Cardiac fibrosis are central to various cardiovascular diseases. Research on the mechanisms and therapeutic targets for cardiac fibrosis has advanced greatly in recent years. However, while many anti-fibrotic treatments have been studied in animal models and seem promising, translation of experimental findings into human patients has been rather limited. Thus, several potential new treatments which have shown to reduce cardiac fibrosis in animal models have either not been tested in humans or proved to be disappointing in clinical trials. A majority of clinical studies are of small size or have not been maintained for long enough periods. In addition, although some conventional therapies, such as renin-angiotensin-aldosterone system (RAAS) inhibitors, have been shown to reduce cardiac fibrosis in humans, cardiac fibrosis persists in patients with heart failure even when treated with these conventional therapies, indicating a need to develop novel and effective anti-fibrotic therapies in cardiovascular disease. In this review article, we summarize anti-fibrotic therapies for cardiovascular disease in humans, discuss the limitations of currently used therapies, along with possible reasons for the failure of so many anti-fibrotic drugs at the clinical level. We will then explore the future directions of anti-fibrotic therapies on cardiovascular disease, and this will include emerging anti-fibrotics that show promise, such as relaxin. A better understanding of the differences between animal models and human pathology, and improved insight into carefully designed trials on appropriate end-points and appropriate dosing need to be considered to identify more effective anti-fibrotics for treating cardiovascular fibrosis in human patients. Cardiac fibrosis is a hallmark of various cardiovascular disease such as hypertension, myocardial infarction (MI), and ischemic, dilated, and hypertrophic cardiomyopathies. Cardiac fibrosis not only leads to cardiac diastolic dysfunction, but is also a major determinant of malignant arrhythmias and end-stage systolic heart failure and consequently, increases the risk of cardiac death. There are two types of fibrosis: regional fibrosis  and diffuse fibrosis . Cardiac fibrosis can be definitively diagnosed by endomyocardial biopsies, but they are an invasive evaluation only representative in diffuse fibrosis. Circulating biomarkers, particularly collagen turnover markers, are widely used to noninvasively assess cardiac fibrosis, however they are not reliable and unable to differentiate regional fibrosis from diffuse fibrosis. Cardiac magnetic resonance imaging (CMR) is an emerging technique to accurately and noninvasively evaluate regional and diffuse cardiac fibrosis by late gadolinium enhancement and post-contrast myocardial longitudinal relaxation time (T1) mapping, respectively, but CMR is expensive and not easily accessible. Functional consequences of cardiac fibrosis, particularly impaired left ventricular (LV) relaxation and heart failure, are also potential, albeit nonspecific, markers of fibrosis.It is known that a complex interaction involving a network of growth factors/cytokines/hormones and fibroblasts and other cell types  is responsible for initiating and maintaining fibrotic response , but not with hydrochlorothiazide (n = 17)  and amlodipine (n = 16) given for 1 year in hypertensive patients revealed that only losartan significant decreased both CVF  and the carboxy-terminal peptide of procollagen type I (PICP) , but not in those with nonsevere fibrosis (n = 12)  more effectively suppressed cardiac fibrosis than did enalapril (n = 13) or amlodipine (n = 13)   and noncanonical (TAK/p-38/JNK and NOX4/ROS) signaling pathways. Anti-TGF-β antibodies and ALK5 inhibitors attenuated cardiac fibrosis in animal models, but they were associated with adverse cardiovascular effects , but not furosemide (n = 17), reduced circulating PICP and myocardial collagen in hypertensive patients with symptomatic heart failure , compared with placebo (n = 103), did not improve exercise capacity or clinical status (Redfield et al., Sildenafil inhibits cyclic GMP-specific phosphodiesterase type-5A and it has been used to treat idiopathic pulmonary fibrosis. In the first proof-of-concept human study, 59 patients with isolated diabetic cardiomyopathy randomly treated 3 months with sildenafil showed improved LV contraction parameters and reduced TGF-β and monocyte chemoattractant protein-1, when compared with controls (Giannetta et al., Cardiac fibrosis is associated with activation of MMPs. It has been shown that MMP inhibition attenuates cardiac fibrosis and LV remodeling in experimental models (Heymans et al., in vivo half-life and it is costly to produce. The relatively short duration of relaxin treatment have partly contributed to the failed clinical trials since fibrosis is a slow process in human. Second, it is important to know the expression of relaxin receptors in different tissues and organs and to understand tissue competence to respond to relaxin along with signaling pathways. Nevertheless, relaxin still holds great potential as a therapy for cardiac fibrosis associated with various cardiovascular disease.Relaxin is an intriguing endogenous hormone that is a potent vasodilator with a number of pleiotropic effects. Relaxin inhibits fibrosis through various mechanisms including inhibiting TGF-β and Smad, regulating the balance between MMPs and tissue inhibitors of metalloproteinases, and inhibiting inflammatory response (Samuel et al., The failure of many clinical trials on anti-fibrotic drugs indicates that extrapolating research data from animal models to human requires caution since there are significant species differences in physiology and genetics between animals and human. Compared to mice, fibrosis is a slower condition in humans, which takes decades to develop and require long-term treatment to diminish its progression. Furthermore, many animal models of diseases do not mimic various clinical settings, thus controversial results are likely to be obtained since there are different signaling pathways and mechanisms in cardiac fibrotic processes in various diseases. In addition, the animals used are normally young while patients with cardiac fibrosis are at a more advanced age. In order to improve clinical translation, it is important to design, conduct and analyse animal experiments properly and to summarize data from animal research adequately before conducting clinical trials. Moreover, the failure of previous trials also emphasizes the need for optimal design of future clinical trials including a selection of suitable patients, appropriate dose, and route and timing and length of administration.There are some important areas for future research in this field. First, although CMR is too expensive to be used in large populations, it should be used to investigate potential new treatments with relatively small group sizes since it allows accurate assessment of regional and diffuse fibrosis. Second, anti-fibrotic therapies targeting downstream signaling pathways may improve safety and efficacy of current treatments such as TGF-β inhibitors. In addition to their role in fibrosis, many proteins are involved in other biological processes. Thus, developing more specific agents targeting fibrotic signaling pathways is likely to be beneficial to minimize potential side effects. Third, combined anti-fibrotic therapies seem more effective than single drug treatment. It is shown that spironolactone or relaxin in combination with angiotensin II inhibitors augmented anti-fibrotic efficacy (Kosmala et al., Although many fibrotic therapies on cardiac fibrosis are promising in preclinical models, clinical translation is limited. There is still a lack of effective treatments to regress cardiac fibrosis in patients with various cardiovascular disease. Future optimally designed clinical studies are required to test new potential treatments and currently available drugs with improved safety and efficacy after adequate analysis of evidence from animal research.LF was responsible for assembling and drafting of the manuscript. AM and AD contributed to the drafting of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zinc is also found in zinc containing proteins. It is estimated that the human proteome contains about 3000 zinc-containing proteins serving signaling, catalytic, and structural roles. In zinc-finger proteins, for example, a zinc ion is complexed in a zinc finger motif through four invariant cysteine and/or histidine residues to form a stable structure and conformation, which regulates protein-DNA, protein-RNA, and protein-protein interactions [Zinc is the second most abundant metal in human body, and a relatively large amount of zinc is found in the brain, indicating its essential role in central nerve system. Znractions .Twenty years ago, abnormal zinc accumulation was first observed in ischemic neurons, which leads to neuronal injuries after cerebral ischemia . We receZinc release from ischemic neurons to extracellular matrix could produce a surrounding environment with a high concentration of zinc after cerebral ischemia. Strong activities of zinc-containing presynaptic terminals may transiently increase local synaptic zinc concentrations up to 300μM, making it available for entry or uptake by neighboring cells . MicrodiAstrocytes can take up the excessive extracellular zinc from synaptic cleft or extracellular matrix to maintain zinc homeostasis. However, zinc overload in astrocytes may induce astrocytic cell death. Our study indicated that zinc overload under hypoxic condition caused a dramatic increase in astrocytic cell death in a zinc-concentration-dependent manner . Very inTo date, almost all research of zinc in brain has focused on its effects on neuronal functions. Little is known about the role of zinc in blood-brain barrier (BBB) disruption after cerebral ischemia. BBB is mainly composed of endothelium of brain microvessels, which are associated with astrocytes, pericytes, neurons, and extracellular matrix. At the early stage of ischemic stroke, high level of BBB permeability is one of the main causes for hemorrhage transformation, preventing wide use of thrombolytic therapy in most ischemic stroke patients.in situ microvessels in the brain of ischemic rats. Treating the animals with a specific zinc chelator (TPEN) at 60-min post ischemia onset remarkably attenuated microvessel permeability in the ischemic rats [The interaction between different types of cells plays very important roles in brain physiology and pathophysiology. Neurons are considered as the first responding cells to ischemic stress, because they are very sensitive and vulnerable to the decrease of oxygen and nutrition supply. Therefore, we hypothesized that, as an impaired signal from neurons, zinc release may contribute to BBB disruption during acute ischemic stroke. Our experiments revealed a strong zinc accumulation in isolated microvessels and mic rats . This waMatrix metalloproteinases (MMPs) family is a large family of zinc-containing endopeptidases that play an important role in mediating gelatin degradation and vasculature disruption. Gelatinases MMP-9 and MMP- 2 specifically degrade the tight junction proteins (TJPs), which seal the gap between endothelial cells to keep BBB integrity. Our study further demonstrated that zinc accumulation in microvessels activated the superoxide/ MMP-9/-2 pathway, leading to the loss of tight junction proteins from microvessels and apoptosis of endothelial cells . These fIn summary, research from our lab and others show that cerebral ischemia triggers zinc accumulation in the microvessels, which critically contributes to ischemia-induced BBB disruption via activating ROS-MMPs pathway. Emerging evidence suggests that zinc is a novel target for reducing both neuronal and vascular damage during ischemic stroke.


