In vitro characterization of an artificial dermal scaffold

The treatment of extensive burn injuries has been enhanced by the development of artificial skin substitutes. Integra Artificial Skin, an acellular collagen-glycosaminoglycan (C-GAG) dermal equivalent requires a two-stage grafting procedure. However, preseeding the C-GAG dermal equivalent with cultured fibroblasts and keratinocytes, with the aim of performing a single-stage grafting procedure, may be beneficial in terms of replacing the requirement for traditional split-skin grafts. In this comparative in vitro study, the interactions of cultured human dermal fibroblasts and epidermal keratinocytes in Integra Artificial Skin in comparison to cadaver deepidermalized dermis (DED) was investigated. An increase in cell proliferation and migration in the C-GAG dermal equivalent was observed over time. Cocultures of fibroblasts and keratinocytes on both dermal equivalents showed positive expression of proliferation, differentiation, and extracellular matrix (ECM) protein markers. Organization of keratinocytes in the epidermal layers of DED composites were better compared to the C-GAG composites. Deposition of ECM proteins was enhanced in the presence of keratinocytes in both dermal equivalents. Results demonstrate that in vitro the C-GAG dermal equivalent is biocompatible for cell attachment, migration, proliferation, and differentiation. Preseeding Integra Artificial Skin with cultured autologous fibroblasts and keratinocytes for in vivo application, as a single-stage grafting procedure, warrants testing. A better clinical outcome may be achieved as shown by our in vitro results of the coculture composites.     

Development of a stable chemically defined surface for the culture of human keratinocytes under serum-free conditions for clinical use

Within the field of tissue engineering there is a need to develop new approaches to achieve effective wound closure in patients with extensive skin loss or chronic ulcers. This article exploits the well-known interdependency of epithelial keratinocytes and stromal fibroblasts in conjunction with plasma surface technology. The aim was to produce a chemically defined surface, which with the aid of a feeder layer of lethally irradiated dermal fibroblasts would improve the attachment and proliferation of the keratinocyte cell from which subconfluent cells can be transferred to wound bed models. Plasma copolymers of acrylic acid/octa-1,7-diene have been prepared and characterized by X-ray photoelectron spectroscopy. The fibroblasts and keratinocytes were cultured on plasma polymer-coated 24-well plates. Cell attachment and proliferation were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-eluted stain assay (MTT-ESTA) and DNA assay. Attachment and proliferation of both cell types on plasma polymer surfaces were compared with tissue culture plastic and collagen I, plus a negative control of a pure hydrocarbon layer. A pure acrylic acid surface, fabricated at a power of 10 W and containing 9.2% carboxylate groups, was found to promote both fibroblast and keratinocyte attachment and proliferation and permit the serum-free coculture of keratinocytes and irradiated fibroblasts.     

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.     

Cytotoxicity tests for antimicrobial agents using cultured skin substitutes fixed at interface of air and culture medium

The present study is focussed on a new cytotoxicity test using cultured dermal and epidermal sheets, which are fixed at the air and medium interface as a wound surface model. The cultured dermal sheet is composed of human fibroblasts and a collagen matrix, and the cultured epidermal sheet is composed of human keratinocytes and a collagen matrix. Each cultured sheet was fixed at the air and medium interface, over which a piece of test specimen was placed. The in vitro system created, provides a mimetic wound surface since during wound repair, fibroblasts are embedded in an extracellular matrix, while keratinocytes migrate and proliferate on provisional granulation tissue. The results thus obtained in this cytotoxicity test are useful for determining the efficacious amount of antimicrobial agent used in clinical cases.     

