D L Kreutzer

University of Connecticut, Storrs, Connecticut, United States

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Publications (180)689.98 Total impact

  • C. BARR, J. FRAILEY, U. KLUEH, D.L. KREUTZER
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    ABSTRACT: Objective: Bone loss in the pelvis, spine and long bones is well documented during human pregnancy/lactation, but not for the tooth-supporting mandible. Other studies reported bone loss during pregnancy/lactation in mice, but did not assess bone changes in the tooth-supporting mandible in pregnant / lactating breeders. We hypothesize there is significant mandibular bone loss in pregnant / lactating mice compared to non-pregnant / non-lactating (non-breeder/virgin) mice that was independent of inflammation. To test this hypothesis we compared tooth-supporting bone loss and bone mass in the mandible in breeders compared to non-breeder / virgin mice. Method: Right and left mandibles were obtained from nine breeders, and eight non-breeder C57BL/6 mice and assessed for alveolar bone loss using both 2D and 3D micro-CT images. All specimens were measured for alveolar bone loss at 9 locations on the buccal surface using NIH Image J. Measurements were recorded digitally in millimeters from the cement-enamel junction of the molars to the crest of the tooth supporting bone as well as total peripheral bone loss. Bone volume to trabecular volume (BV/TV) was also recorded. Data was analyzed for differences between the 2 groups using the Student T Test. The degree of inflammation in soft tissue adjacent to the bone and interdental tissue was determined using standard histopathology (H&E). Result: The breeders exhibited severe alveolar bone loss (0.68mm), as compared to the virgin mice (1.17mm) that was highly statistically significant (P=3.18x10-11). The bone volume fraction comparing total bone volume to trabecular volume bone (BV/TV) between breeders, 65% and non-breeders, 78%, indicated a significant reduction in the breeders (P<0081). Inflammation was minimal in both groups. Conclusion: There is significant tooth-supporting bone loss and bone volume in breeder mice compared to virgin / non-breeders that is independent of tissue inflammation.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: It is assumed that MQ are central to glucose sensor bio-fouling and therefore have a major negative impact on continuous glucose monitoring (CGM) performance in vivo. However to our knowledge there is no data in the literature to directly support or refute this assumption. Since glucose and oxygen (O2) are key to glucose sensor function in vivo, understanding and controlling glucose and O2 metabolic activity of MQ is likely key to successful glucose sensor performance. We hypothesized that the accumulation of MQ at the glucose sensor-tissue interface will act as "Cell Based Metabolic Barriers" (CBMB) to glucose diffusing from the interstitial tissue compartment to the implanted glucose sensor and as such creating an artificially low sensor output, thereby compromising sensor function and CGM. Our studies demonstrated that 1) direct injections of MQ at in vivo sensor implantation sites dramatically decreased sensor output (measured in nA), 2) addition of MQ to glucose sensors in vitro resulted in a rapid and dramatic fall in sensor output and 3) lymphocytes did not affect sensor function in vitro or in vivo. These data support our hypothesis that MQ can act as metabolic barriers to glucose and O2 diffusion in vivo and in vitro.
    Biomaterials 01/2014; · 8.31 Impact Factor
  • Y W Novitsky, S B Orenstein, D L Kreutzer
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    ABSTRACT: Biologic mesh (BM) prostheses are increasingly utilized for hernia repairs. Modern BMs are not only derived from different tissue sources, but also undergo various proprietary processing steps-factors that likely impact host tissue responses and mesh performance. We aimed to compare histopathologic responses to various BMs after implantation in a mouse model. Five-mm samples of non-crosslinked [Strattice (ST)], and intentionally crosslinked [CollaMend (CM), Permacol (PC)] porcine-derived biologic meshes were implanted subcutaneously in C57BL/6 mice. 1, 4, 8, and 12 weeks post-implantation, meshes were assessed for inflammation, foreign body reaction (FBR), neocellularization, and collagen deposition using H&E and trichrome stains. All meshes induced early polymorphonuclear cell infiltration (highest in CM; lowest in ST) that resolved by 4 weeks. ST was associated with extensive macrophage presence at 12 weeks. Foreign body response was not seen in the ST group, but was present abundantly in the CM and PC groups, highest at 8 weeks. New peripheral collagen deposition was seen only in the ST group at 12 weeks. Collagen organization was highest in the ST group as well. Both CM and PC groups were associated with fibrous encapsulation and no evidence of integration or remodeling. Inflammation appears to be a common component of integration of all biologic meshes studied. Pronounced inflammatory responses as well as profound FBR likely lead to observed encapsulation and poor host integration of the crosslinked BMs. Overall, ST was associated with the lowest foreign body response and the highest degree of new collagen deposition and organization. These features may be key predictors for improved mesh performance during hernia repair.
