The

Bioelectric
BY ROBERT 0.

Factors Regeneration
BECKER,
State Veterans

in Amphibian-Limb
*
SYRACUSE,
of New

M.D.,
University

NEW
York, Hospital,

YORK
Upstate Syracuse Me4uxil Center,

From

the

Department and

of Orthopaedics, Orthopaedw Section,

Administration

The
regeneration

replacement
is an

of lost ability the the shared,

body

parts

by

a specific extent, the the

growth

process things.

known

as

to a varying

by all living ability regeneration

In gen-

eral, as restricted. only,

erated

one In

follows man,

evolutionary sequence, process is limited to

becomes increasingly of certain tissues

notably skin and bone. provided the periosteal bone can heal Although there is little

In a child the diaphysis tube is intact, whereas of the doubt that missing factors

of a long bone can be regenin an adult a fracture of a with functional this osseous subsequent represent the osseous vascularity, healing re-differtrue tissue healing tissue is of such as nutrition,

long tissue.

by replacement all play important processes of cellular produce a replica of bone human

portion

and immobilization process, the basic

entiation regeneration.

roles in supporting de-differentiation and of the missing portion healing tissues

which

mechanisms

This process in most other gap. It specific control

is in marked contrast to in which fibrous connective that the in other healing

used to bridge the healing must entail tissue factors It

operative
process tion. in

is evident, therefore, factors not operating this type of tissue themselves that the
may furnish

process of regenerative less adequate types these mechanisms of the of human by the phylum. regenerative regeneracomplexity The specific

healing.

To

adequately, controlling

must be identified and is reasonable to expect

for

controlled. same general and that ability, within the

a basis for

are

all

types the

of regeneration, peak of regenerative is present

knowledge
a study

another

vertebrate

Fortunately,

as judged vertebrate

of the dela, which lower

structures or salamanders, the higher in extremities.

regenerated,

uro-

are present-day vertebrates have structure the Nevertheless,

examples been derived. and larval-stage

of the basic vertebrate Their fore and hind to salamander of thirty required smaller stimulated in the mammals few years, can be the human is capable

type from limbs are upper and of regen-

equivalent

anatomical

complexity

erating a perfect replica of one of its limbs in a period adult salamander is similarly capable, although the longer and the regenerated extremity may be slightly 1). This ally and the resisted observations new
process tion
*

to forty days. The time is somewhat than normal (Fig. study regenerative for literprocess

process

is so dramatically

evident basic controffing

that

it has factors in past now

centuries

t.

However,

the

factors operating investigation until have been

to reduce recently. made

Singer

the process Within the the problem

have successfully some significant approached of the from regenerative the amputatype
The

and

aspect. In one
upon

line of inquiry the presence

23,

demonstrated

of a critical Meeting
Miami of the Beach,

amount
is not Florida,
not Orthopaedic

the dependence of nerve tissue

dependent
Research

within
on

stump
Read

He

showed
Surgeons,

that this effect

the
and

of nerve
American their legs-

at

the

Combined

Society

Academy how comes hoped that

VOL. 43-A.

of Orthopaedic

t Spallanzini (Prodromo,
it to pass, they may
5, JULY

that other land animals acquire them by some

8, 1961. 1768): But if the above mentioned animals

January

useful

are

endowed

with

the

same

recover power?

Is it to be
643

disposition?

NO.

1961

644 tissue present. is reduced by either

i)Ut

R.

0.

BECKER

In the salamander, proximal nerve

if the sectioning, the into

normal then

amount of peripheral regeneration will not

nerve
occur

tissue until

the
sufficient

missing nerves in amount,

grow into is grafted can nuder occur these

area or until neural tissue of any type, the area. Under certain specific circumof nerves (parabiotic twin-

stances, ning 27), possibly general,

limb

limb regeneration indicating that

iii limbs devoid circumstances

the

neural

contribution have, process

nerve

may in of
have

be ancillary rather than primary. left little doubt that some neuial
regeneration, the specific responsible

Although these experiments factor is necessary for the

factors associated with the

yet

to be identified. I reported
existence of

Some recent investigations of this neural factor The

.

