Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures.

Iris Pennings,Emilie Barruet,Blanca M Morales,Juliet Huuksloot,Joost O Fledderus,Lukas A Dijk,Debby Gawlitta,Koen Schepers,Marianne C Verhaar,A Koen Braat,Edward C Hsiao,Antoine J.W.P Rosenberg

doi:10.1002/term.2807

Abstract

To introduce a functional vascular network into tissue‐engineered bone equivalents, human endothelial colony forming cells (ECFCs) and multipotent mesenchymal stromal cells (MSCs) can be cocultured. Here, we studied the impact of donor variation of human bone marrow‐derived MSCs and cord blood‐derived ECFCs on vasculogenesis and osteogenesis using a 3D in vitro coculture model. Further, to make the step towards cocultures consisting of cells derived from a single donor, we tested how induced pluripotent stem cell (iPSC)‐derived human endothelial cells (iECs) performed in coculture models. Cocultures with varying combinations of human donors of MSCs, ECFCs, or iECs were prepared in Matrigel. The constructs were cultured in an osteogenic differentiation medium. Following a 10‐day culture period, the length of the prevascular structures and osteogenic differentiation were evaluated for up to 21 days of culture. The particular combination of MSC and ECFC donors influenced the vasculogenic properties significantly and induced variation in osteogenic potential. In addition, the use of iECs in the cocultures resulted in prevascular structure formation in osteogenically differentiated constructs. Together, these results showed that close attention to the source of primary cells, such as ECFCs and MSCs, is critical to address variability in vasculogenic and osteogenic potential. The 3D coculture model appeared to successfully generate prevascularized constructs and were sufficient in exceeding the ~200 μm diffusion limit. In addition, iPSC‐derived cell lineages may decrease variability by providing a larger and potentially more uniform source of cells for future preclinical and clinical applications.

Highlights

Current in vitro tissue engineering strategies are largely confined to the creation of small constructs
Generation of an inherent functional 3D vascular network within the tissue‐engineered construct is considered essential for the creation of larger constructs
We used a matrigel/hydrogel‐based model to create vascular networks in an osteogenic construct, and used these constructs to investigate an important aspect of robustness of experimental outcomes.The model consistently generated 3D prevascular networks supported by mural cells in osteogenically differentiated constructs

Summary

| INTRODUCTION

The reproducibility and standardization of coculture protocols and outcomes of selected release criteria are essential in enabling quality control of the resulting prevascularized tissue constructs for clinical application. At present, it is not known how the choice and combination of cells from one or different donors affects coculture outcomes. The first aim of the present study is to examine the effects of donor variation on the vasculogenesis and osteogenesis of a 3D coculture model of subcultured primary MSCs and ECFCs. Translating 3D cocultures to the clinic by incorporation of (preferably) autologous cells raises another critical challenge: limited cell sources. A Matrigel coculture system in an osteogenic environment was developed in which donor dependency of vasculogenic and osteogenic cells and their behaviour could be assessed

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION

Findings

CONFLICT OF INTEREST