Endothelial Monolayer Formation Research Articles

The promotion of vascular reconstruction by hierarchical structures in biodegradable small-diameter vascular scaffolds

Tissue engineering of small-diameter vessels remains challenging due to the inadequate ability to promote endothelialization and infiltration of smooth muscle cells (SMCs). Ideal vascular graft is expected to provide the ability to support endothelial monolayer formation and SMCs infiltration. To achieve this, vascular scaffolds with both orientation and dimension hierarchies were prepared, including hierarchically random vascular scaffold (RVS) and aligned vascular scaffold (AVS), by utilizing degradable poly(ε-caprolactone)-co-poly(ethylene glycol) (PCE) and the blend of PCE/gelatin (PCEG) as raw materials. In addition to the orientation hierarchy, dimension hierarchy with small pores in the inner layer and large pores in the outer layer was also constructed in both RVS and AVS to further investigate the promotion of vascular reconstruction by hierarchical structures in vascular scaffolds. The results show that the AVS with an orientation hierarchy that consists with the natural vascular structure had better mechanical properties and promotion effect on the proliferation of vascular cells than RVS, and also exhibited excellent contact guidance effects on cells. While the dimension hierarchy in both RVS and AVS was favorable to the rapid infiltration of SMCs in a short culture time in vitro. Besides, the results of subcutaneous implantation further demonstrate that AVS achieved a fully infiltrated outer layer with wavy elastic fibers-mimic strips formation by day 14, ascribing to hierarchies of aligned orientation and porous dimension. The results further indicate that the scaffolds with both orientation and dimension hierarchical structures have great potential in the application of promoting the vascular reconstruction.

Nanotexture and crystal phase regulation for synergistic enhancement in re-endothelialization on medical pure titanium surface

Re-endothelialization has been recognized as a promising strategy to address the tissue hyperplasia and subsequent restenosis which are major complications associated with vascular implant/interventional titanium devices. However, the uncontrollable over-proliferation of smooth muscle cells (SMCs) limits the clinical application of numerous modified strategies. Herein, a novel modified strategy involving with a two-step anodic oxidation and annealing treatment was proposed to achieve rapid re-endothelialization function regulated by regular honeycomb nanotexture and specific anatase phase on the titanium surface. Theoretical calculation revealed that the presence of nanotexture reduced the polar component of surface energy, while the generation of anatase significantly enhanced the polar component and total surface energy. Meanwhile, the modified surface with regular nanotexture and anatase phase produced positive effect on the expression of CD31, VE-Cadherin and down-regulated α-SMA proteins expression, indicating excellent capacity of pro-endothelial regeneration and inhibition of SMCs proliferation and migration. One-month in vivo implantation in rabbit carotid arteries further confirmed that modified tube implant surface effectively accelerated confluent endothelial monolayer formation and promoted native-like endothelium tissue regeneration. By contrast, original titanium tube implant induced a disorganized tissue proliferation in the lumen with a high risk of restenosis. Collectively, this study opens us an alternative route to achieve the function that selectively promotes endothelial cells (ECs) growth and suppresses SMCs on the medical titanium surface, which has a great potential in facilitating re-endothelialization on the surface of blood-contacting titanium implant.

Successful endothelial monolayer formation on melt electrowritten scaffolds under dynamic conditions to mimic tunica intima

