Introduction

Xerostomia, or dry mouth, is a product of reduced quality and quantity of saliva. Common causes include physiologic mouth breathing, polypharmacy, autoimmune disease, including Sjogren disease, and radiation. Drug-induced xerostomia can occur in the elderly population treated with medications that include anticholinergics, antidepressants, or sympathomimetics. Radiation-induced xerostomia occurs in 63–93% of head and neck cancer patients who undergo radiotherapy. The resulting alterations in the rate and volume of saliva production, and salivary pH and viscosity lead to dysphagia, dysphonia, dysgeusia, halitosis, and increased oral infections, such as sialadenitis, candidiasis, and dental caries. Collectively, these result in a decline in the overall quality of life. External beam radiation to the salivary glands generates reactive oxygen species, which damage cell membranes and trigger proteolysis and leakage of granules. Irreversible changes to the muscarinic receptors, aquaporins, and parenchyma severely disrupt glandular salivary secretion. Morphologically, the damage to the salivary glands ranges from partial damage to irreversible fibrosis of the entire glandular parenchyma.

Radiation-induced xerostomia is a continuing problem, despite the interventions undertaken to reduce radiation exposure to the salivary gland, which include surgical relocation of salivary glands prior to radiation and/or intensity-modulated radiotherapy (IMRT). First-line pharmacologic treatments, such as systemic cholinergic agents pilocarpine and cevimeline, provide short-term relief if there is residual functional tissue. Patient compliance is impacted by systemic side effects. Other widely used options include topical pilocarpine, enzyme enriched salivary substitute such as Biotene gel and paste, xanthum gum, lozenges, and acupuncture, all of which are largely ineffective. Systemic injections of the chemoprotectant, amifostine and its active metabolite, WR-1065, given prior to radiotherapy, modestly improve post-radiation salivary secretion and function over time in some patients. Further, localized retrograde delivery of amifostine is being investigated with promising cellular radioprotection and fewer potential side effects. The currently available pharmacologic approaches to stimulate saliva production from residual acini and the artificial salivary substitutes remain largely ineffective. Gene therapies have included transduction of aquaporin genes via adenoviral vector, but this failed clinical trials due to immunogenicity. Safer ultrasound-enabled transfer of aquaporin gene is being explored, as well as CRISPR-Cas9-based methods. Reverse engineering a functional and implantable salivary gland has the ability to provide a permanent and promising solution to alleviate xerostomia and its devastating clinical features. This chapter briefly highlights current progress in tissue engineering approaches and salivary gland regeneration.

Tissue Engineering Inspired by Salivary Gland Structure and Function

Cells in native salivary glands are polarized and employ tight junctions to enable lumen formation and directional flow of saliva ( Fig. 53.1 ). Salivary gland-derived cells in two-dimensional (2D) cultures tend to lose these morphological features and resultant function. Newly designed scaffolds laden with extracellular matrix (ECM) peptides and growth factors have enabled the restoration of these functions. A three-dimensional (3D) fibrin hydrogel system with synthetic peptides from laminin (YIGSR and A99) induced mature lumen formation of the rat parotid cells in vitro. In addition, the rate of functional cluster formation and the morphology was also improved. Moreover, this combination of fibrin hydrogel with laminin peptides demonstrated superior and faster wound healing with functional regeneration of the mouse salivary gland compared to the fibrin hydrogel alone.

Select structural and functional markers present in human salivary gland cells. (A) In human parotid tissue, the tight junction protein occludin (green) is observed at intercellular interfaces, while laminin (red) surrounds and defines the perimeters of acinar lobules and ducts. (B) α-smooth muscle actin (SMA) (green) expression is observed in the myoepithelial cells that surround acini with thin, finger-like projections. (C) In vitro 3D culture of salivary-derived cells, encapsulated within HA hydrogels, demonstrates restoration of tight junction proteins like β-catenin (green) at cell–cell interfaces. Cell nuclei are stained blue in all three panels.

Several synthetic formulations are in development to mimic the chemical and mechanical properties of the native ECM and basement membrane (BM). Poly(lactic- co -glycolic) acid (PLGA) is a traditional scaffold material known to have poor compliance. Yet, integration of PLGA and elastin through blend-electrospinning enhanced epithelial cell reorganization and allowed for apicobasal polarization. Similarly, incorporation of poly(glycerol sebacate) (PGS) into PLGA scaffolds increased the compliance to allow cell penetration and favored similar events. Encapsulation within poly(ethylene glycol) (PEG) hydrogels, functionalized with matrix metalloproteinase (MMP) degradable sites, promoted proliferation and maintained acinar markers in the encapsulated primary epithelial cells. This system allowed for the expression of ECM proteins collagen IV and laminin, and emphasized the need to formulate biodegradable matrices to maintain tissue characteristics.