    Hernia 12/2013; · 1.69 Impact Factor
  • Ulrike Klueh, Yi Qiao, Jackman T Frailey, Donald L Kreutzer
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    ABSTRACT: 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.
    Biomaterials 12/2013; · 8.31 Impact Factor
  • Ulrike Klueh, Omar Antar, Yi Qiao, Donald L Kreutzer
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    ABSTRACT: The concept of increased blood vessel (BV) density proximal to glucose sensors implanted in the interstitial tissue increases the accuracy and lifespan of sensors is accepted, despite limited existing experimental data. Interestingly, there is no previous data or even conjecture in the literature on the role of lymphatic vessels (LV) alone, or in combination with BV, in enhancing continuous glucose monitoring (CGM) in vivo. To investigate the impact of inducing vascular networks (BV and LV) at sites of glucose sensor implantation, we utilized adenovirus based local gene therapy of vascular endothelial cell growth factor-A (VEGF-A) to induce vessels at sensor implantation sites. The results of these studies demonstrated that (1) VEGF-A based local gene therapy increases vascular networks (blood vessels and lymphatic vessels) at sites of glucose sensor implantation; and (2) this local increase of vascular networks enhances glucose sensor function in vivo from 7 days to greater than 28 days postsensor implantation. This data provides "proof of concept" for the effective usage of local angiogenic factor (AF) gene therapy in mammalian models in an effort to extend CGM in vivo. It also supports the practice of a variety of viral and nonviral vectors as well as gene products (e.g. anti-inflammatory and anti-fibrosis genes) to engineer "implant friendly tissues" for the usage with implantable glucose sensors as well as other implantable devices. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
    Journal of Biomedical Materials Research Part A 11/2013; · 2.83 Impact Factor
  • Ulrike Klueh, Omar Antar, Yi Qiao, Donald L Kreutzer
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    ABSTRACT: Glucose-sensor-induced tissue reactions (e.g., inflammation and wound healing) are known to negatively impact sensor function in vivo. The roles of cytokine networks in controlling these tissue reactions (i.e., sensor biofouling) is not understood. In the present study, we investigated the role of interleukin-1 receptor antagonist (IL-1Ra), a key anti-inflammatory antagonist of the proinflammatory interleukin-1 cytokines [i.e. interleukin-1 (IL-1) alpha and IL-1 beta] in controlling continuous glucose monitoring (CGM). To investigate the role of IL-1Ra in long-term CGM in vivo, we compared CGM in transgenic mice that overexpress IL-1Ra [interleukin-1 receptor antagonist overexpresser (IL-1Ra~OE), B6.Cg-Tg(IL1rn)1Dih/J] or are deficient in IL-1Ra [interleukin-1 receptor antagonist knockout (IL-1Ra~KO), B6.129S-IL1rntm1Dih/J] with mice that have normal levels of IL-1Ra (C57BL/6) over a 28-day time period. Mean absolute relative difference (MARD) analysis of CGM results among the mice of varying IL-1Ra levels demonstrated that during the first 21 days, IL-1~KO mice had the greatest tissue inflammation and the poorest sensor performance (i.e., higher MARD values) when compared with normal or IL-1Ra~OE mice. By 28 days post-sensor implantation, the inflammatory reactions had subsided and were replaced by varying degrees of fibrosis. These data support our hypothesis on the importance of the IL-1 family of agonists and antagonists in controlling tissue reactions and sensor function in vivo. These data also suggest that local delivery of IL-1Ra genes or recombinant proteins (anakinra) or other IL-1 antagonists such as antibodies or soluble IL-1 receptors would suppress sensor-induced tissue reactions and likely enhance glucose sensor function by inhibiting inflammation and wound healing at sensor implantation sites.J Diabetes Sci Technol 2013;7(6):1538-1546.
