lnvited Review
Performance of Subcutaneously Implanted
Glucose Sensors: A Review
Martijn Gerritsen, MD, and
John A. Jansen, DDS, PhD
Department of Biomaterials,
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University of Nijmegen,
Nijmegen, The Netherlands
Alexander Kros and
Roeland 1. M. Nolte, PhD
Department of Organic
Chemistry, University of
Nijmegen, Nijmegen,
The Netherlands
For personal use only.
Jos A. Lutterman, MD, PhD
Department of Internal Medicine,
University Hospital St. Radboud,
Nijmegen, The Netherlands
Received 12 May 1998;
accepted 13 May 1998.
This research was supported by the
Dutch Technology Foundation STW,
the applied science division of the
Dutch Science Foundation NWO, and
the technology program of the
Ministry of Economic Affairs.
Address correspondence t o John A.
Jansen, DDS, PhD, Department of
Biomaterials, Dental School, University
of Nijmegen, P.O. Box 9101,6500 HB
Nijmegen, The Netherlands.
E-mail: J.Jansen@dent.kun.nl.
Journal o f Investigative SurgerK
11:163-174.1998
Copyright 0 1998 Taylor 81Francis
0894-1939198 512.00 + .OO
ABSTRACT Despite a considerable amount of research attributed to
the development of an implantable glucose sensor, to date there is no
clinically applicable concept for continuous glucose monitoring. Investi-
gations to validate the subcutaneous tissue for continuous glucose sens-
ing mostly comprise short-term implantations of glucose sensors. Most
implanted glucose sensors showed a significant decay in sensitivity over
the implantation period. This bioinstability was not to be expected from
the in vitro performance of the sensors. In this article, the influence of
possible failure mechanisms on the poor in vivo performance of subcu-
taneously implanted glucose sensors is reviewed.
KEYWORDS continuous glucose sensing, implantable glucose sensor, subcuta-
neous tissue
P
atients with diabetes mellitus have an increased morbidity and
mortality due to specific and nonspecific complications. Recently
the Diabetes Control and Complications Trial Research Group
has shown that there is a clear effect of intensive treatment of insulin-
dependent diabetes mellitus on the development and progression of
specific complications such as diabetic retinopathy, nephropathy, and
neuropathy [l].
Intensive treatment comprises several blood glucose measurements a
day with subsequent adjustment of the insulin dosage. Even with in-
tensive treatment, the number of measurements is still limited and will
only provide information about blood glucose values at intermittent mo-
ments. Furthermore, intensive treatment for diabetes increases the risk
of severe hypoglycemia. As a consequence, continuous glucose sensing
could result in a more adequate insulin administration and could also
be of use in the early detection of hypoglycemia.
Many investigators have already investigated the possibility of contin-
uous in vivo glucose monitoring by an implantable glucose sensor, using
a wide range of different approaches. Despite this considerable effort,
currently there is no clinical application available. The subcutaneous
tissue is regarded as the most appropriate site of implantation, because
of good accessability for surgery and relatively easy replacement of the
163
 Invited Re view
sensor in case of impaired function. However, de-
spite good in vitro sensor performance, it was ob-
served that subcutaneously implanted glucose sen-
sors showed a significant decay in sensitivity over the
implantation period. Various explanations have been
brought forward, but in general there was no struc-
tural approach to assess the contributions of differ-
ent failure mechanisms to the functional instability
of implanted sensors. In this artic:le, the influence
of possible failure mechanisms on the in vivo beha-
vior of subcutaneously implanted glucose sensors is
reviewed.
J Invest Surg Downloaded from informahealthcare.com by McGill University on 02/19/13
DEFINING GLUCOSE SENSORS
Chemical sensors are miniaturized devices that
detect a chemical compound selectively and reversi-
bly. The detection results in a concentration-depen-
dent electrical or optical signal. Chemical sensors
that use biological molecules, such as enzymes, are
For personal use only.
called biosensors [2]. For a sensor to function as a
device for continuously measuring a chemical com-
pound at an intracorporal implantation site, certain
characteristics have to be met. These hnctional re-
quirements can all be related to the biocompatibility
of the sensor, indicating the ability of the implant to
perform with an appropriate host response in a spe-
cific situation [3]. This means that the sensor has to
be specific for the compound to be measured, needs
to retain its stability for prolonged periods of time in
tissue, and has to be able to detect rapid fluctuations
in concentration of the compound at the implanta-
tion site.
Several techniques are available to design an im-
plantable glucose sensor. The most frequently ap-
plied technique is based on the use of immobilized
glucose oxidase, a redox enzyme, in an electrochem-
ical sensor. In nature, this enzyme catalyzes the ox-
idation of glucose by molecular oxygen, producing
gluconolactone and hydrogen peroxide. Employing
this principle, three generations of electrochemical
glucose sensors have been developed. These are cat-
egorized based on the mechanism of charge transfer
between enzyme and electrode [4].
So-called first-generation glucose sensors estimate
the glucose concentration by measuring the amount
of hydrogen peroxide that is produced or the amount
164
of oxygen that is consumed by the oxidation of glu-
cose. The hydrogen peroxide-detecting sensor, most
frequently, has a membrane containing glucose oxi-
dase immobilized to an electrode. Glucose and oxy-
gen diffuse into the membrane, where the enzymatic
reaction produces hydrogen peroxide. This in turn
diffuses to the electrode, where the oxidation of hy-
drogen peroxide to oxygen produces an electrical
current proportional to the glucose concentration.
Important advantages of this type of glucose sensor
are easy fabrication and the possibility of construct-
ing small sensors. The oxygen detecting sensor also
has a membrane containing immobilized glucose ox-
idase coupled to an electrochemical oxygen sensor.
Glucose and oxygen diffuse through a membrane
and react, resulting in a reduction of the amount
of oxygen at the sensor site. The signal current is
related to a reference electrode without glucose ox-
idase. An advantage of the oxygen-based enzyme
electrode is the low electrochemical interference ow-
ing to the fact that by using a nonporous hydropho-
bic membrane, only gaseous molecules can reach
the electrode. Also, hydrogen peroxidase could be
co-immobilized in the membrane, resulting in less
glucose oxidase denaturation.
Instead of oxygen, second-generation amperomet-
ric glucose sensors make use of artificial electron car-
riers to shuttle the electrons from the enzyme to the
electrode. Ferrocene and its derivatives are the most
commonly used mediators in in vivo experiments.
The oxidation of these mediators can be performed
at lower potentials than that of hydrogen peroxide,
thus lowering electrochemical interference.
Third-generation amperometric glucose sensors
are based on the principle of direct electron transfer
between the enzyme and the electrode. Conduct-
ing organic salts or polymers have been used in the
construction of these electrodes. In this case, no co-
substrates such as oxygen or mediators are required.
However, no subcutaneous implantation studies
have been published using sensors based on this
principle.
SENSOR DESIGN
The construction characteristics of the subcuta-
neously implanted glucose sensors as found in the
M. GERRITSEN ET AL.
 lnvited Review

