scholarly journals Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance

2021 ◽  
Vol 8 ◽  
Author(s):  
Anne Metje van Genderen ◽  
Katja Jansen ◽  
Marleen Kristen ◽  
Joost van Duijn ◽  
Yang Li ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Small Diameter ◽  
Organic Cation Transporter ◽  
Collagen Type Iv ◽  
Renal Transport ◽  
Kidney Tubules ◽  
Cell Orientation ◽  
Differentiation Markers ◽  
Fibrous Scaffolds ◽  
Outer Compartment

Introduction: To date, tubular tissue engineering relies on large, non-porous tubular scaffolds (Ø > 2 mm) for mechanical self-support, or smaller (Ø 150–500 μm) tubes within bulk hydrogels for studying renal transport phenomena. To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties.Materials and Methods: A custom-built melt-electrowriting (MEW) device was used to fabricate tubular fibrous scaffolds with small diameter sizes (Ø = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the tubular scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality.Results: Tubular fibrous scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 1 mm and pore sizes of 0.2 mm were achieved and used for subsequent cell experiments. While HUVEC were unable to bridge the pores, ciPTEC formed tight monolayers in all scaffold microarchitectures tested. Well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp).Discussion and Conclusion: Here, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

scholarly journals Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance

2020 ◽  
Author(s):  
Anne Metje van Genderen ◽  
Katja Jansen ◽  
Marleen Kristen ◽  
Joost van Duijn ◽  
Yang Li ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Small Diameter ◽  
Organic Cation Transporter ◽  
Collagen Type Iv ◽  
Kidney Tubules ◽  
Cell Orientation ◽  
Differentiation Markers ◽  
Type Iv ◽  
Outer Compartment ◽  
Proximal Tubular Epithelial Cells

AbstractTo advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties. A custom-built MEW device was used to fabricate tubular fibrous scaffolds with small diameter sizes (Ø = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality. Tubular scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 0.5 mm and pore sizes of 0.2 mm were achieved. CiPTEC formed tight monolayers in all scaffold microarchitectures tested, but well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp). To conclude, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch

1998 ◽  
Vol 274 (5) ◽  
pp. H1532-H1538 ◽  
Author(s):  
Keiji Naruse ◽  
Takako Yamada ◽  
Masahiro Sokabe
Keyword(s):  
Endothelial Cells ◽  
Cell Elongation ◽  
Umbilical Vein ◽  
Orienting Response ◽  
Cyclic Stretch ◽  
Silicone Membrane ◽  
Cell Orientation ◽  
Human Umbilical Vein ◽  
Intracellular Ca ◽  
Dependent Cell

The present work was designed to elucidate the involvement of Ca2+-permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicone membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and >90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+]i) transiently increased in response to uniaxial stretch, and the basal [Ca2+]igradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.

scholarly journals The role of integrins in the maintenance of endothelial monolayer integrity.

1991 ◽  
Vol 112 (3) ◽  
pp. 479-490 ◽  
Author(s):  
M G Lampugnani ◽  
M Resnati ◽  
E Dejana ◽  
P C Marchisio
Keyword(s):  
Umbilical Vein ◽  
Dermal Fibroblasts ◽  
Collagen Type Iv ◽  
Cell Contact ◽  
Endothelial Monolayer ◽  
Integrin Receptors ◽  
Endothelial Lining ◽  
Type Iv ◽  
Cell Cell

This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.

scholarly journals Adhesion, proliferation and viability of human umbilical vein endothelial cells cultured on the surface of biodegradable non-woven matrices modified with RGD peptides

2019 ◽  
Vol 21 (1) ◽  
pp. 142-152 ◽  
Author(s):  
L. V. Antonova ◽  
V. N. Silnikov ◽  
M. Yu. Khanova ◽  
L. S. Koroleva ◽  
I. Yu. Serpokrilova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Laser Scanning ◽  
Cyclic Peptide ◽  
Umbilical Vein ◽  
Small Diameter ◽  
Laser Scanning Microscopy ◽  
Rgd Peptides ◽  
Human Umbilical Vein ◽  
Cells Cultured

Tissue engineering is a promising area for the production of small-diameter vascular grafts. In recent years, a number of strategies have been developed to make the polymer surfaces of vascular prostheses capable to selectively adhesion of endothelial cells. The arginine–glycine–aspartic acid (RGD) sequence (a cell adhesion site that is present on many extracellular matrix proteins) is the promising target for modification. The efficiency of attachment of endothelial cells can be influenced both by the structure of RGD peptide and the extent of linker group.Aim: to determine the optimal method for modification of non-woven matrices of polyhydroxybutyrate/ valerate and polycaprolactone (PHBV/PCL) by RGD-peptides leading to the increasing of adhesion, viability and proliferation of endothelial cells.Materials and methods. Electrospinning was used to produce 4 mm diameter tubular polymer matrices from PHBV/PCL. Modification of surface of polymer scaffolds was performed using 4,7,10-trioxa-1,13-tridekandiamin, hexamethylenediamine, glutaraldehyde, ninhydrin, ascorbic acid, a cyclic peptide c [RGDFK], RGDK, AhRGD. The quality of modification was assessed by ninhydrin test and determination of arginine-containing peptide. The structure of the surface of matrices before and after modification was studied by scanning electron microscopy. Adhesion, viability and proliferation of Human umbilical vein (HUVEC) endothelial cells cultured for 7 days on the surface of matrices in the presence of RGD and without one were examined using fluorescence and laser scanning microscopy after the cells were pre-stained with fluorescent nuclear dyes (ethidium bromide and Hoechst 33342), and also by special kits for proliferation assessment (Click-iTTM Plus EdU Alexa FluorTM 488 Imaging Kit).Results. RGD peptides bound to the matrix surface via a long linker (4,7,10-trioxa-1,13-tridecanediamine) were characterized by the increased bioavailability and activity. High level of cell adhesion, viability and proliferation were noted on the surface of RGDK and c[RGDFK] modified matrices, whereas their paired analogues with a short linker (hexamethylenediamine) showed low results of cellular viability even against satisfactory cell adhesion.Discussion. Non-woven matrices based on PHBV/PCL and modified using 4,7,10-trioxa-1,13-tridecanediamine showed better results in case of adhesion of HUVEC and subsequent preservation of cell viability and proliferation. RGD-containing peptides of RGDK and c [RGDFK] were more tropic to endothelial cell receptors.

scholarly journals Design and Characterization of a Fluidic Device for the Evaluation of SIS-Based Vascular Grafts

Processes ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 1198
Author(s):  
Alejandra Riveros ◽  
Monica Cuellar ◽  
Paolo F. Sánchez ◽  
Carolina Muñoz-Camargo ◽  
Juan C. Cruz ◽  
...  
Keyword(s):  
Flow Regime ◽  
In Silico ◽  
Umbilical Vein ◽  
Vascular Grafts ◽  
Small Diameter ◽  
Clinical Implementation ◽  
Cfd Validation ◽  
Pulsatile Pressure

Currently available small diameter vascular conduits present several long-term limitations, which has prevented their full clinical implementation. Commercially available vascular grafts show no regenerative capabilities and eventually require surgical replacement; therefore, it is of great interest to develop alternative regenerative vascular grafts (RVG). Decellularized Small Intestinal Submucosa (SIS) is an attractive material for RVG, however, the evaluation of the performance of these grafts is challenging due to the absence of devices that mimic the conditions found in vivo. Thereby, the objective of this study is to design, manufacture and validate in silico and in vitro, a novel fluidic system for the evaluation of human umbilical vein endothelial cells (HUVECs) proliferation on SIS-based RVG under dynamical conditions. Our perfusion and rotational fluidic system was designed in Autodesk Inventor 2018. In silico Computational Fluid Dynamics (CFD) validation of the system was carried out using Ansys Fluent software from ANSYS, Inc for dynamical conditions of a pulsatile pressure function measured experimentally over a rigid wall model. Mechanical and biological parameters such as flow regime, pressure gradient, wall shear stress (WSS), sterility and indirect cell viability (MTT assay) were also evaluated. Cell adhesion was confirmed by SEM imaging. The fluid flow regime within the system remains laminar. The system maintained sterility and showed low cytotoxicity levels. HUVECs were successfully cultured on SIS-based RVG under both perfusion and rotation conditions. In silico analysis agreed well with our experimental and theoretical results, and with recent in vitro and in vivo reports for WSS. The system presented is a tool for evaluating RVG and represents an alternative to develop new methods and protocols for a more comprehensive study of regenerative cardiovascular devices.