    Journal of diabetes science and technology 01/2013; 7(6):1538-46.
  • Yue Gao, Yi Qiao, Donald L Kreutzer, Yuri W Novitsky
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    ABSTRACT: Minimally-invasive surgery (MIS) is associated with a decreased activation of both systemic and peritoneal immunity compared with the open technique. However, hepatic response to laparoscopic (LAP) and hand-assisted laparoscopic (HAL) surgery has not been defined well. We postulated that both LAP and HAL approaches are associated with a diminished activation of hepatic inflammatory signaling pathways compared with the traditional open surgery. Eighteen pigs underwent a transabdominal nephrectomy via Open, HAL, or LAP approach. Liver samples were obtained 24 h postoperatively and spot frozen. Frozen tissue samples were then homogenized and the nuclear pellets were separated and stored. Nuclear extracts were analyzed for activation of three nuclear signaling phosphoproteins: nuclear factor-kappaB (NFκB)-p65, heat-shock protein 27 (HSP27), and p38 mitogen-activated protein kinases (p38MAPK) using a standard Bioplex technique. Statistical comparison was performed using ANOVA and Student's t-test. The average expression of HSP27 was significantly higher in the Open versus either the LAP or the HAL groups (P = 0.028 and P = 0.039). The average expression of NFκB-p65 was significantly higher in the Open versus either the LAP or the HAL groups (P = 0.032 and P = 0.049). The average expression of p38MAPK was significantly higher in the Open versus either the LAP or the HAL groups (P = 0.007 and P = 0.036). There was no significant difference in the expressions of HSP27 and NFκB-p65 between LAP and HAL groups (P = 0.38 and P = 0.20), however, detection of p38MAPK generated statistical difference between these two groups (P = 0.018). Hand-assisted laparoscopic surgery has been widely accepted as an effective alternative to traditional laparoscopic procedures. We demonstrated that both laparoscopic and hand-assisted approaches resulted in blunted hepatic stress manifested by diminished expression of hepatic HSP27, NFκB, and p38-MAPK. In addition, the hand-assisted approach was equal to the laparoscopic approach in two of the three phosphoproteins studied. It appears that the use of hand-assisted techniques did not abrogate immunologic benefits of pure laparoscopy. Overall, in addition to the clinical benefits of minimal access, both hand-assisted and pure laparoscopic techniques may also confer an immunologic advantage over laparotomy.
    Journal of Surgical Research 10/2011; 176(2):608-13. · 2.02 Impact Factor
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    ABSTRACT: While synthetic prosthetics have essentially become mandatory for hernia repair, mesh-induced chronic inflammation and scarring can lead to chronic pain and limited mobility. Mesh propensity to induce such adverse effects is likely related to the prosthetic's material, weight, and/or pore size. We aimed to compare histopathologic responses to various synthetic meshes after short- and long-term implantations in mice. Samples of macroporous polyester (Parietex [PX]), heavyweight microporous polypropylene (Trelex[TX]), midweight microporous polypropylene (ProLite[PL]), lightweight macroporous polypropylene (Ultrapro[UP]), and expanded polytetrafluoroethylene (DualMesh[DM]) were implanted subcutaneously in mice. Four and 12 wk post-implantation, meshes were assessed for inflammation, foreign body reaction (FBR), and fibrosis. All meshes induced varying levels of inflammatory responses. PX induced the greatest inflammatory response and marked FBR. DM induced moderate FBR and a strong fibrotic response with mesh encapsulation at 12 wk. UP and PL had the lowest FBR, however, UP induced a significant chronic inflammatory response. Although inflammation decreased slightly for TX, marked FBR was present throughout the study. Of the three polypropylene meshes, fibrosis was greatest for TX and slightly reduced for PL and UP. For UP and PL, there was limited fibrosis within each mesh pore. Polyester mesh induced the greatest FBR and lasting chronic inflammatory response. Likewise, marked fibrosis and encapsulation was seen surrounding ePTFE. Heavier polypropylene meshes displayed greater early and persistent fibrosis; the reduced-weight polypropylene meshes were associated with the least amount of fibrosis. Mesh pore size was inversely proportional to bridging fibrosis. Moreover, reduced-weight polypropylene meshes demonstrated the smallest FBR throughout the study. Overall, we demonstrated that macroporous, reduced-weight polypropylene mesh exhibited the highest degree of biocompatibility at sites of mesh implantation.