The lack of transplantable tissues and organs as well as the limitations of synthetic implants highlight the need for tissue-engineered constructs to obtain safe, long-lasting, and limitless tissue replacements. Scaffolds for cardiovascular applications, such as for a tissue-engineered vascular graft (TEVG), are thus highly required. For TEVGs, tubular scaffolds should support the formation of confluent endothelial layers in particular under dynamic conditions to prevent thrombosis and maintain hemostasis. For that purpose, a porous and highly diffusible scaffold structure is necessary to allow optimal cell adhesion as well as oxygen and nutrient exchange with the surrounding tissue. Here, we present a three-dimensional-printed scaffold made by a combination of fused deposition modeling (FDM) and melt electrowriting (MEW) out of polycaprolactone that enables monolayer formation and alignment of endothelial cells in the direction of medium flow under a shear stress of up to 10 dyn cm-2. Pore size and coating with human fibrin were optimized to enable confluent endothelial layers on the printed scaffold structures. Cell orientation and shape analysis showed a characteristic alignment and elongation of the tested endothelial cells with the direction of flow after dynamic cultivation. In contrast, melt electrospun scaffolds based on the same CAD design under comparable printing and cultivation conditions were not sufficient to form gapless cell layers. Thus, the new scaffold fabricated by MEW/FDM approach appears most suitable for TEVGs as a template for the innermost vascular wall layer, the tunica intima.

Step-wise CAG@PLys@PDA-Cu2+ modification on micropatterned nanofibers for programmed endothelial healing.

Native-like endothelium regeneration is a prerequisite for material-guided small-diameter vascular regeneration. In this study, a novel strategy is proposed to achieve phase-adjusted endothelial healing by step-wise modification of parallel-microgroove-patterned (i.e., micropatterned) nanofibers with polydopamine-copper ion (PDA-Cu2+) complexes, polylysine (PLys) molecules, and Cys-Ala-Gly (CAG) peptides (CAG@PLys@PDA-Cu2+). Using electrospun poly(l-lactide-co-caprolactone) random nanofibers as the demonstrating biomaterial, step-wise modification of CAG@PLys@PDA-Cu2+ significantly enhanced substrate wettability and protein adsorption, exhibited an excellent antithrombotic surface and outstanding phase-adjusted capacity of endothelium regeneration involving cell adhesion, endothelial monolayer formation, and the regenerated endothelium maturation. Upon in vivo implantation for segmental replacement of rabbit carotid arteries, CAG@PLys@PDA-Cu2+ modified grafts (2mm inner diameter) with micropatterns on inner surface effectively accelerated native-like endothelium regeneration within 1 week, with less platelet aggregates and inflammatory response compared to those on non-modified grafts. Prolonged observations at 6- and 12-weeks post-implantation demonstrated a positive vascular remodeling with almost fully covered endothelium and mature smooth muscle layer in the modified vascular grafts, accompanied with well-organized extracellular matrix. By contrast, non-modified vascular grafts induced a disorganized tissue formation with a high risk of thrombogenesis. In summary, step-wise modification of CAG@PLys@PDA-Cu2+ on micropatterned nanofibers can significantly promote endothelial healing without inflicting thrombosis, thus confirming a novel strategy for developing functional vascular grafts or other blood-contacting materials/devices.

Fucoidan and topography modification improved in situ endothelialization on acellular synthetic vascular grafts.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast in situ endothelialization of polyvinyl alcohol, which is not endothelial cell-adhesive. Among three different immobilization approaches compared, conjugation of aminated-fucoidan promoted endothelial monolayer formation while minimizing thrombogenicity in both in vitro platelet rich plasma testing and ex vivo non-human primate shunt assay. Screening of six topographical patterns showed that 2μm gratings increased endothelial cell migration without inducing inflammation responses of endothelial cells. Mechanistic studies demonstrated that fucoidan could attract fibronectin, enabling integrin binding and focal adhesion formation and activating focal adhesion kinase (FAK) signaling, and 2μm gratings further enhanced FAK-mediated cell migration. In a clinically relevant rabbit carotid artery end-to-side anastomosis model, 60% in situ endothelialization was observed throughout the entire lumen of 1.7mm inner diameter modified grafts, compared to 0% of unmodified graft, and the four-week graft patency also increased. This work presents a promising strategy to stimulate in situ endothelialization on synthetic materials for improving long-term performance.

Comparison of conjugating chondroitin sulfate A and B on amine-rich surface: For deeper understanding on directing cardiovascular cells fate

Chondroitin sulfate (CS) is often used as an ideal drug for long-term prevention of cardiovascular diseases with the advantages of anti-inflammation and promoting blood circulation. In the previous work, the natural CS was conjugated onto the amine rich surface to enhance the biocompatibility of cardiovascular stents . In this study, we choose an artificial CS subtype, CS B, as the functional molecule to prepare a functionalized surface on the poly-dopamine (PDA)/hexanediamine (HD) co-deposited stent to direct the cardiovascular cells fate in comparison with the coating prepared with the natural CS subtype, CS A. Although the PDA/HD-CS B coating exhibited no advantage of inhibiting platelet adhesion compared to PDA/HD-CS A coating, it showed stronger functions on inhibiting adhesion of whole blood composition, further reducing the thrombosis, and contributing broader lumen patency and more blood flow. In addition, PDA/HD-CS B was endowed better ability on directing the fate of vascular cells, i.e. smooth muscle cells (SMC), macrophages, endothelial cells (EC) and endothelial progenitor cells (EPC). Surprisingly, as the main natural subtype of CS, CS A didn't contribute to the formation of endothelial monolayer within 30 days like CS, while the conjugated CS B obtained a complete endothelial monolayer benefiting from its stronger function on regulating the cardiovascular cells behaviors in time sequence and space sequence.

A preliminary study on polycaprolactone and gelatin-based bilayered tubular scaffolds with hierarchical pore size constructed from nano and microfibers for vascular tissue engineering

Due to the insufficient endothelialization and the poor colonization of smooth muscle cells (SMCs), small-diameter vascular tissue engineering is still challenging. An ideal vascular graft is expected to effectively support the formation of endothelial monolayer and the colonization of SMCs. In this study, we proposed a bilayered scaffold with hierarchical pore size constructed from nano and microfibers by electrospinning PCL-PEG-PCL (PCE) and a blend of PCE and gelatin (PCEG) sequentially. The structural features of nano and microfibers were tuned by the concentration of PCE and the proportion of PCE/gelatin in electrospun solution respectively. The results demonstrated the best nanofiber morphology and relatively high mechanical properties were achieved in 18% PCE (w/v) (PCE18) and PCE and gelatin with a weight ratio of 7:3 (P7G3) at a concentration of 18% (w/v) electrospun membranes. The in vitro co-culturing studies of cells and membranes indicated all the PCE membranes supported the proliferation and spreading of endothelial cells and the further endothelialization of the membranous surface, while PCEG membranes facilitated the migration inward of SMCs. Taking the porosity and mechanical properties into consideration, PCE18 and P7G3 were chosen to construct the inner and outer layers of the bilayered scaffold with hierarchical pore size respectively. The circumferential ring test demonstrated that the bilayered scaffold has good mechanical property both in dry and wet state. After cells were co-cultured with this bilayered scaffold for 7 days, the results manifested a continuous endothelial monolayer has formed on the luminal surface and the SMCs have started to colonized from outer layers, indicating the vast potential of this bilayered scaffold in vascular remodeling and regeneration.

Collagen immobilization on ultra-thin nanofiber membrane to promote in vitro endothelial monolayer formation

The endothelialization on the poly (ε-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (ε-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (ε-caprolactone) nanofiber using hydrolysis by NaOH treatment and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide reaction as a cost-effective and stable approach. NaOH was first applied to render the poly (ε-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (ε-caprolactone) nanofibers using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide. Scanning electron microscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and fluorescence microscopy were used to verify stable collagen immobilization on the surface of the poly (ε-caprolactone) nanofibers and the maintenance of the original structure of poly (ε-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (ε-caprolactone) nanofiber membrane and expressed tight junction proteins with the increase in transendothelial electrical resistance, which demonstrated the maintenance of the endothelial cell homeostasis on the collagen-immobilized-poly (ε-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (ε-caprolactone) nanofiber scaffolds.

