Co-culture Of Dermal Fibroblasts Research Articles

Growth of Self-Assembled Bio-Organic Nanomatrices for Skin Tissue Engineering — An in vitro Study

In this paper, we have developed self-assembled nanoscale assemblies that were prepared by conjugating furan-2-carboxylic acid-3-aminopropyl amide with the short peptide sequence Gly-His (abbreviated Gly-His-FCAP). To mimic the extracellular matrix of mammalian fibroblasts and keratinocytes, the assemblies were then conjugated with Type I collagen. We then integrated the collagen bound Gly-His-FCAP assemblies with a short peptide sequence derived from salamander skin into the nanoscale assemblies for the first time to impart regenerative and wound healing properties to the composites. The antioxidant, antimicrobial and biodegradable properties were examined and results indicate that the nanocomposites displayed antioxidant properties as displayed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The biodegradability was found to be gradual. The nanocomposites were also found to inhibit the growth of the fungus Rhizopus sporangia over an 18[Formula: see text]h growth period. As proof of concept, to demonstrate the development of three-dimensional (3D) engineered skin in vitro, 3D printed PLA scaffolds of 2.5[Formula: see text]mm thickness were submerged in media containing nanocomposites and co-cultures of dermal fibroblasts with epidermal keratinocytes mimicking three dimensional skin substitute was examined. Our results indicated that the nanocomposites adhered to and supported cell proliferation and mimicked the components of skin and may have potential applications in skin tissue regeneration.

ID: 19: Increased heparanase expression in keratinocytes promotes dermal fibrosis in scleroderma

Interactions between keratinocyte and dermal fibroblast via paracrine loop play an important role in wound repair and keloid formation. In this study, we investigated heparanase expression in activated keratinocyte, and tested its effect on the survival of dermal fibroblasts. Plasma heparanase levels were measured in scleroderma patients, and heparanase expression was evaluated in the skin of bleomycin-induced fibrosis mice and HaCaT keratinocyte (HaCaT). Dermal fibroblasts were cocultured with HaCaT separated by transwell insert under serum starvation, and apoptosis was determined using APOPercentage assay. Plasma heparanase levels were significantly higher in 26 scleroderma patients than in 10 healthy subjects, and positively correlated with plasma TGF- β levels. In bleomycin-induced fibrosis mice, increased heparanase expression was observed in keratinocyte layer, but not in dermal layer. Treatment of HaCaT with hypoxia resulted in significant increase in heparanase expression, and this increase was accompanied by concomitant increase of matrix metalloproteinase-9, both of which are known to degrade epidermal basement membrane components. Coculture of dermal fibroblasts and HaCaT in the presence of hypoxia significantly protected the apoptosis of dermal fibroblasts induced by serum starvation, but it was abolished by anti-heparanase antibody or transfection of HaCaT with heparanase siRNA. Dermal fibroblasts cocultured with HaCaT exposed to hypoxia exhibited increased Akt phosphorylation, and pretreatment of dermal fibroblasts with LY294002, an inhibitor of phosphatidylinositol 3-kinase, significantly abolished anti-apoptotic effect of heparanase on dermal fibroblasts. These data indicate that hypoxia, caused possibly by microvascular alteration, increases heparanase production in keratinocytes, which may promote fibrosis in scleroderma by inhibiting the apoptosis of dermal fibroblasts.

Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells

Loss of gap junctional intercellular communication (GJIC) is a characteristic of cancer cells. Since a coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis, the present study was designed to test the hypothesis that skin-derived tumor cells may modulate homologous and heterologous GJIC. While homologous GJIC of human dermal fibroblasts as well as epidermal keratinocytes was detected, no communication was measured between SCL-1 cells derived from squamous cell carcinoma of human skin. Interestingly, co-cultures of dermal fibroblasts and SCL-1 tumor cells in serum-containing medium resulted in a 52-70% lowering of the number of communicating fibroblasts. Furthermore, incubation of confluent fibroblast cultures with serum-free supernatant fractions (20-30 kDa) from tumor cells, termed the 20/30 fraction, lowered the homologous gap junction communication of fibroblasts by >90%. This novel aspect of down-regulated homologous GJIC of dermal fibroblasts, which is reversible, was neither mediated by alteration of the expression of connexin43, the major gap junctional protein of dermal fibroblasts, nor by aberrant localization of connexin43 in the plasma membrane. Furthermore, post-translational modifications of connexins, such as phosphorylation, was not measured by mobility shift studies. Tumor cell-mediated GJIC down-regulation between fibroblasts was suppressed using EGTA-containing serum-free tumor cell-derived supernatants suggesting that calcium ions (Ca2+) might mediate the transduction of this effect. The involvement of Ca2+ in down-regulation of homologous GJIC of fibroblasts was supported by an increase in fluorescence intensity of the intracellular calcium-sensitive indicator Fura-2 upon treatment of fibroblasts with the active 20/30 fraction. In conclusion, these data establish homologous GJIC of (stromal) fibroblasts as a parameter modulated by a paracrine acting factor(s) of epithelial tumor cells during tumor-stroma interaction of skin cells.

Mast cells enhance contraction of three-dimensional collagen lattices by fibroblasts by cell-cell interaction: role of stem cell factor/c-kit.

Reorganization of the extracellular matrix is important in many biological and pathophysiological processes, including tissue remodelling, wound healing, or cancer metastasis. The ability of cultured fibroblasts to reorganize and contract three-dimensional type I collagen gels is regarded as an in vitro model for this process. In tissue fibrosis, complex interactions among fibroblasts, inflammatory cells and the extracellular matrix are taking place. Mast cells have often been discussed to play a role in several fibrotic conditions including scleroderma, scar formation, or wound healing. In this study, we examined the effects of mast cells on contraction of collagen lattices. The results demonstrate that co-culture of dermal fibroblasts with a human mast cell line (HMC-1) significantly enhanced contraction of the three-dimensional collagen lattices, whereas mast cells alone failed to contract the gel. Addition of culture supernatants of mast cells did not enhance the speed of gel contraction, indicating the importance of cell-cell contact. Morphological analysis showed that mast cells were incorporated into the lattices. Histological examination also demonstrated that within the lattices, mast cells were localized in close contact to, or attached to, fibroblasts. As fibroblasts and mast cells are known to attach via stem cell factor (SCF)/c-kit interaction when co-cultured in monolayers, we also examined the effect of antibodies against SCF and c-kit in this system. Addition of both antibodies inhibited gel contraction up to 70%. In contrast, antibodies against interleukin-4 (IL-4) and IL-4 receptor did not affect gel contraction. These results indicate that mast cells enhance fibroblast-mediated contraction of collagen lattices via direct cell-cell contact, mediated in part by SCF/c-kit interactions.

The Co-Culture of Dermal Fibroblasts with Human Epidermal Keratinocytes Induces Increased Prostaglandin E2 Production and Cyclooxygenase 2 Activity in Fibroblasts

During wound healing, cell-cell interactions between epidermal keratinocytes and dermal fibroblasts contribute to the organization of epidermis, in which prostaglandin E2 (PGE2) is considered to be involved in proliferation and differentiation of keratinocytes. In the current study, we investigated the regulation of PGE2 biosynthesis in co-culture of human epidermal keratinocytes and human dermal fibroblasts. The production of PGE2 was synergistically enhanced in the co-culture at cell ratios of keratinocytes to fibroblasts between 1/8 and 4, whereas the production of PGE2 was negligible in individual monolayer cultures of keratinocytes or fibroblasts. To address the mechanism of PGE2 production induced by the co-culture of keratinocytes and fibroblasts, we asked whether either cell-derived soluble factor(s) or direct cell-cell contact was required to augment the production of PGE2. Neither the fibroblast-conditioned medium nor membrane fractions influenced the production of PGE2 in keratinocytes. Keratinocyte-conditioned medium greatly enhanced the production of PGE2 in fibroblasts, however, whereas the effect of keratinocyte-membrane fractions was weaker. The main soluble fraction in the keratinocyte-conditioned medium contained a precursor of interleukin-1 alpha (proIL-1alpha) by western blot analysis, and PGE2 production was inhibited by anti-IL-1alpha antibody, but not by anti-IL-1beta or by anti-tumor necrosis factor-alpha antibody. The enhanced production of PGE2 in fibroblasts upon culturing with keratinocytes was due to the induction of COX-2 mRNA mediated by proIL-1alpha released from keratinocytes. These results suggest that cell-cell interactions of keratinocytes and fibroblasts augment the production of PGE2 by a mechanism in which the activity of COX-2 in fibroblasts is increased by the keratinocyte-derived proIL-1alpha in a paracrine manner.

