J. Biomedical Science and Engineering, 2010, 3, 917-926
doi:10.4236/jbise.2010.39122 Published Online September 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online September 2010 in SciRes. http:// www.scirp.org/journal/jbise
Prophylactic Bactericidal Orthopedic Implants – Animal
Testing Study
Richard A. W ysk1, Wayne J. Sebastianelli1, Rohan A. Shirwaiker2, Gregory M. Bailey1, Charumani
Charumani2, Mary Kennett2, Amy Kaucher2, Rachel Abrahams2, Thomas A. Fuller1*, Patricia Royer,
Robert C. Voigt1 and Paul H. Cohen1
1ArgentumCidalElectrics (ACE), Inc., Lewistown, USA;
2The Pennsylvania State University, University Park, USA.
Received 3 January 2010; revised 18 February 2010; accepted 22 April 2010.
ABSTRACT
This paper summarizes preliminary rat studies aim-
ed at identifying the effectiveness of using electrically
stimulated silver as a bactericidal agent for indwell-
ing residual hardware devices (RHD). A variety of
bactericidal indwelling devices were designed, fabric-
ated and surgically inserted into the medullary ca vity
of live rats. The rats were inoculated with Staphyloco-
ccus aureus to try and induce osteomyelitis. A total of
37 surgeries were performed by implanting the rats
with both control and potentially bactericidal devices.
As surgical procedures and devices were improved, it
appeared that the implants produced antibiotic effe-
cts in the animals. All of the control animals and all
of the animals where the device failed tested positive
for Staphylococcus aureus growth. Of the rats with
operational bactericidal devices (those that survived
the surgery and incubation period), half tested nega-
tive for Staphylococcus aureus. The device designs are
discussed in this paper along with the test procedures,
operating practices and results. A statistical analysis
of the results, which shows a very high confidence le-
vel in the effectiveness of electrically stimulated silver
as a bactericidal agent/antibiotic, is also presented.
Keywords: Antibacterial; Antimicrobial; Bactericidal; Si-
lver; Ionic Silver; Residual Hardware Devices (RHD);
Animal Testing
1. INTRODUCTION
Replacing arthritic joints has improved the quality of life
for millions of Americans. Over the past decade, there
has been an increase in the number of total hip and knee
replacement surgeries performed in the U.S. In 2005, a
total of 808,000 total hip and knee replacements were
performed in the U.S [1]. By 2030, the total number of
replacements is projected to be more than 4 million [2].
Of these surgeries, 0.3 – 2% result in deep bone infect-
ions (osteomyelitis), according to current data [3,4]. While
this rate is fairly low, the cost associated with mitigating
deep bone infections far exceeds the cost of the initial
replacement surgery.
Patients who develop osteomyelitis must undergo diff-
icult and expensive treatment. Total mitigation of osteo-
myelitic infection is typically achieved after: 1) implant
device removal, 2) local debridement of infected area, 3)
insertion of a spacer prosthetic, 4) an aggressive six we-
ek course of limb protection and aggressive antimicrob-
ial therapy, and 5) a second joint arthroplasty. The prim-
ary objective of this research is to improve the quality of
life by reducing pathogenic bacteria in in vivo joint rep-
lacements using the antimicrobial properties of silver
stimulated by an electric current. The long-term goal is
to augment current implants with antimicrobial surface
technology, thus allowing treatment of RHD associated
osteomyelitic infections without the removal of prosth-
etic implant or hardware device.
Significant research has been performed recently on
antimicrobial colloids composed of silver nano-particles
stabilized by polymers or other agents [5-7]. Many stud-
ies have shown these materials to display antimicrobial
efficacy to a wide spectrum of microorganisms [8-10].
Our in vitro laboratory studies have shown these new
materials to exhibit acceptable early antibacterial prop-
erties; however in water-rich environs, these materials
may quickly lose their effectiveness (typically minutes
to hours). After years of investigating the bactericidal
effects of silver, the proper device configuration required
for silver to be an effective bactericide has been identi-
fied. The key is to continually produce a controlled rele-
ase of silver ions (Ag+) and expose bacteria to these ions
for extended time periods.
