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Effective bleeding control and wound healing are very important and can be life saving. However, traditional wound dressings with structural deficiencies are not effective in controlling bleeding and promoting the regeneration of functional tissues. In this study, a three-dimensional (3D) layered nanofiber sponge was obtained by expanding two-dimensional (2D) nanofiber membranes into the third dimension. This sponge has a layered nanofiber structure, which increases the interfacial interaction between the sponge and blood cells to accelerate hemostasis. Through fine-tuning of structure, the 3D nanofiber sponge acquires properties beneficial to wound healing such as good elasticity and high permeability and fluid absorption ratio. The 3D nanofiber sponges are both highly compressible and resilient, providing tamponade for deep wounds and creating a good 3D dynamic microenvironment to regulate cellular behavior. Further research has demonstrated that the layered nanofiber structure could promote the regeneration of functional dermis and the restoration of differentiated adipocytes during the early repair phase. Experiments using model mice with full-thickness skin defects have shown that the layered nanofiber structure could effectively accelerate wound healing and reduce scar formation. This layered 3D nanofiber sponge design is easy to produce. Due to its excellent wound healing property, this porous nanofiber sponge has great potential for future clinical application as wound dressings., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The elastic behaviors of stratum corneum, viable epidermis, dermis, and whole skin were investigated by nano/microindentation techniques. Insignificant differences in reduced elastic modulus of skin samples obtained from three different porcine breeds revealed breed type independent measurements. The reduced elastic modulus of stratum corneum is shown to be about three orders of magnitude higher than that of dermis. As a result, for relatively shallow and deep indentations, skin elasticity is controlled by that of stratum corneum and dermis, respectively. Skin deformation is interpreted in the context of a layered structure model consisting of a stiff and hard surface layer on a compliant and soft substrate, supported by microscopy observations and indentation measurements., (© 2013 Published by Elsevier Ltd.)

Time-dependent deformation of porcine skin was studied in vitro using specialized microprobe instruments. The deformation behavior of stratum corneum, dermis, and whole skin is examined in the context of results of creep strain, elastic stiffness, and viscoelastic constants obtained in terms of the hold time, loading/unloading rate, and maximum indentation depth (load). Skin time-dependent deformation is significantly influenced by dermis viscoelasticity up to a critical indentation depth (load) beyond which it is controlled by the outermost hard epidermis, particularly stratum corneum. Skin viscoelastic behavior under constant load (creep) and constant displacement (stress relaxation) is interpreted in the light of phenomenological observations and experimental trends., (© 2013 Published by Elsevier Ltd.)