furnished
direct-current

a possible
electricity

clue

to the identity in living organintermit-

isms
tently

was

first since

observed then. This

by
type

Galvani
of

in 1792, and has received electrical activity, designated

attention
as

the

bwelectric

Fin. An example of the regenerative

one week forelimb amputated

1
The other animal on
specimen

the right forelimb removed at the same level fifteen weeks before. The regenerate is somewhat the normal limb, but the completeness of restoration is evident. These forelimhs to the human upper extremity, containing a humerus, radius, ulna, and carpal hones, iii addition to th.e homologous soft structures. The regenerate restores almost completely the aitatonucal complexity and is a functional organ. It will be noted that the long bones, and, particularly, the spine of the specimen with the week-old amputation, are less distinct and appear somewhat less dense than the same structures of the other animal. This observation has been made repeatedly and is not due to any difference in technique. its significance is unknown at l)resent.
THE JOURNAL OF BONE AND JOINT SURGERY

ability of the salamander. prior to the photograph. The

the left h:ul had the left smaller than are homologous

BIOELECTRIC

FACTORS

IN

AMPHIBIAN-LIMB

REGENERATION

645 from the more field

field familiar

or

the

direct-current potential, The

field, activity

must of the

be

carefully nerves and

distinguished muscles. The

action-potential
organisms.

bioelectric

is a steady-state
of living

measured as direct-current action potentials, on the other depolarization by electrical

longitudinally

potentials, on the surface hand, consist of the transthe the and nerve they do fiber since this
.

mission Although
actual

of
flow

pulses of the pulses

of an

membrane are detected

current

along means,
in

or muscle not involve Since the type was action the

fiber. the techpoten-

electrical

niques

of measurement

of action a prime studied work The on concept

potentials

are

simpler,

tials apparently are has been preferentially Much

twenty years

means of data transmission, by most investigators. the direct-current arrived at then field was no more compared assumed potentiaLs that the structural with the to be the

of activity done field ten to con-

of

the ago.

bioelectric complexity caudad portion

sisted
positive animal.

of a simple
polarity

of the

electrical dipole with cephalad portion, of the potential was

than a of the activity

The

source

total

electrical

of all of the

between

cells of the organism. the nose and the tail

The usual (cephalad-caudad)

measurements done at that or between two other many potentials observations and various in animals
,

time were arbitrary were made, biological and ovulato bethe

points only. Despite this indicating some relationship activities such as: fluid

amorphous between in plants

concept, these

transport

91#{176},anesthesia

tion 19, hypnosis 20 and schizophrenia 21 in man. In particular, there appeared be correlations with growth processes 6,15,16#{149} In recent years the relationship tween direct currents and growth were investigated by artificially applying
currents to

the
23

mato
were

plants

organism. Acceleration and, conversely, complete to follow application appeared worthwhile relationship animal

of the rate regression

of regenerative of malignant entire

growth in totumors in mice 12 polarity. problem of the was posi-

reported It therefore

of currents of appropriate to re-evaluate the to growth processes. in view of its regenerative be the moist done with

bioelectric field and its chosen as the experimental tion in the vertebrate mitted direct-current Utilizing lines on
complete

The salamander ability and its

phylum. Furthermore, measurements to

skin of these less technical

animals perdifficulty. a

the techniques of equipotential the surface of the animal which field plot field, field of the bioelectric the interested is a controlling

line plotting connect all

(determining the imaginary points of equal potential),

the the

electrical bioelectric

field can be made. (For further concepts of reader is referred to references 14 and 18.). If factor in growth processes, such control should the on normal in the a determination field plot. of the simple sinks, was found Therefore, the field configuradipole, and a commultiple in

be indicated by initial observations tion plex

negative

some change from were concentrated Rather multiple

sources

in the intact animal. field was found with

areas, or electron

than finding the expected positive areas, or electron (Fig. 2). This pattern

uniformly

over
spatial

fifty

individuals
of

configuration

of several different species. It of the bioelectric field coincided

central nervous system.

became apparent with the gross areas

that the anatomical sinks)

arrangement coincided

the

The

positive

(electron

with the three major cellular accumulations of the neuraxis (cranial, brachial, and lumbar enlargements). The negative areas (electron sources) correlated with the terminations of the major peripheral nerve outflows from these same areas. It was therefore postulated generated within the cellular aggregates with a completion of the circuit made forms, through the environment). This It was
VOL. 43-A.

that the direct-current potentials were and distributed by the peripheral nerves, through the soft tissues (or in the aquatic hypothesis was tested in a number of ways. to the cord) of the nerves to one ex-

noted
NO. 5,

that
JULY

complete
1961

section

(adjacent

646 tremity zero. tion that that By produced applying a prompt the Hall drop effect

R.

0.