A three-dimensional in vitro model for the study of ocular cytotoxicity and irritancy

A three-dimensional human tissue model has been developed in our laboratory and used as a substrate for assessing ocular cytotoxicity and irritancy potential. The full-thickness model is a co-culture of dermal fibroblasts and epithelial cells (keratinocytes). Metabolically and mitotically active fibroblasts are seeded onto medical-grade nylon mesh, where they attach and secrete collagen and extracellular matrix proteins. Then, keratinocytes are seeded onto the top of this submerged stromal tissue, and the tissue grows into a multilayered epithelium. Histological examination of this cellular co-culture system reveals a cellular organization similar to the rabbit/human cornea. The resulting substrate has been used effectively to study the effects of a variety of test compounds (surfactants, powders, creams) at diluted or full-strength concentrations in a time-course assay. The protocol mimics the way in which in vivo animal testing (Draize eye test) is performed. The treated tissue is assayed for cytotoxicity and irritancy (MTT and PGE 2, respectively) over a period of time. The high correlation (R > 0.81) of our in vitro data to existing animal eye data shows the potential usefulness of these tissue substrates as an in vitro alternative for evaluating the toxicity of test agents from diverse chemical classes.

Assessment of the Ocular Irritancy Potential of Hair Conditioners in a Three-Dimensional in Vitro Human Skin Model

AbstractA three-dimensional human tissue model has been developed in our laboratory and used as a substrate for assessing ocular cytotoxicity and irritancy potential. The full-thickness model is a coculture of dermal fibroblasts and epithelial cells (keratinocytes). Metabolically and mitotically active fibroblasts are seeded onto medical-grade nylon mesh, where they attach and secrete collagen and extracellular matrix proteins. Then keratinocytes are seeded onto the top of this submerged stromal tissue, and the tissue grows into a multilayered pithelium. Histologic examination of this cellular coculture system reveals a cellular organization similar to the rabbit/human cornea. The resulting substrate has been used effectively to study the effects of a variety of test compounds (surfactants, powders, creams) at diluted or full-strength concentrations in a time-course assay. The protocol mimics the way in which in vivo animal testing (Draize eye testing) is performed. The treated tissue is assayed for cytot...

Mononuclear cell-fibroblast interactions in scleroderma

We studied cell proliferation and collagen biosynthesis in cocultures of dermal fibroblasts with peripheral blood mononuclear cells (MNC) from scleroderma patients and from age-matched normal controls. Autologous one-way mixed MNC-fibroblast cultures revealed that fibroblasts do not stimulate MNC proliferation. Conversely, MNC stimulate autologous fibroblasts from scleroderma patients as well as from normal controls. This effect is increased in cells from scleroderma patients in which it seems to be mediated both by cell-to-cell interaction and through the production of soluble factors by MNC. In normal control cell systems we found no proliferative effect of supernatants of unstimulated cells or from those stimulated in autologous mixed-lymphocyte reactions. Coculture of fibroblasts with autologous MNC resulted in increased [ 14C]proline incorporation into both collagenic and noncollagenic proteins. This effect was mediated mostly by soluble factors that are released into the culture medium. Protein synthesis by MNC-fibroblast cocultures from scleroderma patients was significantly greater than protein synthesis by those from normal controls. Culture supernatants from unstimulated MNC or from autologous mixed-lymphocyte cultures caused a slight decrease in collagenic protein synthesis by cultured fibroblasts from scleroderma patients but not by those from normal controls. This effect of culture supernatants could be reproduced, and magnified, with purified IL-1 on cells from either patients or controls. Our findings indicate abnormal MNC-fibroblast interactions in scleroderma that could play an important role in the pathogenesis of fibrosis, the hallmark of this condition.