Thus, many scaffold-based research activities have been done to develop new materials, structures and fabrication technologies to mimic native meniscus for cell attachment and subsequent tissue development, and restore functionalities of injured meniscus for long-term effects. This study begins with a synopsis of relevant structural features of meniscus and goes on to describe the critical considerations. Promising advances made in the field of meniscal scaffolding technology, in terms of biocompatible materials, fabrication methods, structure design and their impact on mechanical and biological properties are discussed in detail. Among all the scaffolding technologies, additive manufacturing (AM) is very promising because of its ability to precisely control fiber diameter, orientation, and pore network micro-architecture to mimic the native meniscus microenvironment.Current surgical treatments for meniscal tears suffer from subsequent degeneration of knee joints, limited donor organs and inconsistent post-treatment results. Three clinical scaffolds (Menaflex CMI, Actifit Meniscus provides physical protection to knee cartilage by transmitting loads through the joint, distributing high peak stresses on the underlying surfaces, providing shock absorption. The internal microstructure of a meniscus consists of circumferentially oriented collagen fibers and radial collagen fibers, as shown in The meniscus consists of three parts: the outer vascular/neural area with 80% collagen type I (dry weight), the inner entirely avascular/aneural area with 70% collagen content (both type II and type I) (dry weight), and the junctional area between the two. Meniscal tears are the most common intra-articular injuries in the knee. From the PearlDiver database which represents approximately 9% of the US population under 65 years, a total of 387,833 meniscectomies and 23,640 meniscus repairs were reported between 2005 and 2011 in the US [In regenerative medicine, scaffolds are used as a temporal template to facilitate host tissue integration upon implantation, for new tissue formation and remodeling. From a structural or mechanical perspective, meniscal scaffolds should (1) provide appropriate biomechanical functions after implantation to shield cells from damaging compressive or tensile forces; (2) maintain their shape integrity , mechanical stability and strength on the defect area until enough host tissue has been regenerated; (3) produce mechanical stimuli to promote tissue regeneration .From a biological perspective, meniscal scaffolds should: (1) possess appropriate/acceptable biocompatibility and nontoxicity profiles (due to direct contact with living tissues); (2) provide suitable surface properties  and mechanical properties  for cell attachment, proliferation and differentiation, and facilitate cell–cell contact and cell migration; (3) have sufficient porosity for the cell media and growth factors to permeate and reach the cells in all the layers of the scaffold; and, (4) serve as a delivery vehicle or reservoir for exogenous growth-stimulating signals such as growth factors. In addition, the degradation rate of scaffolds should match the rate of specific tissue regeneration so that they can disappear completely once the tissues are repaired. Degradation byproducts from scaffolds should also be biocompatible and removable with minimal immune or inflammatory responses and without further surgeries .® scaffold from Orteq Ltd. , and NUsurface® Meniscus Implant from Active Implants . Menaflex CMI and Actifit® scaffold are partial meniscal substitutes with equivalents in histological, radiological, and clinical evaluations [® Meniscus Implant is the first total meniscal substitute, and has been used in Europe under CE Mark since 2008 and in Israel since 2011 [Three types of biodegradable and biocompatible scaffolds are available on the market to reconstruct the segmental meniscus defects: Menaflex CMI from ReGen Biologics, Inc. , Actifitluations . They haluations . NUsurfance 2011 . The clince 2011 .Although these scaffolds are designed to mimic the function of the natural meniscus or stimulate the growth of new tissues, they need to be improved in terms of structure design, material design and so on.Both mesenchymal stem cells (MSCs) and costal chondrocytes (CC) have been used in the study of meniscal scaffolds. MSCs which can be readily harvested from numerous adult tissues, can differentiate into cell types indigenous to bone, cartilage, muscle, tendon, ligament, and connective tissues . MSCs, aResearchers around the world are working on various tenets of meniscal scaffolds like material selection, cell sources, novel fabrication technologies and optimal biomimic structure design. Such information is scattered in various publications and websites with different technical focuses. The objective of this paper is to collate, categorize, analyze and summarize the information pertaining to meniscal scaffold design, fabrication and mechanical properties, as well as to provide a critical insight into the direction of their future development.Four types of scaffold materials are reviewed in Four types of materials are widely used for meniscal scaffold fabrication.Tissue-derived materials include periosteal tissue , small iECM components are naturally derived, such as collagen, proteoglycans and elastin molecules . ScientiSynthetic polymers used for meniscal scaffolds include polyurethane (PU), polyglycolic acid (PGA), polylactic co-glycolic acid (PLGA) and polycaprolactone (PCL). They can be easily shaped to scaffolds with varied pore size, fiber dimension, geometry, porosity, better mechanical properties and an adjustable degradation rate .However, their hydrophobic properties, aseptic inflammation, immune responses and side effects from degradation byproducts limit their applications. For example, diisocyanate from degradation of PU is toxic. The degradation of PLGA generates acidic byproducts, which cause inflammatory responses and damage local tissues .® scaffold (20% PU and 80% PCL) was implanted in a dog and, after about two years, viable living “meniscus-like” tissue was observed, i.e., a fibrous tissue containing type I collagen and fibrocartilage-like tissue containing proteoglycans and type II collagen [Multiple synthetic polymers are mixed together in suitable proportions to enhance the cell-adhesive properties and lower foreign-body reaction. When polyethylene terephthalate (PET) was added into hyaluronic acid/PCL scaffolds, more type II collagen was observed . An Acticollagen . Canine collagen . Furthercollagen ,38,39.Biopolymers, such as silk fibroin (SF) derived from silkworms, have also been applied to meniscus repair applications due to better biocompatibility, slow degradability and relatively better mechanical properties.Mandal et al.  used SF Hydrogels are semi-liquid hydrophilic colloids constructed by a network of crosslinked natural or synthetic polymer chains. They can be easily cast into desired shapes and evenly mixed with seed cells and growth factors. The physical properties of hydrogels are determined by their water content. Higher porosity benefits cell diffusion, but it has poor mechanical properties. Thus, double-network (DN) hydrogels were developed, which can offer excellent mechanical properties—even with the water content exceeding 90%—and a dynamic stiffness value comparable to that of the swine meniscus . CrossliA table of comparison of the above four scaffold types against three criteria  is shown in As shown in Structural and architectural properties of the scaffolds play an important role in determining the overall scaffold mechanical properties, in additional to the inherent material strength. They can be obtained by patients’ computer tomography (CT) or magnetic resonance imaging (MRI) datasets. The datasets are processed with CAD/CAM modelling software and the final model is transferred to a system for fabrication. CT datasets performed better than MRI when reconstructed tissues had an intrinsic degree of opacity .The required biological properties of scaffolds can be achieved by proper selection of scaffold materials, bioactive agents, cell source and architecture. The most commonly used meniscal scaffolds are sponge or fibrous structure. Both of them can achieve higher porosities and exhibit mechanical properties (compression Young’s modulus) similar to that of natural cartilage, but the dynamic stiffness of fibrous scaffolds is higher than that of sponge scaffolds for the same material .Sponge scaffold structure is largely controlled by the fabrication process rather than design parameters . This stWelsing et al.  conducteFibrous scaffolds are architecturally characterized by micro features such as fiber diameter, pore size and internal configuration of deposited fibers, which determines the porosity and mechanical properties of fibrous scaffolds. The extrinsic stiffness of scaffolds with the same micro features (porosity and pore architecture) but dissimilar geometries resulted in different mechanical responses . MechaniFiber diameter is a crucial scaffold design parameter. Scaffolds with smaller fiber diameter can lead to higher porosity and lower tensile modulus. Cell adhesion and growth kinetics are also significantly affected by fiber diameter . To promPore size should vary for different kinds of cells and tissues, since appropriate pore size provides a better tissue growth environment. For example, 20–125 μm for skin regeneration , 200–350For the same material, some technologies can produce scaffolds with higher porosity than others. PCL scaffolds fabricated by fused deposition modeling (FDM)  can achiCells are capable of sensing mechanical rigidity in their surroundings at different scales . When thInterconnected structures with open pores can facilitate homogeneous cell seeding and better nutrient dispersion throughout the construct, especially for vascular, bone and other soft tissue-related applications.In the meniscus, dense collagen bundles are locally aligned and this alignment changes gradually along the height. As a result, a circumferential pattern is formed aiding better load transmission across the knee joint. Meniscal scaffold structure should mimic such directional variation so as to provide the necessary circumferential tensile strength. This can be done by modifying the fiber deposition patterns from a homogeneously perpendicular architecture to a more radially oriented configuration.Moroni et al.  used BioBalint et al.  fabricatOur group at National University of Singapore chose five independent parameters (outer radius (R), number of circumferential fibers (N), gap between two circumferential fibers (G), number of radial fibers (n), and angle between the adjacent radial fibers (θ)) to design meniscal scaffolds using Electrohydrodynamic-jetting (EHD-jetting) . As depiIn testing the functionalities of various scaffold structure designs, compression and tensile properties have received more attention. The 3D-woven scaffolds with fiber-reinforced, gradient structure  or orienMeniscal scaffold fabrication can