Abstract Basement membranes (BM) underlie epithelia and endothelia and surround many tissues. In cutaneous BM epithelial cells are attached to the stroma via multiprotein complexes called hemidesmosomes (HD). Collagen XVII and integrin α6β4 are components of HD and they bind to laminin 332, a component of anchoring filaments, extracellularly. The main interest of this study is the function of collagen XVII and its interactions with these proteins. What is known about the function of collagen XVII is mostly derived from its role as an adhesive component in cutaneous HD. Here we demonstrate for the first time that collagen XVII is expressed by podocytes in the human and murine glomerulus and that mutant mice lacking collagen XVII in addition to small size, blisters and diffuse hair loss, also have deficient glomerular development and a high mortality rate. We also show for the first time at the protein level that collagen XVII is expressed, and probably has a functional interaction with laminin 332, in normal colon epithelia. We demonstrate that collagen XVII is expressed by the invasive cells of human colorectal carcinoma (CRC) samples and its immunostaining is increased in metastasis in CRC. The higher proportion of collagen XVII positive tumor cells correlates with decreased disease-free survival and cancer-specific survival times and we also suggest a functional interaction between collagen XVII and laminin 332 in CRC. Previous studies have suggested that collagen XVII participates in keratinocyte migration by affecting the correlation of HD disassembly and assembly, its expression is increased in squamous cell carcinoma (SCC) and it may have a role in cell adhesion and migration in SCC carcinogenesis. Here we demonstrate upregulated collagen XVII, integrin β4 and laminin γ2 expression in actinic keratosis, Bowen’s disease and SCC. The expression of collagen XVII was increased with a high degree of variation, especially in samples taken from areas where SCC is particularly invasive. We also demonstrate in the SCC-25 cell line that lack of collagen XVII or integrin β4 severely disrupts the adhesion, migration and invasivity of these cells. Taken together, in this study we show that collagen XVII is needed for normal glomerular development, is expressed in normal colon epithelia and participates in CRC and SCC carcinogenesis together with laminin 332 and integrin β4. / Tiivistelmä Tyvikalvot sijaitsevat epiteelin ja endoteelin alla ja ympäröivät monia kudoksia. Ihon tyvikalvossa epiteelisoluja alla olevaan verinahkaan kiinnittää rakenne, jota kutsutaan hemidesmosomiksi (HD). Kollageeni XVII ja integriin α6β4 ovat HD:n rakenneproteiineja. Ne kiinnittyvät solun ulkopuolella laminiin 332 nimiseen proteiiniin, joka muodostaa ankkurifilamentit. Kollageeni XVII ilmentyminen ja toiminta yhdessä näiden kahden proteiinin kanssa on tämän tutkimuksen keskeisin kohde. Valtaosa tutkimuksista, jotka käsittelevät kollageeni XVII:ää, koskevat sen toimintaa ihon keratinosyyteissä. Tässä tutkimuksessa osoitimme ensi kertaa, että hiiren ja ihmisen munuaiskerästen podosyyttisolut ilmentävät kollageeni XVII. Geenimanipuloidut hiiret, joilta kollageeni XVII oli poistettu, olivat pieniä, kehittivät rakkuloita ja karvattomuutta, niillä oli korkea kuolleisuus ja niiden munuaiskerästen kehitys oli häiriintynyt. Kollageeni XVII esiintymistä proteiinitasolla, sekä mahdollista toiminnallista yhteyttä laminiin 332:een, ei aiemmin ole osoitettu paksusuolen epiteelissä. Havaitsimme, että paksu- ja peräsuolen adenokarsinooman (CRC) invasiivinen solukko ilmentää kollageeni XVII:ää, kollageeni XVII esiintyminen on merkittävän voimakasta CRC:n metastasoinnin yhteydessä ja lisääntynyt kollageeni XVII esiintyminen lyhentää syöpävapaata aikaa ja heikentää syöpäspesifistä selviytymistä. Myös CRC:ssä kollageeni XVII toiminta voi liittyä laminiini 332:een. Aiempien tutkimusten mukaan kollageeni XVII osallistuu keratinosyyttien migratioon vaikuttamalla toimivien HD:ien määrään. Sen määrän on havaittu olevan korkeampi okasolusyövässä (SCC) ja sen on ehdotettu osallistuvan syöpäsolujen adheesioon ja migraatioon SCC:n kehittyessä. Me osoitimme kohonneen kollageeni XVII, integriini β4 ja laminiini γ2 ilmenemisen aktiinisessa keratoosissa, Bowenin taudissa sekä SCC:ssä. Kollageeni XVII määrä oli korkea, mutta vaihteli paljon, sekä hiiren että ihmisen invasiivisilla SCC alueilla. Havaitsimme myös SCC-25 solulinjalla, että kollageeni XVII tai integriini β4 puutos häiritsee vakavasti solujen adheesiota, migraatiota ja invaasiota. Yhteenvetona tässä työssä osoitimme, että kollageeni XVII:ää tarvitaan munuaiskerästen kehittymisessä, sitä esiintyy paksusuolen epiteelissä, ja että kollageeni XVII osallistuu CRC:n ja SCC:n kehittymiseen yhdessä integriini β4:n ja laminiini 332:n kanssa.

