3D Brain Vascular Niche Model Captures Invasive Behavior and Gene Signatures of Glioblastoma.

Glioblastoma (GBM) is a lethal brain cancer with no effective treatment; understanding how GBM cells respond to tumor microenvironment remains challenging as conventional cell cultures lack proper cytoarchitecture while in vivo animal models present complexity all at once. Developing a culture system to bridge the gap is thus crucial. Here, we employed a multicellular approach using human glia and vascular cells to optimize a 3-dimensional (3D) brain vascular niche model that enabled not only long-term culture of patient derived GBM cells but also recapitulation of key features of GBM heterogeneity, in particular invasion behavior and vascular association. Comparative transcriptomics of identical patient derived GBM cells in 3D and in vivo xenotransplants models revealed that glia-vascular contact induced genes concerning neural/glia development, synaptic regulation, as well as immune suppression. This gene signature displayed region specific enrichment in the leading edge and microvascular proliferation zones in human GBM and predicted poor prognosis. Gene variance analysis also uncovered histone demethylation and xylosyltransferase activity as main themes for gene adaption of GBM cells in vivo . Furthermore, our 3D model also demonstrated the capacity to provide a quiescence and a protective niche against chemotherapy. In summary, an advanced 3D brain vascular model can bridge the gap between 2D cultures and in vivo models in capturing key features of GBM heterogeneity and unveil previously unrecognized influence of glia-vascular contact for transcriptional adaption in GBM cells featuring neural/synaptic interaction and immunosuppression.

An architecturally rational hemostat for rapid stopping of massive bleeding on anticoagulation therapy.

Hemostatic devices are critical for managing emergent severe bleeding. With the increased use of anticoagulant therapy, there is a need for next-generation hemostats. We rationalized that a hemostat with an architecture designed to increase contact with blood, and engineered from a material that activates a distinct and undrugged coagulation pathway can address the emerging need. Inspired by lung alveolar architecture, here, we describe the engineering of a next-generation single-phase chitosan hemostat with a tortuous spherical microporous design that enables rapid blood absorption and concentrated platelets and fibrin microthrombi in localized regions, a phenomenon less observed with other classical hemostats without structural optimization. The interaction between blood components and the porous hemostat was further amplified based on the charged surface of chitosan. Contrary to the dogma that chitosan does not directly affect physiological clotting mechanism, the hemostat induced coagulation via a direct activation of platelet Toll-like receptor 2. Our engineered porous hemostat effectively stopped the bleeding from murine liver wounds, swine liver and carotid artery injuries, and the human radial artery puncture site within a few minutes with significantly reduced blood loss, even under the anticoagulant treatment. The integration of engineering design principles with an understanding of the molecular mechanisms can lead to hemostats with improved functions to address emerging medical needs.

Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

BACKGROUND: Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. METHODS: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. RESULTS: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. CONCLUSIONS: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics.

Infiltrative growth is a major cause of high lethality of malignant brain tumors such as glioblastoma (GBM). We show here that GBM cells upregulate guidance receptor Plexin-B2 to gain invasiveness. Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies establish Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness.

High-resolution tomographic analysis of in vitro 3D glioblastoma tumor model under long-term drug treatment.

Glioblastoma multiforme (GBM) is a lethal type of brain tumor that often develop therapeutic resistance over months of chemotherapy cycles. Recently, 3D GBM models were developed to facilitate evaluation of drug treatment before undergoing expensive animal studies. However, for long-term evaluation of therapeutic efficacy, novel approaches for GBM tissue construction are still needed. Moreover, there is still a need to develop fast and sensitive imaging methods for the noninvasive assessment of this 3D constructs and their response to drug treatment. Here, we report on the development of an integrated platform that enable generating (i) an in vitro 3D GBM model with perfused vascular channels that allows long-term culture and drug delivery and (ii) a 3D imaging modality that enables researchers to noninvasively assess longitudinal fluorescent signals over the whole in vitro model.

Engineering transferrable microvascular meshes for subcutaneous islet transplantation.

The success of engineered cell or tissue implants is dependent on vascular regeneration to meet adequate metabolic requirements. However, development of a broadly applicable strategy for stable and functional vascularization has remained challenging. We report here highly organized and resilient microvascular meshes fabricated through a controllable anchored self-assembly method. The microvascular meshes are scalable to centimeters, almost free of defects and transferrable to diverse substrates, ready for transplantation. They promote formation of functional blood vessels, with a density as high as ~220 vessels mm-2, in the poorly vascularized subcutaneous space of SCID-Beige mice. We further demonstrate the feasibility of fabricating microvascular meshes from human induced pluripotent stem cell-derived endothelial cells, opening a way to engineer patient-specific microvasculature. As a proof-of-concept for type 1 diabetes treatment, we combine microvascular meshes and subcutaneously transplanted rat islets and achieve correction of chemically induced diabetes in SCID-Beige mice for 3 months.

