*4.3.3. Adenosine*

engineering methods can be carried out for months, especially if it involves in vivo implanta‐ tion. Many proteins, peptides, and lipids can be used to stimulate collagen synthesis and deposition. Most of them are involved in fibrosis and need to be carefully handled to avoid

Beta-glucans constitute a family of carbohydrates that stimulates fibroblasts to produce collagen [89, 90]. For instance, laminaran, a glucan from *Saccharina longicruris* seaweed, increased collagen secretion when added to dermal fibroblast culture [91]. An increase in collagen synthesis and secretion was observed in self-assembled tissues. Moreover, thicker stroma could be obtained without significant increase in cell proliferation and alpha-smooth muscle actin content, a hallmark of fibrosis [92]. The authors argued that the aggregation properties of laminaran triggered a net increase of collagen secretion without inducing a

Tissue engineering often relies on glucose-rich media because glucose is the primary source of energy that allows MSCs to produce ECM compounds. Advanced glycation end-products (AGE) result from glucose metabolism and are found in elderly tissues or in diabetic patient tissues [94]. Unfortunately, the glucose concentration used during the production of most selfassembled tissues is too high. Consequently, it was reported that AGE are involved in the process of skin aging, which has an impact on mechanical and biological parameters [95]. New approaches to circumvent this issue are currently being developed and should generate

In addition to mediating glucose entry in cells, insulin also plays an active role in collagen synthesis and deposition [96]. Insulin has a long history of safety use for human therapies and microencapsulated insulin-secreting cells in hydrogels can improve collagen fiber density in diabetic mouse models [97]. Poly-lactic-co-glycolic acid (PLGA) alginate structure that releases insulin in rats was also found to increase collagen deposition and maturation [98]. In a clinical setting, wound healing is problematic for diabetic patients because their insulin metabolism is altered. Also, their tissues are less irrigated because of microvascular network changes caused by the loss of ECs. When capillary networks are altered, the surrounding tissues undergo hypoxia. In such an environment, fibroblasts change to a fibrotic phenotype. Fibrosis is induced by factors that are released by damaged ECs [99], as well as by other unknown mechanisms [96, 100]. Insulin and hypoxia exert a synergic effect on self-assembled tissues. They increase collagen deposition as demonstrated by tests on human and animal cell cultures [101] (unpublished data). Nevertheless, long-term effect of hypoxia exposure (more than 2 weeks) induced acidification of the cell culture medium and a thinning of the engi‐ neered tissues [102]. Hence, cyclic hypoxia seems a better alternative than constitutive hypoxia

production of pathological-like tissue.

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

*4.3.1. Polysaccharides*

fibrotic phenotype [92, 93].

promising alternatives.

*4.3.2. Insulin and hypoxia*

because it produces thicker tissues in vitro.

Adenosine and other derivatives have been used to enhance the rate of wound healing [103]. Their receptors were also found to be involved in fibrosis. Activation of A2B-adenosine receptors resulted in an increase of collagen synthesis and a decrease in MMP-9 activity [104, 105]. This molecule was successfully tested to produce rabbit tissues by the self-assembly approach [101]. Effects of adenosine on human cultures remain to be evaluated.

### *4.3.4. Lysophosphatidic acid*

Lysophosphatidic acid (LPA) is a bioactive lipid found in blood. LPA binds to its receptors at the surface of many cells and activates pathways leading to proliferation, migration, and secretion of cytokines. LPA expression is upregulated in disease conditions such as in fibrosis and cancer or cancer [106, 107]. As LPA is naturally present in human blood, it was used in vitro and approved by regulatory agencies. LPA-cultured fibroblasts showed increased collagen type-I and fibronectin deposition in a dose-dependent manner that could be com‐ pletely reversible. No adverse effects were noted: alpha-smooth muscle actin was not overexpressed and cell proliferation rates remained normal [108]. Thicker stroma and enhanced collagen deposition kinetics suggested that the production time could be reduced by 25% when LPA was added to the cell culture medium.
