Bioengineering from the laboratory to clinical translation in oral and maxillofacial reconstruction.

Background: Conventional techniques used in oral and maxillofacial reconstruction focus mainly on utilizing autologous tissues that have unquestionably improved function and esthetics for many patients, worldwide. However, the success depends on countless factors such as: donor and recipient sites conditions, patient's medical history, surgeon's experience, restricted availability of high-quality autogenous tissues or stem cells, and increased surgical cost and time. Materials and Methods: Lately, teaming researchers, scientists, surgeons, and engineers, to address these limitations, have allowed tremendous progress in recombinant protein therapy, cell-based therapy, and gene therapy. Results: Over the past few years, biomedical engineering has been evolving from the laboratory to clinical applications, for replacement of damaged body tissues due to trauma, cancer, congenital or acquired disorders. Conclusions: This review provides an outlook on the content, benefits, recent advances, limitations, and future expectations of biomedical engineering for salivary glands, oral mucosa, dental structures, and maxillofacial reconstruction.

Gastrointestinal stromal tumor with maxillary metastasis: a case report and literature review.

Gastrointestinal stromal tumors (GISTs) form commonly in the stomach, small intestine, colorectum, and esophagus. Metastatic GIST occurs in up to 50% of patients at presentation. The liver and peritoneal cavity are the most common (93%) metastatic sites; head and neck metastases are extremely rare. This report describes a unique case of a 77-year-old man who was diagnosed with a duodenal GIST that had been completely resected 15 years ago. Eleven years after complete resection, he presented with liver metastases and then received multiple lines of systemic therapy and ablative radiotherapy. In 2015, he presented to our oral and maxillofacial surgery department with a left exophytic maxillary mass that filled the left maxillary sinus. Incisional biopsy confirmed metastatic GIST. Further evaluation revealed extensive metastases in the patient's liver, lungs, spleen, abdominal wall, and lymph nodes. After adequate staging, the patient's condition was deemed palliative, and he was referred to the radiation oncology department for palliative treatment of the symptomatic maxillary lesion. To our knowledge, this is the first reported case of maxillary metastasis from a duodenal GIST. Inclusion of GIST in the differential diagnosis of jaw tumors in patients with nonoral malignancies is recommended. The literature on oral metastasis of GIST is reviewed and discussed in this case report.

Studying Sjögren's syndrome in mice: What is the best available model?

Sjögren's syndrome (SS) is a common autoimmune disease characterized by lymphocytic infiltration and destruction of exocrine glands. The disease manifests primarily in the salivary and lacrimal glands, but other organs are also involved, leading to dry mouth, dry eyes, and other extra-glandular manifestations. Studying the disease in humans is entailed with many limitations and restrictions; therefore, the need for a proper mouse model is mandatory. SS mouse models are categorized, depending on the disease emergence into spontaneous or experimentally manipulated models. The usefulness of each mouse model varies depending on the SS features exhibited by that model; each SS model has advanced our understanding of the disease pathogenesis. In this review article, we list all the available murine models which have been used to study SS and we comment on the characteristics exhibited by each mouse model to assist scientists to select the appropriate model for their specific studies. We also recommend a murine strain that is the most relevant to the ideal SS model, based on our experience acquired during previous and current investigations.

Postoperative Administration of the Acetylcholinesterase Inhibitor, Donepezil, Interferes with Bone Healing and Implant Osseointegration in a Rat Model.

Donepezil is an acetylcholinesterase inhibitor commonly used to treat mild to moderate Alzheimer's disease. Its use has been associated with increased bone mass in humans and animals. However, the effect of postoperative administration of donepezil on bone healing remains unknown. Therefore, this study aimed to assess the impact of postoperative injection of donepezil on bone healing, titanium-implant osseointegration, and soft tissue healing. Twenty-two Sprague-Dawley rats were randomly assigned to receive a daily dose of either donepezil (0.6 mg/kg) or saline as a control. In each rat, a uni-cortical defect was created in the right tibia metaphysis and a custom-made titanium implant was placed in the left tibiae. After two weeks, rats were euthanized, and their bones were analysed by Micro-CT and histology. The healing of bone defect and implant osseointegration in the rats treated with donepezil were significantly reduced compared to the saline-treated rats. Histomorphometric analysis showed lower immune cell infiltration in bone defects treated with donepezil compared to the saline-treated defects. On the other hand, the healing time of soft tissue wounds was significantly shorter in donepezil-treated rats compared to the controls. In conclusion, short-term administration of donepezil hinders bone healing whereas enhancing soft tissue healing.

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.

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.

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.

Immediate implant placement into fresh extraction socket in the mandibular molar sites: a preliminary study of a modified insertion technique.

BACKGROUND: Immediate implant insertion in mandibular molar extraction sockets raises a series of challenges for clinicians. PURPOSE: This preliminary study demonstrates the use of a modified insertion technique of implant placement at the time of mandibular molar extraction. MATERIALS AND METHODS: Immediate implants were placed at the time of molar extraction in 20 patients; a sulcular buccal incision with releasing periosteal incisions were made around the mandibular molar to be replaced, and implant insertion into the interseptal/interradicular bone was performed. The remnants of roots were atraumatically extracted, and the bony defects around the implant were grafted with synthetic resorbable bone substitute ß- Tricalcium phosphate, and the flap was sutured. Three months later, implants were restored with single crown fixed prostheses. Patients were followed up at 6, 12, and 18 months after insertion using periapical standardized radiographs to monitor the changes in the marginal bone level. RESULTS: Our modified insertion techniques showed an implant survival rate of 95%; one implant failed 4 weeks after insertion. No significant marginal bone loss around the implant was recorded at all times of follow-up. Satisfactory soft issue parameters were achieved. CONCLUSIONS: The combination of immediate implant placement with engagement of the interseptal/interradicular bone, atraumatic extraction of remnant roots, and concomitant regenerative therapy showed preliminary favorable outcomes. However, wider application of this technique for longer following up periods is required for further conclusive recommendations.