A côté de leur rôle structural au sein des tissus, les protéines de la matrice extracellulaire sont impliquées dans un grand nombre de processus cellulaires au cours de divers évènements biologiques. En revanche, leur rôle au cours de la régénération reste étonnamment peu étudié à ce jour. Pourtant, mieux comprendre le rôle de la MEC dans la régénération a de nombreuses applications en médecine régénérative et reconstructrice. Mon projet de thèse vise précisément à répondre à cette question. Pour cela, nous avons utilisé le modèle bien établi de la régénération de la nageoire caudale du poisson zèbre qui présente de nombreux avantages tels qu’une structure simple, facile d’accès et un régénération rapide, en seulement quelques jours. Une approche globale de transcriptomique sans a priori a permis d’établir l’importance de la matrice extracellulaire au cours de la régénération. Une première étape a consisté à établir la liste des gènes de la matrice extracellulaire du poisson zèbre par orthologie, appelé matrisome. Notre étude a fait émerger le rôle inattendu d’un collagène dans la reconstruction de la membrane basale de l’épiderme, une structure importante pour l’attachement de l’épiderme au derme dans la peau. Cette protéine, exprimée uniquement chez l’embryon, est ré-exprimée dans l’épiderme en régénération et déposée au niveau de la membrane basale. Par stratégie anti-sens in vivo, j’ai montré par microscopie à force atomique et microscopie électronique que l’absence de ce collagène impacte la structure et les propriétés biomécaniques de cette membrane basale en reconstruction. Ces résultats ont été confirmés sur une lignée de poisson, invalidée pour ce gène que nous avons créée par la technologie CRISPR/Cas9. Cette lignée a permis d’établir que ce collagène agit transitoirement comme un « spacer » moléculaire nécessaire à l’organisation tridimensionnelle des autres composants de la membrane basale pendant la régénération. / In addition to their role within tissues, extracellular matrix proteins are implicated in a large number of cellular processes. However, their role in regeneration is not well studied at the moment. A better understanding of the extracellular matrix proteins involvement in regeneration can have several future applications for regenerative and reconstructive medicine. The aim of my PhD project is to answer this question.To do this, we used the well-established zebrafish caudal fin model which have many advantages such as a simple structure, easily accessible and a quick regeneration in only few days. A global transcriptomic approach without a priori showed us that extracellular matrix proteins are playing a key role in regeneration. A first step of my work was to use an orthology-based approach to create the first list of extracellular matrix genes in zebrafish, called the matrisome. Our study revealed the unexpected role of a collagen during epidermal basement membrane reconstruction, an importance structure for the dermo-epidermal cohesion in skin. This protein which is expressed only during embryogenesis, is re-expressed in the regenerating epidermis and deposited in the basement membrane. Using an anti-sense strategy in vivo, I have demonstrated by atomic force microscopy and electron microscopy that the absence of this collagen impacts the biomechanics of this reconstructing basement membrane. These results were confirmed on a zebrafish line invalidated for this collagen that I have generated using the genome editing CRISPR/Cas9 technic. We showed that this collagen acts as a molecular spacer needed for the correct tridimensional organization of the other basement membrane components during regeneration.

