Activity-based Sensing Research Articles

Xanthene-Based Dyes for Photoacoustic Imaging and their Use as Analyte-Responsive Probes.

Developing imaging tools that can report on the presence of disease-relevant analytes in multicellular organisms can provide insight into fundamental disease mechanisms as well as provide diagnostic tools for the clinic. Photoacoustic imaging (PAI) is a light-in, sound-out imaging technique that allows for high resolution, deep-tissue imaging with applications in pre-clinical and point-of-care settings. The continued development of near-infrared (NIR) absorbing small-molecule dyes promises to improve the capabilities of this emerging imaging modality. For example, new dye scaffolds bearing chemoselective functionalities are enabling the detection and quantification of disease-relevant analytes through activity-based sensing (ABS) approaches. Recently described strategies to engineer NIR absorbing xanthenes have enabled development of analyte-responsive PAI probes using this classic dye scaffold. Herein, we present current strategies for red-shifting the spectral properties of xanthenes via bridging heteroatom or auxochrome modifications. Additionally, we explore how these strategies, coupled with chemoselective spiroring-opening approaches, have been employed to create ABS probes for in vivo detection of hypochlorous acid, nitric oxide, copper (II), human NAD(P)H: quinone oxidoreductase isozyme 1, and carbon monoxide. Given the versatility of the xanthene scaffold, we anticipate continued growth and development of analyte-responsive PAI imaging probes based on this dye class.

Accelerated Activity-Based Sensing by Fluorogenic Reporter Engineering Enables to Rapidly Determine Unstable Analyte.

Accurate detection of labile analytes through activity based fluorogenic sensing is meaningful but remains a challenge because of nonrapid reaction kinetic. Herein, we present a signaling reporter engineering strategy to accelerate azoreduction reaction by positively charged fluorophore promoted unstable anion recognition for rapidly sensing sodium dithionite (Na2S2O4), a kind of widespread used but harmful inorganic reducing agent. Its quick decomposition often impedes application reliability of traditional fluorogenic probes in real samples because of their slow responses. In this work, four azo-based probes with different charged fluorophores (positive, zwitterionic, neutral, and negative) were synthesized and compared. Among of them, with sequestration effect of positively charged anthocyanin fluorophore for dithionite anion via electrostatic attraction, the cationic probe Azo-Pos displayed ultrafast fluorogenic response (∼2 s) with the fastest response kinetic (kpos' = 0.373 s-1) that is better than other charged ones (kzwi' = 0.031 s-1, kneu' = 0.013 s-1, kneg' = 0.003 s-1). Azo-Pos was demonstrated to be capable to directly detect labile Na2S2O4 in food samples and visualize the presence of Na2S2O4 in living systems in a timely fashion. This new probe has potential as a robust tool to fluorescently monitor excessive food additives and biological invasion of harmful Na2S2O4. Moreover, our proposed accelerating strategy would be versatile to develop more activity-based sensing probes for quickly detecting other unstable analytes of interest.

A Transfer Hydrogenation Approach to Activity-Based Sensing of Formate in Living Cells.

Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.

Click chemistry in the design of AIEgens for biosensing and bioimaging

AbstractThe development of rapid, selective, and sensitive fluorescent sensors is essential for visualizing and quantifying biological molecules and processes in vitro, ex vitro, and in vivo, which is important for not only fundamental biological studies but the accurate diagnosis of diseases. The emerging field of activity‐based sensing (ABS), a sensing method that utilizes molecular reactivity for analyte detection possesses many advantages including high specificity, sensitivity and accuracy. The aggregation caused quenching phenomenon which occurs in most conventional fluorophores results in reduced labeling efficiency of the target analytes and low photobleaching resistance, therefore limiting the applications of the ABS strategy. In contrast, aggregation induced emission (AIE) active luminogens (AIEgens) provide exceptional molecular frameworks for ABS. Of the many reaction classes utilized in the AIEgen ABS approach, click chemistry has become increasing popular. In this review, the sensing concepts of the ABS approach with AIEgens and the principles of click chemistry are discussed, followed by a systematic summary of the application of specific click chemistry reactions in AIEgen ABS protocols for the detection of an array of target analytes. Furthermore, the utility of click chemistry in the construction of AIEgens for bioimaging will also be showcased throughout the review.

