Fibronectin-induced ductal formation in salivary gland self-organization model.

BACKGROUND: Recent advances in tissue regeneration approaches including 3D organoids, were based on various 3D organogenesis models. However, 3D models are generally technique-sensitive and time-consuming. Thus, we utilized an existing model of submandibular salivary gland (SMG) to modify a simple and highly reproducible in vitro 3D culture model of primary SMG cells self-organization into a well-developed cell spheroid inside Matrigel substrate. We used this model to observe the collective multicellular behavior during spheroid formation. Further, we applied various quantitative approaches including real-time live imaging and immune histochemical image analysis to dissect the cellular dynamics during tissue patterning. RESULTS: On a time-scale of hours, we observed marked size and shape transformations in the developed 3D spheroid which resulted in a spatially-controlled growth differential from the canter to the periphery of the formed aggregates. Moreover, we investigated the effect of fibronectin (FN) on SMG cells self-organization using our simplified culture model. Interestingly, we discovered a novel role of FN in inducing duct-like elongation during initial stages of SMG bud formation. CONCLUSION: This in vitro model provides an excellent tool for analyzing the intercellular dynamics during early SMG tissue development as well as revealing a novel role of FN in SMG ductal expansion.

Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features.

The salivary ducts of Wharton and Stenson: analysis of normal variant sialographic morphometry and a historical review.

Submandibular and parotid salivary glands acquire lengthy ducts as they migrate during development. No conclusive reports exist on clinically important anatomical normal variants and morphometry of the submandibular duct of Wharton and the parotid duct of Stenson. We retrospectively analyzed 67 normal digital subtraction sialograms on patients ranging from 16 to 85 years of age (M:F=15:52). In 43 sialograms, the mean parotid duct length was 50 mm. The mean width of the proximal, mid, and distal segments of the parotid duct were 1.8 mm, 1.1 mm, and 1.6 mm, respectively. An accessory parotid gland was present in 68% of patients, with a mean angle of confluence of its tributary duct with the parotid duct of 53°. In 24 sialograms the mean length of the submandibular duct was 58 mm. The mean width of the proximal, mid, and distal segments of the submandibular duct were 2.0 mm, 2.7 mm, and 2.1 mm, respectively. The submandibular duct genu had a mean angle of 115°. The effect of independent variables (age, gender, and side) was statistically tested on the dependent variables (length, mean calibre, and angle) using regression analysis. None of the independent variables affected variations in length, size and angulation. These reported comprehensive and detailed morphometrics are useful for therapeutic planning of luminal procedures on the salivary ducts, including sialography, sialoendoscopy, interventional therapies, and lithotripsy.

Lumen formation in three-dimensional cultures of salivary acinar cells.

OBJECTIVE: Development of an artificial salivary gland will benefit patients with xerostomia after radiation therapy for upper respiratory cancer. The goal is to devise a three-dimensional (3D) culture system in which salivary cells differentiate into polarized acini that express essential biomarkers and directionally secrete alpha-amylase. Differentiated acini-like structures in a 3D biomaterial-based scaffold will mimic salivary gland functions. STUDY DESIGN: Cells were seeded onto HA-based hydrogels containing PlnDIV peptide and allowed to differentiate into acini-like structures. Cell viability and phenotype were examined. SETTING: Laboratory-based tissue procurement study. SUBJECTS AND METHODS: Salivary gland tissue was obtained from patients undergoing surgery. Marker expression established the phenotype of salivary gland cells. Perlecan/HSPG2, an important component of the basement membrane, was highly expressed in salivary gland tissue. A culture system consisting of hyaluronic acid (HA) hydrogel and a coupled bioactive peptide derived from domain IV of perlecan (PlnDIV) was used. Prior studies demonstrated differentiation of acinar cells into lobular structures that mimicked intact glands when cultured on PlnDIV peptide-coated surfaces. RESULTS: Lobular acini-like structures formed on hydrogels and expressed tight junction components such as zona occludens 1. Acini-like structures were stained for the presence of alpha-amylase. Live/dead staining revealed the presence of apoptotic cells in the center of the acini-like structures, indicative of lumen formation. CONCLUSION: A novel system supporting acini-like assembly in a 3D culture system was established. Presence of biomarkers and secretion of salivary enzymes confirms functionality in vitro. Future experiments will test the 3D system in an animal model.

Organogenesis of the juxta-oral organ in mice.

The juxta-oral organ is a bilateral organ in the mammalian bucca. It consists of epithelial cords with surrounding mesenchyme. It develops from embryonic oral epithelium, but its macroscopic morphology in mice is less studied and seems to be very different from that of humans. The juxta-oral organ in mice extends more widely from the subcutaneous tissue of the mandible near the lateral fascia of the masseter to the submucosa of the soft palate. In this paper, we report that the mutant mouse allele Bmp7(lacZ) presented intense lacZ expression in the epithelial component of the juxta-oral organ in its homo- and heterozygous states. The main aims of this study were to show that this mutant mouse allele is suitable for observing macroscopic structure of the juxta-oral organ and to describe the development of this organ during embryonic and postnatal stages. Whole-mount beta-gal staining of this strain of mouse showed that the juxta-oral organ in mice appeared at E12.0 from oral epithelium and lost connection with it before E12.5. Then, the juxta-oral organ extended anteriorly to the lateral fascia of the masseter and posteriorly to the submucosal layer of the soft palate via the orbit. The mature juxta-oral organ had no connection to other epithelia such as those of the bucca and parotid duct. It persisted until adulthood and there seemed to be no tendency to regress. Transmission electron microscopy showed that each part of the juxta-oral organ was an epithelial cord surrounded by a basement membrane and mesenchymal tissue.

