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Introduction to 3D scaffolds for tissue engineering
At first, the cell substrate is of particular importance, since in vivo the extracellular space is occupied by the ECM. The chemical composition of the ECM and the resulting mechanical properties are both important aspects, as the transduction of both chemical and physical signals via cellular adhesion molecules affects cell shape, polarization, migration and differentiation (Burute and Thery 2012; Kaivosoja et al. 2012; Peyton et al. 2007). Furthermore, the ECM topography orients tissue polarity and the morphogenesis of new organs (Burute and Thery 2012; Vogel and Sheetz 2006).
Crude in vitro substrates that lack these features are therefore often insufficient for guiding the assembly and coordination of isolated cells to form a coherent tissue with a common global orientation and polarity. New devices that mimic the microscale structures of the ECM are needed and are now being developed thanks to the fast evolving fields of microfabrication and microfluidics.
Microfabrication allows researchers to structure space at the right order of scale (from micrometers to centimeters) and to position individual cells according to the architectures they experienced in vivo.
Microfluidics also provides new tools for controlling the transport and availability of chemical and biochemical signals on such micron scales. The cells can be seeded in an enclosed area with volume restrictions and transport properties favoring autocrine and paracrine communication, allowing users to build more realistic and/or more specific culture conditions than can be achieved in traditional cell culture. The resulting autonomous and functional tissues shaped with a morphology that attempts to recapitulate conditions seen in vivo organs has led to the concept of „organs on chip‟. The first subpart describes the main chemical and mechanical features needed to create an artificial extracellular matrix that recapitulates in vivo ECM as closely as possible, in order to induce cell differentiation. The second section presents the techniques to structure artificial matrix and already existing devices.
Importance of dynamic hydrogels
In vivo, extracellular matrices are dynamic structures constantly permeated by soluble molecules such as growth factors. Adhesion molecules are also constantly renewed, resulting in either modification or maintenance of their amount and distribution. Furthermore, the cell themselves are also able, in many instances, to modify and remodel their surrounding ECM structure and topography. This means that artificial ECMs will need to incorporate dynamic features if they are to accurately mimic in vivo physiology.
Mimicking ECM dynamics, however, is not straightforward. Although growth factors can be trapped
inside a hydrogel, for example, they are usually released all at once by hydrogel swelling (Andreopoulos and Persaud 2006; Sano et al. 1998; van de Wetering et al. 2005). Growth factors can also be directly added to the culture medium, but this often generates a non-homogeneous distribution of growth factors between external and internal parts of the hydrogel, due to the diffusion of molecules from the medium into the gel.
For improved control, the chemistry of some hydrogels, especially PEGDA hydrogels, can be tailored to reproduce ECM dynamics. For example, chemically modified growth factors (growth factor-PEG methacrylate) and matrix metalloproteinases cleavable groups (A-PEG-cleavable structure-PEG-A) can be covalently photo-grafted to PEGDA networks (Phelps et al. 2010; Zhu 2010). Growth factors anchored to the substrate in this way are released only after the cleavage of the hydrogel by invading cells. The resulting diffusion of the growth factors is very slow (about 2 weeks) and is compatible with in vivo levels of VEGF (Phelps et al. 2010). It is particularly efficient in regenerative medicine, for implants scaffold, as the VEGF release enhances vascularization (Phelps et al. 2010). A similar strategy can be applied to control the concentration of nitrogen monoxide (NO), which can be complexed with acrylate-modified proteins and released over a period of months in the hydrogel to reduce the risk of thromboses (Bohl and West 2000). This ability to combine adhesion molecules, growth factors and different types of molecules modified with PEG methacrylate makes PEGDA a powerful “tailorable” support for tissue engineering.
Spatially constrained 3D cultures
Although whole organs are macrostructures, they are built from cellular-scale microstructures. For instance, the diameter of a blood capillary or a kidney tubule is approximately between 8 and 10 μm, an intestinal villus is about 500 μm tall and a muscle fiber has a diameter of 10 to 100 μm. Microfluidics allows the user to structure or constrain the microstructure of hydrogels at the micrometer length scale (Shin et al. 2012). This spatial confinement favors the sorts of cell-cell interaction and paracrine communication experienced by cells in vivo, while maintaining cell-matrix interactions on physiologic dimensions. The use of hydrogels as artificial matrices ensure the cells a physiological environment as previously described. Different microfluidic techniques have been developed to structure hydrogels at the micrometer scale. We briefly review these techniques below (Figure 20). For more details readers may refer to the reviews of Nikkhah et al.(Nikkhah et al. 2012), Annabi et al. (Annabi et al. 2010) and Huang et al. (G. Y. Huang et al. 2011).
3D microstructured hydrogels: towards micro-organs
Here, we restrict our discussion to studies that have succeeded in reproducing physiological 3D structures and in directing cell growth and differentiation to form tissue-like structures. We will not review investigations performed on non-structured 3D aggregates, often called spheroids, nor discuss the use of micropillars as substrates.
