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Method Article

Engineered Vascularized Muscle Flap

8.1K views

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DOI:

10.3791/52984

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January 11th, 2016

 ,  ,  ,  ,  , 
1Department of Plastic Surgery, Kaplan Medical Center, 2Biomedical Engineering, Technion Israel Institute of Technology, 3Interdepartmental Program in Biotechnology, Technion Israel Institute of Technology, 4Medicine Department, Technion Israel Institute of Technology

* These authors contributed equally

In This Article

Summary

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

Abstract

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

Introduction

Abdominal wall defects often arise following severe trauma, cancer treatment, burns and removal of infected mesh. These defects often involve significant tissue loss, requiring complicated surgical procedures and presenting a major challenge for plastic reconstruction surgeons 1-4. Tissue engineering researchers seeking new sources for artificial tissues have explored different materials, cell sources and growth factors. Successful restorations of various tissues, such as trachea 5,6, bladder 7, cornea 8, bone 9and skin 10, by implantation of engineered tissues were previously reported. However, fabrication of a thick vascularized engineered tissue, particularly for reconstruction of large defects, remains a significant challenge in tissue engineering.

One of the main factors limiting the thickness of a viable tissue construct is the control of oxygen supply to its constituent cells. When relying on diffusion, construct thickness is limited to that of a very thin layer. The maximum distance between oxygen- and nutrient-supplying capillaries in vivo is approximately 200 µm, which correlates with the diffusion limit of oxygen11,12. Insufficient vascularization can result in tissue ischemia and escalate to tissue resorption or necrosis 13.

In addition, the ideal material used for tissue reconstruction must be biocompatible and non-immunogenic. It must also be capable of promoting further integration of host cells with the biomaterial, and maintaining structural integrity. Various biological 14-16 and synthetic 1,17,18 matrices have been previously explored for tissue reconstruction, however their use remain limited due to lack of effective blood supply, infections or insufficient tissue strength.

In this study, a biocompatible, cell-embedded scaffold comprised of Food and Drug Administration (FDA)-approved poly L-lactic acid (PLLA)/poly lactic-co-glycolic acid (PLGA), was implanted around the femoral artery and vein (AV) vessels of a nude mouse and separated from the surrounding tissue, ensuring vascularization from the AV vessels only. One week post-implantation, the graft was viable, thick and well vascularized. This thick vascularized tissue with the AV vessels, was then transferred as a pedicled flap to an abdominal full-thickness defect in the same mouse. One week post-transfer, the flap was viable, vascularized and well integrated with the surrounding tissue, bearing sufficient strength to support abdominal viscera. Thus, the engineered thick, vascularized tissue flap, bearing an autologous pedicle, presents a novel method for repairing full-thickness abdominal wall defects.

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Protocol

All animal studies were approved by the Committee of the Ethics of Animal Experiments of the Technion. For this procedure, athymic nude mice were used to avoid immunological rejection. If using another type of mouse, the mice should be shaved prior to the surgical procedure and administration of cyclosporine (or another anti-rejection substitute) is recommended.

1. Scaffold Preparation and Cell Embedding

  1. Prepare scaffolds composed of 1:1 mixture of poly-L-lactic-acid (PLLA) and polylactic-co-glycolic-acid (PLGA), in the following manner:
    1. Dissolve 500 mg of PLLA and 500 mg of PLGA in 10 ml chloroform.
    2. Add 0.24 ml of polymer solution to 0.4 g of sodium chloride particles gathered in a Teflon molds. Use a salt diameter of 212-600 µm. Allow the chloroform to evaporate O/N.
    3. Leach the salt out using distilled water leading to interconnected porous 3D scaffolds.
    4. Cut polymer pieces using a scissors, then soak in 70% ethanol (v/v) for 30 min and wash 3 times in PBS for 2 min before usage.
  2. Prepare a tri-culture of the following cells in 1:1 endothelial cell medium (EGM-2 supplemented with the components of its bullet kit and muscle cell medium (Dulbecco's minimal essential medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 2.5% HEPES buffer, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Pen-Strep Solution).
    1. Use 0.5 x 106 C2C12 myoblasts, 0.9 x 106 Human umbilical vein endothelial cells (HUVECs), and 0.2 x 106 Normal human dermal fibroblasts (NHDF).
  3. Mix the cells together in a microcentrifuge tube and centrifuge at 200 x g for 4 min, aspirate the medium and re-suspend the pellet in a mixture of 4 µl ice cold extracellular matrix solution and 4 µl cell medium.
  4. Cut a piece of 10 mm x 7 mm x 1 mm (length x width x thickness) PLLA/PLGA scaffold and seed the cell suspension in a 6-well plate, allow the extracellular matrix solution to solidify (30 min, 37 °C, 5% CO2) before adding 4 ml cell medium.
  5. Incubate the scaffolds for ten days before implantation and replace the medium every other day.

