Departments of 1 Biomedical Engineering and 4 Plastic Surgery, Duke University Medical Center, Duke University, Durham, North Carolina 27710; 2 Department of Physiology and Pharmacology, Karolinska Institute, Stockholm S-17177; and 3 Department of Medicine, Karolinska Institute, Danderyd Hospital, Stockholm S-18288, Sweden
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ABSTRACT |
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The rat is commonly used to evaluate physiological responses of subcutaneous tissue to implanted devices. In vivo longevity of various devices and the biocompatibility of biomaterials depend on how adjacent tissue interacts. How closely the rat model predicts the human response has not been well characterized. The objective of this study was to compare rat and human subcutaneous foreign body responses by monitoring the biochemical environment at a polymer-tissue interface over 8 days using microdialysis. Polyamide microdialysis probes were implanted subcutaneously in humans and rats (n = 12). Daily microdialysis samples were analyzed for glucose, lactate, pyruvate, glycerol, and urea. Blood glucose was also monitored. Analyte concentrations differed significantly between rats and humans at the implant-tissue interface. There were also qualitative differences in the 8-day trends. For example, over 8 days, microdialysate glucose increased two- to fourfold in humans but decreased in rats (P < 0.001). This study reveals profound physiological differences at material-tissue interfaces in rats and humans and highlights the need for caution when extrapolating subcutaneous rat biocompatibility data to humans.
wound healing and foreign body responses; microdialysis; diffusion; glucose monitoring; biosensor biocompatibility
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INTRODUCTION |
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SUBCUTANEOUS TISSUE is a common target site for implantation of in vivo glucose sensors (8). Several studies have shown that tissue changes caused by the foreign body reaction surrounding an implanted sensor can lead to sensor signal decline and ultimately to sensor failure (8, 9). Two phenomena involved in the foreign body response (membrane biofouling and tissue encapsulation) have been implicated in creating permeability barriers that decrease analyte flux from the tissue into the sensor (19, 26). In addition to the physical barriers of biofouling and encapsulation, metabolic changes near the implant-tissue interface may alter the absolute concentrations of analytes in the vicinity of the sensor (8). Last, the exclusion or proliferation of blood vessels around an implanted device will dictate the availability of blood-borne analytes to the device (20). The extent and duration of inflammation, angiogenesis, and other stages of the wound healing and foreign body responses (which are believed to be governed by material-tissue interactions; see Ref. 1) may alter the concentration and transport of analytes at a sensor surface.
The rat model is frequently used to evaluate biomaterials (3), sensors (16), and other devices (5) because of the ease of handling, the low cost, and the vast amount of data for comparison from past studies. However, the extent to which the rat accurately predicts the human response to a subcutaneous foreign body remains unclear. It has been shown that glucose transport rates through a fibrous encapsulation layer in rat subcutis can be one-half of that through control rat subcutis (19). It is not known whether the tissue response to an implanted sensor in humans would cause the same decrease in glucose transport as in rats. One recent study investigated differences in the foreign body reaction between five strains of rats and three strains of mice (12). Although transport was not characterized, some histological differences were found between strains, and clearly significant differences were found between species. The findings of the mouse-rat study cast doubt upon the assumed similarities of the foreign body reaction in rats and humans. It is hypothesized that sensor signals should decrease more rapidly in rats than in humans because of the supposedly more aggressive foreign body reaction that takes place in rats.
Microdialysis is a technique that collects substances from interstitial
fluid through diffusion across an implanted semipermeable hollow fiber
membrane (21; Fig. 1). Both sensors and
microdialysis probes require analytes to passively diffuse across a
membrane from the interstitium. Any physical or physiological changes
in the adjacent tissue can affect the output of either device. Because sensors have multiple complex components, it is difficult to
deconvolute the contribution of various causes of signal declines
(electrical issues, enzyme degradation, biofouling, encapsulation,
membrane delamination, etc.; see Ref. 25). The simpler
construction of a microdialysis probe will allow changes in glucose
detection to be attributed to material-tissue interactions (as long as
periods of constant blood glucose are maintained).
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The objective of the present study was to compare the response of rat and human subcutaneous tissue with chronically implanted microdialysis probes over 8 days. To date, no such direct species comparison has been performed with the same materials and the same device. Changes in glucose, lactate, pyruvate, urea, and glycerol flux into the microdialysis implant were monitored over 8 days.