    Journal of Surgical Research 10/2011; 176(2):423-9. · 2.02 Impact Factor
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    ABSTRACT: Mast cells (MCs) and their products (e.g., histamine, serotonin, heparin, prostaglandins, cytokines, etc.) play key roles in controlling local inflammation, wound healing, and foreign body reactions in vivo. Investigation of the role of MCs in mediating local tissue responses to synthetic hernia meshes has been very limited to date. We aimed to determine the effects of MCs/MC products in mice undergoing synthetic mesh implantation. Circular samples (5 mm) of heavyweight microporous polypropylene (Trelex), midweight microporous polypropylene (ProLite), lightweight macroporous polypropylene with poliglecaprone (Ultrapro), and 3-dimensional macroporous polyester (Parietex) meshes were implanted subcutaneously in C57BL/6 J mice with and without cromolyn (MC stabilizer/suppressant) treatment (50 mg/kg, daily IP). Two weeks post-implantation, all meshes were explanted and evaluated histologically using H&E and trichrome stains. Chronic inflammation was focused around individual mesh fibers; inter-fiber inflammation and fibrosis diminished as mesh porosity increased. MC accumulation was seen at the periphery of inflammatory reactions, and in association with mesh-induced fibrosis and neovascularization. Cromolyn treatment resulted in significantly decreased fibrotic responses to all four meshes and reduced inflammation induced by Trelex, ProLite, and Parietex meshes but not Ultrapro. We demonstrated that MCs play important roles in mesh-induced host tissue reactions. Blocking MC degranulation decreased early inflammation and fibrosis induced by most synthetic meshes in this study. Further evaluation and understanding of the role of MCs in mesh-induced tissue reactions will provide new therapeutic approaches to enhance the biocompatibility of surgical meshes and ultimately improve clinical outcomes in patients undergoing hernia repair with synthetic biomaterials.
    Hernia 10/2010; 14(5):511-6. · 1.69 Impact Factor
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    Mark D Litt, David M Shafer, Donald L Kreutzer
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    ABSTRACT: The purpose of this study was to determine whether a brief (6-8 sessions) cognitive-behavioral treatment for temporomandibular dysfunction-related pain could be efficacious in reducing pain, pain-related interference with lifestyle and depressive symptoms. The patients were 101 men and women with pain in the area of the temporomandibular joint of at least 3 months duration, randomly assigned to either standard treatment (STD; n=49) or standard treatment+cognitive-behavioral skills training (STD+CBT; n=52). Patients were assessed at posttreatment (6 weeks), 12 weeks, 24 weeks, 36 weeks, and 52 weeks. Linear mixed model analyses of reported pain indicated that both treatments yielded significant decreases in pain, with the STD+CBT condition resulting in steeper decreases in pain over time compared to the STD condition. Somatization, self-efficacy and readiness for treatment emerged as significant moderators of outcome, such that those low in somatization, or higher in self-efficacy or readiness, and treated with STD+CBT reported of lower pain over time. Somatization was also a significant moderator of treatment effects on pain-related interference with functioning, with those low on somatization reporting of less pain interference over time when treated in the STD+CBT condition. It was concluded that brief treatments can yield significant reductions in pain, life interference and depressive symptoms in TMD sufferers, and that the addition of cognitive-behavioral coping skills will add to efficacy, especially for those low in somatization, or high in readiness or self-efficacy.
    Pain 10/2010; 151(1):110-6. · 5.64 Impact Factor
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    Ulrike Klueh, Manjot Kaur, Yi Qiao, Donald L Kreutzer
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    ABSTRACT: 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.
