POLITECNICO DI MILANO Engineering vascular tissue models in vitro

124 Pages·2017·19.05 MB·English

POLITECNICO DI MILANO

SCUOLA INTERPOLITECNICA DI DOTTORATO

Doctoral Program in Bioengineering

Final Dissertation

Engineering vascular tissue models in vitro

Nina Bono

Advisor Co-ordinator of the Research Doctorate Course
Prof. Gianfranco Beniamino Fiore Prof. Andrea Aliverti

Tutor
Prof. Alessandra Pedrocchi

January 27th, 2017

Acknowledgements

My PhD journey has come to an end. There were plenty of ups and downs - definitely more
downs - but no matter what, every challenge pushed me towards the “light at the end of the
tunnel”.
None of this would have ever been possible without the support of extraordinary people, who
helped me and taught me to never give up, that I would like to thank here:

My Advisor, Professor Gianfranco Beniamino Fiore, for his priceless guidance and his
precious support during these years. I know I was a “hard” student to work with, but with
your helps, you continuously stimulate me to do my best.

Professor Diego Mantovani, who gives me the possibility to learn a lot in his Lab and
improve myself as a “young research” and as a person as well.

The Interpolytechnic Doctoral School, for funding the scholarship that allowed me to
accomplish my research abroad.

A thank is due to Professor Gabriele Candiani, for his precious support in the last period, and
for passing me down the belief that, with passion and hard work, anything is possible (at least
at work!).

Professor Alessandra Pedrocchi, for her supervision and her helpful suggestions and
interesting discussions during these years

Professors Monica Soncini, Simone Vesentini, Alberto Redaelli and my lovely colleagues at
the Biomechs Group, beautiful people and valuable researchers, because without their
supports (and amazing parties J), days would have been much more hard in the Lab.

Amazing colleagues I worked with at LBB, for making my stay so pleasant in the lab and for
making me feel like home.

Finally, my family for believing in me, sometimes too much, and for raising me in the belief
to never give up and continue into my dream.

Table of contents
Table of contents

List of abbreviations 3

Abstract 5

1 Introduction 7
1.1. Tissue engineering 8
1.2. Engineering blood vessels in vitro 10
1.2.1. Materials and methods in vTE 11
1.2.2. Mechanical stimulation using bioreactors 13
1.3. Motivation and outline of the PhD thesis 15

2 Unraveling the role of mechanical stimulation on smooth muscle cells: a comparative
study between two-dimensional and three-dimensional models 21
Rationale 22
2.1. Introduction 23
2.2. Materials and Methods 24
2.2.1. Cell culture 24
2.2.1.1. Cell expansion 24
2.2.1.2. 2D cell culture experiments 24
2.2.1.3. 3D cell culture experiments (fabrication of 3D cell-collagen gels) 25
2.2.1.4. Cyclic mechanical stimulation 26
2.2.2. Western blot 26
2.2.3. Histological and immunofluorescence staining 27
2.2.4. Cell alignment measurements 28
2.2.5. Statistical analysis 29
2.3. Results and Discussion 29
2.3.1. Effect of uniaxial mechanical strain on cellular alignment in 2D vs. 3D models 30
2.3.2. Effect of uniaxial mechanical strain on cell phenotype in 2D vs. 3D models 34
Conclusive remarks 38

3 A bioengineering approach to engineer 3D cellularized collagen gels for vascular tissue
applications 43
Rationale 44
3.1. Introduction 45
3.2. Materials and Methods 46
3.2.1. Design of the bioreactor - Specifications 46
3.2.2. Architecture of the bioreactor 46
3.2.3. Design of the culture chamber 47
3.2.4. The hydraulic circuit 48
3.2.5. Monitoring and control system 48
3.2.6. Experimental validation of the bioreactor 49
3.2.6.1. Maintenance of the sterility 49
3.2.6.2. Validation of the M/C system 50
3.2.7. Preliminary culture of cellularized collagen-based gels 50
3.2.7.1. Construct fabrication and perfusion-based culture 50
3.2.7.2. Preliminary biomechanical characterization 51
Table of contents
3.2.8. Statistical analysis 52
3.3. Results 52
3.3.1. Functional assessment and setting of the stimulation program 52
3.3.2. Preliminary biomechanical characterization of collagen-based constructs 54
3.4. Discussion 55
Conclusive remarks 58

