Infiltration of subcutaneous adipose layer into the dermal layer with aging

Abstract

Background

The elasticity of the dermal layer decreases with aging, leading to ulcer formation and wrinkling, but the mechanism of this change is not fully understood, because it is difficult to access the complex three‐dimensional (3D) internal structure of the dermis.

Objective

To clarify age‐dependent changes in the overall 3D structure of the dermal layer by means of 3D analysis technology.

Methods

We observed sun‐protected human skin by means of X‐ray micro CT, identified the layers of the skin, and reconstructed the 3D structure on computer. Age‐dependent structural changes of the dermal layer were evaluated by statistical comparison of young and aged skin.

Results

Histological observations suggested the presence of two types of ectopic fat deposits, namely infiltrated subcutaneous fat and isolated fat, in the lower region of the reticular dermal layer in aged skin. To elucidate their nature, we observed skin specimens by X‐ray microCT. The epidermis, dermal layer, and subcutaneous adipose layer were well differentiated on CT images, and 3D skin was digitally reconstructed on computer. This method clearly showed that the isolated fat observed histologically was in fact connected to the subcutaneous fat, namely all ectopic fat is connected to the subcutaneous adipose layer. Statistical analysis showed that the severity of fat infiltration into dermal layer is significantly increased in aged skin compared with young skin.

Conclusion

Our findings indicate that subcutaneous fat infiltrates into the dermal layer of aged skin. Our 3D analysis approach is advantageous to understand changes of complex internal skin structures with aging.

1. INTRODUCTION

The dermal layer is composed of abundant extracellular matrix, such as collagen and elastic fibers, which contribute to the skin's physical properties, especially elasticity. ¹ Age‐dependent changes of the dermal layer lead to impaired skin elasticity, ² resulting in delayed wound healing, ulcer formation, wrinkling, ³ and sagging. ⁴ Since the dermal layer contains a variety of appendages, such as sweat glands, sebaceous glands, and hair follicles, ⁵ its three‐dimensional (3D) structure is complex. Consequently, the nature of the age‐dependent changes of dermal layer structure is still unclear.

The subcutaneous adipose layer is located just beneath the dermal layer and consists of adipocytes that store fat. We have established that subcutaneous fat physiologically controls the dermal condition via secretory factors, and its effect is different in obese and normal subjects. ⁶ Further, subcutaneous fat contributes negatively to skin elasticity and even to the phenotype of the skin, namely sagging. ⁷ Since there is no basal membrane between the dermal layer and subcutaneous adipose layer, these layers could in principle influence each other's physical condition or structure. However, it is not yet known whether and how the subcutaneous adipose layer influences the structure of the dermal layer.

　Histological observation is usually used to investigate the internal structure of the skin, but two‐dimensional (2D) observation even with multiple serial specimens is not suitable to unpick the complex structure of the dermal layer. ⁸ Specifically, deformation during the cutting of sections and variations in the thickness of the sections make it difficult to accurately reconstruct detailed structures. Further, numerical analysis of skin structures is also difficult.

However, micro CT is an X‐ray imaging method that can nondestructively observe internal tissue structures at an ultrafine resolution of micrometer order. ⁹ , ¹⁰ Recently, we applied this method to analyze the 3D structure of the sweat gland, a complex organ with coiled, and tangled parts, in skin specimens, ¹¹ and we were able to establish the age‐dependent change of the 3D structure of sweat gland quantitatively.

In this study, we first used microCT to visualize the complex dermal layer separately from the epidermis and subcutaneous adipose layer and then analyzed the change of the 3D structure of the dermal layer with aging.

2. METHODS

2.1. Skin samples

Healthy human abdominal skin (surplus skin excised during plastic surgery) was purchased from Biopredic International (Rennes, France). Areas without stretch marks were used for analysis. The donor age range was 25–71 years, and the body mass index range was 20–29 (Table 1). Statistical analysis of structural differences in the dermal layer was conducted by comparing young (<40 years old) and aged (>60 years old) groups. These studies were approved by the ethics committees of Shiseido Co., Ltd. and the International University of Health and Welfare.

TABLE 1.

Characteristics of the subjects

	Number of subjects	Minimum	Age maximum	Average	Minimum	BMI maximum	Average
Young	10	25	39	33.4	21	29	25.2
Old	10	61	71	65.5	20	27	25.2

Abbreviation: BMI, body mass index.

