Wound healing using a collagen matrix: Effect of DC electrical stimulation

Michael G . Dunn,* Charles J. Doillon," Richard A. Berg,+ Robert M. Olson,s and Frederick H. Silver* Biomaterials Center, Departments of Pathology,* Biochemistry, ' and Surgery * and Graduate Program in Biomedical Engineering, Uniuersity of Medicine and Dentistry of Nmu Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Rapid fibroblast ingrowth and collagen de- position occurs in a reconstituted type I collagen matrix that is implanted on full- thickness excised animal dermal wounds. The purpose of this study is to evaluate the effects of direct current stimulation on der- mal fibroblast ingrowth using carbon fiber electrodes incorporated into a collagen sponge matrix. Preliminary results suggest that fibroblast ingrowth and collagen fi-

ber alignment are increased in collagen sponges stimulated with direct currents between 20 and 100 PA. Maximum fibro- blast ingrowth into the collagen sponge is observed near the cathode at a current of 100 PA. These results suggest that elec- trical Stimulation combined with a collagen matrix may be a method to enhance the healing of chronic dermal wounds.

INTRODUCTION

Wound repair involves cellular events such as cell migration, replication, synthesis and deposition of new connective tissue, remodeling, and epider- mal cell migration over dermal repair tissue. Many studies'-7 suggest that these events may be influenced by endogenous and exogenous electric or magnetic fields in both soft and hard tissue.

Reconstituted collagenous matrices have been shown to delay contraction of dermal animal wounds,' coordinate the deposition of organized dermal repair tissue,' and act as a support for cells in cu1ture.l' Collagen matrices containing hyaluronic acid and fibronectin increase fibroblast migration and proliferation and improve the deposition of organized repair tissue in both an animal model and in cell culture."""

Electrical stimulation using direct electrical currents or induced voltages and currents has been shown to affect wound healir~g.l~-'~ The results of dermal wound healing studies using direct currents are summarized in Table I. The accelerating effects of electrical stimulation on wound healing may be a consequence of: (a) modification of endogenous bioelectricity, (b) activation or attraction of inflammatory cells, (c) the presence of electrode

Address correspondence to: Frederick H. Silver, Ph.D., Biomaterials Center, De- partment of Pathology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854.

J. Biomed. Mater. Res.: Applied Biomaterials, Vol. 22, No. A2, 191-206 (1988) 0 1988 John Wiley & Sons, Inc. CCC 0021-9304/88/A20191-16$04.00

TABL

E I

Ele

ctri

cal S

timul

atio

n of

Sof

t T

issu

e W

ound

s

Ele

ctro

de

Ani

mal

C

urre

nt

on W

ound

M

odel

R

efer

ence

R

esul

ts

100

PA

1000

pA

20

pA

30

0 pA

10

- 20

mA

50

- 10

0 /.L

A

200-

1000

/L

A 20

0-10

00 /

.LA

Cat

hode

C

atho

de

Cat

hode

A

node

C

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de,

anod

e C

atho

de

Cat

hode

, an

ode

Cat

hode

, an

ode

Rab

bit d

erm

al w

ound

R

abbi

t fu

ll-th

ickn

ess

wou

nd

Rab

bit

derm

al i

ncis

ion

Pig

derm

al w

ound

D

iabe

tic m

ice

derm

al w

ound

H

uman

ski

n ul

cer

Hum

an s

kin

ulce

r H

uman

ski

n ul

cer

Ten

sile

str

engt

h in

crea

sed

25%

B

acte

ria c

ount

dec

reas

ed

Ten

sile

str

engt

h in

crea

sed

53%

In

crea

se in

col

lage

n sy

nthe

sis,

reep

ithel

ializ

atio

n H

ealin

g si

mila

r to

nond

iabe

tic m

ice

Ulc

ers

heal

ed c

ompl

etel

y 40

% o

f ul

cers

hea

led

com

plet

ely

59%

of

ulce

rs h

eale

d co

mpl

etel

y

16

17

18

19

20

21

22

23

W c Z Z

ENHANCED WOUND HEALING IN A COLLAGEN MATRIX 193

breakdown products, (d) attraction of connective tissue cells, (e) enhanced cell replication, (f) enhanced cellular biosynthesis, or (g) inhibition of prolif- eration of infectious microorganisms. Circumstantial evidence suggests that direct current stimulation appears to enhance repair of wounds.