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development.

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.

The role of cyclic AMP response element-binding protein in testosterone-induced differentiation of granular convoluted tubule cells in the rat submandibular gland.

The postnatal development of granular convoluted tubules (GCT) in the duct system of the rodent submandibular gland is known to be androgen-dependent, but the underlying molecular mechanism is unclear. To test the possible role of the transcription factor, cyclic AMP response element-binding protein (CREB), in the androgen-induced differentiation of GCT, the effect of testosterone on the expression and localization of epidermal growth factor (EGF), a marker of GCT cells, and of CREB was examined in the submandibular glands of immature 3-week-old rats. Northern blotting demonstrated increases in both EGF and CREB mRNA 1-4 days after testosterone administration. Immunoprecipitation also indicated that CREB protein was increased in amount with testosterone administration, and that induced CREB was phosphorylated at the serine residue as in the active form of CREB. In situ hybridization demonstrated that cells with CREB mRNA signal first appeared in the distal portions of striated ducts at 1 day and had increased in number by 4 days after giving testosterone, when cells with EGF mRNA signal became evident in the same duct portions. Immunohistochemistry also showed the occurrence of CREB protein in the nuclei of duct epithelial cells before their differentiation into EGF-positive GCT cells. Finally, pieces of submandibular gland from immature rats were cultured in vitro and their expression of EGF mRNA analysed by the reverse transcriptase-polymerase chain reaction. Testosterone in the medium caused a marked enhancement of EGF expression in the gland in 1-4 days, which was attenuated by simultaneous administration of the antisense oligonucleotide for CREB as well as that for the androgen receptor. These results suggest the CREB is upregulated by androgen and has a crucial role in androgen-induced differentiation of GCT in the duct system of the rat submandibular gland.

Cell death during development of intercalated ducts in the rat submandibular gland.

Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG-C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG-C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG-C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis.

Immunohistochemical localization of carbonic anhydrases I, II, and VI in the developing rat sublingual and submandibular glands.

Carbonic anhydrase has been localized to the acini and ducts of mature rat salivary glands. This enzyme has been associated with ion transport, a prominent function of striated and excretory ducts in salivary glands, suggesting that it might be used as a marker of ductal differentiation. The purpose of this study was to immunohistochemically document developmental changes in carbonic anhydrase in the ducts of the rat sublingual and submandibular glands. Immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II and VI on sections of sublingual and submandibular glands from rats at representative postnatal developmental ages. Reactions were weak in the ducts of both glands at 1 day, then progressively increased. By 42 days, reactions had the adult pattern of virtually none in the mucous or seromucous acini, moderate to strong in the striated and excretory ducts, and none to weak in the intercalated ducts. Weak to moderate reactions were observed in the granular convoluted tubules of the submandibular gland as they became recognizable at age 42 days. Reactions to carbonic anhydrase I and II antibodies also increased from none (1 day) to modest (42 days) in the demilunes of the sublingual gland. The order of reaction intensity of the antibodies was II > I > VI. When localized via these anti-human antibodies, carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat sublingual and submandibular glands.

Increasing frequency of occurrence of tuft cells in the main excretory duct during postnatal development of the rat submandibular gland.

Tuft cells, a widespread cell type that is present in the mucosal epithelia of hollow organs, including the main excretory duct (MED) epithelia of the rat salivary gland, are well documented morphologically. However, studies of their development are few. The purpose of the present study was to examine the perinatal and postnatal development of tuft cells in the main excretory duct of the rat submandibular gland. Main excretory ducts of the submandibular gland were obtained from five male Wistar rats at the ages of 0, 1, 7, 14, 17, 21, 23, 28, and 56 postnatal days and were prepared for scanning and transmission electron microscopy. The tuft cells, which are distinguished easily by their long microvilli protruding into the lumen, were recognizable first at 17 postnatal days. They showed a remarkable increase in number between 3 and 4 postnatal weeks. The percentages of tuft cells were 0.4% at 17 postnatal days and 0.8% at 3 postnatal weeks. The number of tuft cells represented approximately 5% of the total epithelial cells by 4 postnatal weeks. There was a significant difference between 3 and 4 postnatal weeks (P < 0.01). The microvilli of the tuft cells at the time of weaning had almost the same width as in the adult, but they were shorter. Microfilaments extending from the tips of the microvilli and microtubules and many electron-lucent vesicles in the supranuclear cytoplasm also were observed. These results indicate that tuft cells appeared in the MED of the submandibular gland during weaning and had abundant vesicles in their apical cytoplasm.