Keratinocyte Spreading Research Articles

Substrate softness promotes terminal differentiation of human keratinocytes without altering their ability to proliferate back into a rigid environment.

Substrate stiffness is a key regulator of cell behavior. To investigate how mechanical properties of cell microenvironment affect the human keratinocyte, primary cells were seeded on polyacrylamide hydrogels of different compliances (soft: 4kPa, medium: 14kPa, rigid: 45kPa) in comparison with glass coverslip (> GPa). Keratinocyte spreading and proliferation were strongly decreased on the softest hydrogel, while no significant difference was observed between medium, rigid hydrogels and glass coverslip, and cells' viability was comparable in all conditions after 72h. We then performed a RNA-seq to compare the transcriptomes from keratinocytes cultured for 72h on the softest hydrogel or on coverslips. The cells on the soft hydrogel showed a strong increase in the expression of late differentiation marker genes from the epidermal differentiation complex (1q21) and the antioxidant machinery. In parallel, these cells displayed a significant loss of expression of the matrix receptors (integrin α6 and β1) and the EGF receptor. However, when these cells were replated on a plastic culture plate (> GPa), they were able to re-engage the proliferation machinery with a strong colony-formation efficiency. Overall, using low-calcium differentiation monolayers at confluence, the lesser the rigidity, the stronger the markers of late differentiation are expressed, while the inverse is observed regarding the markers of early differentiation. In conclusion, below a certain rigidity, human keratinocytes undergo genome reprogramming indicating terminal differentiation that can switch back to proliferation in contact with a stiffer environment.

Mussel-Inspired Cell/Tissue-Adhesive, Hemostatic Hydrogels for Tissue Engineering Applications.

The combination of multiple physiological (swelling, porosity, mechanical, and biodegradation) and biological (cell/tissue-adhesive, cell proliferation, and hemostatic) properties on a single hydrogel has great potential for skin tissue engineering. Adhesive hydrogels based on polydopamine (PDA) have become the most popular in the biomedical field; however, integrating multiple properties on a single adhesive hydrogel remains a challenge. Here, inspired by the chemistry of mussels, we developed PDA–sodium alginate–polyacrylamide (PDA–SA–PAM)-based hydrogels with multiple physiological and biological properties for skin tissue engineering applications. The hydrogels were prepared by alkali-induced polymerization of DA followed by complexation with SA in PAM networks. The chemical composition of the hydrogels was characterized by X-ray photoelectron spectroscopy. PDA–SA complexed chains were homogeneously dispersed in the PAM network and exhibited good elasticity and excellent mechanical properties, such as a compressive stress of 0.24 MPa at a compression strain of 70% for 0.4PDA–SA–PAM. The adhesive hydrogel also maintained a highly interconnected porous structure (∼94% porosity) along with PDA microfibrils. The hydrogel possesses outstanding swelling and biodegradability properties. Owing to the presence of the PDA–SA complex in the PAM network, the hydrogels show good adhesion to various substrates (plastic, skin, glass, computer screens, and leaves); for example, the adhesive strength of the 0.4PDA–SA–PAM to porcine skin was 24.5 kPa. The adhesive component of the PDA–SA chains in the PAM network significantly improves the cell proliferation, cell attachment, cell spreading, and functional expression of human skin fibroblasts (CCD-986sk) and keratinocytes. Moreover, the PDA chains exhibited good hemostatic properties, resulting in rapid blood coagulation. Considering their excellent cell affinity, and rapid blood coagulation ability, these mussel-inspired hydrogels have substantial potential for skin tissue engineering applications.

Synthesis of D,L-Lactide–ε-Caprolactone Copolymers and Preparation of Films Based on Them

D,L-Lactide–e-caprolactone copolymers were synthesized by polymerization in the bulk. Films of these copolymers were prepared, and their strain properties and degradation rate under physiological conditions were studied. Introduction of e-caprolactone into the copolymer increases the film elasticity and decreases the degradation rate compared to poly(D,L-lactide) homopolymer, and also favors spreading of human keratinocytes. The films obtained can be used for treatment of wounds and burns.

Fibrin gel as a scaffold for skin substitute – production and clinical experience.