BECKER

of the

potential

along

the

extremity involving the

to nearly interacshown and

(a physical

measurement

between a steady there was actual this flow ceased

magnetic field flow of electrical after nerve

and a moving direct charges longitudinally Finally, an

current) it was in an extremity device,

section.

analogue

or model,

of the central nervous system was constructed and was found to produce the same field plot and to show the same behavior as the living prototype. It must be emphasized, however, that although these experiments show a distinct relationship between in the the the central central nervous has not nervous system been system and responsible identified. At
-tO

the for this

bioelectric field, the specific the generation and transmission time we cannot say whether

tissue of the

currents

FIG. The
to

2 with
body

bioelectnc
central outline

field,
nervous drawing

previous
system. on the

dipole
left

concept
the

compared
total

organized
potential as

field

concept
by

as related
previous

the
The

indicates

measured

investigators (tip of nose reference fluid medium). The equipotential postulated. Such field determinations field plot I expected to find when
The middle outline drawing

electrode, tip of tail recording electrode, lines as drawn represent the theoretical were never reported in the literature. the project was first begun.
the field plot that was actually found

animal immersed dipole field that This is the type

(using the two

in
was

of
elec-

indicates

trode tion). about

equipotential line-plotting technique on an animal kept moist, but not immersed, in soluThe numerical values inserted along the extremities are typical ones found on an animal to recover from anesthesia when the head sink areas are used as the reference electrode site and the recording electrode is located at the other appropriate sites. The outline drawing at the right represents the gross arrangements of the central nervous system in the salamander. It will be noted that the neuraxis contains three major cellular accumulations, with major peripheral nerve trunks extending distally from each. The correspondence between the cellular accumulations and the positive sink areas, and between the nerve-trunk terminations and the negative sources, is apparent.

neurons Schwann These activity,

system. It

themselves, cells-are observations generated

appeared

the the within

that,

neuropil, important on the and

as such,

or the structures. field transmitted

it could

supporting indicate by elements that

elements-the it is a highly of the central

glia
organized

and

bioelectric

nervous

mission

and

control

system.

This

concept

function permits

as a very primitive a restudy of many

data translife processes,

including growth, which are obviously under systems have not yet been identified. In this regeneration, perhaps the highest expression

precise control but for which control paper I shall be concerned with limb of organized growth in the adult
THE
JOURNAL OF BONE AND JOINT

SURGERY

BIOELECTRIC

FACTORS

IN

AMPHIBIAN-LIMB

REGENERATION

647

animal. The the bioelectric tion that

demonstrated field would

relationship between the appear to furnish a logical tissue Singer neural field results to regeneration. observed was

central basis

nervous system and for Singers observa-

relating any type of nerve the basic phenomenon that than specific the bioelectric encouraging

It appeared nerve-related

quite possible direct-current

relacom-

electricity rather tionship between pleted and some

function. A preliminary study of this and growth-control processes has neen obtained.
METHODS

Several species of A. tigrinum, A. opacuum, related amphibian, the animals. scribed been toring
same
2

salamanders with regenerative ability (T. viridescens, and N. maculosus) and a non-regenerating, but closely grass frog (R. pipiens), were used as the experimental of measurement discussed here. recording voltages used were the The only change same as in this previously technique dehas moniof the

The and

techniques will not

be

a refinement in the of the direct-current

apparatus, permitting the simultaneous from as many as eight different areas direct current divider type One electrode as the reference

animal. In the animals, a precision with solid, silver-silver in a pad soaked

experiments direct-current chloride solution and with agents

in which voltage electrodes. to serve

was administered to the of supply was used along was large and encased electrode. The other was

in saline

small and glass mounted Animals were anesthetized effects anesthesia, reversal; negative. and the polarity deepest of other anesthetic regardless that is, the Despite field plot correlates anesthesia this

was applied directly to the amputation stump. tricaine for most of the experiments, although the on the field potentials were also studied. Deep by a complete field cellular areas become lines remain the same

of the inducing agent, is accompanied extremities become positive and the polarity reversal, the equipotential

remains constant. Interestingly, the degree of reversal of field well with the depth of anesthesia, being fully reversed with the and slowly returning to normal as anesthesia diminishes. preceding of normal in making done when full recovery from polarity and potenthe measurements. just as the animals restraint was used

During the anesthesia, tial. These

period (five to ten minutes) immediately the animals demonstrated a field plot factors were taken into consideration

Determinations on wound were fully recovered from on non-anesthetized animals,

and regenerate potentials were anesthesia. In some instances, similar results

RESULTS

were

obtained.

