Introduction
Salivary glands play an essential role in maintaining oral homeostasis by secreting saliva under unstimulated (resting) and stimulated (neuronal-regulated) conditions. For example, saliva functions as a barrier for bacterial infestations due to its antibacterial composition. It also plays the essential roles of oral lubricant, protecting tooth enamel, providing enzymes for digestion of food, and is an indicator of overall health. Unfortunately, cancer treatments (radiation and/or chemotherapy), certain drug medications, and/or immune system disorders, such as Sjögren syndrome, can all lead to severe hyposalivation. Radiation in particular propagates a multitude of chronic oral health-related ailments such as xerostomia or dry mouth, which entails difficulty in swallowing, increased risk for dental caries, oral fungal infections, and overall poor quality of life. Combatting these conditions and/or syndromes requires a deep understanding of factors and mechanisms regulating glandular physiology, development, homeostatic maintenance and/or repair by stem/progenitor cells, which are described below.
Salivary Gland Biochemistry and Physiology
Both humans and mice harbor three major salivary glands: submandibular (SMG), sublingual (SLG), and parotid (PAR) glands, and numerous minor salivary glands (see Chapter 1 ). Unlike the human situation ( Fig. 2.1A ), the SLG and SMG in the rodent model are found together in the anterior space of the neck, encapsulated with common fascia ( Fig. 2.1B ). Rodent SMGs are triangular in shape and the round-shaped SLGs are located on the latero-rostral one fourth of the SMG. Rodent PAR glands are found embedded in subcutaneous adipose tissue posterior inferiorly to the ear, bordering the submandibular gland ( Fig. 2.1B ). While minor salivary glands in mice have not been well described, there are seven subtypes of human minor salivary glands: the buccal, incisive, labial, anterior and posterior lingual, molar, and palatine glands. These minor salivary glands are labeled based on their anatomic location in the oral cavity.
In both the human and murine situation, the three major salivary glands are responsible for producing >90% of total saliva. Less than 10% of saliva is secreted from mucosal minor salivary glands. Saliva secreted from the minor salivary glands is mostly mucosal in content, which serves mainly as a protective lubricant. More specifically, saliva from minor salivary glands, except the von Ebner glands, plays a crucial role in dental biofilm formation, which coats the oral and tooth enamel surfaces. Cumulative saliva secretion from major salivary glands is unique in composition per gland. SMG, SLG, and PAR glands are composed of acinar cells that are secretory units responsible for serous or mucous saliva production. Every acinar cell produces saliva composed of proteins, enzymes, ions, and water, and secretes it into the central lumen. The serous and mucous acini (cluster of acinar cells) can be characterized based on their secretion of specific types of granules. Mucous-rich secretory acini contain granules with large amounts of mucin-type glycoproteins, which compose the viscous saliva. Serous-rich acini contain granules high in ions, water, amylases, secretory immunoglobulins, and proline-rich proteins, but very low levels of mucin-type glycoproteins. As such, both types of acini contribute differently to the overall functions of saliva. Mucous rich saliva acts more as a lubricant and protective barrier for the oral cavity. In contrast, serous saliva mainly aids in food digestion. Histologically, the human PAR glands are exclusively composed of serous acini. In contrast, the SLGs are predominantly made of mucous acini. The SMGs contain a mix of serous and mucous acini where variable levels of serous acini may be dominating.
Also part of the epithelial compartment are contractile myoepithelial cells, which surround the acini and the ducts that directly connect to the acini ( Fig. 2.2 ). Both murine and human salivary glands comprise of three major ductal structures: intercalated (ID), striated (SD), and excretory ducts (ED). Ducts play a large role in modifying saliva before it is secreted to the oral cavity. IDs are connected directly to acini from which they receive primary saliva. Saliva is then pushed to the lumen of SDs for ion reabsorption and then transported to the EDs for excretion to the oral cavity via an additional connecting duct ( Fig. 2.2A ). More in-depth histologic information can be found in Chapter 5 .
Initial saliva by acini is generated by the transmission of water via the basement membrane and aquaporin-5 water channels. This electrochemical gradient is maintained by the adenosine-triphosphatase (ATPase) and sodium-potassium pumps in the basolateral membrane. In brief, the acinar cells are concentrated in K + and Cl − above the electrochemical equilibrium. Autonomic neuronal stimulation opens Ca +2 sensitive ion channels, allowing permeability of potassium ions to the basolateral membrane and interstitium, as well as chloride ions into the lumen of acini. The negative charge created by the Cl − draws Na + through the tight junctions between acinar cells to the lumen. As a result of NaCl accumulation in the lumen, water moves to the lumen via osmotic pressure ( Fig. 2.2B ). While this is a brief and more commonly accepted method of ion transfer in the acinar cells, a more detailed description on additional exchange mechanisms has been previously described.
