**3. Extracellular matrix-inspired biomaterials**

TEM is nearly concomitant of its apparition in the late 1930s. Native collagen fibrils extracted from tissues and stained negatively with phosphotungtsic acid present a typical cross-striated pattern with a series of dark and light bands, spaced with a regular period of 67 nm. These observations have allowed the establishment of the assembly model of collagen molecules into collagen fibrils, known as the quarter-stagger model from Hodge and Petruska (1963). This model proposes a lateral stacking of collagen molecules, creating overlaps that exclude phosphotungstic dye and appear light, and a longitudinal collinear succession of collagen molecule spaced with a constant gap filled by Tungsten dye and appears dark under electron beam [48]. The cross-striated pattern is characteristic of fibrillary collagen, that are collagen type I, type II, and type III. On the other hand, network forming collagen type IV do not present any bands on TEM but is seen as a meshwork of hexagonal structures [49]. The resolution (roughly 1–5 nm) of TEM allows analyzing single macromolecules deposited on carbon film and stained by rotary-shadowing, creating a 3D electron sensitive replica of the specimen [50]. This method has revealed the semiflexible rod structure of collagen molecules terminated by a globular C-term pro-peptide and the cross-shaped triple chain structure of laminin mole‐ cules. TEM is particularly accurate to measure length of ECM molecules and diameters of fibrils and fibers assembly. These last parameters are important when analyzing a tissue because ECM fibrils diameters appear to be tissue-specific and modification of their size can be induced by pathologies such as diabetes, fibrosis, cancer, or aging and consequently impair tissue organization and function [51]. Compact bone tissue which supports most of the load of the body and muscles anchorage has to resist strong mechanical solicitations, but it is surprisingly light in weight structure if compared to human-engineered buildings. Bone tissue is made of an abundant organic ECM, strengthened with a mineral phase, and has highly hierarchical structure with length scales ranging from meters to nanometers that give its overall mechanical properties [52]. The shaft of long bones is organized in cylindrical osteons formed by successive concentric lamellae, themselves constituted by compact assemblies of collagen fibrils. An oblique transverse section of successive concentric lamellae made with ultra-microtome and observed with TEM revealed coexistence of two patterns of organization for collagen fibrils [53]. One is an alternation of parallel and orthogonal fibrils, with a regular 90° shift of fibrils orientation from one lamellae to another. The second is seen under TEM as arced structures, as if collagen fibrils were bent in between two series of longitudinal fibrils. The arced pattern is the consequence of the oblique sections into succession of collagen fibrils rotating with a tiny and constant angle from one lamellae to another, creating the illusion of bend structures [54]. From these TEM observations, a twisted plywood model of collagen fibril organization in bone has been proposed. This particular constant angle twist recall the organization observed in some liquid crystal phase, and it has been suggested that collagen molecules could have a liquid crystal behavior and autoassemble in higher-scales structures [21,55]. This finally underlines the potentials of transmission electron microscopy (TEM) to address ECM archi‐ tectures in tissues. As mentioned above, similar techniques and processing of samples used in optical microscopy are also applied with TEM. The different components of the ECM can be identified by immunolabeling with the same limitations for the necessity to retrieve antigens from the fixation and embedding processes. The antibodies are covalently linked to a gold

304 Composition and Function of the Extracellular Matrix in the Human Body

The deep exploration of ECMs composition, organization, and biological functions associated with the development of methods to produce new biocompatible materials has enabled material scientists to recreate *ex vivo* some of the key characteristics of ECM [59]. This section focuses on how the structural and functional characteristics derived from the knowledge of the native cell microenvironment have been applied to design biologically relevant biomate‐ rials. Different strategies currently exist to build 3D models of the ECM: tissue-derived ECM, use of natural or synthetic polymers, and formulation into hydrogel or porous 3D materials. Some biomaterials are designed to recreate the composition of the ECM and thus offering the right environment for studying cell adhesion and anchorage-associated cell phenotypes. Other materials are developed to recreate the 3D architectures of ECM, proposing fibrillary structures with similar organization and mechanical properties of native tissues. These examples represent preliminary attempts of *ex vivo* models of ECM that will most likely be improved and increase with an overcoming of technical hurdle faced by material scientists and with rising interest of cell biologists for 3D models that will ask for more refined and specific materials to answer fundamental questions on cell biology.

