Name: surgical porcine model of chronic myocardial ischemia treated by exosome-laden collagen patch and off-pump coronary artery bypass graft
Uploaded: 2023-09-15T13:40:00.000+00:00
Duration: 5 min 25 s
Description: Watch this Scientific Journal Video about Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft at JoVE.com

This work was supported by the VA Merit Review #I01 BX000760 (RFK) from the United States (U.S.) Department of Veterans Affairs BLR&#38;D and U.S. Department of Veterans Affairs grant #I01 BX004146 (TAB). We also gratefully acknowledge the support of the University of Minnesota Lillehei Heart Institute. The contents of this work do not represent the views of the U.S. Department of Veterans Affairs of the United States Government.

The Institutional Animal Care and Use Committees (IACUC) of the Minneapolis VA Medical Center and the University of Minnesota have approved all of the animal studies. The current National Institutes of Health (NIH) guidelines for the use and care of laboratory animals were followed.
1. Isolation of mesenchymal stem cells and preparation and characterization of exosomes
<ol>
	<li>Isolation of bone marrow derived mesenchymal stem cells (MSCs)
	<ol>
		<li>Obtain 30-50 mL of sterile bone marrow from the sternum or tibia of a 20-week-old female Yorkshire-Landrace swine. To do this, introduce a 25 mm 15G interosseous needle into the sternum or tibia and draw the sample into a 60 mL syringe with 10 mL of heparin. 
		NOTE: For further details on the collection of bone marrow refer to Pittenger et al. and Hocum-Stone et al.12,16.</li>
		<li>In brief, pass the bone marrow specimen through a Vacutainer&#160;CPT tube with heparin for 30 min at 1800 x&#160;g.</li>
		<li>Remove the buffy coat containing the mononuclear cells and wash with Hank&#39;s balanced salt solution. Pellet mononuclear cells by centrifugation and resuspend in growth medium (10% fetal bovine serum [FBS]).</li>
		<li>Transfer the mononuclear cells to cell culture flasks for adherent growth. Isolate the MSCs from the mononuclear fraction by their adherent nature.</li>
		<li>Wash all non-MSCs within 24 h, leaving a monolayer of MSCs in the tissue culture flask. Confirm the MSC phenotype by flow cytometry, ensuring negativity for CD45, a hematopoietic marker, and positivity for CD90 and CD105, markers of MSCs.</li>
	</ol>
	</li>
	<li>Preparation and characterization of exosomes from porcine mesenchymal stem cells
	<ol>
		<li>Seed 1 x 104 H9C2 rat cardiomyocytes and culture in 1x DMEM+ 10% FBS and 1x Pen/strep. Seed 2 x 104 porcine MSCs in advanced DMEM&#160;+ 5% FBS and 1x Pen/strep.</li>
		<li>Once both cell lines are at least 80% confluent, change the media to exosome depleted H9C2 and MSC media.</li>
		<li>Expose H9C2 cardiomyocytes to mild hypoxia (1% O2 for 24 h). Remove flasks from hypoxia after 24 h and pipette out H9C2 media.</li>
		<li>Remove and discard the MSC media from MSC flask. Add purified H9C2 media to MSC flask. Incubate the flask for 6 h in normoxic conditions (5% CO2, 20% O2, and 37 &#176;C).</li>
		<li>Extract the exosomes from the co-cultured conditioned media using the total exosome isolation reagent following manufacturer&#39;s instructions.</li>
		<li>Verify the identification of exosomes by western blot detection of common exosomal proteins with antibodies against CD-63 (1:1000)17.</li>
		<li>Perform nanoparticle tracking analysis (NTA) to quantify the exosomes and the assessment of nanoparticle size and its distribution. To do this, dissolve total protein (50 &#956;g) of exosomes in 500 &#956;L of PBS to determine the concentration and size distribution of exosomes by using nanoparticle tracking analyzer.</li>
		<li>Analyze the data using nanoparticle tracking software.</li>
	</ol>
	</li>
</ol>
2. Off pump coronary artery bypass graft surgery
<ol>
	<li>Animal preparation
	<ol>
		<li>Weigh the animal (16-week-old female Yorkshire-Landrace pigs) 3 days before scheduled for surgery. Fast the animal for 12 h before surgery while having access to water during fasting.</li>
		<li>Give buprenorphine 0.18 mg/kg via intramuscular route 2-4 h before surgery.</li>
	</ol>
	</li>
	<li>Induction of the animal
	<ol>
		<li>Sedate the animal by giving intramuscular injection of 6.6 mg/kg tiletamine-zolazepam/xylazine.</li>
		<li>Wait for 15 min to ensure adequate sedation by assessing the jaw tone followed by 22G catheter placement in the central ear vein. 
		NOTE: Another peripheral vein may be considered (i.e., cephalic vein) if ear vein is inadequate.</li>
		<li>Administer ophthalmic ointment topically to each eye. Administer 1-2 mg/kg of propofol via intravenous route to induce general anesthesia. Jaw tone most reliably reflects the depth of anesthesia and should be assessed throughout the procedure.</li>
		<li>Intubate the animal with an endotracheal tube of appropriate size.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Shave the sternum and groin of the animal in preparation for surgical procedure.</li>
		<li>Set mechanical ventilation at 10-15 breaths per min, oxygen 1-4 L/min, and isoflurane 1.0-3.0% as needed to maintain deep anesthesia for surgery. Check for absent eye or jaw reflex to confirm deep anesthesia.</li>
		<li>Position monitoring equipment (Electrocardiogram, end tidal CO2, heart rate, Oxygen saturation, blood pressure, and temperature) on the animal.</li>
		<li>Connect the IV catheter to a bag of normal saline or lactate ringers&#39; solution to administer maintenance fluids continuously.</li>
		<li>Prepare the skin using aseptic technique with povidone Iodine scrub and solution 3x for adequate sterility and to minimize the risk of surgical site infection.</li>
		<li>Give Lidocaine via intravascular route (loading dose of 2 mg/kg or continuous infusion at dose of 50 mcg/kg/min) to prevent arrhythmias.</li>
		<li>Position the animal dorsally and drape with sterile towels.</li>
		<li>Perform either left or right femoral artery cut down for arterial line placement by Seldinger technique followed by connecting the catheter to the transducer for continuous blood pressure monitoring at the time of surgery.</li>
		<li>Use monopolar electrocautery to make a 20 cm incision extending from the sternal notch proximally down to the xyphoid process distally, and to incise layers of muscles, subcutaneous fat, and connective tissue down to the sternum.</li>
		<li>Perform median sternotomy by using oscillating saw. 
		NOTE: Standard saw is avoided for repeat sternotomy as it carries higher risk for myocardial injury from previous pericardial adhesions from left thoracotomy procedure done to place the LAD constrictor.</li>
		<li>Divide the posterior sternal plate using a pair of scissors. Use a specialized chest retractor for adequate visualization of the mediastinum.</li>
		<li>Dissect adhesions using either monopolar electrocautery or the Metzenbaum scissors. Carefully dissect the peristernal muscle and fat to expose the left internal mammary artery (LIMA).</li>
