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Salivary gland destruction occurs as a result of various pathological conditions such as radiation therapy for head and neck cancer and Sjögren's syndrome. As saliva possesses self-cleaning and antibacterial capability, hyposalivation is known to deteriorate dental caries and periodontal disease. Furthermore, hyposalivation causes mastication and swallowing problems, burning sensation of the mouth and dysgeusia. Currently available treatments for dry mouth are prescription for artificial saliva, moisturizers and medications which induce salivation from the residual tissue. Unfortunately, these treatments cannot restore the acini functions. This review focuses on various efforts to restore the function of damaged salivary gland. First, the possibility of salivary gland regeneration and tissue engineering is discussed with reference to stem cells, growth factors and scaffold materials. Second, the current status of gene transfer to salivary glands is discussed.

Patients with dry mouth have been treated with salivary substitutes and/or medications such as pilocarpine or cevimeline hydrochloride. These treatments temporarily relieve their symptoms and induce salivation from residual tissue. However, no treatment is available for the purpose of regenerating an atrophic gland. In this study, the feasibility of a cell transplantation therapy for the atrophic submandibular glands was investigated in rats. Further, the potential of cell differentiation into a useful phenotype was assessed by immunohistochemistry together with cell tracking with the fluorescent dye PKH 26. Rat submandibular glands were excised, and the salivary gland epithelial cells were cultured for 3 weeks with 3T3 cells as a feeder layer. Ductal ligation of the submandibular gland was employed to generate an atrophic gland. One week after the operation, the ligation was removed, and the cultured cells labeled with PKH 26 were injected into the atrophic submandibular glands. As a control, the cultured cells were also injected into normal submandibular glands. Two weeks after cell transplantation, the transplanted cells were detectable in both the experimental and control groups. The cells were clustered in the connective tissue between the lobules. Four weeks after transplantation, the labeled cells were detectable in the experimental group but not in the control group. In the atrophic glands, the scattered transplanted cells were observed over a broad area of the gland but localized mainly around the acini and ductal region. Immunostaining results showed a possible involvement of the transplanted cells in ductal regeneration, while neither myoepithelial nor acinar differentiations were observed within the 4 weeks since transplantation. This study demonstrated that cell transplantation to the salivary gland is feasible, and that the transplanted cells were selectively attracted to and remained in the damaged area without affecting normal tissue.

The salivary gland is considered to be a reservoir of many growth factors in rodents. In humans, the epidermal growth factor, basic fibroblast growth factor, and insulin and insulin-like growth factor family have also been detected in this gland, but their physiological role remains unclear. In this study, we focused on bFGF, which is a well-known mitogen for various types of cells, and is present in the salivary gland as well as in saliva. The roles of bFGF in the salivary gland were investigated by three different procedures. First, the effects of bFGF on the salivary gland cells were investigated with a monolayer culture of normal submandibular gland cells. The effects of different concentrations of bFGF on the second passage of these cultured cells were examined. In both human and rat cultured submandibular gland cells, bFGF accelerated the cell proliferation at a concentration of 100 ng/mL or higher. Next, an atrophic model of the rat submandibular gland was used to examine the ability of bFGF to accelerate tissue repair. Two weeks after ductal ligation, the ligature was removed, and various amounts of bFGF, isoproterenol, or saline were administered via a retrograde duct instillation. Both isoproterenol and bFGF increased acinar and ductal cell proliferation significantly. To determine the role of bFGF in saliva, we investigated its effect on the healing process of oral mucosal defects. Four-millimeter mucosal defects were made to the depth of the periosteum in the rat palate under anesthesia. bFGF or vehicle alone was applied once only at the time of surgery as a suspension. At days 3, 5, and 7 in the bFGF group, significant increases in the degree of re-epithelialization were found in treated groups. These results indicate that its action as a mitogen stimulus is the major effect of bFGF on salivary gland cells and mucosal epithelium.

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Because of their easy access, and important role in oral homeostasis, mammalian salivary glands provide a unique site for addressing key issues and problems in tissue engineering. This manuscript reviews studies by us in three major directions involving re-engineering functions of salivary epithelial cells. Using adenoviral-mediated gene transfer in vivo, we show approaches to i) repair damaged, hypofunctional glands and ii) redesign secretory functions to include endocrine as well as exocrine pathways. The third series of studies show our general approach to develop an artificial salivary gland for clinical situations in which all glandular tissue has been lost.