PO-174 Evaluation of immunohistochemical expression of microfibrillar-associated protein 5 (MFAP5) in invasive breast carcinoma of no special type

IntroductionBreast cancer is one of the most malignant cancer types which is the second leading cause of cancer-associated deaths in women. Breast cancer begins as a local disease which can then metastasize to lymph nodes and distant sites. The metastasis of breast cancer is mainly to bone, lung and liver. Being the leading cause of cancer related deaths, it is essential to understand the complex metastasis phenomenon to develop new diagnostic and therapeutic strategies. In this study, we aim to develop a lab-on-a-chip (LOC) system to detect the metastatic capacities of breast cancer cells to different target tissues.Material and methodsThe triple negative breast cancer cell line MDA-MB-231 was selected based on its high metastatic capacity, while normal lung, liver and bone fibroblasts WI-38, BRL3A and HS-5 and human umbilical vein endothelial cell line HUVEC-C were used to generate target tissue microenvironments and endothelial monolayer, respectively. A three-channel LOC system, one for endothelial monolayer formation, one for mimicking target tissue microenvironment and one for media supply, was used. The seeding cell densities, common medium composition and ECM components for microenvironment were optimised for the LOC system. Following addition of cancer cell lines to the endothelial monolayer channel, invasion and extravasation capacities to different target sites were determined by tracking 3D distribution of fluorescently labelled cancer cells up to four days.Results and discussionsMDA-MB-231 cells were shown to invade and extravasate preferentially to lung and liver microenvironments, while their movement to bone or cell-free target sites was limited. The observed results were in agreement with the previously reported phenotype of MDA-MB-231 cell lines.ConclusionOur results show that tissue- specific cell lines in a LOC system can be used to mimic target tissue microenvironments and evaluate different invasion and extravasation capacities of breast cancer cell lines to different target sites.

PO-189 Optimisation of a lab-on-a-chip device and method for quantification of extravasation

IntroductionCancer is a worldwide disease, 90% deaths of which are due to metastasis. Metastasis comprises of intravasation, extravasation and new tumour formation at a secondary site in the body. Understanding the mechanism of metastasis and developing new platforms to study metastasis play a critical role in both the diagnosis and the treatment of cancer. Microfluidic technology provides the ability to mimic the in vivo microenvironment spatially and temporally. Lab-on-a-chip (LOC) devices produced with this technology can be used in metastasis studies.Material and methodsSoft lithography was used for the fabrication of LOC devices. The design consists of a blood vessel channel, extracellular matrix/target tissue matrix channel and a medium reservoir. LOC devices were coated with APTES (3-aminopropyltriethoxysylan) followed by fibronectin, collagen or laminin to enhance adherence of endothelial cells for the construction of the endothelial barrier. Hydrogel mixture comprising of collagen:matrigel:target tissue cells (9:10:1) was loaded into the matrix channel. After the polymerization step, fluorescently labelled endothelial cells suspended in medium with or without 8% of 450–600 kDa dextran were introduced into the blood vessel channel. Media was added into the medium reservoir. 70 kDa fluorescent dextran was introduced into the blood vessel channel in static and flow conditions to determine the integrity of the endothelial barrier. Fluorescently labelled breast cancer cells were then introduced into the blood vessel channel. 3D images were acquired with a confocal microscope and analysis was performed using Fiji.Results and discussionsLaminin was better at promoting endothelial monolayer formation compared to fibronectin and collagen. Number of endothelial cells to be seeded was optimised to be 19736 cells/ height of the blood vessel channel (µm) to form a continuous monolayer. Presence of dextran in the endothelial cell suspension prevented formation of cluster. Real time imaging and analysis showed that 70 kDa flourescent dextran did not diffuse from the flow channel into the matrix channel in static and flow conditions. Interactions of fluorescently labelled breast cancer cells with the endothelial monolayer and their extravasation into the matrix channel was quantitatively determined.ConclusionWe have optimised surface coating, cell density and medium composition for successful mimicking of a blood vessel in a LOC device. The optimised method was used to quantitatively determine extravasation of cancer cells.