2.2. MicroCT observation of skin layers

The methods were described previously. ¹¹ Briefly, skin samples were dissected into cubes of about 5 mm³, which contain epidermis, dermal layer, and subcutaneous adipose layer. The cubes were examined with an X‐ray micro CT (D200RSS270; Comscan Techno, Kanagawa, Japan) under the following conditions: voltage 80 kV, current 100 microA, and resolution 2.6 micrometers. Then, each layer was identified, and a 3D image was reconstructed with Amira (Thermo Fisher Scientific, MA, USA).

2.3. Histological observation

The AmeX procedure was used for histology. ¹² Briefly, skin specimens were fixed, embedded in paraffin, and 5‐μm sections were cut and stained with hematoxylin‐eosin.

2.4. Statistical analysis

Differences between groups were evaluated by means of Student's t‐test. p < 0.05 was considered significant. Data were expressed as mean ± SEM.

3. RESULTS

3.1. Histological observation of the dermal layer in aged skin

Histological observation of aged skin samples revealed the presence of ectopic fat in the lower part of the dermal layer, compared to young skin (Figure 1A,B). Some of the fat deposits were connected to the subcutaneous adipose layer and appeared to consist of round‐shaped adipocytes. In addition, isolated fat (not connected to the subcutaneous adipose layer) was also observed at the bottom or in the middle of the dermal layer and also appeared to consist of adipocytes (Figure 1C). To understand the nature of these ectopic fat deposits, we next conducted 3D analysis of the dermal structure.

FIGURE 1.

Ectopic fat in the dermal layer in aged skin. Histological observation of vertical sections of hematoxylin‐eosin (HE)‐stained abdominal skin. Compared to (A) young skin (31 years old), (B) aged skin (65 years old) shows adipocytes at the bottom of the dermal layer, which is connected to the subcutaneous adipose layer. (C) Another sample of aged skin (64 years old) shows an apparently isolated (from the subcutaneous adipose layer) fat deposit in the middle of the dermal layer

3.2. Digital reconstruction of 3D structure of skin on computer

Digitally reconstructed 3D skin enabled us to differentiate the three layers of skin and to visualize the 3D structure of the ectopic fat deposits at the bottom of the dermal layer (Figure 2A). As mentioned above, the 2D histological observations showed apparently isolated fat deposits in the dermal layer, which appeared to be independent of the subcutaneous adipose layer (Figure 1C). However, the 3D observations clearly showed that all of these apparently isolated fat deposits observed in the 2D sections are actually linked to the subcutaneous adipose layer (Figure 2B‐G). This result supports the idea that the dermal ectopic fat represents infiltration of subcutaneous adipose tissue into the dermal layer.

FIGURE 2.

Subcutaneous adipose layer infiltrates into dermal layer. (A) Illustration of sectioning for 2D histological observation. (B) When the skin specimen is vertically sectioned at (A), then (C) surface rendering or (D) volume rendering shows an apparently isolated fat deposit in the dermal layer. (E) If the skin specimen is vertically sectioned at (B), then (F) surface rendering or (G) volume rendering shows that the ectopic fat is connected to the subcutaneous fat layer, namely it represents infiltrated fat. White scale bars indicate 0.5 mm

3.3. Age‐dependent infiltration of subcutaneous fat into the dermal layer

To analyze the infiltration of subcutaneous fat into the dermal layer with aging statistically, we quantified its severity by computer analysis of 3D skin. However, as shown in Figure 3A,B, the boundary between the dermal layer and subcutaneous adipose layer is not flat in either young or aged skin, making it difficult to set a baseline of fat infiltration in 3D images, compared to 2D images. Thus, we measured proximity to the epidermis (Figure 3A,B), namely the vertical distance to the epidermis from the nearest point of the top of the infiltrated fat. This distance significantly decreased with aging (Figure 3C). In other words, the fat infiltration expanded with aging, reaching closer to the epidermis.

FIGURE 3.

Severity of fat infiltration increases with aging. (A) Young and (B) aged 3D skin, showing the dermal‐subcutaneous boundary. White arrows illustrate the method used to evaluate the severity of the infiltration of subcutaneous fat, based on measurement of the vertical distance from the top of the fat to the epidermis in the 3D structure. (C) The proximity significantly decreases with aging, showing an increase in the severity of fat infiltration with aging. White scale bars indicate 0.5 mm. ***p < 0.001 by Student's t test (mean ± SEM, n = 10)

Furthermore, we evaluated the frequency of fat infiltration into the dermal layer by means of digital dissection of the skin on computer. The skin was horizontally dissected at 1‐mm depth (Figure 4A,B), in order to avoid the natural angulation of the boundary between subcutaneous fat and the dermal layer. In the aged skin, the number of fat infiltration was 21.3 ± 1.9 areas/cm² in the aged skin, which is significantly higher than in young skin (Figure 4C).