The purpose of this study was to create a fibrin-based human skin substitute in vitro with epidermal and dermal component and to assess its healing potential in deep partial and full thickness burns. Fibrin scaffolds were prepared from commercial fibrin glue kits. Human fibroblasts were cultured in fibrin gel. Human keratinocytes were seeded on the top of the gel. Viability of cells was determined fluorimetrically. Scanning electron microscope and immunocytochemistry analysis of cultured cells were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for skin substitutes. Progress of healing was documented using visual estimation and photos. Scanning electron microscope images showed good cell attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaff old. Immunofluorescent staining of cell cultures on fibrin scaffold showed expression of vimentin, a marker of fibroblast cells, cytokeratin 19, a marker of epithelial stem cells, as well as involucrin, a marker of differentiated keratinocytes. Clinical results clearly showed that appearance of the skin did not differ significantly from the areas of transplanted skin using split-thickness skin graft techniques. In conclusion, using these fibrin-cultured autografts on massive full-thickness burn resulted in good healing.

The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites.

PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN.

Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films

Bacterial cellulose-chitosan (BC-C) films were developed by immersing purified BC pellicles in 1.5 ~ 2.0% (w/v) acetic acid solutions containing chitosan of varying molecular weights. Effects of different molecular weight of chitosan on physical, biological and antimicrobial properties of the composite films were investigated. The cumulative chitosan absorption capacities with Mw of 141,000, 199,000, and 263,000 were 38.43, 24.65, and 23.89 mg/cm3 of dry BC film, respectively. The cumulative release profiles of chitosan from the films strongly depended on molecular weight of chitosan and pH of solution. The order of release of chitosan from the BC-C films was dependent on molecular weight as follows: Mw 141,000 > Mw 199,000 > Mw 263,000. All BC-C films showed the antimicrobial abilities against Staphylococcus aureus and Aspergillus niger but had no inhibitory effect on the growth of Escherichia coli. The BC-C films supported for adhesion, spreading and proliferation of both human skin keratinocytes and fibroblasts. The antibacterial activity against S. aureus of the BC-C with the highest Mw chitosan (263,000) was higher than those of the others. On the other hand, the BC-C films with the lowest Mw chitosan (141,000) promoted the growth of human skin cells more than those of the others.

Human Neutrophil Defensins and Their Effect on Epithelial Cells

The aims of the present study include: 1) to localize human neutrophil defensin-1 (HNP-1) through HNP-3 in gingiva and in neutrophil extract-treated epithelial cell monolayers; 2) to determine the effects of HNP-1 on the epithelial cell viability, attachment, and spreading; and 3) to analyze the effect of HNP-1 on the bacterial adherence to epithelial cells. For localization of HNP-1 through HNP-3 in gingival tissue and in preincubated cell monolayers, immunohistochemical and immunocytochemical methods were used. Viability and proliferation of epithelial cells were determined with commercial kits after incubating the keratinocytes with different HNP-1 concentrations (low, 1 to 5 μg/mL; moderate, 10 μg/mL; high, 20 to 50 μg/mL). Attachment and spreading of keratinocytes on fibronectin-coated surfaces in the presence of HNP-1 were evaluated under microscope. Attachment of Fusobacterium nucleatum ATCC25586 and Prevotella intermedia ATCC25611 on keratinocytes preincubated with HNP-1 were determined with a standard antibiotic test. HNP-1 through HNP-3 localized in the junctional epithelium of clinically healthy gingiva and in the pocket epithelium of gingiva with periodontitis. When keratinocyte monolayers were incubated with neutrophil extracts, HNP-1 through HNP-3 were bound to the periphery of the growing cell colonies. In low HNP-1 concentrations, the keratinocyte proliferation enhanced. Moderate and high concentrations of HNP-1 increased the cellular death significantly. HNP-1 increased the attachment and spreading of keratinocytes on fibronectin-coated surfaces and bacterial attachment to keratinocytes in a concentration-dependent manner. HNP-1 plays a role in the integrity of keratinocytes by stimulating their proliferation, attachment, and spreading, whereas higher doses increase the bacterial attachment and keratinocyte death.

Mathematical Modeling of the Impact of Actin and Keratin Filaments on Keratinocyte Cell Spreading

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Collagen XXIII, Novel Ligand for Integrin α2β1 in the Epidermis

Cellular receptors for collagens belong to the family of β(1) integrins. In the epidermis, integrin α(2)β(1) is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α(2)β(1) receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α(2)β(1) in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α(2)β(1).

Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of β1 integrin

Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R(-/-) mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R(-/-) keratinocytes, and 4.1R(-/-) mice exhibited defective epidermal wound healing. Cultured 4.1R(-/-) keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin.

