The role of human fibronectin‐ or placenta basement membrane extract‐based gels in favouring the formation of polarized salivary acinar‐like structures

Head and neck cancer patients treated with radiotherapy commonly experience hyposalivation and oral/tooth infections, leading to a reduced quality of life. Clinical management is currently unsatisfactory for dry mouth. Thus, there is a need for growing salivary fluid‐secreting (acinar) cells for these patients. However, functionally‐grown salivary acinar cells are cultured in Matrigel, a product that cannot be used clinically, owing to its source from a mouse sarcoma. Therefore, finding a gel suitable for clinical use and possessing properties similar to that of Matrigel would allow biopsied salivary cells to be expanded in vitro and transplanted into the mouths of xerostomic patients. This study tested gels made with human placenta basement membrane extract (BME) or fibronectin for the growth and differentiation of human salivary biopsies into acinar cells. We report here that, following expansion of primary human salivary gland epithelial cells (huSGs) in serum‐free medium, using these gels (made from human proteins) allowed morphological and functional differentiation of salivary ductal cells into acinar‐like cells. These (human) gels gave comparable results to Matrigel, such as differentiation into polarized acinar 3D units or monolayers with tight junction proteins (claudin‐1, ‐2, ‐3) and exhibiting adequate transepithelial electrical resistance, acinar proteins (AQP5, α‐amylase, mucin‐1, NKCC1) and acinar adhesion‐related cell markers (CD44, CD166). Ultrastructural, mRNA and protein analyses confirmed the formation of differentiated acinar polarized cells. The mitotic activity was highest with human placenta BME gel. This human culture model provided a reproducible approach to studying human salivary cell expansion and differentiation for tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

A magnetic three‐dimensional levitated primary cell culture system for the development of secretory salivary gland‐like organoids

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva‐secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva‐secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell‐based approaches in three‐dimensional (3D) scaffold‐based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non‐human‐based substrates. Herein, saliva‐secreting organoids/mini‐glands were developed using a new scaffold/substrate‐free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG‐derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG‐like organoids in well‐established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro‐mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG‐specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E‐cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α‐smooth muscle actin); and neuronal (β3‐tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α‐amylase activity assays showed functional organoids with SG‐specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG‐like mini‐glands for SG secretory repair.     

Evaluation of salivary gland acinar and ductal cell-specific promoters in vivo with recombinant adenoviral vectors.

Adenoviral vectors efficiently deliver exogenous genes to salivary glands. There are two general epithelial cell types, with very different functions, in salivary glands--acinar and ductal. To determine if gene expression can be restricted in vivo to either general cell type using a relatively cell/tissue-specific promoter in conjunction with adenovirus-mediated gene transfer, we tested the human amylase and kallikrein promoters. For initial studies the sensitive reporter gene luciferase was used in two adenoviral constructs. The adenovirus AdAMY-luc contains the human salivary gland amylase promoter (-1003 to +2)(AMY1C) and AdKALL-luc contains the human tissue kallikein promoter (-315 to -1)(KLK1). The adenovirus AdKALL-hAQP1 was also used to test a therapeutic gene, human aquaporin-1 (hAQP1), potentially of importance in treating surviving ductal cells in irradiation-damaged glands. Luciferase expression after AdAMY-luc delivery in vivo directly to the parotid, submandibular, and sublingual glands, as well as to the lungs, and intravenously via the femoral vein, was restricted to the three salivary glands and the pancreas. AdKALL-luc delivery via the same routes resulted in a more general distribution of luciferase expression, although greatest luciferase activity was seen in salivary glands and lung. Luciferase activity after AdAMY-luc delivery was proportionally greater (approximately 14-fold) in acinar cells, whereas luciferase activity after AdKALL-luc delivery was proportionally greater (approximately 9-fold) in ductal cells. The expression of hAQP1 after AdKALL-hAQP1 gene transfer was mainly observed in ductal cells in vivo. AdKALL-hAQP1 was as useful as AdCMV-hAQP1 in increasing salivary flow rates of irradiated rats. This study demonstrates that adenoviral vectors containing the relatively cell/tissue-specific AMY1C or KLK1 promoters may be useful for targeting therapeutic gene expression in salivary glands.     