BTBR ob/ob mouse model of type 2 diabetes exhibits early loss of retinal function and retinal inflammation followed by late vascular changes.

AIMS/HYPOTHESIS: Diabetic retinopathy is increasing in prevalence worldwide and is fast becoming a global epidemic and a leading cause of visual loss. Current therapies are limited, and the development of effective treatments for diabetic retinopathy requires a greater in-depth knowledge of disease progression and suitable modelling of diabetic retinopathy in animals. The aim of this study was to assess the early pathological changes in retinal morphology and neuronal, inflammatory and vascular features consistent with diabetic retinopathy in the ob/ob mouse model of type 2 diabetes, to investigate whether features similar to those in human diabetic retinopathy were present. METHODS: Male and female wild-type (+/+), heterozygous (+/-) and homozygous (-/-) BTBR ob/ob mice were examined at 6, 10, 15 and 20 weeks of age. Animals were weighed and blood glucose was measured. TUNEL and brain-specific homeobox/POU domain protein 3A (BRN3A) markers were used to examine retinal ganglion cells. We used immunostaining (collagen IV and platelet endothelial cell adhesion molecule [PECAM]/CD31) to reveal retinal vessel degeneration. Spectral domain optical coherence tomography was used to reveal changes in the thickness and structure of the retinal layer. Vitreous fluorophotometry was used to investigate vascular permeability. A-waves, b-waves and oscillatory potentials were measured under photopic and scotopic conditions. Concanavalin A leucostasis and immunostaining with glial fibrillary acidic protein (GFAP) and ionised calcium-binding adapter molecule 1 (IBA-1) identified differences in inflammatory status. Paraffin sections and transmission electron microscopy were used to reveal changes in the thickness and structure of the retinal layer. RESULTS: Following the development of obesity and hyperglycaemia in 2-week-old and 3-week-old ob-/ob- mice, respectively (p < 0.001), early functional deficits (p < 0.001) and thinning of the inner retina (p < 0.001) were identified. Glial activation, leucostasis (p < 0.05) and a shift in microglia/macrophage phenotype were observed before microvascular degeneration (p < 0.05) and elevated vascular permeability occurred (p < 0.05). CONCLUSIONS/INTERPRETATION: The present characterisation of the development of diabetic retinopathy in the ob/ob mouse represents a platform that will enable the development of new therapies, particularly for the early stages of disease.

Printing of Three-Dimensional Tissue Analogs for Regenerative Medicine.

Three-dimensional (3-D) cell printing, which can accurately deposit cells, biomaterial scaffolds and growth factors in precisely defined spatial patterns to form biomimetic tissue structures, has emerged as a powerful enabling technology to create live tissue and organ structures for drug discovery and tissue engineering applications. Unlike traditional 3-D printing that uses metals, plastics and polymers as the printing materials, cell printing has to be compatible with living cells and biological matrix. It is also required that the printing process preserves the biological functions of the cells and extracellular matrix, and to mimic the cell-matrix architectures and mechanical properties of the native tissues. Therefore, there are significant challenges in order to translate the technologies of traditional 3-D printing to cell printing, and ultimately achieve functional outcomes in the printed tissues. So it is essential to develop new technologies specially designed for cell printing and in-depth basic research in the bioprinted tissues, such as developing novel biomaterials specifically for cell printing applications, understanding the complex cell-matrix remodeling for the desired mechanical properties and functional outcomes, establishing proper vascular perfusion in bioprinted tissues, etc. In recent years, many exciting research progresses have been made in the 3-D cell printing technology and its application in engineering live tissue constructs. This review paper summarized the current development in 3-D cell printing technologies; focus on the outcomes of the live printed tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions of 3-D cell printing technology are also discussed.

Generation of Multi-Scale Vascular Network System within 3D Hydrogel using 3D Bio-Printing Technology.

Although 3D bio-printing technology has great potential in creating complex tissues with multiple cell types and matrices, maintaining the viability of thick tissue construct for tissue growth and maturation after the printing is challenging due to lack of vascular perfusion. Perfused capillary network can be a solution for this issue; however, construction of a complete capillary network at single cell level using the existing technology is nearly impossible due to limitations in time and spatial resolution of the dispensing technology. To address the vascularization issue, we developed a 3D printing method to construct larger (lumen size of ~1mm) fluidic vascular channels and to create adjacent capillary network through a natural maturation process, thus providing a feasible solution to connect the capillary network to the large perfused vascular channels. In our model, microvascular bed was formed in between two large fluidic vessels, and then connected to the vessels by angiogenic sprouting from the large channel edge. Our bio-printing technology has a great potential in engineering vascularized thick tissues and vascular niches, as the vascular channels are simultaneously created while cells and matrices are printed around the channels in desired 3D patterns.

Creating perfused functional vascular channels using 3D bio-printing technology.