		<li>Once LIMA is exposed lateral to the sternal edge, gently separate it from the chest wall using blunt dissection with electrocautery tip. Use the LIMA as a skeletonized graft.</li>
		<li>Start dissection at the level of 3rd intercostal space. Gently elevate the left sternal border for optimal visualization.</li>
		<li>Use gentle traction on the adventitia to expose the arterial and venous branches of LIMA. Clip the LIMA side of the branches using hemoclips and cauterize the chest wall side of the branches. 
		NOTE: Care must be taken not to cauterize the clip on the LIMA, because this may cause conduit narrowing.</li>
		<li>Once an initial segment of LIMA has been mobilized, continue the dissection proximally toward the level of subclavian vein and distally until the LIMA bifurcation.</li>
		<li>Once dissection is finished, administer heparin via intravenous route at a dose of 100-300 U/kg. Wait for 3 min after the heparin is administered.</li>
		<li>After 3 min, clip the distal end of the LIMA, just before the level of the LIMA bifurcation, and divide the conduit. Sew the distal end with a free 2-0 silk suture tie.</li>
		<li>Prepare the proximal end for grafting. Inspect the flow quality visually by letting the graft bleed for a few seconds.</li>
		<li>Gently clamp the distal end of LIMA conduit with an atraumatic bulldog clamp to avoid bleeding. Open the pericardium with an inverted-T making an approximately 5-6 cm incision. Place 3-0 size sutures on the pericardium for traction at both sides of the slit.</li>
		<li>Stabilize the LAD with silicone retraction tapes and tissue stabilizer, which is secured to the sternal retractor. Make an arteriotomy in LAD artery distal to the stenosis (caused by constrictor band) with an 11-blade and extend with an iris scissors.</li>
		<li>Place an appropriately sized coronary shunt in the LAD. Perform the LIMA to LAD anastomosis with 7-0 running non-absorbable suture using an off-pump bypass technique. Release the bulldog occluder on the LIMA and confirm the hemostasis.</li>
	</ol>
	</li>
	<li>Preparation of Mesenchymal Stem Cell (MSC)-derived exosome patch
	<ol>
		<li>Following successful isolation of exosomes from MSCs, suspend roughly 3 x 108 exosomes in 3 mL of normal saline and add to collagen sponge.</li>
		<li>Bring 3 mL of exosome suspension to room temperature at around 22 &#176;C for 10 min. Place 2 absorbable collage sponges (each 1.27 cm x 2.54 cm) into a medium Petri dish.</li>
		<li>Use a 5 mL syringe with an 18G needle to gently mix exosome suspension. Slowly pipette 1.5 mL suspension onto each collagen sponge and wait for 5 min for full absorption.</li>
	</ol>
	</li>
	<li>Placement of exosome patch
	<ol>
		<li>Place the Exosome-laden sponge upside down onto the hibernating region of the heart, which is the epicardium of the anterior septal region in the distribution of the LAD.</li>
		<li>Gently place two sponges to cover the hibernating region of the heart. Use one 3.5 cm x 1.0 cm polyglactin mesh to cover each collagen sponge.</li>
		<li>Sew the mesh onto the epicardium with fine 7-0 interrupted sutures.</li>
	</ol>
	</li>
	<li>Chest tube placement
	<ol>
		<li>Place a chest tube through separate stab incision, near the inferior aspect of the sternotomy incision. Place the chest tube cautiously over the anterior aspect of the heart.</li>
		<li>Once the tube is in place, place a purse string suture with 3-0 suture using a horizontal mattress stitch to allow for closure of the wound upon removal of the tube.</li>
		<li>The chest tube is maintained until complete chest closure.</li>
	</ol>
	</li>
	<li>Chest closure
	<ol>
		<li>Approximate the sternum with non-absorbable sutures using a figure eight pattern. Administer 1 mg/kg bupivacaine via intramuscular route along the entire length of the incision. 
		NOTE: Suture is used rather than wires to avoid interference with MRI imaging.</li>
		<li>Close layers of muscle and skin in the standard fashion using 2-0 and 3-0 absorbable suture, respectively.</li>
		<li>Perform a breath hold and suction to evacuate all the air out of the thoracic cavity. Monitor the airway pressure on the ventilator cautiously and maintain the pressure between 15-22 mmHg and release when complete.</li>
		<li>Once all the air is evacuated, remove the chest tube while closing the wound using the purse string suture. Apply adhesive glue topically to cover the sternal incision.</li>
	</ol>
	</li>
	<li>Post operative care after surgery
	<ol>
		<li>Gradually wean the animal off the ventilator as skin incision is being closed. Ensure that the animal is able to spontaneously breathe and protect reflexes before disconnecting the animal from anesthesia equipment.</li>
		<li>Remove the endotracheal tube after confirming that animal is able to protect its airway. Cover the skin incision with sterile and non-adherent dressing embedded with antibiotic ointment to minimize surgical site infection.</li>
		<li>Continue to monitor vital signs including heart rate, respiratory rate, body temperature every 15 min until animal is able to hold its position without assistance.</li>
		<li>Ensure animal is not left unattended until able to lift and hold its head up and can stand without assistance. Administer meloxicam at a dose of 0.2 mg/kg via subcutaneous route before transporting the animal to the recovery unit.</li>
		<li>Transport the animal to the recovery unit when animal is stable. Keep the surgical site dressing on the incision until postoperative day 3. Replace the dressing if it becomes soiled.</li>
		<li>Continue to monitor the level of the pain, skin incision and overall well-being of the animal for the first 5 days after surgery. Administer half a dose of meloxicam (0.1 mg/kg) as needed once daily for breakthrough pain.</li>
		<li>Single house the animal for first 5 days after surgery while the incision(s) heal to reduce the risk of surgical site infection by another animal. Return the animal to group housing after 5 days.</li>
		<li>Report any complications or changes in the animal&#39;s condition (fever, ascites, weight loss, inappetence etc.) to the veterinarian or appropriate staff.</li>
	</ol>
	</li>
</ol>
3. Coronary angiography using femoral access
<ol>
	<li>Secure the animal on the operating table in the dorsal recumbency. Initiate mechanical ventilation at 10-15 breaths per min. Set oxygen at 2-4 L/min, isoflurane at 1% and 4%, as needed to maintain a deep plane of anesthesia.</li>
	<li>Place ECG leads on the animal&#39;s limb to monitor for heart rhythm. Evaluate the animal for the depth of anesthesia. Consider the animal deeply anesthetized when the eye or jaw reflex is absent.</li>
	<li>Clean the chest and neck area with povidone iodine scrub and then drape the animal with towels.</li>
	<li>Access the femoral artery via surgical cut-down and expose the femoral artery and vein. Make a 1-2 mm longitudinal incision with a no. 11 blade in femoral artery and cannulate the artery using an 11 Fr introducer sheath in the vessel lumen.</li>
	<li>After obtaining access, advance the catheter to perform coronary angiography to assess the anatomy patency of LIMA-LAD graft.</li>
</ol>

