Mesenchymal Cells Affect Salivary Epithelial Cell Morphology on PGS/PLGA Core/Shell Nanofibers.

Engineering salivary glands is of interest due to the damaging effects of radiation therapy and the autoimmune disease Sjögren's syndrome on salivary gland function. One of the current problems in tissue engineering is that in vitro studies often fail to predict in vivo regeneration due to failure of cells to interact with scaffolds and of the single cell types that are typically used for these studies. Although poly (lactic co glycolic acid) (PLGA) nanofiber scaffolds have been used for in vitro growth of epithelial cells, PLGA has low compliance and cells do not penetrate the scaffolds. Using a core-shell electrospinning technique, we incorporated poly (glycerol sebacate) (PGS) into PLGA scaffolds to increase the compliance and decrease hydrophobicity. PGS/PLGA scaffolds promoted epithelial cell penetration into the scaffold and apical localization of tight junction proteins, which is necessary for epithelial cell function. Additionally, co-culture of the salivary epithelial cells with NIH3T3 mesenchymal cells on PGS/PLGA scaffolds facilitated epithelial tissue reorganization and apical localization of tight junction proteins significantly more than in the absence of the mesenchyme. These data demonstrate the applicability of PGS/PLGA nanofibers for epithelial cell self-organization and facilitation of co-culture cell interactions that promote tissue self-organization in vitro.

Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization.

Development of electrospun nanofibers that mimic the structural, mechanical and biochemical properties of natural extracellular matrices (ECMs) is a promising approach for tissue regeneration. Electrospun fibers of synthetic polymers partially mimic the topography of the ECM, however, their high stiffness, poor hydrophilicity and lack of in vivo-like biochemical cues is not optimal for epithelial cell self-organization and function. In search of a biomimetic scaffold for salivary gland tissue regeneration, we investigated the potential of elastin, an ECM protein, to generate elastin hybrid nanofibers that have favorable physical and biochemical properties for regeneration of the salivary glands. Elastin was introduced to our previously developed poly-lactic-co-glycolic acid (PLGA) nanofiber scaffolds by two methods, blend electrospinning (EP-blend) and covalent conjugation (EP-covalent). Both methods for elastin incorporation into the nanofibers improved the wettability of the scaffolds while only blend electrospinning of elastin-PLGA nanofibers and not surface conjugation of elastin to PLGA fibers, conferred increased elasticity to the nanofibers measured by Young's modulus. After two days, only the blend electrospun nanofiber scaffolds facilitated epithelial cell self-organization into cell clusters, assessed with nuclear area and nearest neighbor distance measurements, leading to the apicobasal polarization of salivary gland epithelial cells after six days, which is vital for cell function. This study suggests that elastin electrospun nanofiber scaffolds have potential application in regenerative therapies for salivary glands and other epithelial organs. STATEMENT OF SIGNIFICANCE: Regenerating the salivary glands by mimicking the extracellular matrix (ECM) is a promising approach for long term treatment of salivary gland damage. Despite their topographic similarity to the ECM, electrospun fibers of synthetic polymers lack the biochemical complexity, elasticity and hydrophilicity of the ECM. Elastin is an ECM protein abundant in the salivary glands and responsible for tissue elasticity. Although it's widely used for tissue regeneration of other organs, little is known about its utility in regenerating the salivary tissue. This study describes the use of elastin to improve the elasticity, hydrophilicity and biochemical complexity of synthetic nanofibers and its potential in directing in vivo-like organization of epithelial salivary cells which helps the design of efficient salivary gland regeneration scaffolds.

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts.