Physiological Fluid Flow Research Articles

3D-bioprinted all-inclusive bioanalytical platforms for cell studies

Innovative drug screening platforms should improve the discovery of novel and personalized cancer treatment. Common models such as animals and 2D cell cultures lack the proper recapitulation of organ structure and environment. Thus, a new generation of platforms must consist of cell models that accurately mimic the cells’ microenvironment, along with flexibly prototyped cell handling structures that represent the human environment. Here, we adapted the 3D-bioprinting technology to develop multiple all-inclusive high throughputs and customized organ-on-a-chip-like platforms along with printed 3D-cell structures. Such platforms are potentially capable of performing 3D cell model analysis and cell-therapeutic response studies. We illustrated spherical and rectangular geometries of bio-printed 3D human colon cancer cell constructs. We also demonstrated the utility of directly 3D-bioprinting and rapidly prototyping of PDMS-based microfluidic cell handling arrays in different geometries. Besides, we successfully monitored the post-viability of the 3D-cell constructs for seven days. Furthermore, to mimic the human environment more closely, we integrated a 3D-bioprinted perfused drug screening microfluidics platform. Platform’s channels subject cell constructs to physiological fluid flow, while its concave well array hold and perfused 3D-cell constructs. The bio-applicability of PDMS-based arrays was also demonstrated by performing cancer cell-therapeutic response studies.

Axisymmetric mixed convective propulsion of a non-Newtonian fluid through a ciliated tubule

Thousands of chemical reactions occur in the human body when certain biological fluids, such as blood, semen, mucus, and synovial joint materials, move in various organs. These reactions play a vital role in regulating the life sustaining metabolic processes in the body. Analysis of thermal effects on these chemical reactions is relatively a new area in modern clinical medications. The present study investigates a simulation of the combined response due to heat and mass transport mechanisms taking place in the human body during the flow of physiological fluids. In particular, we focus our attention on the human male reproductive system, wherein the semen transports through the ductus efferentes due to metachronal waves of cilia. The constitutive relations of the robust Jeffrey viscoelastic fluid are used to model the human semen. The mathematical model of the present problem constitutes the axisymmetric flow of a Jeffrey fluid inside a vertical tubule under the influence of mixed convective heat and mass transfers. The inner side of the tubule is covered with ciliated structures. The influence of thermal behaviors of various metabolic processes in the human body due to an external heat source or sink is also taken into account. The mathematical formulation consists of using the approach of lubrication theory approximation; the nonlinear momentum, energy, and concentration equations are simplified to get analytical solutions. Explicit expressions for temperature, concentration, velocity, pressure gradient, and volume flow rate of the proposed bodily fluid (i.e., human semen) are formulated. The expression for the volume flow rate is used to estimate the volume flux of the semen under the influence of various parameters. A comparison between the theoretical and experimentally obtained values of the flow rate of the human semen is also made. It is noted that our calculated values are very close to the estimated values. Industrial applications of the present results are obvious in the fabrication of artificial cilia pumping systems for microfluidic flow systems.

Analysis of Cerebrospinal Fluid Flowmetry in Evaluating the Efficiency of Endoscopic Third Ventriculostomy in Aqueductal Stenosis