Abstract Collagen XIII is a type II transmembrane protein, which has a short intracellular domain and a large, mainly collagenous ectodomain. It is located at many cell-matrix junctions and in focal adhesions in cultured cells and it has a function in cell adhesive processes. Overexpression of collagen XIII molecules with an 83 amino acid deletion in part of the ectodomain leads to fetal lethality in Col13a1del transgenic mice. Doppler ultrasonography was performed at 12.5 days of gestation on fetuses resulting from heterozygous matings and matings between heterozygous and wild-type mice. Some fetuses had atrioventricular valve regurgitation (AVVR) and all of them were transgene positive. In addition, fetuses had pathological changes in functional parameters. Histological analysis showed the trabeculation of the ventricles to be reduced and the myocardium to be thinner in the fetuses with AVVR. Based on in situ hybridization (ISH), collagen XIII mRNA are normal constituents of these structures. Overexpression of mutant collagen XIII results in mid-gestation cardiac dysfunction in fetuses, and these disturbances in cardiac function may lead to death in utero. The heterozygous mice that were initially of normal appearance had an increased susceptibility to develop B cell lymphomas, which originated in the mesenteric lymph node. Collagen XIII protein was not detected in normal lymph nodes or in the lymphomas. The incidence of lymphomas was higher in conventional conditions than in a specific pathogen-free facility. In addition, the expression of collagen XIII was localized in the intestine and the basement membrane was highly abnormal. These findings suggest that collagen XIII is a critical determinant of lymphanogenesis. Using ISH, antibody staining and RT-PCR techniques collagen XIII expression was analyzed during carcinogenesis in mice and in man. Collagen XIII expression increased during carcinogenesis in mice and in man. In the malignant process collagen XIII mRNA localized in the basal epithelium and in the invasive cells. According to antibody staining malignant invasive cells were positive. Results may reflect the disturbed adhesion of epithelial cells and ECM and that may affect the behaviour of the malignant cells, suggesting that collagen XIII has a significant role in the initiation of the invasion.

Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.

Abstract Collagen XVIII is a basement membrane proteoglycan, which has three variant N-termini. These variants are coded by two promoters; promoter 1 directs the synthesis of a short variant and promoter 2 directs the synthesis of two longer variants, of which the middle variant is generated from the longest by splicing. The longest variant contains a cysteine-rich domain in its N-terminus, which shows homology to the frizzled receptors of the Wnt molecules and can inhibit Wnt/beta-catenin signalling in vitro. The C-terminal domain of collagen XVIII, endostatin, is an inhibitor of tumor growth and angiogenesis. Lack of collagen XVIII accelerates cutanous wound healing and wound angiogenesis. Overexpression of endostatin leads to delayed wound healing and the presence of morphologically abnormal wound capillaries. Moreover, endostatin overexpression leads to delayed formation of the wound epidermal basement membrane and impaired maturation of hemidesmosomes. Endostatin treatment decreases osteoblast proliferation in vitro. Moreover, osteoblast proliferation and mineralization of the matrix by osteoblasts are inhibited when cells are treated with endostatin together with VEGF. In vivo, lack of collagen XVIII leads to delayed formation of secondary ossification centers in mouse femurs, whereas overexpression of endostatin leads to a slower growth of bone length. However, both of these changes are transient and mild, suggesting that collagen XVIII/endostatin is not essential for skeletal development. The growth of hair follicles is delayed in the mice overexpressing endostatin. This delay in growth is preceded by an impaired hair follicle associated angiogenesis. Lack of collagen XVIII causes an accelerated onset of the first hair cycle. A similar change can be seen in mice lacking the long variants of collagen XVIII. Lack of the short variant causes mild acceleration in the catagen of the first cycle, and anagen is also significantly accelerated in these mice. The long variants were located in the bulge region, which contains the hair follicle stem cells, and in the basement membrane surrounding the dermal papilla. As it is known that several Wnt-inhibitors are upregulated in the bulge, our results suggest that the longest variant of collagen XVIII may have a role as a regulator of Wnt-signalling in hair follicles.