#### **3.1. Strategies to engineer 3D models of ECMs**

#### *3.1.1. Tissue-derived ECM: the gold standard Matrigel®*

A basement membrane-derived tissue isolated from Engelberth–Holm–Swarm (EHS) mouse sarcoma is commercially available under the brand name Matrigel® (BD Biosciences) and has become widely used to evaluate cell migration, cancer cells behavior, and to create organoids *in vitro*. EHS-sarcoma produces a large amount of ECM rich in collagen type I, laminin-111, heparin sulfate proteoglycans (Perlecan), and Nidogen that are the main constituents of BM [60]. This basement membrane extract is liquid at 4°C and turns into a gel at 37°C under physiological pH and ionic strength. Matrigel® is currently the gold standard in most of the 3D assays performed in cell biology. Indeed, there is not yet any material in the market that is able to better reproduce the composition and partially the organization of BM, in particular because Matrigel® is obtained directly from animals and is not an *ex vivo* engineered ECM. It is a ready-to-use solution that allows user-defined utilization, even if manipulating Matrigel® requires skills and experience. The success of Matrigel® is also due to its biological activity that allows under normal conditions of culture the differentiation of several cell types and the formation of complex structures like vessels or mammary glands acinar structures [61,62]. Besides structural ECM molecules, Matrigel® contains soluble growth factors such as fibro‐ blast growth factors (FGF), epidermal growth factor (EGF), and transforming growth factor-β 19 (TGF-β) and matrix metalloproteinases (MMPs) including MMP-2 and -9. The unique coexistence in an *ex vivo* substrate for cell culture of native and organized fibrous ECM proteins associated with soluble factors explains its genuine bioactivity. Concomitantly, this represents its main drawback to serve as an ECM model for 3D cell cultures. Because Matrigel® is produced and purified from an animal, there is a lack of control on its exact composition and a batch-to-batch variability of its content [63]. Moreover, the presence of growth factors in an unknown and uncontrollable amount can interfere (positively or negatively) with the intended parameters to be studied, like with the evaluation signaling molecule or a drug, and invalidate the use of Matrigel® in any experiments where the role of a growth factors in a cellular process would like to be addressed. The work of Edna Cukierman has demonstrated the dramatic changes in cell morphology in-between 2D and 3D cell culture systems with a massive reorganization of cell cytoskeleton and a modification of integrins expression [64]. This has increasingly recognized the importance of studying cells, in particular their adhesion and migration, within a 3D environment. Thus, Matrigel® should be considered with caution and with the full awareness of its limitations when it is chosen for a 3D model and so not creating a "black-box" during the switch of cell biology from 2D to 3D.

#### *3.1.2. Biological polymers*

Biological polymers were first used as a coating of tissue culture dishes to favor cell adhesion and spreading, and then incorporated into 3D materials under different forms (hydrogels, freeze-dried materials, and surface coating of bulk inorganic materials). Most of the biological polymers used in biomaterials are structural molecules derived from mammalian ECM such as collagens (type I–IV), elastin, fibronectin, laminin (mainly laminin-111), fibrin, and glycoa‐ minoglycans (Hyaluronan, Chondroitin sulfate, and Heparan sulfate) [65]. They are classically purified from animals' ECM-rich tissues such as dermis and tendons (collagen type I, elastin), cartilages (collagen type II and GAGs), tumors (laminins and collagen IV) or directly from blood (fibrin and fibronectin). Nowadays, it is possible to obtain some of these molecules from DNA-recombinant sources. This allows to work with human ECM molecules, produced with a high degree of purity and free of many pathogens [66]. However, DNA-recombinant production of multimeric high molecular weight molecules is still a challenge and it minimizes the number and the amount of molecules available from this source and raises dramatically their prices. Nonmammalian ECM molecules are also widely used in the design of biomate‐ rials, mainly for their ability to self-assemble in 3D structures. Numerous materials are engineered in the form of hydrogel from chitin/chitosan (polysaccharide purified from fungi or arthropods exoskeleton), agarose or alginate (both polysaccharides purified from algae). Silk fibroin, a protein found in silkworm cocoon and spiders, is also used to produce fibrous materials [67]. Because of their origin, nonmammalian biological polymers lack many of the cell adhesion cues on their structures and should be most often supplemented with adhesive molecules or peptides to obtain a biologically active material. Their inherent abilities to form 3D materials remain their best interests. Last type of biological polymers usable in the design of 3D materials are growth factors (FGF, EGF, platelet derived growth factor [PDGF], and vascular endothelial growth factor [VEGF]) that give important biological signals to the cells and enzymes (MMPs, proteolytic enzymes) that can favor a remodeling and a progression with time of the material, like that is observed in the healing processes and during tumor invasion. However, as mentioned in the first section of this chapter, these molecules are not structural proteins and thus no 3D material can be raised from them. Nevertheless, both growth factors and enzymes represent essential molecules to reinforce the basic 3D scaffold in a relevant ECM model. For that purpose, there are strategies to incorporate these soluble molecules into a material and trigger their appropriate release at specific time points or location in the material [59].

