Cerium- and Iron-Oxide-Based Nanozymes in Tissue Engineering and Regenerative Medicine

Nanoparticulate materials displaying enzyme-like properties, so-called nanozymes, are explored as substitutes for natural enzymes in several industrial, energy-related, and biomedical applications. Outstanding high stability, enhanced catalytic activities, low cost, and availability at industrial scale are some of the fascinating features of nanozymes. Furthermore, nanozymes can also be equipped with the unique attributes of nanomaterials such as magnetic or optical properties. Due to the impressive development of nanozymes during the last decade, their potential in the context of tissue engineering and regenerative medicine also started to be explored. To highlight the progress, in this review, we discuss the two most representative nanozymes, namely, cerium- and iron-oxide nanomaterials, since they are the most widely studied. Special focus is placed on their applications ranging from cardioprotection to therapeutic angiogenesis, bone tissue engineering, and wound healing. Finally, current challenges and future directions are discussed.

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title: Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II).
abstract: Nanozymes are nanomaterials with enzyme-like characteristics ( 2013, 42, 6060-6093). They have been developed to address the limitations of natural enzymes and conventional artificial enzymes. Along with the significant advances in nanotechnology, biotechnology, catalysis science, and computational design, great progress has been achieved in the field of nanozymes since the publication of the above-mentioned comprehensive review in 2013. To highlight these achievements, this review first discusses the types of nanozymes and their representative nanomaterials, together with the corresponding catalytic mechanisms whenever available. Then, it summarizes various strategies for modulating the activity and selectivity of nanozymes. After that, the broad applications from biomedical analysis and imaging to theranostics and environmental protection are covered. Finally, the current challenges faced by nanozymes are outlined and the future directions for advancing nanozyme research are suggested. The current review can help researchers know well the current status of nanozymes and may catalyze breakthroughs in this field.

title: A Step into the Future: Applications of Nanoparticle Enzyme Mimics.
abstract: We describe elementary concepts, up-to-date developments, and perspectives of the emerging field of nanoparticle enzyme mimics (so-called "nanozymes") at the interface of chemistry, biology, materials, and nanotechnology. The design and synthesis of functional enzyme mimics is a long-standing goal of biomimetic chemistry. Metal complexes, polymers and engineered biomolecules capturing the structure of natural enzymes or their active centers have been made to achieve high rates and enhanced selectivities. Still, the design of new "artificial enzymes" that are not related to proteins but with capacity of production and stability at industrial level, remains a goal. Inorganic nanoparticles bear this potential. Although it seems counterintuitive to compare nanoparticles and natural enzymes because they appear very different they share many common features: nano-size, irregular shape, and rich surface chemistry. These features enable nanomaterials to mimic reactions of natural enzymes. Representative examples with biomedical and environmental applications are given.

title: Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase.
abstract: Survival of rats exposed to 100% oxygen was increased from 69.5 +/- 1.5 to 118.1 +/- 9.9 h (mean +/- SEM, P less than 0.05) when liposomes containing catalase and superoxide dismutase were injected intravenously before and during exposure. The increased survival time in 100% oxygen was also associated with significantly less fluid in the pleural cavity. Rats injected with catalase- and superoxide dismutase-containing liposomes, which had increased survival in 100% oxygen, had increased lung wet weight upon autopsy compared with saline-injected controls (2.9 +/- 0.2 g/lung vs. 4.8 +/- 0.4 g/lung, mean +/- SE, P less than 0.05). Intravenous injection of control liposomes along with catalase and superoxide dismutase in the suspending buffer decreased the mean pleural effusion volume 89% and had no significant effect on survival time. Lung catalase and superoxide dismutase activities were increased 3.1- and 1.7-fold, respectively, 2 h after a single intravenous injection of liposomes containing catalase or superoxide dismutase. Superoxide dismutase activity was also significantly greater than controls in both air- and 100% oxygen-exposed rat lungs, when enzyme activity was assayed 24 h after cessation of injection of control and oxygen-exposed rats with enzyme-containing liposomes every 12 h for 36 h. Free superoxide dismutase and catalase injected intravenously in the absence of liposomes did not increase corresponding lung enzyme activities, affect pleural effusion volume, lung wet weight, or extend the mean survival time of rats exposed to 100% oxygen. The clearance of liposome-augmented 125I-labeled catalase from lung and plasma obeyed first order kinetics according to a one-compartment model. When clearance of liposome-augmented catalase activity or radioactivity were the parameters used for pharmacokinetic studies, the half-life of augmented lung catalase was 1.9 and 2.6 h, respectively. The half-life of liposome-entrapped catalase and superoxide dismutase activity in the circulation was 2.5 and 4 h, respectively, while intravenously injected catalase and superoxide dismutase had a circulation half-life of 23 and 6 min, respectively.

title: Catalytic Properties and Biomedical Applications of Cerium Oxide Nanoparticles.
abstract: Cerium oxide nanoparticles (Nanoceria) have shown promise as catalytic antioxidants in the test tube, cell culture models and animal models of disease. However given the reactivity that is well established at the surface of these nanoparticles, the biological utilization of Nanoceria as a therapeutic still poses many challenges. Moreover the form that these particles take in a biological environment, such as the changes that can occur due to a protein corona, are not well established. This review aims to summarize the existing literature on biological use of Nanoceria, and to raise questions about what further study is needed to apply this interesting catalytic material to biomedical applications. These questions include: 1) How does preparation, exposure dose, route and experimental model influence the reported effects of Nanoceria in animal studies? 2) What are the considerations to develop Nanoceria as a therapeutic agent in regards to these parameters? 3) What biological targets of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are relevant to this targeting, and how do these properties also influence the safety of these nanomaterials?

title: A Multinuclear Metal Complex Based DNase-Mimetic Artificial Enzyme: Matrix Cleavage for Combating Bacterial Biofilms.
abstract: Extracellular DNA (eDNA) is an essential structural component during biofilm formation, including initial bacterial adhesion, subsequent development, and final maturation. Herein, the construction of a DNase-mimetic artificial enzyme (DMAE) for anti-biofilm applications is described. By confining passivated gold nanoparticles with multiple cerium(IV) complexes on the surface of colloidal magnetic Fe3 O4  /SiO2 core/shell particles, a robust and recoverable artificial enzyme with DNase-like activity was obtained, which exhibited high cleavage ability towards both model substrates and eDNA. Compared to the high environmental sensitivity of natural DNase in anti-biofilm applications, DMAE exhibited a much better operational stability and easier recoverability. When DMAE was coated on substratum surfaces, biofilm formation was inhibited for prolonged periods of time, and the DMAE excelled in the dispersion of established biofilms of various ages. Finally, the presence of DMAE remarkably potentiated the efficiency of traditional antibiotics to kill biofilm-encased bacteria and eradiate biofilms.

title: Synthetic Polymers with Enzyme‐like Activities
abstract: For much of this century, biochemists have used chemical models as a basis for elucidating the behavior of naturally occurring macromolecules. In the last few decades, structural biochemistry has made such remarkable strides in interpreting function that it should now be possible to inaugurate a complementary program: biological molecules ought to serve as models for the design of new chemical entities. For example, chemists are intensively engaged in searches for new catalysts. The preeminent catalysts are enzymes. The molecular structure and behavior of many enzymes is known in great detail. Should it not be feasible to use this information to construct catalysts from nonliving sources?

title: Dendrimers as artificial enzymes.
abstract: Dendrimers are regular tree-like macromolecules accessible by chemical synthesis from a variety of building blocks. Their topology enforces a globular shape that offers a unique opportunity to design artificial enzymes. Catalytic groups such as metal complexes and cofactors can be placed at the dendrimer core to exploit microenvironment and selectivity effects of the dendritic shell. In a second approach, attaching catalytic groups in multiple copies at the end of the dendritic branches may lead to cooperativity effects. Finally, exploration of dendritic structural space by screening combinatorial libraries of peptide dendrimers for catalytic activity can lead to discovery of functional dendrimers with enzyme-like properties, in a process mimicking natural selection.

title: Selective chemical catalysis by an antibody.
abstract: The immunoglobulin MOPC167, which binds the transition state analog p-nitrophenylphosphorylcholine with high affinity, catalyzed the hydrolysis of the corresponding carbonate 1. MOPC167 catalysis displayed saturation kinetics with catalytic constant (kcat) = 0.4 min-1 and Michaelis constant (Km) = 208 microM, showed substrate specificity, and was inhibited by p-nitrophenylphosphorylcholine. The rate of the reaction was first order in hydroxide ion concentration between pH 6.0 and 8.0. The lower limit for the rate of acceleration of hydrolysis by the antibody above the uncatalyzed reaction was 770. This study begins to define the rules for the generation of catalytic antibodies.

title: Nanomaterials Exhibiting Enzyme-Like Properties (Nanozymes): Current Advances and Future Perspectives
abstract: Biological enzymes are macromolecular catalysts that catalyze the biochemical reactions of the natural systems. Although each enzyme performs a particular function, however, holds several drawbacks, which limits its utilization in broad-spectrum applications. Natural enzymes require strict physiological conditions for performing catalytic functions. Their limited stability in harsh environmental conditions, the high cost of synthesis, isolation, and purification are some of the significant drawbacks. Therefore, as an alternative to natural enzymes, recently several strategies have been developed including the synthesis of molecules, complexes, and nanoparticles mimicking their intrinsic catalytic properties. Nanoparticles exhibiting the properties of an enzyme are termed as “nanozymes.” Nanozymes offer several advantages over natural enzymes, therefore, a rapid expansion of the development of artificial biocatalysts. These advantages include simple methods of synthesis, low cost, high stability, robust catalytic performance, and smooth surface modification of nanomaterials. In this context, nanozymes are tremendously being explored to establish a wide range of applications in biosensing, immunoassays, disease diagnosis and therapy, theranostics, cell/tissue growth, protection from oxidative stress, and removal of pollutants. Considering the importance of nanozymes, this article has been designed to comprehensively discuss the different enzyme-like properties, such as peroxidase, catalase, superoxide dismutase, and oxidase, exhibited by various nanoparticles.

title: Integrated nanozymes: facile preparation and biomedical applications.
abstract: Nanozymes have been viewed as the next generation of artificial enzymes due to their low cost, large specific surface area, and good robustness under extreme conditions. However, the moderate activity and limited selectivity of nanozymes have impeded their usage. To overcome these shortcomings, integrated nanozymes (INAzymes) have been developed by encapsulating two or more different biocatalysts (e.g., natural oxidases and peroxidase mimics) together within confined frameworks. On the one hand, with the assistance of natural enzymes, INAzymes are capable of specifically recognizing targets. On the other hand, nanoscale confinement brought about by integration significantly enhances the cascade reaction efficiency. In this Feature Article, we highlight the newly developed INAzymes, covering from synthetic strategies to versatile applications in biodetection and therapeutics. Moreover, it is predicted that INAzymes with superior activities, specificity, and stability will enrich the research of nanozymes and pave new ways in designing multifunctional nanozymes.

title: Emerging Biomedical Applications of Enzyme-Like Catalytic Nanomaterials.
abstract: Nanomaterials have been developed for many biomedical applications, including medical imaging, drug delivery, and antimicrobial coatings. Intriguingly, nanoparticles can display 'enzyme-like' activity and have been explored as alternatives to natural enzymes in several industrial and energy-related applications. Recently, these catalytic nanomaterials with enzyme-mimetic properties have found new biomedical applications, from biofilm disruption to protection against neurodegeneration and tumor prevention. In this review we focus on recent in vivo studies demonstrating potential therapeutic uses of catalytic nanomaterials. We also provide insights about the relationships between catalytic activity, therapeutic efficacy, and biocompatibility that are critical for clinical translatability. Finally, we discuss current challenges and future directions for the use of these nanomaterials as novel platforms for the development of sustainable, affordable, and safe therapeutics.

title: Nanozyme applications in biology and medicine: an overview
abstract: Nanozymes, in nature, are artificial enzymes. Innovated by Ronald Breslow to mimic enzymes. Nanozymes have widespread applications including targeted cancer therapy, diagnostic medicine and bio-sensing even environmental toxicology. However, these applications are a novel research field in biomedicine, but are growing fast. Enzyme-based applications such as immune-absorbent assay (ELIZA) are expensive because of the complexity of producing enzymes and antibodies. Not only, some nanoparticles can mimic these enzymes such as superoxides, but also they can manipulate biological pathways directly like autophagy. These abilities make them a suitable alternative for both therapy and diagnosis. In this review, we opted on metal nanoparticles and application of this cutting edged technology into modern medicine.

title: Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons.
abstract: This paper describes the evaluation of the auto-catalytic anti-oxidant behavior and biocompatibility of cerium oxide nanoparticles for applications in spinal cord repair and other diseases of the central nervous system. The application of a single dose of nano-ceria at a nano-molar concentration is biocompatible, regenerative and provides a significant neuroprotective effect on adult rat spinal cord neurons. Retention of neuronal function is demonstrated from electrophysiological recordings and the possibility of its application to prevent ischemic insult is suggested from an oxidative injury assay. A mechanism is proposed to explain the auto-catalytic properties of these nanoparticles.

title: Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine model of cardiomyopathy.
abstract: Cerium oxide (CeO2) nanoparticles have been shown to protect cells in culture from lethal stress, but no protection in vivo has been reported. Cardiac-specific expression of monocyte chemoattractant protein (MCP)-1 in mice causes ischemic cardiomyopathy associated with activation of endoplasmic reticulum (ER) stress. The aim of this study was to assess the effects of CeO2 nanoparticles on cardiac function and remodeling as well as ER stress response in this murine model of cardiomyopathy.



MCP-1 transgenic mice (MCP mice) and wild-type controls were administered intravenously 15 nmol of CeO2 nanoparticles or vehicle only twice a week for 2 weeks. Cardiac function, myocardial histology, nitrotyrosine formation, expression of cytokines, and ER stress-associated genes were evaluated.



Treatment with CeO2 nanoparticles markedly inhibited progressive left ventricular dysfunction and dilatation in MCP mice and caused a significant decrease in serum levels of MCP-1, C-reactive protein, and total nitrated proteins. The infiltration of monocytes/macrophages, accumulation of 3-nitrotyrosine, apoptotic cell death, and expression of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 in the myocardium were markedly inhibited by CeO2 nanoparticles. Expression of the key ER stress-associated genes, including glucose-regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and heat shock proteins (HSP25, HSP40, HSP70), were also suppressed by CeO2 nanoparticles.



CeO2 nanoparticles protect against the progression of cardiac dysfunction and remodeling by attenuation of myocardial oxidative stress, ER stress, and inflammatory processes probably through their autoregenerative antioxidant properties.

title: Pharmacological potential of cerium oxide nanoparticles.
abstract: Nanotechnology promises a revolution in pharmacology to improve or create ex novo therapies. Cerium oxide nanoparticles (nanoceria), well-known as catalysts, possess an astonishing pharmacological potential due to their antioxidant properties, deriving from a fraction of Ce(3+) ions present in CeO(2). These defects, compensated by oxygen vacancies, are enriched at the surface and therefore in nanosized particles. Reactions involving redox cycles between the Ce(3+) and Ce(4+) oxidation states allow nanoceria to react catalytically with superoxide and hydrogen peroxide, mimicking the behavior of two key antioxidant enzymes, superoxide dismutase and catalase, potentially abating all noxious intracellular reactive oxygen species (ROS) via a self-regenerating mechanism. Hence nanoceria, apparently well tolerated by the organism, might fight chronic inflammation and the pathologies associated with oxidative stress, which include cancer and neurodegeneration. Here we review the biological effects of nanoceria as they emerge from in vitro and in vivo studies, considering biocompatibility and the peculiar antioxidant mechanisms.

title: Nanozymes in bionanotechnology: from sensing to therapeutics and beyond
abstract: In the past few decades, researchers have developed lots of artificial enzymes with various materials to mimic the structures and functions of natural enzymes. Recently, nanozymes, nanomaterials with enzyme-like characteristics, are emerging as novel artificial enzymes, and attracting researchers’ enormous interest. Remarkable advances have been made in the area of nanozymes due to their unique properties compared with natural enzymes and classic artificial enzymes. Until now, lots of nanomaterials have been studied to mimic various natural enzymes for wide applications. To highlight the recent progress of nanozymes (especially in bionanotechnology), here we discuss the diverse applications of nanozymes, which range from sensing, imaging, and therapeutics, to logic gates, pollutant removal, water treatment, etc. Finally, we address the current challenges facing nanozyme research as well as possible directions to fulfill their great potential in future.

title: Teaching the fundamentals of electron transfer reactions in mitochondria and the production and detection of reactive oxygen species
abstract: Mitochondria fulfill a number of biological functions which inherently depend on ATP and O2−•/H2O2 production. Both ATP and O2−•/H2O2 are generated by electron transfer reactions. ATP is the product of oxidative phosphorylation whereas O2−• is generated by singlet electron reduction of di-oxygen (O2). O2−• is then rapidly dismutated by superoxide dismutase (SOD) producing H2O2. O2−•/H2O2 were once viewed as unfortunately by-products of aerobic respiration. This characterization is fitting considering over production of O2−•/H2O2 by mitochondria is associated with range of pathological conditions and aging. However, O2−•/H2O2 are only dangerous in large quantities. If produced in a controlled fashion and maintained at a low concentration, cells can benefit greatly from the redox properties of O2−•/H2O2. Indeed, low rates of O2−•/H2O2 production are required for intrinsic mitochondrial signaling (e.g. modulation of mitochondrial processes) and communication with the rest of the cell. O2−•/H2O2 levels are kept in check by anti-oxidant defense systems that sequester O2−•/H2O2 with extreme efficiency. Given the importance of O2−•/H2O2 in cellular function, it is imperative to consider how mitochondria produce O2−•/H2O2 and how O2−•/H2O2 genesis is regulated in conjunction with fluctuations in nutritional and redox states. Here, I discuss the fundamentals of electron transfer reactions in mitochondria and emerging knowledge on the 11 potential sources of mitochondrial O2−•/H2O2 in tandem with their significance in contributing to overall O2−•/H2O2 emission in health and disease. The potential for classifying these different sites in isopotential groups, which is essentially defined by the redox properties of electron donator involved in O2−•/H2O2 production, as originally suggested by Brand and colleagues is also surveyed in detail. In addition, redox signaling mechanisms that control O2−•/H2O2 genesis from these sites are discussed. Finally, the current methodologies utilized for measuring O2−•/H2O2 in isolated mitochondria, cell culture and in vivo are reviewed.

title: Engineering Nanoceria for Enhanced Peroxidase Mimics: A Solid Solution Strategy
abstract: Nanoceria has been developed as a biocompatible and robust enzyme mimic (called ceria nanozyme) and applied in bioanalysis and therapeutics. However, its catalytic activity such as peroxidase mimic is still moderate, which in turn has limited its promising applications. To tackle this issue, herein we reported an effective strategy to modulate the peroxidase mimicking activity of nanoceria by doping transition metals. We discovered that doping of the first row of transition metals (i. e., Mn, Fe, Co, Ni, and Cu) modulated the activity of nanoceria and with the same doping ratio the activities followed the below order: Mn1Ce10>Co1Ce10>Fe1Ce10>Cu1Ce10>CeO2>Ni1Ce10. By further varying the Mn/Ce ratio, Mn1Ce5 with the highest peroxidase mimicking activity was obtained (13‐fold higher compared with the pristine CeO2). Meanwhile we found that the homogenous doping of Mn into the nanoceria matrix without phase segregation ensured the formation of Mn1Ce5 solid solution. From our experiments and discussion, we concluded that the surface oxygen (Oβ) and the synergistic effect between Ce and Mn played key roles in the enhancement of Mn1Ce5 peroxidase mimicking activity. This study not only demonstrates that Mn‐doped nanoceria exhibits higher active peroxidase mimics but also provides a promising strategy to modulate the catalytic activity of nanozymes.

title: Nanoceria exhibit redox state-dependent catalase mimetic activity.
abstract: In this study we have found that cerium oxide nanoparticles exhibit catalase mimetic activity. Surprisingly, the catalase mimetic activity correlates with a reduced level of cerium in the +3 state, in contrast to the relationship between surface charge and superoxide scavenging properties.