Adjunctive Exosome Therapy: Enhancing Myocardial Recovery Post-CABG Surgery

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The authors have nothing to disclose.

This study presents the first porcine model of chronically ischemic myocardium, in which it was shown that treatment with an MSC-derived exosome laden collagen patch during surgical revascularization recovers diastolic and systolic function upon inotropic stimulation potentially by targeting mitochondrial recovery. Previously, it was demonstrated that in a large animal model of HIB the diastolic and systolic function, as measured by cardiac MRI, remains impaired and only slightly improves with revascularization without complete recovery6,19. The dysfunction occurred despite preserved left ventricular ejection fraction. These findings accurately mimic the clinical experience seen in patients with chronic ischemic myocardium in a single vessel territory with preserved left ventricular function.
There are several critical and technical challenges during revascularization surgery especially in the setting of previous thoracotomy. Cardiovascular injury on sternal entry is a risk as the pericardium has already been breached and adhesions may be present. Sternotomy may cause cardiac injury due to proximity or adherence to the sternum. This risk can be mitigated by use of an oscillating saw, which has been shown to promote an uneventful sternal re-entry.
A key aspect to obtain successful CABG is graft quality. Meticulous LIMA harvesting is an important technical aspect in successfully performing high-quality CABG and is associated with improved graft patency. LIMA can be harvested using two techniques: pedicled and skeletonized. Pedicled technique includes dissecting the LIMA from the sternum along with its veins, fascia, fat, and lymphatics. Skeletonized technique includes dissecting the LIMA free of all surrounding tissue, and therefore yielding the artery only20. In this model, the skeletonization technique was implemented as it can minimize sternal ischemia and the graft is longer than a pedicled LIMA20. LIMA is a delicate structure, any undue stretching, clamping, or misplaced clips can result in vascular injury and unsatisfactory results. During dissection, the cautery tip should be used with caution and at low voltage. When separating the artery from its perforating branches, the LIMA side of the branches are clipped using hemoclips. Care must be taken not to cauterize the clips as it can result in narrowing of the conduit. Confirm pulsatile flow prior to grafting.
Ensure that reasonable amounts of anesthesia and paralytics are maintained to minimize movement during the surgery especially while sewing the anastomosis. It is crucial to use the appropriate doses of lidocaine and heparin (200-300 units/kg) to eliminate the risk of arrhythmia and thrombosis, respectively. A second dose of lidocaine might be indicated if the animal experiences any arrhythmias during surgery. Use of femoral arterial line allows continuous hemodynamic monitoring. When performing the anastomosis, it is useful to either put 1-2 surgical sponges behind the heart or place sutures on either side of pericardium to lift the left ventricle upward. In this model, we use silastic tapes and the tissue stabilizer that utilizes suction pressure to effectively immobilize the target site. A mild drop in arterial blood pressure in addition to ST depression on the EKG may be noted once the stabilizer is placed and the heart is lifted. These hemodynamic derangements are usually well tolerated without requiring any interventions. In situations where hemodynamic instability is significant, a dose of phenylephrine (5-20 &#181;g/kg) via intravenous route may be administered to raise the arterial blood pressure. If hemodynamic instability is life threatening, a dose of Epinephrine (0.1 &#181;g/kg; diluted 1:10,000) can be administered via intravenous route as an emergency rescue drug. Once the LAD is exposed with a beaver blade, an arteriotomy is performed with an 11-blade and completed with microsurgical scissors. One must be careful not to injure the posterior wall of the LAD during this maneuver. It is critical to keep the arteriotomy site bloodless during off-pump CABG to allow for accurate suturing and several techniques have been described including intermittent irrigation with saline solution, use of CO2 blower, and intraluminal coronary shunts21. In this study, the CO2 blower along with an appropriately sized intraluminal coronary shunt were used as both are routinely used in the off-pump CABG operations. A potentially lethal complication of the CO2 mister blower is an air embolism. However, the risk of air embolism can be negated by using coronary shunt, which can act as a physical barrier within the arteriotomy. Additionally, the use of coronary shunt helps to keep the surgical field bloodless, which permits the use of a lower gas flow and further minimizes the risk of air embolism. Shunts also improve&#160;the technical precision for anastomosis and prevent&#160;the inadvertent injury to back wall of the artery while suturing22.
In this well-established porcine model, an off-pump rather than on-pump technique during CABG surgery was employed. The advantages of using this technique, instead of the on-pump, is to minimize the operative time and avoid the central cannulation of the aorta and right atrium with full heparinization. In addition, it helps in faster recovery of the animal after surgery by reducing the risk of post-operative bleeding and/or cardiac temponade. These are presumed advantages based on clinical experience in patients undergoing CABG on both on and off pump.
This exosome-laden collagen patch is novel in that it can be quantified and surgically secured to the region of ischemia that has been revascularized. This allows sustained release of exosomes from the patch over the course of several days resulting in continuous and direct treatment of the ischemic region. Histopathology of the hibernating tissue 4 weeks following treatment with CABG and exosome demonstrated lack of inflammatory response of myocardium to the patch itself, although some inflammation was noted at the site of sutures as evident by staining for inflammatory cells. While a variety of methods has&#160;been suggested for exosome delivery into the myocardium, common techniques such as direct injection of exosome result&#160;in low retention of therapeutic product in the injured area, as up to 90% of exosomes&#160;wash away or disperse after injection23. Analysis of exosomes retention following injection has been completed for up to 3 h post injection and has shown significant decreases in the exosome content24. Exosomes are easy to isolate and have more flexibility in storage conditions over long periods of time, presenting an opportunity for off-the-shelf products that can be used in the acute setting making it more translatable to patients.
This study has several limitations including age and sex of animals. Given surgical and logistic limitations, considerations around animal welfare regulations and staff safety, only juvenile female swine were studied. Although, CABG surgery adds to the complexity of the model, it was a required intervention as other less invasive interventions (percutaneous coronary intervention or PCI) would not allow to open the stenotic region of LAD due to the rigid nature of the constrictor18. Furthermore, this model of single vessel stenosis without any comorbidities does not fully simulate the extent and effects of long-standing coronary atherosclerosis as seen in human population. Future studies will focus on using a multivessel disease model of hibernating myocardium by surgically placing the constrictor on the circumflex artery and LAD. However, this two-vessel disease model would result in hibernating myocardium with reduced ejection fraction. Animal mortality would likely increase, and revascularization surgically is more complex and requires on-pump bypass support. In future, if there were problems with the practical applications of patch type, other scaffold material options will be explored, such as decellularized extracellular matrix, or alternate form of hydrogels.

