Name: implantation of electrospun vascular grafts with optimized structure in a rat model
Uploaded: 2018-06-27T13:00:00.000+00:00
Duration: 8 min 46 s
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), at room temperature (RT) for 12 h.</li>
	<li>Load the PCL solution into a 10-mL glass syringe.</li>
	<li>Place the syringe with a 21-G needle.</li>
	<li>Place the stainless-steel mandrel (2 mm in diameter, 25 cm in length) on the collection instrument.</li>
	<li>For thicker-fiber grafts, use the PCL solution of 25 wt%, working distance of 17 cm from needle tip to collector, flow rate of 8 mL/h, and voltage of 11 kV as the electrospinning parameters. For thinner-fiber grafts, use the PCL solution of 10 wt%, working distance of 20 cm from needle tip to collector, a flow rate of 2 mL/h, and voltage of 18 kV as the electrospinning parameters.</li>
	<li>Ensure that the obtained grafts are placed in vacuum overnight to remove the residual solvent.&#160;Sterilize all instruments prior to the procedure and maintain aseptic technique throughout.</li>
	<li>Prior to the implantation, disinfect the grafts by immersing them in 10 mL of 75% ethanol for 30 min and then exposing them to UV light overnight.</li>
	<li>Fiber and pore size measurements: Calculate the average fiber diameter using ImageJ software based on scanning electron microscopy (SEM) images.</li>
	<li>Mechanical testing of scaffolds:
	<ol>
		<li>Cut the tubular scaffolds into 3 mm sections in length using a razor blade. Measure the thickness of scaffolds using a micrometer.</li>
		<li>Place the tubular scaffolds on a tensile-testing machine with a load capacity of 100 N.</li>
		<li>Clamp the scaffolds with a 1 mm inter-clamp distance and pull longitudinally at a rate of 10 mm/min until rupture. Measure the tensile strength and ultimate elongation at break. Calculate Young&#39;s modulus from the initial linear region (up to 5% strain) of the stress-strain curve.</li>
	</ol>
	</li>
</ol>
2. Rat Abdominal Aorta Implantation Model
NOTE: All materials and instruments used in surgery are sterile. During the surgery, make sure that the operator wears a gauze mask and sterile gloves to avoid infections. Ensure the room temperature is kept at 27 - 30 &#176;C to maintain the animal body temperature. Follow local IACUC guidelines regarding analgesia.&#160;
<ol>
	<li>Use male Sprague Dawley rats weighing 240 - 270 g as recipients of vascular graft. Ensure the rat has fasted 24 h before surgery.&#160;The aim of fasting rats for 24 h&#160;is to empty&#160; the faeces in the intestinal tract sufficiently, thereby broaden the operator&#8217;s horizon.</li>
	<li>Grasp the rat&#39;s back neck and keep its head downwards, insert the springe needle into the abdominal cavity of the lower abdomen. Induce the rat for anesthesia with chloral hydrate (330 mg/kg) by an intraperitoneal injection.</li>
	<li>Confirm adequate anesthetization by ensuring that the rat has relaxed muscles and steady breathing. Place the rat under the operating microscope in a supine position.</li>
	<li>Apply petrolatum ophthalmic vet ointment on the eyes to prevent dryness while under anesthesia. Administer anticoagulation (100 UI/kg) with heparinized physiological saline solution (50 UI/mL) by tail vein injection before surgery.</li>
	<li>Shave off the fur in the anterior abdominal wall using a razor blade, and clean the skin using Iodine solution and medical alcohol solution.</li>
	<li>Perform a midline laparotomy incision with surgical scissors and ensure that the incision is about 4 - 5 cm long, and then expose the abdominal cavity.</li>
	<li>Retract and wrap the intestines with gauze moistened with saline solution preferentially.</li>
	<li>Dissect the abdominal aorta carefully.</li>
	<li>Identify and ligate all small branches using 9-0 monofilament nylon sutures.</li>
	<li>Clamp the isolated section (up to 1 cm in length) of the aorta using two vascular clamps.&#160;The aorta can remain clamped for 20-30 min.</li>
	<li>Transect the abdominal aorta between two clamps using micro-scissors to create the anastomotic sites.</li>
	<li>Flush the two ends of aorta using heparinized saline (50 UI/mL) solution to remove the residual blood.</li>
	<li>Peel off the adventitia using micro-scissors.</li>
	<li>Anastomose the graft with 2 mm inner diameter and 1 cm in length to the rat&#39;s abdominal aorta with a figure-of-eight suture pattern using 9-0 monofilament nylon sutures.</li>
	<li>Firstly, construct four anastomoses according to the sequence of 9, 3, 12, and 6 o&#39;clock positions at the proximal side, then anastomose the cut edges in 4 stitches between two sutures. After finishing the proximal suture, suture the distal side by the same method. 
	NOTE: Every stitch is required to ensure the native side is slightly embedded in the graft.</li>
	<li>Remove the distal clamp to allow the blood to flow into the graft, then remove the proximal clamp.</li>
	<li>Press the suture ends to stop the bleeding using a sterile cotton ball or a small gauze sponge. Press for about 3 min, until hemostasis.</li>
	<li>Return the intestines into the abdominal cavity.</li>
	<li>Flush the abdominal cavity using warm physiological saline solution with gentamicin (320 U/mL).</li>
	<li>Sew up the abdominal wall using a 3-0 Nylon suture in the muscle and skin layer, respectively.</li>
	<li>Place the rat into a clean and dry cage and put a heating pad under the cage to maintain animal body temperature; then wait for the rat to recover from anesthesia. Attend to the animal until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>After it regains consciousness, put the rat in a single cage with food and water. Apply iodine on the wound to prevent infection&#160;after surgery. Return the rat to the company of other animals until it fully recovers.</li>
	<li>Euthanize rats according to institutional guidelines at predetermined time points.</li>
</ol>