title: Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles.
abstract: In this report ceria nanoparticles are shown to act as catalysts that mimic superoxide dismutase (SOD) with the catalytic rate constant exceeding that determined for the enzyme SOD.

title: Regulating the regulator: NF-κB signaling in heart
abstract: The Nuclear Factor-kappaB (NF-kappaB) signaling pathway has been linked to several pathologic processes in the myocardium including cardiomyocyte proinflammatory cytokine release, ischemia/reperfusion injury, hypertrophy and apoptosis. However, very little is known about the intracellular mechanisms that govern NF-kappaB activity in the myocardial cells. Recent advances in our understanding of the regulation of NF-kappaB signaling in non-myocyte systems suggest that the activity of the NF-kappaB pathway is tightly regulated by a diversity of stress-activated signaling intermediates through direct post-translational modification of various components of the NF-kappaB pathway. In this review, we will focus on these recent revelations and their implications not only in cardiac pathologies, but in the development of new therapeutic strategies to manage heart disease.

title: Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism.
abstract: Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 microg/cm(2) ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.

title: Cerium oxide nanoparticles protect cardiac progenitor cells from oxidative stress.
abstract: Cardiac progenitor cells (CPCs) are a promising autologous source of cells for cardiac regenerative medicine. However, CPC culture in vitro requires the presence of microenvironmental conditions (a complex array of bioactive substance concentration, mechanostructural factors, and physicochemical factors) closely mimicking the natural cell surrounding in vivo, including the capability to uphold reactive oxygen species (ROS) within physiological levels in vitro. Cerium oxide nanoparticles (nanoceria) are redox-active and could represent a potent tool to control the oxidative stress in isolated CPCs. Here, we report that 24 h exposure to 5, 10, and 50 μg/mL of nanoceria did not affect cell growth and function in cardiac progenitor cells, while being able to protect CPCs from H(2)O(2)-induced cytotoxicity for at least 7 days, indicating that nanoceria in an effective antioxidant. Therefore, these findings confirm the great potential of nanoceria for controlling ROS-induced cell damage.

title: Protective effect of C70-carboxyfullerene against oxidative-induced stress on postmitotic muscle cells.
abstract: Satellite muscle cells play an important role in regeneration of skeletal muscle. However, they are particularly vulnerable to oxidative stress. Herein, we address our efforts on the cytoprotective activities of carboxyfullerenes with different cage size (C60 vs C70) and adduct number on postmitotic muscle cell (C2C12 cell). The correlation of the structural effect on the cytoprotective capability of carboxyfullerenes was evaluated. We find that quadri-malonic acid C70 fullerene (QF70) exhibits higher capability on protecting cells from oxidative-induced stress among these tested carboxyfullerenes. The accumulation of intracellular superoxide dismutase (SOD) is proposed to play an important role in their diverse antioxidative ability. Moreover, the pretreatment of QF70 could also obviously enhance the viability of myotubes originated from oxidative-stressed C2C12 cells, which facilitates the future application of carboxyfullerenes in tissue engineering and nanomedicine.

title: Carbon-nanotube-embedded hydrogel sheets for engineering cardiac constructs and bioactuators.
abstract: We engineered functional cardiac patches by seeding neonatal rat cardiomyocytes onto carbon nanotube (CNT)-incorporated photo-cross-linkable gelatin methacrylate (GelMA) hydrogels. The resulting cardiac constructs showed excellent mechanical integrity and advanced electrophysiological functions. Specifically, myocardial tissues cultured on 50 μm thick CNT-GelMA showed 3 times higher spontaneous synchronous beating rates and 85% lower excitation threshold, compared to those cultured on pristine GelMA hydrogels. Our results indicate that the electrically conductive and nanofibrous networks formed by CNTs within a porous gelatin framework are the key characteristics of CNT-GelMA leading to improved cardiac cell adhesion, organization, and cell-cell coupling. Centimeter-scale patches were released from glass substrates to form 3D biohybrid actuators, which showed controllable linear cyclic contraction/extension, pumping, and swimming actuations. In addition, we demonstrate for the first time that cardiac tissues cultured on CNT-GelMA resist damage by a model cardiac inhibitor as well as a cytotoxic compound. Therefore, incorporation of CNTs into gelatin, and potentially other biomaterials, could be useful in creating multifunctional cardiac scaffolds for both therapeutic purposes and in vitro studies. These hybrid materials could also be used for neuron and other muscle cells to create tissue constructs with improved organization, electroactivity, and mechanical integrity.

title: Cerium Dioxide Nanoparticle Exposure Improves Microvascular Dysfunction and Reduces Oxidative Stress in Spontaneously Hypertensive Rats
abstract: The elevated production of reactive oxygen species (ROS) in the vascular wall is associated with cardiovascular diseases such as hypertension. This increase in oxidative stress contributes to various mechanisms of vascular dysfunction, such as decreased nitric oxide bioavailability. Therefore, anti-oxidants are being researched to decrease the high levels of ROS, which could improve the microvascular dysfunction associated with various cardiovascular diseases. From a therapeutic perspective, cerium dioxide nanoparticles (CeO2 NP) hold great anti-oxidant potential, but their in vivo activity is unclear. Due to this potential anti-oxidant action, we hypothesize that injected CeO2 NP would decrease microvascular dysfunction and oxidative stress associated with hypertension. In order to simulate a therapeutic application, spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats were intravenously injected with either saline or CeO2 NP (100 μg suspended in saline). Twenty-four hours post-exposure mesenteric arteriolar reactivity was assessed via intravital microscopy. Endothelium-dependent and –independent function was assessed via acetylcholine and sodium nitroprusside. Microvascular oxidative stress was analyzed using fluorescent staining in isolated mesenteric arterioles. Finally, systemic inflammation was examined using a multiplex analysis and venular leukocyte flux was counted. Endothelium-dependent dilation was significantly decreased in the SH rats (29.68 ± 3.28%, maximal response) and this microvascular dysfunction was significantly improved following CeO2 NP exposure (43.76 ± 4.33%, maximal response). There was also an increase in oxidative stress in the SH rats, which was abolished following CeO2 NP treatment. These results provided evidence that CeO2 NP act as an anti-oxidant in vivo. There were also changes in the inflammatory profile in the WKY and SH rats. In WKY rats, IL-10 and TNF-α were increased following CeO2 NP treatment. Finally, leukocyte flux was increased in the SH rats (34 ± 4 vs. 17 ± 3 cells/min in the normotensive controls), but this activation was decreased following exposure (15 ± 2 vs. 34 ± 4 cells/min). These results indicated that CeO2 NP may alter the inflammatory response in both SH and WKY rats. Taken together, these results provide evidence that CeO2 NP act as an anti-oxidant in vivo and may improve microvascular reactivity in a model of hypertension.

title: Oxidative stress in hypertension: mechanisms and therapeutic opportunities.
abstract: Hypertension is a highly prevalent disease worldwide. It is known for being one of the most important risk factors for developing cardiovascular disease, including acute myocardial infarction and stroke. Therefore, during the last decades there have been multiple efforts to fully understand the mechanisms underlying hypertension, and then develop effective therapeutic interventions to attenuate the morbidity and mortality associated with this condition. In this regard, oxidative stress has been proposed as a key mechanistic mediator of hypertension, which is an imbalance between oxidant species and the antioxidant defense systems. A large amount of evidence supports the role of vascular wall as a major source of reactive oxygen species. These include the activation of enzymes, such as NADPH oxidase and xanthine oxidase, the uncoupling eNOS and mitochondrial dysfunction, having as a major product the superoxide anion. Among the stimuli that increase the production of oxidative species can be found the action of some vasoactive peptides, such as angiotensin II, endothelin-1 and urotensin II. The oxidative stress state generated leads to a decrease in the biodisponibility of nitric oxide and prostacyclin, key factors in maintaining the vascular tone. The knowledge of the mechanisms mentioned above has allowed generating some therapeutic strategies using antioxidants as antihypertensives with different results. Further studies are required to position antioxidants as key agents in the treatment of hypertension. The current review summarize evidence of the role of oxidative stress in hypertension, emphasizing in therapeutic targets that can be consider in antioxidant therapy.

title: Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture).
abstract: Endothelial cells synthesize and release various factors that regulate angiogenesis, inflammatory responses, hemostasis, as well as vascular tone and permeability. Endothelial dysfunction has been associated with a number of pathophysiological processes. Oxidative stress appears to be a common denominator underlying endothelial dysfunction in cardiovascular diseases. However, depending on the pathology, the vascular bed studied, the stimulant, and additional factors such as age, sex, salt intake, cholesterolemia, glycemia, and hyperhomocysteinemia, the mechanisms underlying the endothelial dysfunction can be markedly different. A reduced bioavailability of nitric oxide (NO), an alteration in the production of prostanoids, including prostacyclin, thromboxane A2, and/or isoprostanes, an impairment of endothelium-dependent hyperpolarization, as well as an increased release of endothelin-1, can individually or in association contribute to endothelial dysfunction. Therapeutic interventions do not necessarily restore a proper endothelial function and, when they do, may improve only part of these variables.

title: Cerium oxide nanoparticles attenuate monocrotaline induced right ventricular hypertrophy following pulmonary arterial hypertension.
abstract: Cerium oxide (CeO2) nanoparticles have been posited to exhibit potent anti-oxidant activity which may allow for the use of these materials in biomedical applications. Herein, we investigate whether CeO2 nanoparticle administration can diminish right ventricular (RV) hypertrophy following four weeks of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Male Sprague Dawley rats were randomly divided into three groups: control, MCT only (60 mg/kg), or MCT + CeO2 nanoparticle treatment (60 mg/kg; 0.1 mg/kg). Compared to the control group, the RV weight to body weight ratio was 45% and 22% higher in the MCT and MCT + CeO2 groups, respectively (p < 0.05). Doppler echocardiography demonstrated that CeO2 nanoparticle treatment attenuated monocrotaline-induced changes in pulmonary flow and RV wall thickness. Paralleling these changes in cardiac function, CeO2 nanoparticle treatment also diminished MCT-induced increases in right ventricular (RV) cardiomyocyte cross sectional area, β-myosin heavy chain, fibronectin expression, protein nitrosylation, protein carbonylation and cardiac superoxide levels. These changes with treatment were accompanied by a decrease in the ratio of Bax/Bcl2, diminished caspase-3 activation and reduction in serum inflammatory markers. Taken together, these data suggest that CeO2 nanoparticle administration may attenuate the hypertrophic response of the heart following PAH.

title: Cardioprotective effect of cerium oxide nanoparticles in monocrotaline rat model of pulmonary hypertension: A possible implication of endothelin‐1
abstract: Cerium oxide nanoparticles (CeO2NPs) have been recently introduced into the medical field for their antioxidant properties. The ability of CeO2NPs alone or in combination with spironolactone (SP) to attenuate monocrotaline (MCT)‐induced pulmonary hypertension and associated right ventricular hypertrophy was studied in rats. A special emphasis was given to endothelin‐1 pathway.  Pulmonary hypertension was induced in albino rats by a single subcutaneous injection of MCT (60 mg/kg). Rats received either single CeO2NPs therapy or combined therapy with SP for 2 weeks.  CeO2NPs improved pulmonary function tests with concomitant decrease in serum endothelin‐1 and pulmonary expression of endothelin‐1 and its receptor ETAR. Besides, CeO2NPs diminished MCT‐induced right ventricular hypertrophy and reduced cardiac oxidative stress and apoptosis.  CeO2NPs could improve pulmonary hypertension and associated right ventricular hypertrophy with no additive value for SP. Besides being an antioxidant, CeO2NPs work through endothelin‐1 pathway to improve pulmonary hypertension.

title: Pathogenic mechanisms of pulmonary arterial hypertension.
abstract: Pulmonary arterial hypertension (PAH) is a complex disease that causes significant morbidity and mortality and is clinically characterized by an increase in pulmonary vascular resistance. The histopathology is marked by vascular proliferation/fibrosis, remodeling, and vessel obstruction. Development of PAH involves the complex interaction of multiple vascular effectors at all anatomic levels of the arterial wall. Subsequent vasoconstriction, thrombosis, and inflammation ensue, leading to vessel wall remodeling and cellular hyperproliferation as the hallmarks of severe disease. These processes are influenced by genetic predisposition as well as diverse endogenous and exogenous stimuli. Recent studies have provided a glimpse at certain molecular pathways that contribute to pathogenesis; these have led to the identification of attractive targets for therapeutic intervention. We will review our current understanding of the mechanistic underpinnings of the genetic and exogenous/acquired triggers of PAH. The resulting imbalance of vascular effectors provoking pathogenic vascular changes will also be discussed, with an emphasis on common and overarching regulatory pathways that may relate to the primary triggers of disease. The current conceptual framework should allow for future studies to refine our understanding of the molecular pathogenesis of PAH and improve the therapeutic regimen for this disease.

title: Elimination of Intravenously Injected Endothelin‐1 from the Circulation of the Rat
abstract: The rate of elimination and the fate of endothelin-1 (ET-1) from the circulating blood was studied in urethane-anesthetized rats by intravenous injection of [125I]-labeled ET-1. The vasoconstrictor activities of the iodinated ET-1 were confirmed to be similar to those of native ET-1. Following i.v. bolus injection of 30 pmol/kg of [125I]-ET-1 into the femoral vein, the total radioactivity of the right atrial blood decayed rapidly, with a half-life of 7 min. At 5 min after the injection, the administered radioactivity distributed chiefly to the parenchyma of the lungs, kidneys, and liver. The analysis of the chemical form of labeled peptides from the plasma by reverse-phase high-performance liquid chromatography (HPLC) demonstrated no appreciable amount of degraded forms of [125I]-ET-1 in the blood for up to 60 min. [125I]-ET-1 was also stable for up to 60 min upon incubation in vitro with heparinized rat blood at 37$$C. Even when the same amount of labeled ET-1 was injected together with a pressor dose (1,500 pmol/kg) of cold ET-1, the half-life of the radioactivity in the bloodstream was exactly identical to that for [125I]-ET-1 alone. Nevertheless, the pressor response continued for more than 90 min after i.v. bolus injection of 1500 pmol/kg of ET-1 to the rat. These results clearly indicate that the elimination of ET-1 from circulating blood and the ET-1-induced pressor response are not in parallel, and the relatively rapid disappearance of ET-1 from the bloodstream is mostly due to the removal of the peptide by the parenchymal tissues, in the anesthetized rat. The long-lasting pressor action of ET-1 may be ascribed to our previous finding that the dissociation of ET-1 from its specific binding sites on vascular smooth muscle cells is extremely slow.

title: Role of Rho-kinase signaling and endothelial dysfunction in modulating blood flow distribution in pulmonary hypertension.
abstract: Rho-kinase-mediated vasoconstriction and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). However, their contribution to the adverse changes in pulmonary blood flow distribution associated with PAH has not been addressed. This study utilizes synchrotron radiation microangiography to assess the specific role, and contribution of, Rho-kinase-mediated vasoconstriction and endothelial dysfunction in PAH. Male adult Sprague-Dawley rats were injected with saline (Cont-rats) or monocrotaline (MCT-rats) 3 wk before microangiography was performed on the left lung. We assessed dynamic changes in vessel internal diameter (ID) in response to 1) the Rho-kinase inhibitor fasudil (10 mg/kg iv); or 2) ACh (3 μg · kg⁻¹ · min⁻¹), sodium nitroprusside (SNP, 5 μg · kg⁻¹ · min⁻¹), and N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg iv). We observed that MCT-rats had fewer vessels of the microcirculation compared with Cont-rats. The fundamental result of this study is that fasudil improved pulmonary blood flow distribution and reduced pulmonary pressure in PAH rats, not only by dilating already-perfused vessels (ID > 100 μm), but also by restoring blood flow to vessels that had previously been constricted closed (ID < 100 μm). Endothelium-dependent vasodilation was impaired in MCT-rats primarily in vessels with an ID < 200 μm. Moreover the vasoconstrictor response to l-NAME was accentuated in MCT-rats, but only in the 200- to 300-μm vessels. These results highlight the importance of Rho-kinase-mediated control and endothelial control of pulmonary vascular tone in PAH. Indeed, an effective therapeutic strategy for treating PAH should target both the smooth muscle Rho-kinase and endothelial pathways.

title: Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction.
abstract: Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies.

title: Myocardial ischemia-reperfusion injury: a neglected therapeutic target.
abstract: Acute myocardial infarction (MI) is a major cause of death and disability worldwide. In patients with MI, the treatment of choice for reducing acute myocardial ischemic injury and limiting MI size is timely and effective myocardial reperfusion using either thombolytic therapy or primary percutaneous coronary intervention (PPCI). However, the process of reperfusion can itself induce cardiomyocyte death, known as myocardial reperfusion injury, for which there is still no effective therapy. A number of new therapeutic strategies currently under investigation for preventing myocardial reperfusion injury have the potential to improve clinical outcomes in patients with acute MI treated with PPCI.

title: Bone Marrow Mesenchymal Stem Cells Stimulate Cardiac Stem Cell Proliferation and Differentiation
abstract: The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow–derived mesenchymal stem cells (MSCs) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes.  Here, we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire.  Female Yorkshire pigs (n=31) underwent experimental myocardial infarction (MI), and 3 days later, received transendocardial injections of allogeneic male bone marrow–derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit+ CSCs increased 20-fold in MSC-treated animals versus controls (P<0.001), there was a 6-fold increase in GATA-4+ CSCs in MSC versus control (P<0.001), and mitotic myocytes increased 4-fold (P=0.005). Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit+ CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2–5 and troponin I.  MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.

title: Mechanisms of action of mesenchymal stem cells in cardiac repair: potential influences on the cardiac stem cell niche
abstract: Clinical and basic studies of cell-based myocardial therapy have proceeded at a rapid pace. Cell therapy could lead to successful cardiac regeneration or repair by any of three general mechanisms: differentiation of the administered cells into all of the cellular constituents of the heart; release of factors capable of paracrine signaling from the administered cells; and fusion of the administered cells with the existing constituents of the heart. Here, we argue that a fourth general mechanism could be operative: stimulation of endogenous repair by injected cells, which and might cause the regeneration of stem cell niches. In a porcine model of myocardial infarction, allogeneic mesenchymal stem cells stimulated substantial improvement in the ejection fraction, reduction of infarct size, and the growth of a rim of new cardiac tissue in the region in which the mesenchymal stem cells were injected. These effects occurred in the absence of definitive cardiac myocyte differentiation. After myocardial infarction, porcine hearts exhibit evidence of cardiac myocytes that have entered the cell cycle, neovascularization, and reduced levels of apoptosis. These data, in addition to new insights regarding the presence of endogenous cardiac stem cells, strongly support the concept that the heart could contain stem cell niches. Effective cell therapy could lead to restoration of these niches through multifaceted cell–cell interactions.

title: Direct cell-cell interaction of cardiomyocytes is key for bone marrow stromal cells to go into cardiac lineage in vitro.
abstract: Cardiac environmental factors are thought to be powerful inducers in cardiomyogenic differentiation. In this study we simulated the cardiac environment using coculture and evaluated the cardiomyogenic differentiation in bone marrow stromal cells.



In group 1 only bone marrow stromal cells derived from transgenic mice expressing green fluorescent protein (GFP-BMCs) were cultured (n = 5). In group 2 cardiomyocytes from neonatal rats were grown on inserts, which we applied to culture dishes seeded with GFP-BMCs (n = 5). In group 3 GFP-BMCs were cocultured with cardiomyocytes on the same dishes (n = 5). We cultured these cells for 7 days and evaluated the synchronous contraction and the cardiomyogenic differentiation of GFP-BMCs by means of immunostaining.



