Skin lesions are normal to all species, regardless of gender or age. The skin, the largest organ of the body, has function as a primary barrier to the chemical, physical and biological aggressions of the environment. In animals, these lesions may be due to fights and/or predations, also as in humans, there is a very common cause of dermal lesions that are caused by burns and carcinomas. Looking for new techniques of tissue bioengineering, studies have been shown promising results for formulations of acellular biological scaffolds from tissue decellularization for the reconstitution of these lesions. The decellularization has its proof by a varied range of tests such as scanning electron microscopy and residual genomic DNA tests. Subsequently the tissue can go through the process of recellularization using cells of interest, even the animal that will receive this tissue, reducing the risks of rejection and improving the response to tissue transplantation. Thus, this manuscript aimed at the decellularization of the tissue with the use of chemical and physical means followed by sterilization and the establishment of a protocol for the recellularization of a decellularized scaffold from the Wistar rat dermis using murine fibroblasts and mesenchymal stem cells from canine adipose tissue for 7 days. After efficacy tests, the tissue recellularization were confirmed by immunofluorescence assays and scanning electron microscopy where the adherence of the cells in the biological scaffold was observed.

Several studies have reported on certain aspects of the characteristics of different sites within a keloid lesion, but detailed studies on the keloid dermis at different depths within a keloid lesion are scarce. The aim of this study was to investigate the histology of the keloid dermis at different depths. This study included 19 keloid tissue samples that were collected from 19 patients and 19 normal skin samples, which were harvested from subjects without keloids or hypertrophic scar. Samples were studied by light microscopy using routine hematoxylin and eosin histochemical staining, and immunohistochemistry to detect CD20-positive B-lymphocytes and CD3-positive T-lymphocytes. Sirius Red histochemical staining was used to determine the type of collagen in keloid tissue and normal skin samples. The migratory properties of fibroblasts within the keloid dermis at different depths was compared, using an in vitro migration assay. The findings of this study showed that although the papillary and reticular dermis could be clearly distinguished in normal skin, three tissue layers were identified in the keloid dermis. The superficial dermis of keloid was characterized by active fibroblasts and lymphocytes; the middle dermis contained dense extracellular matrix (ECM) with large numbers fibroblasts, and the deep dermis was poorly cellular and characterized by hyalinized collagen bundles. In the keloid samples, from the superficial to the deep dermis, type I collagen increased and type III collagen decreased, and fibroblasts from the superficial dermis of the keloid were found to migrate more rapidly. In conclusion, the findings of this study showed that different depths within the keloid dermis displayed different biological features. The superficial dermis may initiate keloid formation, in which layer intralesional injection of pharmaceuticals and other treatments should be performed for keloid.

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis (TED). To solve these problems, we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet (PBMCS) and pre-vascularized fibroblasts cell sheet (PFCS) by cell sheet technology, and then superimposed or folded them together to construct a pre-vascularized TED (PTED), aiming to mimic the real dermis structure. The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1, 7 and 14 via the methods of histochemistry and immunohistochemistry. The results showed that PTED could rapidly promote the wound closure, especially at Day 14, and the wound-healing rate of three-layer PTED could reach 97.2% (P < 0.01), which was faster than the blank control group (89.1%), PBMCS (92.4%), PFCS (93.8%) and six-layer PTED (92.3%). In addition, the vessel density in the PTED group was higher than the other groups on the 14th day. Taken together, it is proved that the PTED, especially three-layer PTED, is more conducive to the full-thickness dermis-defect repair and the construction of the three-dimensional vascular networks, indicating its potential application in dermis-defect repair.

Water absorption by decellularized dermis was investigated and compared with biopolymer and synthetic polymer hydrogels (glutaraldehyde-crosslinked gelatin and crosslinked poly(acrylamide) hydrogel, respectively). Porcine dermis was decellularized in an aqueous sodium dodecyl sulfate (SDS) solution. Histological evaluation revealed that the SDS-treated dermis has much larger gaps between collagen fibrils than non-treated dermis, and that water absorption depends on these gaps. Decellularized dermis has low water absorptivity and the absorption obeys Fick's second law. During absorption, the water diffusion rate decreases with time and occurs in two steps. The first is rapid absorption into the large gaps, followed by slow absorption by the collagen fiber layer. Because of the gaps, decellularized dermis can absorb more water than native dermis and shows different water absorption behavior to glutaraldehyde-crosslinked gelatin and crosslinked poly(acrylamide) hydrogels.

