Enriching Stem/Progenitor Cells from Dental Pulp Cells by Low-density Culturing.

BACKGROUND/AIM: Clonogenicity is a key feature of stem/progenitor cells. The present study aimed to enrich stem/progenitor cells from dental pulp cells by means of culturing the cells at a low clonal density with spatial separation and the evaluate differentiation potential of the surviving cells. MATERIALS AND METHODS: Pulp cells derived from wisdom teeth were seeded into wells of a 96-plate at a mean density of 1 cell/well and cultured for 2 weeks. Surviving cells were harvested, pooled together and subjected to differentiation into adipocytes, osteoblasts and neurons using respective inducing conditions for 3 weeks. The former two types of cells were examined by staining with Oil Red O and Alizarin Red, respectively. Neuron-like cells were inspected for their morphology and immunostained for microtubule-associated protein 2 and ß-tubulin III. RESULTS: Vital cells were obtained in eight wells of a 96-well plate, corresponding to a survival rate of 8%. Since fewer than two wells would be expected to contain more than four cells at seeding, the majority of surviving cells likely grew from 1-3 cells, which is a very low density. These cells differentiated into functional adipocytes and osteoblasts, and morphologically neuron-like cells. CONCLUSION: Low-density seeding with spatial separation enables statistical estimation of cell number in wells and provides an effective strategy for enriching stem/progenitor cells and for isolating clonal dental pulp cells.

Accuracy of Bone Measurements in the Vicinity of Titanium Implants in CBCT Data Sets: A Comparison of Radiological and Histological Findings in Minipigs.

PURPOSE: The aim of this animal study was the determination of accuracy of bone measurements in CBCT (cone-beam computed tomography) in close proximity to titanium implants. MATERIAL AND METHODS: Titanium implants were inserted in eight Göttingen minipigs. 60 implants were evaluated histologically in ground section specimen and radiologically in CBCT in regard to thickness of the buccal bone. With random intercept models, the difference of histologic measurements and CBCT measurements of bone thickness was calculated. RESULTS: The mean histological thickness of the buccal bone was 5.09 mm (CI 4.11-6.08 mm). The four raters measured slightly less bone in CBCT than it was found in histology. The random effect was not significant (p value 1.000). Therefore, the Intraclass Correlation Coefficient (ICC) was 98.65% (CI 100.00-96.99%). CONCLUSION: CBCT is an accurate technique to measure even thin bone structures in the vicinity of titanium implants.

Postoperative bleeding risk for oral surgery under continued rivaroxaban anticoagulant therapy.

OBJECTIVES: The purpose of this study was to assess the risk of postoperative bleeding complications after oral procedures performed under continued mono or dual anticoagulation therapy with rivaroxaban (and aspirin). METHODS: This retrospective single-center observational study included 52 oral procedures performed under continued oral anticoagulant therapy with rivaroxaban (20 mg/day). Among them, two procedures were performed under continued dual therapy with aspirin (100 mg/day) added to the regimen. Postoperative bleeding events were compared with 285 oral procedures in patients without any anticoagulation/antiplatelet therapy. RESULTS: Postoperative bleeding complications after oral surgery occurred significantly more often in patients under continued rivaroxaban therapy (11.5 %) than in the control cases without anticoagulation/antiplatelet medication (0.7 %). All of the bleeding events were manageable: Two of them were treated with local compression, three by applying new fibrin glue with (one case) or without (two cases) secondary sutures, one occurred during a weekend and was therefore treated under inpatient conditions with suture replacement. All postoperative bleeding episodes occurred during the first postoperative week. CONCLUSIONS: According to our data, continued anticoagulation therapy with rivaroxaban significantly increases postoperative bleeding risk for oral surgical procedures, although the bleeding events were manageable. CLINICAL RELEVANCE: Oral surgeons, cardiologists, general physicians, and patients should be aware of the increased bleeding risk after oral surgical procedures. Close observation up to 1 week postoperatively is advisable to prevent excessive bleeding.

Increased Postoperative Bleeding Risk among Patients with Local Flap Surgery under Continued Clopidogrel Therapy.