We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young's moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Repair of deep burn by use of the dermal equivalent relies strongly on the angiogenesis and thereby the regeneration of dermis. To enhance the dermal regeneration, in this study plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165)/N,N,N-trimethyl chitosan chloride (TMC) complexes were loaded into a bilayer porous collagen-chitosan/silicone membrane dermal equivalents (BDEs), which were applied for treatment of full-thickness burn wounds. The DNA released from the collagen-chitosan scaffold could remain its supercoiled structure but its degree was decayed along with the prolongation of incubation time. The released DNA could transfect HEK293 cells in vitro with decayed efficiency too. Human umbilical vein endothelial cells (HUVECs) in vitro cultured in the scaffold loaded with TMC/pDNA-VEGF complexes expressed a significantly higher level of VEGF and showed higher viability than those cultured in the controls, i.e. blank scaffold, and scaffolds loaded with naked pDNA-VEGF and TMC/pDNA-eGFP, respectively. The four different BDEs were then transplanted in porcine full-thickness burn wounds. Results showed that the TMC/pDNA-VEGF group had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of the dermis. RT-qPCR and western blotting found that the experimental group also had the highest expression of VEGF, CD31 and α-SMA in both mRNA and protein levels. Furthermore, ultra-thin skin grafting was performed on the regenerated dermis 14 days later, leading to complete repair of the burn wounds with normal histology. Moreover, the tensile strength of the repaired tissue increased along with the time prolongation of post grafting, resulting in a value of approximately 70% of the normal skin at 105 days., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Engineered skin must be mechanically strong to facilitate surgical application and prevent damage during the early stages of engraftment. However, the evolution of structural properties during culture, the relative contributions of the epidermis and dermis, and any correlation with tissue morphogenesis are not well known. These aspects are investigated by assessing the mechanical properties of engineered skin (ES) and engineered dermis (ED) during a 21-day culture period, including correlations with cellular metabolism, cellular organization and epidermal differentiation. During culture, the epidermis differentiates and begins to cornify, as evidenced by immunostaining and surface electrical capacitance. Tensile testing reveals that the ultimate tensile strength and linear stiffness increase linearly with time for ES, but are relatively unchanged for ED. ES strength correlates significantly with epidermal differentiation (p < 0.001) and a composite strength model indicates that strength is largely determined by the epidermis. These data suggest that strategies to improve ES biomechanics should target the dermis. Additionally, time-dependant changes in average ES strength and percent elongation can be used to set upper bound limits on mechanical stimulation profiles to avoid tissue damage., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study was based on two dimensional (2D) high frequency elastography to describe quantitatively the mechanical behavior of the human dermis in vivo. The study was conducted on the forearm skin and elastographic tests were performed using a combination of two devices: an extensiometer developed for the in vivo study of the mechanical behavior of the skin using uniaxial stretching stress, and a 20MHz real time sonographer (Dermcup 2020™) for ultrasound skin imaging. The staggered strain estimation algorithm (SSE) was used to produce elastograms. A temporal cumulative technique was applied to improve elastogram quality and to monitor variations in skin strain during stretching. The influence of the natural skin tension controlled by arm bending was studied and distinctive mechanical behavior was observed for low and high mechanical stress levels. In a preliminary analysis, the reproducibility of measurements was assessed by means of coefficient of variation (CV) in 5 selected healthy volunteers.Finally, two hypotheses linked to the geometrical and structural properties of the dermis are proposed to account for the new findings described in this study., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Recently, a new generation of dermal equivalents (DE) was presented which are solely generated on a human fibroblast-derived dermal matrix. Since human mesenchymal stem cells from bone marrow (BM-MSC) and Wharton's Jelly of the umbilical cord (UC-MSC) are characterised by a distinct biosynthetic and paracrine activity, they are an appealing alternative approach for generating cell-based DE. This study compares the epithelial-mesenchymal interaction and extracellular matrix (ECM) remodelling of cell-based and collagen-based DE using fibroblasts, BM-MSC or UC-MSC, respectively, in co-culture with the keratinocyte cell line HaCaT. While fibroblast-based DE exhibit normal matrix synthesis, proliferation and differentiation of keratinocytes, mesenchymal stem cell-based DE resulted in excessive production of inhomogenous matrix aggregates, loss of polarisation of the epidermal cell layer and an inconstant paracrine activity. In contrast, collagen-embedded MSC revealed a homogenous growth pattern as well as a constant expression of growth factors and ECM proteins without a negative influence on the epidermal layer as shown by histology, electron microscopy, immunohistochemistry and realtime-RT-PCR. These results indicate the necessity of an instructive biomaterial-based scaffold to direct stem cell differentiation, proliferation, paracrine activity as well as regulation of ECM deposition., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Angiogenesis of dermal equivalent is one of the key issues for treatment of full thickness skin defects. To develop a gene-activated bilayer dermal equivalent (BDE), N,N,N-trimethyl chitosan chloride (TMC), a cationic gene delivery vector, was used to form complexes with the plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165), which was then incorporated into a collagen-chitosan/silicone membrane scaffold. To evaluate the angiogenesis property in vivo, full thickness skin defects were made on the back of pigs, into which the TMC/pDNA-VEGF complexes loaded BDE and other three control BDEs, i.e. the blank BDE, and the BDEs loaded with pDNA-VEGF and TMC/pDNA-eGFP complexes, respectively, were transplanted. Biopsy specimens were harvested at day 7, 10 and 14 after surgery for histology, immunohistochemistry, immunofluorescence, real-time quantitative PCR (RT-qPCR) and western blotting analyses. The results showed that the TMC/pDNA-VEGF group had the strongest VEGF expression in mRNA and protein levels, resulting in the highest densities of newly-formed and mature vessels. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the TMC/pDNA-VEGF complexes loaded BDE at day 10 and well survived. At 112 days grafting, the healing skin had a similar structure and approximately 80% tensile strength of the normal skin.