Forbes [11] reported that $30 billion is spent annually

R. A. Wysk et al. / J. Biomedical Science and Engineering 3 (2010) 917-926
918
to fight infections, and that drug resistant infections kill
more Americans every year than AIDS and breast cancer
combined. Our hypothesis is that ionized antimicrobial
silver and silver forms can be used to kill a wide variety
of bacteria, i.e., both standard strains of bacteria as well
as methicillin-resistant bacteria. We base this hypothesis
on the long history of bactericidal metals, particularly
silver, gold and copper, that have been used in medicine
for years as antimicrobial agents in the form of wound
dressings and debriding agents [8,9,12-14]. The histori-
cal problem has been that these bactericidal metals seem
to work in certain situations while showing little effect
in others. Based on three plus years of in vitro testing,
we have identified the key science and engineering ele-
ments that allow bactericidal metals, particularly silver,
to work effectively. The use of antimicrobial silver is
particularly promising because in spite of being used for
more than 100 years, there has been no evidence of a
definitive pattern for silver resistant infections [15].
The principal focus of this research is to describe a new
system designed to make replacement prostheses antiba-
cterial. In the following section, we will describe the pr-
oposed system that is identified as effective against most
bacteria which cause osteomyelitic infections. A few
device design iterations along with a final device design
that was used in our animal testing are also described.
Finally, the protocol used for animal testing and the re-
sults that were obtained in the animal tests are presented.
2. THE ANTIMICROBIAL SYSTEM
A system was developed to provide prophylactic and an-
tibiotic protection for both soft tissues as well as bone
against common bacteria, e.g., strains of Escherichia co-
li, Staphylococcus aureus, Pseudomonas aeruginosa,
Enterococcus faecalis, and methicillin-resistant Staphy-
lococcus aureus (MRSA) and fungi e.g. Candida albi-
cans. Our in vitro and early animal testing has shown
that a properly configured device that is stimulated by a
very small amount of electrical current is both an effecti-
ve bactericide as well as a fungicide. The central hypot-
hesis behind the proposed research is that electrically
ionized antimicrobial silver and its alloys can be used as
a safe and effective means of eliminating microorgan-
isms associated with biofilms on RHDs. The key to this
technology is using the bacteria-rich environment to
carry silver ions and complete an electrical circuit in the
device configuration shown in Figure 1. This configura-
tion creates a regional antibiotic environment around the
RHD (inhibition zone diameter ≈ 20mm in a petri dish),
and appears to work for different concentrations of bac-
terial colonies.
3. IN VITRO STUDIES
For more than three years, we have experimented with si
lver stimulated by small currents to produce silver ions.
We have demonstrated that our configuration is effective
against all common harmful bacteria and fungi that were
tested in the laboratory. Our system shows reproducible
results for a variety of bacteria and fungi, as shown in
Figure 2. In another test, we inserted our device in the
medulary cavity of a rat tibia and then embedded the
tibia in Mueller Hinton agar (MHA) inoculated with Ps-
eudomonas aeruginosa to determine if our system wor-
ked in this simulated in vivo environment. The results of
this test are shown in Figure 3. These results are sign-
ificant in that the bactericidal kill region that is created
from within the rat tibia extends well into the agar. This
demonstrates that our Ag+ system can penetrate the bone
– a problem for most antibiotics.
4. IN VIVO STUDIES—ANIMAL TESTING
The properties of silver have been known for quite some
time. Chronic exposure to silver, termed argyria, is man-
ifested by an irreversible gray or blue-gray discoloration
of the skin and mucous membranes. When the body is
Figure 1. Proposed antimicrobial system concept.
(a) (b)
(c) (d)
Figure 2. In vitro testing results of the proposed system on (a)
Escherichia Coli; (b) Pseudomonas Aeruginosa; (c) Staphylo-
coccus Aureus (d) MRSA.