2. Before Starting the Surgical Procedure

  1. Prepare and sterilize (autoclave) the following surgical tools: a small, fine, straight scissors, a spring scissors, a straight, fine-tipped forceps, a serrated forceps and a needle holder.
  2. Prepare a mixture of ketamine:xylazine solution in an microcentrifuge tube by mixing 425 µl ketamine and 75 µl xylazine in a 1 ml syringe.
  3. Dilute 0.3 mg/ml buprenorphine in sterile saline (1:20).

3. Flap Construction (Graft Implantation)

  1. Using an insulin syringe, anesthetize the mouse by an intraperitoneal injection of ketamine:xylazine (35 µl per 20 g of body weight).
  2. Subcutaneously inject buprenorphine (final concentration of 0.05-0.1 mg/kg) before the opperation.
  3. Place the mouse on a stage heated to 37 °C, to ensure normal body temperature, and then secure the mouse using an adhesive bandage.
  4. Using cotton tips, administer lubricating eye ointment to the mouse's eyes (to avoid dehydration).
  5. Using cotton tips, clean the incision site with iodine and then with 70% ethanol, to establish an aseptic working field.
  6. Using small scissors and forceps, make an incision through the skin, from the level of the knee to the inguinal ligament, parallel to the femoral vessels, until the femoral artery and vein (AV) vessels are exposed.
  7. Hold the skin down using a retractor, to gain better access to the tissue.
  8. Using spring scissors and forceps, carefully isolate the femoral artery and vein vessels from the surrounding tissue. Start from the level of the inguinal ligament to the bifurcation of the superior epigastric vessels. Leave the profunda untouched, in order to preserve blood flow to the leg and prevent subsequent ischemia.
  9. Fold the graft (from Section 1) around the exposed femoral AV, below the profunda and above the bifurcation to the tibial and proneal AV, and join its ends using 8-0 silk sutures.
  10. To ensure implant vascularization by the capillaries sprouting from the femoral AV vessels only, wrap a piece of sterilized latex around the graft, and suture with 6-0 sutures.
  11. Close the overlying skin using 4-0 silk sutures.
  12. Monitor the mouse closely until recovery from the anesthesia and every day thereafter, until the graft is transferred as a flap. Subcutaneously inject buprenorphine (final concentration of 0.05-0.1 mg/kg) twice a day for 2-3 days.
    NOTE: Use saline in any stage, to avoid dehydration of the exposed tissue.

