Collagen degradation in the rabbit skin during short-term tissue culture

Full thickness rabbit skin explants were cultured on plastic dish for 1 week and the sequential morphological changes were examined daily by light and electron microscopy. During the cultured period, bundles of dermal collagen fibres gradually loosened and were removed from the upper dermis and from the cut margin of the explant, which was covered by a sheet of migrating epidermal cells. In these areas, cells containing phagocytosed collagen fibrils were observed from the 3rd day to the end of the culture period. These cells containing phagocytosed collagen fibrils included dermal fibroblasts and macrophages, epidermal keratinocytes and endothelial cells lining blood vessels. The presence of acid phosphatase activity in vacuoles containing the collagen fibrils suggested that intracellular degradation of collagen was occurring. In addition, extracellular collagen degradation was recognized around fibroblasts and beneath the migrating epidermis by the high collagenolytic activity at these sites. These findings suggest that both intra- and extracellular collagen degradation may participate in collagen removal from dermal connective tissue in cultured skin explants.     

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.     

Identification of functional markers in a self-assembled skin substitute in vitro

Some functional parameters were identified and assessed in a tissue-engineered self-assembled skin substitute. This skin substitute was produced using fibroblasts and keratinocytes isolated from adult human skin. Keratinocytes were seeded on a dermal layer, composed of two fibroblast sheets cultured for 35 d. The epidermal cells formed a stratified and cornified epidermis and expressed differentiation markers, notably involucrin and transglutaminase. Interestingly and for the first time, the receptor for vitamin D3 was detected in all of the epidermal cell layers of the skin substitute, as it is reported for normal human skin. This observation suggests that keratinocytes retain key receptors during their differentiation in the skin model. A network of collagen fibers was observed by electron microscopy in the dermal layer of the model. In the dermis, collagen fibers remodeling and assembly is dependent on enzymes, notably prolyl-4-hydroxylase. For the first time in a skin construct, the expression of prolyl-4-hydroxylase was detected in dermal fibroblasts by in situ hybridization. The secretion of collagenases by the cells seeded in our skin substitute was confirmed by zymography. We conclude that the self-assembly approach allows the maintenance of several functional activities of human skin cells in a skin model in vitro.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Expression of laminin by human fibroblasts, HT1080 fibrosarcoma cells and MCF-7 breast adenocarcinoma cells. Lack of regulation by the cell density and extracellular matrix.

We have cultured normal fibroblasts, fibrosarcoma HT1080 cells and breast adenocarcinoma MCF-7 cells on various substrates (plastic, collagen type I, laminin). All cell types used adhered on the three substrates with, however, a delayed attachment on laminin. On all substrates, cell grew as monolayer with the exception of MCF-7 cells that formed clusters on laminin. The epithelial MCF-7 cells as well as mesenchymal cells (fibroblasts and tumoral HT1080 cells) synthesized laminin and expressed mRNA coding for laminin B1 chain and for the 67 kD laminin binding protein. The levels of these mRNAs were not modulated by culture conditions which affect cell morphology nor by cell density.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

In vitro differentiation of chicken embryo skin cells transformed by Rous sarcoma virus

The epidermal cells isolated from 14-day chicken embryo shank skin epidermis were infected in vitro with Rous sarcoma virus (RSV). Within a few weeks, rapidly growing colonies of epithelial cells appeared among the sea of transformed fibroblastic cells. When isolated and subcultured, these cells were found to possess typical markers of skin epidermis. The presence of major keratin and typical epithelial cell type morphology strongly suggested that these cells were transformed epidermal cells retaining their differentiated characteristics but having the capacity to propagate in cell culture. If RSV tsNY68, an RSV mutant having a temperature lesion in the src gene, was used, similar transformed epidermal cells were obtained at 36 degrees C (permissive temperature). At the nonpermissive temperature (41 degrees C) the growth rate of these cells decreased and additional keratin species appeared. At 41 degrees C the cells were flattened and lost the refractivity in their peripheries. All the keratins which are synthesized at the nonpermissive temperature were present in normal differentiated shank skin of 19-day old chick embryo. These cells also had "cornified envelop," indicating extensive differentiation. Viral production was as efficient as transformed fibroblasts during the rapid growth phase, while it declined significantly after the cells reached confluency, exhibiting the differentiated characteristics. Since no normal epidermal cells could be cultured under our experimental conditions, these results represent examples in which the src gene is essential for propagation of differentiated cells in cell culture while it abolishes only a part of differentiated characteristics.     

Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.     

Reformation of organized epidermal structure by transplantation of suspensions and cultures of epidermal and dermal cells

Summary The development of epidermis and epidermal appendages from dissociated cells of neonate mouse skin was examined by transplantation of cell suspensions to subdermally prepared, protected graft beds. Using Ficol gradients and culture procedures, we prepared subfractions of primary cell suspensions consisting of essentially pure epidermal cells or fibroblasts.Reformation of an epithelium structurally similar to the epidermis was observed from transplanted epidermal-cell suspensions, but formation of hair follicles and development of normal epidermal microarchitecture was observed only when epidermal cells were transplanted together with cells of dermal origin. This pattern was observed following transplantation of either fresh-cell isolates or cells cultured up to 7 days prior to transplantation.Part of the work was performed when one of us (I.C. Mackenzie) was guest scientist at the DKFZ.     

Expression of catalytically active matrix metalloproteinase‐1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

Increased expression of matrix metalloproteinase‐1 (MMP‐1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP‐1‐mediated collagen fibril fragmentation is a key driver of age‐related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP‐1 mutant (MMP‐1 V94G) in adult human skin in organ culture and fibroblasts in three‐dimensional collagen lattice cultures. Expression of MMP‐1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP‐1 V94G in dermal fibroblasts cultured in three‐dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP‐1 siRNA‐mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP‐1 V94G‐fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF‐β pathway, and reduced collagen production. These results support the concept that MMP‐1‐mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age‐related decline of fibroblast function.     

Morphological and functional characteristics of human temporal-bone cell cultures

Morphology and calcium metabolism have been studied on five different cell cultures from human normal adult temporal-bone biopsies obtained during five stapedectomies. Control cell cultures were obtained from normal human skin. Four different cell types were observed in the bone biopsies: 1) osteoblast-like cells; 2) osteoclast-like cells; 3) fibroblast-like cells; 4) intermediate cells. However, morphology by itself is inadequate for clear differentiation of the four cell types. Hormonal stimulation with calcitonin and dibutyryl-cAMP in presence of 45Ca++ showed a clear-cut difference in 45Ca++ uptake between cultured cells deriving from bone and skin. Functional responses to hormonal stimulation are therefore more specific than cell shape and morphology in differentiating fibroblasts from bone cells. Since responses to hormonal stimulation confirm that temporal-bone cell cultures actually contain bone cells, such cultures seem to be a good experimental model for the study of bone morphology and physiology.     

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β₁ integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.     

Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of alpha1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the alpha1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.     

Cutaneous cell and extracellular matrix responses to ultraviolet-B irradiation

The present study examined fibroblasts and keratinocytes in monolayers and cultured within dermal and skin substitutes and their use in assessing the effect of UVB irradiation on cutaneous cells and extracellular matrix organization. Dermal substitutes (DS) were produced by incorporating normal fibroblasts into a collagen lattice and skin substitutes (SS) were obtained by seeding normal keratinocytes onto the DS. Keratinocyte monolayers, fibroblast monolayers, DS, and SS were exposed once a day to a UVB source (10 ml/cm²). The irradiation protocol was stopped when the keratinocytes of the non-irradiated cultures (control groups) had reached confluence. Microscopic observations revealed that UVB radiation decreased both fibroblast and keratinocyte growth and enhanced their differentiation resulting in (1) less fibroblasts in the DS and SS, and (2) incomplete coverage of the DS by keratinocytes. Microscopic observations and histological analyses revealed major morphological changes. Both cell types became bigger and presented wide nuclei and vacuoles in the cytoplasm. No organized deep epidermal layer was observed in irradiated compared to non-irradiated SS. Irradiated DS and SS extracellular matrices showed an irregular aggregating collagen fiber organization with serious discrepancies suggesting large defects in the structural properties of the extracellular matrix. The present study demonstrated that exposure to a UVB source led to profound morphological and functional disturbances in both cutaneous cells and in the extracellular matrices of the DS and SS. The present technology would be of great interest for step-by-step studies of UVR effects on cutaneous cell morphology and functional properties, and could be an alternative to using animals for pharmacological and toxicological evaluations. © 1996 Wiley-Liss, Inc.     

Characterization of a type VI collagen-related Mr-140 000 protein from cutis-laxa fibroblasts in culture.

The precise biochemical defects in connective-tissue metabolism that are responsible for the laxity of skin seen in the syndrome of cutis laxa are largely unknown. We have studied fibroblasts cultured from skin explants of a 2-year-old male with the syndrome. Electron-microscopic examination of this skin revealed decreased amounts of amorphous elastin and an increase in elastin-associated microfibrils. Although the cultured fibroblasts were similar to control skin fibroblasts in morphology, growth rate and total protein synthesis, there was a 4-6-fold increase in accumulation of a collagenous protein of Mr 140 000 in both the culture medium and in the cell layer. This protein was structurally distinct from collagen types I, III, IV, V and VIII. It was found to be related to a cell-surface-associated glycoprotein, GP140, by both antigenic cross-reactivity and peptide mapping. Our data support observations that GP140 is a precursor of at least one form of pepsin-extracted type VI collagen.     

In vitro maturation of collagen fibrils modulates spreading, DNA synthesis, and collagenolysis of epidermal cells and fibroblasts

Collagen fibrils were maturated in vitro by incubating them in a serum-containing culture medium at 37 degrees C for varied lengths of time. Epidermal cells and fibroblasts were cultured on these maturated collagen gels to see the effects of maturation on cellular morphology and physiology. The spreading and DNA synthesis of both types of cells on the maturated collagen gels were significantly enhanced compared to those on fresh gels. The maturation did not affect the cellular adhesiveness to the substrate. The secretion of collagenase by epidermal cells was suppressed on the maturated collagen gels, the extent of the suppression being related to the length of maturation of the gels. These maturation-related effects of collagen were also observed when collagen was incubated in the medium without serum, indicating that the effects are not due to deposition of serum proteins to collagen gels during maturation. Physical and chemical characterizations of the maturated collagen were performed: the mechanical strength of collagen gels increased in maturated collagen gels, the amounts of insoluble collagen increased with the maturation. These changes in the chemical and physical nature of the maturated collagen gel strongly suggested that there was an increase in intermolecular crosslinks during the process of maturation. These maturation-induced changes in collagen were marked when collagen gels were incubated in the presence of glucose, indicating that a glucose-protein reaction such as the Maillard reaction is involved in this phenomenon.     

Tryptophan hydroxylase expression in human skin cells

We attempted to further characterize cutaneous serotoninergic and melatoninergic pathways evaluating the key biosynthetic enzyme tryptophan hydroxylase (TPH). There was wide expression of TPH mRNA in whole human skin, cultured melanocytes and melanoma cells, dermal fibroblasts, squamous cell carcinoma cells and keratinocytes. Gene expression was associated with detection of TPH immunoreactive species by Western blotting. Characterization of the TPH immunoreactive species performed with two different antibodies showed expression of the expected protein (55-60 kDa), and of forms with higher and lower molecular weights. This pattern of broad spectrum of TPH expression including presumed degradation products suggests rapid turnover of the enzyme, as previously reported in mastocytoma cells. RP-HPLC of skin extracts showed fluorescent species with the retention time of serotonin and N-acetylserotonin. Immunocytochemistry performed in skin biopsies localized TPH immunoreactivity to normal and malignant melanocytes. We conclude that while the TPH mRNA and protein are widely expressed in cultured normal and pathological epidermal and dermal skin cells, in vivo TPH expression is predominantly restricted to cells of melanocytic origin.     

Coculture of newborn rat skin epidermal cells with Swiss 3T3 cells: effect of the phases of 3T3 cells on attachment, growth and keratin synthesis of epidermal cells.

Swiss albino mouse 3T3 cells in various states were inoculated onto one side of Millipore filters. The other side of the filter was then coated with type I collagen and inoculated with newborn rat skin epidermal cells. On coculture of these cells, the attachment, growth and keratin synthesis of epidermal cells were found to depend on the state of the 3T3 cells: 3T3 cells in the stationary phase of growth were the most effective, followed by those in the logarithmic growth phase, those in the lag phase and plasmolyzed fibroblasts being only slightly effective. The effects of 3T3 cells in different states correlated well with their abilities to synthesize type IV collagen, but not type I collagen: with an increase in type IV collagen synthesis by the 3T3 cells, attachment of epidermal cells to the cell support, and their growth and synthesis of keratins increased. This culture system is concluded to mimic conditions in skin in vivo, and therefore to be suitable for studies on the effects of fibroblasts on the growth of epidermal cells.