Continuous oxygen monitoring to enhance ex-vivo organ machine perfusion and reconstructive surgery.

Continuous oxygenation monitoring of machine-perfused organs or transposed autologous tissue is not currently implemented in clinical practice. Oxygenation is a critical parameter that could be used to verify tissue viability and guide corrective interventions, such as perfusion machine parameters or surgical revision. This work presents an innovative technology based on oxygen-sensitive, phosphorescent metalloporphyrin allowing continuous and non-invasive oxygen monitoring of ex-vivo perfused vascularized fasciocutaneous flaps. The method comprises a small, low-energy optical transcutaneous oxygen sensor applied on the flap's skin paddle as well as oxygen sensing devices placed into the tubing. An intermittent perfusion setting was designed to study the response time and accuracy of this technology over a total of 54 perfusion cycles. We further evaluated correlation between the continuous oxygen measurements and gold-standard perfusion viability metrics such as vascular resistance, with good agreement suggesting potential to monitor graft viability at high frequency, opening the possibility to employ feedback control algorithms in the future. This proof-of-concept study opens a range of research and clinical applications in reconstructive surgery and transplantation at a time when perfusion machines undergo rapid clinical adoption with potential to improve outcomes across a variety of surgical procedures and dramatically increase access to transplant medicine.

Versatile, in-line optical oxygen tension sensors for continuous monitoring during ex vivo kidney perfusion.

Integration of physiological sensing modalities within tissue and organ perfusion systems is becoming a steadily expanding field of research, aimed at achieving technological breakthrough innovations that will expand the sites and clinical settings at which such systems can be used. This is becoming possible in part due to the advancement of user-friendly optical sensors in recent years, which rely both on synthetic, luminescent sensor molecules and inexpensive, low-power electronic components for device engineering. In this article we report a novel approach towards enabling automated, continuous monitoring of oxygenation during ex vivo organ perfusion, by combining versatile flow cell components and low-power, programmable electronic readout devices. The sensing element comprises a 3D printed, miniature flow cell with tubing connectors and an affixed oxygen-sensing thin film material containing in-house developed, brightly-emitting metalloporphyrin phosphor molecules embedded within a polymer matrix. Proof-of-concept validation of this technology is demonstrated through integration within the tubing circuit of a transportable medical device for hypothermic oxygenated machine perfusion of extracted kidneys as a model for organs to be preserved as transplants.

Development and in-vivo validation of a portable phosphorescence lifetime-based fiber-optic oxygen sensor.

Oxygenation is a crucial indicator of tissue viability and function. Oxygen tension ([Formula: see text]), i.e. the amount of molecular oxygen present in the tissue is a direct result of supply (perfusion) and consumption. Thus, measurement of [Formula: see text] is an effective method to monitor tissue viability. However, tissue oximetry sensors commonly used in clinical practice instead rely on measuring oxygen saturation ([Formula: see text]), largely due to the lack of reliable, affordable [Formula: see text] sensing solutions. To address this issue we present a proof-of-concept design and validation of a low-cost, lifetime-based oxygen sensing fiber. The sensor consists of readily-available off-the shelf components such as a microcontroller, a light-emitting diode (LED), an avalanche photodiode (APD), a temperature sensor, as well as a bright in-house developed porphyrin molecule. The device was calibrated using a benchtop setup and evaluated in three in vivo animal models. Our findings show that the new device design in combination with the bright porphyrin has the potential to be a useful and accurate tool for measuring [Formula: see text] in tissue, while also highlighting some of the limitations and challenges of oxygen measurements in this context.

A Patient-Ready Wearable Transcutaneous CO2 Sensor.

Continuously monitoring transcutaneous CO2 partial pressure is of crucial importance in the diagnosis and treatment of respiratory and cardiac diseases. Despite significant progress in the development of CO2 sensors, their implementation as portable or wearable devices for real-time monitoring remains under-explored. Here, we report on the creation of a wearable prototype device for transcutaneous CO2 monitoring based on quantifying the fluorescence of a highly breathable CO2-sensing film. The developed materials are based on a fluorescent pH indicator (8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt or HPTS) embedded into hydrophobic polymer matrices. The film's fluorescence is highly sensitive to changes in CO2 partial pressure in the physiological range, as well as photostable and insensitive to humidity. The device and medical-grade films are based on our prior work on transcutaneous oxygen-sensing technology, which has been extensively validated clinically.

Quantitative Luminescence Photography of a Swellable Hydrogel Dressing with a Traffic-Light Response to Oxygen.

Sensor-integrated wound dressings are emerging tools applicable to a wide variety of medical applications from emergency triage to at-home monitoring. Uncomfortable, unnecessary wound dressing changes may be avoided by providing quantitative insight into tissue characteristics related to wound healing such as tissue oxygenation, pH, and exudate/transudate volume. Here, a simple cost-effective methodology for quantifying oxygen and pH in a swellable hydrogel dressing using a single photograph is presented. The red and green luminescence of a novel dendritic polyamine Pt-porphyrin and fluorescein conjugate quantitatively responds to oxygen and pH, respectively, and enables robust sensing. The porphyrin conjugate, when combined with a four-arm star polyethylene glycol (PEG) amine polymer, rapidly crosslinks at room temperature with an N-hydroxysuccinimide (NHS)-PEG crosslinker to form a color-changing hydrogel dressing with tunable swelling capabilities applicable to a variety of wound environments. An inexpensive digital single-lens reflex (DSLR) camera modified with bandpass filters captures the hydrogel luminescence using simple macroscopic photography, and conversion to HSB colorspace allows for intensity-independent image analysis of the hydrogels' dual modality response. The hydrogel formulation exhibits a robust and validated visible red-orange-green "traffic light" spectrum in response to oxygen changes, regardless of swelling state, pH, or autofluorescence from skin, thereby enabling the clinician friendly naked-eye feedback.

Portable Oxygen-Sensing Device for the Improved Assessment of Compartment Syndrome and other Hypoxia-Related Conditions.

Measurement of intramuscular oxygen could play a key role in the early diagnosis of acute compartment syndrome, a common condition occurring after severe trauma leading to ischemia and long-term consequences including rhabdomyolysis, limb loss, and death. However, to date, there is no existing oxygen sensor approved for such a purpose. To address the need to improve the assessment of compartment syndrome, a portable fiber-optic device for intramuscular oxygen measurements was developed. The device is based on phosphorescence quenching, where the tip of an optical fiber was coated with a poly(propyl methacrylate) (PPMA) matrix containing a brightly emitting Pt(II)-core porphyrin. The optoelectronic circuit is highly portable and is based on a microspectrometer and a microcontroller readout with a smartphone. Results from an in vivo tourniquet porcine model show that the sensor is sensitive across the physiological oxygen partial pressure range of 0-80 mmHg and exhibits an appropriate and reproducible response to changes in intramuscular oxygen. A commercial laboratory oxygen sensor based on a lifetime measurement did not respond as expected.

A paintable phosphorescent bandage for postoperative tissue oxygen assessment in DIEP flap reconstruction.

Flaps are common in plastic surgery to reconstruct large tissue defects in cases such as trauma or cancer. However, most tissue oximeters used for monitoring ischemia in postoperative flaps are bulky, wired devices, which hinder direct flap observation. Here, we present the results of a clinical trial using a previously untried paintable transparent phosphorescent bandage to assess the tissue's partial pressure of oxygen (pO2). Statistical analysis revealed a strong relationship (P < 0.0001) between the rates of change of tissue oxygenation measured by the bandage and blood oxygen saturation (%stO2) readings from a standard-of-care ViOptix near-infrared spectroscopy oximeter. In addition, the oxygen-sensing bandage showed no adverse effects, proved easy handling, and yielded bright images across all skin tones with a digital single-lens reflex (DSLR) camera. This demonstrates the feasibility of using phosphorescent materials to monitor flaps postoperatively and lays the groundwork for future exploration in other tissue oxygen sensing applications.

Wearable device for remote monitoring of transcutaneous tissue oxygenation.

Wearable devices have found widespread applications in recent years as both medical devices as well as consumer electronics for sports and health tracking. A metric of health that is often overlooked in currently available technology is the direct measurement of molecular oxygen in living tissue, a key component in cellular energy production. Here, we report on the development of a wireless wearable prototype for transcutaneous oxygenation monitoring based on quantifying the oxygen-dependent phosphorescence of a metalloporphyrin embedded within a highly breathable oxygen sensing film. The device is completely self-contained, weighs under 30 grams, performs on-board signal analysis, and can communicate with computers or smartphones. The wearable measures tissue oxygenation at the skin surface by detecting the lifetime and intensity of phosphorescence, which undergoes quenching in the presence of oxygen. As well as being insensitive to motion artifacts, it offers robust and reliable measurements even in variable atmospheric conditions related to temperature and humidity. Preliminary in vivo testing in a porcine ischemia model shows that the wearable is highly sensitive to changes in tissue oxygenation in the physiological range upon inducing a decrease in limb perfusion.

Humidity-Insensitive Tissue Oxygen Tension Sensing for Wearable Devices†.

Quantification of tissue oxygen partial pressure (pO2 ) at the skin surface is crucial for diagnostic applications in burns, reconstructive surgeries, diabetic ulcers, etc. Further, current advances in wearable and communications technologies have widened the use of transcutaneous oxygen monitors (TCOM) for home care or even enhance athletic performance. For TCOM technology to find widespread use, devices must function reliably yet independently of changes in environmental conditions, humidity in particular. To this end, we have explored the incorporation of an oxygen-sensing metalloporphyrin within different host matrix materials of different compositions with the goal of overcoming the humidity sensitivity of previously explored oxygen-sensing materials. We developed a tetraethyl orthosilicate (TEOS)-based, highly breathable, oxygen-sensing metalloporphyrin polymer film which responds to changes in oxygenation independent of humidity.

Theranostic biocomposite scaffold membrane.

Acute and chronic wounds affect millions and are associated with billions of dollars in healthcare costs. The use of healing markers, biochemical cues from biocompatible matrices and materials, and their correlation with wound healing has the potential to generate valuable diagnostic, prognostic, and therapeutic information. In this study, we developed a collagen-dextran oxygen-sensing biocomposite scaffold membrane in which a phosphorescent oxygen sensor was incorporated to monitor physiological oxygen using in vivo phosphorescence imaging in a preclinical mouse model of wound healing. The oxygen-sensing biocomposite scaffold membrane enabled the noninvasive and longitudinal monitoring of oxygenation changes in vivo in an approach compatible with commercially available preclinical in vivo imaging system instruments. This study provides a new and novel capability where a biocomposite material can serve as a biocompatible, biodegradable theranostic platform to promote and assess tissue oxygenation during wound healing.

Systemically Administered Hemostatic Nanoparticles for Identification and Treatment of Internal Bleeding.

Internal bleeding is an injury that can be difficult to localize and effectively treat without invasive surgeries. Injectable polymeric nanoparticles have been developed that can reduce clotting times and blood loss, but they have yet to incorporate sufficient diagnostic capabilities to assist in identifying bleeding sources. Herein, polymeric nanoparticles were developed to simultaneously treat internal bleeding while incorporating tracers for visualization of the nanoparticles by standard clinical imaging modalities. Addition of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD; a fluorescent dye), biotin functionality, and gold nanoparticles to hemostatic polymeric nanoparticles resulted in nanoparticles amenable to imaging with near-infrared (NIR) imaging, immunohistochemistry, and X-ray computed tomography (CT), respectively. Following a lethal liver resection injury, visualization of accumulated nanoparticles by multiple imaging methods was achieved in rodents, with the highest accumulation observed at the liver injury site, resulting in improved survival rates. Tracer addition to therapeutic nanoparticles allows for an expansion of their applicability, during stabilization by first responders to diagnosis and identification of unknown internal bleeding sites by clinicians using standard clinical imaging modalities.

Oxygen-Sensing Paint-On Bandage: Calibration of a Novel Approach in Tissue Perfusion Assessment.

BACKGROUND: Knowledge of tissue oxygenation status is fundamental in the prevention of postoperative flap failure. Recently, the authors introduced a novel oxygen-sensing paint-on bandage that incorporated an oxygen-sensing porphyrin with a commercially available liquid bandage matrix. In this study, the authors extend validation of their oxygen-sensing bandage by comparing it to the use of near-infrared tissue oximetry in addition to Clark electrode measurements. METHODS: The oxygen-sensing paint-on bandage was applied to the left hind limb in a rodent model. Simultaneously, a near-infrared imaging device and Clark electrode were attached to the right and left hind limbs, respectively. Tissue oxygenation was measured under normal, ischemic (aortic ligation), and reperfused conditions. RESULTS: On average, the oxygen-sensing paint-on bandage measured a decrease in transdermal oxygenation from 85.2 mmHg to 64.1 mmHg upon aortic ligation. The oxygen-sensing dye restored at 81.2 mmHg after unclamping. Responses in both control groups demonstrated a similar trend. Physiologic changes from normal to ischemic and reperfused conditions were statistically significantly different in all three techniques (p < 0.001). CONCLUSIONS: The authors' newly developed oxygen-sensing paint-on bandage exhibits a comparable trend in oxygenation recordings in a rat model similar to conventional oxygenation assessment techniques. This technique could potentially prove to be a valuable tool in the routine clinical management of flaps following free tissue transfer. Incorporating oxygen-sensing capabilities into a simple wound dressing material has the added benefit of providing both wound protection and constant wound oxygenation assessment.

Two-photon microscopy measurement of cerebral metabolic rate of oxygen using periarteriolar oxygen concentration gradients.

The cerebral metabolic rate of oxygen ([Formula: see text]) is an essential parameter for evaluating brain function and pathophysiology. However, the currently available approaches for quantifying [Formula: see text] rely on complex multimodal imaging and mathematical modeling. Here, we introduce a method that allows estimation of [Formula: see text] based on a single measurement modality-two-photon imaging of the partial pressure of oxygen ([Formula: see text]) in cortical tissue. We employed two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen-sensitive nanoprobe PtP-C343 to map the tissue [Formula: see text] distribution around cortical penetrating arterioles. [Formula: see text] is subsequently estimated by fitting the changes of tissue [Formula: see text] around arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline [Formula: see text] in anesthetized rats and modulated tissue [Formula: see text] levels by manipulating the depth of anesthesia. This method provides [Formula: see text] measurements localized within [Formula: see text] and it may provide oxygen consumption measurements in individual cortical layers or within confined cortical regions, such as in ischemic penumbra and the foci of functional activation.

Bright, "Clickable" Porphyrins for the Visualization of Oxygenation under Ambient Light.

A new group of "clickable" and brightly emissive metalloporphyrins has been developed for the visualization of oxygenation under ambient light with the naked eye. These alkynyl-terminated compounds permit the rapid and facile synthesis of oxygen-sensing dendrimers through azide-alkyne click chemistry. With absorption maxima overlapping with the wavelengths of common commercial laser sources, they are readily applicable to biomedical imaging of tissue oxygenation. An efficient synthetic methodology, featuring the stable trimethylacetyl (pivaloyl) protecting group, is described for their preparation. A paint-on liquid bandage containing a new, click-synthesized porphyrin dendrimer has been used to map oxygenation across an ex vivo porcine skin burn model.

Oxygen-Sensing Methods in Biomedicine from the Macroscale to the Microscale.

Oxygen monitoring has been a topic of exhaustive study given its central role in the biochemistry of life. The ability to quantify the physiological distribution and real-time dynamics of oxygen from sub-cellular to macroscopic levels is required to fully understand the mechanisms associated with both normal physiology and disease states. This Review will present the most significant recent advances in the development of oxygen-sensing materials and techniques, including polarographic, nuclear medicine, magnetic resonance, and optical approaches, that can be applied specifically for the real-time monitoring of oxygen dynamics in cellular and tissue environments. As some of the most exciting recent advances in synthetic methods and biomedical applications have been in the field of optical oxygen sensors, a major focus will be on the development of these toolkits.

Non-invasive transdermal two-dimensional mapping of cutaneous oxygenation with a rapid-drying liquid bandage.

Oxygen plays an important role in wound healing, as it is essential to biological functions such as cell proliferation, immune responses and collagen synthesis. Poor oxygenation is directly associated with the development of chronic ischemic wounds, which affect more than 6 million people each year in the United States alone at an estimated cost of $25 billion. Knowledge of oxygenation status is also important in the management of burns and skin grafts, as well as in a wide range of skin conditions. Despite the importance of the clinical determination of tissue oxygenation, there is a lack of rapid, user-friendly and quantitative diagnostic tools that allow for non-disruptive, continuous monitoring of oxygen content across large areas of skin and wounds to guide care and therapeutic decisions. In this work, we describe a sensitive, colorimetric, oxygen-sensing paint-on bandage for two-dimensional mapping of tissue oxygenation in skin, burns, and skin grafts. By embedding both an oxygen-sensing porphyrin-dendrimer phosphor and a reference dye in a liquid bandage matrix, we have created a liquid bandage that can be painted onto the skin surface and dries into a thin film that adheres tightly to the skin or wound topology. When captured by a camera-based imaging device, the oxygen-dependent phosphorescence emission of the bandage can be used to quantify and map both the pO2 and oxygen consumption of the underlying tissue. In this proof-of-principle study, we first demonstrate our system on a rat ischemic limb model to show its capabilities in sensing tissue ischemia. It is then tested on both ex vivo and in vivo porcine burn models to monitor the progression of burn injuries. Lastly, the bandage is applied to an in vivo porcine graft model for monitoring the integration of full- and partial-thickness skin grafts.

Two-photon antenna-core oxygen probe with enhanced performance.

Recent development of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen enabled first noninvasive high-resolution measurements of tissue oxygenation in vivo in 3D, providing valuable physiological information. The so far developed two-photon-enhanced phosphorescent probes comprise antenna-core constructs, in which two-photon absorbing chromophores (antenna) capture and channel excitation energy to a phosphorescent core (metalloporphyrin) via intramolecular excitation energy transfer (EET). These probes allowed demonstration of the methods' potential; however, they suffer from a number of limitations, such as partial loss of emissivity to competing triplet state deactivation pathways (e.g., electron transfer) and suboptimal sensitivity to oxygen, thereby limiting spatial and temporal resolution of the method. Here we present a new probe, PtTCHP-C307, designed to overcome these limitations. The key improvements include significant increase in the phosphorescence quantum yield, higher efficiency of the antenna-core energy transfer, minimized quenching of the phosphorescence by electron transfer and increased signal dynamic range. For the same excitation flux, the new probe is able to produce up to 6-fold higher signal output than previously reported molecules. Performance of PtTCHP-C307 was demonstrated in vivo in pO2 measurements through the intact mouse skull into the bone marrow, where all blood cells are made from hematopoietic stem cells.

Click-assembled, oxygen-sensing nanoconjugates for depth-resolved, near-infrared imaging in a 3D cancer model.

Hypoxia is an important contributing factor to the development of drug-resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click-assembled oxygen-sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click-based ligation of poly(amidoamine)-like subunits for rapid assembly. Near-infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof-of-concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth-resolved imaging in tissue and tissue models.

Direct measurement of local oxygen concentration in the bone marrow of live animals.

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Label-Free, Longitudinal Visualization of PDT Response In Vitro with Optical Coherence Tomography.

A major challenge in creating and optimizing therapeutics in the fight against cancer is visualizing and understanding the microscale spatiotemporal treatment response dynamics that occur in patients. This is especially true for photodynamic therapy (PDT), where therapeutic optimization relies on understanding the interplay between factors such as photosensitizer localization and uptake, in addition to light dose and delivery rate. In vitro 3D culture systems that recapitulate many of the biological features of human disease are powerful platforms for carrying out detailed studies on PDT response and resistance. Current techniques for visualizing these models, however, often lack accuracy due to the perturbative nature of the sample preparation, with light attenuation complicating the study of intact models. Optical coherence tomography (OCT) is an ideal method for the long-term, non-perturbative study of in vitro models and their response to PDT. Monitoring the response of 3D models to PDT by time-lapse OCT methods promises to provide new perspectives and open the way to cancer treatment methodologies that can be translated towards the clinic.