We developed a methodology using 3D bio-printing technology to create a functional in vitro vascular channel with perfused open lumen using only cells and biological matrices. The fabricated vasculature has a tight, confluent endothelium lining, presenting barrier function for both plasma protein and high-molecular weight dextran molecule. The fluidic vascular channel is capable of supporting the viability of tissue up to 5 mm in distance at 5 million cells/mL density under the physiological flow condition. In static-cultured vascular channels, active angiogenic sprouting from the vessel surface was observed whereas physiological flow strongly suppressed this process. Gene expression analysis was reported in this study to show the potential of this vessel model in vascular biology research. The methods have great potential in vascularized tissue fabrication using 3D bio-printing technology as the vascular channel is simultaneously created while cells and matrix are printed around the channel in desired 3D patterns. It can also serve as a unique experimental tool for investigating fundamental mechanisms of vascular remodeling with extracellular matrix and maturation process under 3D flow condition.

Mesoscopic fluorescence molecular tomography of reporter genes in bioprinted thick tissue.

Three-dimensional imaging of thick tissue constructs is one of the main challenges in the field of tissue engineering and regenerative medicine. Optical methods are the most promising as they offer noninvasive, fast, and inexpensive solutions. Herein, we report the use of mesoscopic fluorescence molecular tomography (MFMT) to image function and structure of thick bioprinted tissue hosted in a 3-mm-thick bioreactor. Collagen-based tissue assembled in this study contains two vascular channels formed by green fluorescent protein- and mCherry-expressing cells. Transfected live cell imaging enables us to image function, whereas Flash Red fluorescent bead perfusion into the vascular channel allows us to image structure. The MFMT optical reconstructions are benchmarked with classical microscopy techniques. MFMT and wide-field fluorescence microscopy data match within 92% in area and 84% in location, validating the accuracy of MFMT reconstructions. Our results demonstrate that MFMT is a well-suited imaging modality for fast, longitudinal, functional imaging of thick, and turbid tissue engineering constructs.

The integration of 3-D cell printing and mesoscopic fluorescence molecular tomography of vascular constructs within thick hydrogel scaffolds.

Developing methods that provide adequate vascular perfusion is an important step toward engineering large functional tissues. Meanwhile, an imaging modality to assess the three-dimensional (3-D) structures and functions of the vascular channels is lacking for thick matrices (>2 ≈ 3 mm). Herein, we report on an original approach to construct and image 3-D dynamically perfused vascular structures in thick hydrogel scaffolds. In this work, we integrated a robotic 3-D cell printing technology with a mesoscopic fluorescence molecular tomography imaging system, and demonstrated the capability of the platform to construct perfused collagen scaffolds with endothelial lining and to image both the fluid flow and fluorescent-labeled living endothelial cells at high-frame rates, with high sensitivity and accuracy. These results establish the potential of integrating both 3-D cell printing and fluorescence mesoscopic imaging for functional and molecular studies in complex tissue-engineered tissues.

Intravenous acetaminophen.

Acetaminophen has unique analgesic and antipyretic properties. It is globally recommended as a first-line agent for the treatment of fever and pain due to its few contraindications. Acetaminophen lacks the significant gastrointestinal and cardiovascular side effects associated with nonsteroidal anti-inflammatory drugs and narcotics. An intravenous formulation of acetaminophen is available in Europe and is currently undergoing extensive clinical development for use in the United States. This use may have important implications for management of postoperative pain and fever. This review summarizes recent clinical trial experiences with intravenous acetaminophen for the treatment of postoperative pain and fever in adult and pediatric subjects.

Computer-based insulin infusion protocol improves glycemia control over manual protocol.

OBJECTIVE: Hyperglycemia worsens clinical outcomes in critically ill patients. Precise glycemia control using intravenous insulin improves outcomes. To determine if we could improve glycemia control over a previous paper-based, manual protocol, authors implemented, in a surgical intensive care unit (SICU), an intravenous insulin protocol integrated into a care provider order entry (CPOE) system. DESIGN: Retrospective before-after study of consecutive adult patients admitted to a SICU during pre (manual protocol, 32 days) and post (computer-based protocol, 49 days) periods. MEASUREMENTS: Percentage of glucose readings in ideal range of 70-109 mg/dl, and minutes spent in ideal range of control during the first 5 days of SICU stay. RESULTS: The computer-based protocol reduced time from first glucose measurement to initiation of insulin protocol, improved the percentage of all SICU glucose readings in the ideal range, and improved control in patients on IV insulin for > or =24 hours. Hypoglycemia (<40 mg/dl) was rare in both groups. CONCLUSION: The CPOE-based intravenous insulin protocol improved glycemia control in SICU patients compared to a previous manual protocol, and reduced time to insulin therapy initiation. Integrating a computer-based insulin protocol into a CPOE system achieved efficient, safe, and effective glycemia control in SICU patients.