Background: Endoscopic third ventriculostomy (ETV) approach was gaining popularity over shunt placement in obstructive hydrocephalus. Postoperative ETV assessment and patients follow up remains a matter of controversy and challenge to ensure efficiency and patency of ventriculostomy.Aim of the work: To assess whether cerebrospinal fluid (CSF) flow is restored after ETV and similarity to flow in normal aqueduct with patients clinical and ventricular size changes assessment.Patients and Methods: From April 2016 to April 2019, 30 patients with hydrocephalus due to aqueductal stenosis (AS) were treated with ETV. They were followed up for ventriculostomy patency within the 1st, 6th and 9th month after surgery and compared to 22 persons as a control group of normal aqueductal flow with phase contrast magnetic resonance imaging (PC MRI).Results: Twenty-three (23) patients restored pulsatile bidirectional pattern of CSF flow across the ventriculostomy similar to that of aqueductal flow. While one patient had no flow during the 1st follow up and 6 patients showed initial pulsatile flow then developed flow disturbance during the 2nd follow up period. Absolute stroke volume values showed significant statistical difference between ETV and control groups (p value < 0.001). The value of 85 μl showed sensitivity and specificity of 84.3% and 81.7%, respectively as a cutoff value of ETV efficiency.Conclusion: ETV is an efficient technique to restores the physiological pulsatile Cerebrospinal fluid flow. Absolute stroke volume was a good predictor of ETV efficiency.

Tetrafluoroethylene-Propylene Elastomer for Fabrication of Microfluidic Organs-on-Chips Resistant to Drug Absorption.

Organs-on-chips are microfluidic devices typically fabricated from polydimethylsiloxane (PDMS). Since PDMS has many attractive properties including high optical clarity and compliance, PDMS is very useful for cell culture applications; however, PDMS possesses a significant drawback in that small hydrophobic molecules are strongly absorbed. This drawback hinders widespread use of PDMS-based devices for drug discovery and development. Here, we describe a microfluidic cell culture system made of a tetrafluoroethylene-propylene (FEPM) elastomer. We demonstrated that FEPM does not absorb small hydrophobic compounds including rhodamine B and three types of drugs, nifedipine, coumarin, and Bay K8644, whereas PDMS absorbs them strongly. The device consists of two FEPM layers of microchannels separated by a thin collagen vitrigel membrane. Since FEPM is flexible and biocompatible, this microfluidic device can be used to culture cells while applying mechanical strain. When human umbilical vein endothelial cells (HUVECs) were subjected to cyclic strain (~10%) for 4 h in this device, HUVECs reoriented and aligned perpendicularly in response to the cyclic stretch. Moreover, we demonstrated that this device can be used to replicate the epithelial–endothelial interface as well as to provide physiological mechanical strain and fluid flow. This method offers a robust platform to produce organs-on-chips for drug discovery and development.

A Minimized Valveless Electromagnetic Micropump for Microfluidic Actuation on Organ Chips

In recent years, organ chips have become an ideal alternative for physiological and pathological studies as well as drug screening. To more realistically simulate the in vivo physiological environment, micropumps are often needed as power sources to achieve dynamic culture in organ chips. In this study, we present a valveless electromagnetic micropump that can be used in organ chips. Fluid flow is actuated by the vibration of a PDMS membrane through a varying magnetic field. This micropump uses a nozzle/diffuser structure instead of valves, which simplifies the structure. By reducing the volume of the coil and magnet, the size of the electromagnetic micropump is minimized so that it can be integrated on a microfluidic chip. The micropump and the chip can be portably packaged by using a small signal generation module and a dry battery to supply the coil with a square wave, reducing the dependence on external devices. In addition, multiple electromagnetic micropumps can be integrated on a single chip. These micropumps can be connected in series or in parallel, which can achieve complicated flow conditions and can simulate physiological fluid flow more realistically in different types of organ chips. The flow rate was measured to characterize the actuating performance of the micropump. We established the dynamic coculture of a liver and breast cancer model on the chip, with medium actuated by the electromagnetic micropump. Cell viability, albumin and IL-6 were analyzed. The results indicated that dynamic coculture, which was actuated by the electromagnetic micropump, was beneficial for cell growth and function compared with the static control group.

