• unlimited access with print and download
    $ 37 00
  • read full document, no print or download, expires after 72 hours
    $ 4 99
More info
Unlimited access including download and printing, plus availability for reading and annotating in your in your Udini library.
  • Access to this article in your Udini library for 72 hours from purchase.
  • The article will not be available for download or print.
  • Upgrade to the full version of this document at a reduced price.
  • Your trial access payment is credited when purchasing the full version.
Buy
Continue searching

Regulation of gene expression in response to continuous low intensity direct current electrical fields

Dissertation
Author: Jessica Amber Jennings
Abstract:
In the normal process of wound healing, small electric fields are generated at the wound site due to a disturbance of the potential difference maintained by a continuous epithelium. Although the roles of EFs in healing are unclear, physiological strength fields promote such processes as cellular migration and protein synthesis. Furthermore, the application of exogenous fields in various forms improves repair of chronic wounds. In these studies, the transcriptional response of dermal cells exposed to physiological strength electric fields for one hour is measured using microarrays, followed by the use of bioinformatics tools to interpret the implication of results in wound healing. The identification of 555 significantly increased transcripts and 550 significantly decreased transcripts in microvascular endothelial cells led to the recognition of activity within mitogen activated protein kinase, transforming growth factor-beta, and apoptotic signaling cascades. Transcripts related to TGF-β, MAPK, and apoptotic pathways were also identified within the 164 increased and 302 decreased transcripts in dermal fibroblasts exposed to EFs. In epidermal keratinocytes, 161 transcripts were significantly increased while 245 were significantly decreased. There were 18 genes that were regulated above a two-fold level but not reaching statistical significance in keratinocytes, including primarily chemokines and inflammatory response genes. Several genes were selected to confirm results using real time RT-PCR and to elucidate the field- and time-dependent characteristics of gene expression in EFs. The expression of genes related to growth, inflammation, and adhesion reinforces the impact of EFs in the process of repair.

vii TABLE OF CONTENTS

Page

ABSTRACT.......................................................................................................................iii

DEDICATION.....................................................................................................................v

ACKNOWLEDGEMENTS...............................................................................................vi

LIST OF TABLES.............................................................................................................ix

LIST OF FIGURES...........................................................................................................xi

LIST OF ABBREVIATIONS..........................................................................................xiii

INTRODUCTION...............................................................................................................1

Wounds and injury currents...........................................................................................2 EF-induced cellular effects............................................................................................4 Galvanotaxis............................................................................................................4 Physical Orientation.................................................................................................7 Growth Factors and Receptors.................................................................................8 Protein synthesis and cellular function....................................................................9 Summary of EF-induced cellular effects...............................................................10 Wound Treatment Strategies........................................................................................11 Electric fields.........................................................................................................12 Overall effects of ES..............................................................................................17 Functional Genomics and the Study of Healing Response..........................................19 Project Scope...............................................................................................................22

ELECTRIC FIELDS ELICIT REGULATION OF GENE EXPRESSION IN DERMAL MICROVASCULAR ENDOTHELIAL CELLS.............................................24

TRANSCRIPTIONAL RESPONSE OF DERMAL FIBROBLASTS IN DIRECT CURRENT ELECTRIC FIELDS.......................................................................55

EFFECTS OF DIRECT CURRENT ELECTRIC FIELDS ON GENE EXPRESSION IN EPIDERMAL KERATINOCYTES....................................................84

SUMMARY.....................................................................................................................113

viii TABLE OF CONTENTS (cont.)

Page

GENERAL LIST OF REFERENCES.............................................................................125

APPENDIX

A ENDOTHELIAL TRANSCRIPTS STATISTICALLY DIFFERENT FROM CONTROLS IN MICROARRAY STUDIES..........................................134

B FIBROBLAST TRANSCRIPTS STATISTICALLY DIFFERENT FROM CONTROLS IN MICROARRAY STUDIES..........................................175

C KERATINOCYTE TRANSCRIPTS STATISTICALLY DIFFERENT FROM CONTROLS IN MICROARRAY STUDIES..........................................193

ix LIST OF TABLES

Table Page

ELECTRIC FIELDS ELICIT REGULATION OF GENE EXPRESSION IN DERMAL MICROVASCULAR ENDOTHELIAL CELLS

1 Primers...........................................................................................................................44