Allogeneic Descemet's Membrane Transplantation Enhances Corneal Endothelial Monolayer Formation and Restores Functional Integrity Following Descemet's Stripping.

To characterize the differences in corneal endothelial wound healing in the presence or absence of Descemet's membrane (DM), in vivo. New-Zealand white rabbits were subjected to 7-mm endothelial wound either by scraping (n = 8; DM intact), peeling (n = 6; DM removed), or a combinatory scrape/peel wound (n = 6). In a second experiment, rabbits underwent peel wound with immediate transplantation of pure decellularized human DM (n = 4). In vivo endothelial migration was assessed via trypan blue staining. Recovery of corneal clarity and thickness was performed by using slit-lamp biomicroscopy and optical coherence tomography. Cell proliferation, phenotype, and morphology were assessed by using immunofluorescence and scanning electron microscopy. In vivo wound closure was faster in the presence of DM; 25.4% ± 1.4%/d versus 5.5% ± 0.6%/d (P < 0.0001). At day 8, complete wound closure was seen in all of the scrape samples but none of the peel group, with wound closure preceding clinical recovery by approximately 6 days in the scrape group. Endothelial cells in the scraped areas reformed functional monolayers capable of restoring corneal thickness and transparency whilst those in the peeled area underwent mesenchymal-like transformation resulting in scar formation. Transplanting decellularized DM in animals receiving a peel wound resulted in clarity and thickness comparable to the scrape group. Endothelial proliferation (Ki67 +ve cells) was higher in scraped versus peeled areas: 54.7% ± 3.5% vs. 8.8% ± 0.7%, (P < 0.01). The presence of DM promoted endothelial wound healing, proliferation, and maintenance of a normal phenotype. DM transplantation recovered the abnormal peel phenotype back to that observed after endothelial scraping.

Controlling Molecular Weight of Hyaluronic Acid Conjugated on Amine-rich Surface: Toward Better Multifunctional Biomaterials for Cardiovascular Implants.

The molecular weights (MWs) of hyaluronic acid (HA) in extracellular matrix secreted from both vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) play crucial roles in the cardiovascular physiology, as HA with appropriate MW influences important pathways of cardiovascular homeostasis, inhibits VSMC synthetic phenotype change and proliferation, inhibits platelet activation and aggregation, promotes endothelial monolayer repair and functionalization, and prevents inflammation and atherosclerosis. In this study, HA samples with gradients of MW (4 × 103, 1 × 105, and 5 × 105 Da) were prepared by covalent conjugation to a copolymerized film of polydopamine and hexamethylendiamine (PDA/HD) as multifunctional coatings (PDA/HD-HA) with potential to improve the biocompatibility of cardiovascular biomaterials. The coatings immobilized with high-MW-HA (PDA/HD-HA-2: 1 × 105 Da; PDA/HD-HA-3: 5 × 105 Da) exhibited a remarkable suppression of platelet activation/aggregation and thrombosis under 15 dyn/cm2 blood flow and simultaneously suppressed the adhesion and proliferation of VSMC and the adhesion, activation, and inflammatory cytokine release of macrophages. In particular, PDA/HD-HA-2 significantly enhanced VEC adhesion, proliferation, migration, and functional factors release, as well as the captured number of endothelial progenitor cells under dynamic condition. The in vivo results indicated that the multifunctional surface (PDA/HD-HA-2) created a favorable microenvironment of endothelial monolayer formation and functionalization for promoting reendothelialization and reducing restenosis of cardiovascular biomaterials.