    Biomaterials 03/2010; 31(16):4540-51. · 8.31 Impact Factor
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    ABSTRACT: While porcine-based biologic meshes are increasingly used for hernia repair, little data exist on tissue responses to such products. Host foreign body reaction, local inflammation, and wound healing are principally controlled by monocytes/macrophages (M/MØs). Exaggerated activation of M/MØs may deleteriously influence mesh integration and remodeling. We hypothesized that common porcine meshes induce the differential activation of M/MØs in vitro. Samples of four acellular porcine-derived meshes, CollaMend (CM; C.R. Bard/Davol), Permacol (PC; TSL/Covidien), Strattice (ST; LifeCell), and Surgisis (SS; Cook Biotech), were exposed to mononuclear cells derived from the peripheral blood of six healthy subjects. Following a 7-day incubation period, supernatants were assayed for interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using a multiplex bead-based immunoassay system. The four groups were compared using analysis of variance (ANOVA) and Student's t-test. Each mesh type induced differential mononuclear cell activation in vitro. The mean IL-1beta expressions for CM (7,195 pg/ml) and PC (4,215 pg/ml) were significantly higher compared to ST and SS (123 and 998 pg/ml, respectively; P < 0.05). Similar trends were also seen for IL-6 (range 445-70,729 pg/ml), IL-8 (range 11,640-1,045,938 pg/ml), and VEGF (range 686-7,133 pg/ml). For the first time, we demonstrated that porcine meshes induce M/MØ activation in vitro. CM and PC (chemically crosslinked dermis) induced significantly higher cytokine expression compared to ST (non-crosslinked dermis) and SS (small intestine submucosa). These differences are likely related to proprietary processing methods and/or the extent of collagen crosslinking. Further understanding of immunologic effects of porcine-derived biologic meshes will not only allow for a comparison between existing products, but it may also lead to mesh modifications and improvement of their clinical performance.
    Hernia 02/2010; 14(4):401-7. · 1.69 Impact Factor
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    ABSTRACT: The purpose of this study was to determine whether cognitive-behavioral treatment (CBT) operates by effecting changes in cognitions, affects, and coping behaviors in the context of painful episodes. Patients were 54 men and women with temporomandibular dysfunction-related orofacial pain (TMD) enrolled in a study of brief (6 weeks) standard conservative treatment (STD) or standard treatment plus CBT (STD+CBT). Momentary affects, pain, and coping processes were recorded on a cell phone keypad four times per day for 7 days prior to treatment, and for 14 days after treatment had finished, in an experience sampling paradigm. Analyses indicated no treatment effects on general retrospective measures of pain, depression, or pain-related interference with lifestyle at post-treatment. However, mixed model analyses on momentary pain and coping recorded pre- and post-treatment indicated that STD+CBT patients reported greater decreases in pain than did STD patients, significantly greater increases in the use of active cognitive and behavioral coping, and significantly decreased catastrophization. Analyses of experience sampling data indicated that post-treatment momentary pain was negatively predicted by concurrent active coping, self-efficacy, perceived control over pain, and positive-high arousal affect. Concurrent catastrophization was strongly predictive of pain. Active behavioral coping and self-efficacy reported at the prior time point (about 3h previously) were also protective, while prior catastrophization and negative-high arousal mood were predictive of momentary pain. The results suggest that CB treatment for TMD pain can help patients alter their coping behaviors, and that these changes translate into improved outcomes.
    Pain 07/2009; 145(1-2):160-8. · 5.64 Impact Factor
  • Journal of Surgical Research 02/2009; 151(2):290-290. · 2.02 Impact Factor
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    ABSTRACT: 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). 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. 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. 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.
    Journal of diabetes science and technology 07/2007; 1(4):496-504.
  • Nanomedicine-nanotechnology Biology and Medicine - NANOMED-NANOTECHNOL BIOL MED. 01/2007; 3(4):353-354.
