Inorganic-Organic Interpenetrating Network Hydrogels as Tissue-Integrating Luminescent Implants: Physicochemical Characterization and Preclinical Evaluation.

Sensors capable of accurate, continuous monitoring of biochemistry are crucial to the realization of personalized medicine on a large scale. Great strides have been made to enhance tissue compatibility of long-term in vivo biosensors using biomaterials strategies such as tissue-integrating hydrogels. However, the low level of oxygen in tissue presents a challenge for implanted devices, especially when the biosensing function relies on oxygen as a measure-either as a primary analyte or as an indirect marker to transduce levels of other biomolecules. This work presents a method of fabricating inorganic-organic interpenetrating network (IPN) hydrogels to optimize the oxygen transport through injectable biosensors. Capitalizing on the synergy between the two networks, various physicochemical properties (e.g., swelling, glass transition temperature, and mechanical properties) are shown to be independently adjustable while maintaining a 250% increase in oxygen permeability relative to poly(2-hydroxyethyl methacrylate) controls. Finally, these gels, when functionalized with a Pd(II) benzoporphyrin phosphor, track tissue oxygen in real time for 76 days as subcutaneous implants in a porcine model while promoting tissue ingrowth and minimizing fibrosis around the implant. These findings support IPN networks for fine-tuned design of implantable biomaterials in personalized medicine and other biomedical applications.

Detection of flap tissue ischemia in a rat model: Real-time monitoring of changes in oxygenation and perfusion through injectable biosensors.

BACKGROUND: The success of surgical flaps is improved by timely correction of vascular compromise. Current monitoring methods are labor or cost intensive or have limited clinical benefit. We hypothesize that injectable oxygen sensors can identify acute vascular compromise. The purpose of this study was to use a long-term, real-time method of tissue oxygenation detection in a rat flap model with vascular manipulation. METHODS: Sensors incorporated benzo-porphyrin dye into a microporous hydrogel and were injected intradermally 1 day before flap elevation. Inspired oxygen was modulated between 100% and 12% to confirm sensor O2 sensitivity. Eight random flaps (4 cm wide, 8 cm long) were elevated. Sensor and clinical observation to temporary clamping of the flap vascular pedicle was recorded. Sodium fluorescein in saline was injected intraperitoneally on postoperative days 0, 3, and 7 with subsequent perfusion area analysis. RESULTS: Tissue oxygen tension measurements reflected the changes in inspired oxygen levels. Clinical observation of the flaps did not show any significant change in color or temperature with pedicle clamping. However, clamping of the pedicle resulted in a significant decrease in sensor tissue oxygen tension within 70 seconds. CONCLUSION: Oxygen monitoring of myocutaneous flaps is sensitive and can detect acute vascular occlusion. This technique is faster than current methods and offers a cost-effective and accurate means of monitoring surgical tissues.

Near-Infrared Optical Nanosensors for Continuous Detection of Glucose.

BACKGROUND: Continuous glucose monitors (CGMs) enable people with diabetes to proactively manage their blood glucose and reduce the risk of hypoglycemia. Commercially available CGMs utilize percutaneous electrodes that, after days to weeks of implantation, are subjected to the foreign body response that severely reduces sensor accuracy. The previous work demonstrated the use of hydrogels containing a glucose-responsive viologen that quenches a nearby fluorophore. Here, we investigate the immobilization of this sensing motif onto a nanoparticle surface and optimize local surface concentrations for optical glucose sensing. METHODS: A viologen quencher-fluorescent dye system was incorporated into poly(2-hydroethyl methacrylate) hydrogels in varying quantities to assess the effect of quencher-fluorophore concentration on glucose responsiveness. The sensing motif was then immobilized onto silica nanoparticles by carbodiimide chemistry. Nanosensors with a range of dye and quencher concentrations were challenged for glucose responsiveness to determine the optimal sensor formulation. RESULTS: When incorporated into a hydrogel, high concentrations of viologen quencher and fluorophore were required to permit electron transfer between the two components and yield a detectable glucose response. Immobilization of this glucose-responsive system onto a silica nanoparticle facilitated this electron transfer to yield detectable responses at even low concentrations. Increasing quencher concentration on the nanoparticle, relative to the fluorophore, resulted in the greatest apparent glucose response. CONCLUSION: The nanoparticle sensors demonstrated excellent glucose response in the physiological range and are a promising tool for real-time glucose tracking.