Globally, severe CAD affects over a hundred million patients, and although the mortality rate has decreased, it remains one of the leading causes of death1,2. CAD has a wide clinical spectrum from myocardial infarction (MI) to ischemia with preserved viability. Most pre-clinical research focuses on MI, characterized by the presence of infarcted tissue as it is possible to study in small and large animal models. However, that model does not address patients with preserved viability and amenable to revascularization. Most patients undergoing CABG have decreased blood supply and limited function while maintaining variability in contractile reserve and viability3. Without treatment, these patients can progress to advanced heart failure and sudden death, especially during increased workload4. Among these patients, coronary artery bypass graft (CABG) is an effective therapy but may not result in complete functional recovery5. Importantly, diastolic dysfunction, which is a marker for worse clinical outcomes, fails to recover after revascularization suggesting the need for novel adjuvant therapies during CABG6,7. Currently, there are no clinically available adjuvant interventions used with CABG to restore cardiomyocytes to full functional capacity. This is a major therapeutic gap given that many patients progress to advanced heart failure despite appropriate revascularization8.
An innovative porcine model of chronic myocardial ischemia that is amenable to CABG, to mimic clinical CAD experience was created9. Swine provide a good model of heart disease over other large animals as they do not have epicardial bridging collaterals so stenosis of the LAD alone results in regional ischemia10. In this study, 16-week-old female Yorkshire-Landrace pigs were used. In this model, the LAD was revascularized with off-pump CABG using the left internal mammary artery (LIMA) graft (Supplementary Table 1). Percutaneous coronary intervention (PCI) is not possible to open the stenosis as the constrictor is a rigid device. Cardiac magnetic resonance imaging (MRI) is used to assess global and regional function, coronary anatomy, and tissue viability. Cardiac MRI analysis showed diastolic function, characterized by peak filling rate (PFR) remains impaired despite CABG6. The mechanism of diastolic dysfunction likely relates to impaired mitochondrial bioenergetics and collagen formation in HIB that persist following CABG11.
Mesenchymal stem cells (MSC) provide therapeutic signaling through exosomes to improve myocardial recovery when applied during CABG. In this swine model and parallel in vitro studies, it was shown that placement of an epicardial MSC vicryl patch during CABG recovers contractile function with increase in key mitochondrial proteins namely PGC-1&#945;12, an important regulator of mitochondrial energy metabolism13. The in vitro model allowed us to investigate the signaling mechanism of MSCs on impaired mitochondrial function. Exosomes are secreted stable microvesicles (50-150 nm) that contain protein or nucleic acids including microRNA (miRNA)14. Recent in vitro data suggest that MSC-derived exosomes are an important signaling mechanism necessary for recovery of mitochondrial respiration.
Stem cell derived exosomes are promising adjunctive therapeutics as they are readily accessible, can be commercially produced, and lack ethical conflicts. In consideration of clinical translation, a collagen patch embedded with MSC-derived exosomes was created that can be surgically sutured to the hibernating region of myocardium. It was demonstrated that there is sustained delivery of exosomes using this patch and it provides a cell-free regenerative therapy with paracrine signaling mechanism that targets mitochondrial recovery and enhance mitochondrial biogenesis15. This procedure provides the pre-clinical model to study the impact of MSC-derived therapies to improve cardiac function by means of enhancing mitochondrial function and reducing inflammation at the time of revascularization and reverse the myocyte adaptations to chronic ischemia.
In this study, a surgical method of off-pump CABG using LIMA to LAD anastomosis to bypass the area of proximal LAD stenosis mimicking the standard treatment for patients with CAD is shown. As an adjunctive therapy with CABG, the surgical application of MSC-derived exosome embedded collagen patch on the ischemic region of the myocardium was demonstrated. This surgical model can be used to study the physiologic responses to the paracrine effect seen with use of an exosome patch as well as the molecular mechanisms of recovery.