R. A. Wysk et al. / J. Biomedical Science and Engineering 3 (2010) 917-926
919
Kill Zone
Figure 3. Simulated in vivo testing results of the proposed
system on Pseudomonas Aeruginosa.
exposed to silver challenge the liver acts as a filter mec-
han ism, collecting over 90% of the absorbed silver and
eliminating it in the feces via liver and biliary tract excr-
etion [16,17]. As such the liver cells will experience the
greatest levels of toxic elements borne by the blood. The
cellular toxic levels of hepatocytes were determined to
be both time and concentration dependant but could be
appreciated at 30 M [18]. These results were confirmed
by Rungby in 1990 when he proposed a range of 30-70
M silver concentration as that needed to show appreci-
able cytotoxic effects within the hepatocytes [19]. With-
in all of our designs we will keep local ionic concentrate-
ons at or below toxic concentrations by controlling the
electrical circuitry associated with the implant device.
Two separate sets of animal testing studies were con-
ducted to investigate the effectiveness of an in vivo elec-
trically stimulated silver device that was inserted into the
tibia’s medullary cavity as a bactericidal agent. The pro-
tocol for generating osteomyelitis in the animals paral-
leled that described by Lucke et al. [20]. As described in
this reference, rat tibias were inoculated with Staphylo-
coccus aureus to try to create osteomyelitis. Surgical
procedures were developed to install and retain the pro-
posed bactericidal devices within the rat tibia.
The protocol to grow the inocula was adopted from
Lucke et al. [21]. Staphylococcus aureus (ATCC number
29213), used as the pathogen, was grown overnight in 9 ml
tryptic soy broth (TSB) (caseinpepton–soybean flour–
peptone–solution; Oxoid Ltd., Basingstoke, Hampshire,
UK). From this culture, 100 µl aliquots were transferred
to sterile tubes containing 3 ml of TSB. These tubes
were then incubated for 3 h at 37°C to obtain log-phase
growth. After incubation, the tubes were centrifuged for
10 min at 3000 rpm, the supernatant was decanted, and
the remaining pellet was washed twice with phosphate-
buffered saline (PBS). Under spectrophotometric control
the bacterial sediment was added to PBS until a McFar-
land standard of six was obtained. Colony- forming units
(CFU) per ml were confirmed by several plate counts
with the use of a spiralplater (Spiral System Inc., Cincin-
nati, OH). This procedure was repeated 20 times. Suspe-
nsions were split into portions and deep frozen at –80°C
until the day of surgery. To quantify a possible loss of
viable bacteria following the freeze–thaw cycle the
CFU/ml were reconfirmed after each cycle of defrosting.
In addition, each time a rat was inoculated, a 10 µl sam-
ple of inoculant was analyzed to determine its bacterial
concentration. The concentration was adjusted to fit into
the desired range (102 - 104 CFU/ml) using McFarland
Standards and was confirmed by plate counts.
Surgeries in both sets of studies were performed at the
Penn State Central Biology Labs. All through the testing
studies, several different bactericidal devices designed
were inserted into the tibial canal of 4-month-old Spra-
gue Dawley rats through a proximal incision site and
tibial cannulation. A similar surgical protocol was empl-
oyed for all the hardware insertions with only slight de-
vice-specific modifications being made to the procedures
as necessary. For each of the surgeries, one leg was sha-
ved and scrubbed with betadine and alcohol prep. Each
animal was placed on sterile drapes and their bodies were
covered with sterile sheets; the prepped leg was sepa-
rately draped in a sterile manner. A small incision (~ 5
mm) of skin and fascia at the proximal tibial metaphysis
was created to provide access to the tibial periosteum.
The medullary cavity of the proximal metaphysis was ex-
posed using a 1 mm surgical drill, leaving the surround-
ing periosteum intact. A steel Kirschner wire (Ø 0.8 mm)
was inserted into the medullary cavity and pushed forw-
ard distally for smooth dilatation of the cavity for a leng-
th of approximately 8-10 mm distally, and removed. Sta-
phylococcus aureus was injected into the medullary cav-
ity to initiate osteomyelitis. Animals were closely monit-
ored post-surgery and analgesics were administered ini-
tially on a regular schedule. No antibiotics were given to
any of the animals since they would have masked the re-
sults of the test.