Adenoviral delivery of Tousled kinase for the protection of salivary glands against ionizing radiation damage

Oral complications of salivary hypofunction often afflict cancer patients undergoing radiotherapy for head and neck cancers. Dry mouth or xerostomia is an undesirable consequence of radiotherapy that compromises normal oral functions in addition to causing odynophagia and increasing the patient's risk of oral infections and dental caries. Radiation-induced xerostomia is irreversible, and palliative measures to provide symptomatic relief remain the mainstay of treatment. Previously, we identified a splice variant of a cellular kinase, Tousled-like kinase 1B (TLK1B), which when overexpressed protects normal epithelial cells against ionizing radiation (IR)-induced cell death. To address the need to protect salivary glands in patients undergoing regional radiotherapy, we investigated whether preemptive expression of TLK1B in salivary glands protects against IR. In stably-derived salivary cell lines in vitro, TLK1B expression increased cell survival after IR. Cells expressing exogenous TLK1B were less radiosensitive (A5-TLK1B, α/β=0.67?Gy; ParC5-TLK1B, α/β=4.3?Gy) compared to control cells (A5-BK, α/β=1.7?Gy; ParC5-BK, α/β=32.7?Gy). Using a recombinant adenovirus serotype 5 viral vector for TLK1B gene transfer into rat submandibular salivary glands in vivo, we demonstrated that TLK1B protects the saliva-secreting acinar cells and better preserves salivary gland function against IR relative to control glands. After a single fraction of 16?Gy, the decline in salivary function at 8 weeks was less pronounced in TLK1B-treated animals (40%) as compared to saline-treated controls (67%). Histopathological analysis demonstrated increase in acinar atrophy, decrease in acinar cell number, and increase in inflammatory infiltrate and fibrosis in irradiated control tissues relative to TLK1B-treated glands. These results show the radioprotective benefits of TLK1B and implicate its usefulness in the management of regional radiotherapy-induced xerostomia.     

Enhanced transduction of mouse salivary glands with AAV5-based vectors

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2beta-galactosidase (betagal), rAAV4betagal, or rAAV5betagal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more betagal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.     

Three‐dimensional cultures of mouse submandibular and parotid glands: a comparative study

Freshly isolated salivary cells can be plated on an extracellular matrix, such as growth factor‐reduced Matrigel (GFR‐MG), to induce the formation of three‐dimensional (3D) structures. Cells grown on GFR‐MG are able to form round structures with hollow lumina, capable of sustaining amylase expression. In contrast, cells grown on plastic do not exhibit these features. Our recent studies have used mouse parotid gland (PG) cells, grown on different extracellular matrices, as a model for acinar formation. However, PG cells were not able to respond to the secretory agonist carbachol beyond 5 days and did not sustain polarity over time, regardless of the substratum. An alternative option relies in the use of mouse submandibular glands (SMG), which are more anatomically accessible and yield a larger number of cells. We compared SMG and PG cell clusters (partially dissociated glands) for their ability to form hollow round structures, sustain amylase and maintain secretory function when grown on GFR‐MG. The results were as follows: (a) SMG cell clusters formed more organized and larger structures than PG cell clusters; (b) both SMG and PG cell clusters maintained α‐amylase expression over time; (c) SMG cell clusters maintained agonist‐induced secretory responses over time; and (d) SMG cell clusters maintained secretory granules and cell–cell junctions. These results indicate that mouse SMG cell clusters are more amenable for the development of a bioengineered salivary gland than PG cell clusters, as they form more organized and functional structures. Copyright © 2014 John Wiley & Sons, Ltd.     

Specific complexes derived from extracellular matrix facilitate generation of structural and drug‐responsive human salivary gland microtissues through maintenance stem cell homeostasis

