Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Summary

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Abstract

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Introduction

Fabricating functional vascularized tissue using a tissue engineering approach is an emerging paradigm in regenerative medicine.^1,2 Many approaches to engineer new and healthy tissue for the replacement of injured tissue or defective organs have been developed,^3-6 experimentally in small animal models with promising clinical potential.^7,8 However, vascularization remains one of the great challenges for tissue engineering limiting its potential to grow tissues of clinically relevant size.⁹

Current approaches to vascularize tissue follow either an extrinsic pathway where new vessels grow from the recipient vascular bed and invade throughout the implanted tissue constructs¹⁰ or an intrinsic vascularization pathway where the vasculature grows and expands in unison with the newly developing tissue.¹¹ The extrinsic approach traditionally involves seeding cells onto a scaffold in vitro and implanting the complete construct into the living animal with the expectation that nutrients, previously supplied by culture media, will be sourced from the circulation.^12,13 The concept is simplistic as vascular ingrowth is too slow and only very thin implants (<1-2 mm thick) will remain viable. Providing nutrients and oxygen by means of a sufficient and rapid vascularization is at the heart of any successful attempts to grow more complex and larger tissue-engineered substitutes such as bone, muscle, fat and solid organs.^14,15 Intrinsic vascularization offers the potential for larger constructs to develop by progressive tissue growth commensurate with its expanding blood supply. One design is the in vivo implantation into a chamber of a vascular pedicle with or without a cell seeded scaffold.^5,6 This has paved the way to new procedures for the generation of thicker intrinsically vascularized tissues.^16,17

More recently, strategies have been developed to pre-vascularize tissue grafts, prior to implantation. These incorporated blood vessel networks are aimed to inosculate with host vessels at implantation allowing for the rapid provision of a complete blood supply to improve the survival of all parts of a transplanted thick tissue graft.¹⁸

We pioneered an in vivo vascularized tissue engineering model in small animals that involves a subcutaneously implanted semi-rigid enclosed chamber containing a perfused vascular pedicle and cell-containing biomaterials. The chamber creates an ischemic environment that stimulates angiogenic sprouting from the implanted vessels.³ The vascular pedicle can either be a reconstructed arteriovenous loop or an intact flow-through artery and vein.^3-6,19 This vascular pedicle sprouts a functioning and extensive arterio-capillary-venous network that links at both arteriole and venous ends with the vascular pedicle.^3,20 Furthermore, the surrounding hollow support chamber protects the developing tissue from potentially deforming mechanical forces and prolongs the ischaemic drive to enhance vascularization.^3,21,22 If the vessel pedicle is simply implanted into normal tissue and not inside the protected space of the chamber, angiogenic sprouting ceases along the same timeline as a normal wound and no new tissue will accumulate around the pedicle. Investigators have used this in vivo configuration to produce three-dimensional functional vascularized tissue constructs with supportive vasculature and of clinically relevant size.^4,23 Furthermore, the engineered vascularized tissue constructs with its intact vascular pedicle can be harvested for subsequent transplantation at the injury site.^24,25 A more clinically feasible scenario would be creating the chamber at the definitive site for reconstruction such as the breast. Thus, this de novo tissue engineering approach could have clinical potential to provide a new source of functional target tissue for reconstructive surgery.^26-28

The following protocol will provide a general guide to construct an in vivo vascularized tissue engineering chamber in the rat, which could be adapted in different animal models and employed to examine the intricate processes of angiogenesis, matrix production, and cellular migration and differentiation.

Protocol

The protocols described here have been approved by the Animal Ethics Committee of St. Vincent's Hospital Melbourne, Australia, and were conducted under strict adherence to the Australian National Health and Medical Research Council Guidelines.

NOTE: Two chamber protocols are described below. The two different models and their specific chamber designs are illustrated in Figure 1. Chamber (1) is made of polycarbonate (for rat arteriovenous loop chamber model). It is cylindrical with an internal diameter 13 mm and height 4 mm. A window at one point in the wall allows unimpeded access for the pedicle. In the second model (for rat flow-through pedicle chamber model), the chamber is made of polyacrylic and is rectangular (10 x 8 x 4 mm³ internal dimensions). It has two 1.5 mm openings on opposite sides to accommodate the femoral artery and vein as they transgress the chamber.

1. Rat Arteriovenous Loop Chamber Model (One Chamber Per Animal)

NOTE: Prior to starting surgery, make sure all the instruments have been properly sterilized. Likewise, ensure the instruments rest on sterile towels and are at a reasonable distance from the surgical field to avoid contamination during the procedure.

