Ulrike Klueh

Metabolic Biofouling of Glucose Sensors in Vivo: Role of Tissue Microhemorrhages

Objective: Based on our in vitro study that demonstrated the adverse effects of blood clots on glucose sensor function, we hypothesized that in vivo local tissue hemorrhages, induced as a consequence of sensor implantation or sensor movement post-implantation, are responsible for unreliable readings or an unexplained loss of functionality shortly after implantation. Research Design and Methods: To investigate this issue, we utilized real-time continuous monitoring of blood glucose levels in a mouse model. Direct injection of blood at the tissue site of sensor implantation was utilized to mimic sensor-induced local tissue hemorrhages. Results: It was found that blood injections, proximal to the sensor, consistently caused lowered sensor glucose readings, designated temporary signal reduction, in vivo in our mouse model, while injections of plasma or saline did not have this effect. Conclusion: These results support our hypothesis that tissue hemorrhage and resulting blood clots near the sensor can result in lowered local blood glucose concentrations due to metabolism of glucose by the clot. The lowered local blood glucose concentration led to low glucose readings from the still functioning sensor that did not reflect the systemic glucose level.

Critical role of tissue mast cells in controlling long-term glucose sensor function in vivo.

Little is known about the specific cells, mediators and mechanisms involved in the loss of glucose sensor function (GSF) in vivo. Since mast cells (MC) are known to be key effector cells in inflammation and wound healing, we hypothesized that MC and their products are major contributors to the skin inflammation and wound healing that controls GSF at sites of sensor implantation. To test this hypothesis we utilized a murine model of continuous glucose monitoring (CGM) in vivo in both normal C57BL/6 mice (mast cell sufficient), as well as mast cell deficient B6.Cg-Kit(W-sh)/HNihrJaeBsmJ (Sash) mice over a 28 day CGM period. As expected, both strains of mice displayed excellent CGM for the first 7 days post sensor implantation (PSI). CGM in the mast cell sufficient C57BL/6 mice was erratic over the remaining 21 days PSI. CGM in the mast cell deficient Sash mice displayed excellent sensor function for the entire 28 day of CGM. Histopathologic evaluation of implantation sites demonstrated that tissue reactions in Sash mice were dramatically less compared to the reactions in normal C57BL/6 mice. Additionally, mast cells were also seen to be consistently associated with the margins of sensor tissue reactions in normal C57BL/6 mice. Finally, direct injection of bone marrow derived mast cells at sites of sensor implantation induced an acute and dramatic loss of sensor function in both C57BL/6 and Sash mice. These results demonstrate the key role of mast cells in controlling glucose sensor function in vivo.

In Vitro Activation of Human Peripheral Blood Mononuclear Cells Induced by Human Biologic Meshes

BACKGROUND Inflammation and wound healing play critical roles in the integration of biologic meshes (BMs) at sites of hernia repair. Monocytes/macrophages (M/MQs) are key cells involved in mesh integration. Interleukin-1beta (IL-1beta) is one of the major M/MQ-derived cytokines, and its expression is a reflection of the degree of M/MQ activation. We hypothesized that BMs induce M/MQ activation in vitro and that IL-1beta expression by M/MQ varies among various BMs. MATERIALS AND METHODS Acellular human dermis-derived BM samples (AlloDerm, AlloMax, FlexHD) were placed in 48-well plates and cultured with peripheral blood mononuclear cells (PBMCs) from three healthy human subjects for 7 d. The resulting supernatants were assayed for IL-1beta levels by enzyme-linked immunosorbent assay (ELISA), and the BMs were evaluated histologically. RESULTS IL-1beta expression varied among donors as well as the BMs [AlloDerm (2.11-38.25pg/10(6) PBMCs); AlloMax (13.12-715.40pg/10(6) PBMCs); and FlexHD (116.69-665.40pg/10(6) PBMCs)]. Analysis of this data indicated that AlloMax and FlexHD induced significantly more M/MQ activation compared with AlloDerm (P<0.05). Histologic evaluation of the BMs indicated adherence of M/MQs on BM surface, however no degradation was detected. CONCLUSION For the first time, we have demonstrated that M/MQs are activated to varying levels by human BMs in vitro. These differences may be related to BM processing technologies and/or the biologic variation between donors. Our results raise the possibility that these differences in M/MQ activation could result in varying intensity of inflammation and wound healing that control the integration of BMs at sites of hernia repair.

