According to our data, intrinsic aging has a dramatic effect on human skin composition as reflected by reduced epidermal thickness, flattened dermo-epidermal junction, and the increase in papillary dermis thickness, probably due to the accumulation of GAG. Additionally, reticular dermis thickness decays with age, mirroring GAG and elastic fibers decline. Lastly, the number of vessels, cells, and appendages is diminished in dermis throughout a person’s lifetime (Fig. 6).

Changes in skin composition due to intrinsic aging. A schematization of skin histological structure evolution in photoprotected regions only affected by chronological aging. This model clearly compares variations in all studied parameters between the age groups: group 1 (0–12 years; n = 5), group 2 (13–25 years; n = 5), group 3 (26–54 years; n = 10), group 4 (≥ 55 years; n = 5)

Skin is the largest human organ, extending over more than 2 m2 of surface and representing about 15% of total body weight (Kanitakis 2002). It provides the principal barrier against dehydration, mechanical damage, and biological (i.e., microorganisms and toxins) and physical agents, including UV radiation and temperature changes. Furthermore, the skin plays an active role in thermoregulation, the secretion of waste products through sweat, as well as in immunological, sensory, and endocrine regulation.

In terms of microscopic structure (Khavkin and Ellis 2011), the skin is stratified into three layers, from superficial to deep: epidermis, dermis, and hypodermis. The epidermis, ectoderm-derived, is the outermost layer of the human body, and its predominant cells are keratinocytes (producers of keratin for skin semipermeability maintenance), but it also contains melanocytes, Merkel cells, and Langerhans cells. The dermis, in contrast, is made up of fibers, ground substance, and cells, and contains vascular and nervous plexuses.

Macroscopic changes in human skin due to aging are easily identifiable (Haydont et al. 2019), including wrinkles, atrophy, irregular pigmentation, and laxity (Zhang and Duan 2018). The aging process is regulated by two different mechanisms: extrinsic and intrinsic aging. The former is induced by external factors, known as the exposome: mainly ultraviolet radiation (Berneburg et al. 2000), although others such as environmental pollution and smoking can also play a role. Conversely, intrinsic or chronological aging is a consequence of cellular senescence (Csekes and Račková, 2021), a reduced capacity for cell proliferation, seen especially in basal cells (Zhang and Duan 2018); oxidative stress (Fisher et al. 2002) (a dysfunction of mitochondrial molecules that leads to the formation of reactive oxygen species), and even the activity of some metalloproteinases.

The present research is focused on examining chronological skin aging, thus leaving aside the effect of external factors such as photoaging, given that skin samples are taken from a photoprotected body region, the periumbilical area using morphometric analysis. This technique has been previously employed to determine the microscopic changes in skin parameters due to different interventions (Costello et al., 2023; Demyashkin et al. 2023; Gogly et al. 1997).

Turning to changes in epidermal thickness due to intrinsic aging, a decrease over age, especially notable in the oldest group, was detected. Firstly, one might think that this reduction could be due to a drop in the number of epidermal cells. To corroborate this hypothesis, total epidermal cellularity and mitotic index (reflecting the relationship between dividing and total cells) were morphometrically assessed. In terms of the mitotic index, epidermal regeneration has been shown to lessen with age, leaving a smaller number of keratinocytes in older subjects. Several mechanisms by which the mitotic index of keratinocytes is altered with age have been previously reported, such as decreasing levels of epidermal growth factor (Wang et al. 2020), and rising intracellular calcium (Micallef et al. 2009). Likewise, the augmentation in keratinocyte apoptosis found with aging (Wang et al. 2020) would also help explain the reduced count observed in our results.

However, paradoxically, our data showed no variation in total epidermal cellularity, although a decrease in keratinocytes was suggested. To explain this observation, we also measured other epidermal cell types (i.e., melanocytes, Langerhans cells, and Merkel cells), observing a modest increase with age. These results might account for the stable total cell count in the epidermis. Melanocytes are cells that produce melanin, the pigment that protects against UV rays (Brenner and Hearing 2008). However, although these increase with age, this does not imply greater photoprotection if one takes into account the alterations in its functions (including impaired melanosome transport or glucose metabolism) demonstrated in some studies (Park et al. 2023). Contrariwise, Langerhans cells are cutaneous dendritic cells that exert immunological protection, being mainly antigen presenters (Romani et al. 2003). These are also elevated in the oldest group, probably due to immune system deregulation in these older individuals (Fülöp et al. 2019; Pawelec 2018). Lastly, although no significant changes were found in Merkel cells (sensory neuroendocrine cells), our results are in line with those reported by Moll et al. (1984), who observed these cells in high concentration at the fetal level, they were decreased during childhood, and in older ages their production may increase again, probably due to greater exposure to harmful stimuli (Wright et al. 2017).