Fabrication of multilayer tubular scaffolds with aligned nanofibers to guide the growth of endothelial cells

2020 ◽  
Vol 35 (4-5) ◽  
pp. 553-566 ◽  
Author(s):  
Qingxi Hu ◽  
Caiping Su ◽  
Zhaoxiang Zeng ◽  
Haiguang Zhang ◽  
Rui Feng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Tissue ◽  
Umbilical Vein ◽  
Thermoplastic Polyurethane ◽  
Small Diameter ◽  
Functional Expression ◽  
Water Contact ◽  
Aligned Nanofibers ◽  
Umbilical Vein Endothelial Cells ◽  
Random Composite

Aligned electrospun fibers used for the fabrication of tubular scaffolds possess the ability to regulate cellular alignment and relevant functional expression, with applications in tissue engineering. Despite significant progress in the fabrication of small-diameter vascular grafts (SDVGs) over the past decade, several challenges remain; one of the most problematic of these is the fabrication of aligned nanofibers for multilayer SDVGs. Furthermore, delamination between each layer is difficult to avoid during the fabrication of multilayer structures. This study introduces a new fabrication method for minute delamination four-layer tubular scaffolds (FLTSs) that consist of an interior layer with highly longitudinal aligned nanofibers, two middle layers composed of electrospun sloped and circumferentially aligned fibers, and an exterior layer comprising random fibers. These FLTSs are used to simulate the structures and functions of native blood vessels. Here, thermoplastic polyurethane (TPU)/polycaprolactone (PCL)/polyethylene glycol (PEG) were electrospun to fabricate FLTSs or tubular scaffolds with completely random fibers layer (RLTSs). The surface wettability of the TPU/PCL/PEG tubular scaffold was tested by water contact angle analysis. In particular, compared with RLTSs, FLTSs showed excellent mechanical properties, with higher circumferential and longitudinal tensile properties. Furthermore, the high viability of the human umbilical vein endothelial cells (HUVECs) on the FLTSs indicated the biocompatibility of the tubular scaffolds comparing to RLTSs. The aligned and random composite structure of the FLTSs are conducive to promoting the growth of HUVECs, and the cell adhesion and proliferation on these scaffolds was found to be superior to that on RLTSs. These results demonstrate that the fabricated FLTSs have the potential for application in vascular tissue regeneration and clinical arterial replacements.

scholarly journals Three-dimensional printing of cell-laden bioink for blood vessel tissue engineering: Influence of process parameters and components on cell viability

2021 ◽  
Author(s):  
CONGCONG ZHAN ◽  
Yasong Hu ◽  
ANDUO ZHOU ◽  
SHANFENG ZHANG ◽  
Xia Huang
Keyword(s):  
Tissue Engineering ◽  
Blood Vessel ◽  
Cell Viability ◽  
Process Parameters ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Small Diameter ◽  
3D Bioprinting ◽  
Influence Of Process Parameters ◽  
Composite Gels

