In vitro three-dimensional bone tissue models: from cells to controlled and dynamic environment.

TLDR: This review focuses on state-of-the-art and the current advances in the development of 3D culture systems for bone biology research, and details main characteristics and challenges associated with its three main components, that is, scaffold, cells, and perfusion bioreactor systems.

Abstract: Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates However, these traditional monolayer cell cultures do not reproduce the complex and dynamic 3-dimensional (3D) environment experienced by cells in vivo Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology These in vitro engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical and biomechanical cues that define the in vivo bone environment, while allowing their monitoring This review focuses on state of the art and the current advances in the development of 3D culture systems for bone biology research It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, ie scaffold, cells and perfusion bioreactor systems Finally, future challenges for non-invasive imaging technologies are addressed

Figures

Figure 2. Models of bioreactors. (A) Example of perfusion bioreactor in which the culture medium is forced through the internal interconnected porous network of the seeded scaffold. This can override diffusion limitations and enhance nutrient delivery and waste removal from the cultured cells. The setup includes a medium reservoir, a pump and a tubing system which feeds the culture chamber; (B) Example of perfusion bioreactor allowing the application of controlled dynamic compression to the scaffolds perpendicularly to fluid flow in a dissociated or combined manner.

Table 1: Effects of shear stresses on bone cells seeded on glass in parallel plate flow chambers

Figure 3. Actors and mechanisms of cellular mechanosensing. Mechanical forces can be sensed and transduced by different means through membrane and/or organelle deformation or cell/ECM interactions.

Table 2: Numerical models prediction of shear stress values induced by flow within scaffolds

Figure 4. Schematic representation of the key elements of in vitro 3D culture model including: cell type choice depending on the goal of the study, main scaffold and bioreactor features needed, as well as characterization tools.

Table 3: Direct mechanical loading of cells seeded scaffolds – Modalities and effects Bouet.Table3

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In vitro 3D bone tissue models, from cells to controlled
and dynamic environment
Guénaëlle Bouët, David Marchat, Magali Cruel, Luc Malaval, Laurence Vico
To cite this version:
Guénaëlle Bouët, David Marchat, Magali Cruel, Luc Malaval, Laurence Vico. In vitro 3D bone tissue
models, from cells to controlled and dynamic environment. Tissue Engineering: Parts A, B, and C,
Mary Ann Liebert, 2015, 21 (1), pp.133-156. �10.1089/ten.teb.2013.0682�. �hal-01282701�

1
In vitro 3D bone tissue models, from cells to controlled and dynamic
environment
Guenaelle Bouet
1
, David Marchat
2*
, Magali Cruel
3
, Luc Malaval
1
, Laurence Vico
1
1
Laboratoire de Biologie du Tissu Osseux and Institut National de la Santé et de la Recherche
Médicale - U1059, Université de Lyon - Université Jean Monnet, Saint-Etienne, France
2
Center for Biomedical and Healthcare Engineering, Ecole Nationale Supérieure des Mines,
CIS-EMSE, CNRS:UMR 5307, F-42023 158 cours Fauriel Saint-Etienne cedex 2, France
3
University of Lyon, LTDS, UMR CNRS 5513, Ecole Centrale de Lyon, 36 avenue Guy de
Collongue, 69134 Ecully Cedex, France
Full mailing address and contact information for EACH author:
* Corresponding author: David Marchat, Tel.: +33 4 77 49 97 01; fax: +33 4 77 49 96 94. E-
mail: marchat@emse.fr
Guenaelle Bouet, Tel.: +33 4 77 42 14 47; fax: +33 4 77 57 55 72. E-mail:
guenaelle.bouet@gmail.com
Magali Cruel, Tel.: +33 4 72 18 62 15; fax: +33 4 77 49 96 94. E-mail:
magali.cruel@ec-lyon.fr
Luc Malaval, Tel.: +33 4 77 42 14 44; fax: +33 4 77 57 55 72. E-mail: luc.malaval@univ-st-
etienne.fr
Laurence Vico, Tel.: +33 4 77 42 18 57; fax: +33 4 77 57 55 72. E-mail: vico@univ-st-etienne.fr