The purpose of this study is to evaluate the effects of direct current stimu- lation on the repair of guinea pig dermal wounds using a well characterized collagen sponge model.9-'* Electrical stimulation is achieved using carbon fiber electrodes embedded in the collagen sponge during fabrication. Direct currents of 0 to 100 p A are investigated as well as a comparison of the effects of anodic and cathodic stimulation. Preliminary results suggest that maxi- mum ingrowth occurs by cathodic stimulation using a current of 100 PA.

MATERIALS AND METHODS

Collagen sponge preparation

Collagen sponges containing carbon fiber electrodes were prepared using the following procedures. Pairs of small holes were punched on opposite sides of a styrene plastic tray (Clear-View Packing, Inc., Clifton, NJ). The dimensions of the tray used to form collagen sponges were 12 x 8 x 1 cm. Trays were placed on a piece of plywood and each hole on the tray was lined up with a nail on the plywood. The nails were spaced every 1.0 cm on the plywood as diagrammed in Figure 1. A twisted braided tow of carbon fiber containing 3,000 individual filaments of approximately 7 pm in di- ameter (Hercules, Wilmington, DE) was threaded onto a large needle, passed through distilled water, and threaded through the holes in the tray (see Fig. 1). When the entire tray was threaded, the fiber tow was pulled taut and tacked down. This process resulted in parallel carbon fibers sepa- rated by 1.0 cm in the plastic tray.

Insoluble collagen type I from fresh uncured bovine corium was ob- tained from Devro, Inc. (Somerville, NJ). The corium was limed, fragmented, swollen in acid, precipitated, washed with distilled water and isopro- panol, lyophilized, and stored at -30°C. 24 Collagen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis as typical of type I collagen without noncollagenous protein ~ontamination.~~

A 1% (W/V) dispersion of type I collagen in dilute HCL pH 3.0 was prepared and mixed for 2 min at room temperature at high speed using a blender as previously described.26 Seventy milliliters of the deaerated dis- persion was poured into each plastic tray and air bubbles were removed by placing the tray in an oven at a vacuum of less than 0.4 millitorr at room temperature. The plastic tray containing the dispersion was cooled to -30°C for at least 3 h and the frozen dispersion was lyophilized until dry.

The sponge was removed from the tray and crosslinked by severe dehy- dration using a process termed dehydrothermal crosslinking (DHT) at a temperature of 110°C and vacuum of less than 0.4 millitorr for 3 days. The

194 DUNN ET AL.

I STYRENE TRAY

Figure 1. Diagram showing the method used to fabricate the collagen sponge containing carbon fiber electrodes. Carbon fiber was pulled through the holes in the plastic tray, wrapped around the nails, pulled taut, and fixed in place with tacks. A 1% (W/W) collagen dispersion in the HCl pH 3 was then poured into the tray, frozen, freeze-dried, and crosslinked as described in Methods.

sponge was further crosslinked by placing it in a 1% (W/V) solution of cyanamide for 24 h at pH 7.2,26 and then it was washed thoroughly with at least three changes of distilled water over a 24-h period. The final step was to freeze and then lyophilize the sponge.

A 2-cm width was cut from the 3-mm-thick sponge, and excess carbon fibers were dissected off the strip under a dissection microscope and blown off with air. From this strip, a 2 x 2 cm piece of sponge was cut, with free carbon fiber ends of about 1.0 cm length outside of the sponge. Using a crimping tool and solderless connectors, both of the two free carbon ends were connected to a 5-cm piece of insulated wire (with the ends stripped). Male/female connectors were soldered to the other end of these wires. A thin coating of medical grade silicone (Dow-Corning) was spread over the "air side" of the sponge to act as a moisture barrier for the graft and to provide a tough surface through which anchoring sutures could be applied (see Fig. 2). The silicone layer was allowed to dry overnight or until all the acetic acid was vaporized. Silicone was also used to electrically insulate the crimped connectors at the wirelcarbon fiber interface. Sponges and con- nectors were sealed in a plastic bag and sterilized by exposure to 2.5 Mrad of gamma radiation,

ENHANCED WOUND HEALING IN A COLLAGEN MATIUX 195

GUINEA

COLLAGEN SPONGE

CARBON FIBER ELECTRODES

WIRE

CONNECTORS

Figure 2. Illustration of method used to implant collagen sponge with carbon fiber electrodes on full-thickness excised wound on the back of the guinea pig. The sponge was sutured onto the excision and the circuitry was zonnected to the appropriate male or female connector. In the first model de- vice the cathode and anode were in one sponge while in the second model device they were in different sponges.

