**3. Using AM in chronic wound healing**

Once granulation of the wound is finalized, the process of epithelialization by using AM can be initiated. The source of AM for wound healing is donated placenta. AM has been used for wound healing either as intact AM without epithelium removal or as denuded AM, without the epithelium, [60, 61]. In some cases, AM was used fresh, and in others AM was preserved. Nowadays, it is known that the use of fresh AM is not practical for clinical use [62]. Methods

**Figure 4.** Amniotic membrane fixed to sterile petrolatum gauze (Tulgrasum®) ready for its application.

factor (TNF)‐α, transforming growth factor (TGF)‐α, TGF‐β, basic fibroblast growth factor (b‐ FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatic growth factor (HGF), interleukin‐4 (IL‐4), IL‐6, IL‐8, natural inhibitors of metalloproteases, β‐defensins, and prostaglandins among others [29–31]. Moreover, AM is a biomaterial that can be easily obtained, processed, and transported. On the other hand, AM may function as a substrate where cells can easily proliferate and differentiate [32]. When compared to skin transplanta‐ tion, AM treatment offers considerable advantages. Its application does not produce rejection because it has low immunogenicity and does not induce uncontrolled proliferation [33]. All these effects are related to its capacity for the production and release of biologically active

AM has been applied in medicine for more than 100 years. In 1910, Davis [34] reported a comprehensive review of 550 cases of skin transplantation to various types of burns and wounds using natural AM obtained from labor and delivery at the Johns Hopkins University. In 1913, Sabella [35] and Stern [36] separately reported on the use of preserved AM in skin grafting for burns and ulcers. Since then, there have been several reports of the uses of AM in the treatment of wounds of different etiologies and other applications: first, in the reconstruc‐ tive surgery of different tissues and organs including the mouth, tongue, nasal mucosa, larynx, eardrum, vestibule, bladder, urethra, vagina, and tendons [37–43]; second, as a peritoneum substitute in reconstruction procedures of pelvic exenteration surgery; third, in adherence prevention in the abdomen and pelvic surgery; and finally, as a covering of onphaloceles and

In ophthalmology, the use of AM was reported for the first time in 1940 by De Rötth, who used fresh fetal membranes, namely amnion and chorion, at the ocular surface as a biological dressing in the management of conjunctival alterations [45]. Later, Sorsby et al. [46] used preserved AM as a temporary coating in the treatment of acute caustic ocular lesions. Even though the results were favorable, its use was abandoned for almost four decades. In 1995, with the reconstitution assays of rabbit corneas with limbic disorder using human preserved AM, by Kim and Tseng [47], there was a renewed widespread interest in the use of AM in ophthalmology. Several publications appeared related to the efficacy of the AM in various ocular surface conditions and in diseases like epidermolysis bullosa [44, 48, 49]. Nowadays, AM is a resource widely used in ophthalmology [49–51] and to a lesser degree in the treatment of wounds, burns lesions, and chronic ulcers of the legs [48, 52–54] and in other surgical and

Once granulation of the wound is finalized, the process of epithelialization by using AM can be initiated. The source of AM for wound healing is donated placenta. AM has been used for wound healing either as intact AM without epithelium removal or as denuded AM, without the epithelium, [60, 61]. In some cases, AM was used fresh, and in others AM was preserved. Nowadays, it is known that the use of fresh AM is not practical for clinical use [62]. Methods

substances (see above).

422 Wound Healing - New insights into Ancient Challenges

the like [34–37, 44].

nonsurgical procedures [38–43, 55–59].

**3. Using AM in chronic wound healing**

to remove the epithelium or preserve AM are very diverse and exceed the scope of this chapter. In our case, the placenta is obtained from an uncomplicated elective cesarean of a healthy mother, excluding patients with positive human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) serology. Using an aseptic technique, AM is sepa‐ rated from the subjacent chorion by blunt dissection and stored in saline solution or phosphate buffered saline with antibiotics (cotrimoxazol, tobramycin, vancomycin, and amphotericin B). In this solution, AM is taken to the clean room [55]. Then, its processing, under sterile condi‐ tions, is carried out in a type II vertical laminar flow cabinet (HEPA filter). Then it is cut up into fragments measuring 10 × 10cm, which are then placed on a sterile scaffold of sterile petrolatum gauze (Tulgrasum®) and fixed with silk points at their ends (**Figure 4**). Finally, individual fragments are introduced into a bag with cryopreservative solution to freeze them in liquid nitrogen. These fragments cannot be used in the clinical practice until 3 months have passed, when there is a certainty that their donor has not been seroconverted to HIV, HBV, or HCV. After its defrosting in a 37°C bath, they are taken back to the surgical area and are applied on the wounds of the selected patient [55] (**Figures 5** and **6**).