The aim of this study was to develop a method for efficient production of folliculoid keratinocyte-dermal papilla (DP) microtissues to facilitate epithelial-mesenchymal interaction. The behavior of DP cells and adult keratinocytes from hairless skin on poly(ethylene-co-vinyl alcohol) (EVAL) surface was investigated. Keratinocytes, poorly adherent both to substrate and between homotypic cells, become suspended disperse cells after homotypic cell seeding. Seeded simultaneously, keratinocytes and DP cells are able to aggregate into spheroidal microtissues. Dynamical analysis shows that DP cells act as a carrier in the process due to the heterotypic intercellular adhesion. DP cells attach faster to EVAL and start to aggregate. Keratinocytes adhere to DP cells and are then carried by DP cells to form initial hybrid aggregates. Due to the high motility of DP cells, these hybrid aggregates move collectively as clusters and merge into larger spheroids which subsequently detach from the substratum and can be easily collected. Compared with random cell distribution in spheroids generated in hanging drops, these hybrid spheroids have a preferential compartmented core-shell structure: an aggregated DP cell core surrounded by a keratinocyte shell. In addition to ameliorated DP signature gene expression, keratinocytes show down-regulated epidermal terminal differentiation and enhanced follicular differentiation. Functionally, these microtissues are able to grow hairs in vivo. This work sheds light on the complex effects and dynamics of cell-cell and cell-substratum interaction in the patterning of heterotypic cells into tissue forms and is of potential to be applied to mass generation of other epithelial organ primordia in vitro., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

In the past few years, the plasticity of adult cells in several post-natal tissues has attracted special attention in regenerative medicine. Skin is the largest organ in the body. Adult skin consists of epidermis, dermis and appendages such as hair and glands that are linked to the epidermis but project deep into the dermal layer. Stem cell biology of skin has been a focus of increasing interest in current life science. Committed stem cells with a limited differentiation potential for regeneration and repair of epidermis have been known for decades. Recent studies further found that adult skin tissues contain cell populations with pluripotent characteristics. Multipotent stem cells from skin with and without hair follicles, both in epidermal and dermal tissues, can differentiate and generate multiple cell lineages. Especially, the hematopoietic system in epidermal and dermal tissue, like skin, may be a local, acceptable reservoir of various adult stem cell populations. Given their easy accessibility, such stem cells can provide an experimental model not only for skin biology but also for studying the epithelial-mesenchymal cell interactions of organs other than the skin. This review presents an overview of recent advances in research into skin repair and regeneration involving stem cells from epidermis, dermis, and bone marrow. In particular, we focus on the possible use of blood stem cells as an alternative resource for research advances in skin biology.

Dermis is a heterogeneous tissue in which extracellular matrix components change in relative amount and spatial assembly across the tissue thickness. The effect of the microstructural and compositional heterogeneities on the overall mechanical response of dermis is, however, largely ignored. In this work, we aimed at gaining a better insight into the effect of extracellular matrix microstructure and composition on the mechanical behaviour of different dermal strata by combining mechanical analysis and selective enzymatic digestion of extracellular matrix components. The dynamical-mechanical tests we performed on bovine dermal splits show that the upper dermal stratum, which is richer in papillary dermis, is characterized by higher mechanical properties than the lower one, which is almost composed of reticular dermis. Moreover, the depletion of interfibrillar proteins, proteoglycans and glycosamminoglycans decreases the dynamic moduli of dermis, especially at small frequencies. Of the two dermal layers tested, the upper dermal layer is more sensitive to the enzymatic treatment than the lower layer. Interestingly, the disruption of the elastic network greatly influenced the viscoelastic properties of upper dermis, inducing a dramatic decrease of both storage (G') and loss (G'') moduli, suggesting that the spatial assembly of the elastin and collagen networks as well as their mutual interactions dominate the dynamical mechanical response of the tissue.