2 Transcripts significantly increased in HMVECs...........................................................45

3 Transcripts significantly decreased in HMVECs...........................................................47

TRANSCRIPTIONAL RESPONSE OF DERMAL FIBROBLASTS IN DIRECT CURRENT ELECTRIC FIELDS

1 Primers...........................................................................................................................73

2 Transcripts significantly increased in HDFa..................................................................74

3 Transcripts significantly decreased in HDFa.................................................................76

EFFECTS OF DIRECT CURRENT ELECTRIC FIELDS ON GENE EXPRESSION IN EPIDERMAL KERATINOCYTES

1 Primers.........................................................................................................................103

2 Transcripts significantly increased in HEKa...............................................................104

3 Transcripts significantly decreased in HEKa...............................................................105

4 Transcripts regulated at a greater than 2-fold level.....................................................107

x LIST OF TABLES (cont.)

Table Page

APPENDIX A: ENDOTHELIAL TRANSCRIPTS STATISTICALLY DIFFERENT FROM CONTROLS IN MICROARRAY STUDIES

1 Transcripts significantly increased in HMVECs.........................................................135

2 Transcripts significantly decreased in HMVECs.........................................................154

APPENDIX B: FIBROBLAST TRANSCRIPTS STATISTICALLY DIFFERENT FROM CONTROLS IN MICROARRAY STUDIES

1 Transcripts significantly increased in HDFa................................................................176

2 Transcripts significantly decreased in HDFa...............................................................182

APPENDIX C: KERATINOCYTE TRANSCRIPTS STATISTICALLY DIFFERENT FROM CONTROLS IN MICROARRAY STUDIES

1 Transcripts significantly increased in HEKa...............................................................194

2 Transcripts significantly decreased in HEKa...............................................................200

xi LIST OF FIGURES

Figure Page

INTRODUCTION

1 Trans-epithelial potential.................................................................................................1

2 Injury currents..................................................................................................................2

ELECTRIC FIELDS ELICIT REGULATION OF GENE EXPRESSION IN DERMAL MICROVASCULAR ENDOTHELIAL CELLS

1 Schematic diagrams of stimulation chamber.................................................................48

2 KLF6 expression............................................................................................................49

3 FN1 expression..............................................................................................................50

4 FOS expression..............................................................................................................51

5 RGS2 expression............................................................................................................52

6 TGF- β interactions in EFs..............................................................................................53

7 Apoptotic gene activity in HMVECs.............................................................................54

TRANSCRIPTIONAL RESPONSE OF DERMAL FIBROBLASTS IN DIRECT CURRENT ELECTRIC FIELDS

1 Experimental setup.........................................................................................................77

2 KLF6 expression............................................................................................................78

3 FN1 expression..............................................................................................................79

4 RGS2 expression............................................................................................................80

5 JMJD1C expression.......................................................................................................81

xii LIST OF FIGURES (cont.)

Figure Page

6 Interactions between TGF- β and regulated transcripts in fibroblasts............................82

7 Apoptotic gene expression in fibroblasts.......................................................................83

EFFECTS OF DIRECT CURRENT ELECTRIC FIELDS ON GENE EXPRESSION IN EPIDERMAL KERATINOCYTES

1 Experimental setup.......................................................................................................108

2 NFKBIZ expression.....................................................................................................109

3 CCL20 expression........................................................................................................110

4 Gene expression in keratinocytes related to IL-1 signaling.........................................111

5 Statistically significant interactions.............................................................................112

xiii LIST OF ABBREVIATIONS

AC API5 ATP b-FGF BMP CASP CCL20 CEC CFLAR CXCL ConA cDNA CRIM1 DC ECM EF EGF EGFR ES FDA Alternating current Apoptosis inhibitor 5 Adenosine tri-phosphate Basic Fibroblasts Growth Factor Bone morphogenetic protein Caspase Chemokine (C-C motif) ligand 20 Corneal epithelial cell Caspase 8 and FADD-like apoptosis regulator Chemokine (C-X-C motif) ligand Concavalin A Complementary DNA Cysteine-rich transmembrane BMP regulator 1 Direct current Extracellular matrix Electric field Epidermal growth factor Epidermal growth factor receptor Electrical stimulation Federal Food and Drug Administration