**Figure 5.** Complex and traumatic soft‐tissue wound. Application of the amniotic membrane.

**Figure 6.** Complex and traumatic soft‐tissue wound. Complete epithelialization after amniotic membrane treatment.

### **3.1. Molecular mechanisms underlying AM‐induced skin reepithelialization**

The molecular mechanisms underlying AM‐induced skin reepithelialization are largely unknown. AM might have a wound healing effect by improving keratinocyte migration from the wound edge and stimulating its differentiation, thereby generating an intact epithelium [63]. Niknejad et al. [64] reflected that the stimulatory effect on epithelialization from the wound bed and/or the wound edge is facilitated by growth factors and progenitor cells released by AM. In addition, it has been described that the preservation of the integrity of the basement membrane and stromal matrix increases the healing potency of AM and is crucial in promoting a fast reepithelialization [65].

Insausti et al. [55] had previously worked on HaCaT cells, a spontaneously immortalized human keratinocyte cell line, as a model to comprehend the molecular consequences of AM application on human wounds [66]. This research showed that HaCaT cells exhibited different molecular reactions upon stimulation with AM that were attributed to the effects of soluble AM‐released factors on HaCaT cells [55]. The application of AM to keratinocytes induced the activation of the phosphorylation of ERK1/2, JNK1/2, and p38 [55]. Also, AM‐conditioned medium induced similar responses, suggesting a trans‐effect of AM on the triggering of these events. Additionally, the authors reported that HaCaT cells stimulated with AM showed an increased expression of *c‐JUN*. Members of the AP1 family had been involved in keratinocyte migration and the wound healing process [67–70]. AM induced the phosphorylation of Jun N‐ terminal kinase (JNK)1 and two kinases in HaCaT cells [55]; JNK1 is a positive regulator of c‐ JUN, contributing to its phosphorylation and stabilization [71, 72]. Finally, the expression of c‐Jun in the wounds treated with AM was very strong, and particularly evident at the basal epithelium near the leading edge and at the dermal leading edge or keratinocyte tongue, indicating that c‐Jun expression might be an important event for epithelialization occurring at the AM‐stimulated wound borders [55].

### **3.2. Chronic wound healing, AM, and TGF‐β**

Wound fluid derived from chronic venous leg ulcers is rich in pro‐inflammatory cytokines such as TNF‐α, interleukin‐1β (IL‐1β), and TGF‐β1 [73]. In addition, the quantities of these cytokines drop as the chronic wound commences to heal, denoting a strong correlation between non‐healing wounds and an increased level of pro‐inflammatory cytokines [74]. TGF‐ β has a critical role in regulating multiple cellular responses that occur in all phases of wound healing [75]. Of the many cytokines shown to influence the wound healing process, TGF‐β has the broadest spectrum of action because it affects the behavior of a wide variety of cell types and mediates a diverse range of cellular functions [76]. Platelets are thought to be the primary source of TGF‐β at the wound site; also, activation of latent TGF‐β occurs immediately after wounding [75]. The TGF‐β signaling pathway is considered as a promising target for the treatment of many pathological skin conditions including chronic non‐healing wounds [75]. Keratinocytes, fibroblasts, and monocytes are among the targeted cells in the TGF‐β manage‐ ment of the wound [76]. Monocytes/macrophages and fibroblasts then contribute to autocrine‐ perpetuated high concentrations of TGF‐β at the wound site [76].

**Figure 6.** Complex and traumatic soft‐tissue wound. Complete epithelialization after amniotic membrane treatment.

The molecular mechanisms underlying AM‐induced skin reepithelialization are largely unknown. AM might have a wound healing effect by improving keratinocyte migration from the wound edge and stimulating its differentiation, thereby generating an intact epithelium [63]. Niknejad et al. [64] reflected that the stimulatory effect on epithelialization from the wound bed and/or the wound edge is facilitated by growth factors and progenitor cells released by AM. In addition, it has been described that the preservation of the integrity of the basement membrane and stromal matrix increases the healing potency of AM and is crucial