TABLE 1 Construction of glucose sensors

Group Enzyme Inner Outer
[ref] Mediator Configuration Electrodes Isolation immobilization membrane membrane
Bessman [63] Oxygen Two electrodes Galvanic Nylon, GA None PP
Shichiri Peroxide Needle Pt, Ag, Ag Glass C-di-A None PUIPVA
[26,51,52, Ferrocene Needle Pt, Ag Resin Carboxaldehyde PU PVAI
68-71,741 MPCcoBMA
Fischer Peroxide Cannula Pt, Ag Resin Sepharosel PEICA CNPUIPE
[24,25,27, HSA, GA
36,44,53,
59,76,77]
Pickup Peroxide Cannula Pt, Ag Polyester CA PU
[28,72,73] Ferrocene Cannula Pt, Ag Epoxy Sepharose None C
Ferrocene Needle Gr, Ag Epoxy None PU
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Moussy Peroxide Needle, skin Pt, Ag Vernace PPD Nafion

[14-161 button
Pfeiffer Peroxide Cannula Pt, Ag Epoxy CA, GA None PU
[22,38,75]
Updike Peroxide Plastic housing Pt, Ag, Pt Epoxy PU None Bioprotective
[10-12] membrane
Reach, Peroxide Wireheedle Pt, Ag Epoxy CNGA None PU/CA nafion
Wilson catheter BSNPBQ
[29-34,37,
42,43,
For personal use only.

