**4. Summary**

ß [85]. The effect of AM on TGF‐β‐regulated genes is not indiscriminate, and not all genes are affected by the presence of AM. Interestingly, genes that positively participate in wound healing such as *SNAI‐2* and *PAI‐1* were synergistically up‐regulated by the presence of AM and TGF‐β [85]. Finally, the expression of c‐Jun was maximal when both TGF‐β and AM were

It has been suggested that AM might exert its wound healing effect by increasing keratinocyte migration speed from the wound edge [63]. Growth factors and progenitor cells released by AM [64] are supposed to mediate the epithelialization stimulatory effect. AM induces cell migration in a wound healing assay in keratinocytes and mesenchymal cells [85]. Furthermore, in keratinocytes, inhibition of cell proliferation with mitomycin C, affected the migrating properties of AM. In the same study, the use of JNK1 inhibitors prevented AM‐induced cell migration in both cell types. Moreover, a closer inspection of the margins of the scratch wound healing assays showed a high expression of c‐JUN in the AM‐stimulated cells engaged in the migratory wave. The AM‐induced high expression of c‐JUN at the wound border was prevented by inhibitors SP600125 and PD98059, which is consistent with the fact that AM induces the activation of a signaling cascade that produces the phosphorylation of ERK1/2 and JNK1/2. A local increase of c‐JUN was observed in the patient wound border when the wound had been treated with AM. This is coherent with the AM effect on cell migration. In fact, in the examination of patient wound borders a few days after AM application, a clear proliferation/ migration was observed [85]. This correlates well with the robust expression of c‐Jun at the wound border, which is particularly robust at the *stratum basale* of the epidermis that overlaps the keratinocyte tongue, the area where the migration of keratinocytes happens to epithelialize the wound [85]. Additionally, in that investigation, the authors revealed that the application of AM promotes healing in chronic wounds by refashioning the TGF‐β‐induced genetic program, stimulating keratinocyte migration and proliferation [85]. Additionally, there might be a synergy of AM and TGF‐β signaling for the resolution of chronic wounds [85, 87]. Thus, stimulation of keratinocytes with both AM and TGF‐β was synergistic when compared to both stimulus being added separately [85]. Moreover, the treatment of cells with TGF‐β signaling inhibitors hampered the effect of AM, indicating that both AM and TGF‐β signaling positively contribute to cell migration [87]. The down‐regulation of Smad3 has been suggested as a possible way of improving wound healing [76]. In this sense, the effect of R‐Smads, Smad2 or 3, seems to be different given that the overexpression of Smad2 increased AM‐induced cell migration while the overexpression of Smad3 prevented it [87]. Notably, the ability of kerati‐ nocytes to sense TGF‐β through Smad3 prevents the cell proliferation of keratinocytes and consequently prevents wound healing resolution when the levels of TGF‐β are high [88].

Presently, in order to evaluate the effect of AM on chronic post‐traumatic wounds, a clinical trial is being conducted in our hospital, with exceptional results. The TGF‐β‐stimulated Smad pathway has also been involved in the production of fibrosis and inflammation in response to TGF‐β. Thus, interfering with TGF‐β signaling may be a good way of interfering with fibrosis and improving the evolution of wound healing [76]. In different experimental models, the application of AM is able to ameliorate fibrosis [89–92]. Currently, we are exploring whether

the application of AM is able to reduce fibrosis and inflammation in chronic wounds.

present in either HaCaT or primary keratinocyte cells [85].

426 Wound Healing - New insights into Ancient Challenges

To summarize, AM is a biological dressing that stimulates proper epithelialization in chronic wounds. It has several advantages; among them, it is economical, easy to obtain, and in endless supply. Additionally, AM can be cryopreserved at a low temperature while preserving all its biological functions. Finally, it can be used as a treatment in the outpatient clinic, which reduces costs even more. Thus, AM must be taken into account as a consolidated treatment option for chronic wounds.