4. Flap Transfer

  1. At one or two weeks postimplantation, anesthetize the mouse by an intraperitoneal injection of ketamine:xylazine (35 µl/20 g), using an insulin syringe.
  2. Subcutaneously inject buprenorphine (final concentration of 0.05-0.1 mg/kg) before the opperation.
  3. Place the mouse on a stage heated to 37 °C, to ensure normal body temperature, and then secure the mouse using an adhesive bandage.
  4. Using cotton tips, apply lubricating eye ointment to the mouse's eyes (to avoid dehydration).
  5. Using cotton tips, clean the incision site (from the knee area to the abdomen) with iodine and then with 70% ethanol, to establish an aseptic working field.
  6. Using a scissors and forceps, carefully open the sutures in the skin and, using a scissors, make an incision through the skin parallel to the inguinal ligament, continuing through the ventral skin.
  7. Carefully remove the latex piece with forceps and expose the vascularized flap.
  8. Using a scissors and forceps, dissect the tissue flap from the surrounding tissue.
  9. Using a forceps and a needle holder, ligate the distal end of the femoral AV with 8-0 silk sutures to prevent bleeding from the AV. Then, cauterize the AV at the level of the knee, distally to the folded implanted tissue and the 8-0 sutures.
  10. Gently transfer the femoral AV enveloped by the graft, towards the ventral abdominal wall, avoiding damage to the artery, to determine at which distance the full thickness defect should be made.
    CAUTION: Pulling the artery too far will damage the artery.
  11. Using a fine scissors, make an incision in the ventral abdominal wall.
  12. Using a spring scissors, make an incision in the rectus abdominus muscle. Remove an approximately 1 cm x 0.8 cm segment of the rectus abdominus muscle with the overlying skin.
  13. Transfer the femoral AV, enveloped by the vascularized graft, as an axial flap, to the full-thickness defect in the ventral abdominal wall.
  14. Suture the flap to the surrounding muscle tissue, using 8-0 silk sutures, to prevent hernia.
  15. To avoid suturing the internal organs, raise the muscle with a forceps when suturing the flap to the abdominal muscle. Leave the ventral skin exposed to mimic the effect of a full abdominal wall defect.
  16. Cover the wound in the skin with iodinated gauze and a sterile bandage to prevent contamination of the exposed area.
  17. Suture the skin of the leg using 4-0 silk sutures.
  18. Monitor the mouse closely until recovery from the anesthesia and every day thereafter, until retrieval of the flap.  Subcutaneously inject buprenorphine (final concentration of 0.05-0.1 mg/kg) twice a day for 2-3 days.
  19. NOTE: Use saline in any stage to avoid dehydration of the exposed tissue.

5. Ultrasound Determination of Vascular Perfusion of the Graft

NOTE: Before transfer, vascular perfusion of the graft is measured at one and two weeks after implantation, by ultrasonography.

  1. Anesthetize the mouse using 2% isoflurane.
  2. Place the mouse on a movable stage heated to 38 °C, to ensure normal body temperature, and then secure the mouse using an adhesive bandage.
  3. Find the femoral artery and vein vessels with non-linear transducer set on the B-mode.
  4. Examine the patency of the femoral vessels of the graft using the transducer set on the color Doppler mode.
  5. Prepare non-targeted contrast agent according to the manufacturer’s instructions.
  6. Connect a tail vein catheter (12 cm tubing with a 27 G needle) to a 1 ml syringe filled with saline. Secure the syringe next to the mouse on the heating stage (to avoid movement of the syringe).
  7. Connect the catheter to the mouse tail vein and secure the needle.
  8. Inject some of the saline to both ensure that injections are being made into the vein and to fill the tubing with saline (to avoid air bubbles).
  9. Fill a 1 ml syringe with 70 µl of contrast agent and replace the saline syringe with the contrast agent syringe.
  10. Set up the ultrasound to image injection of the contrast agent. In properties of the non-linear mode set up the number of frames to 1,500 and the disruption pulse to 30% of the frames.
    NOTE: After the disruption pulse, the microbubbles re-fill the vessels at a rate that depends on the flow rate of the microbubbles in the capillaries and is independent of the injection rate of the microbubbles. The number of frames can be changed according to the filling time 19,20.
  11. Slowly inject the contrast agent into the tail vein.
  12. Capture images in the non-linear contrast mode of the high-resolution ultrasound system.
  13. Analyze the results using the software, as previously described 19,20.
    1. Draw a region of interest (ROI) on the image obtained immediately after the disruption pulse in the contrast-mode. The ROI should outline the area filled by the contrast agent only.
    2. Calculate the peak enhancement (PE), which is the ratio of the mean intensity of the non-linear signal after the injection of the microbubbles versus after the disruption pulse, and is a measure of the perfusion volume.
    3. Calculate the flow rate, which is determined from the time (T) that elapses until the peak signal (P).

6. Determination of the Extent of Graft Vascularization

NOTE: The extent of tissue graft vascularization is determined one or two weeks after implantation.