<table><tbody><tr><td>5 Ethibond</td><td>Ethicon</td><td>MG46G</td><td>Suture</td></tr><tr><td># 40 clipper blade</td><td>Oster</td><td>078919-016-701</td><td>Remove hair from surgery sites</td></tr><tr><td>0 Vicryl</td><td>Ethicon</td><td>J208H</td><td>Suture</td></tr><tr><td>1 mL Syringe</td><td>Medtronic/Covidien</td><td>1188100777</td><td>Administer injectable agents</td></tr><tr><td>1&quot; medical tape</td><td>Medline</td><td>MMM15271Z</td><td>Secure wound dressing and IV catheters</td></tr><tr><td>1000mL 0.9% Sodium chloride</td><td>Baxter</td><td>2B1324X</td><td>IV replacement fluid</td></tr><tr><td>12 mL Syringe</td><td>Medtronic/Covidien</td><td>8881512878</td><td>Administer injectable agents</td></tr><tr><td>18 ga needles</td><td>BD</td><td>305185</td><td>Administration of injectable agents</td></tr><tr><td>20 ga needles</td><td>BD</td><td>305175</td><td>Administration of injectable agents</td></tr><tr><td>20 mL Syringe</td><td>Medtronic/Covidien</td><td>8881520657</td><td>Administer injectable agents</td></tr><tr><td>2-0 Vicryl</td><td>Ethicon</td><td>J317H</td><td>Suture</td></tr><tr><td>250 mL 0.9% saline</td><td>Baxter</td><td>&amp;nbsp;UE1322D</td><td>Replacement IV Fluid</td></tr><tr><td>3 mL Syinge</td><td>Medtronic/Covidien</td><td>1180300555</td><td>Administer injectable agents</td></tr><tr><td>3-0 Vicryl</td><td>Ethicon</td><td>VCP824G</td><td>Suture</td></tr><tr><td>36&amp;rdquo; Pressure monitoring tubing</td><td>Smith&amp;rsquo;s Medical</td><td>MX563</td><td>Connect art. Line&amp;nbsp; to transducer</td></tr><tr><td>4.0 mm ID endotracheal tube</td><td>Medline</td><td>DYND43040</td><td>Establish airway for Hibernation</td></tr><tr><td>4-0 Tevdek II Strands</td><td>Deknatel</td><td>7-922</td><td>Suture to secure constrictor around LAD</td></tr><tr><td>48&amp;rdquo; Pressure monitoring tubing</td><td>Smith&amp;rsquo;s Medical</td><td>MX564</td><td>Connect art. Line&amp;nbsp; to transducer</td></tr><tr><td>500mL 0.9% Sodium chloride</td><td>Baxter</td><td>2B1323Q</td><td>Drug delivery, Provide mist for Blower Mister</td></tr><tr><td>6&amp;nbsp; mL Syringe</td><td>Medtronic/Covidien</td><td>1180600777</td><td>Administer injectable agents</td></tr><tr><td>6.0 mm ID endotracheal tube</td><td>Mallinckrodt</td><td>86049</td><td>Establish airway for Revasc,MRI and Termination</td></tr><tr><td>6.5 mm ID endotracheal tube</td><td>Medline</td><td>DYND43065</td><td>Establish airway for Revasc,MRI and Termination</td></tr><tr><td>6&amp;rdquo; pressure tubing line</td><td>Smith&amp;rsquo;s Medical</td><td>MX560</td><td>Collect bone marrow</td></tr><tr><td>60 mL Syringe</td><td>Medtronic/Covidien</td><td>8881560125</td><td>Administer injectable agents</td></tr><tr><td>7.0 mm ID endotracheal tube</td><td>Medline</td><td>DYND43070</td><td>Establish airway for Revasc,MRI and Termination</td></tr><tr><td>7-0 Prolene</td><td>Ethicon</td><td>M8702</td><td>Suture</td></tr><tr><td>Advanced DMEM (1X)</td><td>ThermoFisher Scientific</td><td>12491023</td><td></td></tr><tr><td>Alcohol Prep pads</td><td>MedSource</td><td>MS-17402</td><td>Skin disinfectant</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit</td><td>Millipore Sigma</td><td>UFC910024</td><td></td></tr><tr><td>Anesthesia Machine</td><td>Drager</td><td>Fabious Trio</td><td>maintains general anesthesia</td></tr><tr><td>Anesthesia Machine + ventilator</td><td>DRE Drager- Fabius Tiro</td><td>DRE0603FT</td><td>Deliver Oxygen and inhalant to patient</td></tr><tr><td>Anesthesia Monitor</td><td>Phillips&amp;nbsp; Intellivue</td><td>MP70</td><td>Multiparameter for patient safety</td></tr><tr><td>Arterial Line Kit</td><td>Arrow</td><td>ASK-04510-HF</td><td>Femoral catheter for blood pressure monitoring</td></tr><tr><td>Artificial Tears</td><td>Rugby</td><td>0536-1086-91</td><td>Lubricate eyes to prevent corneal drying</td></tr><tr><td>Bair Hugger</td><td>3M</td><td>Model 505</td><td>Patient Warming system</td></tr><tr><td>Basic pack</td><td>Medline</td><td>DYNJP1000</td><td>Sterile drapes and table cover</td></tr><tr><td>Blood Collection Tubes- green top</td><td>Fisher Scientific</td><td>02-689-7</td><td>Collect microsphere blood samples</td></tr><tr><td>Blower Mister Kit</td><td>Medtronic/Covidien</td><td>22120</td><td>Clears surgical field for vessel anastomosis</td></tr><tr><td>BODIPY TR Ceramide</td><td>ThermoFisher Scientific</td><td>D7540</td><td></td></tr><tr><td>Bone marrow needle- 25mm 15 ga IO needle</td><td>Vidacare</td><td>9001-VC-005</td><td>Collect bone marrow</td></tr><tr><td>Bone Wax</td><td>Medline</td><td>ETHW31G</td><td>Hemostasis of cut bone</td></tr><tr><td>Bovie Cautery hand piece</td><td>Covidien</td><td>E2516</td><td>Hemostasis</td></tr><tr><td>Bupivicaine</td><td>Pfizer</td><td>00409-1161-01</td><td>Local Anesthetic</td></tr><tr><td>Buprenorphine 0.3 mg/mL</td><td>Sigma Aldrich</td><td>B9275</td><td>Pre operative Analgesic for survivial procedures</td></tr><tr><td>Cell Scrapers</td><td>Corning</td><td>353085</td><td></td></tr><tr><td>Cephazolin 1 gr</td><td>Pfizer</td><td>00409-0805-01</td><td>Antibiotic</td></tr><tr><td>Chest Tube</td><td>Covidien</td><td>8888561043</td><td>Evacuates air from chest cavity</td></tr><tr><td>Cloroprep</td><td>Becton Dickenson</td><td>260815</td><td>Surgical skin prep</td></tr><tr><td>CPT tube</td><td>BD</td><td>362753</td><td>MSC isolation from bone marrow</td></tr><tr><td>Delrin Constrictor</td><td>U of MN</td><td>Custom made</td><td>Creates stenosis of LAD</td></tr><tr><td>Dermabond</td><td>Ethicon</td><td>DNX12</td><td>Skin adhesive</td></tr><tr><td>DMEM (1X) Dulbecco&#039;s Modified Eagle Medium, HEPES</td><td>ThermoFisher Scientific</td><td>12430062</td><td></td></tr><tr><td>Dobutamine 12.5 mg/mL</td><td>Pfizer</td><td>00409-2344-01</td><td>Increases blood pressure and heart rate during the second microsphere blood collection</td></tr><tr><td>ECG Pads</td><td>DRE</td><td>1496</td><td>Monitor heart