Three‐dimensional cultured salivary glands (SGs) microtissues hold great potentials for clinical research. However, most SGs microtissues still lack convincing structure and function due to poor supplementation of factors to maintain stem cell homeostasis. Extracellular matrix (ECM) plays a crucial role in regulating stem cell behavior. Thus, it is necessary to model stem cell microenvironment in vitro by supplementing culture medium with proteins derived from ECM. We prepared specific complexes from human SG ECM (s‐Ecx) and analyzed the components of the s‐Ecx. Human SG epithelial and mesenchymal cells were used to generate microtissues, and the optimum seeding cell number and ratio of two cell types were determined. Then, the s‐Ecx was introduced to the culture medium to assess its effect on stem cell behavior. Multiple specific factors were presented in s‐Ecx. s‐Ecx promoted maintenance of the stem cell and formation of specific structures resembling that of salivary glands and containing mucins, which suggested stem cell differentiation potential. Moreover, treatment of the microtissues with s‐Ecx increased their sensitivity to neurotransmitters. On the basis of the analysis of components, we believed that the presented growth factors are able to interact with stem cell they encountered in vivo, which promote the capacity to maintain stem cell homeostasis. This work provided foundations to study molecular mechanism of stem cell homeostasis in SGs and develop novel therapies for dry mouth through new drug discovery and disease modeling.     

Protective effects of curcumin on radioiodine‐induced salivary gland dysfunction in mice

Radioiodine (RI) treatment is widely used in patients with differentiated thyroid cancer. However, RI exposure often induces salivary gland (SG) dysfunction. In this study, we investigated the effect of curcumin on RI‐induced SG dysfunction in mice. Mice were assigned to one of four groups (n = 6 per group) as follows: normal control, RI only, RI + curcumin, and RI + amifostine group. Salivary flow rate, lag time, and changes in ^99mTc (technetium)‐pertechnetate uptake and excretion were measured, and changes in SG morphology and histology were analysed. Salivary epidermal growth factor content, amylase, and superoxide dismutase (SOD) activities were also measured. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess SG apoptosis, and the expression of apoptosis‐related protein was determined by western blotting. The reduced salivary flow rate and prolonged lag time in the RI group was restored by treatment with curcumin or amifostine. In the histological analysis, compared with the RI group, RI + curcumin and RI + amifostine groups had more mucin‐rich acini and less periductal fibrosis. Compared with the RI group, RI + curcumin and RI + amifostine groups showed evidence of tissue remodelling, with a greater number of salivary epithelial cells (AQP‐5‐positive), SG ductal cells (CK18‐positive), endothelial cells (CD31‐positive), and myoepithelial cells (α‐SMA‐positive). RI + curcumin and RI + amifostine groups alleviated RI‐induced cell death, demonstrating antiapoptotic effect, compared with the RI group. Both SOD activity and the protein expression levels of SOD2 were higher in the RI + curcumin and RI + amifostine groups than in the RI group. Our results demonstrate that curcumin ameliorates RI‐induced SG dysfunction in mice.     

Cell extracts from spleen and adipose tissues restore function to irradiation‐injured salivary glands

A cell extract from whole bone marrow (BM), which we named “BM Soup,” has the property to restore saliva secretion to irradiation (IR)‐injured salivary glands (SGs). However, BM cell harvesting remains an invasive procedure for the donor. The main objective of this study was to test the therapeutic effect of “Cell Soups” obtained from alternate tissues, such as adipose‐derived stromal cells (ADSCs) and spleen cells to repair SGs. BM Soup, Spleen Soup, ADSC Soup, or saline (vehicle control) was injected intravenously into mice with IR‐injured SGs (13Gy). Results demonstrated that all three cell soups restored 65–70% of saliva secretion, protected acinar cells, blood vessels, and parasympathetic nerves, and increased cell proliferation. Although protein array assays identified more angiogenesis‐related growth factors in ADSC Soup, the length of its therapeutic efficiency on saliva flow was less than that of the BM Soup and Spleen Soup. Another objective of this study was to compare “Fresh” versus “Cryopreserved (?80 °C)” BM Soup. It was found that the therapeutic effect of 12‐month “Cryopreserved BM Soup” was comparable to that of “Fresh BM Soup” on the functional restoration of IR‐injured SGs. In conclusion, both Spleen Soup and ADSC Soup can be used to mitigate IR‐damaged SGs.     