Cultured human keratinocytes on type I collagen membranes to reconstitute the epidermis

The development of new techniques and modifications to overcome some of the disadvantages in cultured keratinocyte grafting has been motivated by several well-known drawbacks in the use of cultured epithelial autografts such as long culture periods, lack of adherence, difficulty in handling, lack of dermal substrates, and high costs. Two recent insights have influenced further research. On the one hand, it has been shown that the use of undifferentiated proliferative cells in fibrin glue suspensions is effective in epithelial reconstitution. On the other hand, the enzymatic release of cells from the culture surfaces is a critical step leading to at least temporary destruction of anchoring structures of the cultured cells. In this study, we tried to combine these two aspects in an attempt to modify common modalities of keratinocyte transplantation. To avoid dispase dissolving of the cultured cells, keratinocytes were seeded onto bovine collagen type I membranes without feeder layers and under serum-free culture conditions. Subconfluent monolayers of cultured human keratinocytes were transplanted as an upside-down graft on collagen membranes (keratinocyte collagen membrane grafts [KCMG], n = 12) after 3 days of culture or as membrane grafts alone (n = 12) onto standard nude mice full-thickness wounds. Fully differentiated epidermis was found at 21 days after grafting KCMG with persistence of human keratinocytes. This study demonstrates that upside-down grafts of undifferentiated monolayers of keratinocytes on non-cross-linked bovine type I collagen membranes do lead to an early reconstitution of multilayered squamous epithelium with enhanced wound healing compared to the control group. The upside down KCMG grafting technique is able to transfer actively proliferative keratinocytes and simplifies the application compared to conventional epithelial sheet grafting.     

In vitro cytotoxicity testing of a nanocrystalline silver dressing (Acticoat) on cultured keratinocytes

Acticoat is a polyethylene mesh coated with nanocrystalline silver. It has been used widely as a dressing for chronic wounds, acute partial-thickness burn wounds and donor sites. In this study, the in vitro cytotoxicity of Acticoat on cultured keratinocytes is tested. Human keratinocytes are cultivated on a pliable hyaluronate-derived membrane (Laserskin) using dermal fibroblasts as the feeder layer. When the cultured Laserskin (CLS) is subconfluent it is covered by Acticoat, which is exposed to water (Group 1), phosphate-buffered saline (Group 2) or culture medium (Group 3). The control group is not exposed to the Acticoat. After 30 minutes incubation at 37 degrees C, the inhibitory effect of the nanocrystalline silver on keratinocyte growth is measured by an MTT assay. Compared with the control, the relative viability of the CLS dropped to 0%, 0% and 9.3%, respectively. Thus, Acticoat is cytotoxic to cultured keratinocytes and should not be applied as a topical dressing on cultured skin grafts.     

Human skin cell cultures onto PLA50 (PDLLA) bioresorbable polymers: influence of chemical and morphological surface modifications

Poly(alpha-hydroxy acid)s derived from lactic and glycolic acid are bioresorbable polymers which can cover a large range of thermal, physical, mechanical, and biological properties. Human keratinocytes have been shown as able to grow on a poly(DL-lactic acid) film. However the keratinocyte growth was delayed with respect to culture on standard tissue culture polystyrene, even though the same plateau level was observed after 2 weeks. In order to improve the performance of poly(DL-lactic acid) films as skin culture support, their surface was modified by creating tiny cavities using a method based on the leaching out of poly(ethylene oxide) from poly(lactic acid)-poly(ethylene oxide) heterogeneous blends. The surface of the films was also chemically modified by alkaline attack with sodium hydroxide and by type-I collagen coating. Murine fibroblast cell line and primary cultures of human fibroblasts and of two types of keratinocytes were allowed to adhere and to grow comparatively on the different films. The presence of cavities affected neither the adhesion of dermal fibroblasts nor that of keratinocytes. Only keratinocyte proliferation was significantly reduced by the presence of cavities. Collagen coating improved skin cell adhesion and proliferation as well, except in the case of murine fibroblasts. In the case of the NaOH treatments, similar trends were observed but their extent depended on the treatment time. In the case of chemical modifications, fluorescence microscopy bore out adhesion and proliferation tendencies deduced from MTT tests.     