xiv LIST OF ABBREVIATIONS (CONTINUED) FGF FN1 FOS GAPDH GPCR GRO HMVEC-ad HSP70 HUVEC HVPC IL JMJD1C KLF MAPK mRNA mV µA NFKBIZ PDGF PEMF PKA PKC Fibroblast growth factor Fibronectin 1 c-fos Glyceraldehyde-3-phosphate dehydrogenase G-protein coupled receptor Growth related oncogene Human microvascular endothelial cells-adult Heat shock protein 70 Human umbilical vascular endothelial cells High voltage pulsed current Interleukin Jumonji domain containing 1C Krüppel like factor Mitogen-activated protein kinase Messenger ribonucleic acid Millivolts Microamps Nuclear factor of kappa light polypeptide gene enhancer in B- cells inhibitor, zeta Platelet-derived growth factor Pulsed electromagnetic fields Protein kinase A Protein kinase C

xv LIST OF ABBREVIATIONS (CONTINUED) PLAUR RGS2 RMS A

RT-PCR SCS TEP TGF- β TGFBR1 TNF TNFR UV VEGF VEGFR Plasminogen activator, urokinase receptor Regulator of G-protein signaling Root Mean Square, average Reverse transcriptase-polymerase chain reaction Spinal Cord Stimulation Trans-epithelial potential Transforming growth factor-beta Transforming growth factor-beta receptor 1 Tumor necrosis factor Tumor necrosis factor receptor Ultraviolet Vascular endothelial growth factor Vascular endothelial growth factor receptor

1

INTRODUCTION The skin is the largest organ of the body and provides a barrier against environ- mental factors, serving as a first line of defense against pathogens and irritants. Skin also plays a vital role in maintaining homeostasis, aiding in temperature regulation and preventing dehydration. Anatomically the skin is divided into two layers: the epidermis, comprised mainly of keratinocytes, and the dermis, containing connective tissue pro- duced by fibroblasts and rich in blood vessels. Epidermal cells maintain a potential dif- ference between epidermis and dermis, with the dermis positive (Fig. 1). This trans- epithelial potential (TEP) known as the “skin battery” is the product of sodium and potas- sium pumps [Barker et al., 1982; Foulds and Barker, 1983]. Figure 1. Trans-epithelial potential.

2

Wounds and injury currents Wounds interrupt the continuity of the epidermis and cause the trans-epithelial potential to short circuit, generating electric fields (EFs) within the wound ranging from 40-100 mV/mm [Nuccitelli, 2003]. This “current of injury” has been confirmed by measurements of up to 10 mA/cm 2 emanating from the accidentally amputated fingertips of children using the highly sensitive vibrating probe method [Illingworth and Barker, 1980]. The polarity of the exposed dermis at the center of the wound is positive with re- spect to the adjacent epidermal tissues and the void containing conductive matrix and wound fluid, generating an electric field (Fig. 2). EFs generated upon wounding may be much more than a meaningless side effect of disrupting epithelium—it could be one of the first signals to cells that the barrier has been breached and that repair mechanisms must be activated. This idea is corroborated by animal studies showing that healing is Figure 2.

Injury currents.

3

impaired when injury currents are eliminated by allowing wounds to dry out or interfer- ing with sodium channels [Eaglstein et al., 1987; Stump and Robinson, 1986; Rajnicek et al., 1988]. Since EFs develop immediately upon wounding and are strongest at the edge of the wound, they directly interact with the fibroblasts, endothelial cells, and keratino- cytes at the site of injury. Because EFs persist and slowly diminish until epithelialization is complete, they may influence cellular behavior throughout the inflammatory and pro- liferative phases of healing. Wounds also interfere with protective functions of the skin and leave the body vulnerable to infection. Skin injuries can be categorized by causative and inhibitory fac- tors. Acute skin wounds result from direct trauma and heal within an acceptable time frame. Chronic wounds normally develop after repeated trauma to tissue and fail to heal in a timely manner because repair mechanisms are impaired due to disease or injury, such as diabetes, spinal cord injury, or poor blood supply. Normal wound healing is a complex sequence of events that can be divided into overlapping phases of hemostasis, inflammation, proliferation, and remodeling [Schilling, 1976]. Repair is initiated by the cells within wounded tissue, which quickly become acti- vated following injury to initiate the immune defense response and to close the defect. Cells remaining at the injury site include endothelial cells in damaged microvasculature, fibroblasts embedded in the connective tissue of the dermis, and keratinocytes at the wound edge. These remaining cells, in addition to other cells recruited to the site of in- jury, are crucial to the completion of specific tasks in healing. Endothelial cells migrate into newly formed tissue and form new vasculature during angiogenesis [Folkman, 1984]. Fibroblasts form new connective tissue by migrating into the wound and secreting

