Pulsed electric fields in biology and medicine

 

An acute wound is created each time an incision is made in the skin whether the wound results from an operative procedure, or from a traumatic or burn injury.  The human body regrows tissue generally with some scar formation after the wound has completely healed.  The process of scar formation is initiated in the early phases of wound healing and, in most cases, the wound is replaced by new functional scar tissue that shows amounts of collagen, proteoglycans, and water that are similar to normal skin. Abnormal wound healing, especially in burn patients, can result in the formation of proliferative scar, most notably hypertrophic scar that forms as a raised, red, itchy, and inelastic mass of tissue.  Various procedures have been tested over the years to treat scars of any etiology, but no one therapy has been found to be consistently effective.  MGH investigators are developing novel techniques using intermittently delivered pulsed electric fields to regulate fibroblast proliferation, collagen production, and scar contraction in efforts to “turn off” proliferative scar formation and substantially improve the quality of the mature scar.   Read More

The wound repair process

The inflammation phase of wound repair

The inflammation phase of wound repair

Human skin is a complicated structure made up of the epidermis (multiple outer covering layers and includes specialized cells, for example, that produce melanin), the dermis (composed of layers of collagen and elastin and includes blood vessels and nerves, hair follicles, sweat glands, and oil glands), and finally the subcutaneous tissue (a layer of fat and connective tissue in which larger blood vessels and nerves are found).  Exactly how a wound heals after a break in the skin is dependent upon the depth of the wound.  Factors that can affect wound healing include the patient’s age and nutritional status, presence of infection or associated illnesses, and current treatment with steroids, chemotherapy drugs, or radiation.

Illustration of cells undergoing mitosis (cell growth and division)

Illustration of cells undergoing mitosis (cell growth and division)

Wound repair can be described in four sequential and overlapping phases: hemostasis, inflammation, proliferation, and remodeling.  Briefly, any active bleeding is stopped by constriction of the damaged blood vessels and formation of clot composed of aggregated platelets (hemostasis).  Bacteria and cell debris are removed from the wound by the white blood cells (phagocytosis) at the initiation of the inflammation phase.  From the release of various cytokines and growth factors, the migration and division of cells needed for the proliferation phase is initiated.  New blood vessel formation, collagen deposition, granulation tissue formation, epidermal re-epithelialization, and wound contraction take place in the proliferation phase.  In the final maturation or remodeling phase, re-organization of the closed wound environment takes place to include collagen remodeling and realignment.  During ‘normal’ wound healing, the cells that are no longer needed for wound repair undergo the biological process of apoptosis or programmed cell death. Cell growth and apoptosis work hand-in-hand to promote tissue reconstruction in the healing process. In wound healing, apoptosis takes charge of the removal of the inflammatory cells and evolution of granulation tissue into scar.

Scar formation in wound healing is inevitable

The human body regrows tissue with scar formation after the wound has completely healed.  This is unlike some other animal species that have the enhanced capacity to regenerate or regrow tissue or even an entire limb without scar formation, for example, a salamander. It appears that regeneration is an option for some species whereas for others (e.g. mammals), the presence of microbes is a much greater threat thereby making the role of inflammation is more essential to ensure survival.

The process of scar formation is initiated in the early phases of wound healing, which is characterized by increased collagen deposition and elevated concentrations of the many secretory proteins that make up the extracellular matrix or ECM.  The ECM is the space lying outside and between cells that is filled with physiologically active material composed of proteoglycans, water, minerals, and fibrous proteins.

Chest burn showing hypertrophic scars and contractures

Chest burn showing hypertrophic scars and contractures

In most cases over time, the wound is replaced by new functional scar tissue that shows amounts of collagen, proteoglycans, and water that are the same as normal skin.  This ‘normal’ scar tissue is flat, almost the same color as the intact skin, not itchy, and pliable.  Abnormal wound healing, especially in burn patients, can result in the formation of proliferative scar, most notably hypertrophic scar that forms as a raised, red, itchy, and inelastic mass of tissue.  Hypertrophic scar tissue looks the way it does because of the presence of a larger amount of ECM than in normal skin or scar tissue. During hypertrophic scar formation, there is an abnormally high increase in the number of cells present (hypercellularity) and much of the new and immature collagen is physically disorganized in the form of ‘nodules’ instead of thick fibers.  In other words, too many cells are too active for too long.  The exact mechanism that induces proliferative scar tissue formation instead of healthy scar tissue is not known.

Electroporation-based technologies

Electroporation is a phenomenon that occurs when short bursts of high voltage pulsed electric fields (PEFs) cause membrane permeability.  It can be used to (a) reprogram cell and tissue function by introducing external molecules that affect different cellular pathways, (b) load cells with new materials, and (c) cause cell damage and death.  PEF technology has been applied in the food preservation industry for microbial inactivation and cell disintegration and in environmental applications for bacterial decontamination of waste water.  In medical applications using PEF, several successful studies in tissue cultures, animal models, and patients have emerged over the past four decades.  Human and veterinary medical applications of electroporation include electrochemotherapy (reversible electroporation to deliver chemotherapy drugs directly into cells), transdermal delivery of drugs, DNA vaccination, and irreversible electroporation to destroy tumors inside the body.

Applying tissue engineering approaches to wound healing

Various procedures such as surgical excision, intralesional steroid injection, cryotherapy, laser therapy, irradiation, mechanical compression dressing, silicone sheet application, intralesional interferon injection, or combination of techniques have been used over the years to treat acute and chronic wounds, but no one therapy has been found to be consistently effective.  Application of tissue engineering solutions to solve wound healing problems is not new to investigators at MGH.  Our interests began with Dr. John Burke who worked in the 1970’s with Massachusetts Institute of Technology colleague Dr. Ioannas Yannas to pioneer the first, successful tissue-engineered artificial skin to replace lost skin after serious burn injury. The study of wound healing in burn injured patients makes sense as their challenges to wound healing are the most dramatic of all areas of medicine.