Apart from the electrochemical gradient that allows for transport of saliva, myoepithelial cells also contribute in an indirect manner. Myoepithelial cells surrounding the acini can contract to push the saliva out, and aid in the movement of saliva to the connected ducts where the saliva gets modified. IDs contain microvilli projections facing the lumen where it aids in initiating the absorption of a small portion of chloride ions out of the acinar product, thereby changing the electrochemical gradient of saliva. The second intraglandular duct, the striated duct, functions in regulating the secretion and absorption of electrolytes. In a bidirectional way, it absorbs sodium chloride (NaCl) and secretes potassium (K + ) and bicarbonate (HCO 3 − ). Saliva is then transported to the lumen of excretory ducts, where there is a small contribution to the ion exchange ( Fig. 2.2C ). This entire process makes the initial isotonic saliva, which contains similar ionic concentrations as plasma, into a hypotonic fluid before it enters the mouth. The latter is established through three major ducts: Bartholin’s, Stensen’s, and Wharton’s from the SLG, PAR, and SMG, respectively. It is worth noting that ducts are water-impermeable, thereby limiting fluid release in injured states, such as radiation-induced xerostomia, in which acini bundles are lost and several ducts remain. A current gene therapy clinical trial with water-channel aquaporin-1 gene (AQP-1) is making segue into delivering long-term water release from remaining ducts in irradiated glands.
Rodent glands are similar to human glands in terms of their histologic appearance and physiologic functions. An exception to this is the SMG, which is predominantly comprised of serous acini. Also, an additional granular convoluted tubule (GCT) is found between the ID and SD in rodent glands, which secretes growth factors into the saliva. It is important to note, for research purposes, that sexual differences occur in rodents where GCTs are more abundant in males compared with females. Such dimorphism has not been reported for the human system.
While cellular entities play unique roles in manipulating and excreting saliva, regulation of saliva flow and composition is mediated by the autonomic nervous system. Both sympathetic and parasympathetic innervate the salivary glands with the parasympathetic system playing a dominant role. In both human and rodent glands, it was noted that the parasympathetic nerves are present at gland ontogenesis, while the sympathetic nervous system innervates the gland later when branching, cell differentiation, and lumen formation has been initiated. Hence, stimulus for glandular development, growth, vasodilation, and saliva formation and flow are mediated by parasympathetic innervation. In contrast, exocytosis, protein composition, and secretion are dependent on sympathetic innervation. While these systems have unique roles, both can have additive and synergistic effects on fluid secretion by contraction of myoepithelial cells. In brief, saliva flow is regulated by neurotransmitter receptors on the basolateral membranes of the acini and ducts. More specifically, neurotransmitter acetylcholine binds to the muscarinic cholinergic M 3 receptors. This leads to the activation and binding of heterotrimeric guanine nucleotide-binding proteins. The ultimate product is formation of inositol triphosphate and the release of a second messenger, Ca +2 . Ca +2 plays an intimate role in saliva and ion flow to the ductal lumen. Additional movement of Ca +2 is induced by norepinephrine binding to alpha-receptors. While Ca +2 release via acetylcholine parasympathetic stimulation induces saliva flow, beta-adrenergic sympathetic stimulation from binding of norepinephrine to beta-adrenergic receptors mediates saliva composition and exocytosis. In brief, it activates the second messenger, cAMP, leading to exocytosis of proteins such as amylases and mucins. The sympathetic nerves also align with the blood vessels to induce vasoconstriction, although this does not influence the saliva reflex.
Salivary Gland Embryogenesis
Ontogenesis of all glands is initiated by interactions between epithelial cells from the oral lining and the surrounding environment ( Table 2.1 ). In short, a thickening of the oral epithelium (termed “bud”) in the cheek towards the ear initiates the development of the PAR at 5–6 weeks of intrauterine gestation. In humans, this is the first gland to initiate and will form the largest one (25–30 g). Canalization of the PAR is completed by gestational age of 6 months. The ontogenesis of the second largest gland, the SMG (7–15 g), starts at gestational weeks 6–7. Hereto, epithelial outgrowth into the mesenchyme starts at the floor of the mouth where it rapidly proliferates to form numerous branching structures. Lastly, epithelial thickening for the SLG (3 g weight in adulthood) is initiated at gestational week 7–8 in the linguogingival groove, leading to individual canals from the small epithelial thickenings. Minor glands develop later around the 3rd month of gestation. The time frame for gland ontogenesis in rodents is different. For example, mouse SMG initiates first around embryonic day (E)11.5, and the SLG and PAR follow later at E12.5 and E13.5. Interestingly, it is still debatable whether the epithelia of the salivary glands are ectodermal or endodermal in origin. At least in the mouse model, research revealed that the major glands are not derived from the ectoderm. In contrast, some minor mucous glands of the tongue and palate were fully or partially ectodermal-derived, respectively.
