Culture of dermal fibroblasts and protein adsorption on block conetworks of poly(butyl methacrylate-block-(2,3 propandiol-1-methacrylate-stat-ethandiol dimethacrylate)).

Amphiphilic block terpolymer conetworks composed of butyl methacrylate (BMA), 2,3 propandiol-1-methacrylate (GMMA) and ethandiol dimethacrylate (EDMA) were synthesized. Telechelic oligomers with the carboxylic acid end groups were made via ozonolysis of poly(BMA-co-butadiene) and then these were reacted with glycidyl methacrylate to obtain cross-linkable vinyl groups at both chain ends. Networks were then formed via free radical copolymerization with EDMA and GMMA or 2-methyl-acrylic acid 2,2-dimethyl-[1,3]dioxolan-4-ylmethyl ester (GMAc). The acetonide groups of the GMAc units were then removed, by reaction with selenium dioxide and hydrogen peroxide, to give networks with the same molecular structure as the GMMA terpolymers but different cell adhesion and protein adsorption properties. Protein adsorption was maximised in networks prepared with GMMA rather than with GMAc followed by removal of the acetonide. Block conetworks that were synthesised with GMAc were poor substrates for cell proliferation whilst the GMMA class support good levels of both cell viability and proliferation. It is suggested that the difference in behaviour is derived from changes in the surface composition.

A chemically defined surface for the co-culture of melanocytes and keratinocytes.

Patients with stable vitiligo can be helped surgically using transplantation of autologous cultured melanocytes, but there is a need for a culture methodology that is free from xenobiotic agents and for a simple way of delivering cultured melanocytes to the patient to achieve pigmentation with good wound healing. The aim of this study was to develop a chemically defined surface, suitable for the co-culture of melanocytes and keratinocytes which could be used in the future for the treatment vitiligo patients to achieve both restoration of pigmentation and good wound healing. Two keratinocyte growth media and two melanocyte growth media were compared; two of these were serum free. Cells were seeded on a range of chemically defined substrates (produced by plasma polymerisation of acrylic acid, allylamine or a mixture of these monomers) either as mono- or co-cultures. Melanocytes and keratinocytes attached and proliferated on both acid and amine substrates (without significant preferences), and co-cultures of cells proliferated more successfully than individual cultures. One media, M2, which is serum free, supported expansion of melanocytes and to a lesser extent keratinocytes on several plasma polymer substrates. In conclusion, these data indicate that a combination of a chemically defined substrate with M2 media allows serum-free co-culture of melanocytes and keratinocytes.

Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes.

Melanoma invasion is a complex multi stage process involving changes to the cell/extracellular matrix (ECM) and cell/cell interactions. We have previously shown using an in vitro model of reconstructed human skin (consisting of human dermis with a basement membrane [BM] and populated with human skin cells) that some melanoma cells (HBL cell line) invade more actively in the presence of adjacent normal skin cells. The aim of the present study was to further investigate the relationship between melanoma cells, skin cells and ECM proteins during melanoma cell invasion through reconstructed skin, extending this to a study of three melanoma cell lines. We also examined whether such cell/cell induced invasion is due to increased expression and activation of matrix-metalloproteinase-2 (MMP-2) and MMP-9, or due to increases in general protease activity for keratinocytes, fibroblasts or melanoma lines. Addition of skin cells dramatically altered the invasive behaviour of the three metastatic melanoma cell lines (HBL, C8161 and A375SM) used; they increased the invasive ability of HBLs which were unable to invade on their own; they potentiated the invasion of C8161 cells which were invasive in their own right, but reduced the invasion of A375-SM cells which were aggressive invaders in the absence of skin cells. Latent forms of MMP-2, and MMP-9, were clearly expressed by the normal skin cells whereas all three melanoma lines weakly expressed these proteases. Fibroblast and keratinocyte MMPs were activated specifically by culture on type I collagen and on dermis which retained an intact basement membrane. These findings demonstrate that while there is an active communication between melanoma cells and adjacent skin cells, the invasive process is dictated by the melanoma cells and not the skin cells. However, activation of skin cell derived MMPs may play an important role in facilitating invasion by particular melanoma phenotypes.

Establishing a transport protocol for the delivery of melanocytes and keratinocytes for the treatment of vitiligo.

We have previously developed a cell delivery and transfer technology for delivering autologous keratinocytes and melanocytes to patients with vitiligo. However, for this technology to benefit many patients geographically distant from the cell culture facility transportation issues need to be overcome. In this study we begin to investigate this by looking at what role surface chemistry and medium supplements, including fetal calf serum, CO2 gassing, and temperature, play in influencing cell viability. Cells were maintained on carriers for up to 48 h outside of a CO2 incubator at 37 °C and their subsequent ability to adhere and become organized into a new epithelium with appropriately located melanocytes was assessed. Consistently good viability and performance on an in vitro wound bed model was achieved by maintaining cells for 48 h adherent to a 20% acrylic acid coated carrier at lower (around 23 °C rather than 37 °C) temperatures in the medium preperfused with CO2 before transport. Under these circumstances fetal calf serum was not required. In summary, the surface chemistry of the transport substrate and an appropriately CO2 buffered medium at near room temperature can extend the effective performance life of these cultured cells to at least 48 h from when they leave standard incubator conditions.

Simplifying the delivery of melanocytes and keratinocytes for the treatment of vitiligo using a chemically defined carrier dressing.

Obtaining pigmentary function in autologous skin grafts is a current challenge for burn surgeons as is developing reliable robust grafting strategies for patients with vitiligo and piebaldism. In this paper, we present the development of a simple methodology for delivering cultured keratinocytes and melanocytes to the patient that is of low risk for the patient but also user friendly for the surgeon. In this study, we examined the ability of keratinocytes and melanocytes to transfer from potential cell carriers under different media conditions to an in vitro human wound bed model. The number of melanocytes transferred, their location within the neoepidermis, and their ability to pigment were evaluated as preclinical end points. Two inert substrates (polyvinyl chloride and silicone sheets) and three candidate plasma-polymerized coatings with controlled surface chemistry deposited on these substrates were explored. Two media for expansion of cells, Greens, currently used clinically (but which contains fetal calf serum), and a serum-free alternative, M2 (melanocyte medium), were explored. Reproducible transfer of physiologically relevant numbers of melanocytes capable of pigmentation from the coculture of melanocytes and keratinocytes was obtained using either Greens medium or M2 medium, and a silicone carrier pretreated with 20% carboxylic acid deposited by plasma polymerization.

Use of an in vitro model of tissue-engineered human skin to study keratinocyte attachment and migration in the process of reepithelialization.

To produce a stable epidermis, keratinocytes need to be firmly attached to the basement membrane. However, following wounding, keratinocytes are required to develop a migratory phenotype in order to reepithelialize the wound. To investigate some of the issues underlying reepithelialization, we have developed a three-dimensional in vitro model of tissue-engineered skin, comprising sterilized human dermis seeded with human keratinocytes and dermal fibroblasts. Using this model, we have shown that the inclusion of fibroblasts within the model increases the stability of keratinocyte attachment. We have also demonstrated that keratinocyte migration occurs most effectively in the absence of a basement membrane and following the inclusion of fibroblasts in the model. In addition, subjecting the keratinocyte layer to mechanical trauma induces a migratory phenotype. We conclude that this three-dimensional in vitro wound model can be used to increase our understanding of the factors that enhance keratinocyte migration and hence wound healing in vivo.

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis.

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.