Flow induced stability of pluronic hydrogels: Injectable and unencapsulated nucleus pulposus replacement

Poloxamers, or pluronics, have been proposed as biomimetic substitutes for physiological gels. Concern regarding their ability to resist swelling under fluid flows has impeded their implementation. Using a combination of techniques including cryo-TEM and rapid X-ray imaging, we found that rapid flow rates stabilized the gels against dissolution. Energy balance calculations confirmed that disentanglement of individual micelles was not possible at time scales faster than the reptation time when the system response was that of a solid which dissipated the hydrodynamic force field via cooperative deformation. In-vivo tests were performed where the hydrogel was injected as a substitute for the nucleus pulposus following discectomy in dogs. The results indicated that the gel was still present after 3 months, and radiographs indicated that compression of the disc space was prevented despite the gel being exposed to constant perfusion. Statement of SignificanceThis paper demonstrates a highly unexpected result and counter intuitive result, namely the inverse dependence of the dissociation rate of a physical hydrogel on the flow velocity of the liquid medium. Using cryo-electron microscopy we demonstrate that the gel responds like deformable solid in high flow rates, with minimal dissociation. Since these gels are thermoreversible, they were injected into dogs, where we show that they were a viable alternative to the nucleus pulposus, without dissolution in physiological fluid flows for at least three months.

Physiological fluid flow analysis by means of contraction and expansion with addition of hybrid nanoparticles

In this research, we inspected the physiological flow of a viscous fluid with addition of hybrid nanoparticles, generated by means of contraction and expansion. We presented a mathematical model for a vertical annulus, and the two-dimensional equations were shortened under the hypothesis of small inertial forces and large wavelength approximation. The flow analysis is performed for different shapes of the nanoparticles, and mixed convection and wall properties effects are also taken into account. The properties of the different wave shapes were explored with particular interest. The effects of some evolving physical parameters on peristaltic flow features were shown graphically and debated concisely. Finally, streamline designs showed size, and a number of investigated boluses were presented and debated for several involved parameters.

Pulsatile flow of Jeffrey fluid in a porous elastic tube with variable cross-section under the effect of magnetic field

Pulsatile flow of a conducting Jeffrey fluid through a porous elastic tube with variable cross-section is investigated in this paper. The Jeffrey fluid flow under the effect of transverse magnetic field is considered to understand the nature of conductive physiological fluids such as movement of blood in small arteries of heart muscle. The problem is formulated by considering the radial motion of the tube wall. The analytical expressions of the axial and transverse velocity for both steady and fluctuating parts are derived. The influence of different pertinent parameter on axial velocity distribution for different instants of times is analyzed through graphs. The variation in the velocity field for two different geometries is discussed. The results reveal that the effect of frequency parameter on fluctuating axial velocity is significant. The influence of magnetic field and Jeffrey parameter has pronounced effect on variation of fluctuating axial velocity. Also it is observed that the geometry has a remarkable effect on axial velocity distribution. The results obtained for the present oscillatory flow characteristics reveal many interesting behaviors that warrant further study of conductive physiological fluid flows in an elastic tube with variable cross-section. In the absence of Magnetic field and Jeffrey parameter the present results are similar to the observations of Misra and Ghosh [8].

Abstract LB-035: 3D microfluidic tumor model for investigating metastatic tumor migration and colonization of secondary sites