Inflammation and Glucose Sensors: Use of Dexamethasone to Extend Glucose Sensor Function and Life Span in Vivo

Background: It has been generally accepted that the acute loss of sensor function is the consequence of sensor biofouling as a result of inflammation induced at sites of sensor implantation, as well as tissue trauma induced by the sensor and its implantation. Because anti-inflammatory therapies are used routinely to control inflammation in a wide variety of diseases, we hypothesized that anti-inflammatory therapy would likely extend glucose sensor function in vivo. To test this hypothesis, we utilized our recently developed mouse model of implantable glucose sensors and the potent anti-inflammatory steroid dexamethasone (DEX). Method: For this study, glucose sensors were implanted subcutaneously into the head and neck area of mice and sensor function was determined up to 14 days postimplantation. These mice received a daily intraperitoneal injection of DEX at a dose of 1, 6, or 10 mg/kg body weight. Results: Mice not treated with DEX lost sensor functionality very rapidly, usually within the first 24 hours postimplantation. Mice treated with DEX at the various doses had an increased sensor life span of up to 2 weeks postimplantation. Additionally, sensitivity was maintained in DEX-treated mice as compared to control mice (non-DEX treated). Histologic evaluation of tissue surrounding the site of sensor implantation had almost no inflammatory cells in DEX-treated mice, whereas control mice had an intense band of inflammation surrounding the site of sensor implantation. Conclusion: To our knowledge this is the first study directly demonstrating that anti-inflammatory therapy can extend glucose sensor function in vivo and supports the key role of inflammation in loss of sensor function in vivo, as well as the uses of anti-inflammatory therapy as a potential key adjuvant in enhancing glucose sensor function and life span in vivo.

Impact of macrophage deficiency and depletion on continuous glucose monitoring in vivo

Although it is assumed that macrophages (MQ) have a major negative impact on continuous glucose monitoring (CGM), surprisingly there is no data in the literature to directly support or refute the role of MQ or related foreign body giant cells in the bio-fouling of glucose sensors in vivo. As such, we developed the hypothesis that MQ are key in controlling glucose sensor performance and CGM in vivo and MQ deficiencies or depletion would enhance CGM. To test this hypothesis we determined the presence/distribution of MQ at the sensor tissue interface over a 28-day time period using F4/80 antibody and immunohistochemical analysis. We also evaluated the impact of spontaneous MQ deficiency (op/op mice) and induced-transgenic MQ depletions (Diphtheria Toxin Receptor (DTR) mice) on sensor function and CGM utilizing our murine CGM system. The results of these studies demonstrated: 1) a time dependent increase in MQ accumulation (F4/80 positive cells) at the sensor tissue interface; and 2) MQ deficient mice and MQ depleted C57BL/6 mice demonstrated improved sensor performance (MARD) when compared to normal mice (C57BL/6). These studies directly demonstrate the importance of MQ in sensor function and CGM in vivo.

Binding and orientation of fibronectin to silanated glass surfaces using immobilized bacterial adhesin-related peptides.

Previously, we have demonstrated the suitability of bacterial adhesin-related peptides, directly immobilized on polystyrene surfaces, to bind and orient fibronectin (FN). For these studies a method to bind the large protein FN in a desired orientation on a solid substratum was developed which utilizes a bacterial adhesin-related peptide (designated BRP-A), which is known to bind specifically to the NH3-terminus end of FN. Glass substrata was first coated with an amine-terminated silane, followed by streptavidin (SA), which was used as an intermediate tether to bind the biotinylated bacterial adhesin-related peptide. The BRP-A peptide, used for these studies was synthesized with a terminal biotin to assure irreversible coupling of the BRP-A to the streptavidin. The biotinylated BRP-A was next immobilized on the SA-silanated glass surfaces. 125I-FN was used to quantify the amount of FN binding to the (BRP-A):SA-silanated glass surface. Monoclonal antibodies, which react with specific epitopes at either the NH3- or -COOH-termini of FN, were used to quantify the binding and orientation of FN. The results of these studies indicated: (1) FN bound to the BRP-A:SA-silanated glass surface; and (2) the bound FN was oriented such that NH2-terminal region of FN was bound towards the glass surface and the COOH-terminus was oriented away from the glass surface. These studies demonstrate that small peptides can be used to specifically bind and orient large proteins such as FN on the surfaces.

Blood-Induced Interference of Glucose Sensor Function in Vitro: Implications for in Vivo Sensor Function

