A new autologous keratinocyte dressing treatment for non-healing diabetic neuropathic foot ulcers.

AIMS: To evaluate the use of a new cell-tailored carrier surface (TranCell) for delivery of autologous keratinocytes to promote wound healing in patients with chronic neuropathic foot ulcers. METHODS: TranCell is a sterile medical grade polymer coated with a plasma-polymerized functional surface containing 20% carboxylic acid which enables keratinocytes to attach and proliferate. Six diabetic patients with neuropathic ulcers resistant to conventional therapy were treated with weekly applications of autologous keratinocytes delivered on TranCell. A split-thickness skin biopsy was taken from each patient followed by isolation, expansion and freezing down of keratinocytes. Keratinocytes were thawed and seeded on TranCell 48 h prior to application. This procedure was repeated weekly in addition to conventional therapy until wound healing was achieved. RESULTS: Complete healing was achieved in six out of nine ulcers in six patients, a reduction in ulcer size was achieved in one ulcer and no response was seen in one ulcer. Treatment was discontinued in one patient due to development of Methicillin-Resistant Staphylococcus aureus (MRSA) after only three applications of TranCell. Wound healing took 6-17 applications over 6-20 weeks. There were no recurrences in the healed ulcers after a follow-up of 6 months. CONCLUSIONS: TranCell delivery of autologous cells is a promising treatment for chronic diabetic foot ulcers with no side-effects and no recurrence in the healed ulcers.

Latanoprost-induced pigmentation in human iridial melanocytes is fibroblast dependent.

The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pigmentation in irises of patients with hazel or heterochromatic eye colour. The mechanism by which LT induces pigmentation in the iris is not yet established, although it does not appear to induce proliferation of iridial melanocytes. The purpose of this study was to develop an in vitro model in which to investigate this mechanism. The pigmentary responses to LT and prostaglandin F(2alpha) (PGF(2alpha)) were examined in human iridial melanocytes alone or in co-culture with epithelial cells (non-ocular human epidermal keratinocytes and iris pigment epithelial cells) or mesenchymal cells (non-ocular dermal fibroblasts or iridial fibroblasts). Melanogenesis was assessed after 4 days culture with prostanoids, using dopa oxidase activity. Prostaglandin FP expression on human iridial fibroblasts and melanocytes was investigated using an immunofluorescent technique employing antibody to PGF(2alpha) receptor and RT-PCR. Iridial melanocytes did not show a convincing increase in dopa oxidase when cultured alone but in the presence of fibroblasts (ocular or non-ocular) there was a significant increase (25-30%) in dopa oxidase activity in response to 10(-7)-10(-5)m LT and PGF(2alpha). Co-culture of melanocytes with epithelial cells, while leading to increased dopa oxidase activity, did not lead to any melanogenic response to LT or PGF(2alpha). FP receptor expression was detected on fibroblasts but not iridial melanocytes by immunocytochemistry and RT-PCR. The melanocyte/fibroblast co-culture model developed in this study also showed that LT and PGF(2alpha) increased dopa oxidase activity in melanocytes from donors with brown but not blue eyes. These results suggest that LT may be inducing pigmentation in the human iris indirectly through the FP receptor on adjacent fibroblasts.

Plasma-polymerized surfaces for culture of human keratinocytes and transfer of cells to an in vitro wound-bed model.

The aim of this study was to develop plasma-polymerized surfaces suitable for the attachment and culture of human keratinocytes and that would allow their subsequent transfer to a wound-bed model. Keratinocyte attachment has been assessed on a carrier polymer, either untreated or treated with a hydrocarbon plasma polymer, collagen I, or carboxylic-acid-containing plasma copolymers. Cell attachment was poor on the "bare" carrier polymer and hydrocarbon plasma polymer (PP) surfaces. Cell attachment was good and comparable on collagen I-coated carrier polymer and carrier polymer plasma coated with carboxylic acid functionalities. After 24 h of cell culture, surfaces were inverted so that cells were adjacent to a de-epidermalized dermis (DED) for 4 days. After 4 days in contact with DED, the surfaces were removed and the level of residual cells and cells transferred to DED were assessed using a cell viability assay. Cell transfer from the collagen I-coated surface was on the order of 90%. Transfer from the carrier polymer surface and the hydrocarbon-coated surface was poor while cells cultured on acid-containing surfaces showed high levels of transfer. Cell transfer was greatest from those surfaces containing the highest level of acid functionality (ca. 21%). Cell transfer was not significantly affected by the choice of carrier polymer material although some sample-to-sample variation was seen. To determine that plasma-polymerized surfaces could be used clinically, selected samples were sterilized with ethylene oxide. Subsequent analysis and cell culture indicated that the surface chemistry and cell-transfer capability of these plasma-polymerized surfaces were unaffected by the sterilization procedure. Plasma-polymerized carboxylic-acid-containing surfaces show great promise in the field of wound healing, encouraging keratinocyte attachment and permitting keratinocyte transfer to a wound bed.