FIGURE 4.

Fat infiltration into the dermal layer. Horizontal dissection at the depth of 1 mm from the skin surface on computer: (A) Young skin and (B) aged skin. (C) The frequency of fat infiltration in young and aged skin (number of fat infiltration per cm²). White scale bars indicate 1 mm. ***p < 0.001 by Student's t test (mean ± SEM, n = 10)

4. DISCUSSION

In this study, we have established an X‐ray microCT analysis method for quantitative analysis of the 3D structure of the dermal layer and used it to analyze female abdominal skin. Our findings show that ectopic fat deposits appear in the dermal layer with aging. These ectopic fat deposits are all connected to the subcutaneous adipose layer, suggesting that subcutaneous adipose tissue infiltrates into the dermal layer with aging.

　Since the dermal layer contains a variety of internal organs, it is difficult to evaluate age‐dependent changes of its structure. For example, there are conflicting reports as to whether or not the dermal layer becomes thinner with aging. ¹³ , ¹⁴ The disagreement is likely due to the inherent limitations of 2D observation. In the present work, for example, the apparently isolated ectopic fat observed histologically (Figure 1C) proved to be connected to the subcutaneous layer when examined in 3D. This clearly illustrates the value of our microCT‐based approach for studies of the structure of the dermal layer and its age‐dependent changes.

Replacement of organs by fat with aging or obesity has been reported in bone marrow, ¹⁵ skeletal muscle, ¹⁶ and liver. ¹⁷ We previously reported aging‐related defects of the dermal layer that contained fat, but the origin of the fat was unclear. ¹⁸ This ectopic adipocyte formation has been proposed to arise from the differentiation of stromal cells ¹⁹ , ²⁰ in the organs. However, the present study revealed that all of the ectopic fat in the dermal layer is in fact connected to the subcutaneous layer. Our previous measurements of the defects were based on 2D histological analysis, which is hard to apply to this type of complex structure, as we showed in Figure 2. Furthermore, our quantitative measurement of fat infiltration in the 3D structure in the present work established that the extent of the infiltration increases with aging. Thus, it seems likely that fat infiltrates from the subcutaneous layer, rather than arising by cell differentiation in the dermal layer.

Although the dermal layer and subcutaneous fat are clearly separate, there is no specific structure that divides these two layers, in contrast to the basement membrane, which physically separates the epidermis and dermal layer. This may explain why the dermal layer is vulnerable to the infiltration of subcutaneous fat.

We previously showed that in obese subjects, the adipocytes are enlarged to store the excess fat, and these adipocytes secrete matrix metalloproteinase (MMP), which degrades the extracellular matrix (elastic fibers) in the dermal layer. ²¹ However, in the present study, enlargement of adipocytes was not observed in aged subcutaneous fat, suggesting the involvement of a different mechanism in the fat infiltration. The amount of tissue inhibitor of metalloproteinases (TIMP) in the dermal layer is known to decrease with aging. ²² Since the boundary between subcutaneous fat and the dermal layer might be regulated by the balance of MMP/TIMP, loss of TIMP with aging could be related to the increase of fat infiltration with aging, even though the MMP level does not change. Therefore, modulation of these degradation and prevention systems could be a potential solution to prevent the increase of fat infiltration with aging. Further study is necessary to elucidate the precise molecular mechanism of the fat infiltration.

In this study, we used sun‐protected skin to elucidate the age‐dependent structural change of the dermal layer in order to eliminate the influence of photo aging, caused by UV light. Since UV damages the dermal layer, the sun‐exposed dermal layer might be more vulnerable to fat infiltration. Further work to evaluate this idea could lead to new solutions for improving issues such as wrinkles, which are caused by natural aging and enhanced by UV light, based on targeting fat infiltration.

The loss of the dermal layer as a result of fat infiltration with aging decreases type 1 collagen and elastin, which are major contributors to skin stiffness and elasticity. Indeed, the elasticity of the dermal layer has been reported to decrease with aging, ²³ especially in the lower dermal layer. ²⁴ Thus, the age‐dependent fat infiltration into the dermal layer may be a key contributor to the loss of elasticity of the skin with aging. Therefore, these fat infiltrates are a potential target to improve dermal disorders such as ulcers, sagging, and wrinkling.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Ezure T, Amano S, Matsuzaki K. Infiltration of subcutaneous adipose layer into the dermal layer with aging. Skin Res Technol. 2022;28:311–316. 10.1111/srt.13133