Reciprocal epithelio-mesenchymal interactions between the prospective epidermis and the underlying dermis are the major driving forces in the development of skin appendages. Feather development is initiated by a still unknown signal from the dermis in feather-forming skin. The morphological response of the ectoderm to this signal is the formation of an epidermal placode, which signals back to the mesenchyme to induce dermal condensations. Together, epidermal and dermal components constitute the outgrowing feather bud. The bHLH transcription factor cDermo-1 is expressed in developing dermis and is the earliest known marker of prospective feather tracts. To test its function during feather development, we forced cDermo-1 expression in embryonic chicken dermis using a retroviral expression vector. In featherless (apteric) regions, cDermo-1 misexpression induced dense, thickened dermis normally observed in feathered skin (pterylae), and leads to the development of regularly spaced and normally shaped ectopic feather buds. In pterylae, cDermo-1 misexpression enhanced feather growth. In hindlimb skin, according to the local skin identity, misexpression of cDermo-1 induced ectopic scale formation. Thus, we show that forced cDermo-1 expression in developing dermis is sufficient to launch the developmental program leading to skin appendage formation. We propose a role of cDermo-1 at the initial stages of feather induction upstream of FGF10.

Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow-derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow-derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow-derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271(+) and SSEA-4(+) cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271(+) population. The differentiation potential of dermal SSEA-4(+) cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.

The adult human skin harbors a variety of leukocytes providing immune surveillance and host defense, but knowledge about their ontogeny is scarce. In this study we investigated the number and phenotype of leukocytes in prenatal human skin (dermal dendritic cells (DDCs), macrophages, T cells (including FoxP3(+) regulatory T cells), and mast cells) to unravel their derivation and to get a clue as to their putative function in utero. By flow cytometry and immunofluorescence, we found a distinction between CD206(+)CD1c(+)CD11c(+) DDCs and CD206(+)CD209(+)CD1c(-) skin macrophages by 9 weeks estimated gestational age (EGA). T cells appear at the end of the first trimester, expressing CD3 intracytoplasmatically. During midgestation, CD3(+)FoxP3(-) and CD3(+)FoxP3(+) cells can exclusively be found in the dermis. Similarly, other leukocytes such as CD117(+) (c-kit) mast cells were not identified before 12-14 weeks EGA and only slowly acquire a mature phenotype during gestation. Our data show at which time point during gestation antigen-presenting cells, T cells, and mast cells populate the human dermis and provide a step forward to a better understanding of the development of the human skin immune system.

Dermis spheroids from different donors (40 and 50 years old) were developed from primary fibroblasts to demonstrate their capacity to synthetize and organize the main dermal structural components when cultured in 3D microenvironment, forming endogenous de novo ECM according to their potential metabolic activity.

Finding appropriate seed cells for bone tissue engineering remains a significant challenge. Considering that skin is the largest organ, we hypothesized that human bone morphogenetic protein receptor type IB (BmprIB)+ dermal cells could have enhanced osteogenic capacity in the healing of critical-sized calvarial defects in an immunodeficient mouse model. In this study, immunohistochemical staining revealed that BmprIB was expressed throughout reticular dermal cells; the positive expression rate of BmprIB was 3.5% ± 0.4% in freshly separated dermal cells, by flow cytometry. Furthermore, in vitro osteogenic capacity of BmprIB+ cells was confirmed by osteogenic-related staining and marker gene expression compared with unsorted dermal cells. In vivo osteogenic capacity was demonstrated by implantation of human BmprIB+ cell/coral constructs in the treatment of 4-mm diameter calvarial defects in an immunodeficient mouse model compared with implantation of unsorted cell/coral constructs and coral scaffold alone. These results indicate that the selective cell population BmprIB from human dermis is a promising osteogenic progenitor cell that can be a large-quantity and high-quality cell source for bone tissue engineering and regeneration. Stem Cells Translational Medicine 2017;6:306-315.

Soft-tissue reconstruction for a variety of surgical conditions, such as abdominal wall hernia or pelvic organ prolapse, remains a challenge. There are numerous meshes available that may be simply categorized as either synthetic or biologic. Within biologic meshes, porcine dermal meshes have come to dominate the market. This review examines the current evidence for their use and the limitations of knowledge. Although there is increasing evidence to support their safety, long-term follow-up studies that support their efficacy are lacking. Numerous clinical trials that remain ongoing may help elucidate their precise role in soft-tissue reconstruction.