Insausti et al. [55] had previously worked on HaCaT cells, a spontaneously immortalized human keratinocyte cell line, as a model to comprehend the molecular consequences of AM application on human wounds [66]. This research showed that HaCaT cells exhibited different molecular reactions upon stimulation with AM that were attributed to the effects of soluble AM‐released factors on HaCaT cells [55]. The application of AM to keratinocytes induced the activation of the phosphorylation of ERK1/2, JNK1/2, and p38 [55]. Also, AM‐conditioned medium induced similar responses, suggesting a trans‐effect of AM on the triggering of these events. Additionally, the authors reported that HaCaT cells stimulated with AM showed an increased expression of *c‐JUN*. Members of the AP1 family had been involved in keratinocyte migration and the wound healing process [67–70]. AM induced the phosphorylation of Jun N‐ terminal kinase (JNK)1 and two kinases in HaCaT cells [55]; JNK1 is a positive regulator of c‐ JUN, contributing to its phosphorylation and stabilization [71, 72]. Finally, the expression of c‐Jun in the wounds treated with AM was very strong, and particularly evident at the basal epithelium near the leading edge and at the dermal leading edge or keratinocyte tongue, indicating that c‐Jun expression might be an important event for epithelialization occurring at

Wound fluid derived from chronic venous leg ulcers is rich in pro‐inflammatory cytokines such as TNF‐α, interleukin‐1β (IL‐1β), and TGF‐β1 [73]. In addition, the quantities of these

**3.1. Molecular mechanisms underlying AM‐induced skin reepithelialization**

in promoting a fast reepithelialization [65].

424 Wound Healing - New insights into Ancient Challenges

the AM‐stimulated wound borders [55].

**3.2. Chronic wound healing, AM, and TGF‐β**

TGF‐β exerts its effect on cells by increasing the phosphorylation of members of the receptor activated (R‐)Smad family (Smad2 and 3). Additionally, non‐Smad pathways are also activated, including the extracellular‐signal‐regulated kinase (ERK), JNK, and p38 mitogen‐activated protein (MAP) kinase pathways, the tyrosine kinase Src, and phosphatidylinositol 3‐kinase (PI3K) [77, 78]. Once receptor‐induced phosphorylation has taken place, R‐Smads form complexes with the common‐mediator (Co‐) Smad4, which are translocated to the nucleus [79] where they, in cooperation with other transcription factors, co‐activators, and corepressors, regulate the transcription of specific genes [80].

The effects of TGF‐β on full‐thickness wound reepithelialization have been studied in a transgenic mouse. The study in the ear mouse model suggests that TGF‐β has an inhibitory effect on epithelialization when the wound involves all the layers of the skin [81]. Also, the overexpression of TGF‐β, at the epidermis level, causes a decrease in reepithelialization [82, 83]. Abolishing part of the TGF‐β signaling pathway has been suggested as a way to improve wound healing, so abolishing part of the TGF‐β‐stimulated Smad pathways may enhance wound healing and benefit the effect of TGF‐β signaling over matrix synthesis by fibroblasts, for instance [76]. TGF‐β causes the growth arrest of epithelial cells. The mechanisms, which differ somewhat between different cell types, involve the inhibition of the expression of the transcription factor Myc and members of the Id family, and the transcriptional induction of the cell cycle inhibitors *CDKN2B* (*p15*) and *CDKN1A* (*p21*) [84].

In order to further unravel the molecular mechanism by which AM may contribute to the epithelialization and wound border proliferation in chronic post‐traumatic wounds, Alcaraz et al. [85] analyzed the association between TGF‐β signaling and AM regulation in wound healing using keratinocytes. Strikingly, AM was capable of attenuating the TGF‐β‐induced phosphorylation of Smad2 and Smad3 in HaCaT cells. Both the strength and duration of TGF‐ β signaling, expressed as sustained phosphorylation of Smads, are essential to achieve proper cell responses to TGF‐β; the impossibility to do so produces a loss of the cell cycle arrest in response to TGF‐β [86]. AM attenuates TGF‐β‐induced Smad2 and Smad3 phosphorylation and hence attenuates *CDKN2B (p15)* and *CDKN1A (p21)* expression [85], which has been connected to cell cycle regulation [86]. Therefore, the presence of AM counteracts the cell cycle arrest induced by TGF‐β on keratinocytes, releasing them from the restrain imposed by TGF‐ 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 present in either HaCaT or primary keratinocyte cells [85].

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.