The the electrical changes fracture in the Section Limb

lowed

presentation changes the during

of experimental immediately healing results or regenerative

results associated of artificially

will with phase;

be the

divided amputation; electrical the

into

four the changes

sections: subsequent during changes

healing; and the course of regeneration. One amputation

by

augmenting

electrical

in both
a complete

regenerating
reversal

and
of the

non-regenerating
electrical polarity

forms
at the

was

fol-

immediately

ampu-

tation stump the direct-current subsequently seconds) found to probably

voL. 43-A.

(Fig. showed

3). This change levels at other continuous period

was sites

not associated with any major change in measured, except that the head voltages (wave anesthesia. stimuli from the forms lasting These wave ten to forms case, site. fifteen were they The from

fluctuation damaging stimulus

during

the

of recovery

be associated represented
NO. 5, JULY

with any major the continuous

and, in this amputation

1961

648
rapid reversal of electrical polarity

R.

0.

BECKER

at

the

site

of amputation

(with

a shift

from deeto the

It was

negative
tron flow

to positive voltage) may represent in the associated nerve fibers. This

and does not necessitate sectioning

the cessation of direct-current cessation is a neural response

of the nerve fibers themselves.

trauma

noted that any freezing, fracture similar polarity

stimulus of the shift,

producing long bone

cellular trauma-heat coagulation, radiation, of an extremity, or crush injury-produced nerve section. The duration of the direct-

obviously

without

m V CHANNEL

---------

HE A D

+30
-30 LEFT FORELEG +30
-30

AMPUTATION LEFT FORELEG

________________
0 ___________________

BRACHIAL AREA +30

HDLEGC +30 -30 RIGHT

________
+3C0

FORELEG
10 TIME 20 IN MINUTES 30 40

FIG. Direct-current voltages monitored at five sites

3
before and immediately after amputation of the

left foreleg. During deep anesthesia (at left on the time scale) a reversal of normal field pattern is present, with the extremities positive and the central areas negative to some degree. As anesthesia decreases, the voltages swing back through zero to normal polarities and potentials. Amputation of the left foreleg in this stage produces an immediate, lasting reversal of polarity back to the same levels present during deep anesthesia. This positive voltage not only persists to the point of full awakening, but actually increases slightly in magnitude. This positive voltage in the left forelimb is
accompanied by a tendency to reversal of polarity, represented by a slower rate of change, in the

contralateral right foreleg. The left hind limb, however, shows a Steady return to normal value. The three lines immediately above the heavy line representing the head-channel voltage (top) refer to the wave-form patterns seen in this channel with a faster speed of the recording paper (one millimeter per second) and a higher amplification than was used m the scale represented here. In deep anesthesia there is little or no wave-form pattern at this amplification; after amputation, however, continuous wave forms appear and increase in amplitude with time. These are the wave forms typical of the response to trauma; they actually represent variations in direct current since they have frequency periods ranging from five to twenty seconds, In this case, they apparently represent the response to the continuous trauma arising from the amputation.

current we can

only

response appeared to be dependent speculate on the mechanism field system cellular trauma this central

upon relating

the degree of trauma. the cellular or tissue

At present, trauma to be

the changes in the bioelectric sensed in some way by the activity known current direct-current of the appropriate for some time that of injury8. activity Possibly,

itself. It to bring aggregate involving is the nervous

is apparent that about a change (brachial cellular local system.

THE JOURNAL

the trauma must in the direct-current

or lumbar). disruption that there

BONE AND

It has been causes a local influences is some

JOINT

mechanism That
OF

the relaSURGERY

of the

BIOELECTRIC

FACTORS

IN

AMPHIBIAN-LIMB

REGENERATION

649 is evidenced structures by

28

tionship

the

between the decreased current Two The sequence

peripheral of injury

nerve elicited

and from

the injury chronically

potential denervated

Section

of changes

in the

bioelectric

field

during

the

healing

processes in nonchanges process two to 50 per

in regenerating regenerating

forms animals.

was very different from the field-change In the former group, a complex series

sequence of potential

occurred that correlated well with the various stages of the regenerative (Fig. 4). The initial positive polarity fell rapidly in value during the first three days, rose again during the fourth to sixth days to a value of about

