Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Summary

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Abstract

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.

Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

Introduction

Cardiovascular tissues have a prominent load-bearing function. In particular content and organization of collagen fibers in the extracellular matrix contribute to the load-bearing properties and dominate overall tissue strength¹. In tissue engineering mechanical conditioning of the construct is used - typically consisting of (cyclic) straining regimens - to enhance tissue organization and mechanical properties^2,3. Full understanding of strain-induced collagen organization in complex tissue geometries to create tissues with predefined collagen architecture has not yet been achieved. This is mainly due to our limited knowledge of collagen remodelin....

Protocol

1. Culture of Human Vena Saphena Cells

1. Isolate cells from the vena saphena magna, acquired from a donor in accordance to guidelines for secondary use material, according to the protocol by Schnell et al.⁹ and store these in liquid nitrogen. From the part of the vena saphena magna from one donor cut pieces of 2 x 2 mm to culture in a six-well plate. Use 2 pieces per well. Generally enough cells can be obtained to fill about 3 vials with 0.25 x 10⁶ cells in liquid nitrogen. HVSCs are characterized as myofibroblasts, by showing expression of vimentin, no expression of desmin and a subpopulation expressing α smooth muscle ac....

Results

This model system allows for culturing myofibroblast-seeded fibrin gels. Figure 1A shows a tissue cultured first under static biaxial constraints. Tissue constraints are released by cutting the fibrin gel from two constraints, to create uniaxial static constraints, and tissue compacts and remodels afterwards (Figure 1A). For cyclic strain, the tissue is cultured under static biaxial constraints as well. After 5 days cyclic uniaxial strain can be applied (Figure 1B). To i.......

Discussion

The described model system of cell-populated fibrin constructs has great potential for the study of cell and collagen (re)organization (de Jonge et al.¹⁵), e.g. to be used for tissue engineering purposes. By using fibrin as the initial cell carrier, after fibrin degradation, a tissue is created with cells and endogenous matrix only. In this way, cells are stimulated to react to strain, either static or cyclic in nature, by applying contractile forces^16,17, sensing boundary stiffnes.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Culture plastic	Greiner		Includes culture flasks and pipettes
Advanced DMEM	Gibco	12491
Fetal bovine serum	Greiner	758075
Penicillin/streptomycin	Gibco	10378016
GlutaMax	Gibco	35050-079
Elastomer and curing agent	Dow Corning Corporation	3097358-1004	Silastic MDX 4-4210#
Velcro	Regular store		You can buy this at a regular store, only use the soft side
Bioflex culture plates	Flexcell Int	BF-3001U	Untreated
L-Ascorbic Acid 2-phosphatase	Sigma	A8960
ε-Amino Caproic Acid	Sigma-Aldrich	D7754
Bovine thrombin	Sigma	T4648
Bovine fibrinogen	Sigma	F8630
0.45 syringe filter	Whatmann (Schleicher and Scheul)	10462100
Polystyrene microspheres	Invitrogen	F-8829	Blue fluorescent, 10 μm diameter
Flexcell FX-4000T	Flexcell Int		Includes rectangular loading posts
Cell Tracker Orange	Invitrogen Molecular Probes	C2927
CNA35-OG488			Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology
Confocal laser scanning microscope	Carl Zeiss		LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode
Amphotericin	Gibco	15290-018	Needed for cell isolation