The aim of this study was to assess the feasibility of using gene transfer to salivary glands to direct the systemic delivery of therapeutic proteins in vivo. We used a replication-deficient recombinant adenovirus vector (Ad alpha 1AT) that encodes human alpha 1-antitrypsin (h alpha 1-AT), which we used as a marker protein. Ad alpha 1AT (5 x 10(9) pfu) was administered by retrograde ductal instillation to the submandibular glands of male rats. The amount of h alpha 1-AT found in the salivary glands, saliva, serum, and other tissues was analyzed by a sensitive enzyme-linked immunosorbent assay (ELISA). Maximal levels of the marker protein were detected at 24-48 hr post-virus administration for glands (274 ng/mg protein), saliva (approximately 313 ng/ml), and serum (approximately 5 ng/ml). Serum levels remained elevated for 96 hr, whereas the measured half-life for the marker protein was approximately 2 hr. Generally little to no h alpha 1-AT was detectable in most other organs. However, we were able to measure low levels of marker protein in tissues immediately surrounding infected glands. In all animals studied, levels of h alpha 1-AT were higher in the glandular venous effluent than in arterial blood. Similar results were found with parotid glands. The aggregate data demonstrate that salivary glands may be a target for the nonsurgical, systemic delivery of transgene-encoded therapeutic proteins for diseases that require relatively low circulating protein levels.

To establish a selected salivary-gland cell culture and determine the effect of neuropeptides, monolayers were cultured using 3T3 cells as a feeder layer. To confirm the origin of these cultured cells, amylase production was examined by electron microscopy and periodic acid-Schiff staining, together with immunocytochemical analysis of myosin, anti-cytokeratin (CK-1, CK 10/13, CK MNF116, CK LMW, CK HMW and CK-19) and amylase antibody. The cultured cells demonstrated secretion granules containing amylase and presented features characteristic of acinar cells, which they retained until passage two. By using a feeder layer in conjunction with a newly formulated culture medium, the selectability of these cells was improved. Changes in proliferation of cultured salivary-gland cells in the presence of selected neurotransmitters were also examined. Isoproterenol enhanced cellular proliferation. On the other hand, vasoactive intestinal polypeptide and substance P, which increase the weight of salivary glands in vivo, showed no significant enhancement of proliferation.

To examine the effect of aging and medication on xerostomia, the salivary gland function was evaluated in 20 patients with xerostomia using 99mTc-scintigraphy and the measurement of unstimulated whole saliva (USWS). All of the patients showed USWS volume of less than 2ml/10min. The patients were divided into 2 subgroups based on age (under 65 and 65 and older) and medication status (patients who were on medication which reduced salivary secretion and patients who were not on such medication). The scintigraphic results, such as the maximum radioisotope (RI) count, RI secretion velocity and the volume of USWS, were compared between the subgroups. The maximum RI count and the RI secretory velocity in the submandibular gland and the volume of USWS revealed significantly different functional disturbances between relatively younger patients (under 65) and older patients (65 and older). There was no difference when the scintigraphic results and the volume of USWS measurements in medicated patients were compared with the results of similar tests performed on non-medicated patients. When the medicated and non-medicated groups were separated by age, an increase in age still diminished the volume of USWS in medicated patients. This result might be related to an organic change in the submandibular gland in older patients which was suggested by the scintigraphic results.

Objective: The mechanism underlying the onset and development of autoimmune diseases such as Sjogren's syndrome is not well understood. Here, we examined the effects of preceding inflammation of the salivary gland at the onset of autoimmunity against the salivary gland. Materials and methods: One side of the submandibular gland duct was ligated in mice and the effect on the contralateral gland was investigated. After histological evaluation with hematoxylin and eosin staining, the presence of autoantibodies and immune compounds was examined. Results: In all five strains of mice that were used, the salivary gland of the ligated side showed severe inflammation and atrophic change. In two mouse strains (SJL/J and PL/J), mild sialadenitis was observed on the non-ligated side 8 weeks after ligation. Autoantibodies reacting to the salivary gland were detected in three mouse strains (C3H/He, SJL/J, and PL/J). Immune complex was also detected in the duct basement membrane. Conclusion: The results indicate that the autoimmune mechanism is activated by the transient inflammation in the salivary gland under a specific genetic background. [ABSTRACT FROM AUTHOR]