Tissue Engineered Bio‐Blood‐Vessels Constructed Using a Tissue‐Specific Bioink and 3D Coaxial Cell Printing Technique: A Novel Therapy for Ischemic Disease

Endothelial progenitor cells (EPCs) are a promising cell source for the treatment of several ischemic diseases for their potentials in neovascularization. However, the application of EPCs in cell‐based therapy has shown low therapeutic efficacy due to hostile tissue conditions after ischemia. In this study, a bio‐blood‐vessel (BBV) is developed, which is produced using a novel hybrid bioink (a mixture of vascular‐tissue‐derived decellularized extracellular matrix (VdECM) and alginate) and a versatile 3D coaxial cell printing method for delivering EPC and proangiogenic drugs (atorvastatin) to the ischemic injury sites. The hybrid bioink not only provides a favorable environment to promote the proliferation, differentiation, and neovascularization of EPCs but also enables a direct fabrication of tubular BBV. By controlling the printing parameters, the printing method allows to construct BBVs in desired dimensions, carrying both EPCs and atorvastatin‐loaded poly(lactic‐co‐glycolic) acid microspheres. The therapeutic efficacy of cell/drug‐laden BBVs is evaluated in an ischemia model at nude mouse hind limb, which exhibits enhanced survival and differentiation of EPCs, increased rate of neovascularization, and remarkable salvage of ischemic limbs. These outcomes suggest that the 3D‐printed ECM‐mediated cell/drug implantation can be a new therapeutic approach for the treatment of various ischemic diseases.     

Capillary Network‐Like Organization of Endothelial Cells in PEGDA Scaffolds Encoded with Angiogenic Signals via Triple Helical Hybridization

Survival of tissue engineered constructs after implantation depends on proper vascularization. The differentiation of endothelial cells into mature microvasculature requires dynamic interactions between cells, scaffold, and growth factors, which are difficult to recapitulate in artificial systems. Previously, photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs), dubbed PEGDA‐CMP, that can be further conjugated with bioactive molecules via CMP‐CMP triple helix hybridization were reported. Here, it is shown that a bifunctional peptide featuring pro‐angiogenic domain mimicking vascular endothelial growth factor (VEGF) and a collagen mimetic domain that can fold into a triple helix conformation can hybridize with CMP side chains of the PEGDA‐CMP hydrogel, which results in presentation of insoluble VEGF‐like signals to endothelial cells. Presentation of VEGF‐like signals on the surface of micropatterned scaffolds in this way transforms cells from a quiescent state to elongated and aligned phenotype suggesting that this system could be used to engineer organized microvasculature. It is also shown that the pro‐angiogenic peptide, when applied topically in combination with modified dextran/PEGDA hydrogels, can enhance neovascularization of burn wounds in mice demonstrating the potential clinical use of CMP‐mediated matrix‐bound bioactive molecules for dermal injuries.     

Irreversible electroporation attenuates neointimal formation after angioplasty

Restenosis following coronary angioplasty represents a major clinical problem. Irreversible electroporation (IRE) is a nonthermal, nonpharmacological cell ablation method. IRE utilizes a sequence of electrical pulses that produce permanent damage to tissue within a few seconds. Methods and results: The left carotid arteries of eight rats underwent in vivo intimal damage using two Fogarty angioplasty catheters. The procedure was immediately followed by IRE ablation in four rats, while the remaining four were used as the control group. The IRE ablation was performed using a sequence of ten dc pulses of 3800 V/cm, 100 $mu$s each, at a frequency of ten pulses per second, applied across the blood vessel between two parallel electrodes. The electrical conductance of the treated tissue was measured during the electroporation to provide real-time feedback of the process. Left carotid arteries were excised and fixated after a 28-day follow-up period. Neointimal formation was evaluated histologically. The use of IRE was successful in three out of four animals in a way that is consistent with the measurements of blood vessel electrical properties. The integrity of the endothelial layer was recovered in the IRE-treated animals, compared with control. Successful IRE reduced neointima to media ratio ($0.57 pm 0.4$ versus $1.88 pm 1.0, P= 0.02$). Conclusions: We report for the first time the in vivo results of attenuation of neointimal formation using IRE. Our study shows that IRE might be able to attenuate neointimal formation after angioplasty damage in a rodent model of restenosis. This approach may open new venues in the treatment of coronary artery restenosis after balloon angioplasty.      