be as challenging as the scaffold design, with the consideration of bulk material properties, structure design and functional requirements. The fabrication process for either sponge or fibrous structure scaffolds should be reproducible and reliable.Traditional technologies like particulate leaching , gas foaA thermal-induced phase separation process  was usedThe sponge scaffold fabrication techniques are either based on physical or chemical treatment, with associated pressure and/or temperature change. Such a harsh environment makes pre-seed cells impossible to survive or function properly. The residues from the organic solution used during the fabrication process are more or less detrimental for cells and bioactive molecules. The scaffold properties are isotropic and their internal structures are largely controlled by the fabrication process rather than the parameters. In other words, it is very hard to control pore size and geometry, pore distribution, interconnectivity, and porosity. Thus, they suffer more from pore blockage (filtration effect) than the fibrous structures.A fibrous scaffold structure consists of individual fibers either in woven or non-woven patterns with variable pore size, shape and fiber diameter. The advantages of such a structure include a large surface area for cell attachment and rapid diffusion of nutrients in favor of cell survival and growth. Electrospinning and additive manufacturing (AM) are the technologies that are being used for non-woven and woven fibrous scaffold fabrication individually.Electrospinning technology is increasingly being used to process synthetic and natural polymers for fabricating fibrous scaffolds. By controlling the various process parameters like the solution conditions (concentration and solvent type), process conditions , and collection methods (plate versus rotating mandrel and speed of collection), this technique can produce ultrafine disordered fibers from several micrometers to a few nanometers, similar to the size of collagen fibers in the meniscus . DespiteCollagen fibers or uniaxial fiber bundles in the meniscus are organized either in parallel or in perpendicular direction to the surface. To incorporate such spatial cues into meniscal scaffolds, rotating dynamic collectors are integrated into electrospinning set up to align fibers, which could be a disc collector , rotatinBaker et al.  rotated Fisher et al.  rotated The above rotating devices can quickly and roughly align fibers in a limited orientation and create meshes/scaffolds with aligned nanofibers. However, it is difficult to use these electrospun fibers to construct scaffolds with greater architectural complexity . Furthermore, both sponge scaffold and non-woven fibrous scaffold fabrication methods result in random internal structure and vary from part to part and thus lack repeatability.AM is a computer-controlled solid free-form fabrication technique that can reproduce scaffolds with consistent internal and external architecture. A scaffold model based on medical images (MRI or CT) or customized design, can be physically built layer by layer.Common AM technologies such as fused deposition modeling (FDM) and precision extrusion deposition (PED), were used to fabricate synthetic material scaffolds with fiber diameter of about 100–500 µm ,85. In FPED consists of a mini-extruder mounted on a high-precision positioning system. This technique uses granulated materials, which avoid most of the material preparation steps in FDM. Shor et al.  used PEDDue to the extra-large pore size and large fiber diameter (above 100 micrometer), FDM and PED cannot create scaffolds to benefit cell growth. The nozzle blockage is another problem, especially when extruding viscous materials through a small diameter nozzle. To overcome these bottlenecks, AM needs to be integrated with a high-resolution fiber fabrication technology to advance meniscal scaffold fabrication.To generate micron to nanoscale fibers, EHD-jetting, a near-field electrospinning process, is introduced. This process uses a pneumatic or syringe pump to supply material solution to a nozzle at a constant flow rate, and an applied electric field between the substrate and the nozzle to pull very fine fibers out of the nozzle. This technique is capable of producing micron to nanoscale scaffolds with high-resolution patterns, controlled structure and porosity, optimized material formulations and path planning . When thMost importantly, the fabrication parameters can be optimized and standardized, thus improving the repeatability and reliability of scaffold fabrication. A strategic study of cell and scaffold interaction becomes possible, which is particularly important for scaffolds with specially designed physical or biological features.An intelligent process monitoring is expected to optimize this fabrication technology through the control of current/voltage amplitude, waveform, distance between the nozzle and the substrate, solution feed rate and so on. It should not only perform real-time adjustment, but also be able to cope with disturbance from unpredictable factors such as nozzle circularity, substrate material impurities, inhomogeneous solution, as well as voltage and pressure fluctuations.The combination of hydrogel and solid polymers can resemble the biphasic nature of meniscus (water and solid) and produce a cell-friendly environment . The curTo overcome this shortcoming, AM techniques have been used to fabricate hydrogel-based scaffolds with designed patterns and incorporate growth factors, microspheres or nanoparticles into fabrication process. For example, drop-on-demand (DOD) micro-dispensing AM technology, has been used to precisely dispense solid/hydrogel components with customized spatial distribution or varied composition to develop a biomimetic cellular scaffold. Chang et al.  used DODThe electrospinning technique has also been integrated into the AM platform to dispense viable cells or collagens in hybrid scaffold fabrication. Lee et al.  applied Although the above studies are not specifically aimed at meniscus scaffold fabrication, they shed light on the feasibility of using advanced AM techniques to fabricate scaffolds with controlled and designed distribution of cells, hydrogel and bioactive agents.Common fabrication technologies used in meniscal tissue engineering are tabulated in The performance of scaffolds must be evaluated, not only in the relatively controlled in vitro environment, but also in the context of tissue’s physiological function , and ultimately in in vivo conditions. The difference between scaffold degradation behavior in in vitro and in vivo  should aMeniscus is a C-shaped disc of fibrocartilage with a triangular cross-section, where type I and type II collagen fibers are arranged in a circumferential pattern. To document the differences between native meniscus and meniscal scaffolds, scaffolds are usually tested with respect to one or two mechanical properties at the expense of others. Tensile test and compressive test are the most common ones.For native meniscus, tensile properties are cross-section and position dependent due to varied fiber densities and orientations, as a result axial compressive force being shared as circumferential tensile load. Tensile test is to quantify the circumferential tensile properties of scaffolds when the knee bears an axial load. Compressive test is to determine the ability of scaffolds on sharing compressive loads via generating tensile loads. Scaffold structure can significantly influence the results from the tensile and compressive tests. For gradient structure scaffold  or orienScaffolds’ mechanical properties also change with the passage of time because of biomaterial degradation and surrounding ECM formation. The former lowers the mechanical strength of the meniscal scaffold structures and the latter increases it. Overall, the tensile modulus of cell-laden scaffolds is significantly higher than the acellular scaffold .Structural and functional properties of scaffolds can be enhanced under controlled mechanical stimulation (dynamic cell culture). This can modulate the spatial heterogeneity of engineered menisci, with higher stiffness in tension and compression . DynamicThe scaffold degradation is controlled by material properties and composition, local environment (temperature and pH), structure, and mechanical loading conditions. Even the degradation mechanism has not been quantitatively determined: both pore wall thickness (bulk degradation) and surface area (surface degradation) determine scaffolds’ degradation rate. Scaffolds with higher porosity or smaller pore sizes degrade more slowly than those with lower porosity or larger pore sizes . Highly To benchmark the scaffold property tests, the authors have identified three critical issues to be further researched. One is to develop consistent mechanical and biological tests and/or evaluation criteria. Most of the results are based on a variety of testing conditions and criteria. It is very necessary to develop standard evaluation protocols for mechanical and biological assessment. This will be beneficial to compare different scaffold designs and develop better strategies.Testing results published with in vivo studies are considerably fewer in number than that with in vitro. For the same scaffold, the relationship between in vivo and in vitro performance has become another critical issue to be addressed. Last but not least, scaffolds with excellent mechanical properties cannot guarantee the regenerated tissues will have good mechanical properties. Whether a biomimetic design will actually result in a functional  engineered meniscus, is still under research. Further studies are expected to explore the linkage between in vitro and in vivo, and analyze design-based functions.This study summarizes the current research activities on meniscal scaffold in terms of materials, fabrication methods, structure design and their influence on mechanical and biological characteristics. Strategic scaffold designs have been developed using biological and structural knowledge from native meniscus and/or its healing process. The current major challenges are still material property and fabrication capabilities, in terms of biochemical composition and biomechanical properties. To restore an intact meniscus with competent functions, researchers should also explore cell-seeding techniques and medical imaging technology. Of all the fabrication technologies, electrohydrodynamic (EHD)-jetting and drop-on-demand (DOD) micro-dispensing techniques based on the additive manufacturing (AM) platform are more promising for producing multi-material scaffolds with solid framework structure or cell–hydrogel formulation with controlled material distribution. They greatly reduce the steps required for scaffold preparation and fabrication, thus accelerating an economical transition to clinical applications. Last but not least, developing mathematical models to simulate the tissue-formation process and the relationship between scaffold properties and design features would benefit scaffold optimization.