In groups 1 and 2 GFP-BMCs protein did not show any myogenic phenotypes for 7 days. In contrast, in group 3 some GFP-BMCs were incorporated in parallel with cardiomyocytes and revealed myotube-like formation on day 1. On day 2, some GFP-BMCs started to contract synchronously with cardiomyocytes. Myosin heavy chain-positive GFP-BMCs were recognized in 2.49% +/- 0.87% of the total GFP-BMCs on day 5 (P <.0001). Cardiac-specific troponin I-positive GFP-BMCs were in 1.86% +/- 0.53% of the total cells on day 5 (P <.0001). Atrial natriuretic peptide was also seen in GFP-BMCs, and connexin 43 was detected between GFP-BMCs and cardiomyocytes.



Direct cell-cell interaction with cardiomyocytes was important for bone marrow stromal cells to differentiate into cardiomyocytes. This coculture was useful for simulating the cardiac environment in vitro for the research of cell transplantation in the heart.

title: Niche-Dependent Translineage Commitment of Endothelial Progenitor Cells, Not Cell Fusion in General, Into Myocardial Lineage Cells
abstract: Previous studies from our laboratory have shown therapeutic potential of ex vivo expanded endothelial progenitor cells (EPCs) for myocardial ischemia. Our purpose was to investigate the mechanisms regulating EPC contribution to myocardial regeneration. To evaluate niche-dependent expression profiles of EPCs in vitro, we performed coculture using cultured EPCs derived from human peripheral blood and rat cardiac myoblast cell line (H9C2). Reverse-transcription polymerase chain reaction (PCR) disclosed the expression of human-specific cardiac markers as well as human-specific smooth muscle markers. Cytoimmunochemistry presented several cocultured cells stained with human specific cardiac antibody. To prove this translineage differentiation in vivo, human cultured EPCs were injected into nude rat myocardial infarction model. Reverse-transcription PCR as well as immunohistochemistry of rat myocardial samples demonstrated the expression of human specific cardiac, vascular smooth muscle, and endothelial markers. We observed the distribution of colors (Qtracker; Quantum Dot Corp) in coculture to detect the fused cells, and the frequency of cell fusion was <1%. EPCs can contribute to not only vasculogenesis but also myogenesis in the ischemic myocardium in vivo. Transdifferentiation, not cell fusion, is dominant for EPCs commitment to myocardial lineage cells. Ex vivo expanded EPCs transplantation might have enhanced therapeutic potential for myocardial regeneration.

title: Connexin 43 hemichannel regulates H9c2 cell proliferation by modulating intracellular ATP and [Ca2+].
abstract: Connexin 43 (Cx43), known to be the main protein building blocks of gap junctions and hemichannels in mammalian heart, plays an important role in cardiocytes proliferation. Gap junctional intercellular communication has been suggested to be necessary for cellular proliferation and differentiation. However, the effect of Cx43 hemichannel on cardiocytes proliferation and the mechanism remain unclear. In this study, rat heart cell line H9c2 was used. The Cx43 location, the proliferation rate and hemichannel activity of H9c2 cells and Wnt-3a(+)-H9c2 cells were investigated and the changes of intracellular ATP and [Ca(2+)] were determined. Results showed that the inhibited hemichannel induced by 18beta-glycyrrhetinic acid (GA) evoked intracellular ATP and [Ca(2+)] increase and enhanced H9c2 cell proliferation. Wnt-3a(+)-H9c2 cells displayed enhanced hemichannel activity and proliferation rate. Inhibited hemichannel of Wnt-3a(+)-H9c2 cells induced by 18beta-GA decreased intracellular ATP, increased [Ca(2+)], and enhanced the proliferation of H9c2 cells. This study validated the role of hemichannel in H9c2 cell proliferation regulation, and showed a mechanism involved in the regulation of H9c2 cell proliferation. The proliferation could be enhanced by Cx43 hemichannel-mediated ATP release accompanying intracellular [Ca(2+)] change. However, different changes of ATP were observed in Wnt-3a(+)-H9c2 cells. These findings provided new insights into the molecular mechanisms of proliferation regulation in H9c2 cells and the effect of Wnt-3a on intracellular ATP.

title: Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction.
abstract: The goal of this study was to investigate the effect of pre-treatment of mesenchymal stem cells (MSCs) with growth factors (GFs) on cardiomyogenic differentiation, cytoprotective action on cardiomyocytes (CMCs), and their therapeutic efficacy in myocardial infarction.



Mechanisms of myocardial repair with MSC transplantation have not been fully elucidated, and therapeutic efficacy needs to be enhanced.



The MSCs obtained from the bone marrow of Fisher344 rats were treated with fibroblast growth factor-2, insulin-like growth factor-1, and bone morphogenetic protein-2. The expression of cardiac specific markers and the cytoprotective effect of MSCs with its mechanism were evaluated. Efficacy of MSCs transplantation was studied in rat myocardial infarction model.



Treatment of MSCs with cocktails of GFs enhanced expression of cardiac transcription factors and survival. Induction of cardiac specific markers by coculture with CMCs and gap junctional communication with CMCs was more active in GF-treated MSCs than untreated MSCs. The GF-treated MSCs reduced apoptosis of neighboring CMCs in a hypoxic condition and enhanced the phosphorylated Akt and phosphorylated c-AMP response element binding protein expression of CMCs, which was markedly reduced by gap junction blockade. In a rat myocardial infarction model, transplantation of GF-treated MSCs resulted in smaller infarct size and better cardiac function than transplantation of untreated MSCs. Additionally, GF treatment enhanced gap junction formation of transplanted MSCs, which did not aggravate arrhythmia.



Pre-treatment of MSCs with GFs enhanced cytoprotective effects on neighboring CMCs through gap junction and improved the therapeutic efficacy of MSC transplantation for myocardial repair. "Priming of MSCs with GFs" before transplantation might improve the therapeutic efficacy of cell therapy.

title: Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes
abstract: Amniotic fluid‐derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte‐like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2‐week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co‐culture group. Protein expression of cardiac calsequestrin 2 was up‐regulated in direct transmembrane co‐cultures and media control cultures compared to the other experimental groups, but all groups were up‐regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape‐loading dye transfer assay, was significantly increased in direct transmembrane co‐cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co‐culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co‐culture does enhance connexin‐43‐mediated gap junction communication between AFSC.

title: The effects of superparamagnetic iron oxide nanoparticles-labeled mesenchymal stem cells in the presence of a magnetic field on attenuation of injury after heart failure
abstract: Migration of stem cells after transplantation reduces their therapeutic effects. In this study, we hypothesized that superparamagnetic iron oxide nanoparticles (SPION)-labeled mesenchymal stem cells (MSCs) in the presence of magnetic field may have a capability to increase regenerative ability after heart failure (HF). A rat model of ISO (isoproterenol)-HF was established to investigate the effects of SPION-labeled MSCs on tissue regeneration in the presence and absence of magnetic field. Hydrodynamic size, shape, and formation of chemical bonds between SPION and polyethylene glycol (PEG) were measured using dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FTIR). The MRI was used to monitor SPION-labeled MSCs in vivo. Cell and tissue uptake of nanoparticles were determined by Prussian blue staining, atomic absorption spectroscopy (AAS), and inductively coupled plasma spectroscopy (ICP). Purity of the MSCs, heart function, myocardial fibrosis, and histologic damage were evaluated using flow-cytometry, echocardiography, Masson’s trichrome, and H&E staining respectively. Various spectroscopic and microscopic analyses revealed that hydrodynamic size of SPION was 40 ± 2 and their shape was spherical. FTIR confirmed the presence of PEG on the surface of nanoparticles. The presence of magnetic field significantly increased cell homing. Highly purified MSCs population was detected by flow-cytometry. Using SPION-labeled MSCs in the presence of magnetic field markedly improved heart function and myocardial hypertrophy and reduced fibrosis (p < 0.05). Collectively, our results demonstrated that SPION-labeled MSCs in the presence of magnetic field might contribute to regeneration after HF.

title: Long-term tracking of cells using inorganic nanoparticles as contrast agents: are we there yet?
abstract: The use of inorganic nanoparticles as probes to label and track cells in vivo is already a reality. While superparamagnetic nanoparticles have been the subject of clinical studies involving magnetic resonance imaging, quantum dots and gold nanoparticles are starting to be explored for similar goals in pre-clinical studies involving fluorescence and photoacoustic imaging. Although exciting results have been obtained from in vivo investigations, there appears to be a general lack of understanding on the effects of physicochemical properties on the labelling efficiency and toxicity of those nanoparticles, as well as on their stability in the intracellular microenvironment; essential requirements for using them as probes for cellular tracking. In this tutorial review, we look at what the current literature can teach us in respect to cell interactions with these nanoparticles, with the perspective of using them as probes for cell labelling. We also examine the findings obtained in pre-clinical studies that expose potential misinterpretation that can occur when using inorganic nanoparticles for in vivo imaging.

title: Effects of 2,3-dimercaptosuccinic acid modified Fe2O3 nanoparticles on microstructure and biological activity of cardiomyocytes
abstract: Iron oxide nanoparticles (IRONs) have been widely applied in clinical magnetic resonance imaging and in vitro cardiac tissue engineering. However, the underlying effects of IRONs on the microstructures and biological activity of cardiomyocytes remain a controversial issue. In this study, IRONs were modified with 2,3-dimercaptosuccinic acid (DMSA-IRONs) to increase the hydrophobicity. The effects of DMSA-IRONs on the microstructures and biological activity of cardiomyocytes were systematically investigated. DMSA-IRONs were internalized by cardiomyocytes and mainly distributed in the cytoplasm in a dose-dependent manner. Live/dead assay and MTT assay demonstrated that DMSA-IRONs did not affect the survival of cardiomyocytes. The distribution pattern of F-actin and cell shape were also not changed by the incubation of DMSA-IRONs. Further investigations of the mechanism proved that DMSA-IRONs did not interfere with the formation of cell adherens junctions, gap junctions and intracellular Ca2+ transients of cardiomyocytes. However, the intracellular reactive oxygen species (ROS) concentration in cardiomyocytes with incubation of DMSA-IRONs was significantly reduced due to its peroxidase-like activity. In conclusion, DMSA-IRONs are of good biocompatibility and potentially beneficial for cardiomyocytes by decreasing the intracellular ROS. The study here provides a basic investigation of the DMSA-IRONs application in clinical cell-imaging and cardiac disease therapy.

title: Effects of different doses of 2,3-dimercaptosuccinic acid-modified Fe2 O3 nanoparticles on intercalated discs in engineered cardiac tissues.
abstract: Although iron oxide nanoparticles (IRONs) were applied in clinical magnetic resonance imaging in vivo and magnetic tissue engineering in vitro widely, the underlying effects of IRONs on the development of cardiomyocytes especially the intercellular junctions, intercalated discs (IDs), remain an unknown issue. Given the critical role of three-dimensional (3D) engineered cardiac tissues (ECTs) in evaluation of nanoparticles toxicology, it remained necessary to understand the effects of IRONs on IDs assembly of cardiomyocytes in 3D environment. In this study, we first reconstituted collagen/Matrigel based ECTs in vitro and prepared IRONs with 2,3-dimercaptosuccinic acid (DMSA-IRONs). We found that the internalization of DMSA-IRONs by cardiac cells in dose-dependent manner was not associated with the cell distribution in 3D environment by determination of Prussian blue staining and transmission electronic microscopy. Significantly, through determination of western blotting and immunofluorescence of connexin 43, N-cadherin, desmoplakin, and plakoglobin, DMSA-IRONs enhanced the assembly of gap junctions, decreased mechanical junctions (adherens junctions and desmosomes) of cardiac cells but not in dose-dependent manner in ECTs at seventh day. In addition, DMSA-IRONs increased the vesicles secretion of cardiac cells in ECTs apparently compared to control groups. Overall, we conclude that the internalization of DMSA-IRONs by cardiac cells in dose-dependent manner enhanced the assembly of electrochemical junctions and decreased the mechanical related microstructures.

title: At the heart of inter- and intracellular signaling: the intercalated disc
abstract: Proper cardiac function requires the synchronous mechanical and electrical coupling of individual cardiomyocytes. The intercalated disc (ID) mediates coupling of neighboring myocytes through intercellular signaling. Intercellular communication is highly regulated via intracellular signaling, and signaling pathways originating from the ID control cardiomyocyte remodeling and function. Herein, we present an overview of the inter- and intracellular signaling that occurs at and originates from the intercalated disc in normal physiology and pathophysiology. This review highlights the importance of the intercalated disc as an integrator of signaling events regulating homeostasis and stress responses in the heart and the center of several pathophysiological processes mediating the development of cardiomyopathies.

title: The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in intracellular environments.
abstract: Angiogenesis is the formation of new blood vessels from existing blood vessels and is critical for many physiological and pathophysiological processes. In this study we have shown the unique property of cerium oxide nanoparticles (CNPs) to induce angiogenesis, observed using both in vitro and in vivo model systems. In particular, CNPs trigger angiogenesis by modulating the intracellular oxygen environment and stabilizing hypoxia inducing factor 1α endogenously. Furthermore, correlations between angiogenesis induction and CNPs physicochemical properties including: surface Ce(3+)/Ce(4+) ratio, surface charge, size, and shape were also explored. High surface area and increased Ce(3+)/Ce(4+) ratio make CNPs more catalytically active towards regulating intracellular oxygen, which in turn led to more robust induction of angiogenesis. Atomistic simulation was also used, in partnership with in vitro and in vivo experimentation, to reveal that the surface reactivity of CNPs and facile oxygen transport promotes pro-angiogenesis.

title: Enhanced Angiogenesis by Transplantation of Mesenchymal Stem Cell Sheet Created by a Novel Magnetic Tissue Engineering Method
abstract: Therapeutic angiogenesis with cell transplantation represents a novel strategy for severe ischemic diseases. However, some patients have poor response to such conventional injection-based angiogenic cell therapy. Here, we investigated a therapeutic potential of mesenchymal stem cell (MSC) sheet created by a novel magnetite tissue engineering technology for reparative angiogenesis. Human MSCs incubated with magnetic nanoparticle-containing liposomes were cultured, and a magnet was placed on the reverse side. Magnetized MSCs formed multilayered cell sheets according to magnetic force. Nude mice were subjected to unilateral hind limb ischemia and separated into 3 groups. For the control group, saline was injected into ischemic tissue. In the MSC-injected group, mice received magnetized MSCs by conventional needle injections without sheet formula as a control cell group. In the MSC-sheet group, MSC sheet was layered onto the ischemic tissues before skin closure. Blood flow recovery and the extent of angiogenesis were assessed by a laser Doppler blood flowmetry and histological capillary density, respectively. The MSC-sheet group had a greater angiogenesis in ischemic tissues compared to the control and MSC-injected groups. The angiogenic and tissue-preserving effects of MSC sheets were attributable to an increased expression of vascular endothelial growth factor and reduced apoptosis in ischemic tissues. In cultured MSCs, magnetic labeling itself inhibited apoptosis via a catalase-like antioxidative mechanism. MSC sheet created by the novel magnetic nanoparticle-based tissue engineering technology would represent a new modality for therapeutic angiogenesis and tissue regeneration.

title: Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis
abstract: Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.

title: Ginsenoside-Rg1 mediates a hypoxia-independent upregulation of hypoxia-inducible factor-1α to promote angiogenesis
abstract: Hypoxia-inducible factor (HIF-1) is the key transcription regulator for multiple angiogenic factors and is an appealing target. Ginsenoside-Rg1, a nontoxic saponin isolated from the rhizome of Panax ginseng, exhibits potent proangiogenic activity and has the potential to be developed as a new angiotherapeutic agent. However, the mechanisms by which Rg1 promotes angiogenesis are not fully understood. Here, we show that Rg1 is an effective stimulator of HIF-1α under normal cellular oxygen conditions in human umbilical vein endothelial cells. HIF-1α steady-state mRNA was not affected by Rg1. Rather, HIF-1α protein synthesis was stimulated by Rg1. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector p70 S6 kinase (p70S6K), but not extracellular-signal regulated kinase 1/2. We further revealed that HIF-1α induction triggered the expression of target genes, including vascular endothelial growth factor (VEGF). The use of small molecule inhibitors LY294002 or rapamycin to inhibit PI3K/Akt and p70S6K activities, respectively, resulted in diminished HIF-1α activation and subsequent VEGF expression. RNA interference-mediated knockdown of HIF-1α suppressed Rg1-induced VEGF synthesis and angiogenic tube formation, confirming that the effect was HIF-1α specific. Similarly, the angiogenic phenotype could be reversed by inhibition of PI3K/Akt and p70S6K. These results define a hypoxia-independent activation of HIF-1α, uncovering a novel mechanism for Rg1 that could play a major role in angiogenesis and vascular remodeling.

title: Reactive oxygen species driven angiogenesis by inorganic nanorods.
abstract: The exact mechanism of angiogenesis by europium hydroxide nanorods was unclear. In this study we have showed that formation of reactive oxygen species (H(2)O(2) and O(2)·-) is involved in redox signaling pathways during angiogenesis, important for cardiovascular and ischemic diseases. Here we used single-walled carbon nanotube sensor array to measure the single-molecule efflux of H(2)O(2) and a HPLC method for the determination of O(2)·- from endothelial cells in response to proangiogenic factors. Additionally, reactive oxygen species-mediated angiogenesis using inorganic nanorods was observed in transgenic (fli1a:EGFP) zebrafish embryos.

title: Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis.
abstract: Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is an angiogenic cytokine expressed by many human and animal tumors. Hypoxia often up-regulates VPF/VEGF expression further. To better define the role of VPF/VEGF in tumor biology, we screened tumorigenic lines for those expressing minimal constitutive and hypoxia-inducible VPF/VEGF. Human melanoma SK-MEL-2 cells best fit these criteria and formed small, poorly vascularized tumors in immunodeficient mice. We transfected SK-MEL-2 cells stably with sense or antisense mouse VPF/VEGF cDNA or with vector alone. Cells transfected with sense VPF/VEGF (V+) expressed and secreted large amounts of mouse VPF/VEGF and formed well-vascularized tumors with hyperpermeable blood vessels and minimal necrosis in nude/SCID mice. Antisense-transfected VPF/VEGF (V-) cells expressed reduced constitutive VPF/VEGF and no detectable mouse VPF/VEGF, and formed small, minimally vascularized tumors exhibiting extensive necrosis. Vector-alone transfectants (N1 cells) behaved like parental cells. V+ cells formed numerous lung tumor colonies in SCID mice, approximately 50-fold more than N1 cells, whereas V- cells formed few or none. These experiments demonstrate that VPF/VEGF promotes melanoma growth by stimulating angiogenesis and that constitutive VPF/VEGF expression dramatically promotes tumor colonization in the lung.

title: Safety and efficacy of autologous progenitor cell transplantation for therapeutic angiogenesis in patients with critical limb ischemia.
abstract: Therapeutic angiogenesis using cell transplantation (TACT) is a treatment strategy for no-option patients with critical limb ischemia (CLI). However, because one-third of treated patients fail to respond, the present study was an exploration of the characteristics of responders and non-responders to this treatment regimen.



Seven CLI patients (3 with Buerger's disease, 4 with arteriosclerosis obliterans undergoing chronic hemodialysis (ASO-HD)) were treated according to the TACT protocol (n=6: bone marrow-mononuclear cells (MNCs); n=1: peripheral blood-MNCs). Subjective symptoms (visual analog scale) and objective findings (extent of ulcer, ankle-brachial pressure index, transcutaneous oxygen pressure, thermography and angiography) were assessed. Numbers of transplanted CD34+, CD133+ and CD34+ CD133+ cells were counted. Changes in circulating CD34+ and CD133+ cell numbers were also examined before and after the treatment. All responders (n=3) had Buerger's disease, and ASO-HD patients did not respond well. Among the responders, the numbers of circulating CD34+ and CD133+ cells persistently increased for 1 month after the treatment, but not in non-responders.