At the end of a two week incubation period, the anim-
als were euthanized using carbon dioxide. The device was
surgically removed and the anodic tip was used to in-
oculate a sheep blood agar plate. The tip of the device (~
5 mm) was then cut and placed into 10 ml of TSB. The
blood agar plates and TSB were incubated for 24 h at
37°C. Any CFUs found on the blood agar plate were
counted and then analyzed to identify the bacterial spe-
cies obtained from the tibia. The TSB was only visually
examined for the presence of growth (cloudy: positive
growth; clear: no growth). No further testing was perfor-

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920
JBiSE
Length of device
med.
Initial bench top testing revealed that early prototype
device designs performed well; however, biocompatibil-
ity within lab animals remained a question. Iterative tes-
ting of three device designs was performed to determine
the one that was most compatible with the lab rats. Three
separate, but similar, device-specific surgical procedures
were used during this phase of the study.
4.1. Overview of Surgical Procedure for Animal
Testing Studies #1
The first set of animal testing studies was performed in
2007 in which three iterative designs of bactericidal dev-
ices and control devices were fabricated and inserted into
the tibia medullary cavities of 21 living rats. Table 1
identifies the specifics of the iterative design changes.
Sixteen of these animals survived the surgery and the
two week incubation period following the surgery. Of
the 16 rats, 5 were implanted with control devices (titan-
ium wire). The inoculation process proved very succe-
ssful as all 5 of the control animals without a bactericidal
device developed Staphylococcus aureus based bone in-
fections. Of the remaining 11 rats, only 9 animals had
devices which remained in the tibia during the testing
period. 3 of those 9 devices failed during the indwelling
period leaving only 6 animals as prime targets for meas-
uring efficacy of the device. Of these 6 possible cases, 3
of the rats with working devices were found to be staph
free. All of the other animals with non-silver control devi-
ces or failed silver devices that were inoculated and
euthanized cultured positive for Staphylococcus aureus.
This preliminary study showed that the devices worked
in at least half of the cases where they remained in the
tibia and continued to be operational. The quantity of
staphylococcus aureus present in the tibia was not quan-
tified so it is possible that some of the devices judged
ineffective may have significantly reduced the amount of
bacteria present at the end of testing.
During this entire animal testing sequence, the surgi-
cal procedures were continually refined making small
iterative improvements for two purposes: 1) to keep for-
eign-borne microbes from impacting the testing and 2) to
improve the device for animal comfort and performance.
For surgeries performed on the first 6 rats, the staphylo-
coccus (~10 - 20 µl containing 103 CFUs) was injected
via syringe into the medullary cavity prior to inserting
the device. For the remainder of the surgeries, the wire
tip of the device was dipped into the solution of staphy-
lococcus aureus prior to inserting it into the tibia. This
refinement was necessary because the medullary canal
could not hold all of the fluid injected and the overflow
ran into the soft tissue surrounding the incision site. The
overflow of the injection fluid caused, in addition to the
desired osteomyelitic infection, a soft tissue infection
surrounding the incision site. Details of each of the pro-
cedures, blood reports and tibia X-ray reports were re-
corded. Ketmine/Xylazine (100 mg/kg) and IP Isoflurane
(10 mg/kg) were used as anesthetics.
All three device designs used in this set of study and
their surgical results are discussed below. One failure in
this set of testing study was that the epoxy capsule used
to enclose the device battery within the animal became
infected in the majority of the animals.
4.1.1. Antimicrobi al Device #1—Design and Sur g ical
Summary
The first set of surgeries was performed on 6 rats. The
first implant design had a stiff silver wire cathode (Ø 0.8
mm, ~ 30 mm length) and a stiff silver wire anode (Ø
0.8 mm, ~ 40 mm length and then cut to fit the tibia) that
was inserted into the tibial canal. The anodic wire was
connected to the 1.5 V battery via a 35 gage insulated
copper wire. The conductive path for the two wires was
the highly resistive soft rat tissue between the anode and
cathode. The distance between the two wires (anode and
cathode) was ~ 70 - 90 mm when implanted, and the
total length of the flexible wire was as much as 120 mm.