PURPOSE: The purpose of the study was to evaluate the influence of a continued antiplatelet therapy with clopidogrel on postoperative bleeding risk in patients undergoing skin tumor resection and reconstruction with local flaps or skin grafts under outpatient conditions. PATIENTS AND METHODS: The authors designed and implemented a retrospective clinical cohort study at the General Hospital Balingen. The primary endpoint was the bleeding ratio in patients with clopidogrel treatment in comparison to patients without any anticoagulant or antiplatelet therapy. Wound healing was evaluated on days 1, 3, 5, 7, 10, and 14. RESULTS: 650 procedures were performed, 123 of them under continued clopidogrel therapy. There were significantly more postoperative bleeding complications among patients with continued antiplatelet therapy. Regarding the whole study population, malignant lesions, a larger defect size, and skin grafts were accompanied by a higher rate of bleeding incidents. However, there were no significant findings in the univariate analysis of the clopidogrel group. All bleeding incidents were easily manageable. CONCLUSION: Despite an increased bleeding ratio among patients under continued clopidogrel therapy, the performance of simple surgical procedures can be recommended. However, cautious preparation and careful hemostasis are indispensable.

Enrichment and Schwann Cell Differentiation of Neural Crest-derived Dental Pulp Stem Cells.

BACKGROUND/AIM: As already described in previous studies, neural crest stem cells (NCSCs) can be found in adult human dental pulp. The present study investigated the methodology for enrichment and differentiation-induction of the above mentioned cells. MATERIALS AND METHODS: Dental pulp was extracted from human wisdom teeth of four patients and subsequently cultured as explants on fibronectin-coated plates in neurobasal medium supplemented with B27, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, l-glutamine and neuregulin-ß1. The cells were then characterized by immunofluorescence, while their differentiation-potential was tested by the attempt to induce cells into different lineages, i.e. osteogenic, melanocytic and glial. RESULTS: The enriched cell population expressed nestin, CD271 and SOX10, which are well-known markers for NCSCs. Consequently, the cells were successfully induced to differentiate into osteoblasts, melanocytes and Schwann cells, expressing the corresponding differentiation markers. CONCLUSION: Human adult dental pulp contains a population of stem cells with neural crest ontogeny, which can thus be recruited for multiple regenerative therapies.

Is immediate bony microsurgical reconstruction after head and neck tumor ablation associated with a higher rate of local recurrence?

The purpose of this retrospective study was to examine the possible effect of immediate bony microvascular free flap reconstruction of mandibular defects after radical tumor resection of oral squamous cell carcinoma (OSCC) on the rate of local relapse. Our own data regarding recurrence rates for 1-step immediate reconstruction were compared to the published recurrence rates of 2-step reconstructions. A total of 21 patients (aged 45-77 years) with OSCC who underwent a primary surgical therapy with subsequent immediate bony microvascular free flap reconstruction of mandibular defects were followed up for 18-38 months. Four local relapses (19%) were recorded, all in patients with initial tumor stage of T4. Although intraoperative histological findings were R0 in all 21 cases, definitive histology later detected R1 status in the resected bone in 2 cases (10%). Immediate bony free flap reconstruction of mandibular defects after radical surgical resection of OSCC does not seem to increase the risk of local recurrence nor affect patient survival when compared with the 2-step surgical approach.

Postoperative bleeding risk for oral surgery under continued clopidogrel antiplatelet therapy.

OBJECT: To determine the incidence of postoperative bleeding for oral osteotomy carried out under continued monoantiplatelet therapy with clopidogrel and dual therapy with clopidogrel/aspirin. Design. Retrospective single center observatory study of two study groups and a control group. METHODS: A total of 64 and 60 oral osteotomy procedures carried out under continued monoclopidogrel therapy and dual clopidogrel/aspirin therapy, respectively, were followed for two weeks for postoperative bleeding. Another 281 similar procedures were also followed as a control group. All oral osteotomy procedures were carried out on an outpatient basis. RESULTS: We observed postoperative bleeding in 2/281 (0.7%) cases in the control group, in 1/64 (1.6%) cases in the clopidogrel group, and in 2/60 (3.3%) cases in the dual clopidogrel/aspirin group. The corresponding 95% confidence intervals are 0-1.7%, 0-4.7%, and 0-7.8%, respectively, and the incidences did not differ significantly among the three groups (P > 0.09). Postoperative hemorrhage was treated successfully in all cases with local measures. No changes of antiplatelet medication, transfusion, nor hospitalisation were necessary. No major cardiovascular events were recorded. CONCLUSIONS: Our results indicate that minor oral surgery can be performed safely under continued monoantiplatelet medication with clopidogrel or dual antiplatelet medication with clopidogrel/aspirin.