The TACT regimen improved CLI in patients with Buerger's disease but not in those with ASO-HD in this small study. In responders, post procedural circulating CD34+ and CD133+ cells persistently increased for 1 month (ClinicalTrials.gov Identifier: NCT00145262, TACT-NAGOYA).

title: Profoundly Reduced Neovascularization Capacity of Bone Marrow Mononuclear Cells Derived From Patients With Chronic Ischemic Heart Disease
abstract: Cell therapy with bone marrow–derived stem/progenitor cells is a novel option for improving neovascularization and cardiac function in ischemic heart disease. Circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. However, whether this impairment also extends to bone marrow–derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) is unclear. BM-MNCs were isolated from bone marrow aspirates in 18 patients with ICMP (ejection fraction, 38±11%) and 8 healthy control subjects (controls). The number of hematopoietic stem/progenitor cells  CD49d+ (VLA-4) cells, and CXCR4+ cells did not differ between the 2 groups. However, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls (37.3±25.0 versus 113.8±70.4 granulocyte-macrophage colony-forming units; P =0.009). Likewise, the migratory response to stromal cell–derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls (SDF-1, 46.3±26.2 versus 108.6±40.4 cells/microscopic field, P <0.001; VEGF, 34±24.2 versus 54.8±29.3 cells/microscopic field, P =0.027). To assess the in vivo relevance of these findings, we tested the functional activity of BM-MNCs to improve neovascularization in a hindlimb animal model using nude mice. Two weeks after ligation of the femoral artery and intravenous injection of 5×105 BM-MNCs, laser Doppler–derived relative limb blood flow in mice treated with BM-MNCs from patients with ICMP was significantly lower compared with mice treated with BM-MNCs from healthy controls (0.45±0.14 versus 0.68±0.15; P <0.001). The in vivo neovascularization capacity of BM-MNCs closely correlated with the in vitro assessment of SDF-1–induced migration (r =0.78; P <0.001) and colony-forming capacity (r =0.74; P <0.001). BM-MNCs isolated from patients with ICMP have a significantly reduced migratory and colony-forming activity in vitro and a reduced neovascularization capacity in vivo despite similar content of hematopoietic stem cells. This functional impairment of BM-MNCs from patients with ICMP may limit their therapeutic potential for clinical cell therapy.

title: Symmetry-Driven Spontaneous Self-Assembly of Nanoscale Ceria Building Blocks to Fractal Superoctahedra
abstract: A combination of long-term aging studies and molecular dynamics (MD) simulations has been successfully used to explain the time-dependent hierarchical assembly of ceria nanoparticles (CNPs). When the CNPs were aged in as-synthesized condition at room temperature in water, it was observed that the individual 3−5 nm CNPs result in octahedral superstructures through a fractal assembly. This hierarchical fractal self-assembly was observed despite the absence of any surfactant, at room temperature, and under atmospheric pressure. High resolution transmission electron microscopy (HRTEM) and fast Fourier transform (FFT) analysis have been used to explore the assembly of the individual nanoparticles into fractal superoctahedra. Both experimental work and theoretical analysis showed that the initial octahedral and truncated octahedral seeds symmetrically assemble and result in the superoctahedra with intermediate transformation steps.

title: Chromium VI adsorption on cerium oxide nanoparticles and morphology changes during the process.
abstract: In this study, suspended cerium oxide nanoparticles stabilized with hexamethylenetetramine were used for the removal of dissolved chromium VI in pure water. Several concentrations of adsorbent and adsorbate were tested, trying to cover a large range of possible real conditions. Results showed that the Freundlich isotherm represented well the adsorption equilibrium reached between nanoparticles and chromium, whereas adsorption kinetics could be modeled by a pseudo-second-order expression. The separation of chromium-cerium nanoparticles from the medium and the desorption of chromium using sodium hydroxide without cerium losses was obtained. Nanoparticles agglomeration and morphological changes during the adsorption-desorption process were observed by TEM. Another remarkable result obtained in this study is the low toxicity in the water treated by nanoparticles measured by the Microtox(®) commercial method. These results can be used to propose this treatment sequence for a clean and simple removal of drinking water or wastewater re-use when a high toxicity heavy metal such as chromium VI is the responsible for water pollution.

title: Self-assembly of cerium oxide nanostructures in ice molds.
abstract: The formation of nanorods, driven by the physicochemical phenomena during the freezing and after the aging of frozen ceria nanoparticle suspensions, is reported. During freezing of a dilute aqueous solution of CeO2 nanocrystals, some nuclei remain in solution while others are trapped inside micro- and nanometer voids formed within the growing ice front. Over time (2-3 weeks) the particles trapped within the nanometer-wide voids in the ice combine by an oriented attachment process to form ceria nanorods. The experimental observations are consistent with molecular dynamics simulations of particle aggregation in constrained environments. These observations suggest a possible strategy for the templated formation of nanostructures through self-assembly by exploiting natural phenomena, such as voids formed during freezing of water. This research suggests a very simple, green chemical route to guide the formation of one- and three-dimensional self-assembled nanostructures.

title: In Vitro Assay of Angiogenesis: Inhibition of Capillary Tube Formation
abstract: The growth of new blood vessels, or angiogenesis, is a naturally occurring process in both health and disease states. An area of active research, regulation of angiogenesis, is being studied as an approach for the treatment of cancer and a range of other disorders having vascular proliferation as a component. The process of angiogenesis is very complex and occurs in multiple steps, with a major involvement of endothelial cells. Various in vivo models have been developed to assess inhibitors of angiogenesis. As these are generally technically difficult and labor intensive, with observed effects difficult to quantify, they do not lend themselves to compound screening. Rather they are used for confirmatory studies. In contrast, in vitro assays developed to model various steps in the angiogenesis process are easy to perform and lend themselves to high‐throughput analysis. Described in this unit is an in vitro assay that can be employed to investigate endothelial differentiation inhibitors through assessment of their effects on capillary tube formation by endothelial cells on Matrigel. Curr. Protoc. Pharmacol. 43:12.12.1‐12.12.7.

title: Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells
abstract: Although immune mechanisms can suppress tumour growth, tumours establish potent, overlapping mechanisms that mediate immune evasion. Emerging evidence suggests a link between angiogenesis and the tolerance of tumours to immune mechanisms. Hypoxia, a condition that is known to drive angiogenesis in tumours, results in the release of damage-associated pattern molecules, which can trigger the rejection of tumours by the immune system. Thus, the counter-activation of tolerance mechanisms at the site of tumour hypoxia would be a crucial condition for maintaining the immunological escape of tumours. However, a direct link between tumour hypoxia and tolerance through the recruitment of regulatory cells has not been established. We proposed that tumour hypoxia induces the expression of chemotactic factors that promote tolerance. Here we show that tumour hypoxia promotes the recruitment of regulatory T (Treg) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumour tolerance and angiogenesis. Thus, peripheral immune tolerance and angiogenesis programs are closely connected and cooperate to sustain tumour growth.

title: Functionalized nanoceria exhibit improved angiogenic properties.
abstract: The growth of new blood vessels from the pre-existing vasculature known as angiogenesis has a vital role in various physiological and pathological processes. In the present study, we demonstrate the pro-angiogenic properties of functional nanoconjugates of organosilane functionalized cerium oxide (CeO2) nanoparticles (nanoceria). Aqueous dispersible CeO2 and trivalent metal (samarium) ion-doped CeO2 (SmCeO2) nanoparticles conjugated with hydrophilic biocompatible and antifouling (6-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-hexyl)triethoxysilane moieties were prepared. These functional nanoconjugates were prepared via an in situ synthesis and functionalization procedure using an ammonia-induced ethylene glycol-assisted precipitation method. The prepared nanoconjugates were thoroughly characterized using various physico-chemical techniques such as transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis, dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, 13C high-resolution solid-state nuclear magnetic resonance (NMR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The pro-angiogenic properties of the prepared nanoconjugates were evaluated by employing various angiogenesis assays (in vitro and in vivo). The results of the present study illustrate that the functional nanoconjugates of SmCeO2 triggered endothelial cell proliferation and induced the growth of blood vessels in a chick embryo. The enhanced expression of pro-angiogenic markers (p38 MAPK/HIF-1α) by these functional nanoconjugates might be a plausible signaling mechanism underlying their pro-angiogenic properties. Considering all the observations, we believe that (6-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-hexyl)triethoxysilane conjugated SmCeO2 nanoparticles could be developed as potential candidates for the treatment of cardiovascular, ischemic and ocular diseases where angiogenesis is the principal phenomenon.

title: In-situ tissue regeneration through SDF-1α driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect.
abstract: In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required.



Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.

title: Nanoceria Can Act as the Cues for Angiogenesis in Tissue-Engineering Scaffolds: Toward Next-Generation in Situ Tissue Engineering.
abstract: Next-generation tissue engineering exploits the body's own regenerative capacity by providing an optimal niche via a scaffold for the migration and subsequent proliferation of endogenous cells to the site of injury, enhancing regeneration and healing and bypassing laborious in vitro cell-culturing procedures. Such systems are also required to have a sufficient angiogenic capacity for the subsequent patency of implanted scaffolds. The exploitation of redox properties of nanodimensional ceria (nCeO2) in in situ tissue engineering to promote cell adhesion and angiogenesis is poorly investigated. As a novel strategy, electrospun polycaprolactone based tissue-engineering scaffolds loaded with nCeO2 were developed and evaluated for morphological and physicomechanical features. In addition, in vitro and in vivo studies were performed to show the ability of nCeO2-containing scaffolds to enhance cell adhesion and angiogenesis. These studies confirmed that nCeO2-containing scaffolds supported cell adhesion and angiogenesis better than bare scaffolds. Gene-expression studies had shown that angiogenesis-related factors such as HIF1α and VEGF were up-regulated. Overall results show that incorporation of nCeO2 plays a key role in scaffolds for the enhancement of angiogenesis, cell adhesion, and cell proliferation and can produce a successful outcome in in situ tissue engineering.

title: Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector.
abstract: The goal of this study was to modify mesenchymal stem cells (MSCs) cells with a hypoxia-regulated heme oxygenase-1 (HO-1) plasmid to enhance the survival of MSCs in acute myocardial infarction (MI) heart.



Although stem cells are being tested clinically for cardiac repair, graft cells die in the ischemic heart because of the effects of hypoxia/reoxygenation, inflammatory cytokines, and proapoptotic factors. Heme oxygenase-1 is a key component in inhibiting most of these factors.



Mesenchymal stem cells from bone marrow were transfected with either HO-1 or LacZ plasmids. Cell apoptosis was assayed in vitro after hypoxia-reoxygen treatment. In vivo, 1 x 10(6) of male MSC(HO-1), MSC(LacZ), MSCs, or medium was injected into mouse hearts 1 h after MI (n = 16/group). Cell survival was assessed in a gender-mismatched transplantation model. Apoptosis, left ventricular remodeling, and cardiac function were tested in a gender-matched model.



In the ischemic myocardium, the MSC(HO-1) group had greater expression of HO-1 and a 2-fold reduction in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells compared with the MSC(LacZ) group. At seven days after implantation, the survival MSC(HO-1) was five-fold greater than the MSC(LacZ) group; MSC(HO-1) also attenuated left ventricular remodeling and enhanced the functional recovery of infarcted hearts two weeks after MI.



A hypoxia-regulated HO-1 vector modification of MSCs enhances the tolerance of engrafted MSCs to hypoxia-reoxygen injury in vitro and improves their viability in ischemic hearts. This demonstration is the first showing that a physiologically inducible vector expressing of HO-1 genes improves the survival of stem cells in myocardial ischemia.

title: Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies.
abstract: M. Zhang, D. Methot, V. Poppa, Y. Fujio, K. Walsh and C. E. Murry. Cardiomyocyte Grafting for Cardiac Repair: Graft Cell Death and Anti-Death Strategies. Journal of Molecular and Cellular Cardiology (2001) 33, 907-921. Recent studies indicate that cardiomyocyte grafting forms new myocardium in injured hearts. It is unknown, however, whether physiologically significant amounts of new myocardium can be generated. Pilot experiments showed that death of grafted rat neonatal cardiomyocytes limited formation of new myocardium after acute cryoinjury. Time-course studies showed that, at 30 min after grafting, only 1.8(+/-0.4)% of graft cells were TUNEL-positive. At 1 day, however, TUNEL indices increased to 32.1(+/-3.5)% and remained high at 4 days, averaging 9.8(+/-3.8)%. By 7 days, TUNEL decreased to 1.0(+/-0.2)%. Electron microscopy revealed that dead cells had features of both irreversible ischemic injury and apoptosis. To test whether ischemia contributed to poor graft survival, grafts were placed into vascularized 2-week-old cardiac granulation tissue or normal myocardium. TUNEL indices were reduced by 53% and 86%, respectively. Adenoviral infection of graft cells with the cytoprotective kinase Akt, or constitutively active Akt, reduced TUNEL indices by 31% and 40%, respectively, compared to beta -gal-transfected controls. Neither treatment reached statistical significance compared to untreated controls, however. Heat shock reduced cardiomyocyte death in vitro in response to serum deprivation, glucose depletion, and viral activation of the Fas death pathway. When cardiomyocytes were heat shocked prior to grafting, graft cell death in vivo was reduced by 54% at day 1. Therefore, high levels of cardiomyocyte death occur for at least 4 days after grafting into injured hearts, in large part due to ischemia. Death can be limited by activating the Akt pathway and even more effectively by heat shock prior to transplantation.

title: Mesenchymal stem cells and their potential as cardiac therapeutics.
abstract: Mesenchymal stem cells (MSCs) represent a stem cell population present in adult tissues that can be isolated, expanded in culture, and characterized in vitro and in vivo. MSCs differentiate readily into chondrocytes, adipocytes, osteocytes, and they can support hematopoietic stem cells or embryonic stem cells in culture. Evidence suggests MSCs can also express phenotypic characteristics of endothelial, neural, smooth muscle, skeletal myoblasts, and cardiac myocyte cells. When introduced into the infarcted heart, MSCs prevent deleterious remodeling and improve recovery, although further understanding of MSC differentiation in the cardiac scar tissue is still needed. MSCs have been injected directly into the infarct, or they have been administered intravenously and seen to home to the site of injury. Examination of the interaction of allogeneic MSCs with cells of the immune system indicates little rejection by T cells. Persistence of allogeneic MSCs in vivo suggests their potential "off the shelf" therapeutic use for multiple recipients. Clinical use of cultured human MSCs (hMSCs) has begun for cancer patients, and recipients have received autologous or allogeneic MSCs. Research continues to support the desirable traits of MSCs for development of cellular therapeutics for many tissues, including the cardiovascular system. In summary, hMSCs isolated from adult bone marrow provide an excellent model for development of stem cell therapeutics, and their potential use in the cardiovascular system is currently under investigation in the laboratory and clinical settings.

title: Local Delivery of Marrow-Derived Stromal Cells Augments Collateral Perfusion Through Paracrine Mechanisms
abstract: Bone marrow cell therapy is reported to contribute to collateral formation through cell incorporation into new or remodeling vessels. However, the possible role of a paracrine contribution to this effect is less well characterized. Murine marrow-derived stromal cells (MSCs) were purified by magnetic bead separation of cultured bone marrow. The release of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and monocyte chemoattractant protein-1 (MCP-1) was demonstrated by analysis of MSC conditioned media (MSC-CM). MSC-CM enhanced proliferation of endothelial cells and smooth muscle cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partly attenuated these effects. Balb/C mice (n=10) underwent distal femoral artery ligation, followed by adductor muscle injection of 1×106 MSCs 24 hours later. Compared with controls injected with media (n=10) or mature endothelial cells (n=8), distal limb perfusion improved, and mid-thigh conductance vessels increased in number and total cross-sectional area. MSC injection improved limb function and appearance, reduced the incidence of auto-amputation, and attenuated muscle atrophy and fibrosis. After injection, labeled MSCs were seen dispersed between muscle fibers but were not seen incorporated into mature collaterals. Injection of MSCs increased adductor muscle levels of bFGF and VEGF protein compared with controls. Finally, colocalization of VEGF and transplanted MSCs within adductor tissue was demonstrated. MSCs secrete a wide array of arteriogenic cytokines. MSCs can contribute to collateral remodeling through paracrine mechanisms.

title: Mesenchymal stem cell-based gene therapy with prostacyclin synthase enhanced neovascularization in hindlimb ischemia.
abstract: Bone marrow cell therapy contributes to collateral formation through the secretion of angiogenic factors by progenitor cells and muscle cells per se, thereby presenting a novel option for patients with critical limb ischemia. However, some cases are refractory to this therapy due to graft failure. Therefore, we used genetic modification of mesenchymal stem cells (MSCs) to overexpress a vasoregulatory protein, prostacyclin (PGI(2)), to examine whether it could enhance engraftment and neovascularization in hindlimb ischemia.



We engineered the overexpression of PGI(2) synthase (PGIS) within MSCs, which resulted in higher expression levels of phosphorylated Akt and Bcl-2 than in control. Under hypoxic conditions, the overexpression of PGIS led to upregulated expression of cyclooxigenase-2 and peroxisome proliferator-activated receptor delta, following a 40% increased rate of proliferation in MSCs. We then produced unilateral hindlimb ischemia in C57BL6/J mice, which were injected either with MSCs transfected with GFP, with MSCs overexpressing PGIS, or with vehicle. Laser Doppler analyses demonstrated that the administration of MSCs effectively recovered blood perfusion, and that the peak blood flow was reached within 7 days of surgery in mice with MSCs overexpressing PGIS, which was earlier than that in mice with MSCs transfected with GFP. This beneficial effect was correlated to enhanced collateral formation and muscle bundle proliferation.