#### *3.1.3. Synthetic polymers*

become widely used to evaluate cell migration, cancer cells behavior, and to create organoids *in vitro*. EHS-sarcoma produces a large amount of ECM rich in collagen type I, laminin-111, heparin sulfate proteoglycans (Perlecan), and Nidogen that are the main constituents of BM [60]. This basement membrane extract is liquid at 4°C and turns into a gel at 37°C under physiological pH and ionic strength. Matrigel® is currently the gold standard in most of the 3D assays performed in cell biology. Indeed, there is not yet any material in the market that is able to better reproduce the composition and partially the organization of BM, in particular because Matrigel® is obtained directly from animals and is not an *ex vivo* engineered ECM. It is a ready-to-use solution that allows user-defined utilization, even if manipulating Matrigel® requires skills and experience. The success of Matrigel® is also due to its biological activity that allows under normal conditions of culture the differentiation of several cell types and the formation of complex structures like vessels or mammary glands acinar structures [61,62]. Besides structural ECM molecules, Matrigel® contains soluble growth factors such as fibro‐ blast growth factors (FGF), epidermal growth factor (EGF), and transforming growth factor-β 19 (TGF-β) and matrix metalloproteinases (MMPs) including MMP-2 and -9. The unique coexistence in an *ex vivo* substrate for cell culture of native and organized fibrous ECM proteins associated with soluble factors explains its genuine bioactivity. Concomitantly, this represents its main drawback to serve as an ECM model for 3D cell cultures. Because Matrigel® is produced and purified from an animal, there is a lack of control on its exact composition and a batch-to-batch variability of its content [63]. Moreover, the presence of growth factors in an unknown and uncontrollable amount can interfere (positively or negatively) with the intended parameters to be studied, like with the evaluation signaling molecule or a drug, and invalidate the use of Matrigel® in any experiments where the role of a growth factors in a cellular process would like to be addressed. The work of Edna Cukierman has demonstrated the dramatic changes in cell morphology in-between 2D and 3D cell culture systems with a massive reorganization of cell cytoskeleton and a modification of integrins expression [64]. This has increasingly recognized the importance of studying cells, in particular their adhesion and migration, within a 3D environment. Thus, Matrigel® should be considered with caution and with the full awareness of its limitations when it is chosen for a 3D model and so not creating

306 Composition and Function of the Extracellular Matrix in the Human Body

a "black-box" during the switch of cell biology from 2D to 3D.

Biological polymers were first used as a coating of tissue culture dishes to favor cell adhesion and spreading, and then incorporated into 3D materials under different forms (hydrogels, freeze-dried materials, and surface coating of bulk inorganic materials). Most of the biological polymers used in biomaterials are structural molecules derived from mammalian ECM such as collagens (type I–IV), elastin, fibronectin, laminin (mainly laminin-111), fibrin, and glycoa‐ minoglycans (Hyaluronan, Chondroitin sulfate, and Heparan sulfate) [65]. They are classically purified from animals' ECM-rich tissues such as dermis and tendons (collagen type I, elastin), cartilages (collagen type II and GAGs), tumors (laminins and collagen IV) or directly from blood (fibrin and fibronectin). Nowadays, it is possible to obtain some of these molecules from DNA-recombinant sources. This allows to work with human ECM molecules, produced with a high degree of purity and free of many pathogens [66]. However, DNA-recombinant