A small epoxy cap was used to prevent the battery and
wires from eroding through the external skin layers. The
Table 1. Device design summary for 2007 animal testing.
Section of
Text Device number Figure number Anode configuration Cathode configuration Battery encasement
4.11 1 4
Stiff silver wire - connected to battery
by flexible 35 gauge copper wire Stiff silver wire 100-120 mm epoxy
4.12 2 5
Stiff silver wire - connected to battery
by flexible 35 gauge copper wire Flexible copper wire 80-100 mm epoxy
4.13 3 6
Stiff silver wire - connected to battery
by flexible 35 gauge copper wire
Silver painted wire,
wrapped around insulated
portion of anode wire
80-90 mm epoxy

R. A. Wysk et al. / J. Biomedical Science and Engineering 3 (2010) 917-926
921
device design for these surgeries is shown in Figure 4.
The anodic tip of the device was inserted into the tibial
canal; the conductive wire and battery pack was stored
under the dermatological layer of the rat’s flank. This de-
sign was tested at two different levels of current – high
amperage (without resistor) and low amperage (with
resistor), and with a similar control device in which the
battery was removed and the silver wire was replaced
with a titanium wire (2 rats).
Thus, six rats were fit with implants during the first set
of surgical trials; two devices with no resistor, two de-
vices with 1 M-Ω resistor and two devices with titanium
wire (control). All 6 rats survived both the surgery and
the two week incubation period post-surgery. After the
two week period, all 6 rats were euthanized and studied
to determine if the devices had eliminated the bacteria
(especially the staphylococcus aureus). All of the de-
vices were checked for continuity and voltage after re-
moval. Three of the four devices were deemed to be
functional. 2 of these 3 rats with functional devices did
not show any staph growth.
4.1.2. Antimicrobi al Device #2—Design and Sur g ical
Summary
The second set of surgeries was performed on 9 rats. In
this set of surgeries, the cathode of the implanted device
was modified by replacing the stiff sliver wire with a
flexible copper wire. The additional flexibility was
desired for rat comfort as the original design (stiff wire)
dug into the rat’s muscle tissue, causing a point of irrita-
tion. The total length of the implant was reduced to
~80-100 mm in order to facilitate ease of insertion. The
inoculation during the second surgery set was performed
by dipping the wire into the staphylococcus aureus solu-
tion to induce a staph infection within the medullary
cavity. The device used for the second set of surgeries is
shown in Figure 5.
Nine rats were surgically implanted with the follow-
ing: three devices with no resistor (high amperage), three
with a 1 M-Ω resistor (low amperage) and three with a
titanium control. In this group, 2 of 9 rats died. The rem-
aining 7 rats survived the observation period and were
euthanized after two weeks. A few of the devices had
their cathode tipping out of the rat skin, thus breaking
the continuity while in vivo. Devices were checked for
continuity after the animals were euthanized and only
three appeared functional. Unfortunately, the design
changes created a device that could “float” within the
animals, allowing the devices to migrate out of the me-
dullary cavity during the post-surgical phase of the study.
Due to migration, none of the devices remained in the
tibial canal when the postmortems were performed.
Drawbacks of this design included breakage of the cop-
per wire and poking of the cathode through the rat skin.
Resistive soft tissue between the anode-cathode separa-
tion was also a problem.
+
Battery
Silver Cathode
(? 1mm)
Silver A node
Flexible Wire (Insulated)
Resistance
Epoxy Bubble
120 mm
(approx.)
Figure 4. Antimicrobial device #1.
Figure 5. Antimicrobial device #2.