Cellular and hormonal regulation of pigmentation in human ocular melanocytes.

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.

Keratinocyte-driven contraction of reconstructed human skin.

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

Keratinocytes suppress TRP-1 expression and reduce cell number of co-cultured melanocytes - implications for grafting of patients with vitiligo.

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well-integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co-cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down-regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase-related protein-1 (TRP-1) negative in confluent well-integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP-1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP-1 expression.

Characterization of an in vitro model of human melanoma invasion based on reconstructed human skin.

The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three-dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM.

Development of autologous human dermal-epidermal composites based on sterilized human allodermis for clinical use.

The aim of this study was to identify a sterilization technique for the preparation of human allodermis which could be used as a dermal component in wound healing and as the dermal base for production of dermal-epidermal composites for one-stage grafting in patients. We report that it is possible to produce dermal-epidermal composites which perform well in vitro and in vivo using a standard ethylene oxide sterilization methodology. Prevention of ethylene oxide-induced damage to the dermis was achieved using gentle dehydration of the skin prior to ethylene oxide sterilization. The issue of whether viable fibroblasts are required for composite production was examined in comparative studies using glycerol vs. ethylene oxide sterilized dermis. Where good collagen IV retention was achieved following preparation of acellular de-epidermized dermis there was no advantage to having fibroblasts present in vitro or in vivo; however, where collagen IV retention was poor or where keratinocytes were initially expanded in culture then there was a significant advantage to introducing fibroblasts to the composites during their preparative 10-day period in vitro. The requirement for fibroblasts became less evident when composites were grafted on to nude mice. In conclusion, we report a protocol for the successful sterilization of human allodermis to achieve an acellular dermis with good retention of collagen IV. This acellular dermis would be appropriate for clinical use as a dermal replacement material. It can also be used for the production of dermal-epidermal composites using autologous keratinocytes (with or without fibroblasts).

Comparison of proliferation and growth of human keratinocytes on plasma copolymers of acrylic acid/1,7-octadiene and self-assembled monolayers.

Human keratinocytes were cultured on plasma copolymers (PCPs), self-assembled monolayers (SAMs), and tissue culture poly(styrene) (TCPS). Plasma copolymerization was used to deposit films with controlled concentrations of carboxylic acid functional groups (<5%). Human keratinocytes were cultured onto these PCP surfaces, TCPS, and collagen I. A hydrocarbon plasma polymer surface was used as the negative control. Keratinocyte attachment was measured at 24 h and cell proliferation and growth at 3 and 7 days using optical microscopy and DNA concentrations. The PCP surfaces were compared with two SAM systems comprising pure acid and pure hydrocarbon functionalities, and pure gold was used as a control surface. PCP surfaces containing carboxylic acid functionalities promoted keratinocyte attachment. The level of attachment on these surfaces was comparable to that seen on collagen I, a preferred substratum for the culturing of keratinocytes. After several days in culture the cells were well attached and proliferative, forming confluent sheets of keratinocytes. This result was confirmed by DNA assays that suggested the acid PCP surfaces were performing as well as collagen I. Keratinocytes attached well to gold and acid-terminated SAMs but attached poorly to methyl-terminated SAMs. The acid functionality also promoted proliferation and growth of keratinocytes after several days in culture. DNA assays revealed that keratinocyte growth on the acid surface was higher than on collagen I.