In situ image analysis of interactions between normal human keratinocytes and fibroblasts cultured in three-dimensional fibrin gels

The non-invasive investigation of different cells to interact and become spatially organised in a three-dimensional (3D) environment or scaffold is an important challenge in tissue engineering and tissue physiology. The aim of the present study was to develop 3D cell culture systems using fibrin gels, which would allow for the single and co-culture of different cell types with in situ image analysis. Two chambers were constructed for mono-culture and co-culture of human dermal fibroblasts and keratinocytes. During cell culture, in situ imaging and morphological characterisation of cells was assessed using brightfield light and/or fluorescence microscopy, and later confirmed by staining of fixed cells using immunofluorescence microscopy. The results showed that it was possible to investigate fibroblast and keratinocyte interactions in a fibrin scaffold for at least 12 days. Using this model system it was found that when a co-culture of fibroblasts and keratinocytes were plated on top of the fibrin gels, fibroblasts were seen to migrate into the gels within 2-3 days in contrast to keratinocytes, which did not enter. However, keratinocytes were found to retard fibroblast migration into gels when compared to fibroblasts cultured on their own, illustrating the dependency of intracellular communication on cell position for reconstructive approaches.     

Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication

We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell–hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.     

Skin regeneration using keratinocytes and dermal fibroblasts cultured on biodegradable microspherical polymer scaffolds

Bioartificial skin sheet grafts have been utilized to treat large burns and chronic ulcers. However, the trypsinization step to harvest cultured skin grafts from culture dishes damages the cells by breaking the anchoring proteins and lowers their uptake ratio after transplantation. In addition, epidermal sheet grafts require a long fabrication period. To overcome these limitations, we utilized biodegradable poly(lactide-co-glycolide) (PLGA) microspheres as both cell culture matrix and transplantation vehicle of skin cells for skin regeneration in this study. This method could avoid the trypsinization step and have a relatively short preparation period. Human keratinocytes and dermal fibroblasts cultured on PLGA microspheres in spinner flasks proliferated by 3.0-fold and 9.4-fold, respectively, after 10 days. When both types of cells cultured on PLGA microspheres were reinoculated onto culture dishes, the cells migrated from the PLGA microspheres to the culture dish surface, grew, and formed a confluent cell layer within 5 days, showing the growth and migration abilities of the cells cultured on PLGA microspheres. Full-thickness skin wounds created on the back of athymic mice were either treated with transplantation of keratinocytes and dermal fibroblasts cultured on microspheres (cell-transplanted group), treated with PLGA microspheres alone (microsphere-implanted group), or covered with dressing materials without treatment (untreated group). Three weeks after the treatments, differentiated epithelium that stained positively for cytokeratin, a marker of epidermis, was observed in the cell-transplanted group, while the microsphere-implanted group and untreated group showed incomplete reepithelialization. Dermal regeneration with positive staining for vimentin, a marker of dermal fibroblast, was observed in the cell-transplanted group. Regenerated dermis with positive staining for vimentin was partly observed in the microsphere-implanted group and untreated group. These results suggest that transplantation of keratinocytes and dermal fibroblasts cultured on PLGA microspheres could be potentially useful as an alternative to bioartificial skin grafts for the treatment of skin wounds.     

IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes

Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.     

Mixture of fibroblasts and adipose tissue-derived stem cells can improve epidermal morphogenesis of tissue-engineered skin

