A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Summary

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Abstract

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo¹. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased.

Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream^2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream⁵. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

Introduction

A myocardial infarction (MI) is defined as the interruption of blood to a region of the heart caused by the occlusion of a major coronary artery. The damage resulting from an MI is due to the remodeling of the viable heart tissue into non-functional scar tissue, which decreases the ability of the heart or, more specifically, the left ventricle, to beat properly. This results in a decrease in the volume of blood that can be delivered to the body with every heartbeat, known as the stroke volume, and the percentage of blood that is pumped out of the heart with each heartbeat, known as the ejection fraction⁶. These, along with other diminished functions, increases the strain on the rest of the heart to maintain adequate function. Often, this increased strain can become so severe that it causes a second heart attack, a phenomenon seen in approximately 10% of individuals⁷.

While medical practices have evolved to treat the immediate aftermath of an MI, no technique has been developed to halt, slow, or reverse the negative side effects of tissue remodeling. Stem cell therapies have emerged as a possible avenue for such a treatment, however, despite their promising potential, stem cells have not proven successful in the clinical setting. One theory for their shortcomings is the inability to ensure the beneficial cells remain at the site of infarction long enough to generate favorable results⁵. It has been shown that no more than 24% of cells that are simply injected into the site of infarction survived and remained at the damaged site 1 day post-delivery². A possible prospect for addressing this issue of cell retention is to develop biocompatible hydrogel systems that encapsulate either cells or therapeutics, which can be delivered to the damaged site. The hydrogel of choice in this protocol is a poly(ethylene glycol) dimethacrylate due to its previous use in cell encapsulation procedures, however, any hydrogel capable of encapsulation may be used⁸. The delivery of the patch directly to the site of injury ensures cell-to-tissue contact over an extended period of time, increasing the length of time the cells can provide beneficial factors to the underlying myocardium.

A bottleneck to the patch approach is the difficulty of adhering the patch to the heart surface. Many groups have overcome this through a variety of techniques, the most prevalent being a simple suture to tie the construct to the heart surface^9,10. This has proven successful in a number of cases in which the construct is made of a stiffer material, but fails when attempted on a hydrogel system, due to the high water concentration and delicate nature of the patch construct. To overcome this, we have utilized a fibrin glue external adhesive system that mimics the chemistry of clot formation. Fibrin glue has been used in numerous medical surgeries, including dura tears, bronchial fistulas, and corneal transplantation, highlighting the biocompatibility of the product as a wound sealant^11-13. Additionally, fibrin has been used for a variety of cardiac purposes, including surgical treatment of left ventricular ruptures and coronary artery bypass surgeries, however, its use as an adhesion glue for a cardiac patch is not commonly used^14-17. A simple formulation of thrombin and fibrinogen results in a biocompatible glue that can be placed directly on the outside of an external cardiac patch, providing a viable adhesion system to ensure patch to heart interaction.

Protocol

Procedure is in full compatibility with IACUC protocol number 13302 and has been approved by the division of animal resources.

1. Instrument Preparation

1. Autoclave all non-disposable instruments used prior to the surgical procedure to ensure sterility.
2. Sterilize instruments used multiple times in a session with a glass bead sterilizer between uses.

2. Hydrogel Preparation

1. Thaw and plate stem cells at least 24 hr prior to creation of the hydrogel construct.
2. Prepare patches one day prior to the surgical procedure using a stereolithography apparatus (or similar instrument) which has been described in full detail in previous publications^8,18.
3. To create the hydrogel, first generate a computer aided design (CAD) model of the desired patch and export the design to stereolithographic format (STL).
4. Create a pre-gel solution by dissolving poly(ethylene glycol) dimethacrylate (PEGDMA) in sterile 1x PBS to obtain a 20% w/v solution. Concurrently, dissolve the photoinitiator 1-[4-(2-hydroxy- ethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one in DMSO. Add the photoinitiator to the PEGDMA solution at a final w/v of 0.5%. Directly before crosslinking, add 100 ml of the desired density of cells. Note: A cell density between 2.0 x 10⁵-2.0 x 10⁶ cells/ml is recommended.
5. Pipette the pre-gel solution into a dish at the center of the SLA platform and run the SLA software with the previously uploaded STL formatted design.
6. Remove the patches from the apparatus and incubate the hydrogels O/N in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin to allow for cells to adjust to the environment within the patch.

3. Animal Preparation and Oral intubation

1. Evaluate each mouse prior to the procedure to ensure that the animal is in good health, as determined from their grooming habits and interactions with their cage mates. Note: Female 8-10 week old C57BL/6J mice were used for this procedure, however, any mouse age or type can be used.
2. Place animals into an anesthetic chamber and expose to 5% isofluorane with a 1 L/min O₂ support. Monitor the level of anesthesia by toe pinch reflex.
3. Once the animal is properly anesthetized, weigh and place them on the intubation stand.
4. Direct a light source towards the animal’s chest cavity and use forceps to retract the laryngeal surface and expose the vocal cords. Carefully guide a 20 G angiocath between the vocal cords, and smoothly insert into the trachea. Detect proper insertion by a mechanical movement of the chest cavity once the catheter is connected to the small animal ventilator.
5. Adjust ventilation settings to the weight of the animal based on the manufacturer’s guidelines.
6. Once intubated, place the animal in the supine position on a heating pad to prevent hypothermia. Ensure that the heating pad does not exceed 40 °C. If necessary, place a barrier between the animal and the heating pad to minimize the chance of burns.
7. Remove hair from the surgical site through the use of a depilatory cream. Sterilize the site by 3 alternating scrubs of betadine and 75% ethanol to ensure proper sterilization. Place a drape to expose just the surgical site, and place vet ointment on the eyes to prevent dryness during the procedure.