Sustained release of PGI(2) enhanced the proangiogenic function of MSCs and subsequent muscle cell regrowth in the ischemic tissue suggesting potential therapeutic benefits of cell-based gene therapy for critical limb ischemia.

title: iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis
abstract: Angiogenic cell therapy represents a novel strategy for ischemic diseases, but some patients show poor responses. We investigated the therapeutic potential of an induced pluripotent stem (iPS) cell sheet created by a novel magnetite tissue engineering technology (Mag-TE) for reparative angiogenesis. Mouse iPS cell-derived Flk-1+ cells were incubated with magnetic nanoparticle-containing liposomes (MCLs). MCL-labeled Flk-1+ cells were mixed with diluted extracellular matrix (ECM) precursor and a magnet was placed on the reverse side. Magnetized Flk-1+ cells formed multi-layered cell sheets according to magnetic force. Implantation of the Flk-1+ cell sheet accelerated revascularization of ischemic hindlimbs relative to the contralateral limbs in nude mice as measured by laser Doppler blood flow and capillary density analyses. The Flk-1+ cell sheet also increased the expressions of VEGF and bFGF in ischemic tissue. iPS cell-derived Flk-1+ cell sheets created by this novel Mag-TE method represent a promising new modality for therapeutic angiogenesis.

title: Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells
abstract: BackgroundInduced pluripotent stem (iPS) cells are the novel stem cell population induced from somatic cells. It is anticipated that iPS will be used in the expanding field of regenerative medicine. Here, we investigated whether implantation of fetal liver kinase-1 positive (Flk-1+) cells derived from iPS cells could improve angiogenesis in a mouse hind limb model of ischemia.ResultsFlk-1+ cells were induced from iPS cells after four to five days of culture. Hind limb ischemia was surgically induced and sorted Flk-1+ cells were directly injected into ischemic hind limbs of athymic nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk-1+ cells compared with control mice, which were transplanted with vehicle, as evaluated by laser Doppler blood flowmetry. Transplantation of Flk-1+ cells also increased expression of VEGF mRNA in ischemic tissue compared to controls.ConclusionsDirect local implantation of iPS cell-derived Flk-1+ cells would salvage tissues from ischemia. These data indicate that iPS cells could be valuable in the therapeutic induction of angiogenesis.

title: Bone regenerative medicine: classic options, novel strategies, and future directions
abstract: This review analyzes the literature of bone grafts and introduces tissue engineering as a strategy in this field of orthopedic surgery. We evaluated articles concerning bone grafts; analyzed characteristics, advantages, and limitations of the grafts; and provided explanations about bone-tissue engineering technologies. Many bone grafting materials are available to enhance bone healing and regeneration, from bone autografts to graft substitutes; they can be used alone or in combination. Autografts are the gold standard for this purpose, since they provide osteogenic cells, osteoinductive growth factors, and an osteoconductive scaffold, all essential for new bone growth. Autografts carry the limitations of morbidity at the harvesting site and limited availability. Allografts and xenografts carry the risk of disease transmission and rejection. Tissue engineering is a new and developing option that had been introduced to reduce limitations of bone grafts and improve the healing processes of the bone fractures and defects. The combined use of scaffolds, healing promoting factors, together with gene therapy, and, more recently, three-dimensional printing of tissue-engineered constructs may open new insights in the near future.

title: Scaffolds for tissue fabrication
abstract: Tissue engineering is an interdisciplinary and multidisciplinary field. It has shown great promise in generating living alternatives for harvested tissues and organs for transplantation and reconstructive surgery. Materials and fabrication technologies are critically important for tissue engineering in designing temporary, artificial extracellular matrices (scaffolds), which support three-dimensional tissue formation. This review briefly introduces the concept of tissue engineering, and illustrates the relationship between tissue engineering and materials science and engineering. Important scaffold design principles are described. The most frequently used materials and fabrication technologies for scaffolds are reviewed. Some exciting new developments in scaffold materials and fabrication technologies are also discussed.

title: The effect on osteoblast function of colocalized RGD and PHSRN epitopes on PEG surfaces.
abstract: Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg-Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro-His-Ser-Arg-Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD-PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor-ligand interactions initiating signal transduction cascades that down-regulate ECM production.

title: Effect of FGF and Polylactide Scaffolds on Calvarial Bone Healing With Growth Factor on Biodegradable Polymer Scaffolds
abstract: Repair of bone defects remains a major concern in reconstructive surgery. Synthetic biodegradable polymers have been used as scaffolds for guided bone regeneration. Fibroblast growth factors (FGFs) promote cell growth, differentiation, and tissue maintenance factors. They can stimulate the proliferation of osteogenic cells and chondrocytes, and also promote angiogenesis. Acidic and basic fibroblast growth factors (FGF-1 and FGF-2, respectively) are the best known members of this protein family. To evaluate the healing of experimental bone defects using poly-L/D-lactide (PLDLA) 96/4 scaffolds and FGF-1, 18 adult rats were operated on. A 6-mm diameter critical size defect (CSD) was made in the calvarial bone of each rat. The animals were divided into three treatment groups: 1) Neither scaffold nor FGF was used (control group); 2) scaffold only; and 3) scaffold with FGF-1. Follow-up time was eight weeks. Samples were embedded in methylmethacrylate and 5-&mgr;m thick sections from the middle of each specimen were stained with modified Masson-Goldner method. The shape and size of defects were evaluated radiologically. New bone formation was measured histologically and histomorphometrically. Radiologically, in the control group the shape of the defects changed from round to oval and edges were blunt. In the other groups the defects were round with sharp edges. Histomorphometrically, mean surface area of bone trabeculae was 1.05 mm2 (SD ± 0.25) in group 1 (no implant), 1.35 mm2 (SD ± 0.52) in group 2 (implant) and 0.79 mm2 (SD ± 0.34) in group 3 (implant and FGF-1). Histological examinations revealed no or little osteoid in the groups 1 and 2, whereas in the group 3 samples had little or moderate new bone formation. Accordingly, no clear benefit of using knitted PLDLA scaffolds combined with FGF-1 on the healing of calvarial critical size defects in rats could be demonstrated.

title: Modified keratin sponge: binding of bone morphogenetic protein-2 and osteoblast differentiation.
abstract: A keratin sponge was chemically modified to obtain carboxyl and amino sponges by the alkylation of a large amount of active SH group on keratin proteins with iodoacetic acid and 2-bromoethylamine, respectively. The carboxyl sponge having a large amount of carboxyl group was a scaffold that could bind significant amounts of basic bioactive proteins, such as lysozyme and bone morphogenetic protein (BMP)-2, and drugs. Lysozyme (maximum 3.7 mg), a model of basic cytokines such as BMP-2, was absorbed by the carboxyl sponge (4.8 mg), but not by the amino sponge. The lysozyme was rapidly released from the carboxyl sponge in a buffer containing at a concentration higher than 0.5 M, but at 0.15 M, near the physiological ionic strength, after initial burst (only 11%), no significant release was observed (15%, 48 h). BMP-2 also bound the carboxyl sponge. The preosteoblast cells grown inside the BMP-2-loaded sponge differentiated, whereas those grown outside the sponge did not, suggesting that no significant amount of BMP-2 leaked into the surrounding media. The effects of BMP-2 were confined inside modified keratin sponge. Therefore, using in vivo, we expect that only internal osteogenesis will be induced and that no external heterotopis ossification will be induced.

title: Rare earth oxides as nanoadditives in 3-D nanocomposite scaffolds for bone regeneration
abstract: Nanocomposite scaffolds of bioactive glass foams containing tailor made rare earth oxide (i.e. nanoceria) additives were demonstrated to enhance the production of collagen by HMSCs (Human Mesenchymal Stem Cells) compared to bioactive glass scaffolds without nanoceria. The addition of osteogenic supplements was not required for this to occur. Two different preparations of nanoceria were successfully incorporated in 3-D bioactive glass foam scaffolds and were compared with bioactive scaffolds without nanoceria. The nanoparticles had individual particle sizes of 3–5 nm while the agglomerate size varied from 5–15 nm. Preliminary investigations show that nanoceria is non toxic to the cells. After 10 days of culture, nanoceria containing scaffolds showed enhanced osteoblastic differentiation of HMSCs and collagen production compared to the scaffolds without nanoceria even in the absence of any osteogenic supplements (i.e. ascorbic acid, dexamethasone and β-glycerophosphate). This could be due to the incorporation of nanoceria, which acts as an oxygen buffer thereby regulating the differentiation of HMSCs. Further investigations are currently underway to determine the role of the nanoceria controlled oxygen buffering on the HMSC differentiation.

title: Optimising bioactive glass scaffolds for bone tissue engineering.
abstract: A 3D scaffold has been developed that has the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. Sol-gel derived bioactive glasses of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The scaffolds consist of a hierarchical pore network with macropores in excess of 500 microm connected by pore windows with diameters in excess of 100 microm, which is thought to be the minimum pore diameter required for tissue ingrowth and vasularisation in the human body. The scaffolds also have textural porosity in the mesopore range (10-20 nm). The scaffolds were sintered at 600, 700, 800 and 1000 degrees C. As sintering temperature was increased to 800 degrees C the compressive strength increased from 0.34 to 2.26 MPa due to a thickening of the pore walls and a reduction in the textural porosity. The compressive strength is in the range of that of trabecular bone (2-12 MPa). Importantly, the modal interconnected pore diameter (98 microm) was still suitable for tissue engineering applications and bioactivity is maintained. Bioactive glass foam scaffolds sintered at 800 degrees C for 2 h fulfill the criteria for an ideal scaffold for tissue engineering applications.

title: Nodule formation and mineralisation of human primary osteoblasts cultured on a porous bioactive glass scaffold.
abstract: The aim of this study was to analyse human osteoblast responses to a porous bioactive glass scaffold. It was hypothesised that osteoblasts would attach, proliferate and form mineralised nodules in response to culture on the bioactive glass. As dissolution products are a key feature of bioactive glasses, this was measured by inductively coupled plasma optical emission spectroscopy to determine effects of both the glass surface and ion release. Osteoblasts attached and proliferated on the foams as demonstrated by scanning electron microscopy. Nodule formation was also observed in the pores of the glass and also in conditioned medium containing dissolution products at certain concentrations and these nodules were shown to be mineralised by alizarin red staining. Undiluted dissolution products from the foams however caused significant apoptosis suggesting an ion concentration dependent response.

title: The mechanism of mineralization and the role of alkaline phosphatase in health and disease.
abstract: Biomineralization is the process by which hydroxyapatite is deposited in the extracellular matrix. Physiological mineralization occurs in hard tissues, whereas pathological calcification occurs in soft tissues. The first step of mineralization is the formation of hydroxyapatite crystals within matrix vesicles that bud from the surface membrane of hypertrophic chondrocytes, osteoblasts, and odontoblasts. This is followed by propagation of hydroxyapatite into the extracellular matrix and its deposition between collagen fibrils. Extracellular inorganic pyrophosphate, provided by NPP1 and ANKH, inhibits hydroxyapatite formation. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization. Inorganic pyrophosphate, pyridoxal phosphate, and phosphoethanolamine are thought to be the physiologic substrates of TNAP. These accumulate in the event of TNAP deficiency, e.g., in cases of hypophosphatasia. The gene encoding TNAP is mapped to chromosome 1, consists of 12 exons, and possesses regulatory motifs in the 5'-untranslated region. Inhibition of TNAP enzymatic activity suppresses TNAP mRNA expression and mineralization in vitro. Hypophosphatasia is an inherited systemic bone disease characterized by hypomineralization of hard tissues. The phenotype of hypophosphatasia is varied. To date, more than 200 mutations in the TNAP gene have been reported. Knockout mice mimic the phenotypes of severe hypophosphatasia. Among the mutations in the TNAP gene, c.1559delT is frequent in the Japanese population. This frameshift mutation results in the expression of an abnormally long protein that is degraded in cells. DNA-based prenatal diagnosis using chorionic villus sampling has been developed, but requires thorough genetic counseling. Although hypophosphatasia is untreatable at present, the recent success of enzyme replacement therapy offers promise. The problems presented by impaired mineralization in age-related chronic diseases, such as pathologic calcification and decreasing physiological mineralization are growing in importance. Strategies for preventing pathologic calcification using TNAP and NPP1 are in development. A nutrigenomic approach, based on the relationship between TNAP gene polymorphism and bone mineral density, is also discussed.

title: The effect of composition on structural, thermal, redox and bioactive properties of Ce-containing glasses
abstract: The effect of phosphate on the ability of Ce-containing bioactive glasses to inhibit oxidative stress was studied on compositions based on Hench (46.2%SiO224.3%Na2O26.9%CaO2.6P2O5, mol%) and Kokubo (50.0%SiO225.0%Na2O25.0%CaO) glasses. In particular, the reduction of catalase mimetic activity of Ce-containing glasses due to the presence: i) of P2O5 in the glass compositions, and ii) of phosphate groups in the solution employed for catalase mimetic activity tests was explained and rationalized by combining SEM, XPS, XRD, DTA, FT-IR and UV–vis experiments with Molecular Dynamics simulations. 
 
The results suggest that the Ce ions play a different structural role in the two series of glasses. In particular, in phosphate free glasses Ce is coordinated by non-bridging oxygens (NBOs) originated from the disruption of the silicate network, whereas in phosphate containing glasses the NBOs around Ce ions belong to orthophosphate groups. The latter groups stabilize the Ce3+ species subtracting them from the interconversion process between Ce3+ and Ce4+, which is of fundamental importance for the exhibition of the catalase mimetic activity.

title: Electrospun cerium and gallium-containing silicate based 13-93 bioactive glass fibers for biomedical applications
abstract: Fibrous scaffolds have gained increasing attention in tissue engineering applications because of their high porosity, and surface area. In the current study cerium or gallium- containing silicate based 13-93 bioactive glass fibers were prepared using electrospinning approach. Effect of cerium and gallium substitution (up to 5 w%) on the in vitro bioactivity, pre-osteoblast cell response and antibacterial activity of the prepared fibers were assessed. Results revealed that, addition of cerium or gallium has no negative effect on bioactivity and hydroxyapatite forming ability of the glass in long term. The biocompatibility tests on MC3T3-E1 cells using XTT assay revealed no cytotoxicity of the cerium- or gallium-containing bioactive glass fibers. The fibers prepared in the study showed no antibacterial response to the gram positive ( Staphylococcus aureus ) and gram negative ( Escherichia coli ) bacteria. Fibrous scaffolds manufactured in the study may be considered for the use in soft tissue engineering applications in near future.

title: Incorporation of cerium oxide in hollow mesoporous bioglass scaffolds for enhanced bone regeneration by activating the ERK signaling pathway.
abstract: Hierarchically porous structures and bioactive compositions of artificial biomaterials play a positive role in bone defect healing and new bone regeneration. Herein, cerium oxide nanoparticles-modified bioglass (Ce-BG) scaffolds were firstly constructed by the incorporation of hollow mesoporous Ce-BG microspheres in CTS via a freeze-drying technology. The interconnected macropores in Ce-BG scaffolds facilitated the in-growth of bone cells/tissues from material surfaces into the interiors, while the hollow cores and mesopore shells in Ce-BG microspheres provides more active sites for bone mineralization. The cerium oxide nanoparticles in the scaffolds rapidly promoted the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs), as confirmed by the up-regulation of osteogenesis-related markers such as OCN, ALP and COL-1. The enhanced osteoinductivity of Ce-BG scaffolds was mainly related to the activated ERK pathway, and it was blocked by adding a selective ERK1/2 inhibitor (SCH772984). In vivo rat cranial defect models revealed that Ce-BG scaffolds accelerated collagen deposition, osteoblast formation and bone regeneration as compared to BG scaffolds. The exciting results demonstrated that the synergistic effects between hierarchically porous structures and cerium oxide nanoparticles contributed to osteogenic ability, and hollow mesoporous Ce-BG scaffolds would be a novel platform for bone regeneration.

title: The Effects of Cerium Valence States at Cerium Oxide Coatings on the Responses of Bone Mesenchymal Stem Cells and Macrophages
abstract: Ideal orthopedic coatings should trigger good osteogenic response and limited inflammatory response. The cerium valence states in ceria are associated with their anti-oxidative activity and anti-inflammatory property. In the study, we prepared two kinds of plasma sprayed CeO2 coatings with different Ce4+ concentrations to investigate the effects of Ce valence states on the response of bone mesenchymal stem cells (BMSCs) and macrophage RAW264.7. Both the coatings (CeO2-A and CeO2-B) were characterized via XRD, SEM, and X-ray photoelectron spectroscopy. The CeO2 coatings enhanced osteogenic behaviors of BMSCs in terms of cellular proliferation, alkaline phosphatase (ALP) activity and calcium deposition activity in comparison with the Ti substrate. In particular, the CeO2-B coating (higher Ce4+ concentration) elicited greater effects than the CeO2-A coating (higher Ce3+ concentration). RT-PCR and western blot results suggested that the CeO2-B coating promoted BMSCs osteogenic differentiation through the SMAD-dependent BMP signaling pathway, which activated Runx2 expression and subsequently enhanced the expression of ALP and OCN. With respect to either CeO2-A coating or Ti substrate, the CeO2-B coating exerted greater effects on the macrophages, increasing the anti-inflammatory cytokines (IL-10 and IL-1ra) expression and suppressing the expression of the pro-inflammatory cytokines (TNF-α and IL-6) and ROS production. Furthermore, it also upregulated the expression of osteoinductive molecules (TGF-β1 and BMP2) in the macrophages. The regulation of cerium valence states at plasma sprayed ceria coatings can be a valuable strategy to improve osteogenic properties and alleviate inflammatory response.

title: The Effects of Cerium Oxide Incorporation in Calcium Silicate Coating on Bone Mesenchymal Stem Cell and Macrophage Responses
abstract: Ideal coatings for orthopedic implants should be able to induce excellent osseointegration with host bone tissue, which requires good osteogenic responses and limited inflammatory reactions. Cerium oxide (CeO2) ceramics have anti-oxidative properties and can be used to decrease mediators of inflammation, making them attractive for biomedical application. In this study, two kinds of CeO2 incorporated calcium silicate coatings (CS-10Ce and CS-30Ce) were prepared via plasma spraying technique, and the effects of CeO2 addition on the responses of bone mesenchymal stem cells (BMSCs) and RAW264.7 macrophages were evaluated. The CS-10Ce and CS-30Ce coatings were characterized by X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy. An increase in CeO2 content in the coatings resulted in enhanced chemical stability and better BMSCs osteogenic behaviors in terms of cell adhesion, proliferation, ALP activity, and mineralized nodule formation. With respect to either ZrO2-added or unmodified CS coating, the CS-30Ce coating elicited higher effects on the macrophages, suppressing the gene expressions of pro-inflammatory (M1) markers (CCR7, IL-6, and TNF-α), while upregulating the expressions of anti-inflammatory (M2) markers (CD206, IL-1ra, and IL-10); moreover, it could also increase the expression of osteoinductive molecules (BMP2 and TGF-β1) by the macrophages. The results suggested that the regulation of BMSCs behaviors and macrophage-mediated responses at the coating’s surface was related to CeO2 incorporation. The incorporation of CeO2 in CS coatings can be a valuable strategy to promote osteogenic responses and mitigate inflammatory reactions.

title: The effect of cerium valence states at cerium oxide nanoparticle surfaces on cell proliferation.
abstract: Understanding and controlling cell proliferation on biomaterial surfaces is critical for scaffold/artificial-niche design in tissue engineering. The mechanism by which underlying integrin ligates with functionalized biomaterials to induce cell proliferation is still not completely understood. In this study, poly-l-lactide (PL) scaffold surfaces were functionalized using layers of cerium oxide nanoparticles (CNPs), which have recently attracted attention for use in therapeutic application due to their catalytic ability of Ce(4+) and Ce(3+) sites. To isolate the influence of Ce valance states of CNPs on cell proliferation, human mesenchymal stem cells (hMSCs) and osteoblast-like cells (MG63) were cultured on the PL/CNP surfaces with dominant Ce(4+) and Ce(3+) regions. Despite cell type (hMSCs and MG63 cells), different surface features of Ce(4+) and Ce(3+) regions clearly promoted and inhibited cell spreading, migration and adhesion behavior, resulting in rapid and slow cell proliferation, respectively. Cell proliferation results of various modified CNPs with different surface charge and hydrophobicity/hydrophilicity, indicate that Ce valence states closely correlated with the specific cell morphologies and cell-material interactions that trigger cell proliferation. This finding suggests that the cell-material interactions, which influence cell proliferation, may be controlled by introduction of metal elements with different valence states onto the biomaterial surface.

title: Incorporation of Cerium Oxide into Hydroxyapatite Coating Protects Bone Marrow Stromal Cells Against H2O2-Induced Inhibition of Osteogenic Differentiation
abstract: Oxidative stress exerts a key influence in osteoporosis in part by inhibiting osteogenic differentiation of bone marrow stromal cells (BMSCs). With their unique antioxidant properties and reported biocompatibility, cerium oxide (CeO2) ceramics exhibit promising potential for the treatment of osteoporosis resulting from oxidative stress. In this study, protective effects of CeO2-incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) on the viability and osteogenic differentiation of H2O2-treated BMSCs were examined. CeO2-incorporated HA coatings enhanced cell viability and attenuated cell apoptosis caused by H2O2. An increase in CeO2 content in HA coatings better alleviated H2O2-induced inhibition of osteogenic differentiation by increasing alkaline phosphatase (ALP) activity, calcium deposition activity, and mRNA expression levels of osteogenesis markers runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OCN) in BMSCs. Furthermore, the H2O2-induced decrease of gene and protein expressions of β-catenin and cyclin D1 in the Wnt/β-catenin signaling pathway was successfully rescued by the CeO2 incorporated HA coatings. Besides, the decreased expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and the increased ratio of osteoprotegerin (OPG)/RANKL in BMSCs on the CeO2-modified coatings was observed, indicating the inhibition of osteoclastogenesis. The above results were mediated by the antioxidant properties of CeO2. The CeO2-incorporated HA coatings reversed the decreased superoxide dismutase (SOD) activity, reduced reactive oxygen species (ROS) generation, and suppressed the malondiadehyde (MDA) formation. The findings suggested that CeO2-modified HA coatings may be promising coating materials for osteoporotic bone regeneration.

title: Molecular mechanisms and clinical applications of angiogenesis
abstract: Blood vessels deliver oxygen and nutrients to every part of the body, but also nourish diseases such as cancer. Over the past decade, our understanding of the molecular mechanisms of angiogenesis (blood vessel growth) has increased at an explosive rate and has led to the approval of anti-angiogenic drugs for cancer and eye diseases. So far, hundreds of thousands of patients have benefited from blockers of the angiogenic protein vascular endothelial growth factor, but limited efficacy and resistance remain outstanding problems. Recent preclinical and clinical studies have shown new molecular targets and principles, which may provide avenues for improving the therapeutic benefit from anti-angiogenic strategies.