4

matrix proteins to fill the defect [Clark, 1993]. Keratinocytes migrate and reestablish barrier function throughout reepithelialization [Clark, 1985].

EF-induced cellular effects Current generated by disrupting the TEP flows through the conductive connective tissue matrix containing cells. Because lipid bilayers forming the cell membrane have very high resistance, only very minute amounts of current pass through the membrane into the cytoplasm unless they are strong enough to cause damage to the membrane by electroporation [Adair, 1998]. Electroporation at low-level physiological wound EF strengths would occur over a period of weeks or months; therefore, it has been proposed that any rapidly induced EF effects on cells originate at the membrane surface. EFs are capable of affecting enzyme kinetics, conformational states of membrane proteins, or electrophoresis of membrane components [Astumian and Berg, 1991; Poo, 1981]. This in turn could lead to altered ion transport and activation of intracellular signaling cas- cades. EFs induce changes in cellular activities, such as migration, physical orientation, and protein synthesis, which have been reviewed by Robinson [Robinson, 1985].

Galvanotaxis The directional migration of cells in an EF, galvanotaxis, is one of the most nota- ble cellular effects of EFs . Most cells tend to migrate toward the negative pole, although some specific cell types have been observed to migrate toward the positive pole. Embry- onic fibroblasts extend lamellipodia and migrate toward the negative pole in EFs ranging from 10 to 100 mV/mm [Erickson and Nuccitelli, 1984], with tubulin and actin filaments

5

reorienting perpendicularly to the direction of the field [Méthot et al., 2001]. Actin microfilaments are essential to galvanotaxis, but microtubule assembly is not required and has an inhibitory effect [Finkelstein et al., 2004]. Fibroblast galvanotaxis occurs in three-dimensional matrices, and is independent of extracellular calcium, voltage-gated calcium channels, and gradients of cytosolic calcium [Sun et al., 2004; Brown and Loew, 1994]. Although embryonic fibroblasts and transformed fibroblast cell lines migrate in EFs, normal human fibroblasts do not migrate under similar exposure and culture condi- tions, even with a variety of media supplements [Sillman et al., 2003]. This may be a by- product of experimental culture conditions, since the intensity of galvanotaxis in fibro- blasts is dependent on the strength of adherence [Finkelstein et al., 2004]. Keratinocytes begin migrating toward the negative pole within 14 minutes, ob- taining maximal migration speeds at a field strength of 100 mV/mm [Cooper and Schliwa, 1985; Nishimura et al., 1996]. Since keratinocytes form the epidermal layer and are usually closely associated through desmosomes, they exhibit an attraction for one an- other in culture and form colonies or sheets. Groups of keratinocytes also migrate toward the negative pole, although at a slower rate than single cells [Nishimura et al., 1996; Méthot et al., 2001]. In contrast with fibroblast migration, calcium influx is required for keratinocyte galvanotaxis [Trollinger et al., 2002], and actin and tubulin filaments do not reorient significantly [Méthot et al., 2001]. Interaction with specific matrix molecules affects the intensity of galvanotaxis in keratinocytes—cells migrate more rapidly on col- lagens I and IV and plastic than they do on fibronectin or laminin [Sheridan et al., 1996]. This is of significance in healing wounds, where the most predominant matrix molecules secreted by fibroblasts are collagens. Undifferentiated keratinocytes, which are first to