Many studies demonstrate that the type of adjacent mesenchymal cells can affect epidermal morphogenesis of bilayered tissue-engineered skin. However, whether a mixture of different mesenchymal cell types can improve epidermal morphogenesis of bioengineered skin remains unknown. In this study, keratinocytes, dermal fibroblasts and adipose tissue-derived stem cells (ADSCs) were isolated and purified from human skin and subcutaneous fat. Conditioned medium generated from a mixture of dermal fibroblasts and ADSCs at the ratio of 1:1 was superior to that from fibroblasts or ADSCs alone in promoting keratinocyte proliferation, as indicated by MTT assay. Furthermore, ELISA results showed that the cytokine levels of human hepatocyte growth factor and keratinocyte growth factor (also known as FGF7) in the mixed fibroblasts/ADSC group were higher than those in the ADSC or dermal fibroblasts group. To examine the potential roles of mixed fibroblasts and ADSCs on epidermal morphogenesis, a three-dimensional tissue engineered skin system was applied. Histological analyses demonstrated that keratinocytes proliferated extensively over the mixture of fibroblasts and ADSCs, and formed a thick epidermal layer with well-differentiated structures. Keratin 10 (epidermal differentiation marker) was expressed in the suprabasal layer of bilayered tissue-engineered skin in the mixed fibroblasts and ADSCs group. Desmosomes and hemidesmosomes were detected in the newly formed epidermis by transmission electron microscopy analysis. Together, these findings revealed for the first time that a mixture of fibroblasts and ADSCs in bilayered tissue-engineered skin can improve epidermal morphogenesis.     

Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature

To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermo-responsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32 degrees C, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37 degrees C, as on the nongrafted culture dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20 degrees C without need for dispase. No cell remnants were observed on the dishes. Such cell sheet detachment was not observed on nongrafted dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted dishes. Transmission electron microscopy revealed that desmosomes were destroyed in dispase treatment but retained in low-temperature treatment. Use of thermo-responsive dishes was examined as a new tool for tissue engineering to achieve the preparation of artificial epithelium for cell transplantation as well as for the investigation of intact multilayered keratinocyte sheets.     

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.     

Optimization and characterization of an engineered human skin equivalent

Skin equivalents (SEs) have been designed to meet both basic and applied research needs. The successful application of tissue-engineered SEs requires that the reconstituted tissues be endowed with the correct organization and function. A large body of experimental evidence now supports the notion that the inducing effects of mesenchymal tissue on epithelial cell morphogenesis are mediated, at least in part, by extracellular matrix components in addition to cell-cell interactions. A coculture model including both fibroblasts and keratinocytes was used to study the effects of progressive serum reduction on epidermal differentiation, quality of dermal and dermal-epidermal junctions, and expression of extracellular matrix proteins. The cells were successively added to a dermal substrate composed of collagen, glycosaminoglycans, and chitosan. The main aim of this study was to optimize this model for pharmacotoxicological trials. Control skin equivalents were cultured with medium containing 10% serum throughout the production process. Serum content was reduced to 1 and 0% at the air-liquid interface and compared with control skin equivalents. First, we demonstrated that serum deprivation at the air-liquid interface improves keratinocyte terminal differentiation. Second, we showed that, in the absence of serum, the specific characteristics of the SE are maintained, including epidermal and dermal ultrastructure, the expression of major dermal extracellular matrix components (human collagen types I, III, and V, fibronectin, elastin, and fibrillin 1), and the dermal-epidermal junction (laminin, human type IV collagen, alpha6 integrin). Furthermore, our results indicate that coculture models using keratinocytes and fibroblasts have both morphological and functional properties required for biologically useful tissues.     

Proliferation of normal human keratinocytes on silicone substrates.

Several polydimethylsiloxane elastomers and gels were tested as culture substrates for proliferating normal human epidermal keratinocytes. Growth kinetics of normal human keratinocytes (NHK) and dermal fibroblasts were compared on 'very soft', 'soft' and 'hard' silicone gels, as well as on standard cell culture polystyrene dishes. Water contact angles and chemical compositions (IRFT-HATR) of the different silicone surfaces were found to be equivalent, although very different from standard cell culture polystyrene. The topography of the surfaces as well as the shape of the keratinocytes and fibroblasts grown on the different substrates were visualized by scanning electron microscopy, and compared. Although the surface softness and topography of the substrates differed markedly, dermal fibroblasts proliferated in serum-containing medium in equivalent manner on all substrates. Again no correlation could be found between the characteristics and the attachment of the substrates and rapid proliferation of the epidermal keratinocytes in defined medium. The epidermal keratinocytes spread, secreted a structured extracellular matrix network and grew up to confluence on all silicone substrates (elastomers and gels), except the relatively 'hard' silicone gel; this could be due to a direct interference by the waves observed on the silicone gel surfaces.     