Continuous monitoring of interstitial tissue oxygen using subcutaneous oxygen microsensors: In vivo characterization in healthy volunteers.

Measurements of regional tissue oxygen serve as a proxy to monitor local perfusion and have the potential to guide therapeutic decisions in multiple clinical disciplines. Transcutaneous oximetry (tcpO2) is a commercially available noninvasive technique that uses an electrode to warm underlying skin tissue and measure the resulting oxygen tension at the skin surface. A novel approach is to directly measure interstitial tissue oxygen using subcutaneous oxygen microsensors composed of a biocompatible hydrogel carrier platform with embedded oxygen sensing molecules. After initial injection of the hydrogel into subcutaneous tissue, noninvasive optical measurements of phosphorescence-based emissions at the skin surface are used to sense oxygen in the subcutaneous interstitial space. The object of the present study was to characterize the in vivo performance of subcutaneous microsensors and compare with transcutaneous oximetry (tcpO2). Vascular occlusion tests were performed on the arms of 7 healthy volunteers, with repeated tests occurring 1 to 10 weeks after sensor injection, yielding 95 total tests for analysis. Comparative analysis characterized the response of both devices to decreases in tissue oxygen during occlusion and to increases in tissue oxygen following release of the occlusion. Results indicated: (I) time traces returned by microsensors and tcpO2 were highly correlated, with the median (interquartile range) correlation coefficient of r = 0.93 (0.10); (II) both microsensors and tcpO2 sensed a statistically significant decrease in normalized oxygen during occlusion (p < 0.001 for each device); (III) microsensors detected faster rates change (p < 0.001) and detected overshoot during recovery more frequently (38% vs. 4% of tests); (IV) inter-measurement analysis showed no correlation of baseline values between microsensors and tcpO2 (r = 0.03), but comparison of integrated oxygen dynamics showed similar variation in the normalized response to occlusion between devices (p = 0.06), (V) intra-measurement analysis revealed that microsensors detect greater physiological fluctuations than tcpO2 (p < 0.001) and may provide enhanced sensitivity to processes such as vasomotion. Additionally, the functional response of microsensors was not significantly different across time groupings (per month) post-injection (p = 0.61). Although the compared devices have differences in the mechanisms used to sense oxygen, these findings demonstrate that subcutaneous oxygen microsensors measure changes in interstitial tissue oxygen in human subjects in vivo.

Integrated phosphorescence-based photonic biosensor (iPOB) for monitoring oxygen levels in 3D cell culture systems.

Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.

Long-Term In Vivo Oxygen Sensors for Peripheral Artery Disease Monitoring.

Tracking of tissue oxygenation around chronic foot wounds may help direct therapy decisions in patients with peripheral artery disease (PAD). Novel sensing technology to enable such monitoring was tested over 9 months in a Sinclair mini-pig model. No adverse events were observed over the entire study period. Systemic and acute hypoxia challenges were detected during each measurement period by the microsensors. The median time to locate the sensor signal was 13 s. Lumee Oxygen microsensors appear safe for long-term repeated oxygen measurements over 9 months.

Antibiotic-induced changes in the microbiota disrupt redox dynamics in the gut.

How host and microbial factors combine to structure gut microbial communities remains incompletely understood. Redox potential is an important environmental feature affected by both host and microbial actions. We assessed how antibiotics, which can impact host and microbial function, change redox state and how this contributes to post-antibiotic succession. We showed gut redox potential increased within hours of an antibiotic dose in mice. Host and microbial functioning changed under treatment, but shifts in redox potentials could be attributed specifically to bacterial suppression in a host-free ex vivo human gut microbiota model. Redox dynamics were linked to blooms of the bacterial family Enterobacteriaceae. Ecological succession to pre-treatment composition was associated with recovery of gut redox, but also required dispersal from unaffected gut communities. As bacterial competition for electron acceptors can be a key ecological factor structuring gut communities, these results support the potential for manipulating gut microbiota through managing bacterial respiration.

Injectable Phosphorescence-based Oxygen Biosensors Identify Post Ischemic Reactive Hyperoxia.

Novel injectable biosensors were used to measure interstitial oxygenation before, during, and after transient ischemia. It is well known that reactive hyperemia occurs following a period of ischemia. However, increased blood flow does not necessarily mean increased oxygen tension in the tissue. Therefore, the purpose of this study was to test the hypothesis that tissue reactive hyperoxia occurs following release of hind-limb tourniquet occlusions. Rats were injected with bilateral hind-limb biosensors and were simultaneously subjected to a unilateral femoral vessel ligation. After approximately one and three months, the rats underwent a series of oxygenation challenges, including transient hind-limb tourniquet occlusion. Along with the biosensors, near infrared spectroscopy was used to measure percent oxyhemoglobin in capillaries and laser Doppler flowmetry was used to measure blood flow. Post-occlusion reactive hyperemia was observed. It was accompanied by tissue reactive hyperoxia, affirming that the post-occlusion oxygen supply must have exceeded the expected increased oxygen consumption. The measurement of the physiologic phenomenon of reactive hyperoxia could prove clinically beneficial for both diagnosis and optimizing therapy.

Tissue-Integrating Oxygen Sensors: Continuous Tracking of Tissue Hypoxia.

We describe a simple method of tracking oxygen in real-time with injectable, tissue-integrating microsensors. The sensors are small (500 µm × 500 µm × 5 mm), soft, flexible, tissue-like, biocompatible hydrogel s that have been shown to overcome the foreign body response for long-term sensing. The sensors are engineered to change luminescence in the presence of oxygen or other analytes and function for months to years in the body. A single injection followed by non-invasive monitoring with a hand-held or wearable Bluetooth optical reader enables intermittent or continuous measurements. Proof of concept for applications in high altitude, exercise physiology, vascular disease, stroke, tumors, and other disease states have been shown in mouse, rat and porcine models. Over 90 sensors have been studied to date in humans. These novel tissue-integrating sensors yield real-time insights in tissue oxygen fluctuations for research and clinical applications.

Cancer of the gingiva.

Cancer of the gingiva is a rare disease in the Western World. It most commonly affects elderly population. Because of its rarity, the reporting on the disease is sparse and often grouped with other subsites of oral cancer, which makes conclusions difficult to interpret. The aim of this paper is to review the literature on gingival cancer as a specific subsite of oral cancer and report on published prognostic factors as well as treatment of local and regional disease. We also present differences between gingival cancer subgroups, mandibular and maxillary gingival cancer. In addition, both surgical and oncological treatments are reviewed. It seems that surgery is the preferred initial treatment approach for the majority of patients with gingival cancer, although adjuvant radiation, with or without chemotherapy, is commonly recommended to increase locoregional control.

In vivo detection of SERS-encoded plasmonic nanostars in human skin grafts and live animal models.

Surface-enhanced Raman scattering (SERS)-active plasmonic nanomaterials have become a promising agent for molecular imaging and multiplex detection. Among the wide variety of plasmonics-active nanoparticles, gold nanostars offer unique plasmon properties that efficiently induce strong SERS signals. Furthermore, nanostars, with their small core size and multiple long thin branches, exhibit high absorption cross sections that are tunable in the near-infrared region of the tissue optical window, rendering them efficient for in vivo spectroscopic detection. This study investigated the use of SERS-encoded gold nanostars for in vivo detection. Ex vivo measurements were performed using human skin grafts to investigate the detection of SERS-encoded nanostars through tissue. We also integrated gold nanostars into a biocompatible scaffold to aid in performing in vivo spectroscopic analyses. In this study, for the first time, we demonstrate in vivo SERS detection of gold nanostars using small animal (rat) as well as large animal (pig) models. The results of this study establish the usefulness and potential of SERS-encoded gold nanostars for future use in long-term in vivo analyte sensing.

Characterization of Lactate Sensors Based on Lactate Oxidase and Palladium Benzoporphyrin Immobilized in Hydrogels.

An optical biosensor for lactate detection is described. By encapsulating enzyme-phosphor sensing molecules within permeable hydrogel materials, lactate-sensitive emission lifetimes were achieved. The relative amount of monomer was varied to compare three homo- and co-polymer materials: poly(2-hydroxyethyl methacrylate) (pHEMA) and two copolymers of pHEMA and poly(acrylamide) (pAam). Diffusion analysis demonstrated the ability to control lactate transport by varying the hydrogel composition, while having a minimal effect on oxygen diffusion. Sensors displayed the desired dose-variable response to lactate challenges, highlighting the tunable, diffusion-controlled nature of the sensing platform. Short-term repeated exposure tests revealed enhanced stability for sensors comprising hydrogels with acrylamide additives; after an initial "break-in" period, signal retention was 100% for 15 repeated cycles. Finally, because this study describes the modification of a previously developed glucose sensor for lactate analysis, it demonstrates the potential for mix-and-match enzyme-phosphor-hydrogel sensing for use in future multi-analyte sensors.

Preclinical Evaluation of Poly(HEMA-co-acrylamide) Hydrogels Encapsulating Glucose Oxidase and Palladium Benzoporphyrin as Fully Implantable Glucose Sensors.

BACKGROUND: Continuous glucose monitors (CGMs) require percutaneous wire probes to monitor glucose. Sensors based on luminescent hydrogels are being explored as fully implantable alternatives to traditional CGMs. Our previous work investigated hydrogel matrices functionalized with enzymes and oxygen-quenched phosphors, demonstrating sensitivity to glucose, range of response, and biofouling strongly depend on the matrix material. Here, we further investigate the effect of matrix composition on overall performance in vitro and in vivo. METHODS: Sensors based on three hydrogels, a poly(2-hydroxyethyl methacrylate) (pHEMA) homopolymer and 2 poly(2-hydroxyethyl methacrylate-co-acrylamide) (pHEMA-co-AAm) copolymers, were compared. These were used to entrap glucose oxidase (GOx), catalase, and an oxygen-sensitive benzoporphyrin phosphor. All sensor formulations were evaluated for glucose response and stability at physiological temperatures. Selected sensors were then evaluated as implanted sensors in a porcine model challenged with glucose and insulin. The animal protocol used in this study was approved by an IACUC committee at Texas A&M University. RESULTS: PHEMA-co-AAm copolymer hydrogels (75:25 HEMA:AAm) yielded the most even GOx and dye dispersion throughout the hydrogel matrix and best preserved GOx apparent activity. In response to in vitro glucose challenges, this formulation exhibited a dynamic range of 12-167 mg/dL, a sensitivity of 1.44 ± 0.46 µs/(mg/dL), and tracked closely with reference capillary blood glucose values in vivo. CONCLUSIONS: The hydrogel-based sensors exhibited excellent sensitivity and sufficiently rapid response to the glucose levels achieved in vivo, proving feasibility of these materials for use in real-time glucose tracking. Extending the dynamic range and assessing long-term effects in vivo are ongoing efforts.

The First-in-Man "Si Se Puede" Study for the use of micro-oxygen sensors (MOXYs) to determine dynamic relative oxygen indices in the feet of patients with limb-threatening ischemia during endovascular therapy.

OBJECTIVE: Patients with limb-threatening ischemia exhibit uneven patterns of perfusion in the foot, which makes it challenging to determine adequate topographic perfusion by angiography alone. This study assessed the feasibility of reporting dynamic relative oxygen indices and tissue oxygen concentration from multiple locations on the foot during endovascular therapy using a novel micro-oxygen sensor (MOXY; PROFUSA, Inc, South San Francisco, Calif) approach. METHODS: A prospective, 28-day, single-arm, observational study was performed in 10 patients who underwent endovascular therapy for limb-threatening ischemia. At least 24 hours before therapy, four microsensors were injected in each patient (one in the arm, three in the treated foot). The optical signal from the microsensors corresponded to tissue oxygen concentration. A custom detector on the surface of the skin was used to continuously and noninvasively measure the signals from the microsensors. The ability to locate and read the signal from each injected microsensor was characterized. Oxygen data from the microsensors were collected throughout the revascularization procedure. The timing of therapy deployment was recorded during the procedure to assess its relationship with the microsensor oxygen data. Oxygen data collection and clinical evaluation were performed immediately postoperatively as well as postoperatively on days 7, 14, 21, and 28. RESULTS: The study enrolled 10 patients (50% male) with ischemia (30% Rutherford class 4, 70% Rutherford class 5). Patients were a mean age of 70.7 years (range, 46-90 years), and all were Hispanic of varying origin. Microsensors were successfully read 206 of 212 times (97.2%) in all patients during the course of the study. Microsensors were compatible with intraoperative use in the interventional suite and postoperatively in an office setting. In nine of 10 revascularization procedures, at least one of the three MOXYs showed an immediate change in the dynamic relative oxygen index, correlating to deployed therapy. Moreover, there was a statistically significant increase in the concentration of oxygen in the foot in preoperative levels compared with postoperative levels. No adverse events occurred related to the microsensor materials. CONCLUSIONS: This MOXY approach appears to be safe when implanted in patients with limb-threatening ischemia undergoing endovascular recanalization and is effective in reporting local tissue oxygen concentrations over a course of 28 days. Further testing is needed to determine its potential effect on clinical decision making, both acutely on-table and chronically as a surveillance modality, which ultimately can lead to improved healing and limb salvage.

Susceptibility of interstitial continuous glucose monitor performance to sleeping position.

BACKGROUND: Developing a round-the-clock artificial pancreas requires accurate and stable continuous glucose monitoring. The most widely used continuous glucose monitors (CGMs) are percutaneous, with the sensor residing in the interstitial space. Inaccuracies in percutaneous CGM readings during periods of lying on the devices (e.g., in various sleeping positions) have been anecdotally reported but not systematically studied. METHODS: In order to assess the impact of sleep and sleep position on CGM performance, we conducted a study in human subjects in which we measured the variability of interstitial CGM data at night as a function of sleeping position. Commercially available sensors were placed for 4 days in the abdominal subcutaneous tissue in healthy, nondiabetic volunteers (four sensors per person, two per side). Nocturnal sleeping position was determined from video recordings and correlated to sensor data. RESULTS: We observed that, although the median of the four sensor readings was typically 70-110 mg/dl during sleep, individual sensors intermittently exhibited aberrant glucose readings (>25 mg/dl away from median) and that these aberrant readings were strongly correlated with subjects lying on the sensors. We expected and observed that most of these aberrant sleep-position-related CGM readings were sudden decreases in reported glucose values, presumably due to local blood-flow decreases caused by tissue compression. Curiously, in rare cases, the aberrant CGM readings were elevated values. CONCLUSIONS: These findings highlight limitations in our understanding of interstitial fluid physiology in the subcutaneous space and have significant implications for the utilization of sensors in the construction of an artificial pancreas.

Biofouling of polymer hydrogel materials and its effect on diffusion and enzyme-based luminescent glucose sensor functional characteristics.

BACKGROUND: Continuous glucose monitoring is crucial to developing a successful artificial pancreas. However, biofouling and host response make in vivo sensor performance difficult to predict. We investigated changes in glucose diffusivity and sensor response of optical enzymatic glucose sensors due to biological exposure. METHOD: Three hydrogel materials, poly(2-hydroxyethyl methacrylate) (pHEMA), poly(acrylamide) (pAM), and poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (p(HEMA-co-AM)), were tested for glucose diffusivity before and after exposure to serum or implantation in rats for 1 month. Luminescent sensors based on these materials were measured to compare the response to glucose before and after serum exposure. RESULTS: Glucose diffusivity through the pHEMA [(8.1 ± 0.38) × 10(-8) cm(2)/s] slabs was much lower than diffusivity through pAM [(2.7 ± 0.15) × 10(-6) cm(2)/s] and p(HEMA-co-AM) [(2.5 ± 0.08) × 10(-6)]. As expected from these differences, sensor response was highly dependent on material type. The pHEMA sensors had a maximum sensitivity of 2.5%/(mg/dl) and an analytical range of 4.2-356 mg/dl, while the p(HEMA-co-AM) sensors had a higher sensitivity [14.9%/(mg/dl)] and a narrower analytical range (17.6-70.5 mg/dl). After serum exposure, the pHEMA sensors were unaffected, whereas the p(HEMA-co-AM) sensors exhibited significantly decreased sensitivity and increased analytical range. CONCLUSIONS: Decreases in glucose diffusivity in the polymers resulting from in vitro serum exposure and residence in vivo were shown to be similar, suggesting that serum incubation was a reasonable approximation of in vivo fouling. While biofouling is expected to affect the response of flux-based sensors, we have shown that this depended on the type of sensor and matrix used. Therefore, proper design and materials selection may minimize response alterations occurring upon implantation.

Interstitial fluid physiology as it relates to glucose monitoring technologies: symposium introduction.

Nearly all commercially available glucose sensors share the subcutaneous interstitial fluid (ISF) compartment as their preferred implantation site. However, ISF physiology as it relates to glucose sensors is not well understood. This special symposium titled "Interstitial Fluid Physiology as It Relates to Glucose Monitoring Technologies" is intended to help to bridge the gap in our understanding. This symposium is intended to foster a greater understanding of biological factors that impact the success of implantable glucose monitors and to inspire additional research in the area of ISF physiology as it relates to glucose sensing. Recognition that sensor designers need to have an intimate understanding of the biological environment in which their sensor will reside is emphasized. The symposium is published in two parts, with part I published in September 2010 and part II published in May 2011. All articles published in this symposium are summarized herein.

Biomechanics of the sensor-tissue interface-effects of motion, pressure, and design on sensor performance and the foreign body response-part I: theoretical framework.

The importance of biomechanics in glucose sensor function has been largely overlooked. This article is the first part of a two-part review in which we look beyond commonly recognized chemical biocompatibility to explore the biomechanics of the sensor-tissue interface as an important aspect of continuous glucose sensor biocompatibility. Part I provides a theoretical framework to describe how biomechanical factors such as motion and pressure (typically micromotion and micropressure) give rise to interfacial stresses, which affect tissue physiology around a sensor and, in turn, impact sensor performance. Three main contributors to sensor motion and pressure are explored: applied forces, sensor design, and subject/patient considerations. We describe how acute forces can temporarily impact sensor signal and how chronic forces can alter the foreign body response and inflammation around an implanted sensor, and thus impact sensor performance. The importance of sensor design (e.g., size, shape, modulus, texture) and specific implant location on the tissue response are also explored. In Part II: Examples and Application (a sister publication), examples from the literature are reviewed, and the application of biomechanical concepts to sensor design are described. We believe that adding biomechanical strategies to the arsenal of material compositions, surface modifications, drug elution, and other chemical strategies will lead to improvements in sensor biocompatibility and performance.