A Novel Strategy to Engineer Pre-Vascularized Full-Length Dental Pulp-like Tissue Constructs

The requirement for immediate vascularization of engineered dental pulp poses a major hurdle towards successful implementation of pulp regeneration as an effective therapeutic strategy for root canal therapy, especially in adult teeth. Here, we demonstrate a novel strategy to engineer pre-vascularized, cell-laden hydrogel pulp-like tissue constructs in full-length root canals for dental pulp regeneration. We utilized gelatin methacryloyl (GelMA) hydrogels with tunable physical and mechanical properties to determine the microenvironmental conditions (microstructure, degradation, swelling and elastic modulus) that enhanced viability, spreading and proliferation of encapsulated odontoblast-like cells (OD21), and the formation of endothelial monolayers by endothelial colony forming cells (ECFCs). GelMA hydrogels with higher polymer concentration (15% w/v) and stiffness enhanced OD21 cell viability, spreading and proliferation, as well as endothelial cell spreading and monolayer formation. We then fabricated pre-vascularized, full-length, dental pulp-like tissue constructs by dispensing OD21 cell-laden GelMA hydrogel prepolymer in root canals of extracted teeth and fabricating 500 µm channels throughout the root canals. ECFCs seeded into the microchannels successfully formed monolayers and underwent angiogenic sprouting within 7 days in culture. In summary, the proposed approach is a simple and effective strategy for engineering of pre-vascularized dental pulp constructs offering potentially beneficial translational outcomes.

In Vitro Topographical Model of Fuchs Dystrophy for Evaluation of Corneal Endothelial Cell Monolayer Formation

A common indication for corneal transplantation, which is the most transplanted tissue, is a dysfunctional corneal endothelium due to Fuchs' endothelial dystrophy (FED). FED is diagnosed by the presence of in vivo pathological microtopography on the Descemet membrane, which is called corneal guttata. Minimally invasive corneal endothelial cell regenerative procedures such as endothelial cell injection therapy and Rho kinase inhibitor pharmacotherapy have been proposed as alternatives to conventional corneal transplantation for FED patients. However, the effect of guttata on monolayer reformation following such therapies is unknown and there is no equivalent in vitro or animal model to study monolayer reformation. Using a synthetic guttata FED disease model, the formation of the monolayer is investigated to evaluate the efficacy of both therapies. Results obtained suggest that guttata dimensions, density, and spacing greatly affect the fate of corneal endothelial cells in terms of migratory behavior and monolayer reformation. Densely packed synthetic guttata mimicking late-stage FED hinders monolayer reformation, while synthetic guttata of lower height and density show improved monolayer formation. These results suggest that severity of the FED, as determined by height and density of existing guttata, can potentially attenuate corneal endothelial monolayer formation of corneal cell injection therapy and pharmacotherapy.

Blood-Vessel Mimicking Structures by Stereolithographic Fabrication of Small Porous Tubes Using Cytocompatible Polyacrylate Elastomers, Biofunctionalization and Endothelialization.

Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo-curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors.

Evaluation of the corneal endothelial cell monolayer formation on synthetic guttata micro-structures to study the feasibility of corneal cell therapy

