Tissue engineering of a salivary gland requires functional cells that express the vital proteins needed to restore secretion. Here, we report a technique for isolation and maintenance of human salivary gland cells and their differentiation via a human sequence in perlecan that allows for self-assembly of these cells into functional acini-like units, suitable for tissue engineering. We envision that the salivary acinar cells will grow and differentiate, initially, in an artificial scaffold and eventually fully integrate into the patients' subcutaneous buccal mucosa or native salivary gland tissue.
To recreate the native structure of the salivary gland in vitro, biomarkers from the salivary gland tissue were compared in parallel with cells isolated from specimens of the glandular tissue. Tissue cryosections from the human parotid and submandibular glands were obtained for initial histological and immunohistochemical studies. Parallel biomarker studies with CK 19 and α-amylase revealed the presence of ductal and acinar cells, respectively, in both salivary gland tissue and primary cell cultures. Myoepithelial cells were identified in the tissue; however, negative staining for CK 14 revealed their absence from primary cell cultures. Although mixed cultures of acinar and ductal cells were isolated initially, novel culturing techniques were used to obtain acinar-enriched cultures.
The basement membrane, one of the most important components of the ECM of polarized epithelial cells, directs the growth and differentiation of human epithelia.13
In addition to separating the parenchymal cells and connective tissues, the basement membrane also is involved in encasing, organizing, and guiding the behavior of the parenchymal cells that reside on it.14
In the case of the salivary epithelium, a major function of the basement membrane is maintenance of directional secretion. Abundant expression of perlecan and laminin in the tissue reveals the importance of these proteins for maintaining the phenotype of salivary gland cells. The functionality of the cultured salivary acinar cells in vitro
is confirmed by their secretion and organization of their own basement membrane including the critical component perlecan that can support further growth and differentiation by virtue of its ability to interact with cell adhesion molecules that can influence integrin-mediated events.11,15
Uniquely, no non-human-derived material was needed for isolation or culture of the acinar cells.
To aid further differentiation of salivary gland cells into organized glandular structures that express essential biomarkers, acinar cells were cultured on ECM proteins such as perlecan, laminin, and the commonly used BME, Matrigel. Because full-length perlecan cannot be produced in recombinant form because of its large size, and because we wished to avoid animal proteins, human-compatible PlnDIV peptide sequence was used in studies involving perlecan. Evaluation of the behavior of acinar cells grown on Matrigel, laminin, or PlnDIV peptide showed no differences in growth and proliferative capabilities of cells. Morphological analysis revealed that Matrigel and PlnDIV peptide supported gradual differentiation of the cells. Lobe formation with interconnections were observed in cells grown on Matrigel as well as on PlnDIV peptide. Differentiated structures self-assembled into acini-like structures that are reminiscent of salivary units.
α-Amylase, one of the major proteins in saliva that helps the breakdown of starches and initiates digestion, is highly produced and secreted by acinar cells. Differentiated acini-like structures mimicked salivary units and produced and secreted α-amylase. Additionally, α-amylase secretion from differentiated structures increased over time, as seen by amylase activity measurements, suggesting development of functional salivary units.
Tight junctions, vital components in polarized secretory epithelia, hold cells together and form a barrier to maintain transcellular transport. E-cadherin expression confirmed formation of tight junctions in acinar cell cultures grown on plastic, Matrigel, as well as PlnDIV peptide. Additional markers of polarization include AQP, which are important indicators of fluid secretion and play an essential role in saliva production. Lack of AQP5 decreased rates of salivary secretion in mice.16,17
AQP5 expression in apical membrane of acini in salivary gland tissue revealed their secretory abilities. Because epithelial cells must be polarized to secrete fluid unidirectionally, the expression of AQP5 at sites of self-assembly in cultured cells revealed their polarization and secretory potential. Additionally, recent reports have shown that activation of muscarinic acetylcholine receptors is important for translocation of AQP5 to the apical plasma membrane of salivary gland cells.18,19
Self-assembled and differentiated acinar cells expressing AQP5 might receive appropriate signals from stimulatory receptors for AQP5 translocation.
For cells to differentiate and self-assemble, they must receive appropriate signals from the ECM. Stress fiber formation and signals generated from cytoskeletal tension provide essential cues for dynamic cellular organization into higher-order structures.20
Formation of ordered F-actin stress fibers and activation of FAK also are crucial for cell migration.21
The organization of dense stress fiber networks and filopodia in acinar cells cultured on Matrigel or PlnDIV peptide demonstrated their differentiation and migratory potential. Focal adhesions are dynamic protein complexes that form connections between the cell cytoskeleton and the ECM.22
Salivary gland cells grown on Matrigel or PlnDIV peptide supported FAK activation and correctly localized it to adhesion sites, reflecting signal transduction events between the ECM proteins and the cytoskeleton. FAK staining was observed in the cytoplasm of cells grown on plastic, but not many punctate focal adhesions were seen. Thus, although these cells cultured on plastic are expressing some basement membrane proteins on their own, they evidently lack contextual information required to consistently localize FAK to sites of integrin clustering. In contrast, the redistribution of phospho-FAK seen on Matrigel and PlnDIV peptide to the ends of stress fiber assemblies on filopodia can support the organized differentiation of salivary acinar cells.
Information provided by the PlnDIV peptide accelerated the self-assembly of salivary gland cells into lobular structures better than did the intact perlecan produced by the cells themselves. The intact perlecan molecule that is produced by cultured acinar cells is expected to be modified by heparan sulfate chains that in previous studies were found to conceal the adhesive domain IV motifs.11
Fibroblasts cultured on intact perlecan failed to make stable focal adhesion contacts because of the antiadhesive activity of gagosylated perlecan domain I.23
Consistent with this, our studies showed less FAK activation and, more strikingly, a failure to cluster phosphorylated FAK to sites of focal adhesions when cells were cultured on plastic. The mechanism by which PlnDIV peptide triggers self-assembly of salivary gland cells remains unknown. Our earlier studies suggest that adhesion to PlnDIV peptide is mediated through nonintegrin receptors as inhibition of β1 integrins only partially affected adhesion to surfaces modified with the peptide.11
The peptide's origin from an Ig loop on PlnDIV suggests that signaling could be induced via interactions with Ig superfamily members on the surfaces of the acinar cells through homophilic or heterophilic binding events. These interactions can initiate signaling events that alter cytoskeletal dynamics and organization leading to cell migration, differentiation, and ultimately acinar assembly.24
Prior reports of cell transplantation into animal models resulted in regeneration of ductal cells but lacked acinar cell differentiation.25
The cells characterized in this study have the potential to be functional in an artificial scaffold that provides them the appropriate conditions to differentiate. Efforts to devise a three-dimensional culture system with a biomaterial scaffold consisting of PlnDIV peptide are underway. The culture system consisting of PlnDIV peptide reported here will aid the development of an artificial salivary gland that will foster formation of functional salivary units capable of secreting salivary fluid and that can be implanted into patients to relieve xerostomia.