Abstract Introduction: The ability to model the microenvironment of primary and secondary tumor sites is critical for advancing treatment options for metastatic breast cancer. There are currently no 3D tumor models that mimic the in vivo vascular geometry and tumor microenvironment for monitoring extravasation, tissue invasion, and colonization of secondary sites by metastatic tumors. In this study, we report on the development of a 3D tumor model with primary and secondary tumor sites, where metastatic breast cancer cells can escape the primary site, circulate in circulation, adhere to the vascular endothelium and subsequently invade distant tissues. Materials and Methods: Multiple synthetic tumor networks comprising primary and secondary tumor sites were developed using in vivo images and fabricated using soft lithography. Fluorescent dyes (FITC-dextran) was used to test the fluidic integrity while nano and micro-particles were used to test the porosity of the developed devices. Primary vascular endothelial cells were cultured in the vascular channels while GFP-labeled metastatic human breast cancer cell line (MDA-MB-231/GFP) or non-metastatic (MCF-7/GFP) was cultured in the primary tumor site in a 3D environment using Matrigel™. The vascular networks were perfused with endothelial cell media under physiological fluid flow conditions. Real-time monitoring of cellular growth, extravasation and invasion was performed using fluorescence microscopy over a four week period. Results and Discussion: Metastatic MDA-MB-231 breast cancer cells proliferated rapidly in the primary tumor site in contrast to non-metastatic MCF-7. Tumor cells were found to initiate a metastatic cascade as early as 7-10 days following growth in the primary site comprising of intravasation into the vascular channels, rolling and adhesion on the endothelium and extravasation into the secondary site. Significant differences were observed on the tumor behavior based on the flow profiles and morphology of the vascular networks. These results provide a unique perspective of the in vivo realism in an in vitro system for monitoring metastasis Conclusions: We have developed a 3D vascularized model for monitoring the invasive growth and the metastatic potential of tumors thereby mimicking the in vivo microenvironment of solid tumors. This model can be used to investigate tumor-endothelial cell interactions using a combination of real-time imaging techniques and screening of targeted therapeutics that may reduce the metastatic potential of tumors. Citation Format: Deborah Ramsey, Dustin Haithcock, Charles Garson, Ashley Gilbert, Ketan Bhatt, Balabhaskar Prabhakarpandian, Kapil Pant. 3D microfluidic tumor model for investigating metastatic tumor migration and colonization of secondary sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-035.

Mathematical modelling of pressure-driven micropolar biological flow due to metachronal wave propulsion of beating cilia

In this paper, we present an analytical study of pressure-driven flow of micropolar non-Newtonian physiological fluids through a channel comprising two parallel oscillating walls. The cilia are arranged at equal intervals and protrude normally from both walls of the infinitely long channel. A metachronal wave is generated due to natural beating of cilia and the direction of wave propagation is parallel to the direction of fluid flow. Appropriate expressions are presented for deformation via longitudinal and transverse velocity components induced by the ciliary beating phenomenon with cilia assumed to follow elliptic trajectories. The conservation equations for mass, longitudinal and transverse (linear) momentum and angular momentum are reduced in accordance with the long wavelength and creeping Stokesian flow approximations and then normalized with appropriate transformations. The resulting non-linear moving boundary value problem is solved analytically for constant micro-inertia density, subject to physically realistic boundary conditions. Closed-form expressions are derived for axial velocity, angular velocity, volumetric flow rate and pressure rise. The transport phenomena are shown to be dictated by several non-Newtonian parameters, including micropolar material parameter and Eringen coupling parameter, and also several geometric parameters, viz eccentricity parameter, wave number and cilia length. The influence of these parameters on streamline profiles (with a view to addressing trapping features via bolus formation and evolution), pressure gradient and other characteristics are evaluated graphically. Both axial and angular velocities are observed to be substantially modified with both micropolar rheological parameters and furthermore are significantly altered with increasing volumetric flow rate. Free pumping is also examined. An inverse relationship between pressure rise and flow rate is computed which is similar to that observed in Newtonian fluids. The study is relevant to hemodynamics in narrow capillaries and also bio-inspired micro-fluidic devices.

Incubation under fluid dynamic conditions markedly improves the structural preservation in vitro of explanted skeletal muscles.

Explanted organs and tissues represent suitable experimental systems mimicking the functional and structural complexity of the living organism, with positive ethical and economic impact on research activities. However, their preservation in culture is generally limited, thus hindering their application as experimental models for biomedical research. In the present study, we investigated the potential of an innovative fluid dynamic culture system to improve the structural preservation in vitro of explanted mouse skeletal muscles (soleus). We used light and transmission electron microscopy to compare the morphological features of muscles maintained either in multiwell plates under conventional conditions or in a bioreactor mimicking the flow of physiological fluids. Our results demonstrate that fluid dynamic conditions markedly slowed the progressive structural deterioration of the muscle tissue occurring during the permanence in the culture medium, prolonging the preservation of some organelles such as mitochondria up to 48 h.