EXTREMITY POTENTIAL IN mV

10 DAYS

15 FOLLOWING FIG.

20

25
AMPUTATION

30

35

40

The voltage
zero very point

changes
the

in an extremity

of a salamander

during

regeneration
which peak

after

amputation.
with

The
the

day

horizontal scale immediately precedes amputation, high positive peak in the voltage curve. The secondary positive and the negative maximum is on the eighth day. This negative

on

in on the fifth bias of the regenerating limb is

is concurrent this case is

maintained

until

regeneration

(interrupted line). case was practically cent and with

is complete. The normallimb voltage is about ten millivolts negative The lower figures show schematically the stages of regeneration, which in this completed by the thirty-eighth to fortieth day.

of the initial, and then fell, crossing the zero line sometime between the fifth tenth day. This was followed by a prompt negative shift of high amplitude, maximum negativity occurring somewhere between the eighth and the day. This negative polarization normal limb negativity and elevated fortieth was animals. still This the was very high, post-traumatic exceeding positivity. growth and now almost the of the the others voltages, regenerative a group in millivoltage This negative differentiation, completely in more of fourteen they than animals all folare re50

fifteenth both the

polarity remained and as late as the generated per cent extremity of the

during the phase of axial day after amputation the more negative than involved amplitude during there all experiment

and, except for lowed the same The anatomical divided arbitrarily site of amputation, seems
VOL.

minor differences in the sequence of changes. and histological

changes

process

into two phases. Initially, of a mass of undifferentiated in doubt,

1961

is the accumulation, at the cells. The origin of these cells measurements have

to be somewhat
43-A. NO. 5, JULY

although

certain

quantitative

650 recently there evidence

amputation

R.

0.

BECKER

indicated is a movement cell

that mass

they of cells

are from until when

derived the as

from proximal the

remaining tissues It blastema.

tissue into the increases

of the stump in

limb
.

and This

that un-

differentiated

is known

size

without

of differentiation (in the adult), the

suggests

sometime it begins The observed

about twenty to show axial time

to twenty-five days after growth and differentiation of the voltage-change with whereas differentiation which slower
time related.

and
sequence

enters

second

phase.

course

tively negative growth have

trical

non-specific polarity phase. a slower

potential

that the initial healing processes appears It rate was noted

; that

positive polarity may or with the blastema definitely those showed species
processes

be associated formation, with the

relathe

to

be that
is, the

associated and
were

individuals

naturally rate of elec-

of regeneration

a correspondingly

change

two

EXTREMITY 10

POTENTIAL

IN

mV

NOR
0
--

MAL

-20

Voltage changes is again during

4 DAYS

6 FOLLOWING

10 AMPUTATION

12

14

16

FIG.

5 amputation of the forelimb scale immediately precedes

polarity. Unlike the salamander

the The

non-regenerating
which

form).
concurrent

healing process zero point on the

the very high

following horizontal
positive

of a frog amputation,
(Fig.

(a
4)

with

this high positive polarity is maintained for a long period, dropping slowly to nearly normal levels as healing progresses almost to completion. The normal field pattern in this animal is similar to that of the salamander, and the extremities are invariably negative during the last stages of recovery from anesthesia (interrupted line).

Although electrical-polarity sequent sequence The highly

the

non-regenerating reversal following of electrical-potential potentials remained

forms amputation, changes

demonstrated they as the and

the

same

immediate

did not show regenerating fell only slowly

the same subforms (Fig. 5). toward extremity normal zero in

positive

elevated

potential as healing occurred. Only when healing begin to show negative potential and then it was value for some days. so examined showed completed. It must be emphasized even a transient shift

was complete did the generally lower than

that not one of twenty-four to negative voltages until

specimens healing was

THE

JOURNAL

OF

BONE

AND

JOINT

SURGERY

BIOELECTRIC

FACTORS

IN AMPHIBIAN-LIMB

REGENERATION

651 tissue and skin scar edges

The formation (essentially salamanders, regeneration.

healing with the

process gradual

in these epithelialization

frogs

consisted of the

in raw

granulation surface from animals capable However,

the

same healing process as in man). These and in the larval stages are themselves They lose this ability in the adult form. complete stump that necessary
25

are related to the of complete limb the adult frog extra, in functioning the light tissue Electrical of in

can be made neural tissue

Singers

to regenerate a nearly into the amputation findings limb as indicating the level is below

limb This

by grafting is interpreted density

other frog

the

normal to sustain

of nerve

the

adult

regeneration.

