Name: implantation of electrospun vascular grafts with optimized structure in a rat model
Uploaded: 2018-06-27T13:00:00
Description: Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

This work was financially supported by NSFC projects (81522023, 81530059, 91639113, 81772000, 81371699, and 81401534).

The use of experimental animals was approved by the Animal Experiments Ethical Committee of Nankai University and carried out in conformity with the Guide for Care and Use of Laboratory Animals.
1. Fabrication of Electrospun PCL Grafts
NOTE: Herein, an electrospinning technique was utilized to fabricate vascular grafts.
<ol>
	<li>Prepare PCL solutions of 25 wt% and 10 wt%, by dissolving PCL in a mixture of methanol and chloroform, respectively, (1:5 volume ratio),...

<ol>
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L. 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=Femtosecond+laser+ablation+enhances+cell+infiltration+into+three-dimensional+electrospun+scaffolds.">Femtosecond laser ablation enhances cell infiltration into three-dimensional electrospun scaffolds.</a> Acta Biomaterialia. 8 (7), 2648-2658 (2012).</li><li>Rnjak-Kovacina, J., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+pore+size+of+electrospun+scaffolds.">Increasing the pore size of electrospun scaffolds.</a> Tissue Eng Part B Rev. 17 (5), 365-372 (2011).</li><li>Zhong, S., Zhang, Y., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+large+pores+in+electrospun+nanofibrous+scaffolds+for+cellular+infiltration:+a+review.">Fabrication of large pores in electrospun nanofibrous scaffolds for cellular infiltration: a review.</a> Tissue Eng Part B Rev. 18 (2), 77-87 (2012).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Rnjak-Kovacina, J., 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=Tailoring+the+porosity+and+pore+size+of+electrospun+synthetic+human+elastin+scaffolds+for+dermal+tissue+engineering.">Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.</a> Biomaterials. 32 (28), 6729-6736 (2011).</li><li>Wang, Z., 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+effect+of+thick+fibers+and+large+pores+of+electrospun+poly(epsilon-caprolactone)+vascular+grafts+on+macrophage+polarization+and+arterial+regeneration.">The effect of thick fibers and large pores of electrospun poly(epsilon-caprolactone) vascular grafts on macrophage polarization and arterial regeneration.</a> Biomaterials. 35 (22), 5700-5710 (2014).</li><li>Hutcheson, J. D., 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=Genesis+and+growth+of+extracellular-vesicle-derived+microcalcification+in+atherosclerotic+plaques.">Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.</a> Nat Mater. 15 (3), 335-343 (2016).</li><li>Tara, S., 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=Well-organized+neointima+of+large-pore+poly(L-lactic+acid)+vascular+graft+coated+with+poly(L-lactic-co-epsilon-caprolactone)+prevents+calcific+deposition+compared+to+small-pore+electrospun+poly(L-lactic+acid)+graft+in+a+mouse+aortic+implantation+model.">Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-epsilon-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model.</a> Atherosclerosis. 237 (2), 684-691 (2014).</li></ol>

Cell infiltration is critical for the regeneration and remodeling of the vascular graft in vivo16. Limited cell infiltration is often related to the relatively small pores of the graft that hinder the migration of cells into the graft wall. To address this difficulty, we developed a modified method to prepare electrospun PCL vascular grafts with large-pore structure. In detail, the pore size increased with the increase of fiber thickness that could be readily controlled by the processing ...