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METHODS |
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Subjects. Under the approval of the Duke University Institutional Animal Care and Use Committee (protocol no. A071-00-02-1), six male Sprague-Dawley rats (300-400 g) with surgically implanted jugular vein catheters were obtained (Charles River Laboratories, Raleigh, NC). Throughout the study, rats were maintained in the Duke University Vivarium and were given food and water ad libitum.
Six healthy human volunteers were recruited under the approval of the Ethical Research Committee at the Karolinska Institute (Stockholm, Sweden; KI Forskningsetikkommitte Nord Dnr 99-132). The age range was 24-25 yr with a mean age of 24.3 yr. The range of body mass index (BMI) was 19.6-26.3 with a mean BMI of 22.6. A detailed description of the study was given to volunteers before the start of the investigation. All volunteers provided informed consent.Experimental design. Each rat or human received two microdialysis probes that remained implanted for 1 wk. A total of 12 probes was tested in each group (n = 6 subjects × 2 probes). Each day before sample collections, a 15-min equilibration period was allowed consisting of a 6-min flush at 15 µl/min and a 9-min perfusion at 2 µl/min. Steady-state conditions were confirmed, since there was no net increase or decrease in the concentrations of the four subsequently collected microdialysate samples. On the day of the probe insertion (day 0), flow was initiated immediately after insertion, but fluid was not collected until after a 35-min equilibration period (6-min flush at 15 µl/min and 29 min at 2 µl/min). On day 0 and each subsequent day, four 10-min microdialysate fractions were collected immediately after the equilibration period.
Corresponding blood glucose values were collected between dialysate samples 1 and 2 and between samples 3 and 4. Human blood was obtained through standard finger pricks, and rat blood was obtained from the tail. On the 8th day (postoperative day 7), probes were withdrawn. Samples were taken for sterility verification, histology, or scanning electron microscopy (EM). Before probe insertion on day 0 and each day before collection periods, rats were anesthetized with pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, IL) administered intraperitoneally at a dose of 50 mg/kg body wt. On postoperative day 7 after the final microdialysate collection period, but before probe excision, rats were killed with an overdose of pentobarbital sodium. No anesthetic was administered to the human subjects at any time.Microdialysis equipment and perfusion fluid. In the present study, the probes consisted of a 30-mm-long dialysis membrane (polyamide with a 20,000 molecular weight cut-off) with an outer diameter of 0.60 mm (CMA/60; CMA/Microdialysis, Solna, Sweden). The microdialysis probes were connected to sterile microperfusion syringes, which were placed in a microinfusion pump (CMA/107; CMA/Microdialysis, N. Chelmsford, MA). Sterile Ringer solution was perfused through the microdialysis probes.
Insertion of microdialysis probes in healthy volunteers. To maintain a constant level of blood glucose at the time of measurement, healthy volunteers were asked to eat their regular meal 2 h before the start of the experiment, which was preformed with the volunteers in the supine position. Volunteers were asked to maintain a regular sleep schedule. No local anesthesia was used before the insertion of the microdialysis probe. The skin was disinfected with 70% ethanol before probe insertion. Two microdialysis probes were introduced in the periumbilical subcutaneous tissue ~10 cm on both sides of the umbilicus, as per the manufacturer's instruction. Sterile transparent tape (Opsite; Smith & Nephew Medical) was placed over the insertion point and a portion of the inflow and outflow tubing to hold the probes in place. The Opsite was changed after 4 days, or more often if sweat or fluid was observed under the Opsite.
Insertion of microdialysis probes in rats. The dorsum of each anesthetized rat was shaved and disinfected with Providone Iodine Cleansing Solution (Baxter Healthcare, Deerfield, IL). Sterile technique (including sterile probes, gloves, masks, gowns, surgical instruments, drapes, etc.) was used throughout the entire procedure.
Bilateral incisions were made on the dorsum ~4 cm below the left shoulder. Probes were inserted parallel to the spine ~2 cm from the midline on either side. The wing of the probe was sutured under the skin using 4-0 nylon suture (Ethicon, Somerville, NJ). The inflow and outflow tubing was tunneled under the skin to the back of the neck at which point it exited the body. The incisions on the back and neck were sutured and sealed with Vetbond (3M Animal Care Products, St. Paul, MN).Sample analysis. The concentrations of glucose, lactate, pyruvate, urea, and glycerol were analyzed in all microdialysis samples using a CMA/600 Microdialysis Analyzer (CMA/Microdialysis). All blood samples were immediately analyzed after sampling on an ACCU CHEK or ACCU TREND Glucometer (Roche Diagnostics, Mannheim, Germany).