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Abstract:
Most of our knowledge of bone cell physiology is derived from experiments carried out in
vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not
reproduce the complex and dynamic 3-dimensional (3D) environment experienced by cells in
vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for
understanding bone biology. These in vitro engineered systems, less complex than in vivo
models, should ultimately recapitulate and control the main biophysical, biochemical and
biomechanical cues that define the in vivo bone environment, while allowing their
monitoring. This review focuses on state of the art and the current advances in the
development of 3D culture systems for bone biology research. It describes more specifically
advantages related to the use of such systems, and details main characteristics and
challenges associated with its three main components, i.e. scaffold, cells and perfusion
bioreactor systems. Finally, future challenges for non-invasive imaging technologies are
addressed.
Keywords: Bone tissue engineering, Perfusion bioreactor, Cell culture, Scaffold, Experimental
approaches, Mechanical stimuli

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1. CONTEXT
1.1. Tissue Engineering: concepts
Tissue engineering is the application of the principles of biology and engineering to the
development of artificial living tissues (1). It utilises specific combinations of cells, matrices
(referred also as “scaffolds or constructs”), as well as cellular, chemical and mechanical
signals. Generally speaking, current applications of tissue engineering can be divided into
two categories.
The first category consists of elaborating biological substitutes to restore, maintain or
improve tissue functions (1). This therapeutic approach aims to replace/repair damaged
human tissues with manufactured tissue-engineered products. This field is quickly expanding
due to coordinated and converging technological developments in the creation and/or
manipulation of biomolecules, biological materials, cells and tissues, with the goal of
ultimately generating pseudo-tissues and organs for transplant. Such therapy will be
drastically different from classical prosthesis implants, avoiding integration, interface and
wearing-out problems, thus allowing for a longer lived tissue/organ substitution.
The second category of applications of tissue engineering aims to understand the
fundamental aspects of cells working in vivo in 3D controlled systems. In fact, in vivo cells
reside in a complex three dimensional (3D) micro-environment, encompassing several cell
types producing/exchanging many signals, interacting in a dynamic fashion amongst
themselves. These cells also interact with an extracellular matrix whose rigidity varies in time
and space with the nature and/or maturity of the cell type/tissue (2, 3). Moreover, in tissues
like bone, cells experience various mechanical stresses (e.g. compression forces). Therefore,
in vitro 3D systems, based on tissue engineering principles, strive to reproduce (at least
partly) the in vivo environment of cells in a scaffold material. As it was recently

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demonstrated by many studies (4, 5) for review), such systems implies the use of perfusion
bioreactors to control and monitor biochemical and mechanical signals throughout cell
culture.
Such complex models represent the best approach to understand the bone (cell) biology
since they allow study of a multicellular microenvironment whose structure, composition,
topology and perfusion conditions are adaptable and more realistic, respective to the in vivo
situation, than the current two dimensional (2D) standard cell culture method. For instance,
in these systems it will be possible to test the effects of chemical substances or characterise
stimuli acting locally on cell metabolism in order to understand cellular and matrix changes
involved in aging mechanisms and some diseases (e.g. osteoporosis); they will also provide
tools for assessing drugs in development in toxicology or pharmacology studies.
The knowledge gained from such experiments will advance our understanding of
pathophysiology as well as the prevention, diagnosis and treatment of diseases.
1.2. From 2D in vitro cell culture to native bone tissue, the missing link
Bone tissue development and remodeling in living organisms are orchestrated by cascades of
regulatory factors interacting at multiple levels, in both time and space.
Animal models provide the full complexity of biological systems (at least within a given
species), but they offer limited control of the local environment and scanty real time
information. In contrast, traditional cell culture in 2D allows significant control of the cellular
environment and a direct access to cellular processes. However, these culture conditions are
drastically simplified and hardly reproduce the typical bone environment in the
organ/organism, in which (bone) cells develop in a 3D structure subjected to mechanical
stimulation. Moreover, it is well established that signal transduction and many other cellular