VEGF‐Functionalized Polyelectrolyte Multilayers as Proangiogenic Prosthetic Coatings

Stimulation of transprosthetic vascularization represents an interesting strategy in implantology to allow rapid tissue integration and finally to avoid prosthetic rejection. To achieve this goal, we modified the surface of porous titanium implants with polyelectrolyte multilayer (PEM) films functionalized with vascular endothelial growth factor (VEGF). Among the two PEM systems investigated, poly(L‐lysine)/poly(L‐glutamic acid) (PLL/PGA) and poly(allylamine hydrochloride)/poly(sodium 4‐styrenesulfonate) (PAH/PSS), the (PAH/PSS)₄ architecture was selected to functionalize porous titanium, both for its high efficiency to adsorb VEGF and for its biocompatibility toward endothelial cells. In an original way, we unambiguously demonstrated that VEGF adsorbed on (PAH/PSS)₄ maintains its bioactivity in vitro and stimulates endothelial cells proliferation. This effect was correlated with specific activation of intracellular signaling pathways induced by successive phosphorylation of the endothelial VEGF receptor VEGFR2 and mitogen‐activated protein kinases (MAPK) ERK1/2. By clearly demonstrating the proangiogenic activity of the VEGF‐PEM coating in vitro, the present study constitutes a first step toward in vivo application.     

Creating Physicochemical Gradients in Modular Microporous Annealed Particle Hydrogels via a Microfluidic Method

Microporous annealed particle (MAP) hydrogels are an attractive platform for engineering biomaterials with controlled heterogeneity. Here, a microfluidic method is introduced to create physicochemical gradients within poly(ethylene glycol) based MAP hydrogels. By combining microfluidic mixing and droplet generator modules, microgels with varying properties are produced by adjusting the relative flow rates between two precursor solutions and collected layer‐by‐layer in a syringe. Subsequently, the microgels are injected out of the syringe and then annealed with thiol‐ene click chemistry. Fluorescence intensity measurements of constructs annealed in vitro and after mock implantation into a tissue defect show that a continuous gradient profile is achieved and maintained after injection, indicating utility for in situ hydrogel formation. The effects of physicochemical property gradients on human mesenchymal stem cells (hMSCs) are also studied. Microgel stiffness is studied first, and the hMSCs exhibit increased spreading and proliferation as stiffness increased along the gradient. Microgel degradability is also studied, revealing a critical degradability threshold above which the hMSCs spread robustly and below which they are isolated and exhibit reduced spreading. This method of generating spatial gradients in MAP hydrogels can be further used to gain new insights into cell–material interactions, which can be leveraged for tissue engineering applications.     

Near‐Infrared Emitting and Pro‐Angiogenic Electrospun Conjugated Polymer Scaffold for Optical Biomaterial Tracking

Noninvasive tracking of biomaterials is vital for determining the fate and degradation of an implant in vivo, and to show its role in tissue regeneration. Current biomaterials have no inherent capacity to enable tracing but require labeling with, for example, fluorescent dyes, or nanoparticles. Here a novel biocompatible fully conjugated electrospun scaffold is described, based on a semiconducting luminescent polymer that can be visualized in situ after implantation using fluorescence imaging. The polymer, poly [2,3‐bis‐(3‐octyloxyphenyl)quinoxaline‐5,8‐diyl‐alt‐thiophene‐2,5‐diyl] (TQ1), is electrospun to form a fibrous mat. The fibers display fluorescence emission in the near‐infrared region with lifetimes in the sub‐nanosecond range, optimal for in situ imaging. The material shows no cytotoxic behaviors for embryonic chicken cardiomyocytes and mouse myoblasts, and cells migrate onto the TQ1 fibers even in the presence of a collagen substrate. Subcutaneous implantations of the material in rats show incorporation of the TQ1 fibers within the tissue, with limited inflammation and a preponderance of small capillaries around the fibers. The fluorescent properties of the TQ1 fibers are fully retained for up to 90 d following implantation and they can be clearly visualized in tissue using fluorescence and lifetime imaging, thus making it both a pro‐angiogenic and traceable biomaterial.     