Hydrolysis-Resistant Ester-Based Linkers for Development of Activity-Based NIR Bioluminescence Probes.

Activity-based sensing (ABS) probes equipped with a NIR bioluminescence readout are promising chemical tools to study cancer biomarkers owing to their high sensitivity and deep tissue compatibility. Despite the demand, there is a dearth of such probes because NIR substrates (e.g., BL660 (a NIR luciferin analog)) are not equipped with an appropriate attachment site for ABS trigger installation. For instance, our attempts to mask the carboxylic acid moiety with standard self-immolative benzyl linkers resulted in significant background signals owing to undesirable ester hydrolysis. In this study, we overcame this longstanding challenge by rationally designing a new hydrolysis-resistant ester-based linker featuring an isopropyl shielding arm. Compared to the parent, the new design is 140.5-fold and 67.8-fold more resistant toward spontaneous and esterase-mediated hydrolysis, respectively. Likewise, we observed minimal cleavage of the ester moiety when incubated with a panel of enzymes possessing ester-hydrolyzing activity. These impressive in vitro results were corroborated through a series of key experiments in live cells. Further, we showcased the utility of this technology by developing the first NIR bioluminescent probe for nitroreductase (NTR) activity and applied it to visualize elevated NTR expression in oxygen deficient lung cancer cells and in a murine model of non-small cell lung cancer. The ability to monitor the activity of this key biomarker in a deep tissue context is critical because it is associated with tumor hypoxia, which in turn is linked to drug resistance and aggressive cancer phenotypes.

Activity-Based Sensing for Chemistry-Enabled Biology: Illuminating Principles, Probes, and Prospects for Boronate Reagents for Studying Hydrogen Peroxide.

Activity-based sensing (ABS) offers a general approach that exploits chemical reactivity as a method for selective detection and manipulation of biological analytes. Here, we illustrate the value of this chemical platform to enable new biological discovery through a case study in the design and application of ABS reagents for studying hydrogen peroxide (H2O2), a major type of reactive oxygen species (ROS) that regulates a diverse array of vital cellular signaling processes to sustain life. Specifically, we summarize advances in the use of activity-based boronate probes for the detection of H2O2 featuring high molecular selectivity over other ROS, with an emphasis on tailoring designs in chemical structure to promote new biological principles of redox signaling.

Activity-Based NIR Bioluminescence Probe Enables Discovery of Diet-Induced Modulation of the Tumor Microenvironment via Nitric Oxide.

Nitric oxide (NO) plays a critical role in acute and chronic inflammation. NO’s contributions to cancer are of particular interest due to its context-dependent bioactivities. For example, immune cells initially produce cytotoxic quantities of NO in response to the nascent tumor. However, it is believed that this fades over time and reaches a concentration that supports the tumor microenvironment (TME). These complex dynamics are further complicated by other factors, such as diet and oxygenation, making it challenging to establish a complete picture of NO’s impact on tumor progression. Although many activity-based sensing (ABS) probes for NO have been developed, only a small fraction have been employed in vivo, and fewer yet are practical in cancer models where the NO concentration is <200 nM. To overcome this outstanding challenge, we have developed BL660-NO, the first ABS probe for NIR bioluminescence imaging of NO in cancer. Owing to the low intrinsic background, high sensitivity, and deep tissue imaging capabilities of our design, BL660-NO was successfully employed to visualize endogenous NO in cellular systems, a human liver metastasis model, and a murine breast cancer model. Importantly, its exceptional performance facilitated two dietary studies which examine the impact of fat intake on NO and the TME. BL660-NO provides the first direct molecular evidence that intratumoral NO becomes elevated in mice fed a high-fat diet, which became obese with larger tumors, compared to control animals on a low-fat diet. These results indicate that an inflammatory diet can increase NO production via recruitment of macrophages and overexpression of inducible nitric oxide synthase which in turn can drive tumor progression.