*3.1.2. Biological polymers*

Synthetic organic polymers offer a large panel of creativity to produce 3D materials. They inherently lack basic biological activity but possess a great processing flexibility. They are easier to produce as well as purify in large quantities and finally, are free from animal contaminations. Synthetic polymers are suitable for many types of chemical modifications such as chemical grafting of adhesive peptides or incorporation of bioactive molecules and can be processed into 3D materials with many types of techniques (electrospinning, foaming, hydrogel, and sheets), some of them not bearable by biological polymers. The diversity of synthetic polymers used in biomaterials is large, including polyacrylamides, polyacrylates, polyethers (e.g., polyethylene glycol), polyesters (e.g., polycaprolactone), polyhydroxy acids (e.g., poly lactic acid, poly glycolic acid, and copolymers poly lactic-co-glycolic acid), polyfu‐ marates, and polyphosphazenes [68]. Polyesters and polyhydroxy acids have both biodegrad‐ able properties with presence of hydrolysable bonds in their backbone, whereas polyacrylamides and polyacrylates are almost unbreakable under cell culture conditions. Chemically or genetically engineered peptides or protein-like polymers with amphiphilic or autoassembling properties offer a direct incorporation of bioactive cues into fibrillar materials. Inorganic materials such as metal (e.g., titanium, stainless, and cobalt) and mineral alloys (e.g., ceramics) are widely used as bone and dental substitutes, but their bulk and stiff structures associated with poor possibilities of chemical modifications make them rarely used as *ex vivo* engineered ECM [69]. Synthetic polymers are usually functionalized by chemical grafting of peptides which are recognized for cell adhesion such as the well-known Arg-Gly-Asp (RGD) motif found in numerous ECM molecules (fibronectin, collagen, and vitronectin) and bind by several integrins. More specific ECM-derived peptides can also be grafted, such as DGEA and GFPGER sequences found in collagen I and IV, respectively, or IKLLI and YIGSR sequences from laminin alpha-1 and beta-1 chain, respectively. Functionalization strategies use the inherent properties of the synthetic polymers to form a 3D scaffold and to contain active chemical groups (amine, acid, or alcohol functions) sensitive to chemical reactions and so providing an ECM model with well-defined characteristics in order to answer a specific question. Synthetic polymers can also be functionalized with whole proteins such as growth factors or enzymes. Through the mediation of a peptide spacer incorporating a cleavage sequence, the release of the bioactive compounds may be triggered at the required time or progressively by the proper proteolytic action of the cells [70]. In conclusion, synthetic polymers represent an engineering solution to rationalized parameters tested in 3D model of ECM and can introduce a dynamic aspect into the system [71,72].

#### *3.1.4. Hydrogels*

Hydrogels are produced from polymers in solutions which are gelated into 3D materials that possess high water content. To some extent, hydrogels behave in the same way than proteo‐ glycans in ECM and represent a valuable mimicry of soft-tissues ECM structures and physical properties due to its hydrated state. They are most often produced by mild, physiological conditions and, as such, are mainly compatible with cell encapsulation and culture, as water is the natural solvent for living organisms and biochemical reactions. The water content of the gels also facilitates the diffusion of low molecular weight nutriments, oxygen, and metabolic waste. Hydrogel can be produced from a large variety of polymers, including both natural and synthetic substrates, offering a large diversity of biochemical, physical, and mechanical properties. Depending on polymers characteristics, the gelation can be reversible or irrever‐ sible and triggered by different factors such as multivalent ions, chemical covalent crosslinking (including aldehyde fixatives), and physical phase-transition induced by temperature, pH, or concentration [73]. Covalent cross-linking can form hydrogel with almost all types of polymers in solutions, but the toxicity of cross-linking molecules must be considered, and furthermore, most of the cross-links are nondegradable by cells or in physiological conditions. Moreover, the gelating process can be deleterious for cells, and thus it will dictate the ways that cells are associated with the scaffold (i.e., before or after gelation). Mixing the cells with the polymers prior gelation allows a homogenous distribution of the cells inside the material, which could be more difficult to achieve when seeding the cells on an already formed hydrogel [74].