Rapid Self‐Assembly of Macroscale Tissue Constructs at Biphasic Aqueous Interfaces

An entirely new approach to tissue engineering is presented that uses the interfacial forces between aqueous solutions of phase‐separating polymers to confine cells and promote their assembly into interconnected, macroscopic tissue constructs. This simple and inexpensive general procedure creates free‐standing, centimeter‐scale constructs from cell suspensions at the interface between poly(ethylene glycol) and dextran aqueous two‐phase systems in as little as 2 h. Using this method, skin constructs are produced that integrate with decellularized dermal matrices, on which they differentiate and stratify into skin equivalents. It is demonstrated that the constructs produced by this method have appropriate integrity and mechanical properties for use as in vitro tissue models.     

Engineered Extracellular Matrices as Biomaterials of Tunable Composition and Function

Engineered and decellularized extracellular matrices (ECM) are receiving increasing interest in regenerative medicine as materials capable to induce cell growth/differentiation and tissue repair by physiological presentation of embedded cues. However, ECM production/decellularization processes and control over their composition remain primary challenges. This study reports engineering of ECM materials with customized properties, based on genetic manipulation of immortalized and death‐inducible human mesenchymal stromal cells (hMSC), cultured within 3D porous scaffolds under perfusion flow. The strategy allows for robust ECM deposition and subsequent decellularization by deliberate cell‐apoptosis induction. As compared to standard production and freeze/thaw treatment, this grants superior preservation of ECM, leading to enhanced bone formation upon implantation in calvarial defects. Tunability of ECM composition and function is exemplified by modification of the cell line to overexpress vascular endothelial growth factor alpha (VEGF), which results in selective ECM enrichment and superior vasculature recruitment in an ectopic implantation model. hMSC lines culture under perfusion‐flow is pivotal to achieve uniform scaffold decoration with ECM and to streamline the different engineering/decellularization phases in a single environmental chamber. The findings outline the paradigm of combining suitable cell lines and bioreactor systems for generating ECM‐based off‐the‐shelf materials, with custom set of signals designed to activate endogenous regenerative processes.     

Synthesis of Polymerizable Superoxide Dismutase Mimetics to Reduce Reactive Oxygen Species Damage in Transplanted Biomedical Devices

A new polymerizable superoxide dismutase (SOD) mimetic metalloporphyrin macromer was synthesized to minimize inflammatory damage associated with tissue transplantation and biomaterial implantation, such as the use of encapsulated pancreatic islets for the treatment of type I diabetes mellitus (TIDM). This functional SOD mimetic, Mn(III) Tetrakis[1‐(3‐acryloxy‐propyl)‐4‐pyridyl] porphyrin (MnTPPyP‐Acryl), was copolymerized and crosslinked with poly(ethylene glycol) diacrylate (PEGDA) to form hydrogel networks that may actively reduce reactive oxygen species (ROS) damage associated with biomaterial implantation. Solution phase activity assays with MnTPPyP‐Acryl macromers showed comparable SOD activity to MnTMPyP, a non‐polymerizable commercially available SOD mimetic. This work also describes the development of a new, simple, and inexpensive solid phase assay system that was developed to assess the activity of MnTPPyP‐Acryl macromers polymerized within PEGDA hydrogels, which has the potential to fulfill an existing void with the biochemical tools available for testing other immobilized ROS antagonists. With this new assay system, hydrogels containing up to 0.25 mol% MnTPPyP‐Acryl showed significantly higher levels of SOD activity, whereas control hydrogels polymerized with inactive TPPyP‐Acryl macromers showed only background levels of activity. The potential for repeated use of such hydrogel devices to consistently reduce superoxide anion concentrations was demonstrated upon retention of ～100% SOD activity for at least 72 h post‐polymerization. These results demonstrate the potential that polymerizable SOD mimetics may have for integration into medical devices for the minimization of inflammatory damage upon transplantation, such as during the delivery of encapsulated pancreatic islets.     