Expression of heat shock protein 27 with the transition from proliferation to differentiation of acinar precursor cell in regenerating submandibular gland of rats

Heat shock protein 27 (Hsp27) has been suggested to participate in the cell proliferation and differentiation during tissue development. In fact, we have demonstrated the transient occurrence of Hsp27 during the differentiation of salivary gland acinar cells in postnatal rats. The purpose of the present study is to explore the potential role of Hsp27 in the proliferation and differentiation of the acinar cells during regeneration of the salivary gland. Using the experimental regeneration model of the rat submandibular gland after the release of duct ligation, the spatio-temporal localization of Hsp27 was investigated in immunohistochemistry in regenerating acini. No epithelial cells were immunoreactive for Hsp27 immediately after unligation, but Hsp27-immunoreactive cells were observed in regenerating acini located at the end portion of survived ductal tissues on the third day after unligation. The number of Hsp27-immunoreactive cells in regenerating acini reached its peak on the 5th day after unligation, and started to decline on the 7th day. They were undetectable on the 14th day. Importantly, the increase in the number of Hsp27-immunoreactive cells was preceded by the decline in the cell proliferative activity, and Hsp27-immunoreactivity declined and disappeared in conjunction with the progression of acinar cell differentiation, as judged by the double-immunostaining for Hsp27 and proliferating cell nuclear antigen, a cell proliferation marker, or glycine-rich protein-alpha, a specific marker of differentiated acinar cells. All the findings suggest that Hsp27 is expressed with the transition from the cell proliferation to differentiation of the acinar precursor cells during the regenerating process.     

Imaging of the major salivary glands

The major salivary glands, submandibular, parotid and sublingual glands play an important role in preserving the oral cavity and dental health. Patients with problems of the major salivary glands may present with symptoms such as dry mouth, dysphagia and obstruction of duct, inflammation, severe dental caries or swelling. Imaging plays an important role in visualization of morphology and function, to establish a diagnosis, for treatment, and for surgical planning. There are several options for diagnostic imaging: plain radiography, sialography, ultrasound (US), magnetic resonance imaging (MRI), computed tomography (CT), salivary gland scintigraphy and ¹⁸F‐FDG positron emission tomography (PET). We present an overview of the modalities in relation to common salivary gland disease.     

口腔小涎腺肿瘤37例临床分析

目的分析口腔小涎腺肿瘤的临床特点,探讨其诊断方法和治疗原则。方法对1992—2006年间收治的37例小涎腺肿瘤的临床资料进行回顾性分析。结果37例口腔小涎腺肿瘤,良性肿瘤12例,占32．43％;恶性肿瘤25例,占67．57％。良性肿瘤中的混合瘤多见,占良性肿瘤的66．67％。恶性肿瘤中以腺样囊性癌为多,占恶性肿瘤的32．00％,手术加放疗者占恶性肿瘤的64．0％,5年生存率为75．0％。结论小涎腺肿瘤中恶性肿瘤多于良性肿瘤。外科手术是口腔小涎腺肿瘤的主要治疗方法。对于良性小涎腺肿瘤,足够的安全边界是减少复发的关键;对于恶性肿瘤,早期诊断及规范治疗是提高生存率的关键。术后辅助放疗可降低恶性肿瘤复发率。     

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume‐persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self‐assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co‐culture spheroids combining adipose‐derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary‐like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT‐qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary‐like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high‐throughput (pre‐)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co‐culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular‐like structures within engineered tissues in vitro.     

Enhanced tissue remodelling efficacy of adipose‐derived mesenchymal stem cells using injectable matrices in radiation‐damaged salivary gland model

The present study was conducted to introduce the use of a delivery carrier for local transplantation of human adipose tissue‐derived mesenchymal stem cells (AdMSCs) into the salivary gland (SG) and analyse its ability to enhance radioprotection of AdMSCs against irradiation (IR)‐induced damage. An injectable porcine small intestinal submucosa (SIS) matrix was used as a cell delivery carrier, and human AdMSCs were contained within SIS hydrogel (AdMSC/SIS). After local injection into SGs of mice following local IR, morphological and functional changes were evaluated in the sham, vehicle [phosphate‐buffered saline (PBS)], SIS, AdMSC and AdMSC/SIS groups. Local transplantation of AdMSC resulted in less fibrosis, regardless of the use of a carrier, but the AdMSC/SIS group showed more mucin‐producing acini relative to those in the PBS group. Functional restoration of salivation capacity and salivary protein synthesis was achieved in AdMSC and AdMSC/SIS groups, with a greater tendency being observed in the AdMSC/SIS group. AdMSC treatment resulted in tissue remodelling with a greater number of salivary epithelial cells (AQP‐5), SG progenitor cells (c‐Kit), endothelial cells (CD31) and myoepithelial cells (α‐SMA), among which endothelial and myoepithelial cells significantly increased in the AdMSC/SIS group relative to the AdMSC group. AdMSC treatment alleviated IR‐induced cell death, and the anti‐apoptotic and anti‐oxidative effects of AdMSC were enhanced in the AdMSC/SIS group relative to the AdMSC group. These results suggest local transplantation of AdMSC improves tissue remodelling following radiation damage in SG tissue, and that use of a carrier enhances the protective effects of AdMSC‐mediated cellular protection against IR via paracrine secretion. Copyright © 2016 John Wiley & Sons, Ltd.     

Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands

We have previously developed a robust salivary gland‐specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti‐P. falciparum circumsporozoite protein (PfCSP) single‐chain antibody (scFv) fused to DsRed in a secretory form (mDsRed‐2A10 scFv). Fluorescence microscopy showed that the mDsRed‐2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission‐blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed‐2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland‐specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans.     

Up-regulated PAR-2-mediated salivary secretion in mice deficient in muscarinic acetylcholine receptor subtypes

Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.     

Positive selection drives accelerated evolution of mosquito salivary genes associated with blood‐feeding

The saliva of bloodsucking animals contains dozens to hundreds of proteins that counteract their hosts’ haemostasis, inflammation and immunity. It was previously observed that salivary proteins involved in haematophagy are much more divergent in their primary sequence than those of housekeeping function, when comparisons were made between closely related organisms. While this pattern of evolution could result from relaxed selection or drift, it could alternatively be the result of positive selection driven by the intense pressure of the host immune system. We investigated the polymorphism of five different genes associated with blood‐feeding in the mosquito Anopheles gambiae and obtained evidence in four genes for sites with signatures of positive selection. These results add salivary gland genes from bloodsucking arthropods to the small list of genes driven by positive selection.     

涎腺肿瘤的超声诊断

目的分析涎腺肿瘤的声像图特征，以提高超声定性诊断正确率。方法回顾分析62例经手术病理证实的涎腺肿瘤的超声声像图特征。结果超声定性诊断正确率仅40．3％。经手术病理证实的良性肿瘤以多形性腺瘤和腺淋巴瘤为主，声像图具有特征性。结论超声检查对涎腺多形性腺瘤和腺淋巴瘤有较大的诊断价值，但对部分较小的恶性肿瘤和良性肿瘤局部恶变者诊断缺乏特征性，是导致定性困难和误诊的主要原因。     

The response of gingiva monolayer,spheroid, and ex vivo tissue cultures to collagen membranes and bone substitute

Collagen membranes and bone substitute are popular biomaterials in guided tissue regeneration for treatment of traumatized or diseased periodontal tissue. Development of these biomaterials starts in monolayer cell culture, failing to reflect in vivo tissue organization. Spheroid cultures potentially mimic in vivo tissues in structure and functionality. This study aims to compare gingiva cell (GC) monolayers and spheroids to ex vivo gingiva. Human GC monolayers, spheroids and gingiva ex vivo tissues were cultured on plastic surfaces, collagen membranes or bone substitute. Hematoxylin–eosin (HE) staining, immunohistochemistry for KI67 and caspase 3 (CASP3), resazurin‐based toxicity assays, quantitative polymerase chain reaction for collagen I (COL1A1), vascular endothelial growth factor (VEGF), angiogenin (ANG), interleukin (IL)6 and IL8 and ELISA for COL1A1, VEGF, ANG, IL6 and IL8 were performed in all cultures. Morphology was different in all culture set‐ups. Staining of KI67 was positive in monolayers and staining of CASP3 was positive in spheroids. All culture set‐ups were viable. COL1A1 production was modulated in monolayers and ex vivo tissues at mRNA levels, VEGF in monolayers and ex vivo tissues at mRNA levels and in spheroids at protein levels, ANG in spheroids at mRNA levels and in monolayers and spheroids at protein levels, IL6 in monolayers and spheroids at mRNA levels and in spheroids and ex vivo tissues at protein levels and IL8 in monolayers and ex vivo tissues at mRNA levels. Modulations were surface‐dependent. In conclusion, each culture model is structurally and functionally different. Neither GC monolayers nor spheroids mimicked gingiva ex vivo tissue in all measured aspects.