Vascular grafts made from synthetic polymers are widely utilized in clinic for the therapy of cardiovascular diseases (CVDs). Unfortunately, in the case of small-diameter vascular grafts (D &#60;6 mm) there are no successful products available due to the low patency triggered by reduced blood flow velocity, which often leads to thrombosis, intimal hyperplasia, and other complications1.
Tissue engineering provides an alternative strategy to realize long-term patency and homeostasis based on a scaffold-guided vascular regeneration and reconstruction. In detail, the vascular graft, as a three-dimensional template, could provide mechanical support and structural guidance during the regeneration of vascular tissue and influence cellular functions, including cell adhesion, migration, proliferation, and secretion of extracellular-matrix2. Up to now, various synthetic polymers have been evaluated for applications in vascular tissue engineering. Among these polymers, poly(&#949;-caprolactone) (PCL) has been intensively investigated due to good cell compatibility and slow degradation ranging from several months to two years3. In a rat aorta model4,5,6, PCL vascular grafts processed by electrospinning exhibited excellent structural integrity and patency, as well as continuously increased cell invasion and neovascularization in the graft wall for up to 6 months. However, adverse tissue remodeling, including regression of cells and capillaries and calcification, were also observed at longer timepoints, up to 18 months.
Cellularization of the vascular graft is a key factor determining tissue regeneration and remolding7. Electrospinning, as a versatile technique, has been widely employed for the preparation of vascular grafts with nano-fibrous structure8. Unfortunately, the relatively small pore structure often leads to insufficient cell infiltration in the electrospun vascular graft, which limits the subsequent tissue regeneration. To resolve this problem, various techniques have been attempted to increase pore size and overall porosity, including the salt/polymer leaching9,10, modification of collector apparatus, post-treatment by laser radiation11, etc. In fact, the structure of electrospun grafts (including fiber diameter, pore size, and porosity) is closely related to the processing conditions12,13. During electrospinning, the fiber diameter can be readily controlled by changing the parameters, such as the concentration of the polymer solution, flow rate, voltage, etc.14,15, and therefore, the pore size and porosity have been enhanced accordingly.
We recently reported a modified PCL electrospun graft with macroporous structure (fibers with diameter of 5 - 7 &#181;m and pores of 30 - 40 &#181;m). In vivo implantation by replacing rat abdominal aorta showed high rate of patency, as well as good endothelialization and smooth muscle regeneration at 3 months post-surgery16. More importantly, no adverse tissue remodeling including calcification and cell regression could be observed even after one year of implantation.

<table>
	<tbody>
		<tr>
			<td>Poly(&#949;-caprolactone) (PCL) pellets (Mn=80,000)</td>
			<td>Sigma</td>
			<td>704067</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>A1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Tianjin Fengchuan Company</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Laboratory Animal Center of the Academy of Military Medical Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Hebei Tiancheng Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Tianjin Ruijinte chemical company</td>
			<td>2223</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium Injection</td>
			<td>Tianjin Biochem Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin Sulfate Injection</td>
			<td>Jiangsu Lianshui Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-SMA primary antibody</td>
			<td>Abcam</td>
			<td>ab7817</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-smooth MYH primary antibody</td>
			<td>Abcam</td>
			<td>ab683</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-rat elastin antibody</td>
			<td>Abcam</td>
			<td>ab23748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-von Willebrand factor primary antibody</td>
			<td>Abcam</td>
			<td>ab6994</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150117</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>5% normal goat serum</td>
			<td>Zhongshan Golden bridge</td>
			<td>ZLI9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin (H&#38;E)</td>
			<td>Beijing leagene biotech</td>
			<td>DH0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Beijing leagene biotech</td>
			<td>DC0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Verhoeff-van Gieson (VVG)</td>
			<td>Beijing leagene biotech</td>
			<td>DC0059</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Kossa</td>
			<td>Beijing leagene biotech</td>
			<td>DS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,3-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>G3002b</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,9-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>H901</td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation of electrospun vascular grafts with optimized structure in a rat model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

The PCL grafts were explanted at 3 months and 12 months post-operatively and analyzed by standard histological techniques for hematoxylin and eosin (H&#38;E), Masson trichrome, Verhoeff-van Gieson (VVG), Von Kossa, and immunofluorescence staining for &#945;-SMA, MYH, vWF, and elastin. The histological images were taken using an upright microscope, and the immunofluorescence images were taken using a &#64258;uorescence microscope.
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Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