Taken altogether, these findings suggest that the decrease in epidermal thickness caused by intrinsic aging most likely results from a loss in cellular turgor of keratinocytes (which become shorter and wider; Farage et al. 2013). This process is due to dehydration, mainly of the stratum corneum, because of alterations in binding proteins and lipid structures (Waller and Maibach 2006; Rogers et al. 1996). Dehydration would also contribute to maximize epidermal inflammation (Wang et al. 2020), which further supports the results obtained in this field.

Our results from dermal layer analysis highlighted an increase in papillary dermis and a reduction in reticular dermis thickness with aging. To elucidate this, the area occupied by GAG was first quantified. According to our data, intrinsic aging influences the presence of GAG, as reflected by an augmentation in the papillary layer and a reduction in reticular dermis. Admittedly, these results are controversial in comparison with the current literature. Some studies have suggested that intrinsic skin aging induces overexpression of GAG (Timár et al. 2000), while others have reported that photoaging, but not intrinsic aging, provoked increased GAG expression (Waller and Maibach 2006). Further research has uncovered that certain key GAG skin components, like decorin proteoglycan, undergo a reduction in size, leading to a decrease in dermal volume occupied by GAG (Li et al. 2013). Moreover, the increased papillary dermis can be viewed as an indirect consequence of the reduced number and length of ridges and papillae, as a result of which this space becomes occupied by the papillary dermis.

Since fibers are also components of dermal interstitium, modifications in their presence were also determined in subjects at different ages. A previous study from our group reported that the area of the papillary dermis occupied by collagen fibers and the thickness of its bundles were reduced (Marcos–Garcés et al. 2014). In terms of elastic fibers, although some studies suggest no differences in the density of elastic fibers due to intrinsic aging (Timár et al. 2000), our data indicates that the number of elastic fibers within reticular dermis is progressively reduced, especially when reaching adulthood. Contrasting our results with those of other studies, it seems that cutaneous elastin undergoes a series of changes in composition and structure over time (Pasquali-Ronchetti and Baccarani-Contri 1997), while elastic fibers show tortuosity and distortion which cause loss of elasticity (Imayama and Braverman 1989). Thus, unlike the impact of photoaging, which produces an increase in elastic fiber density (Hunzelmann et al. 2001), intrinsic aging therefore leads to progressive degradation (Vitellaro–Zuccarello et al., 1994) as well as cumulative damage in these fibers (Waller and Maibach 2006). In addition, alteration of skin elasticity with age may result from fibroblasts loss (Gunin et al. 2014), reduced biosynthetic activity, and variations in extracellular matrix macromolecules (Frances et al. 1990).

A reduction in dermal cellularity was noted in both the papillary and reticular layers, thus lending credence to the assertion that fibroblast proliferation is decreased (Gunin et al. 2011). Initially, the possibility of an increase in the inflammatory cell population had been suggested (Lee et al. 2021), since other studies have shown a gradual increase in both mast cells and CD45+ cells (Gunin et al. 2011). However, our results regarding mast cell density displayed no changes affecting the older group.

Finally, skin appendages (hair follicles and sweat glands) are notably reduced from youth onwards (Kamberov et al. 2015), which could be explained by a reduction in fibroblast number, circulating growth factors levels, and microvessels density. To corroborate this hypothesis, we subsequently analyzed changes in the number of capillaries at different age stages. According to our data, the number of dermal vessels peaks in children (0–12 years), diminishing notably in group 4 (≥ 55). These results are in line with a previous study demonstrating that a decrease in dermal vascular density is related to downregulation of the vascular endothelial growth factor signaling cascade and lower levels of von Willebrand factor (Gunin et al. 2014). Despite several studies that agree on the reduction of cutaneous blood flow (Waller and Maibach 2005), this finding is a bit controversial as other studies support just the opposite (Chung et al. 2002). Nonetheless, this reduction could explain numerous skin changes attributed to intrinsic aging, being partly a cause and consequence of atrophy, for example, of the appendages.