Lack of evidence for increased postoperative bleeding risk for dental osteotomy with continued aspirin therapy.

OBJECTIVE: Dental osteotomy, the removal of an impacted, ankylosed, or severely destroyed tooth requiring an osteotomy, is more invasive than other minor dental procedures and therefore also has a higher bleeding risk. A considerable number of patients under antiplatelet therapy interrupt their therapy perioperatively, which, however, increases the risk of thromboembolism. STUDY DESIGN: This retrospective study assessed postoperative bleeding incidence for a total of 297 dental osteotomies with continued aspirin therapy, compared with that of 179 similar procedures on patients who were not on any anticoagulation or anti-platelet therapy. All procedures were carried out on an outpatient basis. RESULTS: Postoperative bleeding event was rare in both groups, 5 (1.7%) and 2 (1.1%), respectively, and the difference was not significant (P = .7). CONCLUSIONS: Continued aspirin therapy in patients undergoing dental osteotomies has no effect on the incidence of postoperative bleeding and should not be interrupted.

Bone substitutes enhance osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds.

AIM: The present study aimed to find bone substitutes to enhance osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds in the absence of dexamethasone. MATERIALS AND METHODS: Seven commercial bone substitutes were added to a three-dimensional fibrin-matrix containing rat mesenchymal stem cells in a biocompatible poly-L-lactic-acid mesh. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for three weeks. Expression of bone markers was examined by qualitative evaluation of corresponding transcripts. RESULTS: Six out of the seven bone derivatives exhibited an osteogenic-enhancing effect. CONCLUSION: The osteogenic-enhancing effect of the evaluated bone substitutes suggests their potential clinical application for preparation of autologous bone replacement material in three-dimensional carriers.

Osteogenic differentiation of mesenchymal stem cells in fibrin-hydroxyapatite matrix in a 3-dimensional mesh scaffold.

AIM: To explore the feasibility of culturing mesenchymal stem cells in an hydroxyapatite-fibrin matrix held by a mesh scaffold and inducing osteogenic differentiation of these cells. The aim was to obtain bone-material in vitro in a desired form. MATERIALS AND METHODS: Rat mesenchymal stem cells were mixed with fibrin and nanocrystalline hydroxyapatite in tubular scaffolds constructed from a poly(L-lactic acid) mesh, and cultured under standard and osteogenic differentiating conditions. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for 3 weeks. Living cells and the expression of bone markers were visualized by fluorescence staining and immunofluorescence staining, respectively. Attachment of cells to the scaffold mesh surface was examined by scanning electron microscopy. RESULTS: Cell viability decreased and cytotoxicity increased rapidly during the first day of culture but stabilized gradually afterwards, indicating fast adaptation of the cells in the matrix-scaffold environment. From day 17, cytotoxicity started to decrease, paralleled by an increase in alkaline phosphatase activity, indicating osteogenic differentiation. A large number of living cells were visible in the matrix and on the mesh scaffold. Expression of collagen type I, osteoponin, osteocalcin and core binding factor 1 were evident under osteogenic differentiation conditions. CONCLUSION: The three-dimensional construction of a fibrin-hydroxyapatite matrix in a biocompatible poly(L-lactic acid) as mesh-scaffold provides a promising carrier for producing bone-material in vitro in a desired form for applications in regenerative medicine.

Lack of evidence for increased risk of postoperative bleeding after cutaneous surgery in the head and neck in patients taking aspirin.

Cutaneous surgery in the head and neck carries an intermediate risk of bleeding, so many surgeons stop anticoagulant treatment perioperatively despite the risk of cardiovascular events. We reviewed many procedures done in patients who were taking aspirin, and we did not find evidence of significantly increased postoperative bleeding compared with procedures in patients who were not taking aspirin (10/259=4% compared with 6/209=3%, p=0.6). Our results suggest that cutaneous surgery in the head and neck is safe for patients taking aspirin.

Outcome of microvascular free flaps in a high-volume training centre.