R. A. Wysk et al. / J. Biomedical Science and Engineering 3 (2010) 917-926
922
4.1.3. Antimicrobi al Device #3—Design and Sur g ical
Summary
In order to alleviate the migration of the device within
the test animal, a third device design was created. The an-
ode and cathode were integrated on the same wire and
were separated by a small insulation strip. In this config-
uration, electrons are drawn from the silver paint (cath-
ode) to the anode over the insulation through the cond-
ucting media. This design was accompanied by a change
in electrode polarities resulting in two types of devices –
anodic and cathodic. The length of the flexible wire was
limited to ~80-90 mm. Similar to the previous sets of su-
rgeries, bacterial inoculation was created by dipping de-
vices into the staphylococcus aureus solution. The third
device design is shown in Figure 6.
Within this phase of the study, 6 rats were surgically
implanted using two implants of each kind: three with
high amperage anodic devices, two with low amperage
anodic devices and one with a high amperage cathodic
device. In this group, 3 of the 6 rats died post surgery.
The remaining 3 rats survived for the full observation
period and were euthanized after one week. All the de-
vices were checked for continuity and voltage after re-
moval and all were functional.
Several modifications to the implant device and surg-
ical procedures were made. The device changes appe-
ared to have corrected most of the early drawbacks. The
surgical procedure was modified to include a stitch to
hold the device in place. The result was that none of the
devices floated within the rat’s body. Only 1 of the 3 rats
which survived was staph free. This could have been due
to the limited separation between the electrodes. This
device appeared to be working as evident by the lack of
puss on gross examination during the device removal;
however a quantitative CFU count was not performed.
A few potential device design concerns remained for
this device. It was possible that the amount of fluids sur-
rounding the implant was insufficient to facilitate proper
movement of the antimicrobial Ag+ ions. Another possi-
bility was that the flow of antimicrobial Ag+ ions was
localized since ions take the path of least resistance and
in this case the path of least resistance translated into the
path of least distance over the insulation (about 5 mm).
4.2. Overview of Surgical Procedure for Animal
Testing Studies #2
The second set of animal testing surgeries was perform-
ed in 2008. A total of 16 surgeries were performed with
4 rats used as controls (titanium wire/silver wire without
current) and 12 rats implanted with three iterations of
antimicrobial designs. Ta bl e 2 identifies the specifics of
the iterative design changes. Thirteen of these animals
survived the surgery and an incubation period and could
be harvested for pathology. Three of these thirteen ani-
mals (2 with antimicrobial devices and 1 with control)
were harvested within the one week incubation period
following the surgery due to declining health. Thus, 3 an-
imals with control and 10 animals with the antimicrobial
devices were left as prime targets for measuring the ef-
ficacy of the devices. All control rats tested positive for
Staphylococcus aureus growth. Where the devices failed,
all of the animals also tested positive for Staphylococcus
aureus. Of the 10 rats that had antimicrobial devices
implanted and survived the surgery and incubation pe-
riod, 4 rats were clear of Staphylococcus aureus. The
Staph growth was not quantified in this study.
Battery
Epoxy Bubble 80 mm
(approx.)
Fle x ib le Wir e s
(Insulated)
Insulation
Silver Paint
(Cathode) Silver Wire (Anode)
Figure 6. Antimicrobial device #3.
Table 2. Device design summary for 2008 animal testing.
Section of
Text Device number Figure number Anode configuration Cathode configuration Length of device Battery encasement
4.21 4 7
Stripped end of Teflon
insulated silver wire
Stripped end of Teflon insulated silver wire,
wrapped around insulated portion of anode wire 60 mm epoxy
4.22 5 8
Stiff silver wire connected
directly to battery
Stripped end of copper wire, wrapped around
section of anode, insulated via heat shrink wrap 60 mm Silicone
4.23 6 9
Stiff silver wire connected
directly to battery
Stripped end of Teflon insulated silver wire,
wrapped around section of anode, insulated via
heat shrink wrap
45 mm Silicone

R. A. Wysk et al. / J. Biomedical Science and Engineering 3 (2010) 917-926
923
Refinements in this set of surgeries were focused on
keeping foreign-borne microbes from impacting the tes-
ting, and improving the device for animal comfort and
performance. A surgical protocol similar to the one used
in the first set of surgeries was followed. Two primary
modifications in the surgeries were made: 1) the wire
device was inserted in the primal portion of the tibia so
that a longer portion of the device could be inserted, and
2) the animals were placed in a plastic surgical sack so
that airborne bacteria could be contained. In this set of
surgeries, the medullary cavity of the primal metaphysis
was exposed using a 16 gage needle to produce a hole. 5
µl of Staphylococcus aureus (104 CFU/ml) was injected
via micro-syringe into the medullary cavity prior to inse-
rting the device. The micro-syringe eliminated any over-
flow of the bacteria. Similar to the first set of surgeries,
all animals were closely monitored post-surgery and an-
algesics were administered initially on a regular sched-
ule. No antibiotics were given to any of the animals to
eliminate masking of test results. The epoxy capsule us-
ed to enclose the device battery within the animal in our
early testing was replaced by a silicone shell to minimize
discomfort to the animals.