6

migrate and resurface wounds, migrate toward the negative pole at a faster rate than dif- ferentiated keratinocytes expressing involucrin [Obedencio et al., 1999]. Similarly to fibroblasts, bovine vascular endothelial cells to migrate toward the cathode and display altered expression and distribution of actin filaments in DC EFs [Li and Kolega, 2002]. Human microvascular endothelial cells migrate in a voltage- dependent manner toward the negative pole in field strengths up to 200 mV/mm [Bai et al., 2004]. In contrast, migration toward the positive pole was observed in human um- bilical vascular endothelial cells, aorta smooth muscle cells, and artery-derived fibro- blasts [Bai et al., 2004; Zhao et al., 2004]. The speed of galvanotaxis in endothelial cells is less than that of fibroblasts or neonatal keratinocytes, perhaps correlated to the de- creased distances individual endothelial cells must migrate to initiate angiogenesis. Oscillatory electromagnetic fields induce migration of macrophages, an effect which is negated by blocking β-1 integrins, suggesting the involvement of integrin- dependent signaling pathways in galvanotaxis [Cho et al., 2000]. In constant DC fields macrophages migrate toward the positive pole during 30 minute exposures to high field strengths [Orida and Feldman, 1982]. At lower field strengths significant migration of macrophages does not occur, suggesting that macrophages are less sensitive to electrical fields than fibroblasts or keratinocytes. Increased macrophage infiltration around posi- tively charged beads placed in wounds provides possible in vivo confirmation of the at- traction of macrophages to positive charge [Connors et al., 2000]. Melanocytes contribute to pigmentation of the skin but are not directly involved in wound repair. These cells do not migrate directionally in EFs, which may account for the delay or absence of repigmentation of scar tissue [Grahn et al., 2003].

7

Physical Orientation In studies of galvanotaxis an additional effect of altered physical orientation has been noted in many cell types. Cells tend to orient their long axes perpendicular to the electrical field, resulting in up to a four-fold lower voltage drop across the width of indi- vidual cells [Erickson and Nuccitelli, 1984]. This appears to be related to the redistribu- tion of actin and tubulin filaments, as these structural filaments also orient perpendicu- larly [Méthot et al., 2001]. Keratinocytes have a more rounded morphology, so single cell reorientation is difficult to quantify. Colonies of keratinocytes in culture are often asymmetrical and have been observed to orient the long axes of cell groups perpendicular to the field, even though significant actin and tubulin reorganization is absent [Méthot et al., 2001]. While corneal epithelial cells do not display altered orientation, dividing cells orient their cleav- age planes perpendicularly to an EF [Song et al., 2002]. Endothelial cells display a typi- cally rounded morphology in culture in the absence of physical stresses, although when exposed to electrical fields they elongate and reorient perpendicular to the field, similar to their behavior when exposed to flow shear stress [Zhao et al., 2004]. The orientation of cells could have profound effects on wound repair. EF-induced reorientation of fibroblasts may alter the spatial distribution of collagen fiber deposition, possibly accounting for the more regular deposition of fibers in healed tissue versus nor- mal and the accompanying decrease in mechanical strength. Orientation of epithelial cell cleavage planes in an EF would serve to position the daughter cells to move into the wound. Although EF-induced reorientation of cells in culture is clearly evident, it is diffi-

8

cult to determine whether EFs contribute to significant reorientation in vivo where a three dimensional matrices restrict this movement.

Growth Factors and Receptors Physical changes in cellular activity such as galvanotaxis and orientation are par- tially dependent on the presence of serum, which contains growth factors. Galvanotaxis of CECs is inhibited in serum-free media but is restored by the addition of different com- binations of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor- β (TGF-β) [Zhao et al., 1996]. Although adding individual growth factors restores the directionality of migration, the rate of galvanotaxis in serum is not fully recovered even at field strengths above 150 mV/mm. In this way, it can be af- firmed that growth factors are required for EF effects. Since no single growth factor has been shown to be responsible, multiple independent pathways must exist. Growth factor receptors are likely candidates for the sites of action, since they are found at the membrane surface in direct contact with the field. Before binding growth factors, growth factor receptors must become activated and change conformation; dimeri- zation in most cases. This is accomplished by the phosphorylation of residues within the receptor by enzymes called kinases. Inhibiting tyrosine kinase activity, blocking the autophosphorylation of the EGF receptor (EGFR), diminishes the directionality of migra- tion in keratinocytes [Fang et al., 1999]. Inhibitors of protein kinase A (PKA) also de- crease the galvanotaxis in keratinocytes, while no significant reduction was produced by inhibitors of PKC, calmodulin dependent kinase, or myosin light chain kinase inhibitors [Pullar et al., 2001].