  • Ulrike Klueh, Donald L Kreutzer
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    ABSTRACT: Although implantable glucose sensors have existed for over 30 years, their function deteriorates in hours to days, in large part as a result of tissue responses to the implanted sensor (i.e., acute and chronic inflammation, fibrosis, and vessel regression). Little is known about the mediators and mechanisms that control these tissue responses to implantable glucose sensors. In the present study, we developed and validated a murine model for implantable glucose sensors, which suitably parallel sensor function in humans. Using special care in implantation and implant retaining techniques, we demonstrated that (1) sensor function deteriorates rapidly within days post-implantation and (2) loss of glucose sensor function correlated with tissue reactions at the sites of sensor implantation, especially in the vicinity of the glucose oxidase-based working electrode. These studies establish a murine model that can be used to evaluate implantable glucose sensors in vivo. This model should provide the foundation for future studies to understand the factors and mechanisms that control sensor function in vivo.
    Diabetes Technology &amp Therapeutics 11/2005; 7(5):727-37; discussion 738-40. · 2.21 Impact Factor
  • U Klueh, D I Dorsky, D L Kreutzer
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    ABSTRACT: In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.
    Journal of Biomedical Materials Research Part A 01/2004; 67(4):1072-86. · 2.83 Impact Factor
  • T I Valdes, U Klueh, D Kreutzer, F Moussy
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    ABSTRACT: A major problem with implantable sensors is their short in vivo lifetime, due to strong tissue reactions (i.e., inflammation and fibrosis) caused by the implant and the failure of sensor components. The tissue reactions to the sensor, the biocompatibility of components, and the function of the sensor must be evaluated by using in vivo models. Current methods of in vivo biosensor testing are time- and labor- intensive and expensive. In addition, the results often vary on the basis of the surgical skills of the investigator. The in ova chorioallantoic membrane (CAM) of the developing chicken embryo was previously developed in our laboratory as a novel in vivo system to test biomaterials. In this new article, we describe a novel approach for testing biosensors in vivo using the ex ova CAM model as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 3 days in ova and then transferred into a petri dish (ex ova) for further incubation at 37 degrees C and 80% humidity. After 1 week of incubation, acetaminophen biosensors, used as model sensors, were placed on top of the CAM and allowed to incorporate for 1 week. Biosensors were then tested for their sensitivity to acetaminophen. CAM venules were injected with 0.2 mL of a 3.6 mM acetaminophen solution. The current produced by the sensor reflected the change in blood acetaminophen levels. Sensors were also assessed by using gross and histological evaluations. We previously reported on the similarity of the tissue response of the CAM with the mammalian models. The low cost, simplicity, and possibility to continuously visualize the sensor test site through a cell culture dish make this animal model particularly attractive for the rapid in vivo screening of biosensors.
    Journal of Biomedical Materials Research Part A 11/2003; 67(1):215-23. · 2.83 Impact Factor
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    ABSTRACT: 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.
    Biomaterials 11/2003; 24(22):3877-84. · 8.31 Impact Factor

Publication Stats

5k Citations
689.98 Total Impact Points

Institutions

  • 1987–2014
    • University of Connecticut
      • • Department of Medicine
      • • School of Dental Medicine
      • • Department of Surgery
      Storrs, Connecticut, United States
  • 2003–2010
    • UConn Health Center
      • Division of Behavioral Sciences and Community Health
      Farmington, CT, United States
  • 2000
    • University of California, Los Angeles
      Los Angeles, California, United States
    • Connecticut Children's Medical Center
      Hartford, Connecticut, United States
  • 1996–1999
    • Kuwait University
      • Department of Medical Laboratory Science
      Kuwait, Muhafazat al `Asimah, Kuwait
    • University of Southern California
      • School of Dentistry
      Los Angeles, CA, United States
  • 1997
    • Johns Hopkins University
      • Department of Environmental Health Sciences
      Baltimore, MD, United States
  • 1987–1997
    • Hartford Hospital
      • Department of Pediatrics
      Hartford, Connecticut, United States
  • 1991
    • The Ohio State University
      • Department of Periodontology
      Columbus, OH, United States
  • 1990
    • Keio University
      • Department of Pediatrics
      Tokyo, Tokyo-to, Japan
  • 1984
    • Tufts University
      Georgia, United States
  • 1977–1979
    • University of Kansas
      Lawrence, Kansas, United States