Event Abstract Back to Event Evaluation of the corneal endothelial cell monolayer formation on synthetic guttata micro-structures to study the feasibility of corneal cell therapy Muhammad Rizwan1, 2, Alon Ramos Mendez1, Khadijah Adnan3, Gary S. Peh3, 4, Wui Siew Tan2, Jodhbir S. Mehta4, 5 and Evelyn Yim1, 6, 7 1 National University of Singapore, Department of Biomedical Engineering, Singapore 2 Agency for Science, Technology and Research (A*STAR, Institute of Materials Research and Engineering, Singapore 3 Singapore Eye Research Institute, Tissue Engineering and Stem Cell Group, Singapore 4 Duke-NUS Graduate Medical School, Singapore 5 Singapore National Eye Centre, Singapore 6 National University of Singapore, Department of Surgery, Singapore 7 National University of Singapore, Mechanobiology Institute, Singapore Introduction: Majority of corneal transplants are performed exclusively to replace a dysfunctional corneal endothelium (CE) due to Fuchs’ endothelial dystrophy (FED)[1]. Due to shortage of donor corneas, corneal cell injection therapy has been proposed as a minimally invasive alternative treatment for FED patients[2]-[4]. A significant clinical sign of FED is the presence of excrescences of membrane (DM) which are called corneal guttata (CG)[5]-[7]. Current studies on cell injection therapy are performed in animal model corneas which have unaltered DM and hence, do not recapitulate the topographical micro-environment of FED[2][4],[8],[9]. Studying the monolayer formation on guttata-laden surface will be key to the development of cell therapy as a viable clinical procedure for corneal patients. The aim of this study was to fabricate the synthetic guttata-like micro-structures based on patient guttata characterization and evaluate the feasibility of cell monolayer formation on a synthetic guttata-laden surface for cell therapy applications. Material and Methods: Posterior corneal surfaces of FED patients were analyzed by using specular and transmission electron microscopy to characterize the CG dimensions. Soft lithography and hot embossing were used to fabricate similar dimensions guttata on polystyrene (synthetic guttata). The cell migration and monolayer formation on synthetic guttata was studied by first using B4G12 corneal cell line on various dimensions of guttata followed by primary human corneal endothelial cells (HCECs) on selected guttata. Cell interaction with synthetic guttata was studied with SEM imaging and monolayer formation was studied by using confocal microscopy in terms of ZO1 expression. Cell migration tracks were plotted by using manual tracking plugin in ImageJ and analyzed with Ibidi chemotaxis tool. Results and Discussion: B4G12 cell monolayer formation on top of synthetic guttata pillars gradually increased as the pillar spacing and diameter was increased (Fig. 1). The cell morphology was elongated instead of polygonal on highest density pillars. B4G12 cell migration distance, speed and directness analysis on synthetic guttata pillars showed that the highest density pillars presented the most challenging surface for cell monolayer formation, ZO1 expression and for cell migration. With primary HCECs, the cells were unable to form monolayer on highest density small pillars with migrated cells or with cell seeded on top of pillars. Decreasing the height and the diameter and making the edges round (lenses) led to an increased ZO1 expression on top of the micro-structures which indicated that the cells could form tight junction on synthetic guttata (Fig. 2). HCECs migration analysis showed that the synthetic guttata with round edges and less height (5µm) were relatively more favorable for cell migration and corneal endothelial monolayer formation (Fig. 3). The results suggest that the diseased guttata-laden corneal DM surface might potentially hinder the formation of corneal endothelium. Conclusion: The results of this study shows that the pre-existing DM guttata could interfere with injected cells and corneal endothelial monolayer formation within eye. The surgical removal of preexisting guttata before cell therapy could potentially increase the chance of cell therapy success. Additionally, the characterization of the types of guttata on corneal DM surface of FED patients could also be important for patient-specific cell therapy procedure. MOE AcRF Tier 1 – FRC R-397-000-217-112; National Research Foundation Translational and Clinical Research (TCR) Programme Grant (NMRC/TCR/008-SERI/2013); National Medical Research Council NMRC/NIG/0037/2008; Mechanobiology Institute (MBI) R-714-007-006-271; Biomedical Research Council Translation Clinical Research Partnership (TCRP) Grant (TCR0101673); Singapore International Graduate Award (SINGA) Scholarship by A*STAR Graduate Academy

Studies of endothelial monolayer formation on irradiated poly-l-lactide acid with ions of different stopping power and velocity

In this work we study cell viability, proliferation and morphology of bovine aortic endothelial cells (BAEC) cultured on poly-l-lactide acid (PLLA) modified by heavy ion irradiation. In a previous study comparing ions beams with the same stopping power we observed an increase in cell density and a better cell morphology at higher ion velocities. In the present work we continued this study using heavy ions beam with different stopping power and ion velocities. To this end thin films of 50μm thickness were irradiated with 2MeV/u and 0.10MeV/u ion beams provided the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The results suggest that a more dense and elongated cell shapes, similar to the BAEC cells on the internal surface of bovine aorta, was obtained for stopping power of 18.2–22.1MeVcm2mg−1 and ion velocity of 2MeV/u. On the other hand, for low ion velocity 0.10MeV/u the cells present a more globular shapes.