NORMAL

FRACTURE

--

-1
o
J+IO

DAYS

10

DAYS

0 1F10

I5

DAY S

0 +10

FIG. Voltage junction changes observed

during fracture healing. The simple fracture was produced at the of the lower and middle thirds of the humerus. The normal voltage gradient progressing peripherally along the limb is shown by the upper-line graph. This gradient is measured by placing the reference electrode over the brachial area and sweeping the recording electrode distally. Normally, there is a smooth curve of increasing negativity obtained by this method. The lower four graphs show the disturbances in the limb-voltage gradient induced by the fracture at the times indicated. The sequence of local voltage changes is similar to that shown by the total extremity during regeneration (Fig. 4).

field
similar

measurements
to

on of the

intact salamander,

frogs

demonstrated having the

a direct-current same spatial

field relationship trauma specific did

pattern to the caused sequence not the occur poten-

that

anatomical the same of potential in the tials and erating thesia.

VOL.
43-A,

arrangement polarity reversal changes that was to drive failed

of the central nervous system. Similarly, in the potentials. However, the highly regeneration not had the in the opportunity salamander frog. I have

accompanying regenerating the electrical because

non-regenerating in a frog attempts sequence

to measure

a limb secondary to nerve-grafting potentials of the stump artificially of technical problems related to

procedures, in a regenrepeated anes-

NO.

5,

JULY

1961

652 Section After immediately

break.

R.

0.

BECKER

Three fracture of a long bone in the salamander, the appeared with a skin zone of positive potential subsequent to that (Fig. to the the
between small
+3 CURRENT
GIVEN IN 0

same

polarity

reversal

around healing

the

area

of the fracture lower appear

The

voltage-change appearing 6). The fact normal

various

sequence

during

the

of the somewhat would and

the

was similar in magnitude to be related situated

been made

during difference the limb

stages

limb regeneration, in magnitude part is within gradient.

fracture-healing

although of the potential the Thus soft far,

process

that

injured voltage
of the

tissues no
and

that
voltage-

it is have

along sequence,
of these

correlations good

change
grams

primarily
extremities.

because

of the

difficulty

in obtaining

roentgeno-

GROUP

juA
GROUPI

fl-P

CONTROL

n
0

p A -_---fl
GROUP 2

I?- i-P

+3 CURRENT GIVEN IN uA

23

PERCENT GROWTH INCREASE

I I

n II

+3 CURRENT GIVEN IN iA 0
.

GROUP

3 2 *iA/mm2OF ANIMAL

:
-3 20
-

CONSTANT RANDOM

CURRENT ORIENTATION

PERCENT GROWTH INCREASE

10
I & I I I

00

0 DAYS

5 FOLLOWING

20

25 AMPUTATION

30

35

40

FIG.

7 polarities to the regenerating forelimb of the

The

results

of administration

of current

of various

salamander. The upper three lines (Group 1) refer to the series of animals given positive current during the first five days after amputation. The increase in blastema formation and the slower rate of re-differentiation in the experimental animals are schematically shown. The center two lines (Group 2) refer to the animals given negative current from the fifth to the ninth day inclusive (upper line). The percentage increase in axial growth of the experimental limbs ove, controls is shown on the lower line. A period of growth acceleration appeared to take place from the twentieth to the fortieth day, with a maximum acceleration about the thirty-fifth day (23 per cent). The lower portion (Group 3) refers to the animals exposed to constant current, with random orientation

of the animal permitted. The solitary spurt in axial growth on the twenty-fourth have been the result of changes in ionic strength in the tank. In Groups 2 and were made with an ocular micrometer at five to ten-day intervals.

day is thought 3, all measurements

to

It is an
process
17

apparently
being the

well
case,

accepted
the

fact
observed

that

fracture

healing
type

is a regenerative
of voltage-change

This

regenerative

sequence peripheral

is entirely appropriate. The nerves and the regenerative

relationship process was

THE

between determined
OF BONE

the

density only in
AND JOINT

of the

the case

JOURNAL

SURGERY

BIOELECTRIC

FACTORS

IN

AMPHIBIAN-LIMB

REGENERATION

653 healing nerve itself for the productive is a nor seof

of

limb

regeneration. of speculation.