Biodegradable Polymer Crosslinker: Independent Control of Stiffness,Toughness, and Hydrogel Degradation Rate

Hydrogels are being increasingly studied for use in various biomedical applications including drug delivery and tissue engineering. The successful use of a hydrogel in these applications greatly relies on a refined control of the mechanical properties including stiffness, toughness, and the degradation rate. However, it is still challenging to control the hydrogel properties in an independent manner due to the interdependency between hydrogel properties. Here it is hypothesized that a biodegradable polymeric crosslinker would allow for decoupling of the dependency between the properties of various hydrogel materials. This hypothesis is examined using oxidized methacrylic alginate (OMA). The OMA is synthesized by partially oxidizing alginate to generate hydrolytically labile units and conjugating methacrylic groups. It is used to crosslink poly(ethylene glycol) methacrylate and poly(N‐hydroxymethyl acrylamide) to form three‐dimensional hydrogel systems. OMA significantly improves rigidity and toughness of both hydrogels as compared with a small molecule crosslinker, and also controls the degradation rate of hydrogels depending on the oxidation degree, without altering their initial mechanical properties. The protein‐release rate from a hydrogel and subsequent angiogenesis in vivo are thus regulated with the chemical structure of OMA. Overall, the results of this study suggests that the use of OMA as a crosslinker will allow the implantation of a hydrogel in tissue subject to an external mechanical loading with a desired protein‐release profile. The OMA synthesized in this study will be, therefore, highly useful to independently control the mechanical properties and degradation rate of a wide array of hydrogels.     

Heterotypic Scaffold Design Orchestrates Primary Cell Organization and Phenotypes in Cocultured Small Diameter Vascular Grafts

To facilitate true regeneration, a vascular graft should direct the evolution of a neovessel to obtain the function of a native vessel. For this, scaffolds have to permit the formation of an intraluminal endothelial cell monolayer, mimicking the tunica intima. In addition, when attempting to mimic a tunica media‐like outer layer, the stacking and orientation of vascular smooth muscle cells (vSMCs) should be recapitulated. An integral scaffold design that facilitates this has so far remained a challenge. A hybrid fabrication approach is introduced by combining solution electrospinning and melt electrowriting. This allows a tissue‐structure mimetic, hierarchically bilayered tubular scaffold, comprising an inner layer of randomly oriented dense fiber mesh and an outer layer of microfibers with controlled orientation. The scaffold supports the organization of a continuous luminal endothelial monolayer and oriented layers of vSM‐like cells in the media, thus facilitating control over specific and tissue‐mimetic cellular differentiation and support of the phenotypic morphology in the respective layers. Neither soluble factors nor a surface bioactivation of the scaffold is needed with this approach, demonstrating that heterotypic scaffold design can direct physiological tissue‐like cell organization and differentiation.     

A flexible algorithm for construction of 3-D vessel networks foruse in thermal modeling

A new algorithm for the construction of artificial blood vessel networks is presented. The algorithm produces three-dimensional (3-D) geometrical representations of both arterial and venous networks. The key ingredient of the algorithm is a 3-D potential function defined in the tissue volume. This potential function controls the paths by which points are connected to existing vessels, thereby producing new vessel segments. The potential function has no physiological interpretation, but, by adjustment of parameters governing the potential, it is possible to produce networks that have physiologically meaningful geometrical properties. If desired, the veins can be generated counter current to the arteries. Furthermore, the potential function allows fashioning of the networks to the presence of bone or air cavities. The resulting networks can be used for thermal simulations of hyperthermia treatment     

In Situ Crosslinkable Gelatin Hydrogels for Vasculogenic Induction and Delivery of Mesenchymal Stem Cells

Clinical trials utilizing mesenchymal stem cells (MSCs) for severe vascular diseases have highlighted the need to effectively engraft cells and promote pro‐angiogenic activity. A functional material accomplishing these two goals is an ideal solution as spatiotemporal and batch‐to‐batch variability in classical therapeutic delivery can be minimized, and tissue regeneration would begin rapidly at the implantation site. Gelatin may serve as a promising biomaterial due to its excellent biocompatibility, biodegradability, and non‐immuno/antigenicity. However, the dissolution of gelatin at body temperature and quick enzymatic degradation in vivo have limited its use thus far. To overcome these challenges, an injectable, in situ crosslinkable gelatin was developed by conjugating enzymatically crosslinkable hydroxyphenyl propionic acid (GHPA). When MSCs are cultured in 3D in vitro or injected in vivo in GHPA, spontaneous endothelial differentiation occurs, as evidenced by marked increases in endothlelial cell marker expressions (Flk1, Tie2, ANGPT1, vWF) in addition to forming an extensive perfusable vascular network after 2‐week subcutaneous implantation. Additionally, favorable host macrophage response is achieved with GHPA as shown by decreased iNOS and increased MRC1 expression. These results indicate GHPA as a promising soluble factor‐free cell delivery template which induces endothelial differentiation of MSCs with robust neovasculature formation and favorable host response.     