Cell Compatibility of Fibrin Sealants: In Vitro Study with Cells Involved in Soft Tissue Repair

Fibrin sealants can be used to support tissue regeneration or as vehicles for delivery of cells in tissue engineering. Differences in the composition of fibrin sealants, however, could determine the success of such applications. The results presented in this article show clear differences between Fibrin sealant A (FS A) clots and Fibrin sealant B (FS B) clots with respect to their compatibility with primary human cells involved in soft tissue repair. FS A clots, which are characterized by a physiological coarse fibrin structure, promoted attachment, spreading, and proliferation of keratinocytes, fibroblasts, and endothelial cells. In contrast, FS B clots displaying a fine to medium clot structure failed to support spreading of all three cell types. Adhesion of keratinocytes was decreased on FS B clots compared to FS A clots after 3 h incubation, whereas number of attached fibroblasts and endothelial cells was initially comparable between the two fibrin sealants. However, all three cell types proliferated on FS A clots but no sustained proliferation was detected on FS B clots. We further demonstrate that the observed differences between FS A and B clots are partly based upon 1 M sodium chloride extractable constituents, like thrombin, and partly on nonextractable constituents or the fibrin structure. In conclusion, our in vitro results demonstrate that FS A clots serve as a provisional matrix that encourages adhesion and growth of keratinocytes, fibroblasts, and endothelial cells. Therefore, FS A seems to be well suited for applications in tissue engineering.

Gq-coupled Purinergic Receptors Inhibit Insulin-like Growth Factor-I/Phosphoinositide 3-Kinase Pathway-dependent Keratinocyte Migration

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

Fabrication and characterization of 3-dimensional PLGA nanofiber/microfiber composite scaffolds

Novel three-dimensional (3-D) nano-/microfibrous poly(lactic- co-glycolic acid) (PLGA) scaffolds were fabricated by hybrid electrospinning, involving a combination of solution electrospinning and melt electrospinning. The scaffolds consisted of a randomly oriented structure of PLGA microfibers (average fiber diameter = 28 μm) and PLGA nanofibers (average fiber diameter = 530 nm). From mercury porosimetry, the PLGA nano-/microfiber (10/90) scaffolds were found to have similar pore parameters to the PLGA microfiber scaffolds. PLGA nano-/microfibrous scaffolds were examined and compared with the PLGA microfiber scaffolds in terms of the attachment, spreading and infiltration of normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF). The cell attachment and spreading of both cell types were several times higher in the nano-/microfiber composite scaffolds than in the microfibrous scaffolds without nanofibers. This shows that the presence of nanofibers enhanced the attachment and spreading of the cells on the nano-/microfiber composite scaffolds. Moreover, the nanofibers helped the cells infiltrate easily into the scaffolds. Overall, this novel nano-/microfiber structures has great potential for the 3-D organization and guidance of cells provided for tissue engineering.

Nanotextured titanium surfaces for enhancing skin growth on transcutaneous osseointegrated devices

A major problem with transcutaneous osseointegrated implants is infection, mainly due to improper closure of the implant–skin interface. Therefore, the design of transcutaneous osseointegrated devices that better promote skin growth around these exit sites needs to be examined and, if successful, would clearly limit infection. Due to the success already demonstrated for orthopedic implants, developing surfaces with biologically inspired nanometer features is a design criterion that needs to be investigated for transcutaneous devices. This study therefore examined the influence of nanotextured titanium (Ti) created through electron beam evaporation and anodization on keratinocyte (skin-forming cell) function. Electron beam evaporation created Ti surfaces with nanometer features while anodization created Ti surfaces with nanotubes. Conventional Ti surfaces were largely micron rough, with few nanometer surface features. Results revealed increased keratinocyte adhesion in addition to increased keratinocyte spreading and differences in keratinocyte filopodia extension on the nanotextured Ti surfaces prepared by either electron beam evaporation or anodization compared to their conventional, unmodified counterparts after 4 h. Results further revealed increased keratinocyte proliferation and cell spreading over 3 and 5 days only on the nanorough Ti surfaces prepared by electron beam evaporation compared to both the anodized nanotubular and unmodified Ti surfaces. Therefore, the results from this in vitro study provided the first evidence that nano-modification techniques should be further researched as a means to possibly improve skin growth, thereby improving transcutaneous osseointegrated orthopedic implant longevity.