What

the fibers

relationship stated, need changes.

is in the neither be considered Certainly, the changes

case injury as the

of fracture to the

matter

As previously

proliferation of the nerve quence of electrical-potential sufficient local has transpired. the appropriate alterations. Section Four

prerequisites fracture is

currents of injury The electrical-potential cellular aggregate

to inform

bioelectric field that such an event can be produced functionally by without such anatomical

(brachial

or lumbar)

Three administered salamanders stump current that blastema

separate experiments were performed to regenerating forms after amputation. were given two microamperes of current

in which external current was In the first group, six larval directly to the amputation days. This polarity at a It group. five postoperative occurring positive animals control

for five to ten minutes per day for the first was oriented so as to enhance the normally time. On almost the tenth twice to twelfth as large day, all noted six as that

of these

demonstrated is three-dimenAn observaIn

in a simultaneous since the blastema to precise measurement.

was difficult to quantitate these differences sional and so small that it does not lend itself tion of equal retardation all of the sixteenth four to six importance of regeneration group whereas later. to the increased in the phase there the These developed experimental results are

size of the blastema was the subsequent of axial growth and differentiation. initial digitation group did summarized of the not reach in Figure

control day, days

regenerate on the this stage until 7, upper section. of the microamperes per day from normally subsequent occurring in blastema axial

In the second experiment, six adult animals were given three current directly to the amputation stump for five to ten minutes fifth to the tenth compared day. This at that with current time. was There oriented was control to enhance no discernible group the negative formation polarization

difference

a simultaneous

; however,

growth, as measured by ocular micrometer at intervals ceeded that of the control group by significant amounts The comparisons of growth rate were made by measuring the length of the regenerated limb in the six experimental The average results in each group were determined, increase in axial growth of the experimental animals culated at different time intervals. It was noted that mals showed ing control per a greater length of regenerate animal. The total size of the as compared regenerates,

of five to six days, ex(Fig. 7, center section). by ocular micrometer and six control animals.

and the average percentage over the controls was calall of the experimental most rapidly was small; length. to be anigrowa 25 with the however, in actual appeared

cent difference Digitation,

refers to less than one millimeter an index of differentiation, also group, but encountered be utilized. consistency observation was in this experiment However, despite the of the results observed this

accelerated The small signifigrowth-

in the experimental technical difficulties numbers of animals

cant

difficult to quantitate. necessitated that lack of a statistically suggest that some

sample,

the

accelerating from the was noted the same

effect was obtained. In the animals receiving positive polarity current first to the fifth day after amputation, an increase in tissue resistance on the third day, necessitating twice the previous voltage to deliver amount of current. The same phenomenon was noted on the eighth

day in the series receiving negative polarity current from the fifth to the tenth day. In each case, current of the opposite polarity was easily passed, which would appear to indicate that there was some rectifier type of mechanism in the tissues. The significance of these observations is not known.
VOL. 43-A, NO. 5, JULY

1961

654 A third placed group of six salamanders in a standard aquarium the water in one

R.

0.

BECKER

and flowing

were which direction.

taken had The

on the first day a steady direct current density

after amputation electrical current was regulated to

through

produce two microamperes per square millimeter, and the animals were permitted to assume random orientation in respect to the direction of current flow. There were no changes in the rate of regeneration in this group except for a solitary, but
striking,

measurement
nificant

of about 19 per cent (Fig. 7, lower section). changes in rate, and the group increase

on the twenty-fourth to twenty-fifth Subsequent measurements showed completed regeneration at about the experimental day the water time ago that fields been
study,

day of no sigthe same

time as the control group. In reviewing that two days prior to the twenty-fourth mented. Since Kappers observed some aquatic similar
this

records, in the tank the trophic

it was noted was suppleresponses of

organisms factors are would Since

by

to direct-current presumed to

have

depended in action
strict

upon here.
control

ionic concentration, To evaluate properly

over ionic param-

random-orientation-steady-current

eters

be necessary. completing these similar to the

1909. He

experiments, last one current limbs. current results

to
22

it
a statistically

has was

come done

to with

my large

attention numbers
in increase

that
the

an
rate

experiment,
mals Frazee

described,

of ani-

in

reported

significant

of regeneration salamanders
insufficient

field any
similar
means

was

when direct with amputated to indicate the fragmentary. These frame back
attempts

was passed through tanks housing larval The techniques available at that time were density, and knowledge of the bioelectric therefore could not then be interpreted in It is of interest to note
rate

satisfactory
experimental

of reference.

that

Frazee
by

had

traced

influence

the

of regeneration

electrical

as far

as 1840

DISCUSSION

Growth
spectrum
growth of of

processes
of
an

are and
organism

not

only
from a

complex Whether
single

in

nature

but

cover

a continuous

abilities
entire

potentials.

one fertilized

is considering the embryonic ovum or the simple closure is quite evidently cited as evidence the under of loss in one and work limb not

a skin

defect
control.