62,781
Johnson [35] Peroxide Cannula Pt, Ag, Pt Polyimide BSA, GA None PU
Wilkins [I31 Peroxide Cannula, Pt, Ag, Pt Carbon None PCInafion
rechargeadle
Gough Oxygen Cylinder Pt, Ag, Pt SA, GA
[17,791 tissue chamber
Ward [9] Peroxide Disk shaped Pt, Ag Epoxy BSA, GA None PU (15 4 0 % )
Matthews Ferrocene Needle None
POI
Heller Redox Wire Pt, Ag, Allylamine, PVD None PC
[81,821 polymer gold aziridine
Koudelka Peroxide Planar Pt, Pt, Ag Epoxy BSA, GA None PU
[39-411 microcell
Clark [83] Peroxide Catheter Pt, Ag Cyanoacrylate GA A C
Vadaama Peroxide Needle Pt, SS Epoxy Polyether None PU
[6,56] copolymer
Note. PP, polypropylene; PE, polyethylene; PU, polyurethane; PC, polycarbonate; C, cellulose; A, acetate; GA, glutaraldehyde; (B)(H)SA, (bovine)
(human) serum albumin; PVA, polyvinylalcohol; PPD, poly(o-phenyldiamine); Gr, graphite; PVD, polyvinylpyridine; 55. stainless steel; PBQ, paraben-
zoquinone; MPC-co-BMA, 2-methcryloyloxethyl phosphorylcholine-co-n-butyl methcrylate.

literature are shown in Table 1. As can be seen, most
glucose sensors used in subcutaneous implantation
studies were electrochemical enzyme electrodes ba-
sed on the detection of hydrogen peroxide. Oxygen-
based enzyme electrodes and ferrocene-mediated
sensors were less frequently employed. A schematic
drawing illustrating the general layout, which is ap-
plicable to many glucose sensors, is shown in
Figure 1. In the majority of sensors two electrode
transducers were used, with platinum as working elec-
trode and Ag/AgCl as reference electrode. Epoxy
resin was most frequently used for electrode insula-
tion. Enzyme immobilization (glucose oxidase alone
or mixed with bovine serum albumin) was often ac-
complished by glutaraldehyde cross-linking. Inner
membranes between the working electrode and en-
zyme layer are known to reduce electrochemical in-
terference at the electrode [2,5]. Remarkably, in most
implant designs no inner membrane was used.
Most investigators used an outer interfacial mem-
brane to improve the performance and lifetime of
their subcutaneously implanted glucose sensors.

SUBCUTANEOUS GLUCOSE SENSORS 165

 Invited Review

used to enhance the performance of the implanted

sensors. For example, a rechargeable glucose sen-
sor has been developed that makes it possible to
replace immobilized glucose oxidase with fresh en-
zyme [13]. In another investigation, the implanted
sensors were connected to a skin button connector
to prevent infection and manipulation of the sen-
sor [14-161. The use of a tissue chamber to sup-
port the growth of vascularized subcutaneous tissue
around the implanted sensor was also described [ 171.
Figure 2 shows a percutaneous device as carrier of a
cannula-shaped implantable glucose sensor.
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FIGURE 1 Aschematic drawing showing the general layout

of an implantable glucose sensor. a = anode; b = cathode;
c = electrode insulation; d = inner membrane; e = glucose
oxidase; f = outer membrane.
For personal use only.