title: Cerium Oxide Nanoparticle Modified Scaffold Interface Enhances Vascularization of Bone Grafts by Activating Calcium Channel of Mesenchymal Stem Cells.
abstract: Insufficient blood perfusion is one of the critical problems that hamper the clinical application of tissue engineering bone (TEB). Current methods for improving blood vessel distribution in TEB mainly rely on delivering exogenous angiogenic factors to promote the proliferation, migration, differentiation, and vessel formation of endothelial cells (ECs) and/or endothelial progenitor cells (EPCs). However, obstacles including limited activity preservation, difficulty in controlled release, and high cost obstructed the practical application of this strategy. In this study, TEB scaffold were modified with cerium oxide nanoparticles (CNPs) and the effects of CNPs existed at the scaffold surface on the growth and paracrine behavior of mesenchymal stem cells (MSCs) were investigated. The CNPs could improve the proliferation and inhibit the apoptosis of MSCs. Meanwhile, the interaction between the cell membrane and the nanoparticle surface could activate the calcium channel of MSCs leading to the rise of intracellular free Ca(2+) level, which subsequently augments the stability of HIF-1α. These chain reactions finally resulted in high expression of angiogenic factor VEGF. The improved paracrine of VEGF could thereby promote the proliferation, differentiation, and tube formation ability of EPCs. Most importantly, in vivo ectopic bone formation experiment demonstrated this method could significantly improve the blood vessel distribution inside of TEB.

title: Induction of HIF‐1α expression by intermittent hypoxia: Involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR
abstract: Sleep‐disordered breathing with recurrent apnea (periodic cessation of breathing) results in chronic intermittent hypoxia (IH), which leads to cardiovascular and respiratory pathology. Molecular mechanisms underlying IH‐evoked cardio‐respiratory co‐morbidities have not been delineated. Mice with heterozygous deficiency of hypoxia‐inducible factor 1α (HIF‐1α) do not develop cardio‐respiratory responses to chronic IH. HIF‐1α protein expression and HIF‐1 transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH‐evoked HIF‐1α accumulation. PC12 cells were exposed to aerobic conditions (20% O2) or 60 cycles of IH (30 sec at 1.5% O2 followed by 5 min at 20% O2). Our results show that IH‐induced HIF‐1α accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS‐dependent Ca2+ signaling pathways involving phospholipase Cγ (PLCγ) and protein kinase C activation are required for IH‐evoked HIF‐1α accumulation. IH leads to activation of mTOR and S6 kinase (S6K) and rapamycin partially inhibited IH‐induced HIF‐1α accumulation. IH also decreased hydroxylation of HIF‐1α protein and anti‐oxidants as well as inhibitors of Ca+2 signaling prevented this response. Thus, both increased mTOR‐dependent HIF‐1α synthesis and decreased hydroxylase‐dependent HIF‐1α degradation contribute to IH‐evoked HIF‐1α accumulation. Following IH, HIF‐1α, and phosphorylated mTOR levels remained elevated during 90 min of re‐oxygenation despite re‐activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF‐1α levels during re‐oxygenation indicating that mTOR‐dependent protein synthesis is required for the persistent elevation of HIF‐1α levels during re‐oxygenation.  2008.

title: The calcium channel blocker cilnidipine selectively suppresses hypoxia-inducible factor 1 activity in vascular cells.
abstract: Calcium ion is one of the most important second messengers of cellular signal transduction including hypoxia-elicited signals. In this study, we investigated the effects of the L-type calcium channel blockers such as nifedipine, efonidipine cilnidipine, diltiazem, and verapamil, on the activity of hypoxia-inducible factor-1 (HIF-1), a key transcription factor in control of hypoxia-induced gene expression. Using the lung carcinoma cell line A549 cells, human aortic smooth muscle cells, and human umbilical vein endothelial cells, we demonstrated that cilnidipine exclusively suppressed HIF-1 activity and the expressions of downstream genes in a cell-type specific manner. We also demonstrated that cilnidipine blocked the synthesis of the HIF-1alpha protein not by affecting activity of the intracellular hypoxia-sensing element prolyl hydroxylases but inhibiting activity of Akt and mitogen-activated protein kinase and that the inhibition is not dependent on the effect on calcium homeostasis.

title: NNC 55-0396, a T-type Ca2+ channel inhibitor, inhibits angiogenesis via suppression of hypoxia-inducible factor-1α signal transduction
abstract: Mitochondrial respiration is required for hypoxia-inducible factor (HIF)-1α stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Herein, small molecules that inhibit HIF-1α protein stability by targeting mitochondrial energy production were screened using the Library of Pharmacologically Active Compounds and cell growth assay in galactose or glucose medium. NNC 55-0396, a T-type Ca2+ channel inhibitor, was selected as a hit from among 1,280 small molecules. NNC 55-0396 suppressed mitochondrial reactive oxygen species-mediated HIF-1α expression as well as stabilization by inhibiting protein synthesis in a dose-dependent manner. NNC 55-0396 inhibited tumor-induced angiogenesis in vitro and in vivo by suppressing HIF-1α stability. Moreover, NNC 55-0396 significantly suppressed glioblastoma tumor growth in a xenograft model. Thus, NNC 55-0396, a small molecule targeting T-type Ca2+ channel, was identified by the systemic cell-based assay and was shown to have antiangiogenic activity via the suppression of HIF-1α signal transduction. These results provide new insights into the biological network between ion channel and HIF-1α signal transduction. messageHIF-1α overexpression has been demonstrated in hypoxic cancer cells.NNC 55-0396, a T-type Ca2+ channel inhibitor, inhibited HIF-1α expression via both proteasomal degradation and protein synthesis pathways.T-type Ca2+ channel inhibitors block angiogenesis by suppressing HIF-1α stability and synthesis.NNC 55-0396 could be a potential therapeutic drug candidate for cancer treatment.

title: Role for interleukin 1alpha in the inhibition of chondrogenesis in autologous implants using polyglycolic acid-polylactic acid scaffolds.
abstract: Significant challenges remain in generating tissue-engineered cartilage in immunocompetent animals. Scaffold materials such as polyglycolic acid lead to significant inflammatory reactions, inhibiting homogeneous matrix synthesis. This study examined the generation of tissue-engineered cartilage, using a polyglycolic acid-polylactic acid copolymer (Ethisorb; Ethicon, Norderstedt, Germany) in an autologous immunocompetent pig model. The goals of this study were to determine the role of interleukin 1alpha (IL-1alpha) in this system and to assess the effect of serum treatment on tissue generation. Porcine auricular chondrocytes were seeded onto Ethisorb disks cultured for 1 week in medium supplemented with either fetal bovine serum or serum-free insulin-transferrin-selenium supplement. Specimens were implanted autogenously in pigs with unseeded scaffolds as controls. After 1, 4, or 8 weeks, six specimens from each group were explanted and analyzed histologically (hematoxylin and eosin, safranin O, trichrome, and Verhoeff's staining) and biochemically (glycosaminoglycan content). The presence and distribution of IL-1alpha were assessed by immunohistochemistry. Histology revealed acute inflammation surrounding degrading scaffold. Cartilage formation was observed as early as 1 week after implantation and continued to increase with time; however, homogeneous matrix synthesis was not present in any of the specimens. Strong IL-1alpha expression was detected in chondrocytes at the implant periphery and in cells in the vicinity of degrading polymer. Histologically there was no significant difference between the experimental groups with respect to the amount of matrix synthesis or inflammatory infiltration. The glycosaminoglycan content was significantly higher in the serum-free group. These results suggest that inflammatory reactions against scaffold materials and serum components lead to the production of cytokines such as IL-1alpha that may inhibit cartilage tissue formation in autologous transplant models.

title: A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading
abstract: Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus EY = 277 kPa and unconfined dynamic modulus at 1 Hz G*=7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 × 106 cells/ml seeding density agarose constructs, with EY = 186 kPa, G*=1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.

title: Inhibition of cartilage proteoglycan synthesis by interleukin I.
abstract: We have investigated the mechanism of inhibition of cartilage proteoglycan by interleukin 1. Proteoglycan synthesis was inhibited using lower doses of interleukin 1 than those required to cause cartilage resorption. There was no significant effect on DNA or total protein synthesis. Gel electrophoresis showed a direct inhibitory effect on core protein synthesis while pulse-chase experiments using radiolabelled sulphate showed no alteration in the rate of intracellular transport and secretion of completed proteoglycan. Chondrocytes incubated with cycloheximide showed a first-order decrease in rate of uptake of radiolabelled sulphate (t1/2 = 25 mins) but interleukin 1 induced inhibition showed a delay of at least 1 hr, consistent with a requirement to deplete intracellular pools of protein before effects on post-translational events could be observed. Foetal and neonatal cartilage responded to the cytokine in a similar way to adult cartilage.

title: Differences in interleukin-1 response between engineered and native cartilage.
abstract: Unlike native cartilage explants that are used in autologous tissue transfer procedures, engineered cartilage constructs are typically highly fragile when first formed and must rely on cellular activity to develop over time. However, inflammatory cytokines such as interleukin-1alpha (IL-1alpha) are often present in target joints and may interfere with this development process. Herein we examine to what extent nascent engineered tissue is susceptible to chemical perturbations by IL-1alpha (10 ng/mL), especially when compared to native explants, and whether in vitro preconditioning may promote sufficient integrity to lessen this impact. The studies were carried out using a chemically defined medium supplemented with or without the antiinflammatory steroid dexamethasone. We find that engineered tissue (bovine chondrocytes in agarose hydrogel) at early time points (days 0 and 14) does not grow when exposed to the cytokine even temporarily, but both bovine explants and more developed engineered tissue (day 28) are able to withstand the same exposure without degradation of properties. We argue therefore that some in vitro preconditioning may be necessary to promote both sufficient mechanical integrity and the chemical fortitude without which insufficiently developed engineered constructs will not survive the harsh mechanochemical environment within the joint.

title: Injectable calcium phosphate scaffold with iron oxide nanoparticles to enhance osteogenesis via dental pulp stem cells
abstract: Literature search revealed no systematic report on iron oxide nanoparticle-incorporating calcium phosphate cement scaffolds (IONP-CPC). The objectives of this study were to: (1) use γFe2O3 nanoparticles (γIONPs) and αFe2O3 nanoparticles (αIONPs) to develop novel IONP-CPC scaffolds, and (2) investigate human dental pulp stem cells (hDPSCs) seeding on IONP-CPC for bone tissue engineering for the first time. IONP-CPC scaffolds were fabricated. Physiochemical properties of IONP-CPC scaffolds were characterized. hDPSC seeding on scaffolds, cell proliferation, osteogenic differentiation and bone matrix mineral synthesis by cells were measured. Our data demonstrated that the osteogenic differentiation of hDPSCs was markedly enhanced via IONP incorporation into CPC. Substantial increases (about three folds) in ALP activity and osteogenic gene expressions were achieved over those without IONPs. Bone matrix mineral synthesis by the cells was increased by two- to three folds over that without IONPs. The enhanced cellular osteogenesis was attributed to: (1) the surface nanotopography of IONP-CPC scaffold, and (2) the cell internalization of IONPs released from IONP-CPC scaffold. Our results demonstrate that the novel CPC functionalized with IONPs is promising to promote osteoinduction and bone regeneration. In conclusion, it is highly promising to incorporate γIONPs and αIONPs into CPC scaffold for bone tissue engineering, yielding substantially better stem cell attachment, spreading and osteogenic differentiation, and much greater bone mineral synthesis by the seeded cells. Therefore, novel CPC scaffolds containing γIONPs and αIONPs are promising for dental, craniofacial and orthopaedic applications to substantially enhance bone regeneration.

title: Processing and properties of strong and non-rigid calcium phosphate cement.
abstract: A calcium phosphate cement (CPC) sets to form hydroxyapatite and has been used in dental and craniofacial applications. However, when CPC was used in periodontal repair, tooth mobility resulted in the fracture and exfoliation of the brittle implants. The aim of this study was to develop CPC-chitosan lactate composites with higher strength and increased strain before failure. It was hypothesized that the incorporation of chitosan lactate would render CPC non-rigid with improved properties. Two-way ANOVA showed significant effects of chitosan lactate and powder:liquid ratio (p < 0.001) on flexural strength, strain-at-peak-load, work-of-fracture, and elastic modulus. At powder:liquid = 2, the strength (mean +/- SD; n = 6) at 20% chitosan lactate was 15.7 +/- 1.3 MPa, higher than 4.9 +/- 1.4 MPa of CPC without chitosan lactate. At powder:liquid = 1, the strain-at-peak-load was 0.2% for CPC without chitosan lactate; it increased to 15.8% for CPC containing 15% chitosan lactate. The work-of-fracture was increased by more than ten times. The novel strong and non-rigid CPC may provide compliance for tooth mobility without fracturing the implant, and may also extend the use of CPC into the repair of larger defects in stress-bearing locations.

title: Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.
abstract: Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules, and did not form adipocytes, whereas BMSCs routinely calcified throughout the adherent cell layer with clusters of lipid-laden adipocytes. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSCs formed lamellar bone containing osteocytes and surface-lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

title: Novel magnetic calcium phosphate-stem cell construct with magnetic field enhances osteogenic differentiation and bone tissue engineering.
abstract: Superparamagnetic iron oxide nanoparticles (IONPs) are promising bioactive additives to fabricate magnetic scaffolds for bone tissue engineering. To date, there has been no report on osteoinductivity of IONP-incorporated calcium phosphate cement (IONP-CPC) scaffold on stem cells using an exterior static magnetic field (SMF). The objectives of this study were to: (1) develop a novel magnetic IONP-CPC construct for bone tissue engineering, and (2) investigate the effects of IONP-incorporation and SMF application on the proliferation, osteogenic differentiation and bone mineral synthesis of human dental pulp stem cells (hDPSCs) seeded on IONP-CPC scaffold for the first time. The novel magnetic IONP-CPC under SMF enhanced the cellular performance of hDPSCs, yielding greater alkaline phosphatase activities (about 3-fold), increased expressions of osteogenic marker genes, and more cell-synthesized bone minerals (about 2.5-fold), compared to CPC control and nonmagnetic IONP-CPC. In addition, IONP-CPC induced more active osteogenesis than CPC control in rat mandible defects. These results were consistent with the enhanced cellular performance by magnetic IONP in media under SMF. Moreover, nano-aggregates were detected inside the cells by transmission electron microscopy (TEM). Therefore, the enhanced cell performance was attributed to the physical forces generated by the magnetic field together with cell internalization of the released magnetic nanoparticles from IONP-CPC constructs.

title: Biopolymers - Calcium phosphates composites with inclusions of magnetic nanoparticles for bone tissue engineering.
abstract: Composites based on combination of biopolymers (chitosan, hyaluronic acid and bovine serum albumin or gelatin), calcium phosphates (CP) and magnetic nanoparticles have been prepared by a biomimetic co-precipitation method. The biomimetic strategy is inspired by natural mineralization processes, where the synthesized minerals are usually combined with proteins, polysaccharides or other mineral forms to form composite, in physiological conditions of temperature and pH. The morphology of the magnetic composites, studied using scanning electron microscopy (SEM) indicated a macroporous structure, which influenced the retention of simulated biological fluids. Fourier transformed infrared spectroscopy and X-ray diffraction and Energy-dispersive X-ray spectroscopy (EDX) confirmed the composition of the scaffolds and the formation of various types of calcium phosphates with amorphous nature. The in vitro degradation studies showed a slow degradation process for magnetic composites that confirmed the tightly connection of the polymeric matrix with calcium phosphates, which limits the enzyme access to the degradable components and material disintegration. The magnetic scaffolds exhibited no negative effect on osteoblasts cell, emphasizing a good biocompatibility. Considering the scaffolds properties, some compositions based on calcium phosphates, chitosan, Hya/Bsa and more than 3% of MNPs are recommended for further optimization and in vivo tests.

title: Magnetic Cell-Scaffold Interface Constructed by Superparamagnetic IONP Enhanced Osteogenesis of Adipose-Derived Stem Cells.
abstract: One of the key factors in tissue engineering and regenerative medicine is to optimize the interaction between seed cells and scaffolds such that the cells can grow in naturally biomimetic conditions. Their similarity to macromolecules and many unique properties mean that functional nanoparticles have promising potential for the modification and improvement of traditional scaffolds to obtain excellent biocompatibility, tunable stiffness, physical sensing, and stimulus-response capabilities. In the present study, we report magnetic poly(lactic- co-glycolic acid)/polycaprolactone (PLGA/PCL) scaffolds that were fabricated using a combination of the electrospinning technique and layer-by-layer assembly of superparamagnetic iron oxide nanoparticles (IONPs). PLGA/PCL scaffolds assembled with gold nanoparticles were prepared using the same method for comparison. The results showed that the assembled film of nanoparticles on the surface greatly enhanced the hydrophilicity and increased the elastic modulus of the scaffold, which subsequently improved the osteogenesis of the stem cells. Furthermore, the magnetic property of the IONPs proved to be the key factor in enhancing osteogenic differentiation, which explained the superior osteogenic capacity of the magnetic scaffolds compared with that of the gold nanoparticle-assembled scaffold. These results demonstrated the importance of magnetic nanomaterials as a bioactive interface between cells and scaffolds and will promote the design of biomaterials to improve tissue engineering and regenerative medicine efficacy.

title: Paramagnetic nanofibrous composite films enhance the osteogenic responses of pre-osteoblast cells.
abstract: In this work, a paramagnetic nanofibrous composite film was fabricated with poly lactide, hydroxyapatite and γ-Fe(2)0(3) nanoparticles using the electrospinning technique. The composite film significantly enhanced the proliferation, differentiation and ECM secretion of the osteoblast cells under a static magnetic field, which offers promising application potentials in bone tissue engineering and bone regeneration therapy.

title: Super-paramagnetic responsive nanofibrous scaffolds under static magnetic field enhance osteogenesis for bone repair in vivo
abstract: A novel nanofibrous composite scaffold composed of super-paramagnetic γ-Fe2O3 nanoparticles (MNP), hydroxyapatite nanoparticles (nHA) and poly lactide acid (PLA) was prepared using electrospinning technique. The scaffold well responds extern static magnetic field with typical saturation magnetization value of 0.049 emu/g as well as possesses nanofibrous architecture. The scaffolds were implanted in white rabbit model of lumbar transverse defects. Permanent magnets are fixed in the rabbit cages to provide static magnetic field for the rabbits post surgery. Results show that MNP incorporated in the nanofibers endows the scaffolds super-paramagnetic responsive under the applied static magnetic field, which accelerates new bone tissue formation and remodeling in the rabbit defect. The scaffold also exhibits good compatibility of CK, Cr, ALT and ALP within normal limits in the serum within 110 days post implantation. In conclusion, the super-paramagnetic responding scaffold with applying of external magnetic field provides a novel strategy for scaffold-guided bone repair.

title: Macrophage phenotypic mechanomodulation of enhancing bone regeneration by superparamagnetic scaffold upon magnetization.
abstract: Macrophages are involved in all phases of scaffold induced tissue regeneration, orchestrating the transition from an inflammatory to regenerative phenotype to guide all other cell types to complete the wound healing process when a tissue defect advances beyond the critical size. Therefore, harnessing macrophages by scaffolds is important for facilitating tissue regeneration in situ. In this work we utilized the superparamagnetic scaffold upon magnetization as a mechanostimulation platform to apply forces directly to macrophages grown in the scaffold, aiming to figure out whether the functions of macrophages related to bone tissue regeneration can be mechanomodulated and to elucidate the underlying mechanisms. We showed the first evidence that upon magnetization the interaction of superparamagnetic scaffolds to macrophages drove them to polarize towards an M2-like phenotype by inhibiting TLR2/4 activation and enhancing VEGFR2 activation, thereby inhibiting secretion of the pro-inflammatory cytokines IL-1β, TNF-α and MCP-1, as well as the osteoclast differentiation cytokines MMP-9 and TRAP, and up-regulating VEGF and PDGF. The conditioned media enhanced the osteogenesis of osteoblasts and the angiogenesis of endothelial cells.

