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>
	<li>Coombs, K. E., Leonard, A. T., Rush, M. N., Santistevan, D. A., Hedberg-Dirk, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+effect+of+material+stiffness+on+valvular+interstitial+cell+differentiation.">Isolated effect of material stiffness on valvular interstitial cell differentiation.</a> J Biomed Mater Res A. 105 (1), 51-61 (2017).</li><li>Zhang, L., 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=A+sandwich+tubular+scaffold+derived+from+chitosan+for+blood+vessel+tissue+engineering.">A sandwich tubular scaffold derived from chitosan for blood vessel tissue engineering.</a> J Biomed Mater Res A. 77 (2), 277-284 (2006).</li><li>Thottappillil, N., Nair, P. 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M., 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+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29 (15), 2348-2358 (2008).</li><li>Wang, K., 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=Creation+of+macropores+in+electrospun+silk+fibroin+scaffolds+using+sacrificial+PEO-microparticles+to+enhance+cellular+infiltration.">Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration.</a> Journal of Biomedical Materials Research Part A. 101 (12), 3474-3481 (2013).</li><li>Lee, B. 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 ...

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