Calculations and data analysis.
Transport of glucose through the membrane (Ed) is
characterized by recovery as follows: Ed = (Cdialysate Cperfusate)/(Cextracellular
Cperfusate), where Cdialysate is the
concentration of glucose in the outflow, Cperfusate is the
concentration of glucose in the inflow, and Cextracellular
is the concentration of glucose in the medium surrounding the probe.
Explantation of implants. After the last measurement on the final day in healthy volunteers, the probes were withdrawn. Four probes were preserved for scanning EM. The probes were immediately fixed in 4% buffered gluteraldehyde. Subsequently samples were dehydrated in graded ethanol, dried with a critical point dryer, sputter-coated with gold and paladium, and viewed with a scanning EM (model 501; Philips, Eindhoven, The Netherlands).
In rats, the probes were explanted through dissection. Samples were taken for sterility verification, histology, and scanning EM. The sterility verification was preformed by standard culture on LB agar plates at 37°C for 24 h. The explanted rat probes with surrounding tissue intact were fixed in 10% buffered formalin for light microscopy. Subsequently, specimens were dehydrated, mounted in paraffin, cut into 6-µm sections, and stained with hematoxylin and eosin and Masson's Trichrome. Scanning EM samples were prepared as described above. ![]() |
RESULTS |
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Subcutaneous metabolites detected in the microdialysate.
Comparisons of rat and human metabolites over 8 days are shown in Fig.
2, A-E. Glucose dialysate
concentrations and trends in rats were very different from those from
humans. Rat glucose decreased from 4.3 ± 0.3 (mean ± SE,
n = 12; 6 subjects × 2 probes/subject) to
2.8 ± 0.4 (P < 0.001) nM over the 8 days; in
healthy humans, glucose increased from 0.7 ± 0.1 to 1.9 ± 0.2 (P < 0.001) over the first 4 days and then
remained constant (Fig. 2A). There was very little blood
glucose variation from day to day in either species, and absolute blood
glucose levels in rats and humans were similar. Rat pyruvate on
day 0 (0.112 ± 0.005 mM) was not significantly
different from day 7 (0.180 ± 0.044 mM;
P > 0.05); however, the highly variable peak on
day 3 was significantly different from day 0 (P < 0.05) but not day 7. In humans,
pyruvate increased from 0.049 ± 0.008 mM at day 0 to
0.117 ± 0.021 mM (P < 0.01; Fig. 2B).
In rats, lactate levels rose from 0.8 ± 0.1 to 3.0 ± 0.5 mM
(P < 0.001), and in humans levels rose from 0.3 ± 0.1 to 1.7 ± 0.3 mM (P < 0.001) over the
implantation time (Fig. 2C). Rat urea increased from
3.8 ± 0.5 to 4.7 ± 0.4 mM (P < 0.05), whereas human urea increased from 1.2 ± 0.2 to 3.1 ± 0.2 mM
(P < 0.001; Fig.
3D). Glycerol trends differed
from most other analytes, and this was the only measured analyte where
human values were higher than rat values. In rat dialysate, glycerol
decreased from 0.078 ± 0.007 to 0.025 ± 0.004 mM
(P < 0.001). In humans, glycerol levels increased and
then decreased; however, changes were insignificant because of the
variability observed in this analyte (Fig. 3E).
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Dialysate glucose compared with blood glucose. Figure 2F shows the glucose dialysate-to-blood ratios. These ratios increased initially over the first 24 h in both species. After that time, continuous declines were seen in rats over the next 6 days (from 102 ± 1 to 45 ± 6% recovery). Human glucose recovery increased (from 13 ± 2 to 37 ± 5% recovery) and then plateaued by day 4. To emphasize the slope of the trends of the glucose dialysate-to-blood ratio in both species, curves in Fig. 2F were each plotted on their own axes. If these curves share the same axis, the graph appears similar to the raw dialysate glucose trends in Fig. 2A.
Statistical comparison of rat and human dialysate concentrations.
