**2. Skin: anatomy and physiology**

Skin thickness varies between people and with age and ranges on average from 0.05 to 2 mm. Skin is comprised of three layers: non-vascularized and stratified epidermis, underlying the dermis composed of a connective tissue and the subcutaneous adipose tissue forming the hypodermis including the adnexal structure [34]. The epidermis is preceded by an organized structure the *stratum corneum* formed by as interdigitated dead cells called corneocytes disposed as bricks between multiple lipid bilayers holding the structure defined as "brick and mortar" [35] and represent the first barrier to external factor penetration. The viable layers of the epidermis are *stratum lucidum*, *stratum granulosum, stratum spinosum,* and *stratum germinativum*. Keratinocytes composing these layers undergo progressive differentiation from the basal *stratum germinativum* to the outermost layer. These self-renewal and differentiation processes are important for epidermal regeneration and lead to generation of solid lipid-rich cornified layers [36]. Melanocytes are other cells present in the epidermis; they synthetize the melanin pigment being transferred to mature keratinocytes, providing principal skin protection against UV damages. Merkel cells, dendritic cells, adipocytes, and Langerhans cells are also present within the epidermis.

Fibroblasts are the principal cells constituting the dermis; they are mesenchymal and represent skin scaffolds where they support other epithelial cells and the epidermis through their elongation and shaped form, but especially through secretion of fibrous and elastic components constituting the ECM responsible for cutaneous strength and elasticity [34]. ECM is composed of fibrous proteins and a ground

*Regenerative Medicine*

during wound healing.

regeneration.

cellular and molecular changes leading to progressive reduction in cell proliferation

A recent work has testified that adult multipotent stem cells are present in the dermal sheets and in the interfollicular dermis; they can also be derived from the pericytes [2]. They are expected to play a crucial role in regulating skin function and turnover. Furthermore, these cells were considered as mesenchymal stem cell (MSC)-like expressing the specific mesenchymal markers and differentiating into adipocyte, chondrocyte, osteoblast and myocyte [3]. These cells are identified within the subcutaneous adipose tissue as adipose-derived stem cells (ADSCs) and have been reported to differentiate into skin cells, thus ensuring skin regeneration and maintaining homeostasis [4–6]. Several studies have shown the ability of ADSCs to act through cell-cell contact, but mostly by secreting a panel of cytokines and chemokines, being involved in different biological pathways including cell proliferation, differentiation, homing and migration, senescence, and apoptosis [7–11]. These mechanisms are implicated in the whole process of skin regeneration

ADSC-based therapy is very promising in treating damaged tissues and in completing full-thickness skin replacement. Some clinical applications benefit from its simple and abundant collection from adipose tissue. The capacity of these cells to proliferate and self-renew *in vitro* as well as *in vitro* added to their innate differentiation has targeted more scientific advancements in the field of regenerative medicine. Their immunomodulatory effects also make them more suitable for use compared to their counterpart from bone marrow and umbilical cord blood [12]. These cells have been used for many investigations and are largely used for graft improvement in cosmetic remodeling to prevent fat necrosis [13–15]. ADSCs have presented a great ability to migrate and were recruited rapidly into wounded sites where the process of cell differentiation toward various skin cell components occurred. ADSCs have helped in cicatrization and regulating inflammation and the phases of wound healing [6]. These cells secrete growth factors in their extracellular vesicles [16–18] and produce different amounts of the extracellular matrix (ECM) proteins, thus promoting and accelerating skin regeneration in 3D raft cultures from adult expanded human skin [19, 20]. This suggested that these cells represent a rich source of factors necessary for accelerating wound healing and tissue

Among the secreted growth factors, transforming growth factor-β (TGF-β) and growth differentiation factor 11 (GDF11) are highlighted and both are involved in biology of skin and different organs [5, 21, 22]. Both factors belong to the same superfamily of TGF-β and target similar skin cells including dermal fibroblast (DF), keratinocytes, melanocytes, and dermal microvascular endothelial cells [23–25]. Moreover, these factors activate the intracellular SMAD signaling pathways, thus targeting skin cell properties to repair wounded tissues. Consequently, TGF-β reduces wrinkles and photoaging signs [24, 26, 27]. On the other hand, GDF11 has received more attention with its ability to produce age-reversing effects [25, 28] and

In the current research, many questions have been raised about the involvement

of GDF11 in the inflammatory, proliferative, and remodeling phases of wound healing. Adding to the fact that TGF-β was secreted by utmost epithelial cells and participated extensively in this cascade, an interaction between GDF11 and TGF-β for sustainable skin biology and function has been suggested. Additionally, they share similar intracellular mechanisms involved in healing and aging. Cross-talking with the surrounding cells, mainly resident ADSCs, keratinocytes, DF, melanocytes, and macrophages, these factors might be activated, autoactivated, and/or mediate other cytokines and chemokines to attain and orchestrate even similar

increase skin cell proliferation and functionality [23–25].

and regeneration as a result of increasing cell senescence and apoptosis [1].

**42**

substance. Both these components are rearranged to provide a three-dimensional microenvironment where epithelial cells, stem cells, and the vascular network are closely related to collagen, elastin, and fibronectin fibers [37]. In human skin, collagen fibers, mostly type I, III, and V, are the dominant components in the ECM accounting for 75% of the dry skin weight and confer elasticity and strength. Type I collagen represents 80–90% of the total collagen and type III up to 8–12% while type V collagen represents the remaining minor proportion [38]. ECM not only provides a structural support for skin cells but also plays very critical role in regulating cell behavior in normal conditions and wound healing [39]. This regulation occurs through molecular signaling mediated by integrin cell surface, which orients cells toward proliferation, differentiation, migration, or apoptosis [40].

Additional skin components residing in the dermis and sometimes in the hypodermis are immune cells represented by lymphocytes, macrophages, mast and dendritic cells. Adnexal structures are located in the dermis and hypodermis and include hair follicles, blood vessels, nerves, eccrine glands, sebaceous glands, and apocrine gland.

The hypodermis layer or subcutaneous layer is composed mostly by adipose tissue containing adipocytes, stem cells (ADSCs), and blood and lymph vessels. This adipose tissue is the main actor in regulating skin homeostasis, thermoregulation, metabolism, immune responses, and immunomodulation through a wide range of cytokines and chemokines secreted. This secretory panel conditions the microenvironment of the surrounding ADSCs and consequently their secretome to modulate skin cell proliferation, differentiation, migration, melanin production, and to induce skin rejuvenation [41–43].