Natural polymer hydrogels can be made with collagen, fibrin, hyaluronan, alginate, and agarose. Collagen hydrogels are formed by pH neutralization of acid solutions that trigger the assembly of the collagen molecules into fibrils and fibers which will stabilize the structure of the gel. Mechanical properties of collagen hydrogels can be finely modulated by adjusting their concentrations [75]. Fibrin gels are formed following the same reaction that occurs in the body after an injury or an inflammatory response: fibrinogen is mixed in solution with thrombin, a serine protease, which hydrolyses the N-terminal peptide of fibrinogen to create fibrin monomers that assembles into fibrils that will produce an interconnected fibrous hydrogel. Mechanical properties of fibrin gel are tuned by adjusting fibrinogen and thrombin concen‐ trations. However, fibrin gels are highly sensitive to several proteases, which are secreted by cells, and a gel degradation regularly occurs in long-term cell cultures if no protease inhibitors are added in the culture media [76]. Hyaluronan hydrogels are formed by covalent crosslinking with hydrazide derivatives that react with the carboxylic acid groups of the polymer. They bear inherent biological properties and are susceptible for further chemical modifications which modulate these properties, such as the grafting of fibrous proteins or adhesive peptides. However, hyaluronan hydrogels are mechanically poor, and hyaluronan is more often incorporated into other materials than being the main frame of a hydrogel. Several different types of materials are produced from alginate hydrogels. Alginate polymerization is triggered by addition of divalent cations Ca2+, Mg2+, Ba2+, or Sr2+ that create bridges in-between negative charges of alginate monomers. Processing of alginate hydrogels enables an easy cell encapsulation, but a progressive diffusion and solution-exchange of divalent cation can undermine its structure. Agarose gels are formed by heating (near boiling temperature) of the solution that gelate with cooling. Different porous architectures and mechanical properties are obtained with modulation of agarose concentration.

Hydrogels can be formed with synthetic polymers including polyethylene oxide, polyvinyl alcohol or in a blended solution or copolymer association with poly lactic acid. Again, the interest of synthetic polymers to form hydrogels is the diversity and the reproducibility of materials that they are able to produce, with versatile biophysical, mechanical and biological properties. Poly ethylene glycol (PEG) polymers can be modified to allow *in situ* gelation by visible UV-light induction [77]. Photopolymerization is based on the interaction of light with photo initiators that create free radicals which react with the polymer and initiate cross-links. Compared to other type of chemical cross-linkers, photopolymerization is fast (second or minutes) and allows a spatial control of the polymerization. It works under physiological conditions and the radical species, even harsh for living species, are quickly removed by the polymerization process, making these materials more free of adverse chemical compounds as it may occur with aldehyde or hydrazide derivatives. Photopolymerization of PEG-based solutions was used to encapsulate chondrocytes that were cultured for 14 days with progres‐ sive deposition of a cartilaginous native ECM showing the biocompatibility of the process [77]. Synthetic peptides containing cell adhesion cues can also be designed for autoassembling into supramolecular structures able to form hydrogels. Engineered proteins produced by recombi‐ nant DNA can, in the same way, present adhesive domains and reactive groups such as thiols and amines for being the specific targets of polymerization reactants [72]. Finally, hydrogelforming properties of synthetic polymers can be used to include biological polymers unable to form otherwise materials on their own and then offering a mixed inert 3D structures with biological functions.

#### *3.1.5. Porous material*

motif found in numerous ECM molecules (fibronectin, collagen, and vitronectin) and bind by several integrins. More specific ECM-derived peptides can also be grafted, such as DGEA and GFPGER sequences found in collagen I and IV, respectively, or IKLLI and YIGSR sequences from laminin alpha-1 and beta-1 chain, respectively. Functionalization strategies use the inherent properties of the synthetic polymers to form a 3D scaffold and to contain active chemical groups (amine, acid, or alcohol functions) sensitive to chemical reactions and so providing an ECM model with well-defined characteristics in order to answer a specific question. Synthetic polymers can also be functionalized with whole proteins such as growth factors or enzymes. Through the mediation of a peptide spacer incorporating a cleavage sequence, the release of the bioactive compounds may be triggered at the required time or progressively by the proper proteolytic action of the cells [70]. In conclusion, synthetic polymers represent an engineering solution to rationalized parameters tested in 3D model of

Hydrogels are produced from polymers in solutions which are gelated into 3D materials that possess high water content. To some extent, hydrogels behave in the same way than proteo‐ glycans in ECM and represent a valuable mimicry of soft-tissues ECM structures and physical properties due to its hydrated state. They are most often produced by mild, physiological conditions and, as such, are mainly compatible with cell encapsulation and culture, as water is the natural solvent for living organisms and biochemical reactions. The water content of the gels also facilitates the diffusion of low molecular weight nutriments, oxygen, and metabolic waste. Hydrogel can be produced from a large variety of polymers, including both natural and synthetic substrates, offering a large diversity of biochemical, physical, and mechanical properties. Depending on polymers characteristics, the gelation can be reversible or irrever‐ sible and triggered by different factors such as multivalent ions, chemical covalent crosslinking (including aldehyde fixatives), and physical phase-transition induced by temperature, pH, or concentration [73]. Covalent cross-linking can form hydrogel with almost all types of polymers in solutions, but the toxicity of cross-linking molecules must be considered, and furthermore, most of the cross-links are nondegradable by cells or in physiological conditions. Moreover, the gelating process can be deleterious for cells, and thus it will dictate the ways that cells are associated with the scaffold (i.e., before or after gelation). Mixing the cells with the polymers prior gelation allows a homogenous distribution of the cells inside the material, which could be more difficult to achieve when seeding the cells on an already formed hydrogel