<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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+vascular+regeneration:+current+status.">Scaffolds in vascular regeneration: current status.</a> Vasc Health Risk Manag. 11, 79-91 (2015).</li><li>Pektok, 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=Degradation+and+healing+characteristics+of+small-diameter+poly+(e-caprolactone)+vascular+grafts+in+the+rat+systemic+arterial+circulation.">Degradation and healing characteristics of small-diameter poly (e-caprolactone) vascular grafts in the rat systemic arterial circulation.</a> Circulation. 118 (24), 2563-2570 (2008).</li><li>Innocente, F., 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=Paclitaxel-eluting+biodegradable+synthetic+vascular+prostheses:+a+step+towards+reduction+of+neointima+formation?.">Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?.</a> Circulation. 120 (11 Suppl), S37-S45 (2009).</li><li>de Valence, 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=Advantages+of+bilayered+vascular+grafts+for+surgical+applicability+and+tissue+regeneration.">Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration.</a> Acta Biomater. 8 (11), 3914-3920 (2012).</li><li>Assmann, A., 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=Acceleration+of+autologous+in+vivo+recellularization+of+decellularized+aortic+conduits+by+fibronectin+surface+coating.">Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating.</a> Biomaterials. 34 (25), 6015-6026 (2013).</li><li>Hasan, A., 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=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta Biomater. 10 (1), 11-25 (2014).</li><li>Baker, B. 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>

The authors have no conflicting financial interests.

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 parameters. The results showed that the host cells could easily infiltrate into the wall of this macroporous graft after in vivo implantation and the cellularity remained at a relatively high level without obvious cell regression at 12 months post-surgery.
Native artery mainly consists of three layers, that is, endothelium, tunica media, and adventitia. Endothelium, as an anti-thrombogenic interface, plays a vital role in maintaining the long-term patency of the blood vessel. In our study, full endothelialization on the graft was observed at 3 months. In addition, the tunica media consisting of several layers of smooth muscle cells are very important in regulating blood vessel function and providing appropriate mechanical properties of the artery. The present study revealed that the electrospun PCL grafts with thick-fiber and large pore markedly enhanced the regeneration of functional tunica media. Besides, the structure of regenerated smooth muscle is similar to that of the native tunica media. Immunofluorescence staining showed several layers of MYH+ cells distributed within elastin network, reflecting the contractile phenotype of smooth muscle cells circumferentially. More importantly, regenerated tissues (both endothelium and smooth muscle) kept intact and there was no adverse remodeling even after 12 months due to the imbalance between ECM synthesis and degradation.
Calcification is still a major problem associated with cardiovascular implants, especially in the vascular graft. Vascular smooth muscle cells (VSMCs) lose their original phenotype and experience trans-differentiation towards osteochondrogenic direction, leading to ectopic mineralization during the process of vascular calcification. Our study showed that there was no calcium deposition occurring within the graft wall even after 12 months of implantation. The main reasons for the inhibited calcification in the macro-porous vascular graft include: (1) the structure of the macro-porous graft promotes metabolism, such as ion exchange between cells and blood; (2) the physical cues of the graft structure could regulate or inhibit the differentiation of the VSMC into the osteoblast1, (3) good cell infiltration in the large pores promotes the secretion of the ECM and inhibits its degradation that will trigger calcification17, and (4) normal or functional VSMCs have a potential to prevent the calcium deposition18.
In summary, the long-term evaluation of macro-porous electrospun PCL vascular grafts in the rat abdominal aorta model provides important insight into potential challenges of degradable vascular grafts, which will direct the following research.

