*8.2.3 Mechanical offloading*

Another alternative to limiting fibrosis in cutaneous injuries is mechanical offloading since mechanical tension plays a significant role in the development of fibrosis,

activating numerous mechano-responsive signalling pathways such as the focal adhesion kinase (FAK) [112, 113].

#### **8.3 Cellular targets for scarless wound healing**

Identification of populations of cells contributing to scar formation allows us to explore options that reduce these specific cell populations during wound healing, thereby minimising scarification.

For instance, Dulauroy and team (2012) successfully distinguished a pro- inflammatory subset of perivascular cells that are activated upon acute injury in muscle and dermis by transient expression of a disintegrin and metalloprotease (ADAM12) [114]. Knocking down ADAM12 expression or ablating these cells was shown to decrease fibrosis and resulting scar formation.

This has been concordant with the findings of Rinkevich et al. [115] who found that fibroblasts originating from En1-lineages were the main culprits in the cutaneous scarring. These cells majorly contribute to scar formation in connective tissue.

Selective abrogation of the En1-fibroblast lineage with diprotin A by accompanying CD26 (also known as dipeptidyl peptidase-4, DPP4) surface marker has also been found to reduce cutaneous scarring without compromising the integrity of the healed tissue [115].

The ability of DPP4 inhibitors to curb the fibrogenic phenotypes of keloid-derived fibroblasts and normal fibroblasts have been further verified through various *in vivo* experiments. Observed decrease in collagen production and TGF-β1 expression have also been found to be enabled by underlying mechanisms involving the pro-fibrotic pp38 and pERK1/2 pathways [116].

Further, Myofibroblasts have been identified as the key players in the standard wound healing response, as they contribute to wound contraction and ECM production [117], making them ideal targets for reducing scar formation [118]. However, during the maturation phase of normal wound healing, the majority of these cells undergo apoptosis [119]. Thus, reversal of the myofibroblast phenotype might also help in decreasing this cell population [120, 121].

Unfortunately, like fibroblasts, myofibroblasts (or myofibroblast-like cells) also form a functionally heterogeneous population with potential precursors including fibroblasts, mesenchymal stem cells (MSCs), smooth muscle cells, endothelial cells, and fibrocytes [122]. Although it is still being determined whether fibroblasts and adipocytes share a common progenitor, Schmidt and Horsley [123] have demonstrated that dermal adipocytes are essential for fibroblast recruitment during wound healing mechanisms. Experimental interventions with direct and indirect targeting at both populations have been demonstrated to be responsible for scar formation.

Interestingly, Desai and his team [120] have found indications that the myofibroblast differentiation process is not terminal with basic fibroblast growth factor (bFGF) functioning as a phenotypic reversing agent as it led to diminishing expression of α-Smooth Muscle Actin (SMA), collagen I, and fibronectin, and a loss of focal adhesions and stress fibres being inversely co-related with tenascin-C and vimentin upregulation, in agreement with a more fibroblast-like phenotype [120]. These findings are in tune Rinella et al. [124] work that indicate that extracorporeal shockwaves (ESW), which cause myofibroblast precursors to differentiate into more fibroblastlike cells with lower contractility and higher migration potential, simultaneously reducing α-SMA and type I collagen expression may play a significant role in scar reduction.

#### **8.4 Stem cells**

Stem cells are known to modulate the wound environment and improve healing by reducing inflammation [125–127]. A recent study by Li et al. [128] has demonstrated the potential benefits of conditioned media from umbilical cord (UC)-MSC cultures wherein, dermal fibroblasts under the paracrine influence exhibit characteristics similar to those of foetal fibroblasts: low myofibroblast forming capacity, decreased TGF-β1/TGF-β3 ratio, as well as increased expression of enzymes (matrix metalloproteinases or MMPs) involved in ECM remodelling [128]. Other *in vitro* studies have indicated that human amniotic-fluid-derived MSC-conditioned media also has the potential to inhibit the pro-fibrotic actions of TGF-β1 and even reverse the myofibroblast phenotype to a fibroblast-like state. Conditioned media from ASCs produced similar results, but to a lesser extent [129]. However, this enhanced healing mechanism may not always translate to reduced scar-formation as these experiments do not have similar results when performed *in vivo* [130, 131]. Additionally, ethical, legal as well as practical barriers associated with stem-cell- based therapies might restrict their use in the exploration of scarless wound healing [132].

#### **8.5 Wnt and regeneration**

Various signalling pathways, such as the canonical Wnt/β-catenin have been implicated in the expression of foetal mouse keratinocytes and fibroblasts at embryonic day (E)16 and E18 time points, straddling the transition from scarless to scar-forming repair [117, 133]. While Wnt signalling is a key component of embryological development, it is also involved in various wound healing mechanisms with the canonical Wnt pathway being the most relevant [134]. In this pathway, the Wnt-ligand binding at the cell surface leads to cytoplasmic β-catenin accumulation, which subsequently translocates to the nucleus to exert its effects as a transcriptional co-activator [135].

Additionally, a link between TGF-β and the canonical Wnt/β-catenin pathway has been observed in the case of fibrosis with analysis of pathological scars (hypertrophic scars and keloids) revealing upregulated Wnt signalling secondary to TGF-β [136]. β-catenin levels have been shown to double during the proliferative phase in normal wound healing (with scarring) as well [137]. Keloids in humans also display Wnt-3a over-expression by inducing fibroblasts of endothelial origin to transition to mesenchymal cells that leads to collagen accumulation [138].

#### **8.6 MicroRNA**

MicroRNA (miRNA) gene therapies are a more recent avenue for potential therapeutic interventions as these molecules exert an inhibitory role on mRNA transcription within eukaryotic cells, effectively silencing genes at a post- transcriptional level [139]. Comparison of genome-wide miRNA expression between mid-gestational (E16) and late-gestational (E19) mouse skin discovered global repression of these molecules at the earlier time point where scarless healing is the norm [140]. miR-34 family have been established as potential candidates for scarless wound healing in human foetal keratinocytes, however; expression of these miRNAs was found to be significantly lower as gestation progressed [103].

Regenerative wound healing using miRNAs has been explored to reduce scarification [141–143]. For instance, miR-145 has been found at three times its normal levels in hypertrophic scars and pro-fibrotic TGF-β1-induced myofibroblasts. However,

with the help of a commercial inhibitor of miR-145, Gras et al. (2015) were able to significantly decrease type I collagen expression, TGF-β1 secretion, and contractility in skin myofibroblasts [144].

Similarly, miRNAs have been combined with biomimetic scaffolds to enhance wound healing, and furthering their clinical potential [145]. Future *in vivo* studies will hopefully enumerate the clinical potential for other miRNA-based therapies.