rhythm</td></tr><tr><td>Exosome-Depleted FBS</td><td>ThermoFisher Scientific</td><td>A2720801</td><td></td></tr><tr><td>Falcon Disposable Polystyrene Serological Pipets, Sterile, 10mL</td><td>Fisher Scientific</td><td>13-675-20</td><td></td></tr><tr><td>Femoral and carotid introducer</td><td>Cordis- J&amp;amp;J</td><td>504606P</td><td>femoral and carotis cannulas</td></tr><tr><td>Fetal Bovine Serum, Heat Inactivated, Gibco FBS</td><td>ThermoFisher Scientific</td><td>16140089</td><td></td></tr><tr><td>Flo-thru 1.0</td><td>Baxter</td><td>FT-12100</td><td>used to anastomos LIMA to L</td></tr><tr><td>Flo-thru 1.25</td><td>Baxter</td><td>FT-12125</td><td>FT-12125</td></tr><tr><td>Flo-thru 1.5</td><td>Baxter</td><td>FT-12150</td><td>FT-12150</td></tr><tr><td>Flo-thru 2.0</td><td>Baxter</td><td>FT-12200</td><td>FT-12200</td></tr><tr><td>GlutaMAX Supplement</td><td>ThermoFisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Hair Clipper</td><td>Oster</td><td>078566-011-002</td><td>Remove hair from surgery sites</td></tr><tr><td>Helistat collagen sponge</td><td>McKesson</td><td>570973 1690ZZ</td><td>Sponge for embedding exosomes</td></tr><tr><td>Heparin</td><td>Pfizer</td><td>&amp;nbsp;0409-2720-03</td><td>anticoaggulant</td></tr><tr><td>Histology Jars</td><td>Fisher Scientific</td><td>316-154</td><td>Formalin for tissue samples</td></tr><tr><td>HyClone Characterized Fetal Bovine Serum (FBS)</td><td>Cytiva</td><td>SH30071.03</td><td></td></tr><tr><td>Hypafix</td><td>BSN Medical</td><td>4210</td><td>Secure wound dressing and IV catheters</td></tr><tr><td>Isoflurane</td><td>Sigma Aldrich</td><td>CDS019936</td><td>General Anesthestic- Inhalant</td></tr><tr><td>IV Tubing for Blower Mister</td><td>Carefusion</td><td>42493E</td><td>Adapts to IV Fluids for Blower/Mister</td></tr><tr><td>Jelco 18 ga IV catheter</td><td>Smiths medical</td><td>4054</td><td>IV access in Revasc, MRI and Term</td></tr><tr><td>Lidocaine 2%</td><td>Pfizer</td><td>00409-4277-01</td><td>Local Anesthetic/ antiarrthymic</td></tr><tr><td>Ligaclips</td><td>Ethicon</td><td>MSC20</td><td>Surgical Staples for LIMA takedown</td></tr><tr><td>Long blade for laryngoscope</td><td>DRE</td><td>12521</td><td>Allows for visualization of trachea for intubation</td></tr><tr><td>Meloxicam 5 mg/mL</td><td>Boehringer Ingelheim</td><td>141-219</td><td>Post operative Analgesic</td></tr><tr><td>Microsphere pump</td><td></td><td></td><td>Collect blood samples from femoral introducer</td></tr><tr><td>Monopolar Cautery</td><td>Covidien</td><td>Valleylab&amp;trade; FT10</td><td>Hemostasis</td></tr><tr><td>Nanosight NS 300</td><td>Malvern Panalytical</td><td>MAN0541-03-EN</td><td></td></tr><tr><td>NTA 3.1.54 software</td><td>Malvern Panalytical</td><td>MAN0520-01-EN-00</td><td></td></tr><tr><td>OPVAC Synergy II</td><td>Terumo Cardiovascular System</td><td>401-230</td><td>Heart positioner and Stabilizer</td></tr><tr><td>Oxygen Tank E cylinder</td><td>various</td><td>various</td><td>Used for Blower Mister if anesthesia machine doesn&#039;t have auxiliary flow meter</td></tr><tr><td>PBS, pH 7.2</td><td>ThermoFisher Scientific</td><td>20012050</td><td></td></tr><tr><td>Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td><td>ThermoFisher Scientific</td><td>15640055</td><td></td></tr><tr><td>Pigtail 145 catheter 6 French</td><td>Boston Scientific</td><td>08641-41</td><td>Measure LV pressures</td></tr><tr><td>Pressure Transducer</td><td>various</td><td>Must adapt to anesthesia monitor</td><td>Monitor direct arterial pressures</td></tr><tr><td>Propofol</td><td>Diprivan</td><td>269-29</td><td>Induction agent</td></tr><tr><td>Roncuronium</td><td>Mylan</td><td>67457-228-05</td><td>Neuromuscular blocking agent</td></tr><tr><td>SR Buprenorphine 10 mg/mL</td><td>Abbott Labs</td><td>NADA 141-434</td><td>Post operative Analgesic</td></tr><tr><td>Sterile Saline 20 mL</td><td>Fisher Scientific</td><td>20T700220</td><td>Flush for IV catheters</td></tr><tr><td>Sternal Saw/ Necropsy Saw</td><td>Thermo Fisher</td><td>812822</td><td>Used to open chest cavity</td></tr><tr><td>Stop Cocks</td><td>Smith Medical</td><td>MX5311L</td><td>2 to connect to pig tail</td></tr><tr><td>Succinylcholine 20 mg/mL</td><td>Pfizer</td><td>00409-6629-02</td><td>Neuromuscular blocking agent</td></tr><tr><td>Suction&amp;nbsp; tubing</td><td>Medline</td><td>DYND50223</td><td></td></tr><tr><td>Suction Container</td><td>Medline</td><td>DYNDCL03000</td><td></td></tr><tr><td>Surgery pack with chest retractor</td><td>various</td><td>See pack list</td><td>Femoral cut down and median sternotomy</td></tr><tr><td>Surgical Instruments</td><td>various</td><td>See pack list</td><td>Femoral and carotid cutdowns and sternotomy</td></tr><tr><td>Surgical Spring Clip</td><td>Applied Medical</td><td>A1801</td><td>Clamp end of LIMA after takedown</td></tr><tr><td>Syringe pump</td><td>Harvard</td><td></td><td>Delivers IV Dobutamine infusion</td></tr><tr><td>SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSO</td><td>Millipore Sigma</td><td>S32703</td><td></td></tr><tr><td>Telazol 100 mg/mL</td><td>Fort Dodge</td><td>01L60030</td><td>Pre operative Sedative</td></tr><tr><td>Telpha pad</td><td>Covidien</td><td>2132</td><td>Sterile wound dressing</td></tr><tr><td>Timer</td><td></td><td></td><td>Time collection of blood samples</td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher Scientific</td><td>4478359</td><td></td></tr><tr><td>TPP Tissue Culture Flask, T75, Filter Cap w/ 0.22uM PTFE</td><td>ThermoFisher Scientific</td><td>TP90076</td><td></td></tr><tr><td>Triple Antibiotic Ointment</td><td>Johnson &amp;amp; Johnson</td><td>23734</td><td>Topical over wound</td></tr><tr><td>Vicryl mesh</td><td>Ethicon</td><td>VKML</td><td>Patch for epicardial cell application</td></tr><tr><td>Vortex</td><td></td><td></td><td>Mix microspheres</td></tr><tr><td>Xylazine 100 mg/mL</td><td>Vedco</td><td>468RX</td><td>Pre operative Sedative/ analgesic</td></tr></tbody></table>

