*3.1.2. Immobilized factors*

endothelial cells (hESC-ECs) and enzymatically dispersed hESC-ECs suspended in Matrigel. However, a series of ECM and adhesion molecules-specific genes was considerably downregulated in hESC-EC suspended in PBS (**Figure 2**). These gene-expression data indicated that adding ECM to detached cells could reverse genes down-regulation of ECM pathway, cell

The assignment of cell fate results from a response to sophisticated extracellular signals [29,30]. There is mounting evidence suggesting that ECM could deliver numerous soluble and immobilized factors that play vital roles in making the fate choice between self-renewal and lineage commitment [31]. Further insights and exquisite control of signals transported by ECM could provide opportunities for enhancing the regenerative efficacy in both in vitro and in

The propagation of soluble signalling molecules controls a great variety of cellular responses, including proliferation [32], polarity [33], migration [34], and differentiation [35]. It has been

**Figure 3. Putative model outlining the controlled nitric oxide (NO)-releasing hydrogel enhances the therapeutic ef‐ fect of adipose derived-mesenchymal stem cells (ADSCs) for myocardial infarction.** Encapsulation of ADSCs by NO-releasing hydrogel prevented transplanted cells effusing from injection positions. NO molecule released from the hydrogel catalyzed by β-galactosidase can facilitate angiogenic cytokines secretion of ADSCs, resulting in promoting angiogenesis, ADSCs survival and cardiac function. β-gal, β-galactosidase [9]. Reprinted by permission of the publish‐

vivo and further accelerating the translation of basic science to the clinical setting.

adhesion molecules pathway, ECM and adhesion signalling.

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

**3.1. Biochemical stimulus**

*3.1.1. Release of soluble factors*

er.

Sustained release and improved local retention of regenerative factors, such as growth factors and extracellular substances, are required during tissue regeneration [41]. However, these molecules are suffering from rapid degradation, and therefore they will quickly lose their functionality and clinical efficacy [42]. Additionally, there is evidence that cellular processes are also affected by the interactions between cells and non-soluble constituents of the ECM [43]. For these reasons, immobilization of signalling molecules or functional components to ECM could be suitable for stabilizing these highly reactive molecules, increasing local concentration of biochemical stimuli, and increasing the bioactivity of engineered ECM.

A growing number of studies have utilized short synthetic peptides to mimic the biological properties of full-length growth factors and to substitute parent proteins [44,45]. For example, insulin-like growth factor 1 (IGF-1) is considered as an essential biochemical stimulus in tissue regeneration. The C domain of IGF-1 (IGF-1C), a 12 amino acids sequence, had already been proved as the active region of IGF-1 [46]. IGF-1C has been used as substitute for IGF-1 and applied into hydrogel biomaterials as biomimetic material for tissue engineering and regen‐ erative medicine. The proliferation, apoptosis resistance, and paracrine effects of ADSCs were significantly enhanced after they were seeded on chitosan (CS) hydrogel with immobilization of IGF-1C [47]. When co-transplanted ADSCs with CS-IGF-1C hydrogel into ischemic organ, this biomimetic matrix could create a favourable microenvironment for the survival and adaptation of transplanted cells and further promote functional and structural recovery of injured organ (**Figure 4**).

**Figure 4. Schema of renoprotective effects of ADSCs and synthetic ECM (CS-IGF-1C hydrogel).** When co-trans‐ planted into AKI model, CS-IGF-1C hydrogel could protect delivered ADSCs, facilitated their paracrine and anti-in‐ flammatory effects, and inhibit ECM synthesis in kidney, which result in enhanced angiogenesis, regeneration and alleviated fibrosis after kidney injuries. Consequently, CS-IGF-1C hydrogel therapy leads to improved functional and structural recovery of kidney [47]. Reprinted by permission of the publisher.

#### **3.2. Physical interaction**

Although it has commonly acknowledged that signals transduced by ECM could direct stem cell fate, there is increasing evidence that physical properties of ECM could also make a great impact on cell behaviours [48–50]. Some of these factors are proven to be of great influence, but we still have a long way to go and a lot of work to do to establish a complete theory. For example, in response to injury, the accumulation of ECM is excess and abnormal, which would cause significant changes to the stiffness of ECM and ultimately lead to tissue fibrosis [51,52].

#### *3.2.1. Stiffness and elasticity*

To test the effect of different stiffness (EY) on cell behaviors, substrates with EY ranging from <1 kPa to 30 kPa were synthesized [53]. The results showed that ECM stiffness has influence on cell proliferation as well as cell differentiation. For instance, neural stem/progenitor cells (NSPCs) could proliferated on substrates with EY <10 kPa. On soft substrates (<1 kPa), neuronal differentiation was promoted; whereas, on relatively stiff substrates (>7 kPa), oligodendrocyte differentiation was favoured. This consequence indicated that matrix stiffness had effect on lineage choice and differentiation. In light of previous data that stiffness was a regulator of differentiation, Shih et al. further explore how matrix affects the osteogenic phenotype of MSCs [54]. They found that the matrix rigidity promoted osteogenic commitment through a α2 integrin-ROCK-FAK-ERK1/2 axis.

Fascinatingly, it was observed that mesenchymal stem cells all underwent osteogenic differ‐ entiation on both stiff and soft polydimethylsiloxane (PDMS) substrates; whereas, the osteo‐ blast differentiation of the same cells was promoted on stiff polyacrylamide (PAAm) hydrogels, and more cell differentiated into adipocytes on soft PAAm hydrogels [55]. The different cellular responses to different substrates indicated that stiffness was not an inde‐ pendent stimulus for differentiation. Further data provided in this study suggested that the differentiation of human mesenchymal stem cells on PAAm was also regulated by the elastic modulus. Consistent with the previous study, Xue et al. reported that matrix elasticity was the main physical parameter directing stem cell differentiation at low cell density; with increased cell density, the cell–cell contact force and interactions took priority over the matrix elasticity [56]. Most notably, although cell differentiation was influenced by elastic modulus, recent discovery found that matrix-promoted adipogenic or osteogenic differentiation could not maintain when the cells were re-seeded into tissue culture plastic (TCP) [57]. Furthermore, global gene expression profiles and DNA-methylation profiles revealed no significant impact caused by matrix with different elasticity. These results indicated that matrix elasticity only exerted a transient influence on stem cell lineage commitment.
