The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds

Department of Biological Sciences, University at Albany, State University of New York, 1400 Washington Ave, LSRB 1086, Albany, NY 12222, USA.
Biomaterials (Impact Factor: 8.56). 04/2012; 33(11):3175-86. DOI: 10.1016/j.biomaterials.2012.01.010
Source: PubMed

ABSTRACT Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.

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Available from: Melinda Larsen, Sep 26, 2015
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    • "This leads to the production of micron- and submicron-scale fibers, such that electrospun meshes are characterized by feature sizes that are much smaller than in traditionally extruded meshes. Electrospun meshes have been shown to support cell attachment, alignment, proliferation and differentiation [69,70]. In the context of tissue engineering, however, the small pores limit in vitro cell infiltration as well as in vivo tissue ingrowth and vascularization within the bulk of the electrospun scaffold (ES), as these cellular and tissue ingrowth processes require pore sizes of up to 500 µm [67,71-73]. "
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    ABSTRACT: A paradigm shift is taking place in medicine and dentistry from using synthetic implants and tissue grafts to a tissue engineering approach that uses degradable porous three-dimensional (3D) material hydrogels integrated with cells and bioactive factors to regenerate tissues such as dental bone and other oral tissues. Hydrogels have been established as a biomaterial of choice for many years, as they offer diverse properties that make them ideal in regenerative medicine, including dental applications. Being highly biocompatible and similar to native extracellular matrix, hydrogels have emerged as ideal candidates in the design of 3D scaffolds for tissue regeneration and drug delivery applications. However, precise control over hydrogel properties, such as porosity, pore size, and pore interconnectivity, remains a challenge. Traditional techniques for creating conventional crosslinked polymers have demonstrated limited success in the formation of hydrogels with large pore size, thus limiting cellular infiltration, tissue ingrowth, vascularization, and matrix mineralization (in the case of bone) of tissue-engineered constructs. Emerging technologies have demonstrated the ability to control microarchitectural features in hydrogels such as the creation of large pore size, porosity, and pore interconnectivity, thus allowing the creation of engineered hydrogel scaffolds with a structure and function closely mimicking native tissues. In this review, we explore the various technologies available for the preparation of macroporous scaffolds and their potential applications.
    Journal of periodontal & implant science 12/2013; 43(6):251-261. DOI:10.5051/jpis.2013.43.6.251 · 1.15 Impact Factor
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    • "glands ( SMGs ) were dissected from timed - pregnant female mice ( strain CD - 1 , Charles River Laboratories ) at embryonic day 12 ( E12 ) or 13 ( E13 ) with the day of plug discovery designated as E0 , in accordance with protocols approved by the University at Albany IACUC . The SMGs were cultured as described previously ( Daley et al . , 2009 ; Sequeira et al . , 2012"
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    ABSTRACT: Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK and RSK signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in cytokinesis failure. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.
    Journal of Cell Science 11/2013; 127(3). DOI:10.1242/jcs.133280 · 5.43 Impact Factor
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    • "When peptides representing the epitope were available, peptide preabsorbed antibodies were exposed to salivary gland formalin-fixed, paraffinembedded (FFPE) sections to verify disappearance of the staining pattern (data not shown). All staining patterns on FFPE sections were also verified in whole mount salivary gland tissues fixed in 4% paraformaldehyde and 5% sucrose in 16 PBS, subjected to immunocytochemistry, and imaged using laser scanning confocal microscopy (510 Meta, Zeiss or SP5, Leica) (Larsen et al., 2003; Daley et al., 2009; Daley et al., 2011; Daley et al., 2012; Sequeira et al., 2012). The antibodies used in this study are listed in Table 1, and the order in which the immunohistochemistry steps were performed is listed in supplementary material Table S1. "
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    ABSTRACT: Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells); cytokeratin 7 (ductal cells); and smooth muscle α-actin (myoepithelial cells) and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.
    Biology Open 05/2013; 2(5):439-447. DOI:10.1242/bio.20134309 · 2.42 Impact Factor
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