The overall goal of this surgical intervention is to transplant an electrospun vascular graft into a rat abdominal aorta, and evaluate the longterm performance of the graft. This method has been established by Professor Deling Kong and other colleagues in our group, and it can help answer key questions in the study of electrospun vascular grafts, such as the effect of structure and the surface properties of the graft on the vascular regeneration. The main advantage of this technique is that it has good repeatability and high animal survival rate.Besides, the animal model is easy to manage at a low cost. This work will be conducted by our colleague, Doctor Wang. To begin, prepare the polymer solution by dissolving polycaprolactone in a 1-to-5 mixture of methanol and chloroform, and stirring it at room temperature for 12 hours.Load the polymer solution into a 10 milliliter glass syringe, and equip it with a blunt 21 gauge needle. Then, place a stainless steel mandrel on the collection instrument, and make sure that it is properly grounded. Adjust the working distance from needle tip to collector to 17 centimeters for the 25%polycaprolactone solution.Then, set the flow rate on the syringe pump to eight milliliters per hour. Next, set the voltage on the power supply to 11 kilovolts, and start the mandrel spinning at 40 RPMs in order to collect electrospun fibers for appropriate fiber distribution. With everything now set up, turn on the power supply and the syringe pump to begin collecting the electrospun fibers.After 4.5 minutes, turn off the syringe pump, power supply, and the mandrel motor. Allow the graft to dry in the hood for one hour. Then, remove the graft from the mandrel using absolute alcohol, and place it in a vacuum overnight to remove the residual solvent.Sterilize the graft with ethylene oxide, and allow it to dry in a sterile biological containment hood. Using standard techniques, obtain SEM images of the lumen of the grafts, and import them into ImageJ. Use the measurement tools to measure both the fiber diameter and average pore size in each image, to ensure consistency across samples.To obtain information on the mechanical properties of the graft material, first cut a tubular scaffold into three millimeter sections using a razor blade. Then, use calipers to measure the thickness of the scaffolds. Next, load the scaffolds into the grips of a tensile testing machine, so that there is one millimeter between the grips.Hold the scaffolds axially, at a rate of 10 millimeters per minute, until they rupture. Be sure to record both the tensile strength and ultimate elongation at break for the samples. Prior to surgery, fast male Sprague Dawley rats, weighing 240 to 270 grams, for 24 hours prior to the implantation surgery.Anesthetize the rat and prepare it for surgery as described in the accompanying text protocol. Confirm adequate anesthetization by ensuring that the rat has relaxed muscles and steady breathing. Then, place the rat under the operating microscope in a supine position.Using surgical scissors, perform a four to five centimeter long midline laparotomy incision, and expose the abdominal cavity. Carefully retract and wrap the intestines with gauze that has been moistened with saline solution. While looking through the operating microscope strip the abdominal aorta from the adjacent tissues.Identify and ligate all of the small branches of the descending aorta, using 9-0 monofilament nylon sutures. Next, use vascular clamps to clamp up to a one centimeter isolated section of the aorta. Then, use a pair of micro scissors to transect the abdominal aorta between the clamps.Flush the two ends of the aorta using heparinized saline solution to remove the residual blood. Peel off the adventitia of the clamped aorta using micro scissors. Then, place a one centimeter long section of the sterilized graft in the gap created by the removed section of aorta.Using 9-0 monofilament nylon sutures, construct four anastomoses using a figure eight suture pattern at nine o'clock, three o'clock, 12 o'clock, and then six o'clock positions of the proximal side. Then, construct the other four anastomoses between each of the sutures in the proximal side. Place the graft vertically to show the equal interval among the eight stitches under the operating microscope.After finishing the proximal suture, repeat the process on the distal side. Once the graft has been attached, remove the distal clamp to allow the blood to flow into the graft, then remove the proximal clamp. Using a sterile cotton ball, apply pressure to stop any observed bleeding at the suture points.Press for about three minutes, until hemostasis occurs. Next, return the intestines into the abdominal cavity, and flush the abdominal cavity using warm physiological saline solution containing gentamicin. Then, sew up the abdominal wall using 3-0 nylon suture in the muscle and skin layers.Use iodine on the incision after surgery only once. Return the rat to the company of others once it has fully recovered. The concentration of polycaprolactone in the electrospinning solution can affect both the fiber diameter, and the mechanical properties of the grafts.The higher concentration 25%solution produces vascular grafts with thicker fibers and larger pores than the 10%solution. After three months in vivo, histological staining revealed a layer of new tissue formed on the lumen of the graft, and a significant amount of collagen deposition within the graft itself. This new tissue layer is lined with endothelial cells, below which can be found a layer of both smooth muscle cells and circumferentially aligned elastin.After 12 months in vivo, the new tissue appeared well integrated, and the endothelial and smooth muscle layers remained intact. Once mastered, this technique can be completed in one to two hours, if it is performed appropriately. Following this procedure, other measures like color doppler ultrasound, can be performed in order to answer additional questions, like After watching this video, you should have a good understanding of how to fabricate the electrospun vascular grafts, and evaluate its in vivo performance in a rat abdominal artery replacement model.

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