On each day throughout the implantation time, differences in rat and
human microdialysate metabolite concentrations are large. Table
1 shows the statistical comparison
between rats and humans for each metabolite on each day. All
metabolites were significantly different in rats compared with humans
except glucose and pyruvate on day 7 and glycerol on
day 0. In addition to absolute concentration differences
between rats and humans, the trends over the 1-wk period are
significantly different between the two species. Lactate trends appear
most similar between the two species, but rat lactate increases with a
steeper slope than human lactate.
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Histology and scanning EM. No obvious differences were seen between rat and human scanning EM of explanted probes. Probes from both species showed an accumulation of material (fibrous and cellular in nature) on the surface of the membrane.
Histological sections of control tissue (human control tissue taken from a cadaver) showed vast differences in the morphology of human and rat subcutis (Fig. 3, A and B). Adipose cells dominate the human subcutis, whereas collagen composes the majority of the rat subcutis. In the human subcutaneous sample, the vast majority of the area is occupied by adipose cells, which are massive in size compared with the smaller and more disperse cells of the rat subcutis. Much more interstitial space is visible (white space) between cells and collagen fibers in the rat tissue than between the fat cells of the human tissue. (The interiors of adipose cells are not stained by Masson's Trichrome dyes; hence, the off-white color that surrounds each fat cell is the cell membrane and interstitial space.) Figure 3C shows a probe after 8 days of implantation in rat subcutaneous tissue. The cellular density in the subcutaneous tissue surrounding the probe greatly increased over the 8 days as leukocytes migrate into the area (Fig. 3B). Fewer blood vessels are in the vicinity of the probe. No human probe histology samples were collected. ![]() |
DISCUSSION |
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After implantation of a device such as a biosensor or microdialysis probe, the surrounding tissue undergoes a phase of wound healing and foreign body reactions that alter the environment around the device. Factors that influence analyte concentration and flux in and around implanted devices fall into the following three main categories: 1) physical factors, 2) vascular factors, and 3) metabolic factors.
Physical factors. Physical factors control the diffusion rate of analytes from the interstitium into the device. They include encapsulation, biofouling, tissue void volume, and other characteristics that would affect the diffusion coefficient of a given analyte as it traverses a path through the tissue and into the probe.
Vascular factors. Vascular factors control the supply and removal of substances from the localized area around the probe surface. Angiogenesis in the early stages of wound healing and the exclusion or inclusion of vessels at the implant surface in later stages of the foreign body response have been characterized (1, 3, 19). Some vascular factors that will influence the analyte availability to the probe include vessel proximity, vessel length density, vascular perfusion, and vascular permeability.
Metabolic factors. Metabolic factors control the cellular consumption or production of analytes. It is well known that cells in wounded tissue function in a hypermetabolic state of repair (10). In the typical foreign body response, macrophages populate the area immediately adjacent to the implant surface. It has been shown that activated macrophages in wounded areas consume high levels of glucose and produce extremely high levels of lactate, even under aerobic conditions (4, 7, 24). Because local metabolism fluctuates during the foreign body response, extracellular metabolite concentrations may fluctuate depending on the level of exchange with the local vasculature. Metabolic factors that could affect flux into a device include cellular uptake, cell metabolism, and cellular output of the analytes of interest.
For the three-factor tissue model described here, we assume that the analytes of interest in this study are not consumed, produced, or bound in the extracellular space. The DISCUSSION focuses on explanations of the observed results based on physical, metabolic, and vascular changes that occur in the wound healing and foreign body responses.Significance of selected flow rate. Figure 2F shows dialysate glucose as a ratio of blood glucose (%recovery) in rats and humans. Glucose recoveries <100% indicate that concentrations detected in the subcutaneous microdialysate were less than glucose levels in the blood. It is expected that dialysate glucose should be lower than capillary glucose in this study because of the relatively fast flow rate used. It has been shown that very slow perfusion flow rates (such as 0.16 µl/min) allow complete equilibration of microdialysate in some tissues (at which time microdialysate glucose equals tissue glucose; see Ref. 17). Equilibration is desirable for most microdialysis applications; however, higher, nonequilibrium flow rates yield additional data about how the tissue is changing in response to the implanted microdialysis probes. For instance, with slow flow rates, absolute tissue concentrations are obtained, but changes in transport over time resulting from physical or vascular remodeling of the tissue are not detected. Operating at a faster flow rate of 2.0 µl/min will reveal changes resulting from the combined effects of physical, vascular, and metabolic alterations at the implantation site.
