A human skin equivalent model that mimics the photoproduction of vitamin D3 in human skin

Summary A human skin equivalent was prepared by culturing human keratinocytes on the surface of nylon filtration meshes containing human skin fibroblasts and by growing the epidermal cells at the air-liquid interface. This human skin equivalent model was used to mimic the photoproduction of vitamin D₃ in human skin. It was found that the concentration of 7-dehydrocholesterol and its photoconversion to previtamin D₃ and its subsequent thermal isomerization to vitamin D₃ in the human skin equivalent was essentially identical to that of human skin. The 7-dehydrocholesterol content in the skin equivalent and human skin was 2187±296 and 2352±320 ng/cm², respectively. The percentage of the major photoproducts of 7-dehydrocholesterol in the skin equivalent following ultraviolet B radiation (0.5 J/cm²) was 35% previtamin D₃, 29% lumisterol, and 6% tachysterol; 30% remained as 7-dehydrocholesterol. Similarly, in human skin they were 36%, 29%, 7%, and 28%, respectively. After incubation at 37°C for 30 min, 11% and 12% of the previtamin D₃ had thermally isomerized to vitamin D₃ in the skin equivalent and human skin. In conclusion, compared with cultured keratinocytes or fibroblasts, the human skin equivalent model provides a superior in vitro system that better mimics the physiology and biochemistry of the photosynthesis of vitamin D₃ in human skin.     

Characterization of lymphangiogenesis in a model of adult skin regeneration

To date, adult lymphangiogenesis is not well understood. In this study we describe the evolution of lymphatic capillaries in regenerating skin and correlate lymphatic migration and organization with the expression of matrix metalloproteinases (MMPs), immune cells, the growth factors VEGF-A and VEGF-C, and the heparan sulfate proteogylcan perlecan, a key component of basement membrane. We show that while lymphatic endothelial cells (LECs) migrate and organize unidirectionally, in the direction of interstitial fluid flow, they do not sprout into the region but rather migrate as single cells that later join together into vessels. Furthermore, in a modified "shunted flow" version of the model, infiltrated LECs fail to organize into functional vessels, indicating that interstitial fluid flow is necessary for lymphatic organization. Perlecan expression on new lymphatic vessels was only observed after vessel organization was complete and also appeared first in the distal region, consistent with the directionality of lymphatic migration and organization. VEGF-C expression peaked at the initiation of lymphangiogenesis but was reduced to lower levels throughout organization and maturation. In mice lacking MMP-9, lymphatics regenerated normally, suggesting that MMP-9 is not required for lymphangiogenesis, at least in mouse skin. This study thus characterizes the process of adult lymphangiogenesis and differentiates it from sprouting blood angiogenesis, verifies its dependence on interstitial fluid flow for vessel organization, and correlates its temporal evolution with those of relevant environmental factors.     

Characterization of a new tissue-engineered human skin equivalent with hair

Summary We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.     

Microbial colonization of an in vitro model of a tissue engineered human skin equivalent – a novel approach

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to ' in vivo ' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis , Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10²–10⁶ CFU SE⁻¹ on the surface of replicate SEs. Growth of all species was supported for upto 72–120 h, with recovery densities of between 10⁴–10⁹ CFU SE⁻¹. A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10² to 10⁸ CFU SE⁻¹, as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.     

Characterization of label-retaining cells in the epidermis of a human skin equivalent

Abstract. The human skin equivalent (HSE) is an in vitro reconstructed model that resembles skin morphologically and biochemically. The HSE is formed by overlaying a fibroblast-populated collagen matrix with a suspension of epidermal cells. Basal keratinocytes attach to the dermal equivalent via a newly formed basement membrane and multiply to form a stratified, differentiated epidermis. The aim of the studies described here was to characterize the basal cells of the HSE in terms of their cell cycling potential. The experiments utilized long-term labelling of the cells with tritiated thymidine ([³H]dT), followed by irradiation with ultraviolet light. [³H]dT incorporation was analysed via routine autoradiography. Irradiation with 100 J/m² UV light increased the number of labelled basal cells by 58% over the control, the maximal stimulation observed. Decreased numbers of labelled basal cells were observed at doses of UV light greater than 100 J/m². The maximal number of labelled basal cells was observed on day 14 and decreased over time; the number of labelled suprabasal cells increased concomitantly. Label-retaining cells (12%) persisted in the stratum basale of control HSEs after 32 days in culture. Labelled cells were observed in the apical layers of the stratum granulosum of control HSEs after 22 days in culture. These data suggest that the stratum basale of the HSE contains a population of slow-cycling cells whose characteristics resemble a subpopulation of slowly cycling cells found in normal human skin.     