Abstract PR09: Polymeric mechanical amplifiers of tumor apoptosis

Abstract Regulation of receptor-mediated phenomenon at the nanoscale in a controlled manner remains a challenge for a broad range of fields, including cell biology, medicine, and bioengineering. Fluid forces play a crucial role in receptor-mediated signaling necessary for physiological function, while materials such as nanoparticles have demonstrated potential for spatiotemporal control of cellular signaling. Here, we have developed polymeric mechanical amplifiers that tether to the cell surface and increase receptor-mediated apoptosis in the presence of physiological fluid flow. The mechanical amplifiers consist of both non-degradable and biodegradable polymer nanoparticles that are tethered to the surface of tumor cells via polyethylene glycol (PEG) linkers. Tethered nanoparticles, in the presence of fluid shear forces observed in vivo, increased the therapeutic effect of a TNF superfamily death ligand (TRAIL) on colon and prostate tumor cells by as much as 50% compared to soluble TRAIL treatment alone, while exerting negligible toxic effects on normal cells. Increasing the number of nanoparticles conjugated to the tumor cell surface enhanced TRAIL-mediated apoptosis under fluid shear exposure, while the therapeutic effect under static conditions was not altered. Our results suggest that the enhanced therapeutic response using polymeric nanoparticles is both receptor-mediated apoptosis-specific and does not affect doxorubicin efficacy. Inhibition studies indicate the response is caspase signaling-dependent, and increased tumor cell death receptor expression in the presence of shear forces is also observed. We show that targeted polymeric nanoparticles delivered to tumor cells in vivo mechanically amplified a subsequent treatment of soluble TRAIL, and reduced both circulating tumor cells in blood and overall tumor cell burden in vivo by over 90%. We conclude that surface-bound polymeric nanoparticles enhance receptor-mediated apoptosis in the presence of physiological shear forces, and represents a potentially new application for a broad range of nanotechnologies to maximize the capacity of receptor-mediated signaling and function in the presence of a ligand. This abstract is also being presented as Poster B35. Citation Format: Michael John Mitchell, Robert Langer. Polymeric mechanical amplifiers of tumor apoptosis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR09.

Abstract 3382: A physiological model of the tumor microenvironment for screening drug delivery systems

Abstract Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. Tumor microvasculature has many unique features including unusual transport characteristics, high interstitial pressure, and enhanced permeability and retention (EPR) effect. Current in vitro models of tumor drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. The objective of this study is to develop and characterize a synthetic 3D solid tumor - endothelium model in a novel microfluidic platform that mimics the tumor microenvironment observed in vivo. The novel synthetic tumor model consists of a vascular network of tumor derived endothelial cells forming a complete lumen in communication with 3D solid tumors. Primary human breast tumor associated endothelial cells (HBTAECs) were co-cultured under physiological fluid flow conditions with 3D tumor cells from metastastic (MDA-MB-231) or non-metastatic (MCF-7) origins to study the effect of metastatic potential on the integrity of the adjacent endothelial cell lining. Extravasation of fluorescently labeled liposomes across the endothelium to the tumors was measured following HBTAECs treatment with normal media, tumor conditioned media (TCM), or TNF-α. Tight junction formation was characterized using ZO-1 immunostaining. MDA-MB-231 cells quickly invaded into the vascular network from their primary culture location whereas MCF-7 cells rarely grew beyond the boundary of tumor origin. Permeability of liposomes across the endothelium was significantly higher with TCM treatment compared to both control and TNF-α treated cells (P &amp;lt; 0.01, one-way ANOVA). ZO-1 staining indicated strong tight junction formation when HBTAECs were treated with normal media as compared to TCM or TNF-α. We have successfully established an in vitro 3D tumor - endothelial cell co-culture model in a novel microfluidic platform that closely mimics the tumor microenvironment in vivo. This system reproduces the tumor permeability and retention (EPR) effect on a chip. This realistic in vitro model can have great potential in applications such as cell-cell/cell-drug carrier interaction studies, drug delivery carrier screening, and drug efficacy testing. Citation Format: Yuan Tang, Fariborz Soroush, Sudhir Deosarkar, Bin Wang, Balabhaskar Prabhakarpandian, Mohammad Kiani. A physiological model of the tumor microenvironment for screening drug delivery systems. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3382.

