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Abstract 3D skin models have been explored as an alternative method to the use of animals in research and development. Usually, human skin equivalents comprise only epidermis or epidermis/dermis layers. Herein, we leverage 3D bioprinting technology to fabricate a full-thickness human skin equivalent with hypodermis (HSEH). The collagen hydrogel-based structure provides a mimetic environment for skin cells to adhere, proliferate and differentiate. The effective incorporation of the hypodermis layer is evidenced by scanning electron microscopy, immunofluorescence, and hematoxylin and eosin staining. The transcriptome results underscore the pivotal role of the hypodermis in orchestrating the genetic expression of a multitude of genes vital for skin functionality, including hydration, development and differentiation. Accordingly, we evidence the paramount significance of full-thickness human skin equivalents with hypodermis layer to provide an accurate in vitro platform for disease modeling and toxicology studies.

Abstract Adipose tissues in the hypodermis, the crucial stem cell reservoir in the skin and the endocrine organ for the maintenance of skin homeostasis undergo significant changes during skin aging. Dermal white adipose tissue (dWAT) has recently been recognized as an important organ for both non-metabolic and metabolic health in skin regeneration and rejuvenation. Defective differentiation, adipogenesis, improper adipocytokine production, and immunological dissonance dysfunction in dWAT lead to age-associated clinical changes. Here, we review age-related alterations in dWAT across levels, emphasizing the mechanisms underlying the regulation of aging. We also discuss the pathogenic changes involved in age-related fat dysfunction and the unfavorable consequences of accelerated skin aging, such as chronic inflammaging, immunosenescence, delayed wound healing, and fibrosis. Research has shown that adipose aging is an early initiation event and a potential target for extending longevity. We believe that adipose tissues play an essential role in aging and form a potential therapeutic target for the treatment of age-related skin diseases. Further research is needed to improve our understanding of this phenomenon.

Macrophages are versatile myeloid leukocytes with flexible cellular states to perform diverse tissue functions beyond immunity. This plasticity is however often hijacked by diseases to promote pathology. Scanning kinetics of macrophage states by single-cell transcriptomics and flow cytometry, we observed atopic dermatitis drastically exhausted a resident subtype S1. Characterized by FRβ/CD163 expression, S1 exhibited strong efferocytosis and chemoattracted monocytes and eosinophils. Here we have delineated mechanisms regulating monocyte decision to acquire S1 identity in skin. During M-CSF driven macrophage differentiation in healthy skin, FRβ was expressed via intrinsic control of STAT6 and ALK5 activities, and did not require heterotypic cellular crosstalk. In contrast, CD163 expression required exposure to fibroblastic secretion. This process depended on SHP1 activity and involved STAT5 inactivation. Suppressed STAT5 activity caused CD163 expression and rendered macrophage insensitive to further induction by fibroblasts. Parsing coculture experiments with in silico ligand expression, we identified laminin-α2 and type-V collagen secreted by hypodermal fibroblasts as CD163-driving factors. S1 identity loss in AD followed a stepwise cascade: reduced laminins availability first dampened CD163 expression, IL4 and TGFβ subsequently acted on CD163 lo/- cells to downregulate FRβ. In AD skin, we showed that imitating this fibroblast-macrophage crosstalk with exogenous laminin-211 encouraged monocyte differentiation to S1 macrophages, fostered homeostatic commitment of extravasated eosinophils, and alleviated dermatitis. Hence, we demonstrated that reinforcing a steady-state cue from hypodermal fibroblasts could override maladaptive pressure on macrophage and restored tissue homeostasis., Competing Interests: Declarations. Conflict of interest: KT is a scientific founder, stockholder of and received research funding from StemRIM. ET and YO are employees of StemRIM. All other authors declare they have no competing interests. Human and animal rights: All animal experiments were conducted under the guidelines of and approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine with reference numbers 02-075-003 and 02-075-005. The study did not involve human subjects., (© 2024. The Author(s).)