9

EFs also lead to an asymmetrical distribution of certain receptors and proteins on the membrane surface. EGFRs accumulate at the side facing the negative pole in kerati- nocytes after exposure to 100 mV/mm for 15 minutes [Fang et al., 1999]. Membrane lip- ids also accumulate toward the negative pole in CECs, which may provide part of the mechanism for receptor redistribution, since EGFRs associate with detergent-insoluble lipid rafts [Zhao et al., 2002]. The adhesion molecules concavalin A (Con A) and fi- bronectin receptor integrin α5β1 were also observed to favor accumulation toward the negative pole in fibroblast cell lines [Brown and Loew, 1994]. Several theories have de- veloped as to the mechanism by which this asymmetry develops, one of the most popular being that these charged proteins undergo electrophoresis within the membrane [Jaffe, 1977]. A model for how this asymmetry leads to intracellular signaling and galvanotaxis through a receptor gradient, which acts similarly to a ligand gradient, has been also been proposed [Zhao et al., 2002].

Protein synthesis and cellular function Cells responding to EFs appear to change phenotype—they are activated to per- form functions relevant to wound healing such as migration and signaling. This change in phenotype is reflected by changes in gene expression, protein synthesis and secretion. Human dermal fibroblasts exposed to EFs upregulate the expression of receptors for transforming growth factor- β (TGF-β) [Falanga et al., 1987]. Expression of TGF-β and TGF- βR1 were increased in wounds containing positively charged beads which increase local EF strength [Connors et al., 2000]. Increased expression of TGF- β and activity within the TGF- β pathway have also been identified in studies of bone healing in EFs

10

[Zhuang et al., 1997; Aaron et al., 2006]. Expression of EGFRs is upregulated by CECs prior to their redistribution at the cathodal side of the membrane [Zhao et al., 1999]. Wound response genes such as heat shock protein 70 (HSP 70), c-fos, and c-myc are upregulated in mouse 3T3 fibroblasts by exposure to EFs [Yanagida et al., 2000], which appears to be dependent on specific nCTCTn sequences in promoter regions of DNA [Lin et al., 1999]. Slices of excised rat tissue exposed to DC stimulation up to 100 µA consume 75% more amino acids and have 400% more available ATP than controls [Cheng et al., 1982]. DC stimulation of fibroblasts leads to a 20% increase in both DNA and collagen synthe- sis [Bourguignon and Bourguignon, 1987]. Interrupting the current increases fibroblast collagen synthesis by 100% over controls [Bassett and Herrmann, 1968]. Pulsed EFs promote the differentiation of keratinocytes but do not affect prolif- eration [Hinsenkamp et al., 1997]. Proliferation of vascular endothelial cells is also not affected by normal physiological levels of current and inhibited in fields above 200 mV/mm [Wang et al., 2003]. In sinusoidal electric fields at 10 Hz, increases in prolifera- tive activity of fibroblasts embedded in a collagen matrix were evident with a peak re- sponse of DNA production 70% above controls at 41 mV/mm within 18-24 hours, consis- tent with the timing of the S phase of the cell cycle [Cheng and Goldman, 1998].

Summary of EF-induced cellular effects Exposure to EFs in various forms stimulates changes in cellular activity and be- havior. In other words, EFs induce changes in cellular phenotype. Certain cells become migratory or alter their physical orientation, while others increase protein synthesis and

11

proliferative activities. Recent research has characterized some physical aspects and sev- eral contributing factors to these changes in phenotype, although thorough understanding of these events is lacking. Many of these studies have measured physical effects that are observable microscopically; thus the full range of cellular activity remains unknown. Regardless, the nature of EF induced changes in phenotype provides some justification for the observation that manipulation of EFs can augment the healing response.