Osteoprotegerin (OPG) is a soluble decoy receptor for tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). It belongs to the tumor necrosis factor receptor superfamily (TNFRSF). OPG was initially discovered to contribute to homeostasis of bone turnover due to its capability of binding to receptor activator of nuclear factor-kappaB (NF-kB). However, apart from bone turnover, OPG plays important and diverse role(s) in many biological functions. Besides having anti-osteoclastic activity, OPG is thought to exert a protective anti-apoptotic action in OPG-expressing tumors by overcoming the physiologic mechanism of tumor surveillance exerted by TRAIL. Along with inhibiting TRAIL induced apoptosis, it can induce proliferation by binding to various cell surface receptors and thus turning on the canonical cell survival and proliferative pathways. OPG also induces angiogenesis, one of the hallmarks of cancer, thus facilitating tumor growth. Recently, the understanding of OPG and its different roles has been augmented substantially. This review is aimed at providing a very informative overview as to how OPG affects cancer progression especially breast cancer. At present, one in eight women in the United States will develop breast cancer . Recent The tumor microenvironment (TME) is the cellular environment in which the tumor exists and provides its niche for growth, progression, survival and evolution. The normal microenvironment provides crucial signaling to maintain appropriate tissue architecture, inhibit cell growth and suppress the malignant phenotype, and acts as a barrier to tumorigenesis . IncorreOPG was first identified by sequence homology to the tumor necrosis factor receptor (TNFR) family during a rat intestine cDNA sequencing project and was named based on its function  . OPG wasin vitro . TR. TR37]. pothesis , 38–40 bpothesis . In addie tumors .Apart from inducing angiogenesis and bypassing apoptosis by quenching TRAIL ligand, our study  for the via OPG receptors, such as type II membrane forms of RANKL [Our goal was to decipher the cellular mechanisms of how OPG modulates and reprograms the normal mammary epithelial cells to a tumorigenic state thus suggesting promising avenues for treating IBC as well as highly invasive breast cancer with new therapeutic targets . Our stuof RANKL , 46, TRAof RANKL  and hepaof RANKL , 48. Intn = 400), 40% of samples showed OPG expression that was confined to tumor cells [Recent studies have demonstrated OPG expression in breast cancer tissue samples and in a large cohort of invasive breast cancers  is conducted by numerous physiological and pathological stimuli. These stimuli can be various cytokines, chemokines, and growth factors. Molecular players of angiogenesis have been characterized since the early years of angiogenic studies, and one of the most prominent stimulating growing factors is certainly the vascular endothelial growth factor (VEGF) family. The most prominent member of this family, VEGF, is the foremost controller of physiological and pathological angiogenesis. An increasing number of reports now consider that OPG also has a function in other biological systems, including the vasculature –51. It iin vitro model of angiogenesis , proteins involved in transport and fusion , and proteins involved in gene expression  . Mass spectrometry analysis revealed it to be the protein nucleolin Figure  , which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells Figure  unpubli unpubli  and is a key modulator of metastasis and resistance to TRAIL-induced apoptosis . Shi et OPG is also involved in many other diseases unrelated to cancer. OPG was recently defined as an important cardiovascular (CV) marker in the general population with arterial stiffness, vascular calcification, and carotid intima media thickness . OPG conThe occurrence of single nucleotide polymorphisms (SNPs) in the OPG gene in association with breast cancer has been examined. Ney et al. 2013  for the Kwan et al., 2014 used a meta-analysis of GWAS from FIVE studies comprising > 10 000 individuals from European and Asian origin, and identified two genome-wide significant loci (8q23-q24.1 and 17q11.2) and one locus on chromosome 14 associated with OPG levels with near genome-wide significance . In concDespite the wealth of literature on OPG, there are many questions still unresolved, including the exact role of OPG in bone metastasis of breast cancer and the therapeutic potential of targeting OPG. Besides roles in cancer, OPG plays role(s) in bone metabolism, endometriosis, periodontal disease, thyroid disease and coronary heart disease . Though Conditionally replicating adenoviruses (CRAds) have been used as anticancer agents designed to infect and lyse tumor cells. In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd suggesting the role of OPG . Breast


Parus major). The diversity of the oral microbiota was significantly higher compared to the faecal microbiota, whereas interindividual variation was higher in faecal than in oral samples. We also observed a pronounced difference in taxonomic content between the oral and faecal microbiota. Bacteria belonging to the phyla Proteobacteria, Firmicutes and Actinobacteria typically dominated in both oral and faecal samples. A high abundance of bacteria belonging to Tenericutes was observed only in faecal samples. Surprisingly, we found only a slight correlation between the faecal and oral microbiota at the within-individual level, suggesting that the microbial composition in these body sites is shaped by independent regulatory processes. Given the independence of these two communities at the individual level, we propose that simultaneous sampling of the faecal and oral microbiota will extend our understanding of host vs. microbiota interactions in wild populations.The gastrointestinal tract of vertebrates is inhabited by diverse bacterial communities that induce marked effects on the host physiology and health status. The composition of the gastrointestinal microbiota is characterized by pronounced taxonomic and functional variability among different regions of the vertebrate gastrointestinal tract. Despite the relatively solid knowledge on the among-region variations of the gastrointestinal microbiota in model mammalian species, there are only a few studies concerning among-region variations of the gastrointestinal microbiota in free-living non-mammalian vertebrate taxa. We used Illumina MiSeq sequencing of bacterial 16S rRNA amplicons to compare the diversity as well as taxonomic composition of bacterial communities in proximal vs. distal parts of the gastrointestinal tract  in a wild passerine bird, the great tit ( Animal bodies are inhabited by taxonomically and functionally diverse communities of bacteria –3 that mThe host-associated microbiota exhibits pronounced spatial variation among different parts of the animal body ,9–11. OuMost current studies focusing on the GIT microbiota, use the microbiota of faecal samples, putatively representing the GIT microbiota of the lower intestine, as a proxy ,28–30. HA few studies have analysed host vs. microbiota interactions using microbiota samples from multiple GIT regions ,33,37. HMoreover, most current knowledge on microbial variation along the GIT relies on data from mammalian captive-bred models and humans –42. PattHere we applied high-throughput amplicon sequencing of bacterial 16S rRNA to study the variation in microbiota among samples of proximal and distal parts of the GIT  noninvasively collected in a free-living passerine bird. According to research on mammals, microbial communities of both the proximal and distal GIT are shaped to a large extent by host-intrinsic regulatory mechanisms, while the effect of environmental bacteria on the composition of these communities is usually limited ,50. At tParus major) was selected as a model species. The great tit is an eminent model species for the functionally and evolutionary oriented branches of ecological research  and S-D-Bact-0785-a-A-21 [GACTACHVGGGTATCTAATCC]) were used during the PCR step. Both forward and reverse primers were tagged with 10bp barcodes designed by TagGD software [http://www.ebi.ac.uk/ena/data/view/PRJEB19204 and sample metadata in Following the recommendations of Klindworth et al., , primerssoftware . For thehttp://sourceforge.net/projects/microbiomeutil/files) for the detection and elimination of chimeric sequences. The resulting 16S rRNA sequences were clustered at a 97% similarity threshold using UPARSE [Paired-end Illumina reads were merged using PEAR , and de-g UPARSE  in orderg UPARSE  and the g UPARSE . Represeg UPARSE , the maxg UPARSE . We obseg UPARSE  for statg UPARSE .In order to account for uneven sequencing depth among samples, statistical analyses were calculated based on the rarefied OTU table unless otherwise stated. The number of observed OTUs, Shannon diversity and Chao1 based predictions of total microbial diversity for individual samples were calculated using phyloseq . Linear We further used Principal Coordinate Analysis (PCoA) based on Bray-Curtis, Jaccard, weighted and unweighted UniFrac  distanceThe LME-based approach was further used to identify OTUs whose abundances differed between oral and faecal samples. These analyses were performed on a subset of 240 OTUs  that were detected in at least five samples. For each OTU-specific LME, Box-Cox transformed read counts were used as a response, whereas the effect of GIT regions and individual identity were included as the explanatory variable and random intercept, respectively. In addition, Box-Cox transformed total number of reads per individual samples was included as an offset in LMEs . To account for deviance from a Gaussian error distribution, the significance of the GIT region effect was assessed based on permutations. In particular, observed deviance changes due to the elimination of the GIT region effect for the initial model were compared with the null distribution of deviance changes extracted from LMEs, where both the number of total and OTU-specific read counts were randomly resampled (10 000 permutations). The Qvalue method  was usedDiplorickettsia, Pseudomonas, Erwinia, Escherichia/Shigella, Serratia and Acinetobacter), Alphaproteobacteria  and Actinobacteria (genera Corynebacterium and Pseudonocardia) were the dominating bacterial classes of both the oral and faecal microbiota. However, the abundance of Bacilli (represented by genera Staphylococcus and Lactobacillus) and Betaproteobacteria  was increased in oral compared to faecal samples. At the same time, several faecal samples exhibited high abundances of Mollicutes (represented by genera Ureaplasma and Mycoplasma), Clostridia and Chlamydia, i.e. bacterial classes that were detected in very low abundances in oral samples.Our dataset included 207 497 high-quality reads that were clustered into 1127 non-chloroplast OTUs. There was a significant decrease of alpha diversity in faecal compared to oral microbiota according to the observed number of OTUs and Shannon index, as well as according to Chao1 , Table 1Ureaplasma (phylum Tenericutes), Delftia (phylum Proteobacteria), Carnobacterium (phylum Firmicutes), Deinococcus (phylum Deinococcus-Thermus), Chryseobacterium and Elizabethkingia (both phylum Bacteroidetes) were more abundant in faecal samples. On the other hand, OTUs where the most striking relative abundance increase in oral compared to faecal samples was observed belonged to the genera Pseudomonas, Acinetobacter, Methylobacillus, Herbaspirillum , Catellicoccus, Staphylococcus, Tumebacillus (phylum Firmicutes) and to Arthrobacter, Brevibacterium and the families Intrasporangiaceae and Nocardioidaceae (phylum Actinobacteria).OTU-level analyses identified 33 OTUs (represented by 35.8% reads) whose relative abundances differed between oral and faecal samples . OTUs co2 < 0.02 in all cases).ANCOVA analyses did not reveal any effect of sex or scaled body mass index on the diversity of microbial communities associated with these two GIT regions . In addition, according to adonis analyses, there was no effect of these two variables on the composition of oral or faecal microbiota . Furthermore, we did not detect any within-individual correlation in microbial composition between faecal and oral samples . Finally, the correlation of relative abundances of individual OTUs between oral swabs and faeces was negligible .Many previous studies that focused predominantly on captive bred mammals and humans have found pronounced differences in microbial structure between body sites as well as among different GIT compartments ,81,82. OLactobacilli was detected in a recent study on the quail (Coturnix japonica) [Haemophilus and Streptococcus that dominated the oral microbiota of the kakapo (Strigops habroptilus) [Altogether, the composition of the great tit faecal microbiota was comparable with previous studies on other passerine birds ,60,83–86aponica) , while toptilus)  were lowoptilus) ,43,82,89optilus) ,40,43,90optilus)  values oUreaplasma, Deinococcus, Carnobacterium, Chryseobacterium, Delftia and Elizabethkingia OTUs. Ureaplasma together with another Tenericutes OTU corresponding to the genus Mycoplasma that tended to be increased in the faecal microbiota as well , are common inhabitants of vertebrate gastrointestinal and urogenital tracts. Although these taxa are often asymptomatically present in birds, some of these species are involved in severe pathogenesis [Deinococcus OTU (phylum Deinococcus-Thermus) has previously been detected in several vertebrate species [Carnobacterium OTU (phylum Firmicutes) is a lactic acid bacterium with putative probiotic properties providing protection against various bacterial pathogens [Chryseobacterium and Elizabethkingia OTUs (both from the family Flavobacteriaceae) are related to several pathogenic species of human and other vertebrate taxa [At the OTU level, the faecal microbiota was characterized by increased abundances of ogenesis . The Dei species ,93; howeathogens ,95. On tate taxa –98.Staphylococcus, Acinetobacter, Sphingomonas, Brevundimonas, Dysgonomonas, Hymenobacter, Sphingobium). Many OTUs exhibiting higher abundances in the oral cavity compared to faeces can be involved in interactions with their host's immune system and physiology, or can shape the community composition via interactions with other members of the oral microbiota. This applies, for example, to four Actinobacterial OTUs . Actinobacteria produce a wide variety of bacteriocines and other compounds suppressing the proliferation of bacterial competitors. Therefore, their presence in the oral cavity could be crucial for the defence against bacterial pathogens as well as for the maintenance of overall microbial structure [Arthrobacter abundances in the lower intestine were positively associated with survival rates of passerine species closely related to the great tit [Lysobacter, Pseudomonas and Herbaspirillum. Lysobacter can shape microbial community through the production of bacteriocines and the active predation of other bacteria [Pseudomonas and Herbaspirillum produce extracellular siderophores, i.e. iron chelating compounds, which provide them a competitive advantage over other bacteria by reducing the availability of iron in the environment [Pseudomonas cells secrete exopolysaccharides that make them a difficult target for the host immune system [Pseudomonas species can be pathogenic for birds [Catellicoccus and Lactococcus, which can shape the oral community structure by the modulation of abiotic environmental conditions or by direct interactions with host immune system or other community members [According to the OTU-level analyses, the oral microbiota was characterized by increased abundance of OTUs from genera that commonly colonize the oral cavity, skin or intestine of various vertebrate taxa  host-intrinsic mechanisms such as effects of the immune system and other biotic and abiotic factors operating within the GIT, or (2) extrinsic sources of variation including pools of bacteria present in the diet and other environmental sources.Relatively low interindividual variation of oral microbiota suggests either that environmental bacterial pools colonizing the oral cavity exhibit high homogeneity in space and time, or that there is low interindividual variation in host-specific mechanisms regulating oral microbiota. As previous research has found only limited effects of environmental bacteria on oral microbiota in non-avian vertebrates ,50, and In conclusion, our study is the first to characterize the oral microbial structure and compared it with the faecal microbiota in a free-living bird population. Our results show that the oral and faecal microbiota of passerines represent two distinct bacterial consortia that exhibit marked differences at all levels of community structure, and that the interindividual variation of these communities is likely to be shaped by independent mechanisms. We propose that aside from the effect of environmental bacteria, the structure of both the faecal and oral microbiota is driven to a large extent by mutual interactions among community members or by the host vs. microbiota interactions including immunity. Consequently, given the putative effects of these two microbial communities on the host's heath status, further research focusing on the microbiota in wild vertebrate populations may benefit from simultaneous sampling of these two communities.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Development of clinically relevant, novel drug-nanoparticles capable of accumulating in PDX tumors through the enhanced permeability and retention effect in tumor vasculature may lead to new and effective TNBC treatments.Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer and disproportionately accounts for the majority of breast cancer related deaths. Effective treatment of this disease remains an unmet medical need. Over the past several decades, TNBC cell lines have been used as the foundation for drug development and disease modeling. However, ever-mounting research demonstrates striking differences between cell lines and clinical TNBC tumors, disconnecting bench research and actual clinical responses. In this review, we discuss the limitations of cell lines and the importance of using patients’ tumors for translational research, and highlight the usage of patient-derived xenograft (PDXs) models that have emerged as a clinically relevant platform for preclinical studies. PDX tumors possess tumor heterogeneity with similar cellular, molecular, genetic and epigenetic properties akin to those found within patients’ tumors. Moreover, PDX and clinical tumors possess abnormal vasculature with higher blood vessel permeability, a feature that is not always demonstrated in  Breast cancer remains a leading cause of death in women throughout the world. Triple negative breast cancer (TNBC) accounts for only 15-20% of all breast cancer, but is disproportionally associated with the majority of breast cancer related deaths . As suchin vitro and in vivo preclinical research commonly uses over 27 TNBC cancer cell lines to study cancer pathogenesis, disease advancement, and drug effectiveness. However, a growing disconnection between results generated using TNBC cell lines and clinical trials has been observed. A recent example is the in vitro and in vivo results of PARP inhibitor veliparib. Veliparib is an oral inhibitor of Poly (ADP-Ribose) Polymerase (PARP) 1 and 2, which enhances the activity of DNA damaging agents in DNA repair to promote apoptosis. In vitro, veliparib is capable of suppressing the expression of Snail which promotes epithelial to mesenchymal transition, tumor metastasis and drug resistance. It also sensitizes the MDA-MB-231 TNBC cell line to chemotherapeutic drug doxorubicin, resulting in increased apoptosis ..in vivo


Global health electives offer medical trainees the opportunity to broaden their clinical horizons. Canadian universities have been encouraged by regulatory bodies to offer institutional support to medical students going abroad; however, the extent to which such support is available to residents has not been extensively studied.We conducted a survey study of Canadian universities examining the institutional support available to post-graduate medical trainees before, during, and after global health electives.Responses were received from 8 of 17 (47%) Canadian institutions. Results show that trainees are being sent to diverse locations around the world with more support than recommended by post-graduate regulatory bodies. However, we found that the content of the support infrastructure varies amongst universities and that certain components—pre-departure training, best practices, risk management, and post-return debriefing—could be more thoroughly addressed.Canadian universities are encouraged to continue to send their trainees on global health electives. To address the gaps in infrastructure reported in this study, the authors suggest the development of comprehensive standardized guidelines by post-graduate regulatory/advocacy bodies to better ensure patient and participant safety. We also encourage the centralization of infrastructure management to the universities’ global health departments to aid in resource management. Post-graduate global health (GH) electives are valuable opportunities for residents to increase knowledge,5GH electives call residents to adapt to new challenges; this makes support infrastructure from the home and host institutions an important resource.Given recent initiatives to standardize the GH elective infrastructure available to medical students - particularly in regards to PDTA survey created using Limesurvey™ was sent to the post-graduate deans of the 17 Canadian medical programs to determine the current state of Canadian GH elective infrastructure see . QuestioTwelve complete survey responses were received. These were completed by universities’ global health directors or post-graduate vice-deans; one respondent, belonging to a global health department, self-identified as “physician.” In certain cases, completed surveys were received from multiple individuals involved with global health at a single institution; these were synthesized to one response per university by including positive-over-negative responses . In total, responses were received from eight of 17 universities (47%): McGill University, University of Ottawa, Laval University, University of Toronto, University of Alberta, University of British Columbia, Queen’s University, and one anonymous institution. Responses were randomly coded to maintain anonymity.Six of the eight responding universities offer PDT for residents pursuing GH electives. The training is mandatory at three of these institutions. Of the three universities with non-mandatory PDT, two are aiming to make the training mandatory; the remaining university has its residents sign a guideline document. The content covered during PDT by the six universities is outlined in The locations to which universities send their post-graduate trainees are outlined in Regarding safety, five of the eight universities