Within current research into the development of in vitro micro-organs, one must distinguish between
structures that reproduce the three-dimensional shape of an organ without achieving its functionality, and those with sufficient maturity to achieve a functionality that resembles to some extent that of the of the full organ.
Structures reproducing the three-dimensional shape of organs are usually obtained by first shaping the hydrogel, using, for example, bioprinting (Miller et al. 2012), stereolithographic projection (Gauvin et al. 2012) or molding with sacrificial layers (Esch et al. 2012; J. H. Sung et al. 2011). The microstructures have dimensions comparable to physiological ones and the use of porous hydrogels mimics the composition of the ECM. The biocompatibility of the material induces the growth and proliferation of cells (usually human cell lines).
The development of a confluent monolayer on these 3D hydrogels generates in vitro tissues with a structure very similar to the in vivo ones, such as blood capillaries (Gauvin et al. 2012) and villi (Figure 22) (J. H. Sung et al. 2011). The use of bio-printing has enabled researchers to precisely mimic the structure of blood capillaries and to surround such capillaries with a defined thickness of synthetic ECM containing fibroblasts (Miller et al. 2012; Norotte et al. 2009). Such co-culture increases the potential and realism of organs at the structural level.
In vitro models of intestinal tissues, state of the art.
The intestinal tissue is particularly complex as it is constituted of various cell types: stem cells, transit amplifying cells, differentiated cells comprising: enterocytes, goblet cells, enteroendocrine cells, Paneth cells, Tuft cells and M cells. Therefore, growing intestinal tissue in vitro is more challenging than for other organs as the system should induce the differentiation and maintenance of these 8 different cell types whereas most organs are constituted of only one differentiated cell type. In addition in vitro intestinal tissue should have the ability to self-renew.
Table of contents :
CHAPTER 1: INTRODUCTION
I Introduction to the intestine
I.1.Embryonic morphogenesis of the intestine
I.1.1) Heterotypic cell signaling
I.1.2) Homotypic cell signaling
I.1.3) ECM composition
I.1.4) Mechanical forces
I.2 The maintenance of epithelium homeostasis in the adult small intestine
I 2 1) Stem cells and epithelium renewal
I.2.2) Subepithelial fibroblasts and epithelium homeostasis
I. 2. 3) The role of the epithelial environment on intestinal homeostasis
II Introduction to 3D scaffolds for tissue engineering
II.1 Artificial extracellular matrix
II.1.1) Chemical composition
II.1.2) Hydrogel physical properties
II.1.3) Importance of dynamic hydrogels
II.2 Microstructured 3D environments
II.2.1) Spatially constrained 3D cultures
II.2.2) 3D microstructured hydrogels: towards micro-organs
III In vitro models of intestinal tissues, state of the art.
III.1. Growing intestinal organoids in artificial extracellular matrix
III.2 Gut- on-chip: growing intestinal tissue in microfabricated systems
CHAPTER 2: RESULTS
I How to engineer a scaffold that meets the specifications fixed by in vivo microenvironment?
I. 1 Characterization of collagen I matrix
I 2 Structuring the collagen
I 3 Remodeling of the matrix by epithelial cells and fibroblasts
I. 4 How to strengthen collagen 3D structures?
I. 4.1) Semi-interpenetrating polymer networks
α) Hyaluronic acid/ collagen semi interpenetrating network
β) Fibrin/ collagen semi interpenetrating network
I. 4.2 Chemical cross-linking of collagen fibrils
α) Glycation
β) Glutaraldehyde cross-linking
γ) Genipin cross-linking
II How Caco2 cells behave on a microstructured scaffold
II. 1. Influence of the 3D structure on the spatial location of proliferative cells
II. 2. Location of proliferative cells during the colonization of 3D structures
II. 3Matrix stiffness induced synchronized collective cell colonization of scaffolds
III From in vivo isolated intestinal crypts to an in vitro intestinal epithelium
III.1. Growing primary intestinal epithelium on microstructured collagen scaffold
III.1.1) Coating strategies as basement membrane substitute
III.1.2) Seeding isolated primary cells on collagen structure
III.1.3) Seeding organoids on collagen scaffolds
III.2. Proliferation patterns of primary cells on collagen scaffolds
III.3. Influence of fibroblasts on the primary epithelial cells growth
CHAPTER 3: DISCUSSION
I To which extent can one mimic in vivo environment?
II How the mechanical and physical cues of the matrix affect epithelium behavior
II.1 Evaluation of epithelial tissue forces on the structure
II.2 Emergence of collective coordinated colonization induced by the combination of matrix stiffness and topography
II.3 Local rigidity and geometry sensing integrated at the tissue scale regulates spatial positioning of proliferative cells.
II.4. How is our model useful compared to organoids?
CHAPTER 4: CONCLUSION
CHAPTER 5: MATERIAL AND METHODS
BIBLIOGRAPHY