Natural polymer hydrogels can be made with collagen, fibrin, hyaluronan, alginate, and agarose. Collagen hydrogels are formed by pH neutralization of acid solutions that trigger the assembly of the collagen molecules into fibrils and fibers which will stabilize the structure of the gel. Mechanical properties of collagen hydrogels can be finely modulated by adjusting their concentrations [75]. Fibrin gels are formed following the same reaction that occurs in the body after an injury or an inflammatory response: fibrinogen is mixed in solution with thrombin, a serine protease, which hydrolyses the N-terminal peptide of fibrinogen to create fibrin

ECM and can introduce a dynamic aspect into the system [71,72].

308 Composition and Function of the Extracellular Matrix in the Human Body

*3.1.4. Hydrogels*

[74].

Hydrogels show a good biocompatibility due to their water content but present most often low mechanical properties, high degradation rates, and a compromised deep diffusion of large

molecules like proteins. To overcome these issues, porous materials with interconnected pore networks and surfaces or fibers to sustain cell adhesion as well as cell phenotype have been designed. These materials are discriminated in microporous scaffolds where embedded cells will mainly attach on the surface of pores of >100μm diameter and will more represent curved 2D surfaces, and in nanoporous scaffolds where pore structures are in the range of the cell diameter (roughly 10 μm) and represent more the native 3D environment of ECMs [78]. Nevertheless, microporous structures allow a more effective cell penetration and migration into the material while smaller-sized pores can represent a barrier for cell colonization of the material, and limit the cell interactions at the edges of the material [79]. This particular point of cell colonization represents one of the challenges with nanoporous materials, and definitely a limitation when directly compared with both the microporous and the hydrogel materials. Similar to hydrogels, porous materials can be prepared with both natural and synthetic polymers with a large panel of techniques including, but not restricted to electrospinning, phase separation, templating, and vapor-phase polymerization [79]. Electrospinning forms fibrillar materials with control on the fibers diameters, fibers alignment, and fibers interdis‐ tances that dictate the overall porosity of the material. A polymeric solution is injected through a capillary tube into a high electric field that forms a Taylor cone with application of the electrostatic forces. On the opposite side of the capillary, the collector of the electrospun fibers is charged or grounded. The electric field in-between capillary and collector accelerates the flow of the polymer solution that evaporates the solvent and generate coalescence of the polymers that finally form solid fibers arrays. This technique is mainly used with synthetic polymers (poly lactic/glycolic acid and poly caprolactone) but is also suitable for natural polymers such as collagen and silk fibroin [80]. Electrospinning produces thin materials not exceeding millimeters and with a high-packing density of fibers that can impair a deep colonization by cells, but intensive researches are presently conducted to overcome these issues [81]. Collagen porous materials can be formed by thermally induced phase separation leading to the production of the so-called "collagen sponges" due to their high porous structures. Acidic collagen solutions are frozen which induces a phase separation of collagen molecules from the water-based solvent that is then eliminated by lyophilization. Materials are then made of dense collagen walls with unstriated microfibrils [57] or with native cross-striated fibrils [82]. Size and interconnectivity of the pores can be modified by modulation of the length and temperature of the phase separation, by modulation of the collagen concentration or by mixing the collagen solutions with other natural polymers such as GAGs [82] or with synthetic polymers such as poly lactic acids. Freeze-dried collagen materials have weak mechanical properties and are often strengthened by the addition of GAGs, or cross-linked by chemical species (aldehydes) or dehydrothermal processes [83].

The various polymers and processes presented above enable the engineering of different types of 3D materials designed to study *ex vivo* a large variety of cellular phenomenon such as cell differentiation, drug response, tumor formation, cell migration, cell morphology and cytos‐ keleton organization, cell death and proliferation, tissue architecture, and coculture behavior of cells [84]. For that purpose, 3D materials are built either to mimic ECM composition, ECM organization, or ECM mechanical properties. We present here examples of such engineered 3D models of ECM with description of the material characteristics and cellular outcomes observed.