Differences in absolute concentrations between rats and humans. Before we consider the metabolite trends in each species, we see there are large differences between the absolute metabolite concentrations in the microdialysate from rats and humans. For instance, microdialysate glucose ranges from 2.8 to 5.9 in rats and 0.2 to 1.3 mM in humans on day 0, even though blood glucose levels are comparable. One reason for the differences could be that the whole tissue metabolite concentrations are significantly different in rats and humans. It is unlikely that this is the major factor causing the observed differences. Physical and vascular factors may provide some explanation for the observed differences on day 1. Although the insertion procedure was identical in rats and humans, it is possible that a different amount of trauma or blood was introduced during probe implantation and could have influenced the response.
Fat cells appear densely packed in human tissue with no discernible interstitial space. Adipose tissue has been estimated to be composed of 10% interstitial space (18), which would provide an impediment to metabolite diffusion. Rat subcutaneous tissue had collagen fibers loosely interspersed in a sparsely populated cellular environment with evident interstitial space (Fig. 3, A and B). Using quantitative digital image analysis of histological sections, we estimate rat subcutaneous tissue to be 29 ± 6% (mean ± SE, n = 32 fields; 4 rats × 8 fields/rat) interstitial space. On the basis of these estimates and Fig. 3, it is hypothesized that glucose and other metabolites will more easily diffuse through the interstitium of rat subcutis containing apparently less glycoproteins and water-excluding space to arrive at the probe surface. In fat, the path is more tortuous and restricted between the cells. It is possible that the greater interstitial space seen in rats is a technical artifact. If real, the observed differences in interstitial space would lead to differences in diffusion rates, which could directly influence microdialysis recovery. Although a literature value for a diffusion coefficient through adipose vs. subcutaneous tissue could not be located, diffusion coefficients through adipose tissue were estimated to be more than one order of magnitude less than diffusion coefficients through fibrous mastopathy tissue (whose structure is more like subcutaneous tissue and fibrous encapsulation tissue than adipose tissue; see Ref. 13). On the basis of these data, we cannot speculate on exact magnitudes; however, it seems reasonable that the diffusion coefficient through human fat should be less than that through rat subcutaneous tissue. A lower diffusion coefficient will lead to a lower microdialysis recovery as seen in human fat compared with rat subcutis. Also, vascular factors in rat subcutis may promote greater delivery initially than in human fat. It has been shown that smaller subcutaneous fat layers in humans correspond to larger blood flow per unit weight (14). Rats have an extremely small amount of subcutaneous fat compared with humans (Fig. 3, A and B) and most likely a higher blood flow perfusing their subcutaneous tissues compared with humans. In addition, more vessels seem to populate rat subcutis than human fat, as seen in Fig. 3. Kety (11) reports in a large tissue comparison study that adipose tissue has the lowest capillary density compared with some 10 different tissue types, although subcutaneous tissue was not measured. Nevertheless, less vascularization and less overall blood flow per unit weight in fat tissue most likely contribute to the lower analyte concentrations in humans compared with less fatty rat subcutaneous tissue. Glycerol is the only metabolite that was found at higher concentrations in human microdialysate than in rat microdialysate. Glycerol is formed when fat is split into fatty acids and glycerol during lipolysis (18). Lower glycerol in rat microdialysate is one indication that probes are surrounded by less adipose tissue. Human subcutaneous tissue is mainly composed of adipose tissue, whereas rat subcutis is not; therefore, it is not surprising that the glycerol is more concentrated in human microdialysate.Metabolite trends over 8 days are unstable in rat subcutis.
All metabolites detected in microdialysate from rat subcutis changed
substantially over the course of 8 days. Decreasing glucose flux into
the probes over time is likely to be caused by insufficient vessels in
the local vicinity of the probe surface, increased cellular uptake,
and/or reduced transport resulting from a decreased extracellular
space. Many immune cells migrate in the area by the 8th day (Fig.