require participants to sign waivers outlining the risks of practicing abroad before departure. Bodily fluid exposure protocols are available at four of eight universities. The components offered include incident reports (n=2), home-site contact (n=4), on-site contact (n=0), post-exposure assessment procedure (n=3), HIV prophylaxis (n=1), HCV prophylaxis (n=0), written post-exposure protocol (n=3), on-site testing (n=1), communication with local site (n=1), communication with home centre (n=3), designated support person (n=1), and post-exposure counselling (n=4).Following elective return, seven of eight institutions provide a formal review process of the elective. PRD (post-return debriefing) is provided at three of eight institutions; all three discuss the topics of in-field support, training quality, ethical issues, safety, communication, housing, and cultural acclimatization.GH electives provide the opportunity for medical trainees in diverse settings to develop both their clinical and cultural competencies. The results of this survey are encouraging, suggesting that Canadian residents are completing electives across the globe with more infrastructure than currently mandated by bodies such as the RCPSC; however, there is still room for growth. The present study has identified four potential areas of development regarding Canadian GH elective infrastructure: PDT, professionalism, risk management, and PRD.PDT is available at the majority of institutions with varied coverage of content. This availability likely traces its roots back to the push for mandatory PDT for Canadian medical studentsProfessional guidelines are another resource that could be made more consistently available. During GH electives, residents may be exposed to ethical dilemmas beyond the scope of their home training.Post-exposure resource availability is another important gap in GH elective infrastructure. Since medical trainees under-report bodily fluid exposure,Finally, universities should be encouraged to uniformly offer PRD. PRD is a continuation of a process that begins with PDT that allows for the appraisal of the elective in the context of GH objectives;20Looking towards the future, it is necessary to address the barriers that prevent institutions from addressing gaps in their programming. Such barriers include the absence of guidelines by regulatory bodies, limited funding, lack of buy-in by residents and programs, and scheduling difficulties. To tackle these issues, the authors suggest recruiting organizational support on multiple fronts. Firstly, bodies that represent residents should consider educating trainees about the value of GH elective infrastructure and advocate for greater robustness. Secondly, medical colleges should consider developing standardized guidelines for residents performing GH electives, with specific regards to PDT, professionalism, risk management and PRD. Such a task has already been undertaken by certain colleges in the United States.One limitation of the present study was the response rate. We suspect that programs that do not offer GH electives elected not to respond to the survey. Regardless, the survey was considered geographically representative of Canadian medical institutions (with the exception of Atlantic Canada). A second limitation was the difficulty of assessing GH elective infrastructure by university. Since residency programs often organize GH infrastructure independently, the survey respondent may not have been fully aware of all the programming available at their university. Again, this points to the utility of a centralized GH body to disseminate information within, and eventually amongst, universities. In the meantime, future studies examining the availability of GH elective infrastructure by program would be useful to gauge the growth of elective infrastructure.Canadian medical programs are offering more institutional support to their trainees abroad than mandated; however, there is potential for further development. To address the gaps in GH elective infrastructure, the authors suggest the development of more comprehensive, standardized infrastructure by regulator/advocacy bodies. We also suggest the centralization of this infrastructure to GH departments to ensure widespread infrastructure availability amongst programs.


Trigeminal trophic syndrome is a rare condition that develops from trigeminal nerve damage causing dysesthesias that result in self-mutilation. Facial and nasal destruction develops from self-destructive behavior (repetitive picking or scratching) secondary to the altered skin sensation created by the damaged trigeminal nerve. Early recognition of this condition is crucial to the prevention of the detrimental complications of facial ulceration and nasal tissue necrosis that can lead to corneal ulcerations, full-thickness eyelid defect, and canthal lesions. This case demonstrates a previously unreported complication: orbital cellulitis. She had recently seen her ophthalmologist who noted the swelling and ordered magnetic resonance imaging (MRI), which showed inflammation extending from the right paranasal sinuses into the orbit. She was sent to the ED with concern for an infectious process such as mucormycosis. She had never had nasal, septal, or sinus surgery; however, she did have a remote history of trigeminal neuralgia treated with neurectomy 30 years prior. In the five years prior to this presentation, she had experienced facial paresthesias and pruritus managed by compulsive manipulation of her face, nose and sinuses with cotton tip applicators and fingers.On physical exam, the patient was nontoxic with normal vital signs: temperature 98.1° F, heart rate 94 beats per minute, respiratory rate 18 breaths/min, blood pressure 121/72 mm/Hg. Visual acuity in each eye was 20/20 without visual aids. She was noted to have right eye proptosis, periorbital edema and erythema, in addition to right medial gaze palsy. She had erosive necrosis with complete destruction of the right ala nasi and grossly patent right nasal aperture. While the right inferior turbinate appeared normal, the right middle turbinate was scarred with extensive right septal scarring and scabbing .3/cmm and serum glucose 216 mg/dl. Axial computed tomography (CT)   showed dThe patient was admitted with the diagnosis of orbital cellulitis and treated with parenteral vancomycin and piperacillin-tazobactam. Otolaryngology, ophthalmology, and psychiatry were consulted. Fiber optic endoscopy demonstrated extensive paranasal sinus destruction, with friable mucosa. The tissue destruction seen on CT and subsequent MRI was initially concerning for other serious infections, such as mucormycosis or malignancy; however, frozen pathology showed no evidence of an invasive fungal infection or malignant transformation. Other possible etiologies such as the autoimmune disorders Wegener’s granulomatosis or granulomatous polyangitis were considered.What do we already know about this clinical entity?Trigeminal trophic syndrome (TTS) is a condition that follows trigeminal nerve damage resulting in compulsive pruritic sensations, self-mutilation, and eventual tissue necrosis.What makes this presentation of disease reportable?Orbital cellulitis resulting from TTS has not been reported in the literature; clinicians should be made aware of this potential serious complication.What is the major learning point?Early detection of TTS and recognition that treatment involves medical and psychiatric therapy is necessary to prevent devastating and disfiguring complications.How might this improve emergency medicine practice?Recognition of this disease and the potential serious complications will lead to the early, multidisciplinary management approach necessary for successful treatment, thus improving outcomes.Staphylococcus aureus, and the patient rapidly improved with the appropriate intravenous antibiotics. Psychiatric consultation determined that the patient had a component of an unrecognized OCD. The combination of ongoing facial paresthesia in the V2 division of the trigeminal nerve resulting from the previous trigeminal neuralgia surgery and the newly diagnosed OCD provided the diagnosis of TTS leading to the orbital cellulitis.The required rheumatologic work up to evaluate for these potential causes would include the following: antinuclear antibody; C-reactive protein; erythrocyte sedimentation rate; and anti-neutrophil cytoplasmic antibodies. These lab tests were abandoned when the sinus culture was positive for The patient was ultimately discharged on a course of oral amoxicillin-clavulanic acid and daily fluoxetine to address her previously undiagnosed obsessive-compulsive behavior. In a four-month clinic follow-up, the right ala nasi still appeared necrotic with the right nasal aperture still being widely patent. There was no overt evidence of active infection. The patient did not complain of any visual compromise and no visual acuity was obtained. The patient reported medication non-compliance and continued repetitive manual manipulation of her nose. She refused any further treatment.1) and the maxillary nerve brach (V2).”4TTS is a rare condition that develops due to self-destructive behavior coupled with a damaged trigeminal nerve. The damaged trigeminal nerve most commonly is the result of trigeminal surgical ablation or alcohol injection for treatment of trigeminal neuralgia; however, it has been reported with cerebral meningioma resection, cerebrovascular accidents, herpes zoster ophthalmicus, vertebrobasilar insufficiency, acoustic neuroma, post-encephalitic parkinsonism, syringobulbia, meningioma, astrocytoma, Hansen’s disease, and trauma.The differential diagnosis of patients with facial or nasal ulcerations can be extensive. Herpetic ulceration and other infectious diseases such as syphilis, yaws, leprous trigeminal neuritis, deep fungi, mycobacteria, rhinoscleroma, and leishmaniosis are among the possible infectious etiologies.The time period from trigeminal nerve injury to onset of the ulcer can be from weeks to several years, with a mean of 1–2 years.4Though not previously described, our case demonstrates that orbital cellulitis can also complicate TTS. In this case, the patient admitted to routine manual nose picking and repeatedly using a cotton swab to manipulate her nose for 4–5 years before presentation. This obsessive behavior led to the right alar necrosis and sinus erosion, an unlikely but direct etiology of the orbital cellulitis. Her history of diabetes most likely contributed to the severity of her infection. The management of this patient involved treating the orbital cellulitis with intravenous vancomycin and piperacillin-tazobactam. In an attempt to control her obsessive skin manipulation, she was started on fluoxetine. Sadly, the patient chose to discontinue her fluoxetine and return to self-mutilation, refusing any further attempts to improve her conditionThis case demonstrates a previously undescribed complication of trigeminal nerve injury in a patient with OCD. Trigeminal trophic syndrome is an important consideration when caring for patients with facial ulcerations and/or nasal destructive lesions who have a history of trigeminal nerve injury, even with a 30-year latency. Patients with psychiatric disorders and trigeminal neuralgia requiring surgical intervention, require pre-surgical counseling, extra vigilance in post-surgical follow-up, and consideration of appropriate psychiatric medications to prevent the complications of this syndrome.Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report.