4. Surgical Procedure

1. The thoracotomy is performed on the left side of the animal, therefore, make a skin incision approximately 1 cm to the left of the sternum, that runs the length of the sternum.
2. Use forceps to separate the muscle layer from the underlying ribcage in two steps. Observe a delineating line that represents the pectoralis major muscles of the animal. Lift this muscle slightly, separate it from the underlying external oblique muscle and retract medially. Then, free the external oblique muscle from the underlying ribcage in the same manner, and retract laterally, providing an unobscured view of the second, third, and fourth ribs.
3. Perform the thoracotomy between the third and fourth ribs. Gently lift the fourth rib and use a cauterizer to open the chest cavity between the third and fourth ribs. Place retractors to further open the cavity and expose the heart.
4. Use forceps to rupture the thin pericardium of the heart.
5. Ligate the left coronary artery with an 8-0 monofilament nylon suture. Place the suture approximately 4 mm from the apex of the heart, directly below the bottom tip of the left atrium. Determine proper suture placement by a blanching of the ventricular myocardium and an increase in the size of the left atrium after the suture has been tied.

5. Patch Placement and Glue

1. Keep patches at 37 °C and 5% CO₂ conditions until use.
2. Gently lift the patch using a flat ended spatula and gently place it on the surface of the heart, at the site of infarction.
   Note: To prevent slipping off the left ventricle, the patch can be held in place by lightly maintaining contact using either the spatula or the tip of a pair of forceps.
3. Prepare the glue by mixing a 4:1 ratio of fibrinogen to thrombin. Mix the solution by repeated pipetting until it begins to thicken, usually within 1 min after the beginning of the preparation.
4. Once the solution viscosity reaches the desired level, quickly transfer approximately 10 ml to the patch surface. Note: The clotting time of the fibrin glue is rapid, providing a small window of opportunity for efficient transfer of the glue.

6. Suturing

1. Perform all closing sutures using a 6-0 monofilament nylon suture.
2. Close the rib layer with three to four individual interrupted sutures.
3. Before full closure of the intercostal layer, insert a PE-10 cannula into the incision to evacuate the chest cavity after closure is complete and reestablish proper intrapleural pressure.
4. Close the pectoral muscles with three to four individual interrupted sutures.
5. Seal the skin layer with a continuous suture.
6. Following full closure, attach a 1.0 ml syringe to the end of the cannula and use it to evacuate the chest cavity.
7. Apply a tissue adhesive to the incision site to reinforce the suture site.

7. Post-surgical Treatment

1. Give mice an injection of buprenorphine (0.05-1.0 mg/kg) and carprofen (2.2 mg/kg) subcutaneously immediately following the coronary ligation and least 20 min prior to revival. Administer both drugs 6-8 hr after surgery, and then give twice daily for up to three days to control pain and distress.
2. Continuously monitor the animals until conscious and every hour for the first 4 hr following surgery. Perform a second checkup the night of the surgery (2-4 hr later) to administer a second dose of analgesic.
3. Monitor the animals daily after the second day following the procedure, until animals appear stable.

8. Analysis of Heart Function and Histology

1. Perform echocardiographs on the mice 4 weeks following the procedure to determine the extent of damage following the MI.
2. Anesthetize mice with 4-5% isoflurane within an anesthesia chamber for induction of inhalation anesthesia, and then 2% isoflurane via face mask for maintenance anesthesia.
3. Clear the imaging area with a depilatory cream and use a small animal ultrasound to obtain a 2D M-mode echocardiography scan of the beating heart and record the functional parameters associated with it.
4. Euthanize the mice in a carbon dioxide chamber, then collect the hearts from the animals and place in a 10% formalin solution for 24 hr. After 24 hr, transfer the hearts to 70% ethanol and keep at 4 °C until tissue processing.
5. Process the hearts and embed them in paraffin blocks.
6. Cut 5 mm sections through the ventricular region of the heart, starting at the apex and ending at the atrium.
7. Stain sectioned slices with both Masson’s trichrome and hematoxylin and eosin stain using standard protocols.

Results

During the surgical procedure, ligation of the left coronary artery can be identified by a marked blanching downstream of the occluded artery. As a test before tying the knot, the suture can be tightened briefly to check if it is in the appropriate place. Additionally, since occlusion of the artery results in almost instantaneous decrease in the ability of the left atrium to properly beat, the left atrium will enlarge in response to a backflow of blood in the system.

M-mode echocardiography me...

Discussion

With this approach to the murine MI model, we have developed a system that minimizes the damage to non-myocardium areas that are associated with other murine MI techniques. These areas include damage caused by a tracheostomy, the cutting of the muscular layer, and the breakage of ribs to expose the chest cavity. We believe that these changes improve the overall surgical outcome thanks to the care taken to keep as much of the major structures, including the ribs an...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Harvard Model 687 Mouse Ventilator	Harvard Apparatus	55-0001
Inintech Biosciences LLC Dry Glass Bead Sterilizer	Fisher Scientific	NC9531961
Leica MZ6 surgical microscope	Leica
Cautery Kit	Gemini	GEM 5917
Delicate Forceps - 0.4 mm Tips Angled	Fine Science Tools	11063-07
Agricola Retractor - 3.5 cm Spread	Fine Science Tools	17005-04
Spring Scissors - 2.5 mm Blades Straight	Fine Science Tools	15000-08
Castroviejo Needle Holder - w/Lock Tungsten Carbide 14 cm	Fine Science Tools	12565-14
Iris Scissors - Delicate Straight 10.5 cm	Fine Science Tools	14060-10
8-0 monofilament suture	Ethicon	8730P
6-0 Silk suture	Ethicon	639G
Thrombin	Sigma	T7009
Fibrinogen	Sigma	F3879
Vetbond Tissue Adhesive	3M	1469SB