PURPOSE: Microvascular free tissue transfer allows major ablative defects following oncologic surgical and traumatic reasons to be reliably reconstructed in the head and neck region. A retrospective analysis of the microvascular flap procedures which were performed within one year in a high volume training centre was performed. PATIENTS AND METHODS: The microvascular free flap procedures of the year 2011 were reviewed and followed up until the 31st December 2012. The type and indication of the reconstructive procedure, operation time, operating team, experience and level of training of the surgeons involved, postoperative IMC (intermediate care unit) and/or ICU (intensive care unit) time, inpatient time, flap revisions, further postoperative complications, preoperative and postoperative radiation of the patients, the placement of dental implants were studied. RESULTS: From 1st of January 2011 to 31st of December, 2011, the data of 101 patients with 103 microvascular free flap procedures were analysed of which 72% (84 flaps) were harvested by residents. The patients ranged in age from 14 to 89 years (mean age 59 years, 71 males and 40 females). The mean operation time was 591 min with the longest operation times for scapular flaps (744 min) and the shortest operation times for ALT flaps (455 min). Mean inpatient time was 34.2 days with a minimal time for the fibular flaps of 27.2 days and a maximum of 45.7 days for the latissimus dorsi flaps. 24 flaps (23.3%) in total had to be revised with bleeding being the main cause of immediate revisions (41.7% of all revisions). 5 flaps (4.85% of all flaps) were lost despite a revision procedure meaning a successful revision rate in 79.2% of all revisions. CONCLUSION: Microvascular reconstruction procedures are safe and should be considered as standard procedures for reconstruction of large defects especially in high volume training centres. Intensive flap monitoring and early revisions maximize the flap outcome.

Her2 expression and gene amplification is rarely detectable in patients with oral squamous cell carcinomas.

PURPOSE: Her2 (ErbB2) transforms cells when overexpressed and is an important therapeutic target in breast cancer. Contrary to breast cancer, studies on Her2 overexpression and gene amplification in squamous cell carcinomas of the head and neck region described largely different results. This study was undertaken to learn more on the prevalence and clinical significance of HER2 amplification and overexpression in squamous cell carcinomas of the head and neck. MATERIALS AND METHODS: Her2 expression and gene amplification was analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on two tissue microarrays composed of 427 squamous cell carcinomas of the head and neck region and 222 oral squamous cell carcinomas. Results were compared with clinicopathological features. RESULTS: Her2 expression and gene amplification was rarely detectable in squamous cell carcinomas of the head and neck region and unrelated to tumor phenotype or survival of the patients with oral squamous carcinoma. DISCUSSION: Our results demonstrate that Her2 protein and gene amplification was only detectable in a small subset of squamous cell carcinomas of the head and neck region as well as oral squamous cell carcinomas. However, it can be speculated that those few patients with Her2 overexpressing and gene amplificated tumors may possibly benefit from an anti-Her2 therapy.

Postoperative bleeding risk for cutaneous surgery in the head and neck region with continued phenprocoumon therapy.

In a total of 171 surgical procedures for lesions in the head and neck region in patients in whom phenprocoumon therapy was not stopped, 16 (9%) postoperative bleeding events were observed over a follow-up period of two weeks. Local measures were sufficient in all cases except one severe case where blood transfusion was needed and anticoagulant treatment was stopped for 7 days. The bleeding risk was significantly higher for the surgical procedures of the nose than those in other areas (21% versus 6%, P = 0.014), but was not influenced by the international normalized ratio (INR) of blood coagulation, size, site and type of the lesion, surgical procedure, and sex and age of the patients. The bleeding rate in patients not on any anticoagulation therapy was significantly lower (6/211 = 3%). Across both groups, just over 80% of the bleeding episodes were within the first two days (55% on the same day and 32% on the next day) of the surgery. No bleeding was recorded after 5 days. Our data suggest that cutaneous surgery in the head and neck region can be safely performed with continued phenprocoumon therapy in most cases in an INR range of 1.3-3.4, but rarely severe bleeding does occur and can be managed with a close-contact follow-up and with 24-h on call services during the first two days postoperatively.

Immunohistochemical and FISH analysis of EGFR and its prognostic value in patients with oral squamous cell carcinoma.