Three-dimensional (3D) bioprinting is a potential therapeutic method for tissue engineering owing to its ability to prepare cell-laden tissue constructs. The properties of bioink are crucial to accurately control the printing structure. Meanwhile, the effect of process parameters on the precise structure is not nonsignificant. We investigated the correlation between process parameters of 3D bioprinting and the structural response of κ-carrageenan-based hydrogels to explore the controllable structure, printing resolution, and cell survival rate. Small-diameter (<6 mm) gel filaments with different structures were printed by varying the shear stress of the extrusion bioprinter to simulate the natural blood vessel structure. The cell viability of the scaffold was evaluated. The in vitro culture of human umbilical vein endothelium cells (HUVECs) on the κ-carrageenan (kc) and composite gels (carrageenan/carbon nanotube and carrageenan/sodium alginate) demonstrated that the cell attachment and proliferation on composite gels were better than those on pure kc. Our results revealed that the carrageenan-based composite bioinks offer better printability, sufficient mechanical stiffness, interconnectivity, and biocompatibility. This process can facilitate precise adjustment of the pore size, porosity, and pore distribution of the hydrogel structure by optimising the printing parameters as well as realise the precise preparation of the internal structure of the 3D hydrogel-based tissue engineering scaffold. Moreover, we obtained perfused tubular filament by 3D printing at optimal process parameters.

scholarly journals Bioprinting small-diameter vascular vessel with endothelium and smooth muscle by the approach of two-step crosslinking process

2021 ◽  
Author(s):  
Qianheng Jin ◽  
Guangzhe Jin ◽  
Jihui Ju ◽  
Lei Xu ◽  
Linfeng Tang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Uv Light ◽  
Umbilical Vein ◽  
Vascular Grafts ◽  
Three Dimensional ◽  
Small Diameter ◽  
Tubular Structure ◽  
Flat Structure ◽  
Crosslinking Process ◽  
Umbilical Vein Endothelial Cells

Three-dimensional (3D) bioprinting shows great potential for autologous vascular grafts due to its simplicity, accuracy, and flexibility. 6mm diameter vascular grafts are used in clinic. However, producing small-diameter vascular grafts are still an enormous challenge. Normally, sacrificial hydrogels are used as temporary lumen support to mold tubular structure which will affect the structure’s stability. In this study, we develop a new bioprinting approach to fabricating small-diameter vessel using two-step crosslinking process. ¼ lumen wall of bioprinted gelatin mechacrylate (GelMA) flat structure is exposed to ultraviolet (UV) light briefly for having certain strength, while ¾ lumen wall shows as concave structure remained uncrosslinked. Pre-crosslinked flat structure is merged towards the uncrosslinked concave structure. Two individual structures will be combined tightly into an intact tubular structure by receiving more UV exposure time. Complicated tubular structures are constructed by these method. Notably, the GelMA-based bioink loaded with smooth muscle cells (SMCs) are bioprinted as the outer layer and human umbilical vein endothelial cells (HUVECs) are seeded onto the inner surface. A bionic vascular vessel with dual layers is fabricated successfully and keeps good viability, and functionality. This study may provide a novel idea for fabricating biomimetic vascular network or other more complicated organs.

scholarly journals QuBiT: A quantitative tool for epithelial tubes reveals cell dynamics and unexpected patterns of organization during Drosophila tracheal morphogenesis

2018 ◽  
Author(s):  
Ran Yang ◽  
Eric Li ◽  
Madhav Mani ◽  
Greg J. Beitel
Keyword(s):  
Cell Shape ◽  
Small Diameter ◽  
Cell Orientation ◽  
Cell Geometry ◽  
Cell Dynamics ◽  
Animal Survival ◽  
Tube Shape ◽  
Unexpected Findings ◽  
Cell Intercalation ◽  
Tubular Surfaces

AbstractBiological tubes are essential for animal survival, and their functions are critically dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. We therefore developed QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis during embryonic Drosophila tracheal (airway) development. We find that there are previously unknown anterior-to-posterior (A-P) gradients of cell orientation and aspect ratio, and that there is periodicity in the organization of cells in the main tube. Furthermore, cell intercalation during development dampens an A-P gradient of the number of the number of cells per cross-section of the tube, but these intercalation events do not change the patterns of cell connectivity. These unexpected findings demonstrate the importance of a computational tool for analyzing the morphogenesis of small diameter biological tubes.

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