Plasma copolymer surfaces of acrylic acid/1,7 octadiene: surface characterisation and the attachment of ROS 17/2.8 osteoblast-like cells.

The purpose of this study was: (a) to examine the effect of plasma-gas composition on plasma polymer oxygen/carbon (O/C) ratio, functional group composition and stability in water, and then (b) to examine cell attachment to surfaces containing different concentrations of O/C and functional groups. Oxygen-functionalised surfaces were deposited by means of the plasma copolymerisation of acrylic acid/1,7-octadiene. The use of a diluent hydrocarbon allowed the deposition of surfaces with a range of O/C concentrations. Plasma copolymer surfaces were characterised by X-ray photoelectron spectroscopy (XPS). Changes in functional group composition with % acrylic acid monomer and the non-dispersive and dispersive parts of the surface energy of these plasma copolymers were measured. The solubility of the plasma copolymers was assessed by means of XPS. The degree of attachment of ROS 17/2.8 osteoblast-like cells to plasma copolymer surfaces deemed to be 'stable' in aqueous medium was measured. Tissue culture polystyrene (TCPS) was included as a control. Attachment was found to be greatest to the plasma copolymer surface with an O/C of 0.11. This surface had a carboxylic acid concentration of ca. 3%. Attachment did not correlate with increased surface wettability (i.e. the non-dispersive component of the surface energy).

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

Investigation of the presence and role of calmodulin and other mitogens in human burn blister fluid.

It is unclear whether burn blister fluid is beneficial or deleterious to the healing of the underlying wound. We investigated the calcium binding protein calmodulin in human burn blister fluid and its role in the mitogenicity of this fluid in the culture of human keratinocytes, fibroblasts, and mouse 3T3 fibroblasts. Calmodulin levels in blister fluid were three times greater than in serum (p < 0.005), whereas epidermal growth factor and platelet-derived growth factor concentrations were significantly lower (p < 0.001). Calmodulin in blister fluid was biochemically identified after affinity chromatography, Western blotting, and immunostaining with a monoclonal antibody. Inhibiting calmodulin with either an antagonist or antibody to calmodulin reduced the mitogenic activity of blister fluid in three cell types by 26% to 80%. These in vitro studies suggest that burn blister fluid may promote wound healing, and locally released calmodulin contributes to this effect. In appropriate cases it may be beneficial to leave burn blisters intact.

A calmodulin-like protein as an extracellular mitogen for the keratinocyte.

This study investigated the importance of extracellular calmodulin to the proliferation of the keratinocyte. Normal keratinocytes in culture produced a calmodulin-like protein in their culture media, the level of which increased abruptly and transiently during their growth. This protein was calmodulin-like, in that it specifically bound to a calmodulin affinity column, exhibited calmodulin-like immunoreactivity in both an ELISA and on immunoblots when immunostained with a monoclonal antibody against calmodulin, had an apparent M(r) between 18,000 and 20,000, and stimulated activity in a calmodulin-dependent phosphodiesterase enzyme assay. Addition of exogenous pure calmodulin was of no further mitogenic benefit to the keratinocytes, and slightly reduced proliferation under the culture conditions used. However, addition of either a neutralizing antibody to calmodulin, or W7-agarose, to the culture media of proliferating cells markedly inhibited their proliferation. Accordingly, a calmodulin-like protein was found to satisfy all but one of the criteria for its action as an autocrine growth factor for the keratinocyte. We propose that the lack of mitogenic response to calmodulin in vitro is due to the cell meeting its own requirement for extracellular calmodulin.

Antiproliferative effects on keratinocytes of a range of clinically used drugs with calmodulin antagonist activity.

Thirty-two drugs, including some in use for a variety of clinical disorders, were examined for their ability to inhibit calmodulin activity in vitro. From these, 10 drugs were selected for their inhibition of calmodulin activity and examined for their ability to inhibit proliferation of rapidly dividing human keratinocytes. A significant correlation between antiproliferative activity and calmodulin antagonist potency was found. Of these drugs there were several, including miconazole, dequalinium chloride, bromocriptine and tamoxifen, whose use is well established and well documented. The potential use of these drugs (and others identified in this way) as antipsoriatic agents is discussed.

