Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches

Electrospun freestanding hydrophobic fabric as a potential polymer semi-permeable membrane for islet encapsulation

One of the significant problems associated with islet encapsulation for type 1 diabetes treatment is the loss of islet functionality or cell death after transplantation because of the unfavorable environment for the cells. In this work, we propose a simple strategy to fabricate electrospun membranes that will provide a favorable environment for proper islet function and also a desirable pore size to cease cellular infiltration, protecting the encapsulated islet from immune cells. By electrospinning the wettability of three different biocompatible polymers: cellulose acetate (CA), polyethersulfone (PES), and polytetrafluoroethylene (PTFE) was greatly modified. The contact angle of electrospun CA, PES, and PTFE increased to 136°, 126°, and 155° as compared to 55°, 71°, and 128° respectively as a thin film, making the electrospun membranes hydrophobic. Commercial porous membranes of PES and PTFE show a contact angle of 30° and 118°, respectively, confirming the hydrophobicity of electrospun membranes is due to the surface morphology induced by electrospinning. In- vivo results confirm that the induced hydrophobicity and surface morphology of electrospun membranes impede cell attachment, which would help in maintaining the 3D circular morphology of islet cell. More importantly, the pore size of 0.3–0.6 μm obtained due to the densely packed structure of nanofibers, will be able to restrict immune cells but would allow free movement of molecules like insulin and glucose. Therefore, electrospun polymer fibrous membranes as fabricated in this work, with hydrophobic and porous properties, make a strong case for successful islet encapsulation

FACS analysis for VEGFR2 on the surface of HDMECs at week 1 (A,C) and week 2 (B, D).

<p>The colors are depicted as black: negative control; blue: cells in cocktail media = group I; red: group II (A, B) or, group III (C, D); green: group IV (A, B) or, group V (C, D). The percentages of total cells positive for the dye in group IV (MSC-bioglass) are indicated in A and B; those in group V (MSC-Cu-bioglass) are in C and D.</p

The primers used for real time RT-PCR.

<p>The primers used for real time RT-PCR.</p

At the end of week 2, HDMECs were trypsinized and assayed for tube formation in Matrigel.

<p>(A) group I, (B) group II, (C) group III, (D) group IV, and (E) group V. Similar pictures were observed also at week 1. (F) Schematic diagram showing the scaffold seeded with cells in culture inserts and one layer of HDMECs at the bottom as in groups IV and V.</p

At the end of week 2, acetylated LDL uptake assay is performed in one well seeded with HDMECs.

<p>Though all cells uptake the dye, only in group V cells containing MSC-seeded Cu²⁺-bioglass inserts show tube like forms even in 2D cultures (arrows). (A) group I, (B) group II, (C) group III, (D) group IV, and (E) group V. For comparison, a light microscopic picture of the same well is shown in figure F.</p

At the end of week 2, light microscopic pictures of HDMECs show tube formation only in group V containing both MSC-seeded Cu²⁺-bioglass inserts (arrows).

<p>(A) group I, (B) group II, (C) group III, (D) group IV, and (E) group V.</p