Such an outer membrane has several functions [2,

5-71, First, it is regularly used for entrapment of glu-
cose oxidase at the electrode, thus preventing leak-
age of the enzyme. This is called microencapsula-
tion. Furthermore, its selective permeability slows
the transport of proteins and interferents to the elec-
trode. By restricting glucose diffusion to the elec-
trode, the linearity of the response is increased, and if
the membrane is more permeable to oxygen than to
glucose, the oxygen dependence will also be reduced.
Finally, this membrane is in direct contact with the
subcutaneous tissue and therefore it is an important
determinant for the biocompatibility of the sensor.
Membranes made of cellulose acetate, polycarbon-
ate, polyvinylalcohol, polyurethane, and the perflu-
orinated ionomer Nafion were most commonly used
for these purposes.
Considering the actual configuration of the im-
planted sensor, needle- or cannula-shaped sensors
were most popular. Microfabricated planar thin-film
cells [8] and disc-shaped sensors [9] were also used.
In only three studies, the glucose sensor was fully
implanted together with a transmitter system in an at-
tempt to develop a continuously implantable biosen-
sor [lo-121. FIGURE 2 (a) A cannula-shaped implantable glucose sen-
In addition to variations in Sensor CO~struCtion sor. (b) A percutaneous device in a New Zealand white rabbit
and configuration, various design approaches were as carrier of the sensor.

166 M. GERRITSEN ET AL.

 Invited Review

TABLE 2 Operating characteristics of glucose sensors

~~~ ~

In vitro In vivo
sensitivity sensitivity
Group Species Sterilization Duration Calibration (nA/mM) (nA/mM)
Bessman Dog Ethylene oxide 5 days In vitro 50%
Shichiri Dog/human Ethylene oxide 3-7,14 days In vitro 0.21-0.93, 1.4 0.30-0.69, 103%
Fischer Dog UV light, none 3-96 h In vitro/in vivo 0.20-1.70 0.40-3.48
Pickup Humadpig 2-4,8 h In vitroh vivo 0.4, 2780 0.05
Moussy Dog Dry heat 10-1 4 days In vitro 6-25 3-1 0
Pfeiffer Humadsheep Propanolol 7h In vitro 0.7 0.2-1.5
Updike Dog Thimerosal 20-144 days In vivo/in vitro 0.3 245%
Reach/ Rat/dog/human 2 h-10 days In vivo 0.9-3.6, 0.3-1.5, 4
Wilson 1000-3000
Johnson RabbiUhuman 30,72 h In vitro/in vivo
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Wilkins Sheep lh In vitro 500

Gough Rat Glutaraldehyde 10 days In vitro
Ward Rat 1.5 h In vitro 0.043, 0.24 0.028, 0.080
Matthews Rat/human y-Irradiation 4.5, 6 h In vitro 40-50
Heller Rat/dog Ethylene oxide Hours In vitro/in vivo 0.2-0.3 0.15-0.2
Koudelka Rat y-Irradiation 3 h-8 days In vivo 1.5-3.1 0.6-2.0
Clark Cat/dog 4h In vitro
Vadgama Rat 4h In vitro
For personal use only.

IN VlVO EVALUATION 100% of the plasma glucose concentration. Over-

OF IMPLANTED SENSORS
The in vivo operating characteristics of the glucose
sensors as listed in Table 1 are shown in Table 2.
This table demonstrates that glucose sensors have
been implanted in humans and a number of animal
species like rat, rabbit, dog, pig, and sheep. Notice
that different procedures were employed for steriliza-
tion of the sensors before implantation, like ethylene
oxide, propanolol, thimerosal, dry heat, and y-irra-
diation. Occasionally, no sterilization was performed
prior to implantation. Needle-shaped glucose sen-
sors based on hydrogen detection were most fre-
quently used in subcutaneous implantation studies.
The sensors remained in situ for periods ranging
from 1 h up to 144days. The majority of sensors,
however, only functioned for several hours to sev-
eral days.
In vivo sensitivities to glucose ranged from as low
as 0.2 to over 100% compared to in vitro values
and, in general, were lower. Absolute sensor cur-
rents, if mentioned at all, ranged from 28 pA/mM
to 50 nA/mM. Subcutaneous tissue glucose concen-
trations ranged between approximately 20 and over
all, the subcutaneous glucose concentration in the
various studies closely followed alterations in blood
glucose values during steady-state conditions and
oral glucose tolerance tests. However, rapid changes
in blood glucose concentration, as induced by in-
sulin and glucose infusions, were associated with de-
lays ranging from less than 1 min to approximately
45 min.
Frequently, in vitro calibration of the sensor prior
to implantation and after explantation was used to
interpret the sensor current during implantation. Less
frequently, one- or two-point in vivo calibration pro-
cedures were used to relate variations in sensor cur-
rent to changes in glycemia. Almost all implanted
glucose sensors generally showed a significant decay
in sensitivity during the implantation period. By in
situ calibration or replacement, the lifetime of the
sensor was prolonged, but mostly the duration of
sensor implantation did not even exceed the wound
healing period. Further, taking into account that dif-
ferent processes involved in sensor implantation, like
the wound healing process and the host response to
the implant, are likely to cause a decay in sensor
sensitivity, the physiological relevance of the sensor

SUBCUTANEOUS GLUCOSE SENSORS 167

 Invited Review
signal during this initial period has to be questioned.
In the next section the influences of possible failure
mechanisms are discussed.
FAILURE MECHANISMS
In general, subcutaneous implantation of a glu-
cose sensor resulted in a gradual decrease in sensi-
tivity, and eventually in a complete loss of sensor
function within hours or days. In this section, pos-
sible contributions in sensor failure related to the
properties of implanted sensors, calibration proce-
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dures, healing phenomena, and interfacial reactions
around subcutaneously implanted glucose sensors
are addressed.
Sensor Properties
The observed decay in sensor sensitivity after im-
plantation can be related to the design of the sen-
sor [4,18-211. The main problems associated with
For personal use only.
hydrogen peroxide detection are limitation of the
enzyme reaction due to an oxygen deficit, and elec-
trochemical interference by small endogenous mole-
cules. Oxygen limitation can for the greater part be
avoided by implantation of a reference oxygen sen-
sor, reduction of the reaction area in relation to the
oxygen permeation area, the use of membranes with
high oxygen solubility, or selective reduction of glu-
cose diffusion to the enzyme by a hydrophobic outer
membrane. Electrochemical interference can be re-
duced to a large extent by the use of selective mem-
branes or nonconducting electropolymerized films.
The major disadvantage of oxygen-based enzyme
electrode is that the sensor output depends on the
ambient oxygen concentration at the implantation
site. Implantation of a reference oxygen sensor is
needed to correct for these fluctuations. This, in turn,
leads to problems in the construction and miniatur-
ization of the sensors.
A serious problem associated with second-genera-
tion glucose sensors is mediator leakage and toxi-
city. Improved immobilization of the mediator by
adsorption or covalent binding to the electrode, or
entrapment in a polymer matrix or albumin matrix
are possible solutions. This limited the application
of these sensors for glucose sensing in vivo. Lack of
168
knowledge of the toxicity and carcinogenicity of the
new materials used in third-generation glucose sen-
sors also limited their in vivo application.
Calibration Procedures
For the final clinical application, the output of a
subcutaneously implanted glucose sensor has to be
related to the actual glucose concentration in the
subcutaneous tissue. Frequently, an in vitro calibra-
tion technique is used prior to implantation and after
explantation to interpret the sensor current during
implantation. In this procedure, it is assumed that
the basal sensor current and the sensitivity of the
sensor are identical under both in vitro and in vivo
conditions. This is not realistic, because we know
that after implantation sensor performance changes
[ 11 16,22,23].
Since the final aim is to use a glucose sensor for
long implantation periods, frequent in vitro calibra-
tion is no solution. Consequently, so called one- or
two-point in situ calibration methods have been de-
veloped [10,11,16,24-421. In the one-point calibra-
tion procedure the sensor output at a certain time
afier implantation is related to the simultaneously
measured plasma glucose level, resulting in an in
vivo sensitivity coefficient. In the two-point calibra-
tion method the plasma glucose level and sensor out-
put reach a new plateau following insulin or glucose
infusion. It is possible to calculate an in vivo sen-
sitivity coefficient from the sensor currents during
the two steady-states. The obtained in vivo sensitiv-
ity coefficient is then used to determine an apparent
subcutaneous glucose concentration from the sensor
output and to estimate its variations during changes
of blood glucose concentration.
Generally, the two-point in vivo calibration pro-
cedure is considered to be the most reliable [43].
However, since this method requires the induction of
blood glucose alterations, its feasibility for daily clin-
ical practice can be questioned. As a consequence,
other techniques like linear regression analysis of
paired plasma/sensor current values have been pro-
posed, which would not require induced adjust-
ments [44]. The clinical practability of all these cal-
ibration methods is still unclear.
M.GERRITSEN ET AL.

Healing Phenomena Around
Implanted Devices
The subcutaneous implantation of a sensor will
always result in a tissue response. This response is
a combination of the healing process of the wound
and the host response to the implant [3,45-471. The
healing process comprises two phases, an inflamma-
tory phase followed by a repair phase.
The inflammatory phase, which takes about 3 days,
starts after infliction of the wound. Implantation of
the sensor damages cells, blood vessels, and con-
nective tissue, initiating a number of healing mech-
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anisms. Initially, the wound bed fills with blood,
followed by activation of the coagulation system.
Platelets bind to exposed collagen leading, to throm-
bus formation. The complement system is activated
by exposed collagen and extracellular ATP, induc-
ing vasodilatation, changes in blood flow, and an
increase of the vascular permeability [48]. Th’is re-
For personal use only.
sults in the formation of an extracellular exudate at
the implantation site containing plasma proteins and
other mediators. By increasing the vascular perme-
ability, the compliment system also facilitates the
migration of phagocytic leukocytes to the damaged
tissue [47,48].These cells, mainly polymorphonu-
clear granulocytes and monocytes, are attracted and
activated by complement mediators and chemotac-
tic substances released by platelets and damaged cells.
They start phagocytosis of injured tissue fragments
and microorganisms. Polymorphonuclear granulo-
cytes have a life span of only a few days in the tis-
sue. They are end-state, nondividing cells and obtain
their energy primarily from the anaerobic metabo-
lism of stored glycogen. This makes them able to
persist in areas with highly disturbed metabolic
states. After fulfilling their function, they die rapidly.
The presence of large numbers of live neutrophils
in tissue is evidence of continued inflammatory
challenge.
The monocytes differentiate into macrophages
[47]. The activated macrophage is not an end-state
cell; it has an aerobic glycolysis and is able to un-
dergo a number of transformations. Macrophages
phagocytize injured tissue, debris, and bacteria by
releasing degrading enzymes. Furthermore, they re-
lease substances that stimulate neovascular growth
SUBCUTANEOUS GLUCOSE SENSORS
Invited Review
and the proliferation of fibroblasts, leading to col-
lagen synthesis. These events mark the start of the
repair phase. This form of repair continues until the
defect is closed. The tissue formed is called granu-
lation tissue. Later, remodeling of the wound takes
place, in which a new balance between collagen syn-
thesis and lysis is reached. Myofibroblasts are respon-
sible for reducing the wound surface through wound
contraction. Finally, cell numbers will decrease, leav-
ing scar tissue behind.
The presence of an implant prevents a return to
the original situation. It can provide a continuous
inflammatory stimulus and result in a prolonged in-
flammatory phase, associated with increased cellu-
lar activity. However, it is still possible to achieve
a chronic phase in which no progressive biological
changes occur. This is called resolution [31. The time
required to enter this steady state varies according
to the host site and species, the trauma imposed
during implantation, the variables of normal wound
healing, and the chemical composition, micro- and
macrostructure, and mechanical properties of the
implant. The possible ways by which resolution is
achieved are extrusion, resorption, integration, and
encapsulation. Encapsulation is the usual response
of the host to an implant. The fibrous capsule is
maintained by the presence of the implant and its
thickness is influenced by several factors, like chem-
ical properties of the implant material, shape of the
implant, and mechanical factors at the tissue-implant
interface. Implanted electrodes-for example, glu-
cose sensors-are known to additionally influence
capsule formation through electrical currents,
changes in local pH and oxygen tension, or the re-
lease of corrosion products [3]. The influence of
implant-host interactions and the host response on
the performance of implanted glucose sensors is dis-
cussed next.
Interfacial Reactions Around
Subcutaneously Implanted
Glucose Sensors
Initial Events
The initial step in the complex cascade of interac-
tions between the implant and the host is contact of
169
 Invited Review
the sensor with blood and extracellular tissue fluid.
Biological molecules from the exudate that forms
around the implant, mainly proteins, are attracted to
the implant-tissue interface. Because of their chem-
ical heterogeneity, proteins have the tendency to
adsorb at interfaces [23,46,49,50]. This results in cov-
erage of the implant surface within seconds to min-
utes, followed by a competitive exchange of different
proteins, called remodeling. Mostly, the adsorbed
layer does not exceed a monolayer. In protein ad-
sorption from blood this is called the Vroman effect.
In general, the composition of the adsorption layer is
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strongly dependent on the characteristics of the sur-
face of a particular sensor [23,49]. To what extent this
deposition acts as a diffusive barrier for glucose is un-
known. The influence of the remodeling process on
sensor performance is also unclear. Postimplantation
in vitro evaluation frequently showed sensor sensitiv-
ities similar to preimplantation values [9,11,14-16,
22,26-28,32,36,37,40,41,43,5 1-54]. This indicates
For personal use only.
that the formation ofa protein layer does not prevent
glucose diffusion to the sensor. However, it cannot
be not excluded that the removal of the sensor from
the implantation site disrupted the integrity of the
adsorption layer and, in this way, influenced sensor
performance. Occasionally, the in vitro sensitivity
of explanted probes was lower than before implan-
tation, but returned gradually in buffered glucose
solutions [11,16,221. This could have been caused
by a gradual desorption of adsorbed molecules from
the sensor surface [23].
Open-flow microperfusion has been used to im-
prove sensor stability. This technique uses slow flow
of isotonic phosphate buffer over the sensor tip to
reduce electrode fouling by proteins. It was demon-
strated that upon implantation only a minor reduc-
tion in output occurred, which was not progressive.
After implantation, the in vitro sensitivitywas found
to be equal to the sensitivitybefore implantation, in-
dicating little or no surface fouling [55,56].
Thrombus formation due to damage of small cap-
illaries could further inhibit glucose diffusion by cov-
erage of the sensor surface [16,17]. Manipulations of
the sensor during implantation can cause repetitive
small hemorrhages at the implantation site, with a
subsequent drift in sensor signal [8,22,38]. Since the
needle-type sensor configuration is frequently used
170
in implantation studies, this problem needs further
consideration.
lntermediate Events
In the inflammatory phase of the wound healing
response, the output of implanted glucose sensors
could be influenced by the actions of inflammatory
cells. After attachment to the surface of the implant,
these cells release various reactive oxygen radicals in
the so-called respiratory burst. This is followed by
the secretion of a multitude of proteolytic enzymes,
resulting in a decrease of the tissue pH [48,57]. The
adsorption of cells and proteolytic enzymes on the
sensor surface could prevent glucose from enter-
ing the sensor. Another possibility is that these
products may damage components of the sensor,
like glucose oxidase, and impair sensor function-
ing [23]. O n the other hand, glucose oxidase has
been shown to be very resistent to proteolysis and
pH changes [58].
The observation that most explanted glucose sen-
sors are still capable of glucose detection could also
indicate that low concentrations of analyte were
present at the implantation site. Inflammatory cells,
mainly monocytes and macrophages, are known to
alter the glucose concentration of the exudate fluid
at the implantation site [59]. The damage inflicted
on the subcutaneous tissue upon implantation can
also result in a limitation of the blood supply to
the sensor surroundings, and can cause a decrease in
sensor output through poor availability of glucose