The response to different force load ranges and actuation at low energies is of considerable interest for applications of compliant and flexible devices undergoing large deformations. We present a review of technological platforms based on nitride materials  for the microfabrication of a class of flexible micro-electro-mechanical systems. The approach exploits the material stress differences among the constituent layers of nitride-based (AlN/Mo, Si Inspired by nature, where biological sensors can adapt their mechanical sensitivity to cope with large changes in the environment, flexible functional materials can be used to tune and efficiently control how mechanical information can be detected and transferred in a biomimetic sensor design. Recent developments in thin-film-based MEMS have enabled the cost-effective manufacture of these flexible functional materials.The sensing elements of the MEMS devices we review here typically consist of deformable flexural elements, such as cantilever beams, membranes and bridges, which are suspended above a base layer. These are usually designed and fabricated with silicon as the structural material ,2. MultiThe flexural stiffness of the deformable elements has to be carefully designed and implemented by growth techniques in order to maximize the sensitivity of the sensor while ensuring it does not fail due to mechanical overloading. The micro-scale geometrical design made possible through modern MEMS manufacturing techniques provides the tool required to solve this apparent paradox. The thickness, width, length and curvature of the deformable element can all be determined quantitatively at design time, and the cavities left behind during the release process can provide shelter for the deformable element during periods of overload by distributing the force over the larger and stronger main substrate.This review focuses on approaches to the design and fabrication of “stress-driven” flexible MEMS for sensing applications, providing an overview of some recent technological improvement in microfabrication of nitride-based devices  as a platform for developing a class of compliant and flexible MEMS undergoing and withstanding large deformations. In G, Young’s modulus E and Poisson ratio t) can be combined to design a specific flexural stiffness for flexible MEMS elements such as membranes and beams. Embedding and overlaying thin films with different thermal and elastic properties generates internal mechanical stresses. These forces are caused by multiple factors. The mismatch of atomic sizes in the different materials or the lattice-mismatch due to thermal treatments during material deposition and device microfabrication cause an intrinsic stress, which is usually considered detrimental to MEMS purposes, as it often causes failure due to surface wrinkles [The realization of a stress-driven design requires a careful choice of material characteristics and an appropriate technology for material deposition. The proper choice of mechanical properties  , polycryThe approach described above has been realized by exploiting nitride-based materials, such as Silicon Nitride (SiN) and Aluminum Nitride (AlN), whose intrinsic stress can be controlled via physical sputtering deposition or Low Pressure Chemical Vapor Deposition (LPCVD).The careful control of silicon nitride residual stress has been studied since 1985, when Claassen et al.  studied  1200 μm . The pla 1200 μm ,10,15.Aluminum nitride thin films can be grown by sputtering with excellent polycrystalline quality on different substrates including dielectrics, semiconductors and metallic layers, as well as on flexible substrates, such as polymers and polyimides ,22,23,24 devices . Previou devices ,17,22. I devices  investigIn dependence of which stress-driven-based mechanical element will be fabricated, a proper substrate needs to be chosen. In order to obtain the control of residual stress in a cantilever structure, a silicon dioxide/silicon template can be used. Flexible beam fabrication was demonstrated on a silicon template made up of a 200 ubstrate . A 600 ny 150 μm . Controly 150 μm , commercy 150 μm  and paryy 150 μm  flexiblef 350 μm . Consequf 350 μm ,27,28.The piezoelectric properties of aluminum nitride transform mechanical bending  into electrical signals, turning the bent structure into an efficient transducer. AlN possesses a number of key characteristics that make it very attractive when compared to other piezoelectric materials. ZnO and lead zirconate titanate (PZT) are well-known piezoelectric materials, which are limited in their application by the complexity and compatibility of their fabrication process (contamination risks in standard CMOS lines). Organic polymers such as Polyvinylidene Fluoride (PVDF) and its copolymer Poly(Vinylidene Fluoride-co-Trifluoroethylene) (P(VDF-TrFE)) have been widely used as tactile sensor piezoelectric materials ,30,31. PPoly-crystalline inorganic aluminum nitride thin films can be obtained and easily deposited by reactive sputtering at relatively low temperature (from 160 –300 ∘C) ,33 and dn et al. . Finallyn et al. ,36.The integration of flexible polyimide with AlN thin film deposited by sputtering techniques has made possible the development of a wide range of flexible and compliant devices, from piezoelectric tactile sensors to piezoelectric micromachined Ultrasonic Transducers (pMUTs) and micro power generators, all based on stress differences between the constituent layers. By exploiting the quasi-unavoidable mechanical stress in these films due to the lattice mismatch and difference in thermal expansion coefficients of film and substrate, the sensory performance of structures such as upwards-bent cantilever beams and domed circular membranes can be improved.This section describes the microfabrication process of two different classes of stress-driven MEMS structures, namely piezoresistive upwards-bent cantilever beams and piezoelectric tactile membranes.In the past, different stressor layers were micromachined on a silicon or silicon-on-insulator wafer, which provided the required residual stress to shape cantilevers that bent out of the plane after releasing, such as silicon nitride ,10,15, sIn this section, we review the processes used to fabricate these three stressor layers  and the choice of the manufacturing strategy as described in the previous section is the way to achieve devices that are able to mimic and recreate the capabilities of biological sensors.Over the course of evolution, fish have evolved a special sensory organ ideally adapted to their aquatic environment: the lateral line organ offers valuable information about the adjacent water movement by detecting changes in the pressure and water flow along the body. This so-called “distant touch” forewarns fish of predators, helps to avoid obstacles and may play a role in saving energy during underwater locomotion ,45.The individual sensory receptors in the lateral line organ, hair-like structures called neuromasts, detect the tiniest differences in pressure and perceive fractional changes of the flow velocity . As a geFrom an engineering and biomimetics perspective, the implementation of an artificial lateral line system, inspired by how nature has solved the problem in fish, is a promising approach to explore hydrodynamics and to test the hypothesis that lateral line sensing can improve the propulsion efficiency, sensing capability and feedback control systems in underwater vehicles.Artificial lateral line flow sensing applications require hair-like structures with a length enough to reach into the laminar flow layer above the vehicle’s skin. Production processes employed in microsystems technology allow for sufficient miniaturization of the components required to develop bio-inspired artificial hair cells with the required dimensions. Various piezoresistive-based sensor design methodologies and MEMS fabrication processes for producing hair cell-like flow sensors have been developed in various laboratories world-wide, as thoroughly described in several comprehensive literature reviews ,49,50,51Wang et al.  performeZhang et al.  used deiThe stress-driven artificial hair cell described by Qualtieri et al.  was teste sensor ,38. FiguKlein and Bleckmann  and HerzWang et al.  positionshown in ), the suIn the flow sensing application described by Abels et al. , a lineaThe human tactile sense is an extremely accurate and sensitive active organ. It is responsible for detecting mechanical stimuli in a pressure range from 10–100  texture ,70. Tact texture ,72. Huma texture ,74.From an engineering point of view, humanoid robots need tactile interfaces to perform complex tasks in unstructured environments and for safely interacting with humans in daily and routinely executed activities, such as assistance, support and coexistence. Bio-mimicking abilities of the human tactile system and performing advanced in-hand manipulation tasks are made possible by detecting static forces, as well as dynamic forces, such as normal and tangential contact for gathering spatial and geometrical information from surface exploration. Arrays of pressure-sensitive sensors could be integrated into an electronic skin to complement the desirable properties of flexibility, conformability and stretchability in improving anthropomorphism ,76.In the last decade, the development of flexible materials for tactile sensing applications has attracted an increasing interest in robotics, industrial and manipulation applications, prosthetic and orthotic devices and tools for the evaluation of tissue stiffness and for preventing damage ,33,39,40Piezoelectric micromachined transducers are of great importance as measurement tools for pulse-echo ultrasonics applications in robotics, medical diagnostic and proximity detection ,78,79,80Using flat tactile pressure sensors that exploit the dependence of the piezoelectric voltage on the contact force, Dagdeviren et al.  as show used an 2 mN see . Khan etAn improvement in the sensitivity of piezoelectric tactile sensors can be achieved by adding complexity to the geometry of the sensor. Kim et al.  fabricatAs illustrated in rate see  [27,81,8Dome-shaped devices with different releasing height .The mechanical behavior of the circular membranes was investigated by driving the piezoelectric film by means of a sinusoidal voltage whose frequency sweeps from 0.5 Hz–2 MHz, as presented in  Vpp see d, even iIn this review, we described a stress-driven approach to design and fabricate flexible nitride-based MEMS devices for sensing applications. A controllable stress gradient along the cross-section of thin aluminum nitride and silicon nitride layers can be induced by appropriately designing and micromachining multilayered thin films. By depositing piezoresistive nichrome strain gauges onto nitride-based multilayers or by using direct piezoelectric properties of aluminum nitride-based microstructures, highly sensitive mechanical strain/stress detection can be realized, turning dome-shaped membranes and upwards-bent cantilevers into tactile and flow sensors for various fields of applications. Future development on stress-driven bio-inspired technology will be directed towards large area systems for robotics and ultrasonics applications. Next generation artificial hair cell-like flow sensors might not only approximate the morphological properties of biological neuromasts more precisely, but also bio-mimic physiological functions, such as bidirectional sensitivity and interconnections between individual hair cells. Dome-shaped devices could be designed as an array of sensors and ultrasonics generators for both dynamic and static tactile sensing based on contact and ultrasonic range finding. Future developments will be focused on the experimental evaluation of the contribution of flexoelectricity in a tactile stress-driven device as a function of the AlN material thickness, in order to better control flexoelectric properties.