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine, 2-oxyglutarate, 5-dioxygenase, Plod) catalyzes the hydroxylation of certain lysine residues in collagens and in other proteins with collagenous domains. Three lysyl hydroxylase isoforms have been cloned from human and rat. The importance of lysyl hydroxylase 1 in collagen biosynthesis is demonstrated by the heritable disorder, Ehlers-Danlos syndrome type VI, which is characterized by joint laxity, progressive scoliosis, muscle hypotonia, scleral fragility and rupture of the ocular globe. An alternatively spliced form of lysyl hydroxylase 2 seems to function as a telopeptide lysyl hydroxylase. Lysyl hydroxylase 3 has three enzyme activities, lysyl hydroxylase, hydroxylysyl galactosyltransferase (EC 2.4.1.50), and galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66) activities that have been demonstrated earlier with in vitro experiments. In this thesis study, the cDNAs of mouse lysyl hydroxylase isoforms 1, 2 and 3 were cloned and characterized and the gene structures of lysyl hydroxylase 2, Plod2, and lysyl hydroxylase 3, Plod3, were determined. Mouse lysyl hydroxylase isoforms were found to be highly homologous to the corresponding human isoforms and they were approximately 60% identical with each other. The mouse Plod3 gene has 19 exons as do the human PLOD1 and PLOD3 genes, and mouse Plod2, like the human PLOD2, has 20 exons including one alternatively spliced extra exon. The mouse isoforms were also found to have distinct tissue distributions. Phylogenetic analysis revealed that the lysyl hydroxylase genes have evolved from an ancestral gene through two gene duplication events. Lysyl hydroxylase 3 was demonstrated to be the oldest isoform, which is further supported by the association of glycosyltransferase activities with lysyl hydroxylase 3 and with the only lysyl hydroxylase of Caenorhabditis elegans. The roles of the different enzyme activities of lysyl hydroxylase 3 were determined in vivo by generating three genetically modified lysyl hydroxylase 3 mouse lines. The analysis of these mouse lines demonstrated that lysyl hydroxylase 3 possesses at least lysyl hydroxylase and glucosyltransferase activities in vivo and it functions as the main, if not the only glucosyltransferase during embryogenesis. The absence of lysyl hydroxylase 3 and, especially, its glucosyltransferase activity results in the abnormal glycosylation of type IV collagen, and thus causes a severe basement membrane defect leading to death during early development. By contrast, lysyl hydroxylase activity had no effect on embryonic development, but caused changes in the structure of the epidermal basement membrane and changes in collagen fibril organization and probably in their interactions.

