**4.2. Enzymatic reactions and chemical stimulation**

Although ascorbic acid is an essential element that contributes to collagen deposition, an increase in its concentration does not lead to enhanced collagen deposition. Ascorbic acid is an enzymatic cofactor of prolyl- and lysyl-hydroxylase [86], and its action reaches a plateau when these enzymes achieve their peak of activity. Independently of its role as a cofactor, ascorbic acid is responsible for a certain level of collagen secretion in fibroblast cultures, until it reaches its biological limits [87], albeit it can be toxic for fibroblasts if present in high dose [23].

Chemical inhibitors of MMP could also increase ECM production by restricting the extent of protease activity. Among them, galardin was used to produce self-assembled tissues and it significantly increased the thickness of treated tissues [54]. Currently, the cost associated with the use of galardin is too expensive to be a promising solution.

L-arginine (L-Arg) is converted in ornithine followed by glutamine semialdehyde and finally proline, an important amino acid that is metabolized during collagen synthesis. L-Arg supplementation to culture media was evaluated, when the stroma was produced using the self-assembly method. Although an increase in collagen synthesis and secretion (20% more collagen type-I) was observed, collagen deposition remained unchanged when compared to controls [88]. A plausible explanation would be that enzymes involved in collagen matura‐ tion were not sufficient to process the surplus of this amino acid in vitro.

### **4.3. Biological stimulation**

Biological stimulation of ECM deposition in the field of tissue engineering is a challenge. This complexity is due to pleiotropic roles of multiple bioactive agents and their subtle effects, which could appear after a long period of time, for instance after tissue implantation. In contrary to monolayer culture studies in which experiments rarely exceed days, tissue 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 production of pathological-like tissue.

#### *4.3.1. Polysaccharides*

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 fibrotic phenotype [92, 93].

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 promising alternatives.

#### *4.3.2. Insulin and hypoxia*

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 because it produces thicker tissues in vitro.
