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Research Papers

Nonthermal Irreversible Electroporation for Tissue Decellularization

[+] Author and Article Information
Mary Phillips

Department of Mechanical Engineering, University of California, Berkeley, 6124 Etcheverry Hall, Berkeley, CA 94720mary_phillips@berkeley.edu

Elad Maor

Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720eladmaor@gmail.com

Boris Rubinsky

Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720; Department of Mechanical Engineering, University of California, Berkeley, Berkeley, CA 94720brubinsky@gmail.com

http://www.americanheart.org

J Biomech Eng 132(9), 091003 (Aug 16, 2010) (8 pages) doi:10.1115/1.4001882 History: Received August 26, 2009; Revised April 28, 2010; Posted May 27, 2010; Published August 16, 2010; Online August 16, 2010

Tissue scaffolding is a key component for tissue engineering, and the extracellular matrix (ECM) is nature’s ideal scaffold material. A conceptually different method is reported here for producing tissue scaffolds by decellularization of living tissues using nonthermal irreversible electroporation (NTIRE) pulsed electrical fields to cause nanoscale irreversible damage to the cell membrane in the targeted tissue while sparing the ECM and utilizing the body’s host response for decellularization. This study demonstrates that the method preserves the native tissue ECM and produces a scaffold that is functional and facilitates recellularization. A two-dimensional transient finite element solution of the Laplace and heat conduction equations was used to ensure that the electrical parameters used would not cause any thermal damage to the tissue scaffold. By performing NTIRE in vivo on the carotid artery, it is shown that in 3 days post NTIRE the immune system decellularizes the irreversible electroporated tissue and leaves behind a functional scaffold. In 7 days, there is evidence of endothelial regrowth, indicating that the artery scaffold maintained its function throughout the procedure and normal recellularization is taking place.

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Copyright © 2010 by American Society of Mechanical Engineers
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Figures

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Figure 1

Schematic of the electrode clamps used to induce NTIRE: (a) clamp consists of two printed circuit boards (PCBs) with disk electrodes at the end; (b) the carotid artery is pressed gently between the two electrodes, holding the electrodes apart by approximately 0.4 mm

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Figure 2

Schematic of the model geometry. The artery is modeled as shown (bottom). The 0.4×3 mm2 artery is in direct contact with the 0.1×3 mm2 copper electrodes. The PCBs are modeled using the material properties of flame retardant 4 (FR4) and are 1.6×3 mm2 in size. The artery and electrode clamp construct are surrounded by air (3×3 cm2).

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Figure 3

Maximum temperatures obtained over the course of the simulation. The maximum temperatures obtained at time steps throughout the simulations show a peak maximum temperature obtained after the final electric pulse, followed by a cooling down period for 1 Hz (a) and 4 Hz (b) frequencies. The maximum temperatures stay well below the designated thermal damage limit.

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Figure 4

Effects of NTIRE for 1 Hz treatment. H&E staining of cell nuclei (shown as dark spots) shows that at 3 days the NTIRE-treated artery is largely decellularized when compared with the control artery. At 5 days the NTIRE-artery is decellularized, and repopulation of the endothelial layer can be seen. At 7 days, the NTIRE-treated artery (shown embedded in the surrounding tissue) is still almost completely decellularized when compared with the control artery. Note that the endothelial cells for the treated artery at 7 days are similar in number to those of the control.

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Figure 5

Effects of NTIRE for 4 Hz treatment. H&E staining shows the results for an artery 3 days after NTIRE-treatment and at 7 days post-treatment as compared with the nontreated control. At 3 days, the artery is almost completely decellularized. Seven days after treatment, though still mostly decellularized, cells have begun to repopulate the artery, especially along the endothelial layer.

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Figure 6

DAPI staining. Though DNA (shown by the bright spots) is seen throughout the medial (m) and adventitial (a) layers of the control artery, the medial and adventitial layers of the artery 7 days after NTIRE-treatment are almost completely void of DNA. The NTIRE-treated artery shown here is embedded in the tissue, and DAPI shows DNA throughout the tissue surrounding the artery as well as along the artery intimal layer (i). The white scale bar on the top left corner of each image represents 25 μm.

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Figure 7

Smooth muscle cell removal. Arteries stained for α-SMA (shown as the darker staining in between the elastin fibers of the medial layer) demonstrate a decrease in VSMC at 3 days and a lack of VSMC in the acellularized construct at 7 days after NTIRE-treatment as compared with the control.

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Figure 8

Staining for endothelial cells. Staining for Factor VIII related antigen (shown as dark staining along the lumen surface) was used to identify the cells lining the lumen as endothelial cells for both the control and treated artery at 7 days after applying NTIRE.

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Figure 9

Ablation zone boundary. Marked margination between VSMC-populated and depopulated regions are highlighted in three different examples.

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Figure 10

EVG staining. From the 1 Hz pulse group, EVG staining shows undamaged elastin fibers for the NTIRE-treated artery at 3 days post-treatment and at 7 days post-treatment when compared with the control.

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Figure 11

Further ECM analysis. Movat’s pentachrome stain for an artery 3 days after NTIRE-treatment shows undamaged elastin fibers (black) as well as collagen and reticulum fibers (orange) and proteoglycans (blue and highlighted by arrows).

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