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.

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 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.

Substratum modification by endothelial cells (EC) in culture

1992 ◽  
Vol 50 (1) ◽  
pp. 656-657
Author(s):  
J.C. Lewis ◽  
I. Hermanns ◽  
K.W. Grant ◽  
S. Evans ◽  
C. Gossen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Collagen Type ◽  
Umbilical Vein ◽  
Surface Characteristics ◽  
Collagen Iv ◽  
Collagen Type Iv ◽  
Cell Periphery ◽  
Natural Polymers ◽  
Type Iv ◽  
Grafting Materials

Thrombosis resulting from blood interaction with prostheses is a concern following vascular grafting, and efforts have been made to endothelialize grafting materials and thereby reduce thrombogenicity. These efforts have focused upon altering polymer surfaces to enhance endothelial compatibility following surgery; however, using homologous EC attempts are being made to pre-seed grafting materials having defined surface characteristics. Such surfaces might be pre-absorbed with natural polymers as collagens, fibrin(ogen) or fibronectin. Irrespective of the starting character, these surfaces are rapidly altered following contact with tissues and body fluids, and as an extension by the EC themselves. To address this latter question, human umbilical vein endothelial cells (HUVEC) were seeded for whole-mouat microscopy onto carbon-stabilized, formvar-coated surfaces to which either collagen Type IV or its CB3 fragment had been adsorbed. Following culture for 3 or 24 hrs the cells were fixed in situ with glutaraldehyde, washed and processed for two-stage multiantigen immunogold microscopy. The first stage was comprised of rabbit anti-human fibronectin and mouse antihuman collagen Type IV. These were followed by second stage probes including goat anti-rabbit IgG (5nM gold), goat anti-mouse IgG (15 nM gold) and goat anti-human IgG (10 nM gold) as a control. HUVEC readily attached to and spread upon collagen IV and the CB3 fragment; the degree of spreading was much more pronounced with CB3 at both 3 and 24 hrs. The primary difference between the culture times was in cell density, with more cells at 24 than 3 hrs (Fig. 1). This difference could be explained by the additional generation time. The interaction of cells with the substratum was complex. This included deposition of proteins on the surface and extension of delicate macromolecular bridges from the cell periphery. Through the use of immunogold microscopy, fibronectin, collagen and immunoglobulin could be detected in all samples, including those plated in the absence of serum. Initially, collagen (IV and CB3) was more dense on the substrate than on the cells, and this was consistent with surface coating prior to cell seeding (Fig. 3). Fibronectin and IgG, on the other hand, were derived either from the medium or secreted from the cells, and each was associated with the cells or the cell edge (Fig. 4,5). Notably, detection of all three peptides was reduced over time, and this may be interpreted as secondary conditioning of the surface by HUVEC secreted products.

The use of 1nm silver enhanced gold probes for the detection of skin antigens

1990 ◽  
Vol 48 (3) ◽  
pp. 902-903
Author(s):  
J.P Cassella ◽  
H. Shimizu ◽  
A. Ishida-Yamamoto ◽  
R.A.J. Eady
Keyword(s):  
Type Iv Collagen ◽  
Freeze Substitution ◽  
Collagen Type Iv ◽  
Electron Microscopic ◽  
Normal Human Skin ◽  
Type Iv ◽  
Type Vii Collagen ◽  
Keratin Filaments ◽  
Normal Human ◽  
Phosphate Buffered Saline

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.

scholarly journals Serum Anti-Collagen IV IgM and IgG Antibodies as Indicators of Low Vascular Turnover of Collagen IV in Patients with Long-Term Complications of Type 2 Diabetes

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 900
Author(s):  
Krasimir Kostov ◽  
Alexander Blazhev
Keyword(s):  
Type 2 Diabetes ◽  
Serum Levels ◽  
Vascular Wall ◽  
Collagen Iv ◽  
Collagen Type Iv ◽  
Non Invasive ◽  
Type Iv ◽  
Antibody Levels

Thickening of the vascular basement membrane (BM) is a fundamental structural change in the small blood vessels in diabetes. Collagen type IV (CIV) is a major component of the BMs, and monitoring the turnover of this protein in type 2 diabetes (T2D) can provide important information about the mechanisms of vascular damage. The aim of the study was through the use of non-invasive biomarkers of CIV (autoantibodies, derivative peptides, and immune complexes) to investigate vascular turnover of CIV in patients with long-term complications of T2D. We measured serum levels of these biomarkers in 59 T2D patients with micro- and/or macrovascular complications and 20 healthy controls using an ELISA. Matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) were also tested. In the T2D group, significantly lower levels of CIV markers and significantly higher levels of MMP-2 and MMP-9 were found compared to controls. A significant positive correlation was found between IgM antibody levels against CIV and MMP-2. These findings suggest that vascular metabolism of CIV is decreased in T2D with long-term complications and show that a positive linear relationship exists between MMP-2 levels and CIV turnover in the vascular wall.

Release of collagen type IV degrading activity from C6 astrocytoma cells and cell density

1996 ◽  
Vol 84 (6) ◽  
pp. 1013-1019 ◽  
Author(s):  
Masashi Tamaki ◽  
Warren McDonald ◽  
Rolando F. Del Maestro
Keyword(s):  
Cell Density ◽  
Collagen Type ◽  
Cell Communication ◽  
Collagen Type Iv ◽  
Major Protein ◽  
Content Type ◽  
Type Iv ◽  
Astrocytoma Cells ◽  
C6 Astrocytoma

✓ Type IV collagen is a major protein component of the vascular basement membrane and its degradation is crucial to the initiation of tumor-associated angiogenesis. The authors have investigated the influence of cell density on the release of collagen type IV degrading activity by C6 astrocytoma cells in monolayer culture. The release of collagen type IV degrading activity was assessed biochemically, immunocytochemically, and by Western blot analysis. The results demonstrate that increasing plating density and increasing cell density are associated with decreased collagen type IV degrading activity released per tumor cell. These findings indicate the existence of regulatory mechanisms dependent on cell—cell communication, which modulate release of collagen type IV degrading activity. The extrapolation of these results to the in vivo tumor microenvironment would suggest that individual and/or small groups of invading tumor cells, distant from the main tumor mass, would release substantial collagen type IV degrading activity, which may be crucial to their continued invasion and to angiogenesis.

Collagen type IV in acute rejection of kidney

2000 ◽  
Vol 32 (7) ◽  
pp. 1785 ◽  
Author(s):  
K Utsumi ◽  
T Sawada ◽  
E Adachi ◽  
S Horita ◽  
T Tojimbara ◽  
...  
Keyword(s):  
Acute Rejection ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Type Iv
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