Skin aging is a physiological issue that is still relatively poorly understood. Studies have demonstrated that the dermal extracellular matrix (ECM) plays important roles in skin aging. However, the roles of the changes in ECM characteristics and the molecules that are secreted to the extracellular space and are involved in the formation of the dermal matrix from birth to old age remain unclear. To explore the way in which the ECM microenvironment supports the functions of skin development across different age groups is also poorly understood, we used a decellularization method and matrisome analysis to compare the composition, expression, and function of the dermal ECM in toddler, teenager, adult, and elderly skin. We found that the collagens, glycoproteins, proteoglycans, and regulatory factors that support skin development and interact with these core ECM proteins were differentially expressed at different ages. ECM expression markers occurring during the process of skin development were identified. In addition, our results elucidated the characteristics of ECM synthesis, response to skin development, and the features of the ECM that support epidermal stem cell growth via the basement membrane during skin aging.

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this finding could be clinically relevant. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of dogs without cutaneous disease using two different approaches: traditional cultures and DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS).

Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

The structure of a biological scaffold is a major determinant of its biological characteristics and its interaction with cells. An acellular dermis tissue transplant must undergo a series of processing steps, to remove cells and genetic material and provide the sterility required for surgical use. During manufacturing and sterilization the structure and composition of tissue transplants may change. The composition of the human cell-free dermis transplant Epiflex® was investigated with specific attention paid to its structure, matrix composition, cellular content and biomechanics. We demonstrated that after processing, the structure of Epiflex remains almost unchanged with an intact collagen network and extracellular matrix (ECM) protein composition providing natural cell interactions. Although the ready to use transplant does contain some cellular and DNA debris, the processing procedure results in a total destruction of cells and active DNA which is a requirement for an immunologically inert and biologically safe substrate. Its biomechanical parameters do not change significantly during the processing.

The aim of this study was to adapt a proprietary decellularisation process for human dermis for use with porcine skin. Porcine skin was subject to: sodium chloride (1 M) to detach the epidermis, trypsin paste to remove hair follicles, peracetic acid (0.1% v/v) disinfection, washed in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulphate in the presence of proteinase inhibitors followed by nuclease treatment. Cellular porcine skin, decellularised porcine and human dermis were compared using histology, immunohistochemistry, GSL-1 lectin (alpha-gal epitope) staining, biochemical assays, uniaxial tensile and in vitro cytotoxicity tests. There was no microscopic evidence of cells in decellularised porcine dermis. DNA content was reduced by 98.2% compared to cellular porcine skin. There were no significant differences in the biomechanical parameters studied or evidence of cytotoxicity. The decellularised porcine dermis retained residual alpha-gal epitope. Basement membrane collagen IV immunostaining was lost following decellularisation; however, laminin staining was retained.

Given the potential usefulness of Acellular Dermal Matrices (ADM) for wound healing, we aimed to evaluate the stability, histological characteristics, and effectiveness of ADM compared with cryopreserved dermis (CPD) in rat models.

Resident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR(+) precursors of human foreskin can be ascribed to the Schwann (CD56(+)) and perivascular (CD56(-)) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR(+)CD56(+) Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread.

Sustained epidermal Wnt/β-catenin signalling expands the stem cell compartment and induces ectopic hair follicles (EFs). This is accompanied by extensive fibroblast proliferation and extracellular matrix (ECM) remodelling in the underlying dermis. Here we show that epidermal Hedgehog (Hh) and Transforming growth factor-beta (TGF-β) signalling mediate the dermal changes. Pharmacological inhibition or genetic deletion of these pathways prevents β-catenin-induced dermal reprogramming and EF formation. Epidermal Shh stimulates proliferation of the papillary fibroblast lineage, whereas TGF-β2 controls proliferation, differentiation and ECM production by reticular fibroblasts. Hh inhibitors do not affect TGF-β target gene expression in reticular fibroblasts, and TGF-β inhibition does not prevent Hh target gene induction in papillary fibroblasts. However, when Hh signalling is inhibited the reticular dermis does not respond to epidermal β-catenin activation. We conclude that the dermal response to epidermal Wnt/β-catenin signalling depends on distinct fibroblast lineages responding to different paracrine signals.