Rapid Generation of Biologically Relevant Hydrogels Containing Long‐Range Chemical Gradients

Many biological processes are regulated by gradients of bioactive chemicals. Thus, the generation of materials with embedded chemical gradients may be beneficial for understanding biological phenomena and generating tissue‐mimetic constructs. Here a simple and versatile method to rapidly generate materials containing centimeter‐long gradients of chemical properties in a microfluidic channel is described. The formation of a chemical gradient is initiated by a passive‐pump‐induced forward flow and further developed during an evaporation‐induced backward flow. The gradient is spatially controlled by the backward flow time and the hydrogel material containing the gradient is synthesized via photopolymerization. Gradients of a cell‐adhesion ligand, Arg‐Gly‐Asp‐Ser (RGDS), are incorporated in poly(ethylene glycol)‐diacrylate (PEG‐DA) hydrogels to test the response of endothelial cells. The cells attach and spread along the hydrogel material in a manner consistent with the RGDS‐gradient profile. A hydrogel containing a PEG‐DA concentration gradient and constant RGDS concentration is also shown. The morphology of cells cultured on such hydrogel changes from round in the lower PEG‐DA concentration regions to well‐spread in the higher PEG‐DA concentration regions. This approach is expected to be a valuable tool to investigate the cell–material interactions in a simple and high‐throughput manner and to design graded biomimetic materials for tissue engineering applications.     

Complex 3D‐Printed Microchannels within Cell‐Degradable Hydrogels

3D‐printing is emerging as a technology to introduce microchannels into hydrogels, for the perfusion of engineered constructs. Although numerous techniques have been developed, new techniques are still needed to obtain the complex geometries of blood vessels and with materials that permit desired cellular responses. Here, a printing process where a shear‐thinning and self‐healing hydrogel “ink” is injected directly into a “support” hydrogel with similar properties is reported. The support hydrogel is further engineered to undergo stabilization through a thiol‐ene reaction, permitting (i) the washing of the ink to produce microchannels and (ii) tunable properties depending on the crosslinker design. When adhesive peptides are included in the support hydrogel, endothelial cells form confluent monolayers within the channels, across a range of printed configurations (e.g., straight, stenosis, spiral). When protease‐degradable crosslinkers are used for the support hydrogel and gradients of angiogenic factors are introduced, endothelial cells sprout into the support hydrogel in the direction of the gradient. This printing approach is used to investigate the influence of channel curvature on angiogenic sprouting and increased sprouting is observed at curved locations. Ultimately, this technique can be used for a range of biomedical applications, from engineering vascularized tissue constructs to modeling in vitro cultures.     

Electrospun Photocrosslinkable Hydrogel Fibrous Scaffolds for Rapid In Vivo Vascularized Skin Flap Regeneration

Distal necrosis of random skin flap is always clinical problematic in plastic surgery. The development of 3D functional vascular networks is fundamental for the survival of a local random skin flap. Herein, an effective technique on constructing 3D fibrous scaffolds for accelerated vascularization is demonstrated using a photocrosslinkable natural hydrogel based on gelatin methacryloyl (GelMA) by electrospinning. It is found that the ultraviolet (UV) photocrosslinkable gelatin electrospun hydrogel fibrous membranes exhibit soft adjustable mechanical properties and controllable degradation properties. Furthermore, it is observed that the optimized hydrogel scaffolds can support endothelial cells and dermal fibroblasts adhesion, proliferation, and migration into the scaffolds, which facilitates vascularization. Importantly, a rapid formation of tubes is observed after 3 d seeding of endothelial cells. After GelMA fibrous scaffold implantation below the skin flap in a rat model, it is found that the flap survival rate is higher than the control group, and there is more microvascular formation, which is potentially beneficial for the flap tissue vascularization. These data suggest that GelMA hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues.     