4 A dual-mode bioreactor system for vascular tissue engineering applications 63
Rationale 64
4.1. Introduction 65
4.2. Materials and Methods 66
4.2.1. Architecture of the dual-mode bioreactor system 66
4.2.2. Construct mode 68
4.2.3. Culture mode 69
4.2.3.1. Design of the culture mode 69
4.2.3.2. Mathematical prediction of the strain-pressure relation for the vascular
constructs 69
4.2.3.2.1. Characterization of the silicone sleeve behavior 70
4.2.3.2.2. Compliance tests on native vessels 71
4.2.3.2.3. Experimental validation with native vascular tissues 72
4.2.4. Setting up of the dual-mode bioreactor 72
4.2.4.1. Preparation of tubular constructs 72
4.2.4.1.1. Cell culture 72
4.2.4.1.2. Fabrication of cell-laden collagen gels constructs 73
4.2.4.2. Application of the mechanical stimulation 73
4.2.4.3. Histological and immunofluorescence staining of the constructs 74
4.2.4.4. Quantification of cells density 75
4.2.4.5. Step-wise stress-relaxation testing 75
4.2.5. Statistical analysis 76
4.3. Results 77
4.3.1. Assessment of distensibility behavior of sleeve and native vessels 77
4.3.2. Validation of the mathematical model 78
4.3.3. Performances of the dual-mode bioreactor for fabricating engineering tissues 81
4.3.4. Biological and biomechanical assessment of the constructs 82
4.3.4.1. Effects of culture condition of construct contraction 82
4.3.4.2. Effects of culture condition on construct morphology and cell behavior 83
4.3.4.3. Effects of culture condition on constructs’ biomechanical properties 87
4.4. Discussion 89
Conclusive remarks 91

5 General conclusions and future perspectives 95

ANNEX A 104

List of publications 116
List of abbreviations
List of abbreviations

2D Two-dimensional
3D Three-dimensional
A Initial Cross-sectional Area
0
BCA Bicinchoninic Acid Assay
BP Burst Pressure
BSA Bovine Serum Albumin
CAs Porcine Coronary Arteries
CLM Compressive Linear Modulus
DMEM Dulbecco’s Modified Eagle’s Medium
ECM Extracellular Matrix
ECs Endothelial cells
EGF Epidermal Growth Factor
EtO Ethylene Oxide
FBS Fetal Bovine Serum
FBs Fibroblasts
FGF-b Fibroblast Growth Factor-basic
GFs Growth Factors
HI Human Insulin
HUASMCs Human Umbilical Aortic Smooth Muscle Cells
IF Immunofluorescence
L Gauge Length
0
M/C Monitoring and Control
ON Overnight
PBS Phosphate-buffered Saline
Pen-Strep Penicillin-Streptomycin
PGA Polyglycolic Acid
PLA Polylactic Acid
PMMA Polymethylmethacrylate
PP Polypropylene
pSMCs Porcine Smooth Muscle Cells
RIPA Radioimmunoprecipitation Assay
3
List of abbreviations
ROI Region of interest
RPMI Roosvelt Park Memorial Institute medium 1640
RT Room Temperature
SD Standard Deviation
SM α-actin Smooth Muscle alpha-actin
SMCs Smooth Muscle Cells
SV Saphenous Vein
TBS Tris-buffered saline
TE Tissue Engineering
TEVCs Tissue Engineered Vascular Constructs
UCT Unconfined compression tests
UTS Ultimate Tensile Stress
UTT Uniaxial tensile tests
vSMCs vascular Smooth Muscle Cells
vTE vascular Tissue Engineering

4
Abstract
Abstract

Vascular tissue engineering (vTE) has made significant progress over the past decades
towards the creation of engineered blood vessel-like structures suitable for replacement or
bypass of damaged arteries. More recently, in parallel to the quest for clinical applications,
engineered tissues have been developed as laboratory-oriented tools to study the mechanisms
involved in vascular physiology and pathophysiology.
In this context, the goal of this PhD thesis was to develop a suitable strategy to engineer
3D vascular tissues as in vitro models for mechanobiology investigations. The main
commitment was to capture in vitro some of the complex features of the in vivo vascular
milieu, with particular focus on the replication of the vascular-like hemodynamics and the
way how it influences cell and tissue behaviors. Since vascular smooth muscle cells (vSMCs)
are the predominant cell type in blood vessel wall and primarily responsible for
vasoregulation and vessel remodeling, major efforts have been devoted to the in vitro
development of SMC-based engineered tissues. On the one hand the focus was to demonstrate
that contractile vascular cells respond more physiologically in a 3D environment under cyclic
mechanical stimulation; on the other hand, the focus was on the development and stepwise
optimization of a culture platform for the fabrication of tubular constructs and their
stimulation at once.
In the whole context of the vTE, the general picture drawn in this PhD thesis
highlighted i) the importance of the 3D environment to study the cell behavior, and ii) the
need of mechanically stimulating 3D vascular tissue structures to drive their biological and
biomechanical properties and their maintenance in vitro. This research has led to the
development of a newly, easy-to-use and inexpensive bioreactor useful to produce viable
vascular models. In perspective, this platform will provide new insights into the potential to
drive cell behavior while studying mechanotransduction pathways activated following the
exposure to mechanical strain.