Sucralfate induces proliferation of dermal fibroblasts and keratinocytes in culture and granulation tissue formation in full-thickness skin wounds

Sucralfate is used to induce healing of gastrointestinal tract ulcers. We evaluated its potential utility in the healing of skin wounds. Initial experiments examined the effects of the sucralfate on proliferation of cultured dermal fibroblasts and keratinocytes. Sucralfate induced proliferation in quiescent cultures of both cell types. Additionally, sucralfate enhanced prostaglandin E₂ synthesis in basal keratinocytes and in interleukin-1-stimulated keratinocytes and dermal fibroblasts. Basal interleukin-1 and 6 release were not affected by sucralfate, but the agent enhanced interleukin-1-stimulated interleukin-6 release from fibroblasts. When applied daily to full-thickness wounds in rats, sucralfate increased the thickness of granulation tissue when assessed at day 12.     

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting

Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead(?) assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2-5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3-10 μm) and scaffold thickness 50-70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.     

Human galectins induce conversion of dermal fibroblasts into myofibroblasts and production of extracellular matrix: potential application in tissue engineering and wound repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multifunctionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-β1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum.     

Expression of CD13/aminopeptidase N and CD10/neutral endopeptidase on cultured human keratinocytes

Keratinocytes actively participate in immune response and inflammation by secreting cytokines and chemokines. Membrane-bound peptidases serve as negative loop in controlling concentration of peptide signalling molecules. Recently, they have also been proposed as additional mechanism of cell-to-cell interaction and as signalling molecules. In this study, we examined expression of two membrane-bound peptidases: aminopeptidase N (APN; EC 3.4.11.2; CD13) and neutral endopeptidase (NEP; EC 3.4.24.11; CD10) on nonstimulated cultured human keratinocytes obtained from healthy skin. Membrane expression of CD13 and CD10 was analysed by FACS and fluorescent microscope. Functional properties of CD13 and CD10 were examined by testing their enzymatic activity towards selective substrates. The data were compared to those obtained on cultured nonstimulated human skin fibroblasts expressing both CD13/APN and CD10/NEP. Approximately one-third (i.e. 31.7+/-2.8%; n=3) of cultured keratinocyte express CD13 as compared to fibroblasts which are 100% CD13(+) (n=3). Density of CD13 on keratinocytes is several times lower than on fibroblasts. Membrane CD13 expression on keratinocytes was associated with significant enzyme activity, which on the basis of substrate (L-Ala-betaNA) and inhibitor (bestatin, actinonin) selectivity could be ascribed to aminopeptidase N. Kinetic parameter V(max) revealed lower APN activity expressed on keratinocytes than on fibroblasts (V(max)=1.49+/-0.08 microM/60 min/5 x 10(4) cells for keratinocytes, n=3 versus V(max)=4.09+/-0.76 microM/60 min/5 x 10(4) cells for fibroblasts, n=3). Likewise, K(m) value of APN on keratinocytes was lower as compared to fibroblasts (K(m)=0.307+/-0.090 mM for keratinocytes, n=3 versus K(m)=0.766+/-0.065 mM for fibroblasts, n=3). CD13 demonstrated on cultured keratinocytes, is at least partly due to its constitutive expression since it was also found on freshly prepared epidermal skin cells. Inhibitors of APN, actinonin, bestatin and substance-P, as well as the APN blocking antibody WM-15, decreased keratinocytes growth. In contrast to membrane CD13 associated with APN enzyme activity, neither membrane CD10, nor its enzyme (NEP) activity could be found on the same keratinocyte samples. In conclusion, functional CD13, associated with APN activity, was found on about one third of cultured, non-stimulated keratinocytes, whereas no CD10/NEP was found on the same keratinocyte samples. Role of APN in regulation of keratinocyte growth is suggested, as its inhibition resulted in decreased keratinocyte growth.