  1. Using an insulin syringe, anesthetize the mouse by an intraperitoneal injection of ketamine:xylazine (35 µl/20 g).
  2. Using 1 ml syringe, intravenously inject 200 µl of 10 mg/ml fluorescein isothiocyanate-conjugated dextran (FITC-Dextran, MW = 500,000) into the tail vein.
  3. Ten sec after completion of the injection, euthanize the mouse by cervical dislocation.
  4. Open the leg of the mouse and expose the graft.
  5. Wash the graft area with phosphate buffered saline (PBS) and apply 10% neutral buffered formalin.
  6. Image the graft in the mouse leg, using a confocal microscope.
  7. Quantify functional vessel density (FVD).
    1. Isolate the green channel (excitation = 488 nm) of the confocal microscopic images.
    2. Pass the resulting images through a high-pass filter, in order to accentuate the high-contrast structures.
    3. Use a despeckling filter to remove noise that might have been amplified by the high-pass filter.
    4. Set up the value of threshold on the resulting image; the threshold value is adjusted so that the features and structures in the original image are visible in the binary image.
    5. Apply a size-threshold so that only groups of connected pixels larger than the defined size threshold remain in the binary image.
    6. Outline the skeleton of the vessels in the graft using the Zhang-Suen's algorithm 21.
    7. Calculate the FVD by summing the lengths of the midline of each vessel and dividing the result by the area of the ROI.

7. Immunohistological and Histological Staining of the Graft

  1. Immunohistological Staining
    1. After confocal imaging, retrieve the graft using a small scissors and forceps. Dissect the AV vessels in the two ends of the graft and retrieve the graft.
    2. Fix the grafts in 10% neutral buffered formalin for 30 min - 2 hr.
    3. Place the grafts in histology cassettes and store in 70% ethanol until paraffin embedding.
    4. Embed in paraffin using a standard fixation and embedding protocol 22 and slice the paraffin embedded tissues into 5 µm slices using a microtome. Slice the whole tissue perpendicularly to the AV vessels.
    5. Deparaffinize the paraffin-embedded sections by immersion in 100% xylene twice, for 10 min each.
    6. Arrange the slides in a 250 ml plastic Coplin jar.
    7. Rehydrate by serial immersions in decreasing concentrations of ethanol (100%, 96%, 90%, 80%, 70%, 50%, 30% and double distilled water (DDW)) for 3 min each.
    8. Prepare antigen unmasking solution by diluting 2.35 ml of antigen unmasking solution with 250 mL of DDW. Heat the antigen unmasking solution for 2 min in a microwave set to 95 °C.
    9. Insert the slides into the pre-heated antigen unmasking solution and heat again for 3 min in a microwave set to 95 °C. Cool the slides to 50 °C.
    10. Heat again for 2 min and then cool to RT.
    11. Incubate the slides in 3.3% H2O2solution in methanol for 10 min, at RT to quench the activity of endogenous peroxidase and then rinse twice in PBS.
    12. Prepare an incubation chamber: place wet absorbent sheets inside a histology box and dry the slides using delicate task wipers.
    13. Circle the slice area on the slide using a hydrophobic pen and incubate for 30 min at RT, with approximately 50 µl of goat serum blocking solution (2%, prepared in PBS) per slice.
    14. Dilute the rabbit anti-CD31 antibody 1:50 in blocking solution, apply approximately 50 µl antibody per slice and incubate O/N, at 4 °C. Rinse 3 times with PBS, 5 min each.
    15. Dilute the biotinylated goat anti-rabbit secondary antibody 1:400 in PBS, place over slides approximately 50 µl of antibody per slice and incubate for 30 min at RT.
    16. Rinse 3 times with PBS, 5 min each.
    17. Dilute streptavidin-peroxidase 1:400 in PBS, apply approximately 50 µl per slice and incubate with slides for 30 min, at RT. Rinse 3 times with PBS, 5 min each.
    18. Apply approximately 50 µl of aminoethylcarbazole (AEC) substrate per slice, according to the manufacturer’s instructions. Dip the slides in DDW to stop the reaction.
    19. Stain the nuclei in blue by immersing the sections in approximately 50 µl of Mayer’s hematoxylin solution for 2 min. Gently rinse in water and then cover the sections with mounting medium.
    20. Quantify CD31-positive staining using a double-blind approach. CD31 staining appears as a brown stain. Choose 4-5 different fields in a 40X magnification viewing field under a binocular microscope. Count the number of CD31-positive lumens.
      1. Calculate the average number of CD31-positive lumens per scaffold by dividing the total number of counted lumens with the number of fields and the fieldarea (according to the binocular data).
        NOTE: The applied reagent volume must be sufficient to cover the entire outlined slice.
  2. Histological Staining
    1. Fix grafts in 10% neutral buffered formalin.
    2. Embed grafts in paraffin using standard fixation and embedding procedures.
    3. Remove the paraffin from the paraffin-embedded sections by immersion in 100% xylene twice for 10 min.
    4. Fast wash twice for rehydration in 100% isopropanol.
    5. Fast wash twice 95% Ethanol.
    6. Wash three times in tap water.
    7. Immerse in Hematoxylin for 10 min.
    8. Wash three times in tap water.
    9. Immere in Eosin for 2 min.
    10. Fast wash dehydrate in 95% Ethanol twice.
    11. Fast wash 100% isopropanol twice.
    12. Fast wash 100% xylene twice.
    13. Cover with DPX glue with cover glass 1.5 mm thick.
    14. Image using microscope in 40X magnification.