surgical porcine model of chronic myocardial ischemia treated by exosome-laden collagen patch and off-pump coronary artery bypass graft

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts to reduced oxygen availability by reducing metabolic activity, thereby preventing irreversible cardiomyocyte injury and infarction. This is distinct from myocardial infarction, as HIB has the potential for recovery with revascularization. Patients with significant coronary artery disease (CAD) experience chronic ischemia, which puts them at risk for heart failure and sudden death. The standard surgical intervention for severe CAD is coronary artery bypass graft surgery (CABG), but it has been shown to be an imperfect therapy, yet no adjunctive therapies exist to recover myocytes adapted to chronic ischemia. To address this gap, a surgical model of HIB using porcine that is amenable to CABG and mimics the clinical scenario was used. The model involves two surgeries. The first operation involves implanting a 1.5 mm rigid constrictor on the left anterior descending (LAD) artery. As the animal grows, the constrictor gradually causes significant stenosis resulting in reduced regional systolic function. Once the stenosis reaches 80%, the myocardial flow and function are impaired, creating HIB. An off-pump CABG is then performed with the left internal mammary artery (LIMA) to revascularize the ischemic region. The animal recovers for one month to allow for optimal myocardial improvement prior to sacrifice. This allows for physiologic and tissue studies of different treatment groups. This animal model demonstrates that cardiac function remains impaired despite CABG, suggesting the need for novel adjunctive interventions. In this study, a collagen patch embedded with mesenchymal stem cell (MSC)-derived exosomes was developed, which can be surgically applied to the epicardial surface distal to LIMA anastomosis. The material conforms to the epicardium, is absorbable, and provides the scaffold for the sustained release of signaling factors. This regenerative therapy can stimulate myocardial recovery that does not respond to revascularization alone. This model translates to the clinical arena by providing means of physiological and mechanistic explorations regarding recovery in HIB.