Wound Treatment Strategies The majority of wound care therapies are not designed to improve healing or quality of healed tissue but rather to remove barriers to healing, such as microbial infec- tion and necrotic tissue. However, these treatments which include dressings and oint- ments, contribute to an average of over $15,000-30,000 incurred per year by individuals for wound treatment in addition to costs of treating underlying disorders [Curtin, 1984]. Along with a greater understanding of the role of growth factors and ECM in healing has come a host of new therapeutic strategies that aim to stimulate regenerative repair proc- esses. Tissue engineered wound products combine cells, biomaterials, and bioactive fac- tors delivered to the wound site to support and speed cellular healing events. Despite the scientific basis for these designs, clinical results have been disappointing. The adult human body does not fully regenerate skin, so repair of tissue is accom- panied by the formation of a scar, which is inferior to the original tissue in strength and function. Regeneration of highly developed morphologically similar structure to the damaged tissue requires an extended period of time and significant energy expenditure, leaving the body vulnerable to the environment. Scarless healing does occur in fetal

12

wounds, however, and up to one year of age children can regenerate whole fingertips with morphologically complete structures including nail beds if these are not covered or sutured [Illingworth, 1974; Douglas, 1972]. Reepithelialization of superficial wounds to the epidermis, hematopoeisis after blood loss, and bone healing can also be classified as true regenerative events [Becker and Selden, 1985]. In numerous clinical applications, therapeutic strategies to activate latent regenerative abilities are preferred over those that lead to scarring or require transplantation of tissue.

Electric fields Electric fields have been shown to play a key role in developmental morphogene- sis and regeneration. Endogenous fields exist in developing organisms and have particu- lar association with morphogenetic events such as limb formation and left-right asymme- try formation [Levin, 2003]. In regenerating species of salamanders, injury currents be- gin similarly to those of non-regenerating species, but potentials gradually decrease until around day 9 and then shift polarity until they reach -30 mV [Becker, 1972]. This is pos- sible because of the conductive nature of pond water in contact with wounds and the ab- sence of resistive stratum corneum. Since this appears to be a fundamental difference between regenerating and non-regenerating animals, several experiments followed that verified that manipulation of injury currents has the potential to initiate regeneration. With a simple method of supplying current through a battery constructed of bimetallic wires, investigators were able to induce artificial re-growth of a limb in a non- regenerating species of frog [Smith, 1967]. Using a similar implanted bimetallic battery,

13

researchers claimed to produce partial limb regeneration in a mammal [Becker and Spadaro, 1972]. It appears that careful control of the electrical environment in tissue can determine the type of healing response, with some types of EFs stimulating regeneration and morphogenesis and others leading to scar formation. This is not a new theory. In fact, techniques to augment wound healing with electrical stimulation (ES) were in successful use long before the discovery of injury currents. The first applications of ES in wound healing may date back to the 1600s when gold leaf was applied topically to accelerate healing [Dayton and Palladino, 1989]. After the publication of the Flexner Commission report in 1910 [Flexner, 1910], which stated that the use of ES was not scientifically based, most research in this field was abandoned. With the rediscovery of the current of injury and its possible implication in healing in the 1980s, it became more acceptable to use and study ES. Recent reports on best practices for pressure ulcers and diabetic wounds have selected ES as one of the few adjunctive therapies with documented suc- cess, although there are no clinical devices solely dedicated to ES for wound healing [Bergstrom N, 1994]. However, its use in wound healing has not reached the degree of success or acceptance that it has in orthopedic applications for the promotion of fracture unions [Anglen, 2003]. This is in part due to a lack of comprehension of basic mecha- nisms and a very wide variety of techniques that have been developed based on empirical evidence. Direct Current. The most commonly used form of ES specifically for skin wound healing is direct current (DC) stimulation, which encompasses all methods using unidi- rectional current for at least one second or longer. Typical protocols for DC stimulation

