Ola M. Maria

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.

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.

Paracrine Effects of Bone Marrow Soup Restore Organ Function, Regeneration, and Repair in Salivary Glands Damaged by Irradiation

Background There are reports that bone marrow cell (BM) transplants repaired irradiated salivary glands (SGs) and re-established saliva secretion. However, the mechanisms of action behind these reports have not been elucidated. Methods To test if a paracrine mechanism was the main effect behind this reported improvement in salivary organ function, whole BM cells were lysed and its soluble intracellular contents (termed as “BM Soup”) injected into mice with irradiation-injured SGs. The hypothesis was that BM Soup would protect salivary cells, increase tissue neovascularization, function, and regeneration. Two minor aims were also tested a) comparing two routes of delivering BM Soup, intravenous (I.V.) versus intra-glandular injections, and b) comparing the age of the BM Soup’s donors. The treatment-comparison group consisted of irradiated mice receiving injections of living whole BM cells. Control mice received irradiation and injections of saline or sham-irradiation. All mice were followed for 8 weeks post-irradiation. Results BM Soup restored salivary flow rates to normal levels, protected salivary acinar, ductal, myoepithelial, and progenitor cells, increased cell proliferation and blood vessels, and up-regulated expression of tissue remodeling/repair/regenerative genes (MMP2, CyclinD1, BMP7, EGF, NGF). BM Soup was as an efficient therapeutic agent as injections of live BM cells. Both intra-glandular or I.V. injections of BM Soup, and from both young and older mouse donors were as effective in repairing irradiated SGs. The intra-glandular route reduced injection frequency/dosage by four-fold. Conclusion BM Soup, which contains only the cell by-products, can be advantageously used to repair irradiation-damaged SGs rather than transplanting whole live BM cells which carry the risk of differentiating into unwanted/tumorigenic cell types in SGs.

Human mesenchymal stem cells cultured with salivary gland biopsies adopt an epithelial phenotype.

Sjogrens syndrome and radiotherapy for head and neck cancer result in severe xerostomia and irreversible salivary gland damage for which no effective treatment is currently available. Cell culture methods of primary human salivary gland epithelial cells (huSGs) are slow and cannot provide a sufficient number of cells. In addition, the majority of cultured huSGs are of a ductal phenotype and thus not fluid/saliva secretory cells. Some reports indicated that mesenchymal stem cells (MSCs) possessed the potential to differentiate into epithelial cells. To test this hypothesis with huSGs, a coculture system containing 2 chambers separated by a polyester membrane was used to study the capacity of human MSCs to adopt an epithelial phenotype when cocultured with human salivary gland biopsies. Results were that 20%-40% of cocultured MSCs expressed tight junction proteins [claudin-1 (CLDN-1), -2, -3, and -4; occludin; junctional adhesion molecule-A; and zonula occludens-1] as well as other epithelial markers [aquaporin-5, α-amylase (α-AMY), and E-cadherin], and generated a higher transepithelial electrical resistance. Electron microscopy demonstrated that these MSCs had comparable cellular structures to huSGs, such as tight junction structures and numerous secretory granules. Quantitative real time (RT)-polymerase chain reaction revealed an upregulation of several salivary genes (aquaporin-5, AMY, and CLDN-2). Moreover, the amounts of α-AMY detected in cocultured MSCs were comparable to those detected in huSGs control cultures. These data suggest that cocultured MSCs can demonstrate a temporary change into a salivary gland acinar phenotype.

Cell surface markers CD44 and CD166 localized specific populations of salivary acinar cells

OBJECTIVE Experimental approaches tested to date for functional restoration of salivary glands (SGs) are tissue engineering, gene transfer, and cell therapy. To further develop these therapies, identifying specific cell surface markers for the isolation of salivary acinar cells is needed. To test a panel of cell surface markers [used in the isolation of mesenchymal stem cells, (MSCs)] for the localization of salivary acinar cells. MATERIALS Human submandibular and parotid glands were immunostained with a panel of MSC markers and co-localized with salivary acinar cell differentiation markers [α-amylase, Na-K-2Cl cotransporter-1, aquaporin-5 (AQP5)]. Additional cell markers were also used, such as α-smooth muscle actin (to identify myoepithelial cells), cytokeratin-5 (basal ductal cells), and c-Kit (progenitor cells). RESULTS CD44 identified serous acini, while CD166 identified mucous acini. Cytokeratin-5 identified basal duct cells and 50% of myoepithelial cells. None of the remaining cell surface markers (Stro-1, CD90, CD106, CD105, CD146, CD19, CD45, and c-Kit) were expressed in any human salivary cell. CONCLUSIONS CD44 and CD166 localized human salivary serous and mucous acinar cells, respectively. These two cell surface markers will be useful in the isolation of specific populations of salivary acinar cells.

Apoptotic effect of different self-etch dental adhesives on odontoblasts in cell cultures

OBJECTIVES We aimed to evaluate the potential cytotoxicity (apoptosis-induction) of three types of self-etch dental adhesives: two-component one-step (Xeno III), two-component two-steps (Clearfil Protect Bond) and one-component one-step (Xeno V) on cultured odontoblasts. METHODS Each adhesive was prepared to simulate its clinical manipulation. Cured sterile individual masses were immersed in DMEM and left at 37 °C for 24 h. Then a volume of 100 μL of the extract medium was added to the cultured odontoblasts and incubated for additional 24 h, 48 h and 72 h, respectively. Acridine orange-propidium iodide (AO-PI) labelling was employed to assess the proportion of dead to total number of cells. In addition, an in situ apoptosis detection kit was used to evaluate the DNA cleavage and chromatin condensation employing the immunohistochemical (IHC) technique. Statistical analysis of the data was performed using one-way ANOVA. RESULTS Both apoptosis evaluation methods revealed comparable results with the exception that IHC showed 5-7% less number of dead cells when compared to similar groups evaluated by AO-PI. The percentages of dead to total cells after treatment with Clearfil Protect Bond, Xeno III and Xeno V, were significantly different from the percentage of dead cells after treatment with DMEM alone (-ve control), P value <0.05 and Xeno V dental adhesive had the weakest cytotoxic effect on odontoblasts followed by Xeno III especially after 24 h of incubation. Clearfil Protect Bond had the strongest cytotoxic effect on odontoblasts that was almost closer to that of Staurosporine in DMEM (+ve control). CONCLUSION All tested dental adhesives had remarkable adverse effect on the odontoblasts in vitro; this might be of concern when applied clinically in deep cavities where such cytotoxic chemicals become in close contact to dental pulp. Therefore, further in vivo studies on animal models are recommended to support or refute these in vitro findings.

Mesenchymal Stem Cells Adopt Lung Cell Phenotype in Normal and Radiation-induced Lung Injury Conditions.

Lung tissue exposure to ionizing irradiation can invariably occur during the treatment of a variety of cancers leading to increased risk of radiation-induced lung disease (RILD). Mesenchymal stem cells (MSCs) possess the potential to differentiate into epithelial cells. However, cell culture methods of primary type II pneumocytes are slow and cannot provide a sufficient number of cells to regenerate damaged lungs. Moreover, effects of ablative radiation doses on the ability of MSCs to differentiate in vitro into lung cells have not been investigated yet. Therefore, an in vitro coculture system was used, where MSCs were physically separated from dissociated lung tissue obtained from either healthy or high ablative doses of 16 or 20 Gy whole thorax irradiated rats. Around 10±5% and 20±3% of cocultured MSCs demonstrated a change into lung-specific Clara and type II pneumocyte cells when MSCs were cocultured with healthy lung tissue. Interestingly, in cocultures with irradiated lung biopsies, the percentage of MSCs changed into Clara and type II pneumocytes cells increased to 40±7% and 50±6% at 16 Gy irradiation dose and 30±5% and 40±8% at 20 Gy irradiation dose, respectively. These data suggest that MSCs to lung cell differentiation is possible without cell fusion. In addition, 16 and 20 Gy whole thorax irradiation doses that can cause varying levels of RILD, induced different percentages of MSCs to adopt lung cell phenotype compared with healthy lung tissue, providing encouraging outlook for RILD therapeutic intervention for ablative radiotherapy prescriptions.

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

LASER sintered one-piece early-loaded dental implants for mandibular premolars replacement.

This study was designed to evaluate laser-sintered early-loaded 1-piece implants (OPI) based on clinical and radiographic findings. Thirty OPI were placed in the mandibular premolar area and subjected to early loading after 3 weeks of initial placement; patients were followed up for 6 months. Clinical evaluation included pocket depth, gingival health, implant stability, and esthetics. Periapical radiographs were used to measure the marginal bone loss (MBL). All implants were considered successful resulting in a survival rate of 100%. A remarkable difference (P < 0.01) existed when comparing MBL levels at 1 month with those at 3 and 6 months. Significant differences (P < 0.01) existed when comparing implant stability at 1 month to 3 months and at 3 months to 6 months. Moreover, significant differences (P < 0.01) were observed when comparing peri-implant probing depth at 1 month to that at 3 and 6 months on both the mesial and distal sides. The mean value of pink esthetic score was 11 at time of final restoration. The laser-treated early-loaded OPI design is associated with satisfactory clinical and radiographic follow-up results and it is a good alternative to the 2-piece design.