A Selective Fluorescent Turn-On Probe for Imaging and Activity-Based Sensing of Hydrogen Peroxide in Living Cells

A Selective Fluorescent Turn-On Probe for Imaging and Activity-Based Sensing of Hydrogen Peroxide in Living Cells

Advances in Activity-Based Sensing Probes for Isoform-Selective Imaging of Enzymatic Activity.

Until recently, there were no generalizable methods for assessing the effects of post-translational regulation on enzymatic activity. Activity-based sensing (ABS) has emerged as a powerful approach for monitoring small-molecule and enzyme activities within living systems. Initial examples of ABS were applied for measuring general enzymatic activity; however, a recent focus has been placed on increasing the selectivity to monitor a single enzyme or isoform. The highest degree of selectivity is required for differentiating between isoforms, where the targets display significant structural similarities as a result of a gene duplication or alternative splicing. This Minireview highlights key examples of small-molecule isoform-selective probes with a focus on the relevance of isoform differentiation, design strategies to achieve selectivity, and applications in basic biology or in the clinic.

Ligand-Directed Approach to Activity-Based Sensing: Developing Palladacycle Fluorescent Probes That Enable Endogenous Carbon Monoxide Detection.

Carbon monoxide (CO) is an emerging gasotransmitter and reactive carbon species with broad anti-inflammatory, cytoprotective, and neurotransmitter functions along with therapeutic potential for the treatment of cardiovascular diseases. The study of CO chemistry in biology and medicine relative to other prominent gasotransmitters such as NO and H2S remains challenging, in large part due to limitations in available tools for the direct visualization of this transient and freely diffusing small molecule in complex living systems. Here we report a ligand-directed activity-based sensing (ABS) approach to CO detection through palladium-mediated carbonylation chemistry. Specifically, the design and synthesis of a series of ABS probes with systematic alterations in the palladium-ligand environment (e.g., sp3-S, sp3-N, sp2-N) establish structure-activity relationships for palladacycles to confer selective reactivity with CO under physiological conditions. These fundamental studies led to the development of an optimized probe, termed Carbon Monoxide Probe-3 Ester Pyridine (COP-3E-Py), which enables imaging of CO release in live cell and brain settings, including monitoring of endogenous CO production that triggers presynaptic dopamine release in fly brains. This work provides a unique tool for studying CO in living systems and establishes the utility of a synthetic methods approach to activity-based sensing using principles of organometallic chemistry.

Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper.

Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed, copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the same time copper misregulation is associated with inflammation and neurodegeneration. As such, chemical probes that can track dynamic changes in copper with spatial resolution, especially in loosely bound, labile forms, are valuable tools to identify and characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we designed copper-directed acyl imidazole dyes that operate through copper-mediated activation of acyl imidazole electrophiles for subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively, after exposure to inflammatory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper, an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic signaling and stress analytes in biology.

Activity-Based Sensing: A Synthetic Methods Approach for Selective Molecular Imaging and Beyond.

Emerging from the origins of supramolecular chemistry and the development of selective chemical receptors that rely on lock-and-key binding, activity-based sensing (ABS)-which utilizes molecular reactivity rather than molecular recognition for analyte detection-has rapidly grown into a distinct field to investigate the production and regulation of chemical species that mediate biological signaling and stress pathways, particularly metal ions and small molecules. Chemical reactions exploit the diverse chemical reactivity of biological species to enable the development of selective and sensitive synthetic methods to decipher their contributions within complex living environments. The broad utility of this reaction-driven approach facilitates application to imaging platforms ranging from fluorescence, luminescence, photoacoustic, magnetic resonance, and positron emission tomography modalities. ABS methods are also being expanded to other fields, such as drug and materials discovery.

An Activity-Based Sensing Approach for the Detection of Cyclooxygenase-2 in Live Cells.

Cyclooxygenase-2 (COX-2) overexpression is prominent in inflammatory diseases, neurodegenerative disorders, and cancer. Directly monitoring COX-2 activity within its native environment poses an exciting approach to account for and illuminate the effect of the local environments on protein activity. Herein, we report the development of CoxFluor, the first activity-based sensing approach for monitoring COX-2 within live cells with confocal microscopy and flow cytometry. CoxFluor strategically links a natural substrate with a dye precursor to engage both the cyclooxygenase and peroxidase activities of COX-2. This catalyzes the release of resorufin and the natural product, as supported by molecular dynamics and ensemble docking. CoxFluor enabled the detection of oxygen-dependent changes in COX-2 activity that are independent of protein expression within live macrophage cells.

Activity-Based Sensing and Theranostic Probes Based on Photoinduced Electron Transfer.

Fluorescent probes have become powerful tools in detection, imaging and disease diagnosis due to their high sensitivity, specificity, fast response, and technical simplicity. In the last decades, researchers have made remarkable progress in developing signaling mechanisms to design fluorescent probes such as photoinduced electron transfer (PET), intramolecular charge transfer (ICT), and fluorescence resonance energy transfer (FRET). Typical PET is composed of a multicomponent system in which a fluorophore (electron acceptor) is separately linked with a recognition group (electron donor) via a short spacer. PET probes normally feature a low fluorescence background and significant fluorescence enhancement in response to targets. Recent research revealed that PET probes have also been used as theranostic agents, whose fluorescence and toxicity can be simultaneously activated by cancer-specific parameters. In this Account, we highlight the recent advances of rational design and applications of PET probes, focusing primarily on studies from our research group. For example, different from the case of the traditional single-atom electron donor (O, S, N, Se, Te, etc.) in typical PET, we used more a electron-rich pyrrole ring to "switch off" the fluorescence of the fluorophore more efficiently through an "enhanced PET" effect which provided a lower background fluorescence and higher signal-to-noise ratio. Furthermore, normal PET represents the main principle behind the design of small molecule "off-on" fluorescent sensors. We developed new PET platform through intramolecular space folding (folding PET) to overcome the difficulty of designing PET enzyme-targeting probes. Therefore, based on typical PET and these new PET concepts, we, for instance, reported PET probes for the detection of Zn2+ without proton interference, a BODIPY-based d-PET probe for reporting local hydrophilicity within lysosomes, and an "enhanced PET" fluorescent probe for imaging HClO in cancer cells. We also developed COX-2-specific probe for identifying cancer cells and quantifying cancer-related events, and a KIAA1363-sensitive probe for tracking solid tumors in living mice. Furthermore, we first applied an aminopeptidase N (APN)-sensitive probe based on PET for cancer diagnosis and therapy. We anticipate that further development of PET fluorescent probes providing more sensitivity and selectivity to analytes of interest will be equipped with more functions and play indispensable roles in the studies of pathology, diagnostics, and cancer therapies.

Activity-Based Sensing Methods for Monitoring the Reactive Carbon Species Carbon Monoxide and Formaldehyde in Living Systems.

Carbon is central to the chemistry of life, and in addition to its fundamental roles as a static component of all major biomolecules spanning proteins, nucleic acids, sugars, and lipids, emerging evidence shows that small and transient carbon-based metabolites, termed reactive carbon species (RCS), are dynamic signaling/stress agents that can influence a variety of biological pathways. Recent examples include the identification of carbon monoxide (CO) as an ion channel blocker and endogenous formaldehyde (FA) as a one-carbon metabolic unit formed from the spontaneous degradation of dietary folate metabolites. These findings motivate the development of analytical tools for transient carbon species that can achieve high specificity and sensitivity to further investigate RCS signaling and stress pathways at the cell, tissue, and whole-organism levels. This Account summarizes work from our laboratory on the development of new chemical tools to monitor two important one-carbon RCS, CO and FA, through activity-based sensing (ABS), where we leverage the unique chemical reactivities of these small and transient analytes, rather than lock-and-key binding considerations, for selective detection. Classic inorganic/organometallic and organic transformations form the basis for this approach. For example, to distinguish CO from other biological diatomics of similar shape and size (e.g., nitric oxide and oxygen), we exploit palladium-mediated carbonylation as a synthetic method for CO sensing. The high selectivity of this carbonylation approach successfully enables imaging of dynamic changes in intracellular CO levels in live cells. Likewise, we apply the aza-Cope reaction for FA detection to provide high selectivity for this one-carbon unit over other larger biological aldehydes that are reactive electrophiles, such as acetaldehyde and methylglyoxal. By relying on an activity-based trigger as a design principle for small-molecule detection, this approach can be generalized to create a toolbox of selective FA imaging reagents, as illustrated by a broad range of FA probes spanning turn-on and ratiometric fluorescence imaging, positron emission tomography imaging, and chemiluminescence imaging modalities. Moreover, these chemical tools have revealed new one-carbon biology through the identification of folate as a dietary source of FA and alcohol dehydrogenase 5 as a target for FA metabolism. Indeed, these selective RCS detection methods have been expanded to a wider array of imaging platforms, such as metal-complex-based time-gated luminescence and materials-based imaging scaffolds (e.g., nanotubes, nanoparticles, and carbon dots), with modalities extending to Raman and Rayleigh scattering readouts. This pursuit of leveraging selective chemical reactivity to develop highly specific ABS probes for imaging of RCS provides not only practical tools for deciphering RCS-dependent biology but also a general design platform for developing ABS probes for a broader range of biological analytes encompassing elements across the periodic table.

Aggregation-Induced Emission Luminogens for Activity-Based Sensing.

Fluorescent sensing has emerged as a powerful tool for detecting various analytes and visualizing numerous biological processes by virtue of its superb sensitivity, rapidness, excellent temporal resolution, easy operation, and low cost. Of particular interest is activity-based sensing (ABS), a burgeoning sensing approach that is actualized on the basis of dynamic molecular reactivity rather than conventional lock-and-key molecular recognition. ABS has been recognized to possess some distinct advantages, such as high specificity, extraordinary sensitivity, and accurate signal outputs. A majority of ABS sensors are constructed by modifying conventional fluorogens, which are strongly emissive when molecularly dissolved in solvents but experience emission quenching upon aggregate formation or concentration increase. The aggregation-caused quenching (ACQ) phenomenon leads to a limited amount of labeling of the analyte with the sensor and low photobleaching resistance, which could impede practical applications of the ABS protocol. As an anti-ACQ phenomenon, aggregation-induced emission (AIE) provides a straightforward solution to the ACQ problem. Thanks to their intrinsic advantages, including high photobleaching threshold, high signal-to-noise ratio, fluorescence turn-on nature, and large Stokes shift, AIE-active luminogens (AIEgens) represent a class of extraordinary fluorogen alternatives for the ABS protocol. The use of AIEgen-involved ABS can integrate the advantages of AIEgens and ABS, and additionally, the AIE process offers some unique properties to the ABS approach. For instance, in some cases of water-soluble AIEgen-involved ABS, chemical reaction not only leads to a chang in the emission color of the AIEgens but also causes solubility variations, which could result in specific "light-up" signaling. In this Account, the basic concepts and mechanistic insights of the ABS approach involving the AIE principle are briefly summarized, and then we highlight the new breakthroughs, seminal studies, and trends in the area that have been most recently reported by our group. This emerging sensing protocol has been successfully utilized for detecting an array of targets including ions, small molecules, biomacromolecules, and microenvironments, all of which closely relate to human health, medical, and public concerns. These detections are smoothly achieved on the basis of various reactions (e.g., hydrolysis, boronate cleavage, dephosphorylation, addition, cyclization, and rearrangement reactions) through different sensing principles. In these studies, the AIEgen-involved ABS strategy generally shows good biocompatibility, high selectivity, excellent reliability and high signal contrast, strongly indicating its great potential for high-tech innovations in the sensing field, among which bioprobing is of particular interest. With this Account, we hope to spark new ideas and inspire new endeavors in this emerging research area, further promoting state-of-the-art developments in the field of sensing.