1. Preparation of the Animal for Surgery
   1. Use rats weighing at least 250 g for their large size of vessels for creation of the arteriovenous loop.
   2. Anaesthetize the animal with 4% isoflurane inhalation. Corroborate adequate depth of anesthesia by assessing unresponsiveness to toe-pinch. After anesthesia, keep the animal adequately anesthetized throughout the procedure with 2% isoflurane.
   3. Place the animal in a supine position on a warming pad and apply sterile lubricant to the eyes to prevent desiccation during surgery.
   4. Using an electric razor, shave both groins and remove hair with a piece of moist gauze.
   5. Prep the surgical sites with chlorhexidine/70% ethanol solution and drape the animal with sterile towels. Administer a single dose of Carprofen (5 mg/kg, subcutaneously) as analgesic.
2. Harvest of Femoral Vein Graft
   1. Using a #15 blade, make a 4 cm long skin incision on the left groin parallel to the inguinal ligament. This exposes the inguinal fat pad.
   2. Cut through the fat pad circumferentially with scissors leaving it attached to its vascular pedicle based on the epigastric vessels.
   3. Using micro scissors, free the filmy connective tissue adhesions between the abdominal wall and underlying femoral vessels.
   4. Place a retractor on the abdominal wall and pull medially. This exposes the inguinal ligament and the whole length of the femoral vessels.
   5. Using micro forceps and curved scissors dissect the epigastric vein and isolate it from its surrounding fat by gently pulling and cutting. This vein acts as a tether when constructing the loop.
   6. Using micro forceps and curved micro scissors open the perivascular sheath containing the femoral vessels and nerve all the way from the inguinal ligament to its bifurcation distal to the epigastric branch.
   7. Using micro forceps, pick up the femoral vein by its adventitia and gently separate it from the surrounding tissues and accompanying artery. Do this with micro forceps and curved round-pointed micro scissors by pulling the tissue apart and cutting through it.
      NOTE: Never grab the whole thickness of the vein wall as this might cause trauma to the intima making it prone to thrombosis.
   8. Ligate side branches found during the dissection with 10/0 nylon suture or coagulate them with a bipolar coagulator.
   9. With the femoral vein completely free, ligate its proximal and distal ends with 4/0 silk sutures. Make sure to obtain a vein graft of at least 10 mm length and include approximately 0.5 cm length of the epigastric branch to be used as a guy rope tether to hold the loop open in the chamber.
   10. Using micro forceps and straight micro scissors, trim the adventitia from the graft's ends by gently pulling and cutting. This can also be done later, before microsurgical anastomoses.
   11. Flush the vein graft with heparinized saline solution (10 U/ml of heparin) and leave it to rest in the solution. Close the wound using continuous running 4/0 silk suture plus two or three additional simple interrupted stitches.
3. Creation of Arteriovenous Loop and Implantation of Chamber
   1. Repeat steps 1.2.1 to 1.2.4 in the exact same way on the contralateral limb.
   2. Using micro forceps, dissect and isolate both the epigastric artery and vein form the surrounding fat pad. Do this by gently pulling the tissue away from the vessels
   3. Using micro forceps, pick up the femoral artery by its adventitia and free it from the surrounding tissues. Do this with micro forceps and curved round-pointed micro scissors by pulling the tissue apart and cutting through it. Ligate or coagulate its side branches.
   4. Ligate the femoral artery and vein distal to the emergence of the epigastric vessels using 4/0 silk suture.
   5. Place a single clamp proximally on each of the femoral artery and vein. Using a sharp straight micro scissor, make a clean transverse cut in each vessel distal to the emergence of the epigastric branches. Place a sterile plastic contrast background under the vessels.
   6. Flush the vessels vigorously with generous amounts of heparinized saline until all the blood is removed from the lumen.
   7. Bring the vein graft into the operative field and remove any redundant adventitia from the vessels' ends as per step 1.2.10, if needed.
   8. Perform both microsurgical anastomoses with 10/0 nylon suture. Anastomose the proximal end of the vein graft to the femoral vein and the distal end to the femoral artery. This will allow the blood to flow from the arterial to the venous side without resistance from valves inside the vein graft.
      NOTE: Make sure the femoral vessels and the vein graft rest in their natural position without any twists.
   9. Check for leaks at both anastomotic sites. Resolve small leaks, which look like non-pulsating blood coming out of the anastomotic site, by placing a small piece of fat on top and gently compressing for 5-10 min. Larger pulsating leaks that rapidly flood the entire field will need additional stitches.
   10. Check patency of the arteriovenous loop. Gentle occlusion of the femoral artery should make it shrink while the same in the femoral vein should engorge it.
   11. Place the base of the tissue engineering chamber under the arteriovenous loop with the latter resting in its natural position without twists or kinks.
   12. Secure the base of the chamber to the inguinal ligament and underlying muscle fascia with 6/0 nylon sutures.
   13. Place the lid over the base so that the femoral vessels enter the chamber through a notch (window in the side of the chamber). When closing the lid, make sure it catches the epigastric branches, between the chamber base and lid, which act as tethers to hold the arteriovenous loop into position.
   14. Close the wound using continuous running 4/0 silk suture plus two or three additional simple interrupted stitches.
   15. Allow the animal to recover from anesthesia on a warming pad. 
   16. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Likewise, do not return an animal that has undergone surgery to the company of other animals until fully recovered. 24 hr later, administer another single dose of Carprofen (5 mg/kg, subcutaneously) as analgesic.
   17. Treat the wound with topical antibiotic ointment for 5 days. If the wound is opened, anesthetize the animal as in step 1.1.2 through 1.1.5 and close the wound as in 1.3.14. Monitor the health of animal daily. Euthanize the animal using a lethal dose of intraperitoneal lethabarb injection (163 mg/kg in 0.25 ml by 23 G needle) if the animal shows more than one moderate signs of inacitivity, poor appetite, weight loss and loss of color.