In recent studies, MGH bioengineers and biologists have demonstrated that intermittently delivered pulsed electric fields may be useful as a non-operative, non-chemical method to treat open wounds or scars. The investigators believe that the hypercellularity of fibroblasts and other inflammatory cells observed in scar tissue formation may be due to a delay or reduction in the normal onset of apoptosis.  Fibroblasts, a type of cell that synthesizes the ECM and collagen during wound repair, play a very important role in wound healing and these cells are induced to change their actin gene expression to become myofibroblasts, taking on some of the properties of smooth muscle cells and helping to pull the wound margins together.  Myofibroblasts in particular are known to be resistant to the onset of apoptosis that normally accompanies scar maturation and normal wound healing, which contributes to their increased density in scar tissue.

Tissue engineering of skin using PEFs

Histological analysis 1 week after PEF.  Regeneration of epidermis (left) and normal collagen structure (right)

Histological analysis 1 week after PEF. Regeneration of epidermis (left) and normal collagen structure (right)

Recent published basic science and clinical data on electroporation-based applications have used pulsed electric fields for tissue ablation, a procedure known as irreversible electroporation (IRE). IRE is a tissue ablation method where specific pulsed electric fields cause irreversible damage to cells by affecting their cell membranes, while sparing the neighboring tissue scaffold, large blood vessels, and other accessory structures.  IRE effects on tissues are different from laser-induced tissue damage because IRE allows for extended control of cell density without any significant effect on the surrounding ECM.

MGH investigators using intermittently delivered PEF have successfully controlled human skin fibroblast density in culture and in an animal model by therapeutic induction of apoptosis.  Using non-thermal, PEF controlled ablation, the investigators have demonstrated that PEF preserves the blood supply and ECM structure.  PEF treatment results in acute cellular death, transiently altered skin perfusion, followed by a self-limited local inflammatory response.  Despite early signs of significant tissue damage, complete skin and panniculus carnosus (skin muscle) tissue regeneration without any signs of fibrosis is observed.

The investigators have also developed new animal models of hypertrophic scarring and scarless skin regeneration in normal skin to test whether PEF skin ablation treatment can repair or possibly regenerate skin.  These wound management and scarring studies using animal models will be followed by studies in patients experiencing scarring in the hope that giving PEF during the proliferation phase of wound repair will optimize the remodeling phase of wound repair.  It is anticipated that this novel tissue decellularization using irreversible electroporation will become a new, non-operative, non-chemical, treatment strategy for wound healing and scarless healing.

Relevant publications

Scott PG, Ghahary A, Wang JF, Tredget EE. Molecular and cellular basis of hypertrophic scarring. In: Herndon DN, ed. Burn Care.  3rd ed. New York, NY : Saunders Elvesier; 2007:596-607

Golberg A, Rubinsky B. A statistical model for multidimensional irreversible electroporation cell death in tissue. Biomed Eng Online. 2010 Feb 26;9:13. doi: 10.1186/1475-925X-9-13. PubMed PMID: 20187951; PubMed Central PMCID: PMC2839970

Golberg A, Laufer S, Rabinowitch HD, Rubinsky B. In vivo non-thermal irreversible electroporation impact on rat liver galvanic apparent internal resistance. Phys Med Biol. 2011 Feb 21;56(4):951-63. PubMed PMID: 21248392

Golberg A, Bei M, Sheridan RL, Yarmush ML. Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields. Biotechnol Bioeng. 2013 Jun;110(6):1759-68. PubMed PMID: 23297079

Golberg A, Yarmush ML. Nonthermal irreversible electroporation: fundamentals, applications, and challenges. IEEE Trans Biomed Eng. 2013 Mar;60(3):707-14. Review. PubMed PMID: 23314769

Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavčič D. Electroporation-based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng. 2014 Jul 11;16:295-320. PubMed PMID: 24905876

Golberg A, Broelsch GF, Bohr S, Mihm MC Jr, Austen WG Jr, Albadawi H, Watkins MT, Yarmush ML. Non-thermal, pulsed electric field cell ablation: A novel tool for regenerative medicine and scarless skin regeneration. Technology (Singap World Sci). 2013 Sep;1(1):1-8. PubMed PMID: 24999487; PubMed Central PMCID: PMC4078877

Quinn KP, Golberg A, Broelsch GF, Khan S, Villiger M, Bouma B, Austen WG Jr, Sheridan RL, Mihm MC Jr, Yarmush ML, Georgakoudi I. An automated image processing method to quantify collagen fibre organization within cutaneous scar tissue. Exp Dermatol. 2015 Jan;24(1):78-80. PubMed PMID: 25256009; PubMed Central PMCID: PMC4289465

Golberg A, Broelsch GF, Vecchio D, Khan S, Hamblin MR, Austen WG Jr, Sheridan RL, Yarmush ML. Pulsed electric fields for burn wound disinfection in a murine model. J Burn Care Res. 2015 Jan-Feb;36(1):7-13. PubMed PMID: 25167374; PubMed Central PMCID: PMC4286470

Contact

Martin L. Yarmush, MD, PhD Alexander Golberg, PhD William Gerald Austen, Jr, MD Robert L. Sheridan, MD
myarmush@mgh.harvard.edu agolberg@gmail.com wausten@mgh.harvard.edu rsheridan@mgh.harvard.edu
617-371-4882 617-726-3474 617-724-9922 617-726-3712

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Grace McDonald-SmithPulsed electric fields in biology and medicine