Stimulation of von Willebrand factor antigen release by immunoglobulin from thrombosis prone patients with systemic lupus erythematosus and the anti-phospholipid syndrome.

The level of von Willebrand factor antigen (vWF) released by endothelial cells in response to IgG isolated from 18 patients with systemic lupus erythematosus (SLE), eight patients with the anti-phospholipid syndrome (APS) and 22 controls has been measured. Incubation with IgG from the combined patient group resulted in a significantly greater release of vWF (mean stimulation index +/- SEM, 4.57 +/- 0.78) when compared with controls (1.96 +/- 0.22, P = 0.003). Furthermore, IgG from 17 patients who had had a history of thrombosis induced higher levels of vWF release (5.33 +/- 1.09) when compared with the controls (P = 0.008). These findings suggest that IgG from patients with SLE or APS is capable of stimulating vWF release and that this ability may be implicated in the thrombotic events that are observed in these conditions.

Mitogenic role for extracellular calmodulin-like activity in normal human umbilical vein endothelial cells.

Normal human umbilical vein endothelial cells cultured on gelatin-coated plastic dishes were found to produce a protein in their media which had calmodulin-like immunoreactivity and biological activity. Further identification of the protein was achieved by examining the incorporation of 14C leucine into protein found in the conditioned medium. Cells produced 14C labelled protein in their medium which specifically bound to an affinity column for calmodulin. This latter material stimulated calmodulin dependent phosphodiesterase activity in vitro and this stimulation was inhibited by the addition of the calmodulin antagonist W7. The presence of calmodulin-like activity and immunoreactivity in the media varied as the cells grew from low to high density, a peak of extracellular calmodulin-like activity preceding an increase in cell number. Extracellular calmodulin-like activity did not correlate with the presence of lactate dehydrogenase in the medium. The addition of pure pig brain calmodulin affected the rate of cell proliferation; significant proliferation to pure calmodulin was only seen in cells at low density, at higher density calmodulin either had no effect or inhibited proliferation. Inhibition of extracellular calmodulin activity by a calmodulin antagonist immobilized on agarose beads, or by an antibody to calmodulin significantly decreased proliferation in all dividing cultures. Taken together this data suggests that, in vitro, calmodulin, or a very closely related protein, influences endothelial cell proliferation through an autocrine mechanism.

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids.

In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band comigrating with authentic calmodulin (Mr 17,000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14,000-15,000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0.03 nmol/l in urine to 18.6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0.79, P less than 0.01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products.

An extracellular role for calmodulin-like activity in cell proliferation.

1. Addition of extracellular pure pig brain calmodulin was found to modulate DNA synthesis and cell proliferation in K562 human leukaemic lymphocytes. At lower cell densities calmodulin significantly stimulated [3H]thymidine uptake; at higher densities it decreased it. 2. A protein biochemically indistinguishable from calmodulin was detected in the cell-conditioned media of rapidly dividing K562 cells. The concentration of calmodulin-like activity found in the conditioned media of these and a range of other normal and neoplastic cells (250-1636 ng/ml) was of the same order as would stimulate DNA synthesis in subconfluent cells. 3. Amounts of extracellular calmodulin-like activity and immunoreactivity varied during cell growth from low to high density, a peak of extracellular calmodulin preceding DNA synthesis in synchronized K562 cells. Extracellular calmodulin concentrations did not correlate with the presence of lactate dehydrogenase in the medium. 4. Inhibition of extracellular calmodulin activity by calmodulin antagonist immobilized on agarose beads, or by antibody to calmodulin, significantly decreased DNA synthesis. 5. These data strongly suggest that calmodulin or a very closely related protein can influence mitosis through an extracellular mechanism.

Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry.

Syntheses are described for a range of N-(omega-aminoalkyl)-5-iodo- and -5-cyanonaphthalene-1-sulphonamides. The selective activity of these compounds as inhibitors for calmodulin-dependent phosphodiesterase (EC 3.1.4.17) is compared with their activity for the calmodulin-independent but calcium-dependent enzymes protein kinase C and transglutaminase (EC 2.3.2.13). The results show a drastic improvement in the selectivity of effect for the 5-iodo-compounds compared with the widely-used drug, W7, N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide.