Skin Tissue Engineering Applications Research Articles

Development of bFGF-Chitosan Matrices and Their Interactions with Human Dermal Fibroblast Cells

Chitosan (CH) is a naturally derived, biodegradable polymer of glucosamine with a variable frequency of N-acetyl-D-glucosamine units, and has been demonstrated to have numerous pharmacological and wound-healing properties. Biodegradable chitosan films were fabricated using a solvent casting technique and investigated for skin tissue-engineering applications. Basic fibroblast growth factor (bFGF) was incorporated into the CH matrices (1 μg/film) by 3 methods: adsorption, entrapment and covalent binding. Release rates and biological activity of the incorporated bFGF were monitored. Human dermal fibroblasts (HDF cells) were used as an in vitro model for cell response to CH and bFGF-CH films. Cell attachment, growth and acid-soluble collagen quantification were employed as an assessment of cell function. The fibroblasts were found to remain viable on the chitosan films and scaffolds. CH films without bFGF were compatible with HDF cells; however, the fibroblasts did not proliferate. The release profile of adsorbed and bound bFGF from CH films were similar (indicating that binding was not efficient) while entrapped bFGF was not released in the time frame studied. The concentration of bFGF released to the cell culture medium was not high enough to stimulate HDF proliferation. However, cell attachment was significantly increased in chitosan films with bFGF adsorbed onto the surface as compared to control surfaces. HDF cells grown on CH films produced significantly more collagen than those on control surfaces.

Surface Engineering in Microfluidic Devices for the Isolation of Smooth Muscle Cells and Endothelial Cells

AbstractMicrofluidic cell separation systems have emerged as attractive alternatives to traditional techniques in recent years. These systems offer the advantages of being able to handle small sample volumes and at the same time achieve highly selective separation. Conventional separation techniques, including both fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), typically require a pre-processing incubation step to attach ligated tags (such as fluorescent dyes or magnetic beads) to cell surfaces prior to separation. These techniques are also constrained by infrastructure and high cost. Microfluidic devices with surface-immobilized adhesion molecules eliminate the need for pre-processing incubation and are a low cost alternative.We describe the selective adhesion of smooth muscle cells and endothelial cells in microfluidic devices coated with adhesion peptides. The device geometry is such that the shear stress varies linearly as a function of flow channel length, allowing simultaneous evaluation of the effects of surface chemistry and fluid shear on cell adhesion. The adhesion peptides, val-ala-pro-gly (VAPG) and arg-glu-asp-val (REDV), are known to bind selectively to smooth muscle cells and endothelial cells, respectively. These peptides were tethered to the device surface using silane chemistry and NHS-ester coupling. Cell adhesion was examined in a shear stress range of 1.3-4.0 dyn/cm2. Under these conditions, endothelial cells show significantly higher adhesion to REDV-coated devices compared to smooth muscle cells and fibroblasts. Correspondingly, smooth muscle cell adhesion in VAPG-coated devices is much greater than that of endothelial cells and fibroblasts. This selective binding behavior is also observed when mixed suspensions of the three cell types are flowed into both types of peptide-coated microfluidic devices. These results suggest that microfluidic devices coated with REDV and VAPG can be used as effective separation tools in various applications, such as tissue engineering. Specific examples of applications in cardiac and skin tissue engineering will be discussed.

Generation and Differentiation of Human Embryonic Stem Cell-Derived Keratinocyte Precursors

Human embryonic stem cells (hESC) hold tremendous potential in the future of tissue engineering, offering promise as a source of virtually unlimited quantities of desired cell and tissue types. We have identified soluble chemical and extracellular matrix factors that permit isolation of keratinocyte precursors from hESCs. Culturing embryoid bodies (EB) formed from hESCs in a defined serum-free keratinocyte growth medium on a gelatin matrix generated keratin 14 (K14) expressing cells with an epithelial morphology. These K14 expressing cells could be subcultured in medium supplemented with hydrocortisone and induced to stratify and terminally differentiate by addition of calcium. Optimum times for obtaining K14 expressing cells were found for EB formation and for differentiation and growth of cultures after EB plating. EB formation was not necessary to generate keratinocyte precursors; direct transfer of hESC colonies to keratinocyte growth medium permitted differentiation into the keratinocyte lineage. With further studies to optimize generation and purification of hESC-derived keratinocyte precursors, these cells could provide a source of epidermal cells for skin tissue engineering applications in vitro or in vivo.

Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering

Chitosan, the deacetylated derivative of chitin, is a promising scaffold material for skin tissue engineering applications. It is biocompatible and biodegradable, and the degradation products are resorbable. However, the rapid degradation of chitosan and its low mechanical strength are concerns that may limit its use. In this study, chitosan with 80%, 90% and 100% degree of deacetylation (DDA) was crosslinked with dimethyl 3-3, dithio bis’ propionimidate (DTBP) and compared to uncrosslinked scaffolds. The scaffolds were characterized with respect to important tissue engineering properties. The tensile strength of scaffolds made from 100% DDA chitosan was significantly higher than for scaffolds made from 80% and 90% DDA chitosan. Crosslinking of scaffolds with DTBP increased the tensile strength. Crosslinking with DTBP had no significant effect on water vapour transmission rate (WVTR) or water absorption but had significant effect on the pore size and porosity of the samples. All samples showed a WVTR and pore size distribution suitable for skin tissue engineering; however, the water absorption and porosity were lower than the optimal values for skin tissue engineering. The biodegradation rate of scaffolds crosslinked with DTBP and glutaraldehyde (GTA) were reduced while no significant effect was observed in biodegradation of the samples made from 100% DDA chitosan whether crosslinked or uncrosslinked after 24 days of degradation.

The role of surface zeta potential and substratum chemistry for regulation of dermal fibroblasts interaction

AbstractSynthetic materials considered for biohybrid skin or other tissue engineering applications have to support cellular interaction and colonisation of implants. However, despite the number of studies reported in the literature, there is no agreement on the principal factors applicable to modulate the interaction with cells, such as the wettability of biomaterials, their surface potential and chemistry of the surface. Particularly, in this study we were interested on the impact of surface chemistry and net surface (zeta) potential on dermal fibroblasts. To address this question self assembled monolayers of silanes on glass, further derivatized with different functional groups, were used to study the adhesion and proliferation of human skin fibroblasts in response to these factors. The model surfaces were characterized using streaming potential (zeta potential) and water contact angle measurements. All samples were found to be negatively charged at physiological pH (regardless of the different chemistry) increasing (or equal ∼) the magnitude of the negative potential in the following order: hydroxy (OH) < amine (NH2) ∼ epoxy (EPOXY) < carboxyl (COOH) < three‐fluorocarbon (CF3) < sulfonic (SO3) functionalities. The interaction of fibroblast characterized by the effectiveness of cell adhesion, spreading and actin stress fibres formation decreased almost in the opposite order: NH2 > OH > EPOXY > SO3 > COOH > CF3 functions. The surfaces were found also to be highly wettable, except CF3. Interestingly, the best cellular interaction was found on the moderately wettable NH2 surface representing water contact angle (CA) of 65°, and the worst, on the least wettable CF3 surface (CA 85°) indicating that not only the surface potential but also the type of functional groups may play an important role. The organisation of α5 and β3 integrins generally followed the same trend of less clustering in focal adhesion plaques when the negative potential increased, except on SO3 surface, where β3 but not α5 integrins were greatly expressed. Cell growth however, showed a significant decrease on highly charged COOH and SO3 surfaces, as well as, on non‐polar CF3 functions. The best proliferation response was obtained on surfaces with primary amine groups. The results indicate that the surface (zeta) potential might be a critical parameter for cellular interactions, but also the substratum wettability and the type of functional groups have to be controlled in order to improve the biocompatibility of material surfaces.

Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering

The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage. However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors. To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process. The human (HaCaT, A549) and rat (NBT II, MH1C1) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Adβ-gal adenovirus, encoding for the reporter gene E. coli β-galactosidase, in the presence of various polybrene concentrations. We determined the amount of β-gal positive cells by X-gal staining and the β-gal expression by ONPG-assay after 24 h. In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Adβ-gal resulted in a marked increase of β-gal positive cells and β-gal protein expression. The efficacy of polybrene showed a clear dose dependency. The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent. In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.