Bioactive copper-doped glass scaffolds can stimulate endothelial cells in co-culture in combination with mesenchymal stem cells.

Subha N Rath,Ulrich Kneser,Alexander Hoppe,Andreas Brandl,Daniel Hiller,Uwe Gbureck,Aldo R Boccaccini,Raymund E Horch,Elisabeth Engel

doi:10.1371/journal.pone.0113319

Abstract

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.

Highlights

Bone tissue engineering requires application of suitable biomaterials and cells, which can effectively produce a bone-like tissue matrix in them and, which can substitute the bone defect site [1]
Tests Alamar blue, alkaline phosphatase (ALP) assay, RT-PCR of osteogenic markers RT-PCR of vascular endothelial growth factor (VEGF) Alamar blue, ALP assay, Scanning electron microscopic (SEM) or cell attachment Cu2+ quantification
We have shown that Cu2+-doped 45S5 Bioactive glass (BG) scaffolds exhibit high acellular bioactivity as proven by rapid formation of a carbonated HA layer on BG scaffolds surface [16]

Summary

Introduction

Bone tissue engineering requires application of suitable biomaterials and cells, which can effectively produce a bone-like tissue matrix in them and, which can substitute the bone defect site [1]. Without proper nutrition from a vascular channel, the cells become necrosed and the whole system as a bone replacement product fails to replace the lost tissue. The vascularized bone is crucial for weight-bearing lost bone, substantial bone loss, and for precise healing of the defect site. Bioactive glass (BG) is an important biomaterial for scaffold-based bone tissue engineering applications, as it has shown high osteo-inductive and angioinductive properties compared to other biomaterials [3, 4]. Calcium phosphatebased apatite is produced on the surface of BG in contact with body fluids, and BG represents a suitable substrate to promote bone formation both in vitro and in vivo. The release of inorganic ions in the media affects the cell behavior for osteogenesis [5, 6]

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