DC stimulator circuit construction

The stimulation circuit was designed to deliver a constant, adjustable direct electric current (DC).27 The current was set to 20 or 100 pA via a potentiometer, and was unaffected by changes in the load resistance based on experimental measurements on animals. The transistors were Sylvania ECG 457, or NTE 457 JFETs (field effect transistors). The power source was a 9-V Duracell transistor battery, the load resistor was 1 KR, and the poten- tiometer had a maximum value of 100 KQ. The circuit components were taped onto the power source. The stimulator circuit and power source was approximately the size of a standard 9-V battery. Insulated wire (28 AWG) and standard solder (60% tin, 40% lead) were used for the connections. The cathode lead wire was connected to the negative side of the battery and the anode connected to the transistors.

Animal studies: First experimental model

Female (500-1,000 g) Albino Hartley guinea pigs (Perfection Breeders, Douglassville, PA) were depilated 1 day before surgery by applying a depila-

196 DUNN ET AL.

tory cream (Nair). The day of surgery guinea pigs were anesthesized by injection of Ketamine (Vetalar, Parke Davis) at 35 p g / g body weight and Xylazine (Rompun, Bayret) at 5 p g / g body weight. The shaved and de- pilated back of the guinea pig was disinfected with several washes of alcohol and Povidone. Surgery was performed under sterile conditions. A full thick- ness, 2 X 2 cm square of skin including the panniculus carnosus was re- moved from one side of the back via careful dissection with a scalpel, and hemostasis was achieved. The collagen/carbon sponge was wetted with ster- ile saline for 10 min and then placed into the wound, with the silicone side up. Catgut stitches were applied to secure the sponge in place. The pro- cedure was repeated for a duplicate wound on the opposite side of the spine. One wound was randomly chosen to receive electrical stimulation, and the maleifemale connectors at the end of the electrode wires were attached to the circuit; one wound served as an unstimulated control. The control device consisted of a collagen sponge containing carbon fibers that was connected to a device without a battery. The wounds were wrapped with sterile gauze, then the circuitry was fixed on the back with Elastikon elastic tape (Johnson & Johnson).

Currents of 20 and 100 pA were studied in this model. The current was checked daily by measuring the voltage across the 1 KR resistor via test leads. Current levels remained unchanged during the experiment.

The current density was estimated to be in the range of 1.5 to 15 pA/cm’ and 8 to 80 pA/cm2 for current levels of 20 and 100 pA, respectively. The higher value of current density for each current level was obtained by as- suming that the carbon fibers act as a solid electrode 1 mm in radius while the lower value for each current density was obtained by assuming that the current was delivered from the surface of 3,000 individual carbon fibers 7 pm in diameter.

Animal studies: Second experimental model

In the second experimental model, two collagen sponges were implanted in the same manner as described above for the first experimental model, however each sponge contained only one active electrode; the outer elec- trode of the two in the sponge was always activated. The anode and cathode were randomly chosen to be in the left or right sponge and in this instance there was no control sponge on the animal. The wounds were wrapped with Elastikon tape as described above for the first generation device.

Six days after implantation of a device in the first or second experimental model the guinea pig was anesthesized, and the bandages were carefully removed. The distance between the electrodes was measured. The current through the stimulated wound was determined daily and at sacrifice by measuring the voltage across the 1 KIR resistor with a VoltiAmplOhm meter (Radio Shack). Both of the sponges (control and stimulated) were removed from the animal by dissecting around the wound and the material was placed in Carson’s fixative. The carbon fiber electrodes and silicone layer were

ENHANCED WOUND HEALING IN A COLLAGEN MATRIX 197

carefully removed (and saved for SEM) and the collagen sponge was divided into four equal squares. The specimens were marked cathode 1 ,2 and anode 1,2 where sample 1 was closest to the circuit and sample 2 was from the distal portion of collagen sponge. These four regions were then prepared for histological evaluation.

Currents of 20 and 100 pA were studied in this model and remained unchanged during the course of the experiments.

