Simon D. Tran

Differentiation of human bone marrow-derived cells into buccal epithelial cells in vivo: a molecular analytical study

BACKGROUND Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the intrinsic plasticity of these cells has been questioned by results of in-vitro studies suggesting that such cells might fuse with other cells giving the appearance of differentiation. We aimed to determine whether fusion events are important in vivo. METHODS To test whether BMD cells can colonise an epithelial tissue and differentiate there without fusion, we did in-situ hybridisation with Y and X chromosome probes labelled with 35-sulphur or digoxigenin, or labelled fluorescently. We did immunohistochemistry with anticytokeratin 13 along with fluorescence in-situ hybridisation to identify Y-chromosome positive buccal epithelial cells in cheek scrapings obtained from five females who had received either a bone-marrow transplant or an allogeneic mobilised peripheral-blood progenitor-cell transplant (enriched in CD34+ cells) from male donors. FINDINGS When examined 4-6 years after male-to-female marrow-cell transplantation, all female recipients had Y-chromosome-positive buccal cells (0.8-12.7%). In more than 9700 cells studied, we detected only one XXXY-positive cell (0.01%) and one XXY cell (0.01%), both of which could have arisen when an XY cell fused with an XX cell. INTERPRETATION Male BMD cells migrate into the cheek and differentiate into epithelial cells, an occurrence that does not depend on fusion of BMD cells to recipient cells. This finding might be an example of transdifferentiation of haemopoietic or stromal progenitor cells. Plasticity of BMD cells could be useful in regenerative medicine.

Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation

Treatment for most patients with head and neck cancers includes ionizing radiation. A consequence of this treatment is irreversible damage to salivary glands (SGs), which is accompanied by a loss of fluid-secreting acinar-cells and a considerable decrease of saliva output. While there are currently no adequate conventional treatments for this condition, cell-based therapies are receiving increasing attention to regenerate SGs. In this study, we investigated whether bone marrow-derived cells (BMDCs) can differentiate into salivary epithelial cells and restore SG function in head and neck irradiated mice. BMDCs from male mice were transplanted into the tail-vein of 18Gy-irradiated female mice. Salivary output was increased in mice that received BMDCs transplantation at week 8 and 24 post-irradiation. At 24 weeks after irradiation (IR), harvested SGs (submandibular and parotid glands) of BMDC-treated mice had greater weights than those of non-treated mice. Histological analysis shows that SGs of treated mice demonstrated an increased level of tissue regenerative activity such as blood vessel formation and cell proliferation, while apoptotic activity was increased in non-transplanted mice. The expression of stem cell markers (Sca-1 or c-kit) was detected in BMDC-treated SGs. Finally, we detected an increased ratio of acinar-cell area and approximately 9% of Y-chromosome-positive (donor-derived) salivary epithelial cells in BMDC-treated mice. We propose here that cell therapy using BMDCs can rescue the functional damage of irradiated SGs by direct differentiation of donor BMDCs into salivary epithelial cells.

Transplanted human bone marrow cells generate new brain cells.

Multiple studies have reported that adult cells of bone marrow origin can differentiate into muscle, skin, liver, lung, epithelial cells, and neurons. To determine whether such cells might produce neurons and other cells in the human brain, we examined paraffin sections from female patients who had received bone marrow transplants from male donors. Y-chromosomes were labeled using autoradiography and fluorescent in situ hybridization. Neurons and astrocytes were identified histologically and immunohistochemically in neocortex, hippocampus, striatum, and cerebellum. However, most labeled cells in both gray and white matter appeared to be glia. Others have suggested that such Y-labeling represents fusion between host and donor cells, rather than true transdifferentiation. The possibilities of fusion and microchimerism were therefore examined using buccal epithelial cells as a model system. The female patients in this study had received either bone marrow or stem cell (CD34+ enriched) transplants from their brothers. Double labeling for X- and Y-chromosomes showed that Y-labeled buccal cells could not be explained by fusion. Genotyping studies of one patient, her brother, and her son ruled out the possibility of microchimerism. Whether, and under what circumstances, some form of bone marrow transplantation might provide adequate number of cells capable of replacing lost brain cells or enhancing their function will require additional studies.

Absence of Tight Junction Formation in an Allogeneic Graft Cell Line Used for Developing an Engineered Artificial Salivary Gland

An essential structural feature of fluid-secreting epithelial tissues is the presence of tight junctions. To develop a tissue-engineered organ capable of fluid secretion, the cellular component must establish these structures. As part of efforts to create an engineered artificial salivary gland, we have examined the ability of a candidate allogeneic graft cell line, HSG, to produce several key tight junction proteins, as well as to exhibit functional activities consistent with effective tight junction strand formation. In contrast to results obtained with a control kidney cell line, MDCK-II, HSG cells were unable to synthesize four important tight junction-associated proteins: ZO-1, occludin, claudin-1, and claudin-2. In addition, unlike MDCK-II cells, HSG cell monolayers could not restrict paracellular permeability. HSG cells were, thus, unable to generate significant transepithelial electrical resistance or serve as an effective barrier to osmotically imposed fluid movement. Furthermore, these two functional activities could not be reconstituted via the stable transfection of HSG cells with cDNAs encoding either claudin-1 or claudin-2. We conclude that because of their inability to form tight junctions, HSG cells are unsuitable for use as an allogeneic graft cell in an artificial salivary fluid secretory device. These studies also emphasize the importance of graft cell selection in artificial organ development, as certain required characteristics may be difficult to reengineer.