Following revascularization, coronary angiography is performed to assess for LAD stenosis (greater than 80%) and patency of the LIMA-LAD graft (Figure 1). Four weeks following the revascularization surgery and placement of the exosome-laden collagen patch, cardiac MRI is performed to assess for systolic and diastolic function of the heart at rest and under stress using low-dose dobutamine infusion at 5&#181;g/kg/min. Systolic function is analyzed by measuring wall thickness percentage (wall thickness at end systole - wall thickness at end diastole). Diastolic function is analyzed by measuring peak filling rate over end diastolic volume (PFR/EDV; Figure 2). Delayed contrast imaging was performed to confirm lack of myocardial infarction in the LAD territory. If an infarct in the LAD region is present, it is likely due to the occluded artery secondary to thrombosis caused by the constrictor. Absence of regional wall motion abnormalities demonstrate lack of hibernating phenotype.
At low dose of dobutamine infusion, HIB animals demonstrate significant decrease in diastolic function, measured by PFR/EDV, compared to control group (5.5 &#177; 0.8 vs. 6.9 &#177; 1.5, respectively, p &#60; 0.05). CABG group demonstrates a trend towards improvement in PFR/EDV compared to HIB group (6.3 &#177; 0.9 vs. 5.5 &#177; 0.8, respectively, p = 0.06). However, CABG + MSC group demonstrate a significant increase in PFR/EDV when compared to HIB group (6.6 &#177; 1.1 vs. 5.5&#177; 0.8, respectively, p = 0.03; Figure 3). Cardiac MRI was used to confirm lack of necrosis and patency of the Left internal mammary artery (LIMA) to left anterior descending artery (LAD) bypass graft distal to the area of stenosis18.
At rest, CABG + MSC group does not alter regional systolic function (measured by percentage wall thickness) compared with CABG alone (26.3% &#177; 7.0% vs. 34.9% &#177; 6.3%; p = 0.19). Under stress, CABG + MSC group shows significant improvement in regional systolic function compared with CABG alone (78.3% &#177; 19.6% vs. 39.2% &#177; 5.6%; p = 0.05)12 (Figure 4).
At necropsy, appropriately sized coronary dilators were used to ensure the LAD stenosis and LIMA patency. Myocardium was grossly inspected to ensure the tissue viability is present in all regions especially in the ischemic region. Triphenyltetrazolium chloride (TTC) stain confirmed absence of scar.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65553/65553fig01.jpg" /> 
Figure 1. Cardiac angiogram demonstrating the anatomy. Coronary angiography demonstrates &#62;80% stenosis of proximal LAD artery and patent LIMA-LAD graft anastomosis. Abbreviations: LIMA= Left internal mammary artery, LAD= Left anterior descending <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65553/65553fig01large.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/65553/65553fig02.jpg" /> 
Figure 2. Assessment of diastolic relaxation, global contractile function, and viability using cardiac MRI. (A) Diastolic relaxation: Relationship of left ventricular (LV) volume during a cardiac cycle. The x-axis is time in s; y-axis is volume of left ventricle in mL. The red line indicates peak filling rate (fastest rate at which the LV increases volume). PFR is normalized to the animal&#39;s end-diastolic volume (PFR/EDV) to account for size variance across animals. (B) Global contractile function: Segmental circumferential strain (Circ strain) during the cardiac cycle (x-axis: time in ms; y-axis: percentage change in circumferential length of left ventricle segment compared with end-diastolic measurement). Peak circumferential strain is represented by the most negative value of cycle. (C) Representative cardiac MRI image of LAD distribution: LAD distribution is highlighted in red represents the anteroseptal wall. There was no evidence of infarction based on enhanced gadolinium contrast on 4 chamber (D) long axis and (E) short axis views. Abbreviations: LV= left ventricle/ventricular; LAD= left anterior descending; MRI= magnetic resonance imaging. This figure has been modified from 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65553/65553fig02large.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/65553/65553fig03.jpg" /> 
Figure 3. MRI assessment of Peak filling rate/End diastolic volume. Diastolic function, measured by PFR/EDV, was compared among four groups (Control, HIB, CABG, and CABG + MSC). At rest, PFR/EDV is comparable among four animal groups. However, under stress using low dose dobutamine infusion (5&#181;g/kg/min), HIB group showed a significant decrease in PFR/EDV when compared to control (p &#60; 0.05) with trend towards improvement in CABG group (p = 0.06) and significant increase in CABG + MSC group (p &#60; 0.05). Statistical analyses were performed using one-way analysis of variance (ANOVA) test. Data are presented as means &#177; SD. Abbreviations: CABG= Coronary artery bypass graft, PFR= Peak filling rate, EDV= end diastolic volume; MRI= Magnetic resonance imaging, MSC= Mesenchymal Stem Cells, SD= Standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65553/65553fig03large.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/65553/65553fig04.jpg" /> 
Figure 4. MRI assessment of regional systolic function by wall thickening %. Treatment with MSC patch shows improvement in regional cardiac function compared to sham patch. (A) Regional systolic function, measured by wall thickening percentage, does not significantly improve at rest with MSC patch treatment (n = 6) compared with sham (n = 6). (B) Under stress using low dose dobutamine infusion (5&#181;g/kg/min), there is significant improvement in regional systolic function following treatment with the MSC patch compared with sham animals (P&#60;.05). statistical analyses were performed using Mann-Whitney test. Horizontal bars indicate mean standard deviation. *P&#60;.05. Abbreviations: MSC= Mesenchymal stem cells. This figure has been modified from 12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65553/65553fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary table 1. Overview of procedures and timeline of each procedure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65553/Overview of procedures and timeline.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft at JoVE.com

Author Spotlight: Enhancing Coronary Artery Revascularization

This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery.

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts ...

surgical-porcine-model-chronic-myocardial-ischemia-treated-exosome

Nanyang Technological University

Research

JoVE Journal

Medicine

623 Views.  University of Minnesota. Het algemene doel van deze chirurgische ingreep is het creëren van een chirurgisch model van revascularisatie van de kransslagader met behulp van LIMA naar LAD-bypass, en het gebruik van met exosomen beladen collageenpleister als aanvulling om fysiologische verbetering van de hartfunctie aan te tonen. Ons in-vivo-model is een model met één vat dat geen comorbiditeiten bevat, wat niet representatief is voor de typische klinische situatie voor patiënten die een behandeling ondergaan voor me...

Video: Auteur in de schijnwerpers: verbetering van revascularisatie van de kransslagader

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Bioengineering

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Developmental Biology

Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e., occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.

This paper describes a novel nonocclusive coronary anastomotic connector in a porcine off-pump coronary artery bypass (OPCAB) model. This easy-to-use coronary connector has intrinsic potential to facilitate minimally invasive OPCAB surgery.

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft

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