A series of in situ gelable hydrogels were prepared from oxidized dextran (Odex) and N-carboxyethyl chitosan (CEC) without any extraneous crosslinking agent. The gelation readily took place at physiological pH and body temperature. The gelation process was monitored rheologically, and the effect of the oxidation degree of dextran on the gelation process was investigated. The higher the oxidation degree of Odex, the faster the gelation. A highly porous hydrogel structure was revealed under scanning electron microscopy (SEM). Swelling and degradation of the Odex/CEC hydrogels in PBS showed that both swelling and degradation were related to the crosslinking density of the hydrogels. In particular, the hydrogels underwent fast mass loss in the first 2 weeks, followed by a more moderate degradation. The results of long-term cell viability tests revealed that the hydrogels were non-cytotoxic. Mouse fibroblasts were encapsulated in the hydrogels and cell viability was at least 95% within 3 days following encapsulation. Furthermore, cells entrapped inside the hydrogel assumed round shape initially but they gradually adapted to the new environment and spread-out to assume more spiny shapes. Additionally, the results from applying the Odex/CEC system to mice full-thickness transcutaneous wound models suggested that it was capable of enhancing wound healing.

Self-aggregation is key to hair follicle (HF) induction ability of dermal papilla (DP) cells and neogenesis of HF can be achieved by transplanting DP microtissues. However, there is currently lack of a suitable system that allows efficient production of DP microtissues and analysis of DP self-aggregation in vitro. We demonstrate that, at a higher seeding cell density, poly(ethylene-co-vinyl alcohol) (EVAL) membranes facilitate DP self-assembly into many compact spheroidal microtissues that are able to induce new HFs. This self-assembling process is associated with an enhanced cell movement and a declined cell-substrate adhesivity on EVAL. A compromised cell growth is also revealed on EVAL. On the contrary, a more adherent surface allows faster cell expansion but maintains DP cells in a flat morphology. Dynamically, cell migration, intercellular collision and intercellular adhesion contribute to DP microtissue formation on EVAL. Our results suggest that, for large-scale production of DP microtissues for HF regeneration, an adhesive surface is needed for quick cell expansion and a biomaterial with a lower adhesivity is required for self-aggregation. In addition, this system can be a model for investigation of DP self-aggregation in vitro.

Investigations to determine pH profiles across human stratum corneum (SC), in vivo as well as in vitro, were carried out using the tape stripping technique and a flat surface pH electrode. This method was extended to the deeper skin layers (=viable epidermis+dermis; DSL) in vitro. Statistically significant changes in the pH values were detected in the SC between in vivo and in vitro investigations and also between male and female skin in vivo. For the DSL, no gender-dependent differences in pH were observed. While the results achieved for the SC are in accordance with data already published in the literature, the values for the DSL were surprising: An alkaline pH, with a steep increase of about two pH units in the first 100 microm of the DSL and a plateau of this level was thereafter detected. Research was also done to examine the influence of different in vitro test systems on the results of pH measurements across the skin. A permeation model (Franz diffusion cell; FD-C) and a penetration model (Saarbruecken penetration model; SB-M) were compared. Experiments were carried out concerning the incubation time as well as the pH of the acceptor solution in the FD-C. Independent of the test system used, no change in the pH profiles could be observed for the SC, but a strong effect of the acceptor medium and its pH on the pH profiles across the DSL could be demonstrated using the FD-C, which showed itself partly after 30 min in statistically significant differences between incubated and formerly frozen skin. The results after the use of buffer solutions with different pH values, the pH across the DSL seemed to come into line with the one of the buffer solution, which was investigated for acidic as well as alkaline pH values. The results obtained with the flat surface pH electrode were confirmed using two different dyes: the pH-dependent fluorescent dye carboxy-SNARF-1 and the pH indicator bromthymolblue.

The developmental transition from a residential, immature 'yellow' eel to a migratory, maturing adult 'silver' eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed 'chisel-shaped' snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.