Expression of Polycystins in LLC‐PK1 Cells Does Not Increase Flow‐Activated Calcium Fluxes

The primary cilium is thought to play a critical role as a mechanosensor that detects fluid flow in the kidney. The molecular basis of this mechanosensitivity has yet to be fully elucidated. Portions of the cellular populations of the polycystin 1 (PC1) and polycystin 2 (PC2) proteins, which are encoded by the Pkd1 and Pkd2 genes that are mutated in Autosomal Dominant Polycystic Kidney Disease, localize to the primary cilium. PC1 is a ~450 kDa membrane protein with a large N terminal extracellular domain and 11 predicted transmembrane spans. PC2 spans the membrane six times and is a member of the TRP family of nonselective calcium‐permeable cation channels. PC1 and PC2 form a complex with one another, and this association plays an important role in their trafficking to the primary cilium. It has been suggested that the ciliary population of polycystin proteins functions as a mechanosensitive ion channel in renal epithelia, and that the ciliary polycystins play an obligate role in producing cilium‐dependent flow‐activated cytosolic calcium transients.We generated LLC‐PK1 porcine renal epithelial cell lines that stably express PC1 and PC2. Immunofluorescence analysis reveals that both of these proteins are abundantly detected in the primary cilium. We conducted live cell imaging of the intracellular Ca2+ concentration ([Ca2+]i) using these LLC‐PK1 cells loaded with the fluorescent Ca2+‐sensitive dye Fluo4‐AM. We find that overexpression of ciliary PC1 and PC2 does not increase the magnitude or duration of the [Ca2+]i transients elicited in response to increase in physiological fluid flow (10 μl/s corresponding to a linear velocity above the membrane of 70 μm/s). These data suggest that, at least when expressed in this cell type, exogenous polycystins do not by themselves further add to the flow‐activated [Ca2+]i increase. Previous data have suggested that TRPV4 works together with polycystins and that a complex containing TRPV4 and the polycystins confers mechanosensitivity on the cilium. To test this possibility, we co‐expressed TRPV4 tagged with the S‐Tag at its C‐terminus with PC1 and PC2 in LLC‐PK1 cells. Immunofluorescence using an antibody directed against the S‐Tag revealed that little or no TRPV4 protein was detectable in the primary cilium, but accumulated instead at the basolateral plasma membrane. Interestingly, however, expression of TRPV4 in LLC‐PK1 cells caused much larger flow‐induced and ATP‐induced [Ca2+]i transients than was detected in cells expressing PC1 and PC2 alone. These results demonstrate that exogenously expressed polycystins are not sufficient to further enhance ciliary mechanosensation and suggest that TRPV4, may play an indirect role in this process and in supporting cellular Ca2+ signaling.Support or Funding InformationThe NSF GRFP Graduate Research Opportunities Worldwide (GROW), The Danish Council for Independent Research, The Lundbeck Foundation and the research centre MEMBRANES, The William Townsend Porter Predoctoral Fellowship from the American Physiological Society

Theoretical, Computational, and Experimental Investigations on Activation of Mechanosensitive Channels