Nerve regeneration after correction of its defect with a vascularized autologous nerve transplant

In chronic experiment performed on 6 dogs, by means of some neurohistological techniques peculiarities of regeneration of nerves have been studied, when their defects are substituted for vascularized and common autoneurotransplants. When the vascularized autoneurotransplants are applied, more favourable conditions are made for the nerve regeneration, than at a free plastisity and amount of regenerated fibers distal to the graft reaches 34-88%, more often making 48-52% of the initial number of fibers in the proximal parts of the nerves in 3 months after the operation. When in the same animals in the contralateral side common autoneurotransplants have been applied, during the same time the amount of fibers regenerated into the peripheral parts of the nerves makes 10-26%, more often 17-20% from the initial level, or in 2-2.5 times less than at substitution of the defects for the vascularized grafts. Absence of an active cellular reaction of the surrounding tissues to the sutural material supramid is used.     

Characterization of the living skin equivalent as a model of cutaneous re-epithelialization

The living skin equivalent, a three-dimensional organotypic model, has been widely used to investigate many aspects of cutaneous biology. However, there are relatively few studies assessing how faithfully the skin equivalent reproduces normal skin biology. The skin equivalent was fabricated by seeding human epidermal keratinocytes onto the upper surface of a hydrated collagen lattice populated with human dermal fibroblasts and subsequently raised to the air-liquid interface where keratinocyte stratification and differentiation led to the formation of a tissue which showed many common morphological features to that of normal skin. Histology and immunohistochemical detection of keratinocyte integrins and matrix metalloproteinases (MMPs) were used as cytological markers to assess the accuracy of the model during cutaneous re-epithelialization. Analysis of expression of keratinocyte integrins revealed that whilst there were a number of similarities to normal skin, skin equivalent keratinocytes appeared to be 'activated' and hyper-proliferating. Wounding of the skin equivalent, by complete bisection, induced re-epithelialization from both wound edges within 8-12 h, which completely restored the epidermis within 4 days. This migration, like that in vivo, was associated with nascent expression of MMPs and upregulation of certain integrins. However, whilst integrin expression, was similar to in vivo re-epithelialization, there were subtle differences in the level of expression and distribution of certain integrins.     

Keratinocyte growth factor induces hyperproliferation and delays differentiation in a skin equivalent model system.

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell growth. To examine the direct effects of KGF on the morphogenesis of the epidermis, we generated skin equivalents in vitro by seeding human keratinocytes on the papillary surface of acellular dermis and raising them up to the air-liquid interface. KGF was either added exogenously or expressed by keratinocytes via a recombinant retrovirus encoding KGF. KGF induced dramatic changes to the 3-dimensional organization of the epidermis including pronounced hyperthickening, crowding, and elongation of the basal cells, flattening of the rete ridges, and a ripple-like pattern in the junction of stratum corneum and granular layers. Quantitative immunostaining for the proliferation antigen, Ki67, revealed that in addition to increasing basal proliferation, KGF extended the proliferative compartment by inducing suprabasal cell proliferation. KGF also induced expression of the integrin alpha 5 beta 1 and delayed expression of keratin 10 and transglutaminase. However, barrier formation of the epidermis was not disrupted. These results demonstrate for the first time that a single growth factor can alter the 3-dimensional organization and proliferative function of an in vitro epidermis. In addition to new strategies for tissue engineering, such a well-defined system will be useful for analyzing growth factor effects on the complex links between cell proliferation, cell movement and differentiation within a stratified tissue.     

Development of a dielectric equivalent gel for better impedance matching for human skin

It would be useful to develop a tissue equivalent gel to improve the uniformity of the electromagnetic field in the human body, and for making a tissue equivalent dielectric human phantom. In this study, solid type, water based gelatin-honey gels were developed which have the electrical characteristics of skin tissue. It was demonstrated that a stable and homogeneous gel, with a relative dielectric constant epsilon ' chosen from desired ranges found in skin, can be made for 200-400 MHz.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Summary Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crousing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerved following tramatic injuries and surgical procedures.     

Patterns of regeneration in vessels of human diseased muscle and skin

Regenerating vessels from 36 muscle biopsies and 12 skin biopsies pertaining to patients with the clinical and histological diagnosis of dermatomyositis-polymyositis and other inflammatory myopathies were described ultrastructurally. The following characteristics of vascular regeneration were encountered: a) alternation of thin and thick endothelial cells, b) superimposed segments of endothelial cytoplasm with formation of twisted intercellular junctions, c) long and complex intraluminal endothelial projections, d) discontinuous and porous perivascular basement lamina or multilaminated basement lamina. Some vessels featured only two or three of the mentioned characteristics. Endothelial fenestrations were occasionally encountered in the skin capillaries and were an exception in the muscle capillaries. In the skin there were aspects suggesting that intercalation of perivascular cells in the regenerating endothelial wall may occur. Most capillaries were in the final stage of regeneration.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crossing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerves following traumatic injuries and surgical procedures.     

