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

Podsumowanie

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.

Streszczenie

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 µm) and larger pores (~30 µm) 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.

Wprowadzenie

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 <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(ε-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 model^4,5,6, 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 leaching^9,10, 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 conditions^12,13. 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 µm and pores of 30 - 40 µm). 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.

Protokół

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.

1. 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.
2. Load the PCL solution into a 10-mL glass syringe.
3. Place the syringe with a 21-G needle.
4. Place the stainless-steel mandrel (2 mm in diameter, 25 cm in length) on the collection instrument.
5. 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.
6. Ensure that the obtained grafts are placed in vacuum overnight to remove the residual solvent. Sterilize all instruments prior to the procedure and maintain aseptic technique throughout.
7. 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.
8. Fiber and pore size measurements: Calculate the average fiber diameter using ImageJ software based on scanning electron microscopy (SEM) images.
9. Mechanical testing of scaffolds:
   1. Cut the tubular scaffolds into 3 mm sections in length using a razor blade. Measure the thickness of scaffolds using a micrometer.
   2. Place the tubular scaffolds on a tensile-testing machine with a load capacity of 100 N.
   3. 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's modulus from the initial linear region (up to 5% strain) of the stress-strain curve.

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 °C to maintain the animal body temperature. Follow local IACUC guidelines regarding analgesia. 

1. Use male Sprague Dawley rats weighing 240 - 270 g as recipients of vascular graft. Ensure the rat has fasted 24 h before surgery. The aim of fasting rats for 24 h is to empty  the faeces in the intestinal tract sufficiently, thereby broaden the operator’s horizon.
2. Grasp the rat'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.
3. 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.
4. 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.
5. Shave off the fur in the anterior abdominal wall using a razor blade, and clean the skin using Iodine solution and medical alcohol solution.
6. 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.
7. Retract and wrap the intestines with gauze moistened with saline solution preferentially.
8. Dissect the abdominal aorta carefully.
9. Identify and ligate all small branches using 9-0 monofilament nylon sutures.
10. Clamp the isolated section (up to 1 cm in length) of the aorta using two vascular clamps. The aorta can remain clamped for 20-30 min.
11. Transect the abdominal aorta between two clamps using micro-scissors to create the anastomotic sites.
12. Flush the two ends of aorta using heparinized saline (50 UI/mL) solution to remove the residual blood.
13. Peel off the adventitia using micro-scissors.
14. Anastomose the graft with 2 mm inner diameter and 1 cm in length to the rat's abdominal aorta with a figure-of-eight suture pattern using 9-0 monofilament nylon sutures.
15. Firstly, construct four anastomoses according to the sequence of 9, 3, 12, and 6 o'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.
16. Remove the distal clamp to allow the blood to flow into the graft, then remove the proximal clamp.
17. 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.
18. Return the intestines into the abdominal cavity.
19. Flush the abdominal cavity using warm physiological saline solution with gentamicin (320 U/mL).
20. Sew up the abdominal wall using a 3-0 Nylon suture in the muscle and skin layer, respectively.
21. 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.
22. After it regains consciousness, put the rat in a single cage with food and water. Apply iodine on the wound to prevent infection after surgery. Return the rat to the company of other animals until it fully recovers.
23. Euthanize rats according to institutional guidelines at predetermined time points.

Wyniki

The PCL grafts were explanted at 3 months and 12 months post-operatively and analyzed by standard histological techniques for hematoxylin and eosin (H&E), Masson trichrome, Verhoeff-van Gieson (VVG), Von Kossa, and immunofluorescence staining for α-SMA, MYH, vWF, and elastin. The histological images were taken using an upright microscope, and the immunofluorescence images were taken using a fluorescence microscope.

Dyskusje

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 ...

Materiały

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