Mesenchymal Stromal Cells Improve Salivary Function and Reduce Lymphocytic Infiltrates in Mice with Sjogren's-Like Disease

Background Non-obese diabetic (NOD) mice develop Sjögrens-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freunds adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45−/TER119− cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined. Methodology/Principal Findings Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45−/TER119− cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3+ (Treg) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF). Conclusions/Significance The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

Efficacy and Safety of an Intraoral Electrostimulation Device for Xerostomia Relief: A Multicenter, Randomized Trial

OBJECTIVE To evaluate the efficacy and safety of an intraoral electrostimulation device, consisting of stimulating electrodes, an electronic circuit, and a power source, in treating xerostomia. The device delivers electrostimulation through the oral mucosa to the lingual nerve in order to enhance the salivary reflex. METHODS The device was tested on a sample of patients with xerostomia due to Sjögrens syndrome and other sicca conditions in a 2-stage prospective, randomized, multicenter trial. Stage I was a double-blind, crossover stage designed to compare the effects of the electrically active device with the sham device, each used for 1 month, and stage II was a 3-month open-label stage designed to assess the long-term effects of the active device. Improvement in xerostomia severity from baseline was the primary outcome measure. RESULTS A total of 114 patients were randomized. In stage I, the active device performed better than the sham device for patient-reported xerostomia severity (P<0.002), xerostomia frequency (P<0.05), quality of life impairment (P<0.01), and swallowing difficulty (P<0.02). At the end of stage II, statistically significant improvements were verified for patient-reported xerostomia severity (P<0.0001), xerostomia frequency (P<0.0001), oral discomfort (P<0.001), speech difficulty (P<0.02), sleeping difficulty (P<0.001), and resting salivary flow rate (P<0.01). CONCLUSION Our findings indicate that daily use of the device alleviated oral dryness, discomfort, and some complications of xerostomia, such as speech and sleeping difficulties, and increased salivary output. The results show a cumulative positive effect of the device over the period of the study, from baseline to the end of the trial.

Matrigel improves functional properties of human submandibular salivary gland cell line.

Sjogrens syndrome and radiotherapy for head and neck cancers result in irreversible damage to functional salivary tissue, for which no adequate treatment is available. The microenvironment for salivary gland cell cytodifferentiation is critical for the future development of salivary gland regeneration, repair and tissue engineering treatments. Results from this study indicate that human submandibular cell line (HSG) cultured on Matrigel (2mg/ml) could be induced to differentiate into polarized secretory acinar-like cells. The HSG cells grown on Matrigel were evaluated by physiological functional assays, molecular and immunohistochemistry, immunofluorescence, and morphological assessments. The results showed (1) a decrease in cell proliferation; (2) an increase in cell apoptosis; (3) cellular polarization evident by transepithelial electrical resistance (TER), expressions of tight junction proteins (claudin-1, -2, -3, -4, occludin, JAM-A, and ZO-1) and transmission electron microscopy (TEM); (4) an increase in the production and/or secretion of acinar cell proteins, i.e., alpha-amylase, aquaporin-5, cytokeratins, and mucin-1, that were not associated with increases in mRNA transcription; (5) a decrease in vimentin expression; and (6) expression of potential stem cell biomarkers CD44 and CD166. The data indicated that Matrigel provided a suitable microenvironment for morphological and functional differentiation of HSG cells into 3D acinar like cells. This study provides an in vitro model and baseline data on future developments of new strategies for salivary gland regeneration and replacement.

Distribution of Tight Junction Proteins in Adult Human Salivary Glands

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, −3, and −4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland.

Tumor Necrosis Factor-α and Interleukin-6: Potential Interorgan Inflammatory Mediators Contributing to Destructive Periodontal Disease in Obesity or Metabolic Syndrome

Obesity has become a worldwide health burden in the last two decades. Obesity has been associated with increased comorbidities, such as coronary artery disease, diabetes, and destructive periodontal disease. Obesity is also part of a group of risk factors occurring together in an individual, which is referred to as metabolic syndrome. Clinical studies have shown higher risk for destructive periodontal disease in obesity and metabolic syndrome. However, the role of obesity and metabolic syndrome in the onset and development of destructive periodontal disease has not yet been fully understood. In this review, we discuss a working model, which focuses on interorgan inflammation as a common etiological factor for destructive periodontal disease associated with obesity and metabolic syndrome. Specifically, we suggest that elevated levels of tumor necrosis factor-α (TNF-α) or interleukin 6 (IL-6)—both adipokines and known risk factors for destructive periodontal disease—in obesity and metabolic syndrome contribute to the onset and development of destructive periodontal disease. The connections between destructive periodontal disease and systemic conditions, such as obesity or metabolic syndrome, are complex and potentially multidirectional. This review largely focuses on TNF-α and IL-6, inflammatory mediators, as potential common risk factors and does not exclude other biological mechanisms.

Saliva as a diagnostic tool for oral and systemic diseases

Early disease detection is not only vital to reduce disease severity and prevent complications, but also critical to increase success rate of therapy. Saliva has been studied extensively as a potential diagnostic tool over the last decade due to its ease and non-invasive accessibility along with its abundance of biomarkers, such as genetic material and proteins. This review will update the clinician on recent advances in salivary biomarkers to diagnose autoimmune diseases (Sjogrens syndrome, cystic fibrosis), cardiovascular diseases, diabetes, HIV, oral cancer, caries and periodontal diseases. Considering their accuracy, efficacy, ease of use and cost effectiveness, salivary diagnostic tests will be available in dental offices. It is expected that the advent of sensitive and specific salivary diagnostic tools and the establishment of defined guidelines and results following rigorous testing will allow salivary diagnostics to be used as chair-side tests for several oral and systemic diseases in the near future.