017 Laminin 10 in Wound Reepithelization and Angiogenesis

Extracellular matrix laminins are the major noncollagenous components in the basement membrane zones (BMZ) and influence a variety of important biological processes including cell attachment, migration, and tissue development. A newly discovered laminin family member, laminin 10 (alpha 5, beta 1, and gamma 1), is found a major laminin present in the BMZs at the dermal-epidermal junction and dermal microvascular vessels. We examined the roles of laminin 10 in cutaneous wound repair in vitro and in vivo. Purified laminin 10 protein promoted human keratinocyte spreading and attachment, which is mediated by integrins. In human wound lesions, immunoperoxidase staining, with antibody 4C7 specific to laminin alpha 5 chain, stained strongly positive for laminin 10 in newly formed BMZs of epidermis and dermal blood vessels. Using a laminin 10 alpha 5 chain knockout mouse, excisional wounds in this model had abnormal reepithelialization and decreased granulation tissue formation. Transmission electron microscopy analysis of the BMZ of the mutant skin showed discontinuous lamina densa formation in areas between hemidesmosomes. In a porcine burn wound model, Reverse Transcription and Polymerase Chain Reaction analysis found significant higher expression of laminin 10 shortly after wounding and persisted through day 21, peaking at day 10. This expression pattern is consistent with the reconstitution of BMZs in the epidermis and microvascular blood vessels. These studies indicate that laminin 10 plays important roles in wound healing by promoting cell migration, BMZ assembly, reepithelialization, and angiogenesis. This study was supported by grants from NIH/NIAMES, DOD/DARPA and the Dermatology Foundation of South Florida.

Gα(q/11)‐coupled P2Y2nucleotide receptor inhibits human keratinocyte spreading and migration

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

Interaction of cultured skin cells with the polylactide matrix coved with different collagen structural isoforms

These experiments were done to optimize the surface properties of polylactide film designed for human keratinocyte cultivation. The film was covered with type 1 collagen to increase its hydrophilic properties. The method of protein application determines the consistency the matrix coating and the formation of different collagen structures on the film. Substrates modified by collagen affect the growth of cultured keratinocytes. Our data on keratinocyte spreading, morphology and cytoskeleton organization demonstrate that the best form of collagen to use to cover the polymeric surface is the fibrillar isoform.

Integrin α3β1-Dependent Activation of FAK/Src Regulates Rac1-Mediated Keratinocyte Polarization on Laminin-5

Upon epidermal wounding, keratinocytes at the wound edge become activated, deposit newly synthesized laminin-5 into the extracellular matrix, and migrate into the wound bed. The interaction between integrin alpha3beta1 and laminin-5 is essential for establishment of a stable, leading lamellipodium and persistent keratinocyte migration. We previously showed that integrin alpha3beta1 activates the Rho family GTPase Rac1 and regulates Rac1-dependent formation of polarized, leading lamellipodia in migrating keratinocytes. In the present study, we explored the role of focal adhesion kinase (FAK) and src signaling in this process. We show that overexpression of the FAK inhibitor FAK-related non-kinase or of the FAK(Y397F) auto-phosphorylation mutant, induced abnormal, non-polarized spreading of keratinocytes on laminin-5. Integrin alpha3beta1 was required for full FAK auto-phosphorylation at Y397, and subsequent src kinase-dependent phosphorylation of FAK at residues Y861 and Y925, sites responsible for promoting signal transduction downstream of FAK, indicating that alpha3beta1 regulates the coordination of FAK/src signal transduction. Inhibiting either src kinase activity or FAK signaling interfered with alpha3beta1-mediated Rac1 activation and polarized cell spreading. These findings reveal a novel pathway in migratory keratinocytes wherein alpha3beta1-laminin-5 interactions regulate src kinase signaling through FAK, promoting Rac1 activation and polarized lamellipodium extension.

Growth of Human Keratinocytes and Fibroblasts on Bacterial Cellulose Film

Thin films of bacterial cellulose (BC) from a nata de coco culture system were developed, characterized, and investigated for the growth of human keratinocytes and fibroblasts. The average pore diameter and total surface area of the dried BC films estimated by BET were 224 A and 12.62 m(2)/g, respectively. With an film thickness of 0.12 mm, the average tensile strength and break strain of the dried films were 5.21 MPa and 3.75%, whereas those of the wet films were 1.56 MPa and 8.00%, respectively. The water absorption capacity of air-dried film was 5.09 g water/g dried films. For uses in the therapy of skin wounds, the potential biological mechanism of action of BC film was evaluated by using human keratinocytes and fibroblasts. Our results were the first direct demonstration that BC film supported the growth, spreading, and migration of human keratinocytes but not those of human fibroblasts. Expressions of E-cadherin and the alpha-3 chain of laminin confirmed the phenotype of human keratinocytes on BC film.