Role of N-WASP in Endothelial Monolayer Formation and Integrity

Endothelial cells (ECs) form a monolayer that serves as a barrier between the blood and the underlying tissue. ECs tightly regulate their cell-cell junctions, controlling the passage of soluble materials and immune cells across the monolayer barrier. We studied the role of N-WASP, a key regulator of Arp2/3 complex and actin assembly, in EC monolayers. We report that N-WASP regulates endothelial monolayer integrity by affecting the organization of cell junctions. Depletion of N-WASP resulted in an increase in transendothelial electrical resistance, a measure of monolayer integrity. N-WASP depletion increased the width of cell-cell junctions and altered the organization of F-actin and VE-cadherin at junctions. N-WASP was not present at cell-cell junctions in monolayers under resting conditions, but it was recruited following treatment with sphingosine-1-phosphate. Taken together, our results reveal a novel role for N-WASP in remodeling EC junctions, which is critical for monolayer integrity and function.

Reorganization of actin and microtubule systems in human vein endothelial cells during intercellular contact formation

Endothelial cells tightly joined to each other line the interior surface of all vessels in living organisms and ensure the regulation of vascular permeability and exchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro, the endothelial cell monolayer retains the basic barrier function of the native endothelium. We use endothelial cells growing in vitro as a model system to study the interaction between cytoskeleton and adhesion structures in endotheliocytes. In the present paper, cultured human vein endothelial cells were used to quantify cytoskeleton alterations from cell spreading and formation of the first cell–cell contacts to confluent monolayer formation. The actin filament system in vein endothelial cells consists of a cortical network and stress fibers arranged parallel to the substrate. This cells express two isoforms of nonmuscle actin: β- and γ-cytoplasmic actins. Antibodies to β-actin stain actin filament bundles, whereas antibodies to γ-actin visualize cortical and lamellar networks. To evaluate the state of actin structures we analyzed their fluorescence intensity near the free edge and zone of cell–cell contacts. Fluorescence intensity of β-actin structures was higher in the areas of cell–cell contacts. Fluorescence of γ-actin was the highest at the leading edges of lamellae and the weakest in the stable edges of the cells with formed cell–cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and what was more, the quantity of microtubules in the area of the already-formed cell–cell contact always being higher than in the leading edge region.

Reorganization of endothelial cells cytoskeleton during formation of functional monolayer in vitro

Endothelium lining the inner surface of vessels regulates permeability of vascular wall by providing exchange between blood circulation in vessels and tissue fluid and therefore performs a barrier function. Endothelial cells (ECs) in culture are able to maintain the barrier function peculiar to cells of vascular endothelium in vivo. The endothelial monolayer in vitro is a unique model system that allows studying interaction of cytoskeletal and adhesive structures of endotheliocytes from the earliest stages of its formation. In the present work, we described and quantitatively characterized the changes of EC cytoskeleton from the moment of spreading of endotheliocytes on glass and the formation of the first contacts between neighbor cells until formation of a functional confluent monolayer. The main type of intermediate filaments of ECs are vimentin filaments. At different stages of endothelial monolayer formation, disposition of vimentin filaments and their amount do not change essentially, they occupy more than 80% of the cell area. Actin filaments system of endotheliocytes is represented by cortical actin at the cell periphery and by bundles of actin stress fibers organized in parallel. With formation of contacts between cells in native endothelial cells, the number of actin filaments rises and thickness of their bundles increases. With formation of endothelial monolayer, there are also changes in the microtubules system—their number increases at the cell edge. At all stages of EC monolayer formation, the number of microtubules in the region of the already formed intercellular contacts exceeds the number of microtubules in the free lamella region of the cell.