Substrate Microarchitecture Shapes the Paracrine Crosstalk of Stem Cells with Endothelial Cells and Osteoblasts

Francisco Martín-Saavedra,Enrique Gómez-Barrena,Nuria Vilaboa,Lara Crespo,Laura Saldaña,Clara Escudero-Duch

doi:10.1038/s41598-017-15036-x

Abstract

We examined the hypothesis that substrate microarchitecture regulates the crosstalk between human mesenchymal stem cells (hMSC) and cell types involved in bone regeneration. Compared with polyester flat substrates having uniformly distributed homogenous pores (2D), three-dimensional polystyrene substrates with randomly oriented and interconnected pores of heterogeneous size (3D) stimulated the stromal secretion of IGF-1 while lessened the production of VEGFR-1, MCP-1 and IL-6. The medium conditioned by hMSC cultured in 3D substrates stimulated tube formation by human endothelial cells (hEC) to a higher extent than medium from 2D cultures. 3D co-cultures of hMSC and hEC contained higher secreted levels of IGF-1, EGF and FGF-2 than 2D co-cultures, resulting in increased hEC proliferation and migration. Substrate microarchitecture influenced the secretion of factors related to bone remodeling as the ratio RANKL to OPG, and the levels of M-CSF and IL-6 were higher in 3D co-cultures of hMSC and human osteoblasts (hOB) than in 2D co-cultures. Cytokine microenvironment in 3D co-cultures stimulated osteoblast matrix reorganization while demoted the late steps of osteoblastic maturation. Altogether, data in this study may unveil a new role of scaffold microarchitecture during bone regeneration, as modulator of the paracrine relationships that hMSC establish with hEC and hOB.

Highlights

Bone tissue engineering aims to develop substrates that establish proper interactions with MSC to generate ex vivo “tissue intermediates” which, upon implantation in the injured bone site, unlock mechanisms of self-regeneration
Actin filaments arranged in more closely package arrays in cells growing in 3D substrates, as revealed by quantification of Figure 2. 2D and 3D substrates differentially affect the paracrine relationships that human mesenchymal stem cells (hMSC) establish with human umbilical vein EC (HUVEC). (a) Secreted angiopoietin-1, vascular endothelial growth factor (VEGF), VEGFR-1, monocyte chemotactic protein-1 (MCP-1), IL-6 and IGF-1 levels in media conditioned by hMSC cultured for 7 days on 2D or 3D substrates
Data are relative to the levels measured in 2D cultures, which were given an arbitrary value of 100 and correspond to the values shown in the Table 1. (b) Representative phase contrast micrographs of HUVEC incubated in media conditioned by hMSC cultured on 2D or 3D substrates, in EGM-2 or in growth medium (GM). (c) Metabolic activity of HUVEC or hMSC

Summary

Introduction

Bone tissue engineering aims to develop substrates that establish proper interactions with MSC to generate ex vivo “tissue intermediates” which, upon implantation in the injured bone site, unlock mechanisms of self-regeneration. Levels of soluble factors related with inflammation and chemotaxis are substantially lower in co-cultures of human macrophages and hMSC seeded in polystyrene substrates with randomly oriented and interconnected pores of variable size than in co-cultures of hMSC seeded on polyester flat surfaces with uniform porosity. Both types of substrates, named 3D and 2D respectively, were used in the present study to investigate to what extent their topographical features might influence the paracrine relationships that transplanted hMSC maintain with resident cells that participate in bone healing, such as hEC or hOB

Methods

Results

Conclusion