In vitro elastogenesis: instructing human vascular smooth muscle cells to generate an elastic fiber-containing extracellular matrix scaffold

Svenja Hinderer,Elaine C Davis,Nian Shen,Sara Y Brucker,Léa-Jeanne Ringuette,Katja Schenke-Layland,Dieter P Reinhardt,Jan Hansmann

doi:10.1088/1748-6041/10/3/034102

Abstract

Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.

Highlights

The extracellular matrix (ECM) is a complex assembly of structural and functional proteins that are maintained by the resident cells
Neonatal or adult rat vascular smooth muscle cells (VSMCs) and neonatal human dermal fibroblasts are routinely used to synthesize tropoelastin in vitro, and elastin deposition can be shown in two-dimensional (2D) cell cultures [1, 8, 9], there have been no reports of the generation of functional human-based elastic fibers in three-dimensional (3D) tissue-engineered constructs [10,11,12]
VSMC alignment was observed after 6 d of culture, a phenomenon that has been previously described when VSMCs are exposed to laminar shear stress [33]

Summary

Introduction

The extracellular matrix (ECM) is a complex assembly of structural and functional proteins that are maintained by the resident cells. Inducing elastogenesis in a tissue-engineered construct would enable the production of functional implants and the generation of advanced human-based in vitro test systems. These elastic fibercontaining systems could either be used to further understand the process of elastic fiber assembly or to study processes that are attributed to elastic fiber degeneration, including ageing. Neonatal or adult rat vascular smooth muscle cells (VSMCs) and neonatal human dermal fibroblasts are routinely used to synthesize tropoelastin in vitro, and elastin deposition can be shown in two-dimensional (2D) cell cultures [1, 8, 9], there have been no reports of the generation of functional human-based elastic fibers in three-dimensional (3D) tissue-engineered constructs [10,11,12]. It has been previously described that biochemical and biophysical factors impact elastin gene and protein expression in 3D tissue-engineered constructs, elastic fiber assembly in the extracellular space has not been shown [13]

Methods

Results

Discussion

Conclusion