Abstract Transmembrane collagen XVII (BP180) is a structural component of hemidesmosomes that connects the two layers of skin. Collagen XVII is associated with both autoimmune and inherited bullous skin diseases. Mutations in collagen XVII gene cause junctional epidermolysis bullosa, and in the diseases of the pemphigoid group autoantibodies target collagen XVII. In this work, collagen XVII was studied in both junctional epidermolysis bullosa and gestational pemphigoid. Two novel glycine substitution mutations were found in the largest collagenous domain of collagen XVII. Analysis of recombinantly produced mutated proteins showed that these novel mutations and previously described glycine substitution mutations decrease the thermal stability of collagen XVII ectodomain. In addition, these mutations were found to cause intracellular accumulation of the mutated proteins and affect the post-translational modifications of collagen XVII. Meanwhile, an in-frame deletion of nine amino acids had no effect on the thermal stability or secretion of the collagen XVII ectodomain. Gestational pemphigoid autoantigen collagen XVII has been mainly studied in the skin, and its expression and function during pregnancy are so far largely unknown. For the first time, collagen XVII was shown to be expressed by cytotrophoblasts of the first trimester human placenta and by cultured cytotrophoblasts. Transmigration assay of cytotrophoblasts indicated that collagen XVII promotes trophoblast invasion, and may thus have a role in placental formation. In addition, significant amounts of in vivo produced collagen XVII were found in the amniotic fluid throughout pregnancy. Collagen XVII expression was also observed in hemidesmosomes of amniotic membranes and in cells cultured from amniotic fluid. These findings suggest that collagen XVII could have a function, albeit so far unknown, during pregnancy.

La membrane basale (BM) constitue une barrière physiologique entre les tissus et leur microenvironnement. Dans le cas des cancers épithéliaux, au stade de carcinome invasif, la membrane basale est compromise et les cellules cancéreuses envahissent le stroma. Dans cette thèse de doctorat, j’ai proposé d’étudier l’invasion de la membrane basale par les cellules cancéreuses et comment une population de cellules stromales, les fibroblastes, affectent cette invasion. Dans le cadre de cette étude, nous avons utilisé le modèle du cancer colorectal. En collaboration avec l’hôpital Curie, nous avons isolé des fibroblastes à partir de tumeurs de patients opérés. On a nommé les fibroblastes isolés de la partie tumorale « CAF » et ceux venant de la partie du tissu normal, à proximité de la tumeur, « NAF ». Comme modèle de BM nous avons utilisé le mésentère de souris. Afin étudier l’invasion des cellules cancéreuses à travers le mésentère et l’effet des fibroblastes, nous avons mis en place une construction en 3D in vitro. Nous avons montré que les CAFs, et rarement les NAFs, induisent l’invasion des cellules cancéreuse et que cet effet est prononcé quand les CAFs sont physiquement présents sur la membrane. En faisant une étude protéomique comparative entre CAFs et NAFs, on a montré que les CAF expriment plus de protéines composantes de la membrane basale, des protéines impliqués dans le remodelage de la matrice extracellulaire, et des protéines impliquées dans la contraction des cellules. Nous avons ensuite voulu comprendre par quel mécanisme les CAFs induisent l’invasion. Nous avons montré qu’en présence des CAFs, l’invasion ce fait de façon indépendante des métaloprotéinases mais que l’effet contractif des CAFs est nécessaire. En conclusion, l’ensemble de ces résultats mets en évidence l’effet promoteur des CAFs sur l’invasion des cellules cancéreuses et souligne l’importance de leur contractilité dans ce mécanisme. / Basement membrane represents a physiological barrier between epithelial tissues and their microenvironment. In invasive carcinomas, the membrane is breached and cancer cells disseminate in the stroma. In this PhD thesis, I investigated how cancer cells breach the BM and whether a stromal cell population, fibroblasts, assist them in that process. I used colorectal cancer as a model. In collaboration with the Institut Curie Hospital, we isolated human primary fibroblasts from human colorectal cancers, called CAFs and the adjacent normal tissue, NAFs. To study BM invasion, I developed a 3D in vitro assay based on the mouse mesentery. We showed that CAFs, and rarely NAFs, induce cancer cell invasion. This pro-invasive effect is mainly mediated when CAFs are physically present on the membrane, rather than through paracrine ways. To understand how CAFs facilitate invasion, we performed a proteomic comparison between cancer cell-stimulated CAFs and NAFs. Results showed that CAFs produced more proteins-components of the ECM, matrix remodelers and they were more contractile compared to NAFs. Further, we wished to understand the mechanism by which CAFs mediate their effect. We showed that CAFs can induce invasion in a MMP independent way. However, Inhibition of contractility abolished CAFs capacity to induce invasion. Dynamic analysis of cancer cells-fibroblasts co-cultures showed that CAFs could pull on the BM fibers. To directly test this possibility, we created holes in the BM using laser ablations. While in the presence of cancer cells alone, holes remained the same size, in the presence of CAFs, holes widen over time. We further showed that this mechanism is MMP independent but depends on contractility. Altogether, these results demonstrate that CAFs stimulate cancer cell invasion through BM by acting directly on the BM, possibly by depositing ECM components and proteins that remodel ECM and by exerting physical forces on the membrane by contraction.

Experimentally, traditional developmental models and transgenic animals consistently underscore the importance of studying cell behavior in the correct tissue context. However, live animal experimentation is inherently complex, and systematic assessment of the effects of individual variables, such as cell shape and matrix compliance on cell behavior, is extremely difficult at best. Two-dimensional monolayer culture of key individual cell types has provided abundant, fundamental information on cell response, but cannot be used to show the normal phenotype of breast epithelial cells. Furthermore, their results often fail to translate into in vivo and clinical studies. It has been previously established that normal human breast epithelial cells can form their original phenotype as seen in vivo when embedded in or layered on reconstituted basement membrane extract. This phenotype is characterized as a single or double layer of polarized cells in acinar form with a lumen devoid of cells. Most malignant cell lines cultured under the same conditions exhibit severe morphological deformities, including colony overgrowth, luminal filling (hyperplasia), and resistance to apoptosis. It was hypothesized that malignant breast cells can be reverted to a normal phenotype through the manipulation of two factors: control of the environment via extra-cellular matrix proteins, and control of cellular pathways via signaling inhibitors. It was observed that high levels of epidermal growth factor resulted in disrupted multi-acinar formations. Inhibition of the protein complex known as mammalian target of rapamycin is currently being investigated as a potential method for cancer treatment. Exposure of rapamycin, mammalian target of rapamycin's primary inhibitor, led to decreased proliferation and increased caspase activity. Through the exposure to rapamycin in three-dimensional cultures, proliferation was reduced in malignant cells, while normal cells were not significantly affected. Unfixed fluorescent staining with ethidium bromide indicated the presence of luminal cell death. Increased structural organization was observed by immunofluorescent staining of F-actin and β-catenin. Through RT-PCR analysis, increased expression of a number of genes related to polarity and structural organization was detected in malignant cells exposed to rapamycin. Further study will be required to better characterize the reversion effects of rapamycin and its derivatives.

yes / A major challenge in the production of 3D tissue engineered skin is the recreation of the basement membrane region to promote secure attachment and yet segregation of keratinocytes from the dermal substitute impregnated with fibroblasts. We have previously shown that simple electrospun scaffolds provide fibres on which the cells attach, proliferate, and self-sort into epithelium and dermis. In a development of this in this study tri-layered scaffolds were then electrospun from poly L-lactic acid and poly hydroxybutyrate-co-hydroxyvalerate. In these a central layer of the scaffolds comprising nano-porous/nano-fibrous poly hydroxybutyrate-co-hydroxyvalerate fibres was interwoven into the bulk micro-porous poly L-lactic acid microfibers to mimic the basement membrane. Keratinocytes and fibroblasts seeded onto these scaffolds and cultured for 2 weeks showed that neither cell type was able to cross the central nano-porous barrier (shown by SEM, and fluorescence monitoring with CellTracker™) while the micro-fibrous poly L-lactic acid provided a scaffold on which keratinocytes could create an epithelium and fibroblasts could create a dermal substitute depositing collagen. Although cells did not penetrate this barrier the interaction of cells was still evident-essential for epithelial development.