Early-loaded laser-sintered versus acid-etched one-piece dental implants for mandibular premolars replacement: a preliminary study.

AIM: Early loading of dental implants can simplify treatment and increase patient's satisfaction. This 1-year preliminary clinical trial aimed to clinically and radiographically evaluate early-loaded one-piece implants that had either laser-sintered or acid-etched surfaces. MATERIALS AND METHODS: Sixty patients with missing mandibular premolars received 60 implants of one-piece type that were subjected to early loading after 3 weeks of initial placement. Group 1 included 30 implants with laser-sintered surface while group 2 included 30 implants with acid-etched surface. Patients were recalled for follow-up at 1, 3, 6, and 12 months after loading. RESULTS: No significant difference (P > 0.05) was found between the 2 implant groups at 1, 3, 6, and 12 months of implant loading, and all implants were considered successful after 12 months of follow-up. CONCLUSIONS: These preliminary results suggest that these implants are associated with satisfactory clinical and radiographic outcomes. Laser versus acid-etched surface treatments did not show any significant difference among different clinical measures or radiographic evaluations at different follow-up times. However, wider application for longer follow-up periods is required for further conclusive recommendations.

WITHDRAWN: Effect of different dentin cleaning techniques on bond strength and the micromorphology of dentin/self-adhesive resin cement interface.

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WITHDRAWN: Effect of post space conditioning and luting resin on the retentive strength of fiber-reinforced composite resin posts.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

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.

Evaluation of oral tissue response and blood levels of mercury released from dental amalgam in rats.

UNLABELLED: Dental amalgam is the most common restorative material used in dentistry. It was reported that amalgam might constitute potential toxic hazards to pregnant patients and foetuses through mercury release and absorption. The present study aimed to investigate the vital tissue response in contact to dental amalgam plus determination of blood mercury levels in mother and offspring Wistar strain albino rats. Pregnant mothers were divided into two main groups each had dental amalgam implanted into either an oral mucosa incision or a bony socket following extraction. Third and fourth groups included the offspring rats of mothers from the first and second groups, respectively. The blood mercury levels and histopathology of oral tissues were analyzed in mothers at one and six months post-implantation and in offspring rats one day after birth. The blood mercury levels of mothers increased significantly at six months (P<0.01) as compared to levels at one month. However, blood mercury levels were not significant (P>0.05) when the two offspring (third and fourth) groups were compared. Histopathology results from mothers showed inflammatory response at the bottom of the socket, one month after amalgam implantation. At six months, teeth germs showed vacuolation of the abnormal odontoblasts with globular dentine formation. Degenerated periodontal fibres and thin trabeculae forming the bony sockets with large marrow spaces were evident. A fibrous connective tissue capsule surrounded the amalgam mass inside the mucosa of mothers at one month and was evident also at 6 months with a huge inflammatory cell infiltrate. Teeth germs showed elongated odontoblasts with intercellular oedema, thinner dentine and bony trabeculae with wider marrow spaces. Offspring rats showed comparable oral tissue response. CONCLUSIONS: There is a positive correlation between blood mercury levels and oral tissue response in mothers, however, the negative impact of mercury on oral tissues of offspring rats was due to high mercury levels in their mothers' blood during pregnancy. We would recommend that women should - as far as possible - postpone having dental amalgam filling placed or removed during pregnancy to avoid its harmful effect on the foetus. Further clinical studies are recommended to test our findings in man.

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.

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

Sjogren's 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.

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

Sjogren's 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.

Matrigel improves functional properties of primary human salivary gland cells.

Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to <5% of huSG cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.

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.

Bone marrow cells are a source of undifferentiated cells to prevent Sjögren's syndrome and to preserve salivary glands function in the non-obese diabetic mice.

Non-obese diabetic (NOD) mice develop Sjögren's-like syndrome (Ss) and a gradual loss of saliva secretory function. Our previous study showed that injections of matched normal spleen cells with Complete Freund's Adjuvant (CFA) reversed salivary gland dysfunction in 14-week-old NOD mice, which had established Ss. The spleen and bone marrow are closely related organs, and both are among the first sites of hematopoiesis during gestation. Noticing a rapidly increasing number of clinical trials using bone marrow (BM) cells treatments for autoimmune diseases, we tested if BM cells can prevent Ss and restore salivary glands' function. We injected CFA and MHC class I-matched normal BM cells in 7-week-old NOD mice, which had not yet developed Ss. We found at week 52 post-treatment that all NOD mice receiving BM cells and CFA had a recovery of salivary flow and were protected from Ss and diabetes. BM cells-treated mice had their salivary function restored quantitatively and qualitatively. Saliva flow was higher (p<0.05) in BM cells-transplanted mice when compared to control mice, which continued to deteriorate over time. Total proteins, epidermal growth factor, amylase, and electrolytes concentrations in saliva of BM cells-treated mice were not significantly changed at week 44 and 52 post-therapy when compared to pre-therapy (when the mice did not have Ss). Restoration of salivary flow could have resulted from a combination of rescue and paracrine effects from BM cells. This study suggests that a combined immuno- and cell-based therapy can permanently prevent Ss and restored salivary function in NOD mice.