title: Enhanced bone regeneration and visual monitoring via superparamagnetic iron oxide nanoparticle scaffold in rats
abstract: A main challenge for use of scaffolds in bone engineering involves non‐invasive monitoring in vivo and enhanced bone regeneration. The tissue repair effect of superparamagnetic iron oxide nanoparticles (SPIONs) was demonstrated previously by our group. However, testing in vivo is needed to confirm in vitro results. Here, SPIONs loaded gelatin sponge (GS) was used as a scaffold (SPIONs‐GS) and implanted in the incisor sockets of Sprague–Dawley rats. Incisor sockets filled with nothing and filled with GS served as controls. Rats were sacrificed at 2 and 4 weeks. A significant decrease in the signal intensity of T2‐weighted magnetic resonance imaging (MRI) in the SPIONs‐GS group was noted. Changes in image intensity of scaffolds (indicating scaffold degradation and interaction with host tissues) could be visually monitored over time. Microcomputed tomography showed that the SPIONs‐GS group had more newly formed bone (64.44 ± 10.92 vs. 28.1 ± 4.49, p < .0001) and a better preserved alveolar ridge than blank control group at 4 weeks (0.962 ± 0.01 vs. 0.92 ± 0.01, p < .0001). Histology confirmed imaging results, showing good consistency in new bone formation and scaffold degradation. The number of SPIONs decreased rapidly with time due to quick degradation of GS, whereas the number of endocytic SPIONs in cells increased with time. These residual SPIONs, together with newly formed bone, could be detected by MRI at 4 weeks. Therefore, it was clear that SPIONs induced active osteogenesis. In conclusion, good visibility on MRI and enhanced regeneration of bone can be obtained by implanting SPIONs‐GS in vivo without using an external magnetic field.

title: Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis
abstract: Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine.

title: Ultrarapid Engineering of Biomimetic Materials and Tissues: Fabrication of Nano‐ and Microstructures by Plastic Compression
abstract: Currently, the concept of engineered tissues depends on the ability of cultured cells to fabricate new tissue around a scaffold. Ibis is inherently slow and expensive and has had limited success so far. We report here a new process for the cell-independent, controlled engineering of biomimetic scaffolds by rapid removal of fluid from hyperhydrated collagen gel (or other) constructs, using plastic compression (PC). PC fabrication produces dense, cellular, mechanically strong native collagen structures with controllable nano- and microscale biomimetic structures. The huge-scale shrinkage (> 100-fold) provides the ability to introduce controllable mechanical properties, microlayering, and embossed interface. topography without cell participation, but with high cell viability. Critically, this takes minutes rather than the conventional days and weeks. The rapidity and biomimetic potential of the PC fabrication process at the mesoscale opens a new route for the production of biomaterials and patient-customized tissues. It also represents a new concept in 'engineering' tissues.

title: Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition.
abstract: Bone is distinguished from other tissues by its mechanical properties, in particular stiffness. However, we know little of how osteoblasts react to the stiffness of their microenvironment; in this study we describe their response to a dense (>10 wt.%) collagenous 3D environment. Primary pre-osteoblasts were seeded within a novel form of native collagen, dense collagen, and cultured for up to 14 days in the presence and absence of osteogenic supplements: analysis was via Q-PCR, histology, fluorescent in situ zymography, MMP loss-of-function and tensile testing. Differentiation as measured through the up-regulation of Bsp (247-fold), Alp (14.2-fold), Col1A1 (4.5-fold), Mmp-13 (8.0-fold) and Runx2 (1.2-fold) transcripts was greatly accelerated compared to 2D plastic at 7 and 14 days in the same medium. The scale of this enhancement was confirmed through the use of growth factor stimulation on 2D via the addition of BMP-6 and the Hedgehog agonist purmorphamine. In concert, these molecules were capable of the same level of osteo-induction (measured by Bsp and Alp expression) as the dense collagen alone. Mineralisation was initially localised to remodelled pericellular regions, but by 14 days embedded cells were discernible within regions of apatite (confirmed by MicroRaman). Tensile testing of the matrices showed that this had resulted in a significant increase in Young's modulus at low strain values, consistent with a stiffening of the matrix. To determine the need for matrix remodelling in the mineralisation event the broad spectrum MMP Inhibitor Ilomastat was used. It was found that in its presence mineralisation could still occur (though serum-specific) and the apoptosis associated with MMP inhibition in hydrated collagen gels was abrogated. Analysis of gene expression indicated that this was due to the up-regulation of Mmp-13 in the presence of Ilomastat in dense collagen (400-fold), demonstrating a powerful feedback loop and a potential mechanism for the rescue from apoptosis. Osteoid-like matrix (dense collagen) is therefore a potent stimulant of osteoblast differentiation in vitro and provides an environment that enables survival and differentiation in the presence of MMP inhibition.

title: Enhanced in vitro biocompatibility and osteogenesis of titanium substrates immobilized with dopamine-assisted superparamagnetic Fe3O4 nanoparticles for hBMSCs
abstract: Titanium (Ti) is an ideal bone substitute due to its superior bio-compatibility and remarkable corrosion resistance. However, in order to improve the osteoconduction and osteoinduction capacities in clinical applications, different kinds of surface modifications are typically applied to Ti alloys. In this study, we fabricated a tightly attached polydopamine-assisted Fe3O4 nanoparticle coating on Ti with magnetic properties, aiming to improve the osteogenesis of the Ti substrates. The PDA-assisted Fe3O4 nanoparticle coatings were characterized by scanning electron microscopy, energy dispersive spectroscopy, atomic force microscopy and water contact angle measurements. The cell attachment and proliferation rate of the human bone mesenchymal stem cells (hBMSCs) on the Ti surface significantly improved with the Fe3O4/PDA coating when compared with the pure Ti without a coating. Furthermore, the results of in vitro alkaline phosphatase (ALP) activity at 7 and 14 days and alizarin red S staining at 14 days showed that the Fe3O4/PDA coating on Ti promoted the osteogenic differentiation of hBMSCs. Moreover, hBMSCs co-cultured with the Fe3O4/PDA-coated Ti for approximately 14 days also exhibited a significantly higher mRNA expression level of ALP, osteocalcin and runt-related transcription factor-2 (RUNX2). Our in vitro results revealed that the present PDA-assisted Fe3O4 nanoparticle surface coating is an innovative method for Ti surface modification and shows great potential for clinical applications.

title: Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.
abstract: Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration.



The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs, and the IOP-MC coatings with outer distributed IOPs (O-IOPs-MC) shows the maximized mechanical stimuli to cells with enhanced osteogenic differentiation under static magnetic field. The upregulated expression of the associated genes reveals that the enabled mechanotransduction signaling pathway is responsible for the promoted cellular osteogenic differentiation. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface to promote osseointegration.

title: Response of MAPK pathway to iron oxide nanoparticles in vitro treatment promotes osteogenic differentiation of hBMSCs.
abstract: Iron oxide nanoparticles (IONPs) are generally used in multiple biomedical applications. The tissue repair effect of IONPs had been demonstrated in the previous studies of our group, but the underlying mechanism is unclarified. It is well known that stem cell-based therapies show promising prospect in tissue engineering and regenerative medicine, however, whether IONPs could modulate stem cell fate to promote tissue repair is still unclear. Herein, we found that IONPs could promote osteogenic differentiation of human bone-derived mesenchymal stem cells (hBMSCs) in vitro. To insightfully understand the molecular mechanisms, we performed systematic analyses by use of gene microarray assay and bioinformatics analysis, which revealed that gene expression was widely regulated and classical mitogen-activated protein kinase (MAPK) signal pathway was activated by IONPs treatment. As a result, downstream genes of this pathway were regulated to promote osteogenic differentiation. In summary, the present study elucidates a molecular basis explaining how IONPs effect on hBMSCs, which could have many meaningful impacts for stem cells application in regenerative medicine.

title: Magnetic assembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies
abstract: Here we reported an interesting phenomenon that the field-induced assemblies of magnetic nanoparticles can promote the differentiation of primary mouse bone marrow cells into osteoblasts. The reason was thought to lie in the remnant magnetic interaction inside the assemblies which resulted from the magnetic field-directed assembly. Influence of the assemblies on the cells was realized by means of interface effect rather than the internalization effect. We fabricated a stripe-like assemblies array on the glass plate and cultured cells on this surface. We characterized the morphology of assemblies and measured the mechanic property as well as the magnetic property. The cellular differentiation was measured by staining and quantitative PCR. Finally, Fe uptake was excluded as the reason to cause the phenomenon.

title: PERSPECTIVE ARTICLE: Growth factors and cytokines in wound healing
abstract: Wound healing is an evolutionarily conserved, complex, multicellular process that, in skin, aims at barrier restoration. This process involves the coordinated efforts of several cell types including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. The migration, infiltration, proliferation, and differentiation of these cells will culminate in an inflammatory response, the formation of new tissue and ultimately wound closure. This complex process is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines and chemokines. Of particular importance is the epidermal growth factor (EGF) family, transforming growth factor beta (TGF‐β) family, fibroblast growth factor (FGF) family, vascular endothelial growth factor (VEGF), granulocyte macrophage colony stimulating factor (GM‐CSF), platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF), interleukin (IL) family, and tumor nerosis factor‐α family. Currently, patients are treated by three growth factors: PDGF‐BB, bFGF, and GM‐CSF. Only PDGF‐BB has successfully completed randomized clinical trials in the Unites States. With gene therapy now in clinical trial and the discovery of biodegradable polymers, fibrin mesh, and human collagen serving as potential delivery systems other growth factors may soon be available to patients. This review will focus on the specific roles of these growth factors and cytokines during the wound healing process.

title: Oxidative stress in normal and impaired wound repair.
abstract: A large percentage of the population suffers from wound healing abnormalities, in particular aged individuals, patients with diabetes, and those treated with immunosuppressive drugs, chemo- or radiotherapy. The mechanisms underlying the impaired healing response are still poorly understood. Recent studies provided strong evidence for a role of oxidative stress in the pathogenesis of non-healing ulcers. Therefore, it is of major importance to identify and functionally characterize the factors involved in the generation and detoxification of reactive oxygen species (ROS). This will provide the basis for the development of new strategies for therapeutic intervention. In this review we summarize the current information about the roles of low molecular weight antioxidants and ROS-detoxifying enzymes in normal and impaired wound repair, and we report on the consequences of their modulation at the wound site.

title: Stem Cell Aligned Growth Induced by CeO2 Nanoparticles in PLGA Scaffolds with Improved Bioactivity for Regenerative Medicine
abstract: Hybrid 2D polymeric-ceramic biosupports are fabricated by mixing a nanostructured CeO2 powder with 85:15 poly(D,L-lactic-co-glycolic acid) (PLGA)/dichloromethane solutions at specific concentrations, followed by solvent casting onto pre-patterned molds. The mold patterning allows the orientation of ceramic nanoparticles into parallel lines within the composite scaffold. The ability of the produced films to host and address cell growth is evaluated after 1,3, and 6 days of culturing with murine derived cardiac and mesenchymal stem cells (CSCs and MSCs), and compared with PLGA films without ceramics and loaded with nanostructured TiO2. Aligned cell growth is observed only for scaffolds that incorporate oriented ceramic nanoparticles, attributed to the nanoceramic ability to modulate the roughness pitch, thus improving cell sensitivity towards the host surface features. Better CSC and MSC proliferative activity is observed for CeO2 composites with respect to either TiO2-added or unfilled PLCA films. This evidence may be related to the nanostructured CeO2 antioxidative properties.

title: Effects of cerium oxide nanoparticles on the growth of keratinocytes, fibroblasts and vascular endothelial cells in cutaneous wound healing.
abstract: Rapid and effective wound healing requires a coordinated cellular response involving fibroblasts, keratinocytes and vascular endothelial cells (VECs). Impaired wound healing can result in multiple adverse health outcomes and, although antibiotics can forestall infection, treatments that accelerate wound healing are lacking. We now report that topical application of water soluble cerium oxide nanoparticles (Nanoceria) accelerates the healing of full-thickness dermal wounds in mice by a mechanism that involves enhancement of the proliferation and migration of fibroblasts, keratinocytes and VECs. The Nanoceria penetrated into the wound tissue and reduced oxidative damage to cellular membranes and proteins, suggesting a therapeutic potential for topical treatment of wounds with antioxidant nanoparticles.

title: Antioxidant status in delayed healing type of wounds
abstract: This investigation studied the contribution of antioxidants in delaying healing in excision cutaneous wounds (8 mm) in diabetic, aged and immunocompromised animals. Skin levels of catalase, glutathione (GSH), ascorbic acid (AA) and vitamin E in streptozotocin‐induced diabetic rat were lower as compared to nondiabetics. The 7‐d wound tissue of diabetic rats showed an increased vitamin E level along with depleted GSH content. In aged rats (18 months old), higher levels of skin superoxide dismutase (SOD), glutathione peroxidase (Gpx) and thiobarbituric acid reactive substances (TBARS) and lower levels of catalase and GSH were found as compared to their values in young rats (3–4 months old). The levels of SOD, GPx, catalase, AA, GSH and vitamin E in 7‐d wound tissue of aged rats were significantly lower in comparison to those in young rats. However, TBARS were elevated in these wound tissues. The non‐wounded skin of immunocompromised (athymic) mice showed lower levels of SOD, catalase, and TBARS and higher GSH and GPx levels in comparison to those present in normal mouse skin. Surprisingly, the analysis of 7‐d wound tissue showed higher levels of SOD, catalase, GPx, and GSH and lower TBARS level in athymic mice compared to the wound tissue of normal mice.

title: Cerium oxide nanoparticles scavenge nitric oxide radical (˙NO).
abstract: In this study we have obtained evidence that cerium oxide nanoparticles (CeO(2) NPs) are able to scavenge nitric oxide radical. Surprisingly, this activity is present in CeO(2) NPs with a lower level of cerium in the 3+ state (CeO(2) NPs with low 3+/4+ ratio and therefore a reduced number of oxygen vacancies), in contrast to the superoxide scavenging properties which are correlated with an increased level of cerium in the 3+ state (CeO(2) NPs with high 3+/4+ ratio and therefore an increased number of oxygen vacancies).

title: Chitosan-coated cerium oxide nanocubes accelerate cutaneous wound healing by curtailing persistent inflammation
abstract: Inflammation is the initial phase in the healing of cutaneous wounds; however, persistent inflammation will hamper the healing process by generating excess inflammatory cytokines and reactive oxygen species (ROS). Therefore, preventing persistent inflammation and clearing redundant ROS are important strategies in accelerating wound healing. Owing to their unique redox activity, cerium oxide (CeO2) nanoparticles have shown promising potential as antioxidative and anti-inflammatory agents for the treatment of various diseases resulting from oxidative stress. In the present study, we prepared chitosan-coated CeO2 nanocubes (CCNs) and evaluated their cutaneous wound healing potential when topically applied to open excision wounds on adult Sprague Dawley (SD) rats. CCN application significantly increased the wound healing rates and showed superior wound healing capabilities compared to a clinically applied wound healing agent, recombinant human epidermal growth factor (rhEGF). We attribute this superior wound healing ability to their anti-inflammatory ability by decreasing the expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and increasing the expression of the anti-inflammatory cytokine interleukin-10 (IL-10), as well as to their antioxidative ability by increasing antioxidant enzyme levels. These results suggest that CCNs hold therapeutic potential in treating refractory wounds characterized by persistent inflammation caused by oxidative-stress related diseases such as diabetes.

title: Polyurethane foam containing rhEGF as a dressing material for healing diabetic wounds: Synthesis, characterization, in vitro and in vivo studies.
abstract: Diabetic wounds are a major health issue associated with diabetes mellitus. To surmount this issue, we developed polyurethane foams (PUFs) incorporating varying amounts of recombinant human epidermal growth factor (rhEGF) (rhEGF-PUFs) as a wound dressing for diabetic wounds. From electron microscopy images, it was found that the pore size of the rhEGF-PUFs surface (the wound contact layer) was less than 100μm, regardless of rhEGF content. The release of rhEGF from the PUFs was evaluated using an enzyme-linked immunosorbent assay. The result showed that the release of rhEGF was time and concentration dependent, i.e., the amount of released rhEGF significantly increased as the immersion time and the rhEGF content of the PUFs increased. In vitro cytotoxicity testing showed that rhEGF-PUFs increased the viability of HaCaT human keratinocytes and CCD986-sk human fibroblasts, which indicated that the incorporated rhEGF maintained its biological activity. In an in vitro scratch wound healing assay, the wound closure rate was faster in CCD986-sk human fibroblasts than in HaCaT human keratinocytes. Finally, the rhEGF-PUFs were evaluated as an in vivo treatment in a full-thickness wound model in diabetized Sprague-Dawley rats. The result indicated that compared with PUFs, rhEGF-PUFs accelerated wound healing by promoting wound contraction, re-epithelialization, collagen deposition and the formation of a skin appendage. These findings demonstrate that rhEGF-PUFs are a promising dressing for diabetic wounds.

title: Use of Cerium Oxide Nanoparticles Conjugated with MicroRNA-146a to Correct the Diabetic Wound Healing Impairment.
abstract: Diabetic wounds have become one of the most challenging public health issues of the 21st century, yet there is no effective treatment available. We have previously shown that the diabetic wound healing impairment is associated with increased inflammation and decreased expression of the regulatory microRNA miR-146a. We have conjugated miR-146a to cerium oxide nanoparticles (CNP-miR146a) to target reactive oxygen species (ROS) and inflammation. This study aimed to evaluate the consequences of CNP-miR146a treatment of diabetic wounds.



Eight-millimeter wounds were created on the dorsal skin of Db/Db mice and treated with PBS or differing concentrations of CNP-mir146a (1; 10; 100; or 1,000 ng) at the time of wounding. Rate of wound closure was measured until the wounds were fully healed. At 4 weeks post-healing, a dumbbell-shaped skin sample was collected, with the healed wound in the center, and an Instron 5942 testing unit was used to measure the maximum load and modulus.



Our data showed that diabetic wounds treated with PBS or 1 ng CNP-miR146a took 18 days to heal. Treatment with 10, 100, or 1,000 ng of CNP+miR-146a effectively enhanced healing, and wounds were fully closed at day 14 post-wounding. The healed skin from the CNP-miR146a-treated group showed a trend of improved biomechanical properties (increased maximum load and modulus), however it did not reach significance.