by
Tumor

precise

epithelial and

proliferation, keloid formation

the process are usually

or disturbance are operative view

of the control mechanisms. in all of these conditions can

Whether or not only be speculated is such

same mechanisms upon at this time process that

of the extreme Philosophically,

paucity of information. the phenomenon of growth primitive control some interrelationship. between However,

a basic

would expect that exceedingly that all of these processes bear of Singer, regeneration, establishing is an the important

mechanisms The the

are operative aforementioned nerves effect and should

relationship beginning.

peripheral this neural

be interpreted Since any type action property anism. a true

ters

as involving any of the of nerve tissue is equally cannot nervous evidence activity

known functions potent, such

of the nervous known parameters

system. as the

potential of the From data the

be involved. It appears system, or its associated presented of some portion in this paper, of the

more likely that some very basic tissues, is the controlling mechthe thesis nervous one is proposed system that constitutes the central and that processes

direct-current
of the

system

transmission is the

and control system control of the growth

of the involved

output paramein regeneration

and repair. Since the electrical-field produced action potentials and may Schwann-cell syncytium, the observation

activity is not related to the system that even be a function of such tissues as the that any type of nerve tissue will bring
THE JOURNAL OF BONE AND JOINT SURGERY

BIOELECTRIC

FACTORS

IN

AMPHIBIAN-LIMB

REGENERATION

655 linkage mech-

about anisms input system

in

regeneration between or sensory inputs, field the the potentials

becomes cells fashion

more of the and

logical.

There

must

exist

certain

organism and as an output demonstrated to on be mediated

the bioelectric field itself, both in an or control mechanism. Considering between by the trauma current and of injury the changes generated

relationships appear Regarding potential

26#{149} In

the the

at
state

cellular level. direct-current

outputs cells

of the system, the has been previously the by

influence of a steadydemonstrated in the

central nervous system of cortical neurons can ing in the environment

cells
of

this case be modulated of the neurons. have normal direct-current influence specific fashion, state

rhythmic activity (as action potentials) changing the direct-current level existMore recently, it was demonstrated that charges environmental bioelectric site field of an injury that are different from for all generated accumulaelecfield control probcells cells in An parameter potential so as to cause

of

a regenerating

tissue in its is the

electrical
.

the the

same area.

tissue This field

of their tion trical

organism of cell types potentials.

or the

could

bearing In this

electrical charges at the output portion data must

points having specific of the direct-current transmission be stressed. and In all

system
system,

can be linked is established The preliminary

to cells, and a true closed ioop, for the growth process. nature of these experiments

ability, the electrical field changes are much more complicated (both here. Nevertheless, it would appear electric field accompany not appear in the case the process of less adequate

accompanying growth and healing processes temporally and spatially) than are reported that certain specific alterations in the bioof regeneration and healing processes. that these alterations It is surprising that do the

application of intermittent electrical currents produced an acceleration in the regenerative process. If the field potentials are the control mechanism, they must be considered to be operative in a continuous fashion. In these experiments, it must normally
tials in

be assumed has the this case that the

that the observed alterations were capacity to regenerate an extremity, reinforced continuous successful those currents attempt which were would to inhibit to

produced in an animal and that the added occurring. Although

which potenit

naturally

would appear ral potentials, mittent The processes beings.

have to be applied malignant-tumor the study both and

to reverse natugrowth by intercontrol and the of precise growth measmdibearwas

field reversals application in man Only recently

must be noted 12 of similar techniques much has more a technique been

requires

investigation evolved

in animals

in human

permitting

urement of the direct-current field in the human being. Initial measurements cated the presence of a human field system similar to that of the salamander, ing the same spatial relationships to the central nervous system. Anesthesia

found to produce a similar tendency toward reversal of the field potentials. However, the cranial portions of the bioelectric field in man show a much greater complexity than in animals, apparently being influenced by the emotions and sensory
inputs.
ian

Despite

this,

the
are

general

relationship

between

the

human

and

the

amphib-

bioelectric In summary,

field

remarkably

similar. potentials system. between have been shown to conand control established Relationships the direct-current indicatfield

bioelectric

direct-current

stitute an organized data transmission ing some control function have been and the process of limb regeneration.

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VOL. 43-A,

C. H.,

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D. C. Measurements
NO. 5, JULY
1961

BECKER, R. in the Human

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656
2. 3. 4.
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R. R. S.; M. W.
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THE

JOURNAL

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AND

JOINT

SURGERY