Among others, various types of biomaterials and transfection method have been employed as key tools for development of the in vitro settings. This review focuses on the current strategies to improve stemness maintenance, direct differentiation, and reprogramming using biomaterials and genetic factors without any aids from additional biochemicals and growth factors.Stem cells have recently emerged as an important candidate for cell therapy. However, some major limitations still exist such as a small quantity of cell supply, senescence, and insufficient differentiation efficiency. Therefore, there is an unmet need to control stem cell behavior for better clinical performance. Since native microenvironment factors including stem cell niche, genetic factors, and growth factors direct stem cell fate cooperatively, user-specified  Stem cell therapy possesses significant advantages compared to conventional cell therapy using mature cells, as stem cells are more accessible and obtainable, are easy to culture and expand, and enable avoiding graft-versus-host rejection –3. With in vitro culture, the efficacy of their proliferation and differentiation decreases due to a progressive loss of stemness driven by senescence. To overcome such problems, state-of-the-art technologies using biomaterials, genetic factors, and growth factors which can mimic a native microenvironment or improve stem cell behavior have been employed recently. In conventional studies, various growth factors or cytokines were pretreated to stem cells during in vitro cultivation to induce a specific direction of differentiation for transplanting in a damaged tissue ..ε-caprolFurthermore, nanofibrous scaffolds provided a 3D microenvironment to stem cells and thus enhanced stemness. For example, ASCs displayed an improved adhesion capacity with high rates of bioactivity and proliferation when cultured on emu oil-loaded nanofibers . MesenchFinally, a chitosan film induced spheroid formation and triggered a cell-cell interaction of hASCs, thereby enhancing stemness. After spheroid formation, the spheroid-forming hASCs expanded efficiently, formed a colony, and upregulated the expression of pluripotency marker genes compared to the monolayer-cultured control condition , 57.In conclusion, the aforementioned types and formats of biomaterials were found to enhance or maintain stemness. We summarize the three major mechanisms by which the biomaterials enhanced or maintained stemness as follows: (1) reduction of cytoskeletal tension by reducing matrix stiffness, (2) spheroid formation by reduction of integrin-binding sites and consequent promotion of cell-cell interaction, and (3) antioxidative effects by radical scavenging activity. These strategies have potential to effectively improve stemness of MSCs in various biomaterial formats.While most methods known to induce mesenchymal lineage differentiation of MSC depend on exposure to one or more soluble growth factors, a growing body of evidence suggests that it is possible to control MSC differentiation in the absence of soluble factors. MSCs exhibit the ability to differentiate towards specific lineages through biomaterials with modification of mechanical or biochemical properties, matrix composition, topography, and surface stiffness. This approach would simplify the tissue engineering procedure and be cost-effective. Here, we summarize recent studies that employed such approaches to induce direct differentiation of MSC via biomaterial technologies .Collagen and glycosaminoglycan (GAG), major components of natural ECM, play a key role in osteochondral regeneration. Hence, their combined (CG) scaffolds have been used successfully in tissue engineering applications for regeneration of cartilage and bone , 58. A pβ when the particles were used as a cell carrier [Recent studies investigated whether chondrogenic differentiation of MSC could be directed by biomimetic or decellularized tissue-derived materials. Biomimetic polyacrylate substrates functionalized with the RGD integrin-binding peptide promoted chondrogenesis of MSC in the absence of any soluble growth factors . They su carrier .Calcium phosphate- (CaP-) based ceramics play a significant role in bone repair due to their osteoconductive potential for filling lost bone volumes, when CaP nanoparticles and demineralized bone matrix (DBM) were fabricated as an injectable bone graft by incorporating polymerized high internal phase emulsions (polyHIPEs). This injectable bone graft was found to induce direct osteogenesis of MSC . On the Proper regeneration of the myocardium is dependent on scaffold properties and thus can be enhanced by mimicking features of the myocardial ECM. A three-hydroxybutyrate and 3-hydroxyvalerate (PHBHV)/gelatin construct mimicking the myocardial ECM structure was developed to promote cardiac differentiation of MSC and cardiac resident cells without any chemical stimulation . This stin vitro cultures, stiffness of culture substrates was suggested first as a key property in several important studies [β1 played a critical role in this process. Cells sense their mechanical microenvironment via integrin-ligand interactions which form focal adhesions and thereby regulate intracellular signaling [Among the biophysical cues that were identified to regulate cell fate in static  studies –64. An i studies , 61. The studies . This stignaling . Anotherin vitro and in vivo. Our results showed that GHPA hydrogels induced spontaneous endothelial differentiation of MSC without any soluble factors.Although the differentiation potential of MSCs into endothelial cells (ECs) remains unclear , 69, somμm line, 20 μm spacing, and 1 μm height) of fibronectin strips printed on a poly(lactic-co-glycolic acid) (PLGA) thin film were found to direct linage commitment of MSCs [μm) through α2β1 integrin-mediated interactions with cocultured osteoblasts [When cells are cultured on biomaterial substrates, surface topography is known as a key regulator of cell behavior. Several previous studies reported that surface topographical cues induced direct lineage specification of MSCs , 70–72.  of MSCs . In this of MSCs . As a re of MSCs , 72. The of MSCs . It was Induced pluripotent stem cells (iPSCs) were introduced in 2006, which opened a new avenue for stem cell research and regenerative medicine . ObtainiResident stem cells in various tissues were also studied as a promising source of stem cells, but the lack of appropriate markers to define their phenotypes and their low differentiation potential were considered as major hurdles for using these cell sources. Hence, the breakthrough idea of reprogramming somatic cells with ectopic pluripotent markers  to eventually represent embryonic stem cell (ESC) characteristics was undisputedly attractive . Howeverβ receptor antagonist also significantly increased the reprogramming efficiency and kinetics in murine [Substantial progress has been made in the methodologies to improve the efficiency and efficacy of reprogramming somatic cells to iPSCs in the past decade . The typn murine , 96 and n murine .It needs to be investigated further whether the type of vectors used to induce pluripotency contributes to the heterogeneity of produced iPSC lines or not. When retrovirus, Sendai virus, and episomal vectors were used for iPSC generation, different reprogramming strategies were applied to obtain human iPSCs. As a result, very similar global gene expression profiles were displayed but subtle differences were observed in the levels of gene expression, indicating that the heterogeneity of produced iPSC lines resulted from clonal signatures rather than the reprogramming method itself . HoweverAs a summary, random DNA aberrations are most notably caused by viral genome integration, leading to iPSC heterogeneity and unpredictability. Thus, nonintegrating systems should be primarily considered as a basic means for differentiation strategies towards clinical applications.The origin and quality of donor cells are also important factors to ensure successful reprogramming results. In particular, easy access, abundant quantity, and enough replenishment of donor cells after harvesting should be considered when the target donor source is selected.+ peripheral blood mononuclear cells), amniotic fluid cells, cord blood stem cells, endometrial stromal fibroblasts, and dental pulp cells have been reported as reliable sources of somatic cells for reprogramming (Donor somatic cell-specific transcriptional and epigenetic signatures significantly contribute to the heterogeneity of efficiency and efficacy in reprogramming and differentiation of iPSCs . The mosgramming  [100–106If stemness is enhanced, the negative effect of tissue-specific epigenetic signature may be attenuated during the process of serial passaging while losing the epigenetic memory sequentially. Characteristic DNA methylation patterns are originated from the donor cells and thus can be tracked in specific iPSC clones. Consequently, limitations in stemness of iPSCs as opposed to the full pluripotency of embryonic stem cells are inevitable. Experimental techniques to overcome the gap between the epigenetic memory of the donor cell and the stemness of the derived iPSC lines have been described in previous studies , 111. OnAlthough a growing body of evidence suggests stem cells as a promising candidate for cell therapy in the position of replacing somatic cells, the aforementioned issues regarding senescence and low differentiation efficiency must be addressed for successful clinical applications. In this review, we introduced state-of-the-art methods which are currently approached to improve efficiency and efficacy of stemness maintenance, direct differentiation, and iPSC reprogramming, with the minimal use of expensive and side effect-occurring growth factors. Biophysical stimulation, organic compound treatment, genetic transfection, and various types of biomaterials were employed to achieve the purposes. In particular, the effects of matrix stiffness, improving cell-cell interaction, and antioxidant treatment became a major part of interest. Additionally, biomaterials with specific composition, stiffness, and topography can serve as a promising toolbox to guide direct differentiation of stem cells. Finally, several combinations or individual uses of genetic factors to induce reprogramming of somatic cells were introduced as a means of generating iPSCs. Pros and cons of major reprogramming methods were discussed as well. Taken together, selection of biomaterials or other external factors needs to be customized for target-specific developments and application of stem cell therapy towards successful clinical applications.