Noninvasive vascular elastography: theoretical framework

Changes in vessel wall elasticity may be indicative of vessel pathologies. It is known, for example, that the presence of plaque stiffens the vascular wall, and that the heterogeneity of its composition may lead to plaque rupture and thrombosis. Another domain of application where ultrasound elastography may be of interest is the study of vascular wall elasticity to predict the risk of aneurysmal tissue rupture. In this paper, this technology is introduced as an approach to noninvasively characterize superficial arteries. In such a case, a linear array ultrasound transducer is applied on the skin over the region of interest, and the arterial tissue is dilated by the normal cardiac pulsation. The elastograms, the equivalent elasticity images, are computed from the assessment of the vascular tissue motion. Investigating the forward problem, it is shown that motion parameters might be difficult to interpret; that is because tissue motion occurs radially within the vessel wall while the ultrasound beam propagates axially. As a consequence of that, the elastograms are subjected to hardening and softening artefacts, which are to be counteracted. In this paper, the Von Mises (VM) coefficient is proposed as a new parameter to circumvent such mechanical artefacts and to appropriately characterize the vessel wall. Regarding the motion assessment, the Lagrangian estimator was used; that is because it provides the full two-dimensional strain tensor necessary to compute the VM coefficient. The theoretical model was validated with biomechanical simulations of the vascular wall properties. The results allow believing in the potential of the method to differentiate hard plaques and lipid pools from normal vascular tissue. Potential in vivo implementation of noninvasive vascular elastography to characterize abdominal aneurysms and superficial arteries such as the femoral and the carotid is discussed.     

Vapor‐Based Multicomponent Coatings for Antifouling and Biofunctional Synergic Modifications

A general concept is introduced featuring an ideal multifunctional surface that can avoid fouling problems while allowing the installed groups to perform with the high efficacy and accuracy necessary for delivering cascading and spontaneous biological activities. The idea is realized by using a direct synthesis of a multicomponent coating containing the two functionalities of 4‐methyl‐propiolate and 4‐N‐maleimidomethyl that is achieved via chemical vapor deposition copolymerization on various substrates. The novel coating can simultaneously perform specific bio‐orthogonal reactions, including the azide‐alkyne click reaction and a thiol‐maleimide coupling reaction. In the study, azide‐terminated polyethylene glycols are first immobilized on the methyl propiolate groups to impart an antifouling property, while bioactivity is enabled by tethering biotinylated thiols or Cys‐Arg‐Glu‐Asp‐Val (CREDV) peptides on the maleimide groups. The induced antifouling properties and bioactivities are confirmed by quartz crystal microbalance and cell culture studies. Finally, precisely manipulated endothelial cells, namely, human umbilical vein endothelial cells and bovine arterial endothelial cells, are observed on a complex stent substrate and on confined areas of the poly(methyl methacrylate) substrates.     

In vivo Cellular Uptake,Degradation, and Biocompatibility of Polyelectrolyte Microcapsules

Polyelectrolyte microcapsules are made by layer‐by‐layer (LbL) coating of a sacrificial template, followed by decomposition of the template, to produce hollow microcapsules. In this paper, we report on the in vivo cellular uptake, degradation and biocompatibility of polyelectrolyte microcapsules produced from alternating dextran sulphate and poly‐L‐arginine layers on a template of calcium carbonate microparticles. We show that a moderate tissue reaction is observed after subcutaneous injection of polyelectrolyte microcapsules in mice. Within sixteen days after subcutaneous injection, most of the microcapsules are internalized by the cells and start to get degraded. The number of polyelectrolyte layers determines the stability of the microcapsules after cellular uptake.     

Anatomy and flow in normal and ischemic microvasculature based on a novel temporal fractal dimension analysis algorithm using contrast enhanced ultrasound

Strategies for improvement of blood flow by promoting new vessel growth in ischemic tissue are being developed. Recently, contrast-enhanced ultrasound (CEU) imaging has been used to assess tissue perfusion in models of ischemia-related angiogenesis, growth-factor mediated angiogenesis, and tumor angiogenesis. In these studies, microvascular flow is measured in order to assess the total impact of adaptations at different vascular levels. High-resolution methods for imaging larger vessels have been developed in order to derive "angiograms" of arteries, veins, and medium to large microvessels. We describe a novel method of vascular bed (microvessel and arterial) characterization of vessel anatomy and flow simultaneously, using serial measurement of the fractal dimension (FD) of a temporal sequence of CEU images. This method is proposed as an experimental methodology to distinguish ischemic from nonischemic tissue. Moreover, an improved approach for extracting the FD unique to this application is introduced.