4.2.1. Antimicrobi al Device #4—Design and Sur g ical
Summary
The fourth implant design used during the first set of
surgeries in the second study set was a modification of
antimicrobial device #3 used during the first set of study.
In this design, the anode and cathode were integrated on
the same wire. Unlike device #3 where the anode was a
silver wire soldered to 35 gage copper wire, the anode in
this design was the stripped end of Teflon coated silver
wire directly connected to the positive terminal of a 1.5
V battery. Similarly, the silver painted cathode of device
#3 was replaced by the stripped end of another Teflon co-
ated silver wire directly connected to the negative batt-
ery terminal. The electrodes were separated by Teflon
insulation on the anodic wire. The electrons were drawn
from the silver anode to the silver cathode over the insu-
lation by the conducting media. A small silicone cap was
used to encase the battery and its soldered connections.
Length of the device was limited to 60 mm. The design
was used with two combinations of currents – high am-
perage (no resistor) and low amperage (100 KΩ resistor).
Figure 7 shows schematic drawing of the high amperage
device design.
Four rats were implanted with this device and all surv-
ived the surgery and incubation period. One of the 4 rats
implanted with this device tested staph free. This result
could be attributed to the quality of the fabricated device
as all devices seemed to separate at the coiled portion.
Although this design was more robust in construction
than the previous ones, a more effective release of ions
was desired. This could be achieved by replacing one of
the coated electrode wires with a bare one. Commercial
silicone used to encase the battery was not bio-compati-
ble and caused irritation to the rats.
4.2.2. Antimicrobi al Device #5—Design and Su rg ical
Summary
Since anodic device designs were being used, the Teflon
coated silver anode was replaced by a bare silver wire.
Thirty-five gage copper wire was used as the cathode.
Insulated shrink tubing was fitted onto the bare silver an-
ode to separate the electrodes. Commercial silicone was
replaced with medical grade sterile silicone for the bat-
tery encasing. Length of the device was limited to 60
mm. These devices were only used with high amperage
(no resistor). The device design is shown in Figur e 8.
Figure 7. Antimicrobial device #4.
Figure 8. Antimicrobial device #5.

R. A. Wysk et al. / J. Biomedical Science and Engineering 3 (2010) 917-926
924
Of the 4 rats implanted with antimicrobial devices, 1
died before pathology was performed. One of the rem-
aining 3 with working antimicrobial devices tested Staph
free. In analyzing the device used in these surgeries, we
concluded that the 35 gage copper wire should be re-
placed with Teflon coated silver wire as the cathode as
the copper wire seemed to unravel and oxidize. The sili-
cone encasing should be reduced in size by about 20%.
The length of the device should be smaller for ease of
insertion.
4.2.3. Antimicrobi al Device #6—Design and Su rg ical
Summary
This device was slightly modified from the previous ver-
sion. Bare silver wire continued to be used as the anode.
Teflon coated silver wire replaced 35 gage copper wire
as the cathode. In addition to the shrink tubing used as
insulation between the anode and cathode, another piece
of the tubing was introduced to hold the anodic and ca-
thodic wires together. The size of medical grade silicone
encapsulation was reduced. Length of the device was
reduced to 45 mm. These devices were also used with
high amperage (no resistor). The schematic design of the
device is shown in Figur e 9.