Macrophages are versatile myeloid leukocytes with flexible cellular states to perform diverse tissue functions beyond immunity. This plasticity is however often hijacked by diseases to promote pathology. Scanning kinetics of macrophage states by single-cell transcriptomics and flow cytometry, we observed atopic dermatitis drastically exhausted a resident subtype S1. Characterized by FRβ/CD163 expression, S1 exhibited strong efferocytosis and chemoattracted monocytes and eosinophils. Here we have delineated mechanisms regulating monocyte decision to acquire S1 identity in skin. During M-CSF driven macrophage differentiation in healthy skin, FRβ was expressed via intrinsic control of STAT6 and ALK5 activities, and did not require heterotypic cellular crosstalk. In contrast, CD163 expression required exposure to fibroblastic secretion. This process depended on SHP1 activity and involved STAT5 inactivation. Suppressed STAT5 activity caused CD163 expression and rendered macrophage insensitive to further induction by fibroblasts. Parsing coculture experiments with in silico ligand expression, we identified laminin-α2 and type-V collagen secreted by hypodermal fibroblasts as CD163-driving factors. S1 identity loss in AD followed a stepwise cascade: reduced laminins availability first dampened CD163 expression, IL4 and TGFβ subsequently acted on CD163lo/− cells to downregulate FRβ. In AD skin, we showed that imitating this fibroblast-macrophage crosstalk with exogenous laminin-211 encouraged monocyte differentiation to S1 macrophages, fostered homeostatic commitment of extravasated eosinophils, and alleviated dermatitis. Hence, we demonstrated that reinforcing a steady-state cue from hypodermal fibroblasts could override maladaptive pressure on macrophage and restored tissue homeostasis. [ABSTRACT FROM AUTHOR]

In this study, we investigate the mechanical behavior of each skin layer, in terms of the nominal stress-strain curve by uniaxial tensile tests using specimens of porcine skin in two forms: dermis containing epidermis, and all three layers. All tests were performed under cyclic loading at the constant strain rate of 10–3 s–1 at ambient temperature. To measure the precise initial cross-sectional areas of each layer, the thickness of each skin layer was quantified by counting the number of pixels on the photo-image using image-processing software. In the tensile test, force-strain curves of the total skin and dermis with epidermis were obtained. Subsequently, a rule of mixtures was applied to determine the nonlinear mechanical properties of the hypodermis layer. In conclusion, we could define the uniaxial tensile behavior of the hypodermis, and additionally predict the weight effect of the dermis and hypodermis layers in the tensile test.

Photoaging is an important extrinsic aging factor leading to altered skin morphology and reduced function. Prior work has revealed a connection between photoaging and loss of subcutaneous fat. Currently, primary models for studying this are in vivo (human samples or animal models) or in vitro models, including human skin equivalents (HSEs). In vivo models are limited by accessibility and cost, while HSEs typically do not include a subcutaneous adipose component. To address this, we developed an “adipose-vascular” HSE (AVHSE) culture method, which includes both hypodermal adipose and vascular cells. Furthermore, we tested AVHSE as a potential model for hypodermal adipose aging via exposure to 0.45 ± 0.15 mW/cm2 385 nm light (UVA). One week of 2 h daily UVA exposure had limited impact on epidermal and vascular components of the AVHSE, but significantly reduced adiposity by approximately 50%. Overall, we have developed a novel method for generating HSE that include vascular and adipose components and demonstrated potential as an aging model using photoaging as an example.

• The leaf ontogenesis of Myrtoideae species revealed that the subepidermal layer is a hypodermis that originates from periclinal divisions of the ground meristem. • The occurrence of a hypodermis in representatives of Myrtaceae needs to be better studied and discussed in relation to the phylogeny of the family. • This study demonstrates the importance of ontogeny to understand plant structures so they can be properly used in taxonomic and phylogenetic interpretations. The ninth largest family of angiosperms is Myrtaceae, which comprises 127 genera and over 6000 species. The leaf anatomy of Myrtaceae has been widely studied and can be used in phylogenetic analyses and species delimitations in complexes within the family. A multiple epidermis and hypodermis have been described for the leaves of representatives of the family but have not been confirmed using ontogenetic studies, which could lead to incorrect interpretations about these tissues. Thus, we studied the leaf ontogenesis of the following 9 species in subfamily Myrtoideae: tribe Syzigieae - Syzigium cumini ; tribe Myrteae - Myrceugenia alpigena and M. euosma (subtribe Luminae), Psidium sartorianum, P. guajava, Campomanesia adamantium and Pimenta pseudocaryophyllus (subtribe Pimentinae), Myrcianthes pungens and M. gigantea (subtribe Eugeniinae). Shoot apices and leaves up to the fourth node were transversally and longitudinally sectioned following techniques used for plant anatomy. Leaf development in all species is from apical, intercalary, dorsal, marginal and plate meristematic activity. Protodermal periclinal divisions only occur to give rise to secretory cavities. The results show that in all the species the subepidermal layer is a hypodermis, which originates from periclinal divisions of the ground meristem. The occurrence of this layer could have phylogenetic implications according to reports in the literature, reinforcing the relationships among the subtribes in current topologies of Myrteae. However, the occurrence of a hypodermis in representatives of other subtribes of Myrteae and Syzigieae need to be better studied and discussed in relation to the phylogeny of Myrtaceae. [ABSTRACT FROM AUTHOR]