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(&amp;epsilon;-caprolactone) (PCL) pellets (M&lt;sub&gt;n&lt;/sub&gt;=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-&amp;alpha;-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&amp;amp;E)</td><td>Beijing leagene biotech</td><td>DH0006</td><td></td></tr><tr><td>Masson&#039;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.
All data were expressed as mean &#177; SD. A two-tailed paired Student&#39;s t-test was used to compare the differences. A value of p &#60;0.05 was considered to be statistically significant.
Novel electrospun PCL grafts with optimized structure, that is, thicker fibers and larger pores, were successfully fabricated in this study. SEM images demonstrated that the averaged fiber diameter was almost 8 times thicker in the modified grafts (Figure 1A) than in the conventional one (Figure 1B) (5.59 &#177; 0.67 versus 0.69 &#177; 0.54 &#181;m). As a result, the averaged pore size was markedly increased, from ~ 4.66 &#181;m in thinner-fiber graft to ~ 40.88 &#181;m in the thicker-fiber one. Cross-sections showed homogeneous fiber distribution within the wall of the tubular grafts in both thicker-fiber (Figure 1D-F) and thinner-fiber groups (Figure 1G-I). The wall thickness was about 400 - 500 &#181;m. The mechanical properties of grafts were characterized by tensile testing, and the typical stress-strain curves were shown in Figure 1C. The mechanical properties of two grafts were evidently different in terms of elongation. The corresponding value of thicker-fiber grafts was about 3 times higher than the thinner-fiber grafts, suggesting the enhanced toughness.
The prepared vascular grafts (inner diameter of 2.0 mm and length of 1 cm) (Figure 2A) were implanted to replace a segment of native abdominal aorta in rat (Figure 2B). At predetermined time points, the patency of the implanted grafts was examined by ultrasound. Results showed that most of the grafts were patent (Figure 2C). Further, the velocity of blood flow was similar between the graft and adjacent native blood vessels at 12 months. Explanted grafts retained good morphology without aneurysm (Figure 2D), and no stenosis or thrombi could be observed on the luminal surface (Figure 2E).
Tissue regeneration and ECM secretion at 3 months were further assessed by histology analyses. H&#38;E staining showed that a layer of neo-tissue was formed on the lumen of the graft (Figure 3G-H). Furthermore, vWF staining show that the luminal surface was fully covered by newly formed endothelium (Figure 3A), resembling that of the native aorta (Figure 3B). Meanwhile several layers of MYH-positive cells were organized along the circumferential direction, indicating the regeneration of vascular media (Figure 3C-D). Extracellular matrix synthesis was observed by Masson and VVG staining, respectively. A significant amount of collagen and fibrous elastin could be observed within the graft (Figure 3I-J, K-L), which plays an important role in vascular regeneration and remodeling. Immunofluorescence staining further showed the structure of elastin was aligned circumferentially in a pattern like that in the native artery (Figure 3E-F).
Furthermore, the regenerated tissues including endothelium and smooth muscle maintained integrated and did not regress after 12 months of implantation (Figure 4A-C). More importantly, there was no sign of calcification occurring within the graft wall based on the Von Kossa staining (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57340/57340fig1.jpg" /> 
Figure 1: The structure and mechanical property of the PCL graft. SEM images of electrospun PCL mats with thicker fibers (A) and thinner fibers (B). Cross-sections of thicker-fiber tubular grafts (D-F) and thinner-fiber grafts (G-I). The representative strain-stress curve is shown in (C). These figures have been modified from Zhao, et al.16 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig1large.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/57340/57340fig2.jpg" /> 
Figure 2: Implantation of vascular grafts in a rat abdominal aorta model. Electrospun PCL vascular graft of 1 cm in length (A) was surgically interposed into the abdominal aorta in rat (B). The ultrasound image showed that the graft was patent in vivo at 1 year (C). Stereomicroscopic images show that the graft was well-integrated with adjacent native aorta without aneurysm (D), and the luminal surface is clean and free of thrombus (E). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig2large.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/57340/57340fig3.jpg" /> 
Figure 3: Tissue regeneration and deposition of ECM in the explanted grafts at 3 months in comparison with native aorta. Cross-sectional images of the regenerated grafts (A, C, and E) and native artery (B, D, and F) were immunostained to detect the endothelial cells, smooth muscle cells, and elastin. H&#38;E staining shows the tissue regeneration in the explanted grafts (G) compared to native aorta (H). Masson&#39;s staining revealed that the presence of collagen in the explanted grafts (I) and native aorta (J). VVG staining showed the presence of elastin in the explanted grafts (K) and native aorta (L). These figures have been modified from Zhao, et al.16 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig3large.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/57340/57340fig4.jpg" /> 
Figure 4: Histological analysis of the explanted grafts at 12 months. (A) H&#38;E staining showed the tissue regeneration in the explanted grafts. (B) Endothelium was immunostained by vWF antibody. (C) Smooth muscle was immunostained by &#945;-SMA antibody. (D) Calcification was evaluated by Von Kossa staining. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Nanyang Technological University

Research

JoVE Journal

Biology

7.9K Views.  Nankai University. Hier presenteren we een gewijzigde electrospinning methode voor het fabriceren van PCL vaattransplantaten met dikke vezels en grote poriën en een protocol ter evaluatie van de prestaties in vivo in rat model van abdominale aorta vervanging beschrijven.

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

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Medicine

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

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 ...

Bioengineering

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

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 ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

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

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