**Author details**

On 2D surfaces, it has been shown that cells are responsive to surface rigidity and that it influences the commitment of mesenchymal stem cells toward differentiation in a specific lineage [96]. Again, surface stiffness applies forces which are unevenly distributed on the cells (i.e., only located at the cell-surface interface). Developing a 3D material with tuned and controllable mechanical properties will generate a more biologically relevant environment to evaluate the role of ECM mechanobiology on cells functions and differentiation processes. To study the influence of mechanical stiffness on mesenchymal stem cells differentiation, a series of alginate gels with elastic modulus ranging from 2.5 to 110 kPa has been developed [97]. Mechanical properties of alginate gels are modulated by the percentage of alginate polymers in the final hydrogel. Because alginate is not sensitive to the degradation of hydrolytic enzymes of mammalian cells, the elastic modulus of the mechanical properties of the material are expected to remain constant all along the study (7 days of cell culture). RGD-peptides are covalently grafted to alginate polymers prior to hydrogel formation to give to the cells adhesive cues. The more rigid materials trigger mesenchymal stem cells differentiation toward bone lineage with an expression of the bone-related molecular markers such as alkaline phosphatase and osteopontin after 7 days of culture. On the other hand, alginate gels with the lower elastic modulus (softer material) triggers an accumulation of oil-droplets into stem cells, indicating adipose tissue differentiation. The density of RGD-peptides incorporated into these materials did not modify the cell fate related to the elastic modulus of the material, but induce a higher

level of expression of the lineage markers for both bone and adipose-committed cells.

As mentioned at the beginning of this section, the ECM-derived Matrigel® represents currently the most often used material for 3D experiments in cell biology. Despite that Matrigel®-related drawbacks are of importance, Matrigel®is a widely and available model to investigate many fundamental questions in cell biology, from cell adhesion and tumor formation, to drug testing. We have presented in this chapter a large panel of techniques, methodologies, and engineering processes that allow the exploration of ECM organization and permit to recreate *ex vivo* some of their key features. At the conclusion of this chapter and after the review of several studies investigating various 3D materials, it appears that no material can represent the unique and ideal answer for all cell investigations in 3D [63]. A modular approach should be taken by rationalizing the biological question to be studied and the parameter of ECM intended to be recreated. Nevertheless, more and more complex materials are engineered that will finally be able to mimic simultaneously several key factors of ECM composition, architecture, or mechanical properties, and so enabling investigation of multiple parameters for cell biology experiments. An important drawback with engineered 3D materials is to create a "black-box" where undefined and uncontrollable parameters may influence the cellular outcomes to be investigated. To avoid part of this problem, rigorous attention should be paid on the purity of polymers used to build the material, in particular with biological polymers. The development of DNA-recombinant production of ECM proteins can overcome this problem, even if this will raise ultimately the cost of the final material. The structural characteristics of the final material (porosity, polymers distribution, and fibrils diameter) should be consistently reproducible and addressed. To do so, an "easy," meaning straightforward, process of the material should be

**3.4. Conclusion: toward a gold-standard of 3D model of ECMs?**

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

Sylvain Vigier1\* and Tamas Fülöp2\*

\*Address all correspondence to: sylvain.vigier@gmail.com and Tamas.fulop@usherbrooke.ca

1 Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, Québec, Canada

2 Department of Medicine, Geriatric Division Research Center on Aging, Université de Sher‐ brooke, Sherbrooke, Qc, Canada