3C), so they may well consume a significant quantity of
glucose before it has a chance to diffuse into the probe. Rat lactate
and pyruvate also increase (Fig. 2, B and C), as
would be expected when cellular glucose consumption increases. Toward
the end of the implantation time, however, lactate rises disproportionately compared with pyruvate, indicating a shift in the
metabolic pathway to anaerobic metabolism. Figure
4 shows that the lactate-to-pyruvate
ratio at the implant interface in rats is not stable over 1 wk. The
combination of the glucose, lactate, and pyruvate trends in rats
supports the hypothesis that insufficient vessels are present to meet
the local cellular demands. Insufficient delivery of nutrients from
vessels would render the area hypoxic or ischemic, thus
promoting less aerobic and more anaerobic metabolism. In addition,
histological sections of excised rat tissue show macrophages densely
populating the area immediately adjacent to the surface of the probe
(Fig. 3C). Their high level of glucose consumption and
lactate output (4, 7, 24) most likely contributes to the
decreased glucose and increased lactate detected over time in rat
probes.
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Metabolite trends over 8 days in human subcutis are also unstable but differ substantially from those in rats. Not only do absolute concentrations of metabolites differ significantly between rats and humans, but also the trends over 8 days significantly differ. Microdialysate glucose in humans increases over 8 days as opposed to the decreasing trends in rats. Although lactate and pyruvate increase in both humans and rats (indicating that cells at the biomaterial interface become more metabolically active over time or that more cells are present), the lactate-to-pyruvate ratio between the two species is very different (Fig. 4). In humans, the lactate-to-pyruvate ratio remains relatively stable, indicating that the local cells have adequate oxygen supply to meet their needs. Although human cells seem to be in a hypermetabolic state based on increased lactate and pyruvate production, it is hypothesized that more localized angiogenesis and/or less macrophage infiltration compared with rat subcutis are responsible for differences in metabolite trends seen in humans.
It has been shown by Wientjes et al. (22) that, after microdialysis probe insertion, there is a depression in the absolute glucose concentration in the immediate vicinity around the probes for 2-3 days. The depression was attributed to localized trauma of vessels, which regenerate after a few days. Studies carried out using the no-net-flux method (15) and slow flow rates with the same microdialysis probes in humans for 3 wk showed stable glucose concentrations after a few days, leading one to believe that tissue adjacent to the implanted probe stabilizes after an initial healing period (22). In the current study, however, a glucose plateau is also reached, but lactate, pyruvate, and glycerol (not measured in study by Wientjes et al.) continue to change over the 8 days of implantation (Fig. 2). The instability of these metabolites shows that the tissue environment at the interface of the implant is still in a state of change in humans and rats. However, the stability of glucose and urea (which are supplied to the tissue, not produced by the tissue) after day 3 suggests that vascular and physical factors are not changing in humans. Changes in other human metabolites between days 4 and 7 should therefore be attributed to metabolic changes (for which the vasculature is able to meet the demands for oxygen and glucose). Finally, it should be emphasized that the changes in the glucose dialysate-to-blood ratio reported in this study were detected at a dialysis flow of 2 µl/min. At a lower flow, such as 0.3 µl/min, commonly used in clinical applications, a 100% glucose dialysate-to-blood ratio would likely be achieved, and changes in flux resulting from physical and vascular factors of the tissue response would not influence the detected glucose recovery; however, changes resulting from metabolic factors may still be detected at lower flow rates. Glucose and other metabolite trends over 8 days at a biomaterial-tissue interface differ significantly in rats and humans. Disparities may be attributed to physical, vascular, and metabolic differences in the anatomy, wound healing, and foreign body response in rats and humans. Over an 8-day period, the rat subcutaneous tissue-material reaction appears to be a poor model of the human subcutaneous tissue-material reaction, as evidenced by differences in the interfacial biochemical environments of the two species. Caution should be used when extrapolating rat biomaterial data to human applications. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Alan Proia for histology assistance, Pierre Lemelin for assistance in obtaining human cadaver tissue, and Darin Buxbaum for surgical, scanning electron microscopy, and other assistance.
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FOOTNOTES |
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This work was supported by predoctoral fellowships from the National Science Foundation, the National Institutes of Health (NIH) Duke Center for Cell and Biosurface Engineering, and the National Science Foundation-Engineering Research Center for Emerging Cardiovascular Technologies (N. Wisniewski), by the Robert E. Jones Fund (B. Klitzman), NIH Grant DK-54932 (rat studies; W. M. Reichert), and CMA/Microdialysis in Solna, Sweden (human studies; U. Ungerstedt).
Address for reprint requests and other correspondence: B. Klitzman, Box 3906, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: Klitz{at}duke.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 11, 2002;10.1152/ajpendo.00259.2001
Received 14 June 2001; accepted in final form 7 January 2002.
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