Enhanced Nrf2 up-regulation by extracellular basic pH in a human skin equivalent system

Extracellular basic pH regulates cellular processes in wounds, and consequently influenced wound healing. Oxidative defence system modulation in the skin helps heal wounds, inhibits skin ageing and improves the skin condition. Moreover, the role of keratinocyte growth factor (KGF) and nuclear factor erythroid 2-related factor 2 (Nrf2) in antioxidant systems has been reported in various skin models. However, the effects of extracellular basic pH on wound- or skin ageing-related skin damage have not been examined. Thus, we investigated the antioxidant systems affected by extracellular basic pH in a 3D human skin equivalent system (3HSE). Extracellular basic pH decreased KGF expression and enhanced the oxidative defence system, and thus activated Nrf2 in the 3HSE. Additionally, extracellular basic pH and KGF treatment up-regulated Nrf2 activation and its regulation of the oxidative defence system in the 3HSE. This indicates that Nrf2 up-regulation is enhanced by reactive oxygen species production, rather than KGF, and by extracellular basic pH of the skin. The inhibition of skin damage through pH imbalance and KGF regulation suggests that the development of pH-regulating or pH-maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin.     

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet–rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line–derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2′-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.     

The role of human immortal skin keratinocytes-acellular dermal matrix scaffold in skin repair and regeneration

In this study, we aimed to investigate the phenotypic characteristics of human immortal skin keratinocytes (HaCaT) cells and the role of acellular dermal matrix (ADM) in coculture system of HaCaT cells and ADM. Flow cytometry was used to examine the cluster of differentiation (CD) makers of HaCaT cells. Apoptosis analysis was applied to detect the apoptosis rate of HaCaT cells. Morphological observation of ADM isolated from the reticular layer of Sprague-Dawley rat dermis was utilized to evaluate the morphological structure of ADM. Methylthiazolyl tetrazolium (MTT) assay and morphological experiments were further used to confirm the scaffold role of ADM in HaCaT cells. A wound-healing mice model accompanied by HaCaT-ADM scaffold transplantation was performed to further verify the function of HaCaT-ADM scaffold. Our results showed that CD71, CD49f, K19, and CD29 were highly expressed in HaCaT cells, and the percentage of apoptosis cells was significantly increased, which represented that HaCaT cells had much stronger capacities of adhesion and proliferation than normal human keratinocytes. Additionally, the morphological structure of ADM presented many natural microbores, which made cells rapidly grow on ADM. The results exhibited that the HaCaT cells indeed promptly proliferate on ADM and easily grow into the microbores of ADM. Finally, an in vivo experiment further confirmed that the transplantation of the HaCaT-ADM scaffold into the dorsal skin of a wound-healing mice model could gradually repair the injured wound. Thus, these findings indicated that HaCaT cells might be as seed cells to develop skin tissue engineering and the HaCaT-ADM scaffold might be a better candidate to promote skin repair and regeneration.     

Changes in the skin epithelium and nerve elements of the rat sole with directed regeneration of the sciatic nerve

The work has been performed on Wistar rats and non-inbred animals. Their ischiatic nerves have been dissected at the femoral superior third under nembutal narcosis. The end of the sectioned nerve are connected by a fragment of an aorta from rats of the same age. The state of nervous elements and dermal epithelium of the hind extremity sole in the animals is studied by means of general histological and neurohistological techniques. Mitotic activity of cells in the plantar epidermis, thickness as a whole and its separate layers are estimated, keratinization coefficient and correlation of thickness of separate sheaths in the whole layer are calculated. Use of the arterial vessels for connecting the end of the cut ischiatic nerve, trophic ulcers, that usually take place after the nerve section, do not develop. At early stages after the operation mitotic activity in the epidermis decreases by 70%, and the layer thickness--by 40%. Restoration of both indices proceeds slowly. As soon as the regenerating nerve fibers reach the distal part of the ischiatic nerve, the state of the epidermis improves: the mitotic activity differs from the normal by 20-30%, and thickness of the epithelium--by 28-30%. Coordination of thickness of separate layers in the epidermis is not nearly disturbed. It remains in the same state up to complete restoration of receptory structures in the rat plantar skin (during 9-9.5 months after the operation).     

Target-specific nerve regeneration through a nerve guide in the rat

Nerve regeneration across a gap in peripheral nerve has been achieved through various nonneural nerve guides in both lower and primate species. This technique can only be useful if the regenerated nerve cable grows specifically to and reinnervates the appropriate distal target. In this study, the proximal peroneal fascicle of rat sciatic nerve was inserted into the proximal limb of a Y-shaped nerve guide. Distal peroneal and tibial fascicles were placed within the two distal limbs of the same Y. The proximal peroneal nerve grew preferentially by a 2:1 ratio to the appropriate distal peroneal fascicle suggesting that target-specific reinnervation is possible through a nerve guide.