8. Mechanical Properties Assessment

  1. Retrieve the graft using a small scissors and forceps.
  2. Rinse the graft in PBS.
  3. Measure the width and thickness of the graft and calculate the cross-section area as width multiplied by thickness.
  4. Use the test instrument to generate a stress-strain curve of hydrated grafts.
    1. Mount the graft between the system tensile grips and measure the final length between the two grips (i.e., initial length).
    2. Apply a strain rate of 0.01 mm/sec, until failure.
    3. Record the developed force and displacement using the manufacturer’s protocol.
    4. Generate a stress-strain curve in Matlab.
      1. Calculate strain according to displacement divided by the initial length. Stress is calculated as the measured force divided by the cross-sectional area.
      2. Determine stiffness as the slope of the linear-region in the stress-strain curve and the maximum point of the curve is the ultimate tensile strength.

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Results

Graft vascularization and perfusion in vivo

The grafts were implanted one or two weeks prior to their transfer as axial flaps. At one and two weeks post-implantation, gross observation of the graft area revealed viable and vascularized tissue grafts. These grafts proved to be highly vascularized, as determined by positive CD31 immunostaining (Figure 1A), and highly perfused, as evidenced by FITC-dextran tail vein injection and ultrasound measurements. Many vessels wer...

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Discussion

The advances in tissue engineering have been met with a growing demand for substitute tissues for reconstruction of various tissue types. A variety of synthetic 1,17,18 and biological 14-16 materials as well as fabrication methods have been assessed for their capacity to address these demands. However, despite the progress in clinical care and in tissue engineering, the restoration of full-thickness abdominal wall defects remains a challenge. A tissue adequate for reconstruction of such massive defe...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

This research was supported by the FP7 European Research Council Grant 281501, ENGVASC.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
small fine straight scissorsFine Science Tools (FST)14090-09
spring scissorsFine Science Tools (FST)15003-08
straight forceps with fine tipFine Science Tools (FST)11251-20
serrated forceps Fine Science Tools (FST)11050-10
needle holderFine Science Tools (FST)12500-12
Small vessel cauterizer Fine Science Tools (FST)18000-00
DuratearsAlcon5686
SedaxylanEuravetDJ03
Clorketam 1000Vetoquinol4A0726B
BuprenorphinevetmarketB15100
4-0 silk suturesAssut sutures647
6-0 polypropylene suturesAssut sutures9351F
8-0 silk suturesAssut sutures684568
Insulin syringe (6 mm needle)BD324911
Vevo 2100 high-resolution ultrasound systemVisualSonics inc.
MS250 non-linear transducerVisualSonics inc.
Micromarker non-targeted contrast agentVisualSonics inc.VS-11694
tail vein catheterVisualSonics inc.VS-11912
Vevo 2100 softwareVisualSonics inc.
fluorescein isothiocyanate-conjugated dextranSigmaFD500S
MatlabMathworks, MA, USA
KimwipesKimtech34120
antigen unmasking solutionVector laboratoriesH-3300
anti-CD31 antibodyAbcam ab28364
biotinylated goat anti-rabbit (secondary) antibodyVector laboratoriesBA-1000
streptavidin-peroxidaseJackson 016-030-084
Mayer's hamatoxylin solutionSigma-AldrichMHS-16
aminoethylcarbazole (AEC) substrate kitLife technologies, Invitrogen 00-2007
VectamountVector laboratoriesH-5501

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