Background: Although tissue hemorrhages, with resulting blood clots, are associated with glucose sensor implantation, virtually nothing known is about the impact of red blood cells and red blood cell clots on sensor function in vitro or in vivo. In these studies, we tested the hypothesis that blood can directly interfere with glucose sensor function in vitro. Methods: To test this hypothesis, heparinized human whole blood (HWB) and nonheparinized human whole blood (WB) were obtained from normal individuals. Aliquots of HWB and WB samples were also fractionated into plasma, serum, and total leukocyte (TL) components. Resulting HWB, WB, and WB components were incubated in vitro with an amperometric glucose sensor for 24 hours at 37°C. During incubation, blood glucose levels were determined periodically using a glucose monitor, and glucose sensor function (GSF) was monitored continuously as nanoampere output. Results: Heparinized human whole blood had no significant effect on GSF in vitro, nor did TL, serum, or plasma-derived clots from WB. Sensors incubated with WB displayed a rapid signal loss associated with clot formation at 37°C. The half-life was 0.8 ± 0.2 hours (n = 16) for sensors incubated with WB compared to 3.2 ± 0.5 (n = 12) for sensors incubated with HWB with a blood glucose level of approximately 100 mg/dl. Conclusion: These studies demonstrated that human whole blood interfered with GSF in vitro. These studies further demonstrated that this interference was related to blood clot formation, as HWB, serum, plasma-derived clots, or TL did not interfere with GSF in vitro in the same way that WB did. These in vitro studies supported the concept that the formation of blood clots at sites of glucose sensor implantation could have a negative impact on GSF in vivo.

Importance of interleukin-1 and interleukin-1 receptor antagonist in short-term glucose sensor function in vivo.

Background: The importance of the interleukin (IL)-1 cytokine family in inflammation and immunity is well established as a result of extensive in vitro and in vivo studies. In fact, much of our understanding of the in vivo importance of interleukin-1beta (IL-1B) is the result of research utilizing transgenic mice, such as overexpression or deficiencies of the naturally occurring inhibitor of IL-1 known as interleukin-1 receptor antagonist (IL-1RA). For the present studies, we utilized these transgenic mice to determine the role of IL-1B in glucose sensor function in vivo. Methods: To investigate the role of IL-1B in glucose sensor function in vivo, we compared glucose sensor function in transgenic mice that (1) overexpressed IL-1RA [B6.Cg-Tg(II1rn)1Dih/J] and (2) are deficient in IL-1RA (B6.129S-Il1rntm1Dih /J), with mice that have normal levels of IL-1RA (C57BL/6). Results: Our studies demonstrated that, during the first 7 days post-sensor implantation (PSI), mice deficient in IL-1RA had extensive inflammation and decreased sensor function when compared to normal or IL-1RA-overexpressing mice. Conclusion: These data directly support our hypothesis that the IL-1 family of cytokines and antagonists play a critical role in controlling tissue reactions and thereby sensor function in vivo during the first 7 days PSI.

Analysis: On the Path to Overcoming Glucose-Sensor-Induced Foreign Body Reactions

It is generally accepted that unreliable in vivo performance of implantable glucose sensors originates, in large part, from tissue reactions to the implanted sensor, including foreign body reactions (i.e., inflammation, fibrosis, and vessel regression). Development of glucose sensor coatings with increased biocompatibility would contribute to the development of a reliable long-term glucose sensor. In this issue of Journal of Diabetes Science and Technology, Van den Bosch and coauthors report on their initial in vitro results on a candidate biocompatibility coating for sensors (silica nanoparticle- polyethylene-glycol-based coating). Although the initial standard testing is encouraging, it is important that sensor-specific testing protocol be utilized to more accurately predict sensor performance in vivo. The development and application of sensor-specific testing standards will likely speed the development of biocompatible coatings that will increase sensor accuracy and lifespan in the future.

Basement Membrane-Based Glucose Sensor Coatings Enhance Continuous Glucose Monitoring in Vivo.

Background: Implantable glucose sensors demonstrate a rapid decline in function that is likely due to biofouling of the sensor. Previous efforts directed at overcoming this issue has generally focused on the use of synthetic polymer coatings, with little apparent effect in vivo, clearly a novel approach is required. We believe that the key to extending sensor life span in vivo is the development of biocompatible basement membrane (BM) based bio-hydrogels as coatings for glucose sensors. Method: BM based bio-hydrogel sensor coatings were developed using purified BM preparations (ie, Cultrex from Trevigen Inc). Modified Abbott sensors were coated with Cultrex BM extracts. Sensor performance was evaluated for the impact of these coatings in vitro and in vivo in a continuous glucose monitoring (CGM) mouse model. In vivo sensor function was assessed over a 28-day time period expressed as mean absolute relative difference (MARD) values. Tissue reactivity of both Cultrex coated and uncoated glucose sensors was evaluated at 7, 14, 21 and 28 days post–sensor implantation with standard histological techniques. Results: The data demonstrate that Cultrex-based sensor coatings had no effect on glucose sensor function in vitro. In vivo glucose sensor performance was enhanced following BM coating as determined by MARD analysis, particularly in weeks 2 and 3. In vivo studies also demonstrated that Cultrex coatings significantly decreased sensor-induced tissue reactions at the sensor implantation sites. Conclusion: Basement-membrane-based sensor coatings enhance glucose sensor function in vivo, by minimizing or preventing sensor-induced tissues reactions.