2. Flow-through Pedicle Chamber (Two Chambers per Animal)

1. Preparation of the Animal for Surgery
   1. Repeat steps 1.1.1 through 1.1.4. Two chambers can be implanted into both groin regions of a single rat.
2. Isolation of Femoral Vessels and Insertion of the Chamber
   1. Repeat steps 1.2.1 through 1.2.8.
   2. With both artery and vein completely freed of surrounding tissues and their branches ligated, bring the chamber into the operative field.
   3. Place each of the intact femoral vessels on the corresponding slit of the chamber base making sure there are no twists or kinks.
   4. Close the chamber by attaching the lid to the base. Close the wound using continuous running 4/0 silk suture plus two or three additional simple interrupted stitches and allow the animal to recover as previously described.

3. Harvest of Chambers and Tissue Processing

1. Once the experiment's time points (4-6 weeks post-implantation) are reached, anesthetize the animal as in step 1.1.2 and repeat steps 1.1.3 through 1.1.5.
2. Open the wound using a #15 blade and cut through the tissues with scissors until the chamber is completely exposed.
3. Expose the femoral vessels proximal to the construct and test for vascular patency: gently occlude the vessel with two microforceps, then milk the blood in a distal direction and finally release the proximal forceps. If the vessel fills with blood again, this confirms patency. Ligate the femoral vessels proximally in the case of the arteriovenous loop and both proximally and distally in the case of the flow-through configuration, and remove the chambers with the containing tissue en bloc.
4. At the end of the experiment, euthanize the animal using a lethal dose of intraperitoneal lethobarb injection (163 mg/kg in 0.25 ml by 23 G needle).
5. Fix tissues in 4% paraformaldehyde at room temperature for 24 hr. Divide tissues into multiple transverse sections (1-2 mm thick) and embed in paraffin wax or optimal cutting temperature compound for paraffin sections (5 µm) or frozen sections (10 µm), respectively.^{3,4,8,17,22,24}
6. Perform routine histological staining such as hematoxylin and eosin to examine the general morphology of tissues. Perform immunohistochemical staining with specific antibody to identify cell type of interest,^{3,4,8,17,22,24,29} for example cardiac troponin T immunostaining for cardiomyocytes.

Results

The microsurgical creation of tissue engineering chambers was performed as described in the protocol above. Tissues generated inside the chambers can be examined histologically as describe in protocol step 3. Various tissue types have been successfully engineered using the in vivo vascularized chamber (Figure 2). These include cardiac tissue with neonatal rat cardiomyocytes (Figure 2A), muscle tissue with rat skeletal myoblasts (Figure 2B...

Discussion

Engineering of the microcirculation is currently being investigated essentially through two approaches. The first involves developing a highly interconnected vascular network within the construct in vitro so that when implanted, capillaries from the host vascular bed connect with those of the transplanted construct through a process called inosculation, thus ensuring the delivery of nutrients not only to the periphery but also to the core.^21,32,33 This is called pre-vascularization. The second approac...

Materials

Name	Company	Catalog Number	Comments
1 15 Blade Scalpel	Braun	BB515
1 Toothed Adson Forceps	Braun	BD527R
1 Needle Holder	Braun	BM201R
1 Bipolar Coagulator	Braun	US335
1 Micro Needle Holder B-15-8.3	S & T	00763
1 Micro Dilator Forceps D-5a.2	S & T	00125
1 Micro Jeweler's Forceps JF-5	S & T	00108
1 Micro Scissors - Straight SAS-11	S & T	00098
1 Micro Scissors - Curved SDC-11	S & T	00090
2 Single Clamps B-3	S & T	00400
2 10/0 nylon suture	S & T	03199
1 6/0 nylon suture	Braun	G2095469
2 4/0 Silk Sutures	Braun	C0760145
Xilocaine 1%	Dealmed	150733	10 mg/ml
Heparin Sodium	Dealmed	272301	5000 UI / ml
Ringer Lactate	Baxter	JB2323	500 ml
1 dome-shaped tissue engineering chamber	custom made
1 flow-through chamber	custom made
Lectin I, Griffonia Simplicifolia	Vector Laboratories	B-1105	1.67 μg/mL
Troponin T antibody	Abcam	Ab8295	4 μg/mL
Human-specific Ku80 antibody	Abcam	Ab80592	0.06 μg/mL
Desmin antibody	Dako	M0760	2.55 μg/mL
Cell Tracker CM-DiI dye	Thermo Fisher Scientific	C-7000	3 mg/106 cells