We found that a 100-ng dose of CNP-miR146a improved diabetic wound healing and did not impair the biomechanical properties of the skin post-healing. This nanotechnology-based therapy is promising, and future studies are warranted to transfer this therapy to clinical application.

title: Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy.
abstract: Inflammation has a critical role in the pathogenesis of diabetic complications, including diabetic nephropathy (DN). MicroRNAs have recently emerged as important regulators of DN. However, the role of microRNAs in the regulation of inflammation during DN is poorly understood. Here, we examined the in vivo role of microRNA-146a (miR-146a), a known anti-inflammatory microRNA, in the pathogenesis of DN. In a model of streptozotocin-induced diabetes, miR-146a(-/-) mice showed significantly exacerbated proteinuria, renal macrophage infiltration, glomerular hypertrophy, and fibrosis relative to the respective levels in control wild-type mice. Diabetes-induced upregulation of proinflammatory and profibrotic genes was significantly greater in the kidneys of miR-146a(-/-) than in the kidneys of wild-type mice. Notably, miR-146a expression increased in both peritoneal and intrarenal macrophages in diabetic wild-type mice. Mechanistically, miR-146a deficiency during diabetes led to increased expression of M1 activation markers and suppression of M2 markers in macrophages. Concomitant with increased expression of proinflammatory cytokines, such as IL-1β and IL-18, markers of inflammasome activation also increased in the macrophages of diabetic miR-146a(-/-) mice. These studies suggest that in early DN, miR-146a upregulation exerts a protective effect by downregulating target inflammation-related genes, resulting in suppression of proinflammatory and inflammasome gene activation. Loss of this protective mechanism in miR-146a(-/-) mice leads to accelerated DN. Taken together, these results identify miR-146a as a novel anti-inflammatory noncoding RNA modulator of DN.

title: Neuropathic diabetic foot ulcers treated with cerium dioxide nanoparticles: A case report.
abstract: Wound healing in diabetes is frequently impaired and its treatment remains a challenge. The ability of topical application of cerium (Ce) dioxide nanoparticles (CNPs) to accelerate wound healing in an animal model provides a rationale to develop this technology for use in humans affected by traumatic injury, diabetes and burns. We first described a case report of successful topical treatment of neuropathic diabetic foot ulcers with novel gel containing CNPs. The CNPs has bacteriostatic activity, anti-inflammatory properties and can penetrated into the wound tissue and reduced oxidative damage therefore protect regenerative tissue, suggesting a therapeutic potential for topical treatment of diabetic foot ulcers.

title: Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution.
abstract: The current design requirement for a tissue engineering skin substitute is that of a biodegradable scaffold through which fibroblasts can migrate and populate. This artificial "dermal layer" needs to adhere to and integrate with the wound, which is not always successful for the current artificial dermal analogues available. The high cost of these artificial dermal analogues also makes their application prohibitive both to surgeons and patients. We propose a cost-effective composite consisting of a nanofibrous scaffold directly electrospun onto a polyurethane dressing (Tegaderm, 3M Medical) - which we call the Tegaderm-nanofiber (TG-NF) construct - for dermal wound healing. Cell culture is performed on both sides of the nanofibrous scaffold and tested for fibroblast adhesion and proliferation. It is hoped that these studies will result in a fibroblast-populated three-dimensional dermal analogue that is feasible for layered applications to build up thickness of dermis prior to re-epithelialization. Results obtained in this study suggest that both the TG-NF construct and dual-sided fibroblast-populated nanofiber construct achieved significant cell adhesion, growth and proliferation. This is a successful first step for the nanofiber construct in establishing itself as a suitable three-dimensional scaffold for autogenous fibroblast populations, and providing great potential in the treatment of dermal wounds through layered application.

title: Cerium oxide nanoparticle-containing poly (ε-caprolactone)/gelatin electrospun film as a potential wound dressing material: In vitro and in vivo evaluation.
abstract: In the present study, cerium oxide (CeO2) nanoparticles were incorporated into poly (ε-caprolactone)/gelatin films in order to develop a potential wound dressing material. The wound dressings were prepared by electrospinning of poly (ε-caprolactone)/gelatin (1:1 (w/w)) solutions containing 1.50%, 3% and 6% (w/w) CeO2 nanoparticles. The electrospun films were evaluated regarding their morphology, contact angle, water-uptake capacity, water vapor transmission rate, tensile strength and cellular response. The film containing 1.50% CeO2 nanoparticles was chosen as the optimal dressing for the in vivo study on full-thickness excisional wounds of rats. The study showed that after 2weeks, the wounds treated with the CeO2 nanoparticle-containing dressing achieved a significant closure to nearly 100% compared with the sterile gauze with the nearly 63% of wound closure. Our results provided evidence supporting the possible applicability of CeO2 nanoparticle-containing wound dressing for a successful wound treatment.

title: Wound repair and regeneration: Mechanisms, signaling, and translation
abstract: The combination of an advanced scientific understanding of tissue repair and regeneration and the identification of knowledge gaps and the limitations of past clinical trials will provide a strong basis to advance viable clinical approaches for treating patients with wound-healing pathologies. The cellular and molecular mechanisms underpinning tissue repair and its failure to heal are still poorly understood, and current therapies are limited. Poor wound healing after trauma, surgery, acute illness, or chronic disease conditions affects millions of people worldwide each year and is the consequence of poorly regulated elements of the healthy tissue repair response, including inflammation, angiogenesis, matrix deposition, and cell recruitment. Failure of one or several of these cellular processes is generally linked to an underlying clinical condition, such as vascular disease, diabetes, or aging, which are all frequently associated with healing pathologies. The search for clinical strategies that might improve the body’s natural repair mechanisms will need to be based on a thorough understanding of the basic biology of repair and regeneration. In this review, we highlight emerging concepts in tissue regeneration and repair, and provide some perspectives on how to translate current knowledge into viable clinical approaches for treating patients with wound-healing pathologies.

title: Wound repair and regeneration
abstract: The repair of wounds is one of the most complex biological processes that occur during human life. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In human adults, the wound repair process commonly leads to a non-functioning mass of fibrotic tissue known as a scar. By contrast, early in gestation, injured fetal tissues can be completely recreated, without fibrosis, in a process resembling regeneration. Some organisms, however, retain the ability to regenerate tissue throughout adult life. Knowledge gained from studying such organisms might help to unlock latent regenerative pathways in humans, which would change medical practice as much as the introduction of antibiotics did in the twentieth century.

title: Preparing the ground for tissue regeneration: from mechanism to therapy
abstract: Chronic diseases confer tissue and organ damage that reduce quality of life and are largely refractory to therapy. Although stem cells hold promise for treating degenerative diseases by 'seeding' injured tissues, the regenerative capacity of stem cells is influenced by regulatory networks orchestrated by local immune responses to tissue damage, with macrophages being a central component of the injury response and coordinator of tissue repair. Recent research has turned to how cellular and signaling components of the local stromal microenvironment (the 'soil' to the stem cells' seed), such as local inflammatory reactions, contribute to successful tissue regeneration. This Review discusses the basic principles of tissue regeneration and the central role locally acting components may play in the process. Application of seed-and-soil concepts to regenerative medicine strengthens prospects for developing cell-based therapies or for promotion of endogenous repair.

title: Ceria nanocrystals decorated mesoporous silica nanoparticle based ROS-scavenging tissue adhesive for highly efficient regenerative wound healing.
abstract: Restoration of tissue integrity and tissue function of wounded skin are both essential for wound repair and regeneration, while synergistic promotion of the two remains elusive. Since elevated reactive oxygen species (ROS) production in the injured site has been implicated in triggering a set of deleterious effects such as cellular senescence, fibrotic scarring, and inflammation, it is speculated that alleviating oxidative stress in the microenvironment of injured site would be beneficial to promote regenerative wound healing. In this study, a highly versatile ROS-scavenging tissue adhesive nanocomposite is synthesized by immobilizing ultrasmall ceria nanocrystals onto the surface of uniform mesoporous silica nanoparticles (MSN). The ceria nanocrystals decorated MSN (MSN-Ceria) not only has strong tissue adhesion strength, but also significantly restricts ROS exacerbation mediated deleterious effects, which efficiently accelerates the wound healing process, and more importantly, the wound area exhibits an unexpected regenerative healing characteristic featured by marked skin appendage morphogenesis and limited scar formation. This strategy can also be adapted to other wound repair where both ROS-scavenging activity and tissue adhesive ability matter.

title: Wound healing essentials: Let there be oxygen
abstract: The state of wound oxygenation is a key determinant of healing outcomes. From a diagnostic standpoint, measurements of wound oxygenation are commonly used to guide treatment planning such as amputation decision. In preventive applications, optimizing wound perfusion and providing supplemental O2 in the perioperative period reduces the incidence of postoperative infections. Correction of wound pO2 may, by itself, trigger some healing responses. Importantly, approaches to correct wound pO2 favorably influence outcomes of other therapies such as responsiveness to growth factors and acceptance of grafts. Chronic ischemic wounds are essentially hypoxic. Primarily based on the tumor literature, hypoxia is generally viewed as being angiogenic. This is true with the condition that hypoxia be acute and mild to modest in magnitude. Extreme near‐anoxic hypoxia, as commonly noted in problem wounds, is not compatible with tissue repair. Adequate wound tissue oxygenation is required but may not be sufficient to favorably influence healing outcomes. Success in wound care may be improved by a personalized health care approach. The key lies in our ability to specifically identify the key limitations of a given wound and in developing a multifaceted strategy to specifically address those limitations. In considering approaches to oxygenate the wound tissue it is important to recognize that both too little as well as too much may impede the healing process. Oxygen dosing based on the specific need of a wound therefore seems prudent. Therapeutic approaches targeting the oxygen sensing and redox signaling pathways are promising.

title: Biological magnetic cellular spheroids as building blocks for tissue engineering.
abstract: Magnetic nanoparticles (MNPs), primarily iron oxide nanoparticles, have been incorporated into cellular spheroids to allow for magnetic manipulation into desired shapes, patterns and 3-D tissue constructs using magnetic forces. However, the direct and long-term interaction of iron oxide nanoparticles with cells and biological systems can induce adverse effects on cell viability, phenotype and function, and remain a critical concern. Here we report the preparation of biological magnetic cellular spheroids containing magnetoferritin, a biological MNP, capable of serving as a biological alternative to iron oxide magnetic cellular spheroids as tissue engineered building blocks. Magnetoferritin NPs were incorporated into 3-D cellular spheroids with no adverse effects on cell viability up to 1 week. Additionally, cellular spheroids containing magnetoferritin NPs were magnetically patterned and fused into a tissue ring to demonstrate its potential for tissue engineering applications. These results present a biological approach that can serve as an alternative to the commonly used iron oxide magnetic cellular spheroids, which often require complex surface modifications of iron oxide NPs to reduce the adverse effects on cells.

title: Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing.
abstract: A novel asymmetric chitosan membrane has been prepared by immersion-precipitation phase-inversion method and evaluated as wound covering. This new type of chitosan wound dressing which consists of skin surface on top-layer supported by a macroporous sponge-like sublayer was designed. The thickness of the dense skin surface and porosity of sponge-like sublayer could be controlled by the modification of phase-separation process using per-evaporation method. The asymmetric chitosan membrane showed controlled evaporative water loss, excellent oxygen permeability and promoted fluid drainage ability but could inhibit exogenous microorganisms invasion due to the dense skin layer and inherent antimicrobial property of chitosan. Wound covered with the asymmetric chitosan membrane was hemostatic and healed quickly. Histological examination confirmed that epithelialization rate was increased and the deposition of collagen in the dermis was well organized by covering the wound with this asymmetric chitosan membrane. The results in this study indicate that the asymmetric chitosan membrane thus prepared could be adequately employed in the future as a wound dressing.

title: Integration of a Superparamagnetic Scaffold and Magnetic Field To Enhance the Wound-Healing Phenotype of Fibroblasts.
abstract: Most of the existing scaffolds for guiding tissue regeneration do not provide direct mechanical stimulation to the cells grown on them. In this work, we used nanofibrous superparamagnetic scaffolds with applied magnetic fields to build a "dynamic" scaffold platform and investigated the modulating effects of this platform on the phenotypes of fibroblasts. The results of enzyme-linked immunosorbent and transwell assays indicated that fibroblasts cultivated in this platform secreted significantly higher type I collagen, vascular endothelial growth factor A, and transforming growth factor-β1 and did so in a time-dependent manner. At the same time, they produced fewer pro-inflammatory cytokines, including interleukin-1β and monocyte chemoattractant protein-1; this, in turn, accelerated the osteogenesis of preosteoblasts with the help of increased basic fibroblast growth factor as well as balanced extracellular matrix components. Mechanistic studies revealed that the platform modulated the phenotypic polarization of fibroblasts through the activation of components of integrin, focal adhesion kinase, and extracellular signal-regulated kinase signaling pathways and the inhibition of the activation of Toll-like receptor-4 and nuclear factor κB. Overall, the platform promoted the wound-healing phenotype of fibroblasts, which would be of great benefit to the scaffold-guided tissue regeneration.

title: Chasing the Paradigm: Clinical Translation of 25 Years of Tissue Engineering.
abstract: IMPACT STATEMENT
In this Perspective, we discuss the impact of the past 25 years of tissue engineering on the development of clinical therapies. Based on their success and other significant research accomplishments, platforms of innovation were identified. Their discoveries will enable tissue engineering inspired therapies to meet the requirements necessary for large-scale manufacturing and Food and Drug Administration (FDA) approval for a diverse range of indications.

title: The Third Era of Tissue Engineering: Reversing the Innovation Drivers.
abstract: IMPACT STATEMENT
From this perspective, we discuss the different stages of development the tissue engineering (TE) field has gone through in its relatively young history. We discuss how TE is evolving from a technology-driven, science-focused field toward a patient-driven, manufacturing-focused one where patients' needs are translated into production process requirements, and subsequently into technological and biological innovations needed to meet the regulatory and clinical demands.

title: In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles
abstract: Increasing biomedical applications of iron oxide nanoparticles (IONPs) in academic and commercial settings have alarmed the scientific community about the safety and assessment of toxicity profiles of IONPs. The great amount of diversity found in the cytotoxic measurements of IONPs points toward the necessity of careful characterization and quantification of IONPs. The present document discusses the major developments related to in vitro and in vivo toxicity assessment of IONPs and its relationship with the physicochemical parameters of IONPs. Major discussion is included on the current spectrophotometric and imaging based techniques used for quantifying, and studying the clearance and biodistribution of IONPs. Several invasive and non-invasive quantification techniques along with the pitfalls are discussed in detail. Finally, critical guidelines are provided to optimize the design of IONPs to minimize the toxicity.

title: The Yin: An adverse health perspective of nanoceria: uptake, distribution, accumulation, and mechanisms of its toxicity.
abstract: This critical review evolved from a SNO Special Workshop on Nanoceria panel presentation addressing the toxicological risks of nanoceria: accumulation, target organs, and issues of clearance; how exposure dose/concentration, exposure route, and experimental preparation/model influence the different reported effects of nanoceria; and how can safer by design concepts be applied to nanoceria? It focuses on the most relevant routes of human nanoceria exposure and uptake, disposition, persistence, and resultant adverse effects. The pulmonary, oral, dermal, and topical ocular exposure routes are addressed as well as the intravenous route, as the latter provides a reference for the pharmacokinetic fate of nanoceria once introduced into blood. Nanoceria reaching the blood is primarily distributed to mononuclear phagocytic system organs. Available data suggest nanoceria's distribution is not greatly affected by dose, shape, or dosing schedule. Significant attention has been paid to the inhalation exposure route. Nanoceria distribution from the lung to the rest of the body is less than 1% of the deposited dose, and from the gastrointestinal tract even less. Intracellular nanoceria and organ burdens persist for at least months, suggesting very slow clearance rates. The acute toxicity of nanoceria is very low. However, large/accumulated doses produce granuloma in the lung and liver, and fibrosis in the lung. Toxicity, including genotoxicity, increases with exposure time; the effects disappear slowly, possibly due to nanoceria's biopersistence. Nanoceria may exert toxicity through oxidative stress. Adverse effects seen at sites distal to exposure may be due to nanoceria translocation or released biomolecules. An example is elevated oxidative stress indicators in the brain, in the absence of appreciable brain nanoceria. Nanoceria may change its nature in biological environments and cause changes in biological molecules. Increased toxicity has been related to greater surface Ce3+, which becomes more relevant as particle size decreases and the ratio of surface area to volume increases. Given its biopersistence and resulting increased toxicity with time, there is a risk that long-term exposure to low nanoceria levels may eventually lead to adverse health effects. This critical review provides recommendations for research to resolve some of the many unknowns of nanoceria's fate and adverse effects.

title: Metabolic pathway and distribution of superparamagnetic iron oxide nanoparticles: in vivo study
abstract: Experimental tissue fusion benefits from the selective heating of superparamagnetic iron oxide nanoparticles (SPIONs) under high frequency irradiation. However, the metabolic pathways of SPIONs for tissue fusion remain unknown. Hence, the goal of this in vivo study was to analyze the distribution of SPIONs in different organs by means of magnetic resonance imaging (MRI) and histological analysis after a SPION-containing patch implantation.  SPION-containing patches were implanted in rats. Three animal groups were studied histologically over six months. Degradation assessment of the SPION-albumin patch was performed in vivo using MRI for iron content localization and biodistribution.  No SPION degradation or accumulation into the reticuloendothelial system was detected by MRI, MRI relaxometry, or histology, outside the area of the implantation patch. Concentrations from 0.01 μg/mL to 25 μg/mL were found to be hyperintense in T1-like gradient echo sequences. The best differentiation of concentrations was found in T2 relaxometry, susceptibility-sensitive gradient echo sequences, and in high repetition time T2 images. Qualitative and semiquantitative visualization of small concentrations and accumulation of SPIONs by MRI are feasible. In histological liver samples, Kupffer cells were significantly correlated with postimplantation time, but no differences were observed between sham-treated and induction/no induction groups. Transmission electron microscopy showed local uptake of SPIONs in macrophages and cells of the reticuloendothelial system. Apoptosis staining using caspase showed no increased toxicity compared with sham-treated tissue. Implanted SPION patches were relatively inert with slow, progressive local degradation over the six-month period. No distant structural alterations in the studied tissue could be observed.  Systemic bioavailability may play a role in specific SPION implant toxicity and therefore the local degradation process is a further aspect to be assessed in future studies.

title: In vivo clearance and toxicity of monodisperse iron oxide nanocrystals.
abstract: Thermal decomposition of organometallic precursors has been found to generate highly crystalline iron oxide (IO) nanocrystals that display superior MR contrast and lower polydispersity than IO nanocrystals synthesized by aqueous precipitation. In the present study, the in vivo characteristics of IO nanocrystals prepared by the thermal decomposition route and then coated with a phospholipid containing a pendant poly(ethylene glycol) chain are examined. The size and surface chemistry of the IO nanocrystal influence the biodistibution, the rate of biodegradation and bioclearance, and the biodegradation products. We conclude that the in vivo fate of PEGylated monodisperse IO nanocrystals and the iron, phospholipid, and oleic acid biodegradation products may influence the cellular environments in the organs and blood that can determine their safety in the body.

title: Iron oxide nanoparticles for use as an MRI contrast agent: pharmacokinetics and metabolism.
abstract: The pharmacokinetics and metabolism of a new preparation of superparamagnetic iron oxide nanoparticles were evaluated by 59Fe radiotracer studies and histologic examination of mice liver and spleen tissues (light and transmission electron microscopy). In the first 30 min following IV injection of the product half of the dose injected remains in the blood, the other part being sequestered mainly by the mononuclear phagocyte system (MPS). In the first five days following IV administration of the nanoparticles, early metabolization of the iron oxide cores occurs, revealed by modification of their aspect in the lysosomes of Kupffer cells and macrophages of the splenic red pulp. The incorporation of 59Fe is then observed in RBC of the mice. These results are discussed in relation with the physicochemical properties of this new preparation of nanoparticles, and compared with current pharmacokinetic data concerning injectable particle systems.

title: Superparamagnetic iron oxide: pharmacokinetics and toxicity.
abstract: The pharmacokinetics (distribution, metabolism, bioavailability, excretion) and toxicity (acute and subacute toxicity, mutagenicity) of a superparamagnetic iron oxide preparation (AMI-25), currently used in clinical trials, were evaluated by 59Fe radiotracer studies, measurements of relaxation times, the ability to reverse iron deficiency anemia, histologic examination, and laboratory parameters. One hour after administration of AMI-25 to rats (18 mumol Fe/kg; 1 mg Fe/kg), 82.6 +/- 0.3% of the administered dose was sequestered in the liver and 6.2 +/- 7.6% in the spleen. Peak concentrations of 59Fe were found in liver after 2 hr and in the spleen after 4 hr. 59Fe slowly cleared from liver (half-life, 3 days) and spleen (half-life, 4 days) and was incorporated into hemoglobin of erythrocytes in a time-dependent fashion. The half-time of the T2 effect on liver and spleen (24-48 hr) was shorter than the 59Fe clearance, indicating metabolism of AMI-25 into other forms of iron. IV administration of AMI-25 (30 mg Fe/kg) corrected iron-deficiency anemia and showed bioavailability similar to that of commercially available IV iron preparations within 7 days. No acute or subacute toxic effects were detected by histologic or serologic studies in rats or beagle dogs who received a total of 3000 mumol Fe/kg, 150 times the dose proposed for MR imaging of the liver. Our results indicate that AMI-25 is a fully biocompatible potential contrast agent for MR.