14

involve an electrode wrapped in saline soaked gauze in direct contact with the wound, and another electrode on the skin near the wound site [Wolcott et al., 1969; Carley and Wainapel, 1985]. Initially the electrode in contact with the wound is of negative polarity, supplying continuous current ranging from 20 to 100 µA. The stimulation is continued for two-hour sessions repeated 2 to 3 times daily. Many protocols recommend reversing the polarity after a few days, and reversing again every few days or when a plateau in healing is reached. In animal studies, positive DC has been shown to attract inflammatory cells to the wound site [Carey and Lepley, 1962]. Negative current applied to rabbit ear wounds produced 25% acceleration in healing of full-thickness rabbit ear wounds, as well as denser, stronger connective tissue [Assimacopoulos, 1968b]. The rate of epithelialization and collagen synthesis in pig wounds is also increased in DC fields [Alvarez et al., 1983]. Stimulation with an occlusive dressing that provides DC stimulation increased healing rates of full thickness rabbit dorsal wounds as well as increasing regenerative epitheliali- zation relative to scarring contraction [Jennings, 2003]. In human clinical trials, DC stimulation has been shown to produce healing in pa- tients whose chronic ulcers have resisted conventional treatment for several years [Assi- macopoulos, 1968a]. An antimicrobial effect has been observed in addition to improved healing in case studies and controlled clinical trials [Wolcott et al., 1969; Gault and Gat- ens, 1976; Carley and Wainapel, 1985]. Additionally, decreases in perceived pain and more flexible scar tissue have been reported [Barron, 1985]. Pulsed Current. A variation of DC current uses short pulses of unidirectional cur- rent, but is not technically classified as DC. In low intensity pulsed current, 30 to 40 mA

15

of current is pulsed at 128 Hz for 4 hour sessions with 8 hours of rest. In high voltage pulsed current (HVPC) around 200 V is pulsed at around 100 Hz through the active elec- trode wrapped in saline-soaked gauze in direct contact with the wound, with the disper- sive electrode placed on the thigh [Nelson et al., 1999]. Similarly to DC stimulation, ini- tially the active electrode is negative and reversals of polarity are recommended when healing plateaus are observed. Because of the short duration of pulses of high voltage, the root mean square average (RMS A ), and thus the actual charge per second is very low, at around 500 µA [Nelson et al., 1999]. An advantage of this method is that HVPC stimulators are FDA approved for use in pain management, muscle stimulation, and cir- culatory increases. Though their use for wound healing is not approved, clinicians may use them off-label at their discretion. Numerous case studies, randomized clinical trials, and retrospective studies have shown that healing of chronic ulcers improves with treatment by pulsed currents [Alon, 1986; Kloth and Feedar, 1988; Houghton et al., 2003; Gentzkow and Miller, 1991]. This treatment has advantages for treatment of recalcitrant ulcers [Westerhof and Bos, 1983], and in patients with diabetic neuropathy [Peters et al., 1998]. Alterations in collagen deposition patterns in diabetic mice have been noted under pulsed ES treatment [Thawer and Houghton, 2001]. Laser Doppler flowmetry measurements and transcutaneous oxy- gen measurements have been used to demonstrate that HVPC used for wound treatment can also reverse ischemia through vasodilation and angiogenic responses [Goldman et al., 2004]. Alternating Current. Alternating current changes polarity with reference to the zero baseline at least once every second, and eliminates the need for attention to initial

Full document contains 224 pages
Abstract: In the normal process of wound healing, small electric fields are generated at the wound site due to a disturbance of the potential difference maintained by a continuous epithelium. Although the roles of EFs in healing are unclear, physiological strength fields promote such processes as cellular migration and protein synthesis. Furthermore, the application of exogenous fields in various forms improves repair of chronic wounds. In these studies, the transcriptional response of dermal cells exposed to physiological strength electric fields for one hour is measured using microarrays, followed by the use of bioinformatics tools to interpret the implication of results in wound healing. The identification of 555 significantly increased transcripts and 550 significantly decreased transcripts in microvascular endothelial cells led to the recognition of activity within mitogen activated protein kinase, transforming growth factor-beta, and apoptotic signaling cascades. Transcripts related to TGF-β, MAPK, and apoptotic pathways were also identified within the 164 increased and 302 decreased transcripts in dermal fibroblasts exposed to EFs. In epidermal keratinocytes, 161 transcripts were significantly increased while 245 were significantly decreased. There were 18 genes that were regulated above a two-fold level but not reaching statistical significance in keratinocytes, including primarily chemokines and inflammatory response genes. Several genes were selected to confirm results using real time RT-PCR and to elucidate the field- and time-dependent characteristics of gene expression in EFs. The expression of genes related to growth, inflammation, and adhesion reinforces the impact of EFs in the process of repair.