α-terthienyl is an allelochemical derived from the roots of marigold (Tagetes spp.), which is used to suppress plant parasitic nematodes. We investigated the nematicidal activity of α-terthienyl against the model organism Caenorhabditis elegans and the root-knot nematode, Meloidogyne incognita. As reported previously, α-terthienyl action was much higher after photoactivation, but was still effective against C. elegans dauer larvae and M. incognita second stage juveniles, even without photoactivation. Expression induction of two major enzymes, glutathione S-transferase (GST) and superoxide dismutase (SOD), was restricted in C. elegans hypodermis following treatment with α-terthienyl. The susceptibility of nematodes to α-terthienyl changed when the expression of GST and SOD was induced or suppressed. From these results, under dark conditions (without photoactivation), α-terthienyl is an oxidative stress-inducing chemical that effectively penetrates the nematode hypodermis and exerts nematicidal activity, suggesting high potential for its use as a practicable nematode control agent in agriculture.

Photoaging is an important extrinsic aging factor leading to altered skin morphology and reduced function. Prior work has revealed a connection between photoaging and loss of subcutaneous fat. Currently, primary models for studying this are in vivo (human samples or animal models) or in vitro models, including human skin equivalents (HSEs). In vivo models are limited by accessibility and cost, while HSEs typically do not include a subcutaneous adipose component. To address this, we developed an "adipose-vascular" HSE (AVHSE) culture method, which includes both hypodermal adipose and vascular cells. Furthermore, we tested AVHSE as a potential model for hypodermal adipose aging via exposure to 0.45 ± 0.15 mW/cm 2 385 nm light (UVA). One week of 2 h daily UVA exposure had limited impact on epidermal and vascular components of the AVHSE, but significantly reduced adiposity by approximately 50%. Overall, we have developed a novel method for generating HSE that include vascular and adipose components and demonstrated potential as an aging model using photoaging as an example.

Traveling-wave optical coherence elastography (OCE) is a promising technique to measure the stiffness of biological tissues. While OCE has been applied to relatively homogeneous samples, tissues with significantly varying elasticity through depth pose a challenge, requiring depth-resolved measurement with sufficient resolution and accuracy. Here, we develop a broadband Rayleigh-wave OCE technique capable of measuring the elastic moduli of the 3 major skin layers (epidermis, dermis, and hypodermis) reliably by analyzing the dispersion of leaky Rayleigh surface waves over a wide frequency range of 0.1-10 kHz. We show that a previously unexplored, high frequency range of 4-10 kHz is critical to resolve the thin epidermis, while a low frequency range of 0.2-1 kHz is adequate to probe the dermis and deeper hypodermis. We develop a dual bilayer-based inverse model to determine the elastic moduli in all 3 layers and verify its high accuracy with finite element analysis and skin-mimicking phantoms. Finally, the technique is applied to measure the forearm skin of healthy volunteers. The Young's modulus of the epidermis (including the stratum corneum) is measured to be ∼ 4 MPa at 4-10 kHz, whereas Young's moduli of the dermis and hypodermis are about 40 and 15 kPa, respectively, at 0.2-1 kHz. Besides dermatologic applications, this method may be useful for the mechanical analysis of various other layered tissues with sub-mm depth resolution. STATEMENT OF SIGNIFICANCE: To our knowledge, this is the first study that resolves the stiffness of the thin epidermis from the dermis and hypodermis, made possible by using high-frequency (4 - 10 kHz) elastic waves and optical coherence elastography. Beyond the skin, this technique may be useful for mechanical characterizations of various layered biomaterials and tissues., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier Ltd.)