Light microscopy

Fixed specimens were processed using routine paraffin embedding and tissue sectioning procedures. Tissue sections were stained with hematoxylin and eosin (H&E) or picro-Sirius red.2R

The extent of wound tissue deposition in the collagen sponge was quali- tatively determined by examining H&E stained sections under a Leitz Laborlux 12 light microscope. Neovascularization, fibroblast proliferation, collagen deposition, and the inflammatory and foreign body responses were noted. Light micrographs were taken with a Leitz 35 mm camera, with the long axis of the micrograph corresponding to the direction of the electric field between the electrodes, in order to determine the number of fibroblasts/ mm2. Numbers of fibroblasts reported in the results were based on the means of at least three counts per slide per experiment. Cells were considered fibroblasts if they had an elongated nucleus and elongated shape. For the electrically stimulated collagen sponges, fibroblasts were counted in the sponge at a magnification of 100 in the region between the electrodes near the cathode (a) and near the anode (b).

Statistical analysis

Statistical analysis and graphics were performed on an IBM-PC XT using STATGRAPHICS software (Statistical Graphics Corp., Rockville, MD), includ- ing analysis of variance and plotting of 95% confidence level least significant differences.

RESULTS

These studies were directed at evaluating the effect of low direct current electrical stimulation on repair tissue in a well characterized skin excision model. In this model the collagen sponge is infiltrated by fibroblasts and capillaries resulting in the deposition of organized collagen fibers and for- mation of new vasculature as described p r e v i o ~ t s l y . ~ , ~ ~ , ~ ~

Controls consisted of collagen sponges containing carbon fibers that were connected to a wire without a circuit. Control devices were implanted on excised dermal wounds. Sponges in control devices were infiltrated by day 6

198 DUNN ET AL.

postimplantation with unoriented fibroblasts (see Fig. 3). Tissue ingrowth was often accompanied by a slight foreign body reaction within the collagen sponge. The foreign body reaction consisted of accumulation of inflam- matory cells. The number of fibroblasts/unit area at day 6 was observed to be 125 * 18 (see Table 11) for unstimulated sponges.

The electrodes (cathode and anode) in the first animal model were sepa- rated by l cm. At a current of 20 pA the repair tissue within the collagen sponge appeared visually to be denser than in the control. Dense ingrowth of fibroblasts and capillaries was noted near the anode. No evidence of a foreign body reaction was observed. The number of fibroblasts ranged from 130 * 14 to 156 ? 13 at the cathode and anode, respectively (see Table 11).

At a current of 100 pA using the first animal model, anode and cathode regions appeared distinctly dissimilar. The cathode region was well in- filtrated with oriented, mature fibroblasts. However, the anode region was well infiltrated by neutrophils. Fibroblast infiltration was increased sig- nificantly (at a 95% confidence level) at the cathode region compared with controls.

A second model in which the electrodes were in separate sponges (dis- tance between electrodes = 3 cm) was studied at a current of 100 pA to evaluate the effects of the anode and cathode independently. The cathode region was similar in appearance to the cathode region in the first model device stimulated with 100 pA, but the sponge had dense oriented fibro- blasts (see Fig. 4). In comparison, the sponge containing the anode con- tained numerous neutrophils (see Fig. 5) which were grossly visible as pus at sacrifice at 6 days. Fibroblast infiltration was maximized at the cathode (183 2 43) and minimized at the anode (94 ? 4) compared to controls. Figure 6 summarizes the fibroblast ingrowth results.

DISCUSSION

There have been several studies reported in the literature (see Table I) on the use of direct currents to stimulate dermal wound healing. These studies used currents in the range of 10 to 1000 pA and cathodic and/or anodic stimulation. A variety of electrode types were used including stainless s t ~ e l , ~ ~ , ~ ~ copper, 17,22 and silver-nylon. l9 The results of these studies appear to be dependent on the current level and type of metallic electrode employed.

The present study was conducted using a well characterized guinea pig full-thickness excised wound model and a stimulated collagen sponge. Pre- vious studies9~11,25,2Y have characterized the physical structure of the collagen sponge as well as its wound-healing properties. We have used carbon fibers as the electrodes to eliminate the influence of metal ions on inflammation and granulation tissue deposition. Carbon fibers are inert and do not release metallic ions; however, the anode may oxidize at high current densities. No electrode corrosion was evident based on scanning electron microscopy. Low

Figure 3. Light micrograph of unstimulated collagen sponge (arrow) con- taining carbon fibers 6 days postimplantation on a guinea pig excised dermal wound. Fibroblast nuciei are seen as round dark spots scattered throughout micrograph separated by thick collagen fibers. Bar = 50 pm.

