Swati Pradhan-Bhatt

Hyaluronan: a simple polysaccharide with diverse biological functions.

Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of d-glucuronic acid and N-acetyl-d-glucosamine. It is evolutionarily conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Being a simple polysaccharide, HA exhibits an astonishing array of biological functions. HA interacts with various proteins or proteoglycans to organize the ECM and to maintain tissue homeostasis. The unique physical and mechanical properties of HA contribute to the maintenance of tissue hydration, the mediation of solute diffusion through the extracellular space and the lubrication of certain tissues. The diverse biological functions of HA are manifested through its complex interactions with matrix components and resident cells. Binding of HA with cell surface receptors activates various signaling pathways, which regulate cell function, tissue development, inflammation, wound healing and tumor progression and metastasis. Taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, researchers have developed various HA-based biomaterials and tissue constructs with promising and broad clinical potential. This paper illustrates the properties of HA from a matrix biology perspective by first introducing the principles underlying the biosynthesis and biodegradation of HA, as well as the interactions of HA with various proteins and proteoglycans. It next highlights the roles of HA in physiological and pathological states, including morphogenesis, wound healing and tumor metastasis. A deeper understanding of the mechanisms underlying the roles of HA in various physiological processes can provide new insights and tools for the engineering of complex tissues and tissue models.

A Novel In Vivo Model for Evaluating Functional Restoration of a Tissue-Engineered Salivary Gland

To create a novel model for development of a tissue‐engineered salivary gland from human salivary gland cells that retains progenitor cell markers useful for treatment of radiation‐induced xerostomia.

Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture

Radiotherapy for head and neck cancer often has undesirable effects on salivary glands that lead to xerostomia or severe dry mouth, which can increase oral infections. Our goal is to engineer functional, three‐dimensional (3D) salivary gland neotissue for autologous implantation to provide permanent relief. An immediate need exists to obtain autologous adult progenitor cells as the use of embryonic and induced pluripotent stem cells potentially pose serious risks such as teratogenicity and immunogenic rejection. Here, we report an expandable population of primary salivary human stem/progenitor cells (hS/PCs) that can be reproducibly and scalably isolated and propagated from tissue biopsies. These cells have increased expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) compared with differentiation markers of the parotid gland (acinar: MIST1/BHLHA15 and AMY1A; ductal: K19 and TFCP2L1). Isolated hS/PCs grown in suspension formed primary and secondary spheres and could be maintained in long‐term 3D hydrogel culture. When grown in a customized 3D modular hyaluronate‐based hydrogel system modified with bioactive basement membrane‐derived peptides, levels of progenitor markers, indices of proliferation, and viability of hS/PCs were enhanced. When appropriate microenvironmental cues were provided in a controlled manner in 3D, such as stimulation with β‐adrenergic and cholinergic agonists, hS/PCs differentiated into an acinar‐like lineage, needed for saliva production. We conclude that the stem/progenitor potential of adult hS/PCs isolated without antigenic sorting or clonal expansion in suspension, combined with their ability to differentiate into specialized salivary cell lineages in a human‐compatible culture system, makes them ideal for use in 3D bioengineered salivary gland applications. Stem Cells Translational Medicine 2017;6:110–120

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function

Myoepithelial cells are flat, stellate cells present in exocrine tissues including the salivary glands. While myoepithelial cells have been studied extensively in mammary and lacrimal gland tissues, less is known of the function of myoepithelial cells derived from human salivary glands. Several groups have isolated tumorigenic myoepithelial cells from cancer specimens, however, only one report has demonstrated isolation of normal human salivary myoepithelial cells needed for use in salivary gland tissue engineering applications. Establishing a functional organoid model consisting of myoepithelial and secretory acinar cells is therefore necessary for understanding the coordinated action of these two cell types in unidirectional fluid secretion. Here, we developed a bottom-up approach for generating salivary gland microtissues using primary human salivary myoepithelial cells (hSMECs) and stem/progenitor cells (hS/PCs) isolated from normal salivary gland tissues. Phenotypic characterization of isolated hSMECs confirmed that a myoepithelial cell phenotype consistent with that from other exocrine tissues was maintained over multiple passages of culture. Additionally, hSMECs secreted basement membrane proteins, expressed adrenergic and cholinergic neurotransmitter receptors, and released intracellular calcium [Ca2+i] in response to parasympathetic agonists. In a collagen I contractility assay, activation of contractile machinery was observed in isolated hSMECs treated with parasympathetic agonists. Recombination of hSMECs with assembled hS/PC spheroids in a microwell system was used to create microtissues resembling secretory complexes of the salivary gland. We conclude that the engineered salivary gland microtissue complexes provide a physiologically relevant model for both mechanistic studies and as a building block for the successful engineering of the salivary gland for restoration of salivary function in patients suffering from hyposalivation.

Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells

Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i.e., viral vectors and genetic sequences of bacterial origin). In this study, we applied 2 emerging technologies, artificial transcriptional complexes and epigenetic editing, to explore whether AQP1 expression could be achieved by activating the native gene locus in a human salivary ductal cell line and primary salivary human stem/progenitor cells (hS/PCs), as opposed to the conventional approach of cytomegalovirus promoter-driven expression from an episomal vector. In our first study, we used a cotransfection strategy to express the components of the dCas9-SAM system to create an artificial transcriptional complex at the AQP1 locus in A253 and hS/PCs. We found that AQP1 expression was induced at a magnitude comparable to adenoviral infection, suggesting that AQP1 is primarily silenced through pretranscriptional mechanisms. Because earlier literature suggested that pretranscriptional silencing of AQP1 in salivary glands is mediated by methylation of the promoter, in our second study, we performed global, chemical demethylation of A253 cells and found that demethylation alone induced robust AQP1 expression. These results suggest the potential for success by inducing AQP1 expression in human salivary ductal cells through epigenetic editing of the native promoter.

Salivary Gland Tissue Engineering and Repair

Abstract Oral homeostasis is severely compromised upon salivary gland atrophy, which occurs after radiation therapy in head and neck cancer patients. Salivary gland hypofunction typically leads to xerostomia, dysphagia, dental caries, and other oropharyngeal infections, reducing the quality of life in afflicted patients [ 70 ]. Currently, xerostomia has no cure. Palliative therapies such as oral sialagogues and saliva substitutes only improve mild cases of xerostomia. Generation of a functional, tissue-engineered salivary gland will benefit patients suffering from xerostomia. Engineering of glandular tissue requires several essential components, including primary cells that retain biomarkers typical of the native gland, extracellular matrix proteins that can orchestrate the differentiation of primary cells into functional structures, and a scaffold that can hold these components together and recreate the microenvironment found in native glandular tissue. Additionally, vascularization and innervation must reach the implant for it to survive and function in vivo.

Salivary Gland Tissue Engineering and Future Diagnostics

Our study of salivary gland structure and function relies on our ability to study the organ in its native environment. To scale those needs to a laboratory setting, researchers have created in vitro models that replicate the salient features of the gland, but these are inevitably tied to the abilities of current technologies and may require some compromises along the way. In this chapter, we discuss key features of the gland that would be desired in a model and the potential intersection of those needs with advances in the field of tissue engineering. The application of these new technologies, along with improvements in imaging and phenotype reporting, holds the promise of significantly impacting salivary diagnostics through continual improvements in the accuracy and scalability of laboratory models.