on large animals6 before they can master the technique. Because failure is inevitable during initial training, many animals are likely to die of vascular complications, which is concerning regarding animal welfare. Further, during the procedure of end-to-end vascular reconstruction, to avoid mistakes in stitch positions or loose sutures, the surgeon needs at least one assistant to expose the posterior vascular wall and pull the suture. Thus, vascular reconstruction usually cannot be performed by the surgeon individually, and the efficiency of preparation is usually limited by the proficiency of the assistant.
Magnetic anchoring surgery has become a topic of interest in recent years7,8,9,10,11. The clinical trial by Rivas et al.7 showed that with his magnetic surgical instrument and following the principle of magnetic anchoring, surgeons can perform reduced-port laparoscopic cholecystectomy. The use of this instrument also allows for a reduced role for the assistant during open surgery. Through the magnetic field, the magnetic device is adsorbed onto an anchoring point. This magnetic anchoring device can act as a mechanical arm, grasping and retracting the tissue or organ, exposing the surgical field, and simplifying the operation. Based on this rationale, we invented magnetic tractors to retract the vascular wall and suture, and a magnetic suture puller to pull the polypropylene sutures.
The use of a vascular reconstruction training machine was another milestone in this study. It consists of an operating floor and a control panel: the vasculature is fixed on the operating floor, and the trainee can practice on it. After anastomosis, the trainee can set the perfusion parameters on the control panel in order to test the quality of anastomosis. Compared to previous vascular anastomosis training systems6,12,13,14, the use of this system provides two main advantages: First, magnetic devices can be used to expose the surgical field, so that the trainees can practice on it individually. Second, the trainee can check the effect of anastomosis using a perfusion test.
In the present study, we introduce a training and testing system where the trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique and the quality of reconstruction can also be tested. Limited by the design and size of the water inlet and water outlet on the operating floor, the training system can only perform end-to-end reconstruction on vessels with a &#62;5 mm diameter.

<table><tbody><tr><td>Circular permanent magnet</td><td>Hangzhou Permanent Magnet Group Co.LTD</td><td>20*1mm</td><td>Magnetic tractor</td></tr><tr><td>Magnetic balls</td><td>Hangzhou Permanent Magnet Group Co.LTD</td><td>5mm</td><td>Magnetic suture puller</td></tr><tr><td>Magnetic cylinders</td><td>Hangzhou Permanent Magnet Group Co.LTD</td><td>5*5mm</td><td>Magnetic suture puller</td></tr><tr><td>Polypropylene suture</td><td>Johnson and Johnson</td><td>PROLENE 4-0</td><td>Used for anastomosis</td></tr><tr><td>Silk suture</td><td>SILK</td><td>2-0?3-0</td><td>Used for fixing vascular and ligation</td></tr><tr><td>Surgical insturments</td><td>Jinzhong Shanghai</td><td>JZ-2018</td><td>Suture scissors, tissue scissors? forceps, needle and needle holder</td></tr><tr><td>Universal testing machine</td><td>Zwick GmbH&amp;amp;Co</td><td>Z010</td><td>Used for testing the association between the length of traction wire and the traction force</td></tr></tbody></table>

a training and testing system for performing vascular reconstruction in vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

The vascular reconstruction training machine is shown in Figure 1 and includes two main parts: the operating floor and the control panel. The operating floor consists of a water inlet, a water outlet, and a water storage basin. The two ends of the vasculature are tied to the water inlet and water outlet to test the effect of anastomosis. The length of the water outlet is adjustable, and we set the parameters (e.g., the perfusion pressure, duration of peak pressure, temperature, and pressure deviation) on the control panel. In addition, we can observe the pressure curve on the control panel when the vasculature is tested.
The magnetic suture tractor and the magnetic vascular tractor are shown in Figure 3. The length of the traction wire is 30 cm, and the traction force increases with the lengthening of the traction wire (Figure 2). The range of the magnetic tractor&#39;s traction force is 0&#8211;1.8 N, which covers the range of traction force required for suture and vascular traction.
Photos of the magnetic suture puller are shown in Figure 4A,B. The three magnetic balls have a diameter of 5 mm, and the magnetic cylinders have a diameter of 5 mm and a height of 5 mm. These can be replaced by smaller or bigger ones. The suture pulling force will change accordingly.
In testing the effect of anastomosis, a time-perfusion pressure curve was generated and is shown in Figure 5. The perfusion pressure ascended to 2.0 kPa, which we set as the peak pressure. This was maintained for 5 s, which was set as the duration of peak pressure.
Regarding the safety of the magnetic suture puller, we tested whether the magnetic suture puller damaged the polypropylene suture using a breaking strength test and a light microscope. As shown in Figure 6, the breaking strength test results of the three groups were compared pairwise, and no significant difference was observed (p &#62; 0.05). As shown in Figure 7, the control group was less likely to be damaged (p &#60; 0.05), but the number of damaged points in the manual group and the magnetic puller group were similar (p &#62; 0.05).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60141/60141fig1.jpg" /> 
Figure 1: The vascular reconstruction training machine&#39;s two main parts. The operating floor and the control panel. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60141/60141fig2.jpg" /> 
Figure 2: The association between the length of the traction wire and the traction force. The length of the traction wire was 30 cm, and the range of traction force that the magnetic tractor could provide was 0&#8211;1.8 N. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60141/60141fig3.jpg" /> 
Figure 3: The magnetic suture tractor and the magnetic vascular tractor. (A) Magnetic suture tractor. (B) Magnetic vascular tractor. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60141/60141fig4.jpg" /> 
Figure 4: The magnetic suture puller. (A) Front view. (B). Lateral view. The magnetic suture puller consists of a quasi-oval polylactic acid board with a thickness of 2 mm, a major axis diameter of 10 cm, a minor axis diameter of 2 cm, three magnetic balls with diameter of 5 mm, and three magnetic cylinders with a diameter of 5 mm and a height of 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60141/60141fig5.jpg" /> 
Figure 5: Time-perfusion pressure curve. The perfusion pressure ascended to 2.0 kPa, which we set as the peak pressure. It was maintained for 5 s, indicating that the anastomosis was successful. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60141/60141fig6.jpg" /> 
Figure 6: The breaking strength test. (A) The association between the length of the polypropylene suture and the tension. (B). Comparison of the breaking strength among the three groups. There was no significant difference in the three groups (p &#62; 0.05). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60141/60141fig7.jpg" /> 
Figure 7: The light microscope tests. (A) Control group. (B) Manual group. (C) Magnetic puller group. (D) Comparison of the number of damage points among the three groups. The control group had fewer damage points (p &#60; 0.05), but there was no significant difference between the manual group and the magnetic puller group (p &#62; 0.05). The black arrow points to the damage point. The asterisk represents the significant difference. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60141/60141fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Training and Testing System for Performing Vascular Reconstruction In Vitro at JoVE.com

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...

a-training-testing-system-for-performing-vascular-reconstruction

Universidad San Sebastian

Research

JoVE Journal

Medicine

7.9K Views.  First Affiliated Hospital of Xi'an Jiaotong University. Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Video: A Training and Testing System for Performing Vascular Reconstruction In Vitro

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Bioengineering

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Step By Step: Microsurgical training method combining two nonliving animal models

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly laboratory rats. This method although extremely valuable can be economically demanding both for the surgeon and the sponsoring institution; it also requires special training facilities that may not always be available or accessible. Furthermore ethical concerns can limit the use of living animals for training purposes. Alternative training methods, such as inert tubes and gloves have not gained popularity among surgeons since they do not offer an experience similar to that of a clinical situation. Non-living animal models include the use of chicken thighs and wings; they offer a practice experience that resembles a clinical situation to a considerable extent. This type of training is relatively cheap and easily available. The microscope and instruments required can be acquired over the internet, and the chicken pieces can be bought at the local supermarket.
This approach allows a motivated trainee to rehearse different types of surgical techniques several times at a reasonable expense, helping to develop or maintain his surgical expertise if more complex facilities are not available. On the current manuscript we describe how to setup a small practice station, how to dissect the specimens, and how to practice both with the chicken thighs and with the chicken wings in a progressive fashion. This approach takes advantage on the versatility of the chicken thigh model and the small size of the chicken wing Brachial artery.

Here we describe how to set up a small microsurgical practice station and a simple and inexpensive method for the training of microsurgery with non-living animal models.

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly ...

3D Angiography for Venous Compliance Assessment in Sheep

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native vessel into which they are implanted. Compliance matching with the native vessel is emerging as a key property for graft success. The current gold standard for assessing vessel compliance involves the vessel's excision and ex vivo biaxial mechanical testing. We developed an in vivo method to assess venous compliance and distensibility that better reflects natural physiology and takes into consideration the impact of a pressure change caused by flowing blood and by any morphologic changes present. 
This method is designed as a survival procedure, facilitating longitudinal studies while potentially reducing the need for animal use. Our method involves injecting a 20 mL/kg saline bolus into the venous vasculature, followed by the acquisition of pre and post bolus 3D angiograms to observe alterations induced by the bolus, concurrently with intravascular pressure measurements in target regions. We are then able to measure the circumference and the cross-sectional area of the vessel pre and post bolus. 
With these data and the intravascular pressure, we are able to calculate the compliance and distensibility with specific equations. This method was used to compare the inferior vena cava's compliance and distensibility in native unoperated sheep to the conduit of sheep implanted with a long-term expanded polytetrafluorethylene (PTFE) graft. The native vessel was found to be more compliant and distensible than the PTFE graft at all measured locations. We conclude that this method safely provides in vivo measurements of vein compliance and distensibility.

Author Spotlight: Improved Imaging for Neovascular Development in Congenital Heart Disease Research

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native ...

In Vitro Thrombosis Test for Ventricular Assist Devices

The risk of thrombosis remains a significant concern in the development and clinical use of ventricular assist devices (VADs). Traditional assessments of VAD thrombogenicity, primarily through animal studies, are costly and time-consuming, raise ethical concerns, and ultimately may not accurately reflect human outcomes. To address these limitations, we developed an aggressive in vitro testing protocol designed to provoke thrombosis and identify potential high-risk areas within the blood flow path. This protocol, motivated by the work of Maruyama et al., employs a modified anticoagulation strategy and utilizes readily available components, making it accessible to most laboratories conducting in vitro blood testing of VADs. We demonstrated the utility of this method through iterative testing and refinement of a miniature magnetically levitated pediatric VAD (PediaFlow PF5). The method has been effective in identifying thrombogenic hotspots caused by design and manufacturing flaws in early VAD prototypes, enabling targeted improvements before advancing to animal studies. Despite its limitations, including the absence of pulsatile flow and the influence of donor blood characteristics, this protocol serves as a practical tool for early-stage VAD development and risk mitigation.

We present a benchtop protocol to induce thrombosis in ventricular assist devices (VAD) within a recirculating test platform. This method serves to identify thrombogenic hotspots in the blood flow path and can help improve thromboresistance ahead of preclinical testing in animal models.

The risk of thrombosis remains a significant concern in the development and clinical use of ventricular assist devices (VADs). Traditional assessments of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

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Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Tissue Engineering of a Human 3D in vitro Tumor Test System

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

Step By Step: Microsurgical training method combining two nonliving animal models

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography

In Vitro Thrombosis Test for Ventricular Assist Devices