Stem Cell Sources for Salivary Gland Regeneration

Attempts have been made to isolate human stem/progenitor (S/P) cells to serve as building blocks that can be induced to differentiate into functional neotissue. Label-retaining cell studies and in vitro organ morphogenesis reveal several S/P cell populations in salivary glands that can differentiate into functional acinar and ductal cells. Mature, terminally differentiated acinar cells can self-duplicate, potentially maintaining salivary gland homeostasis. A cell-based approach demonstrated that 100–300 cKit+ murine stem cells isolated from salisphere suspension cultures rescued salivary gland hyposalivation using an irradiated mouse model. cKit+ cells isolated from human salivary gland tissue also demonstrated similar stem cell properties and differentiated in vitro into acinar and ductal lineages. Intraglandular injection of salisphere-derived cKit+ cells rescued saliva production after irradiation and improved tissue homeostasis. Salisphere culture for isolation of S/P cells together with the ability of cKit+ cells for functional differentiation into multilineage organoids is a promising cell-based approach for salivary gland regeneration. Salivary gland morphogenesis studies revealed another stem cell population of SOX2+ cells, essential for acinar lineage maintenance along with parasympathetic nerve supply.

Earlier work demonstrated the isolation and culture of human salivary epithelial cells and differentiation into salivary units with the expression of α-amylase, aquaporin 5, and tight junction proteins in a collagen and/or Matrigel based 3D culture system. While most studies used murine xenogeneic Matrigel that cannot be used in humans, a human compatible explant culture system was established to obtain scalable S/P cells without antigenic sorting. The isolated cells expressed and maintained stem cell markers K5, K14, MYC, ETV4, ETV5 even with long-term cultures either in 2D monolayer or in a 3D system ( Fig. 53.2 ). These cells self-organized into organoids when grown in hyaluronate (HA)-based hydrogels and differentiated into acinar-like cells after neurotransmitter stimulation. More importantly, these encapsulated 3D structures retained their acini-like assembly and were viable with salivary biomarkers for 3 weeks in vivo in an athymic rat model. The contractile myoepithelial cells were separately isolated and characterized for co-culture with the preassembled acinar spheroids. The formation of secretory microtissue complexes and the integration of the implanted cell-laden hydrogel scaffolds with the neighboring tissue in the parotid resection rodent model are promising steps towards engineering an implantable salivary gland. Combining a mitotically active feeder layer of mesenchymal stem cells (MSCs) with laminin can enhance the organization of cell clusters and lumen formation presumably by releasing the necessary soluble factors. A separate study demonstrated the potential of MSC feeder cells to promote self-organization and early branch-like structures in 3D cultures of submandibular epithelial cells. Another study suggested that co-culture of the epithelial cells with MSCs could be a viable strategy to induce the apical localization of the tight junction, an essential event for the initiation of polarity. Studies also evaluated the application of embryonic stem cells (ESCs) as an alternate for autologous S/P cells in salivary gland regeneration. However, the concerns related to ethics, teratogenicity, and immunogenicity associated with ESC precludes their application in the near horizon.

Observed expression of K5 (red) and K14 (green) in (A) human parotid gland tissue; (B) 2D culture of salivary-derived human stem/progenitor cells (hS/PCs); (C) 3D culture of hS/PCs in peptide-functionalized HA hydrogels. Each panel is presented as an overlay; green and red signals overlay to give a yellow composite, demonstrating that >99% of K14+ cells are also K5+. Insets show just the K5 red signal, for reference. Cell nuclei are stained blue.

Direct injection of stem cells into a fibrotic, radiation-damaged salivary bed may not provide the ideal tissue microenvironment needed to support long-term salivary gland restoration in vivo. To recreate the native environment in structure and function, several biomimetic scaffolds have been designed with the incorporation of ECM and BM peptides and components. These ECM and BM proteins play vital roles in regulating several cellular signaling and functions to promote organ morphogenesis, proliferation, adhesion, migration, and apico-basal organization. A 3D hydrogel system decorated with two different acrylated peptides from BM proteins laminin (YIGSR, IKVAV) and perlecan/HSPG2 (perlecan domain IV) remarkably increased the proliferation rate, viability, and importantly, the progenitor markers in the human salivary spheroids.