and oxygen. Sensor output was shown to drop to
approximately background levels in the first days of
implantation, after which the output gradually rose
to stabilize at a new level. This increase was attributed
to the development of sufficient blood supply to the
tissue surrounding the sensor [ 10,113.
The rate of glucose oxidation is also dependent
upon the availability of oxygen. For every oxygen
molecule in the interstitial fluid there will be an ex-
cess ofglucose molecules present [60]. This can cause
oxygen limitation of the enzyme reaction. Insuffi-
cient blood supply, resulting in a lack of oxygen at
the implantation site, is likely to further increase the
oxygen dependence ofglucose oxidase based sensors.
In in vitro experiments an oxygen independence of
M. GERRITSEN ET AL.

the implantable sensors was shown down to oxygen
tensions that are thought to be present in the subcu-
taneous tissue. The influence of tissue oxygenation
on in vivo sensor performance has not been studied
extensively. Occasionally, implantable oxygen sen-
sors were used to investigate the oxygen dependence
of implanted sensors. In general, it was found that
the output of the sensors was not influenced by
alterations in oxygen tension at the subcutaneous
implantation site [ 17,60-621. Nevertheless, Bessman
et al. [63] noticed a distinct influence of tissue oxy-
gen tension on sensor readings and stated that all
J Invest Surg Downloaded from informahealthcare.com by McGill University on 02/19/13
glucose electrodes based on glucose oxidase will re-
quire correction for oxygen tension by a reference
oxygen electrode. The fact that second-generation
glucose sensors, where output is not influenced by
variations in oxygen tension, also showed a decay in
sensitivity upon implantation indicates that oxygen
limitation is not the only problem that has to be
overcome.
For personal use only.
Late Events
In the repair phase of the healing process, the for-
mation of a fibrous capsule around the sensor cre-
ates a diffusional barrier to glucose. This is thought
to influence sensor response time, dependent on the
thickness of the capsule [64]. Further, it can be ques-
tioned whether the transsudate within the fibrous
capsule surrounding implanted sensors is at all in dy-
namic equilibrium with glycemia and provides phys-
iologically relevant data [7,65]. By using a difhsion
model, Sharkawy et al. [66] estimated that the for-
mation of a fibrous capsule will increase the lag-time
of a sensor to detect 95% of the blood analyte con-
centration from approximately 20 min (no fibrous
capsule) to 60 min (with a 200-pm-thick fibrous cap-
sule). Clearly, this estimated increase is dependent
on the fibrosity and vascularity of the capsule sur-
rounding the sensor.
In only a few studies the performance of implanted
glucose sensors was related to the structure of the
subcutaneous tissue surrounding implanted sensors.
Relatively thin, well-vascularized fibrous capsules
were found, and a rapid adjustment of the glucose
concentration in the interstitial fluid within the cap-
sule to blood glucose changes was demonstrated
SUBCUTANEOUS GLUCOSE SENSORS
Invited Review
[ 10,16,17,29,67]. Also, sufficient oxygen tensions
have been reported to exist within a fibrous capsule
[10,16]. In one study, a layer of granulation tissue
with numerous small vessels was found surrounding
the sensor several weeks afier implantation, indicat-
ing a persistent inflammatory reaction [ l l ] . From
these results it can be assumed that sufficient vas-
cularization of the fibrous capsule is necessary for a
rapid response of encapsulated glucose sensors. Im-
proved sensor performance is needed to assess the
physiological relevance of this response.
PROSPECTS
Despite a considerable effort, there is no clinical
concept available for continuous subcutaneous glu-
cose monitoring. In general, good in vitro results
were achieved with differently designed implantable
glucose sensors. Upon implantation, however, these
devices showed a progressive loss of sensor function.
This can be regarded as poor bioperformance of the
implanted sensors. The fact that the in vitro function
of explanted sensors was ofien unaffected indicates
that the environment surrounding the sensor plays
an pivotal role in the behavior of implanted sensors.
Questions concerning the influences of wound heal-
ing, host response to the implant, and structure and
blood supply of the surrounding tissue on sensor
performance need to be addressed systematically. In
addition, more insight is needed in the physiological
processes at the sensor-tissue interface.
The achievement of this knowledge implies an in-
terdisciplinary approach at a basic scientific level.
In vitro experiments can reproduce many elements
of the in vivo situation and should be used more
often to bridge the gap with implantation studies. In
vivo experiments can then be reserved for the more
promising approaches. Animal models seem more
suitable in this respect than human subjects, since in-
terventions and histological processing are more
easily performed. Only with a better understanding
of the processes involved in sensor inactivation is it
possible to develop adequate strategies to improve in
vivo sensor performance. It also seems worthwhile to
simultaneously investigate possibilities of influenc-
ing the tissue reaction to implanted devices.
171
 Invited Review
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