Four rats were implanted with this device out of which 3
survived through the post surgery incubation period.
Two of the remaining 3 rats were found to be Staph free.
Of the 10 rats that had antimicrobial implant devices and
survived through the incubation period post surgery in
animal testing studies #2, the rats implanted with these
devices looked the healthiest. This design eliminated all
drawbacks of the previous versions and seemed very
robust.
5. STATISTICAL ANALYSIS
Statistical testing was conducted in order to determine
any significant statistical difference between the devices
used in surgeries. Because several of the animals died pr-
ior to harvest and 3 of the devices did not function after
they were implanted, unequal test samples resulted for
all categories. This made performing a paired test impos-
sible so an F-Test was conducted to determine significa-
nce. The first test conducted compared all effective con-
trol devices to all effective antimicrobial devices. The
term effective here means that the devices remained in
the tibia post-surgery and were extracted from the live
rats after an incubation period. These results are summa-
rized in Table 3. Using a Fischer’s F-Test at 85% confi-
dence level, we can conclude that there is a statistically
signify- cant difference between antimicrobial and con-
trol devices.
Figure 9. Antimicrobial device #6.
Table 3. Summary of all devices for all surgeries (total)
Staph aureus free Staph aureus Total
Antimicrobial device 7 12 19
Control Device 0 7 7
Total 7 19 26

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925
During animal testing studies #1, antimicrobial de-
vices were found to be electrically disconnected proxi-
mal to the tibial cavity insertion point of 2 rats, render-
ing the devices electrically ineffective. If these 2 devices
are considered as control instead of antimicrobial devic-
es, the F-test concludes that there is a statistically sign-
ificant difference between antimicrobial devices and con-
trol devices at 95% confidence level. In addition, each
device design was compared with others to determine
any statistical significance. Strictly statistically speaking,
only antimicrobial device #1 and antimicrobial device #6
showed significant difference from the controls among
all six design iterations. Although 2 out of 3 animals
implanted with working antimicrobial device #1 tested
staph free, the device tip often penetrated the soft tissue
and caused irritation to the animals. On the other hand,
antimicrobial device #6 also resulted in 2 out of 3 ani-
mals being staph free but eliminated the problem associ-
ated with damaging the soft tissue.
6. DISCUSSION AND CONCLUSIONS
An early conjecture in this research was that a bacteri-
cidal environment would be created if we could get bac-
teria to conduct Ag+. The hope was to create this envi-
ronment within a bone because getting traditional antibi-
otics to penetrate the bone while the antibiotics are still
viable can be very difficult. A basic design concept was
developed and had to go through several iterations gov-
erned by rat comfort after implant as well as bactericidal
performance. Interestingly, the first and the last device
design iterations showed the greatest efficacy.
Based on the surgeries, pathology results and statisti-
cal analysis, the rat osteomyelitic model described in lit-
erature is validated, since all animals without an antimic-
robial device were infected. More importantly, the resu-
lts show that properly configured electrically stimulated
silver is an effective bactericidal agent for indwelling
devices. Of all the surgeries performed using the bacteri-
cidal devices, there is a statistically significant difference
between using no device and an antimicrobial device. As
the antimicrobial devices and surgical procedures were
refined throughout the study, the effectiveness of the
devices was found to be improved. In the last set of sur-
geries, 67% of the harvested animals were free of Sta-
phylococcus aureus even after they were inoculated with
the bacteria and given no antibiotics. The bactericidal
device as configured has a definite ability to reduce/
eliminate bacterial infection. Using such a bactericidal
device in conjunction with a standard treatment of anti-
biotics should have a profound effect on the number of
residual hardware associated bacterial infections.
7. ACKNOWLEDGEMENTS
The technology and designs tested within this study are protected
under U. S. Patent as owned by ArgentumCidalElectrics, Inc.. It is only
with their support that device modifications and manufacturing could
be properly completed and controlled.
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