Chemiluminescent Probes for Activity‐Based Sensing of Formaldehyde Released from Folate Degradation in Living Mice

AbstractFormaldehyde (FA) is a common environmental toxin that is also produced naturally in the body through a wide range of metabolic and epigenetic processes, motivating the development of new technologies to monitor this reactive carbonyl species (RCS) in living systems. Herein, we report a pair of first‐generation chemiluminescent probes for selective formaldehyde detection. Caging phenoxy‐dioxetane scaffolds bearing different electron‐withdrawing groups with a general 2‐aza‐Cope reactive formaldehyde trigger provides chemiluminescent formaldehyde probes 540 and 700 (CFAP540 and CFAP700) for visible and near‐IR detection of FA in living cells and mice, respectively. In particular, CFAP700 is capable of visualizing FA release derived from endogenous folate metabolism, providing a starting point for the use of CFAPs and related chemical tools to probe FA physiology and pathology, as well as for the development of a broader palette of chemiluminescent activity‐based sensing (ABS) probes that can be employed from in vitro biochemical to cell to animal models.

Chemiluminescent Probes for Activity-Based Sensing of Formaldehyde Released from Folate Degradation in Living Mice.

Formaldehyde (FA) is a common environmental toxin that is also produced naturally in the body through a wide range of metabolic and epigenetic processes, motivating the development of new technologies to monitor this reactive carbonyl species (RCS) in living systems. Herein, we report a pair of first-generation chemiluminescent probes for selective formaldehyde detection. Caging phenoxy-dioxetane scaffolds bearing different electron-withdrawing groups with a general 2-aza-Cope reactive formaldehyde trigger provides chemiluminescent formaldehyde probes 540 and 700 (CFAP540 and CFAP700) for visible and near-IR detection of FA in living cells and mice, respectively. In particular, CFAP700 is capable of visualizing FA release derived from endogenous folate metabolism, providing a starting point for the use of CFAPs and related chemical tools to probe FA physiology and pathology, as well as for the development of a broader palette of chemiluminescent activity-based sensing (ABS) probes that can be employed from in vitro biochemical to cell to animal models.

Fluorescence Imaging and Engineered Biosensors

Annals of the New York Academy of SciencesVolume 961, Issue 1 p. 196-197 Fluorescence Imaging and Engineered Biosensors Functional and Activity-Based Sensing Using High Content Screening RAVI KAPUR, Corresponding Author RAVI KAPUR Cellomics, Inc., Pittsburgh, Pennsylvania 15219, USAAddress for correspondence: Ravi Kapur, Cellomics, Inc., 100 Technology Drive, Pittsburgh, PA 15219. Voice: 412-826-3600; fax: 412-826-3850; [email protected].Search for more papers by this author RAVI KAPUR, Corresponding Author RAVI KAPUR Cellomics, Inc., Pittsburgh, Pennsylvania 15219, USAAddress for correspondence: Ravi Kapur, Cellomics, Inc., 100 Technology Drive, Pittsburgh, PA 15219. Voice: 412-826-3600; fax: 412-826-3850; [email protected].Search for more papers by this author First published: 24 January 2006 https://doi.org/10.1111/j.1749-6632.2002.tb03081.xCitations: 12Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume961, Issue1REPARATIVE MEDICINE: GROWING TISSUES AND ORGANSJune 2002Pages 196-197 RelatedInformation