We investigate the gating mechanism of large conductance mechanosensitive channels (MscLs) on cellular membranes and vesicles using an integrated approach including theoretical modeling, multiscale simulations and microfluidics experiments. At the channel level, we recently developed a theoretical model to study the MscL gating in vesicles exposed to physiological fluid flows, demonstrating the possibility of gating MscL via flow-generated membrane tension (Pak, et al, PNAS, 112:9822-7, 2015). At the cellular level, membrane tension under different flow conditions is calculated using a 3D whole-cell model based on dissipative particle dynamics (DPD). The simulated tension is passed to the MscL channel model to predict the MscL gating. The system will also be studied with microfluidics experiments including micropipette array aspiration, constricted tube flow and shear flow for comparison with results from theoretical modeling. The results provide guidance for future experiments in inducing MscL opening for intracellular transport.

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170±20nm and a diameter of 2–4nm. Force pulling revealed a strong protein core that can withstand over 100pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890pN and a corresponding Young's modulus of 71MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.

Early in-situ cellularization of a supramolecular vascular graft is modified by synthetic stromal cell-derived factor-1α derived peptides

In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α–specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides.

A novel mechano-enzymatic cleavage mechanism underlies transthyretin amyloidogenesis.

The mechanisms underlying transthyretin-related amyloidosis in vivo remain unclear. The abundance of the 49–127 transthyretin fragment in ex vivo deposits suggests that a proteolytic cleavage has a crucial role in destabilizing the tetramer and releasing the highly amyloidogenic 49–127 truncated protomer. Here, we investigate the mechanism of cleavage and release of the 49–127 fragment from the prototypic S52P variant, and we show that the proteolysis/fibrillogenesis pathway is common to several amyloidogenic variants of transthyretin and requires the action of biomechanical forces provided by the shear stress of physiological fluid flow. Crucially, the non-amyloidogenic and protective T119M variant is neither cleaved nor generates fibrils under these conditions. We propose that a mechano-enzymatic mechanism mediates transthyretin amyloid fibrillogenesis in vivo. This may be particularly important in the heart where shear stress is greatest; indeed, the 49–127 transthyretin fragment is particularly abundant in cardiac amyloid. Finally, we show that existing transthyretin stabilizers, including tafamidis, inhibit proteolysis-mediated transthyretin fibrillogenesis with different efficiency in different variants; however, inhibition is complete only when both binding sites are occupied.

Lysosomal Degradation of CD44 Mediates Ceramide Nanoliposome-induced Anoikis and Diminished Extravasation in Metastatic Carcinoma Cells

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.

Adhesion patterns in the microvasculature are dependent on bifurcation angle

Particle adhesion in vivo is highly dependent on the microvascular environment comprising of unique anatomical, geometrical, physiological fluid flow conditions and cell–particle and cell–cell interactions. Hence, proper design of vascular-targeted drug carriers that efficiently deliver therapeutics to the targeted cells or tissue at effective concentrations must account for these complex conditions observed in vivo. In this study, we build upon our previous results with the goal of characterizing the effects of bifurcations and their corresponding angle on adhesion of functionalized particles and neutrophils to activated endothelium. Our hypothesis is that adhesion is significantly affected by the type of biochemical interactions between particles and vessel wall as well as the presence of bifurcations and their corresponding angle. Here, we investigate adhesion of functionalized particles (2μm and 7μm microparticles) to protein coated channels as well as adhesion of human neutrophils to human endothelial cells under various physiological flow conditions in microfluidic bifurcating channels comprising of different contained angles (30°, 60°, 90°, or 120°). Our findings indicate that both functionalized particle and neutrophil adhesion propensity increase with a larger bifurcation angle. Moreover, the difference in the adhesion patterns of neutrophils and rigid, similar sized (7μm) particles is more apparent in the junction regions with a larger contained angle. By selecting the right particle size range, enhanced targeted binding of vascular drug carriers can be achieved along with a higher efficacy at optimal drug dosage. Hence, vascular drug particle design needs to be tailored to account for higher binding propensity at larger bifurcation angles.