200 DUNN ET AL.

Figure 4. Light micrograph of cathode region with 100 p A stimulated col- lagen sponge containing carbon fibers at 6 days postimplantation on an ex- cised guinea pig dermal wound. The direction of the electric field (E) was approximately parallel to the axis of the collagen fibers (arrow). The carbon fibers are removed during histological preparation and are perpendicular to the plane of the section. Bar = 50 pm.

Figure 5. Light micrograph of the anode region (A) in the collagen sponge described in Figure 4. Polymorphonuclear leukocytes and monocytes are present. The carbon fibers are perpendicular to the plane of the section. Bar = 50 pm.

ENHANCED WOUND HEALING IN A COLLAGEN MATRIX

0

4 -I m 0

ii LL 0 140- (L W m

k 180

160

f 120- z

203

-

-

1 !

Figure 6 . Plot of number of fibroblasts per 1 mm2 versus direct current stimulation in microamps and electrode polarity for implanted collagen sponges containing carbon fibers 6 days postwounding. Means and standard deviations are shown for pooled data for first and second experimental de- vices. Error bars show standard deviations obtained by pooling data from first and second experimental models for observations at the anode and cathode. At a 95% confidence level fibroblast ingrowth at the cathode at 20 pA is significantly greater than the control. At 100 p A the number of fibroblasts at the anode is significantly less than the control.

currents (up to 100 p A ) were employed since they could be delivered with a battery pack for periods of up to 7 days.

Experiments were conducted with the anode and cathode close together (first model) and separated (second model) to evaluate the possibility of synergism between the effects of the two electrodes. For both devices the fibroblast density near the cathode was significantly higher at a 100 p A current than that observed in controls. In the second model the number of fibroblasts at the anode was significantly lower than that of controls. These results suggest that fibroblast ingrowth into the collagen sponge is enhanced by cathodic stimulation and that degradation products formed at the anode may result in decreased numbers of fibroblasts.

A clear trend is observed if the number of fibroblasts within the collagen sponge are plotted versus stimulation current and polarity (see Fig. 6 ) . This trend suggests that 100 p A cathodic stimulation results in about 41% more fibroblasts in the collagen sponge after 6 days. Histological evidence (see Fig. 4) suggests that fibroblasts near the cathode are oriented parallel to the direction of the electric field and lay down collagen fibers that are aligned in direction of the field. No differences are observed in the number of fibroblasts in the sponge that are found near the circuit connector compared

204 DUNN ET AL.

with areas away from the connector. In comparison, the anode at 100 p A appears to attract inflammatory cells possibly due to an electrophoretic effect or release of electrode degradation products.

Increased numbers of fibroblasts seen in cathodic stimulated collagen sponges may reflect the effect of electrical current on cell migration or may be a result of changes at the electrodes of factors such as oxygen tension and pH. Water molecules may be protonated at the anode and deprotonated at the cathode and lead to pH changes depending on the current density.32 Brighton et al.33 attributed the deposition of bone near the cathode to de- creased oxygen tension.

The presence of a collagen matrix has been shown to be chemotatic to fibroblasts in wound tissue. Increased numbers of fibroblasts observed in the presence of direct currents leads to the deposition of aligned collagen indicative of the remodeling phase of wound healing. Rapid remodeling of wound tissue is required to accelerate chronic wound repair.

In conclusion, the results of this study indicate fibroblast migration and collagen fiber alignment are enhanced in dermal wounds stimulated with direct currents between 20 and 100 p A . The anode was observed to attract inflammatory cells and therefore must be removed from the wound site in order to prevent tissue destruction. Cathodic stimulation using a collagen sponge and inert carbon electrodes appears promising in searching for methods to enhance healing of chronic dermal wounds.

The authors would like to acknowledge Ethicon, Inc. for partial support for this project. The authors thank Dr. Harry Stumpf for implant histological analyses.

References 1.

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33.

Received October 8, 1987 Accepted March 31, 1988