OBJECTIVES: To study immunohistochemical expression of the epithelial growth factor receptor (EGFR) in oral carcinomas and the head and neck region to examine possible associations with various features of the tumors and survival of the patients. MATERIALS AND METHODS: Sections were made from two tissue arrays composed of 206 oral squamous cell carcinomas and 427 squamous cell carcinomas of the head and neck region, respectively, and examined for EGFR expression and Ki-67 labeling index by means of immunohistochemistry, and for EGFR gene amplification by means of fluorescence in situ hybridization. Correlation between resulting parameters and with clinical features was evaluated using chi-square test and Kaplan-Meyer analysis. RESULTS: A statistically significant association was observed for strong EGFR immunohistochemical (IHC) expression with advanced lymph node involvement (P = 0.02). EGFR immunohistochemical expression did not significantly correlate with patient disease specific (DS) or overall survival (OS). EGFR gene amplification was not correlated with any of the tumor features nor to survival of the patients (DS and OS). DISCUSSION: Epithelial growth factor receptor IHC expression and gene amplification might be suitable to predict locoregional control in oral squamous cell carcinoma patients but an inappropriate predictor for patients survival.

First description of a hybrid tumor of the sublingual gland.

BACKGROUND: Hybrid tumours of the salivary glands are rare neoplasms. They are composed of at least two different tumour entities located in the same topographic area and account for only 0.1% of all salivary gland tumours. The most common component is an adenoid cystic carcinoma. There are several possible forms of hybrid tumours, which are most commonly located in the parotid gland. CASE REPORT: We report on a 59-year-old female, who presented with a lesion of the caruncula of the left sublingual gland. The biopsy showed an adenoid cystic carcinoma in combination with a salivary duct carcinoma. Treatment consisted of tumour resection, bilateral selective neck dissection and adjuvant radiotherapy. Histopathologically, at least 30% of the tumour mass was composed of a salivary duct carcinoma and 70% of an adenoid cystic carcinoma. At 58 months after treatment, the patient is alive without evidence of recurrent disease. CONCLUSION: To our knowledge, the presented case is the first description of a hybrid tumour of the sublingual gland. Furthermore, the post-therapeutic course is encouraging, as hybrid tumours of the salivary glands usually have a poor prognosis.

The detection of oral pre- malignant lesions with an autofluorescence based imaging system (VELscope™) - a single blinded clinical evaluation.

OBJECTIVE: The disease specific five-year survival rate especially for patients with advanced oral cancer has not improved significantly over the period of time. The most effective way of combating this dilemma is an early detection, diagnosis and eradication of early-stage lesions and their precursors. The use of VELscope® using an autofluorescence as a diagnostic tool might be useful in early detection of oral malignant lesions. MATERIALS AND METHODS: 120 patients with suspicious oral premalignant lesions were examined with two examination methods. They were randomly divided into two groups. Group 1 was examined conventional with white-light and group 2 was examined additionally to the white-light-examination with an autofluorescence visualization device, VELscope®. Biopsies were obtained from all suspicious areas identified in both examination groups (n = 52). The diagnostic strategies were compared regarding sensitivity and specificity. RESULTS: Based upon the result, use of the VELscope® leads to a higher sensitivity (22.0%), but regarding specificity the additional use of the VELscope® is inferior (8.4%). CONCLUSION: The VELscope device is a simple, non-invasive test of the oral mucosa, which can help the experienced clinician to find oral precursor malignant lesions.

Bleeding incidence after oral surgery with continued oral anticoagulation.

OBJECTIVES: The aim of this retrospective study was to assess the incidence of postoperative hemorrhage in patients treated with coumarins without interruption of the anticoagulant treatment undergoing oral surgical procedures, mostly osteotomies for tooth removal, when compared with patients who had never been anticoagulated. MATERIAL AND METHODS: Six hundred thirty-seven patients underwent 934 oral surgical procedures on an outpatient basis. The INR was measured preoperatively being 2.44 in the mean SD 0.61. Local hemostasis was carried out routinely (80%) with collagen fleece, local flap, and acrylic splint. RESULTS: Of these 637 patients, 47 presented with a postoperative hemorrhage (7.4%), 15 of these 47 cases had to be treated in hospital (2.4%). All patients showed up finally with a good wound healing, no administration of blood was necessary, and local measures revealed to be sufficient in all cases except for two patients, where the preoperative anticoagulant treatment had to be changed for 6 days. The bleeding incidence in 285 patients with comparable oral surgical procedures, who had never been anticoagulated, was 0.7%. CONCLUSIONS: The results suggest that oral surgical procedures can be performed safely without alteration of the oral anticoagulant treatment. CINICAL RELEVANCE: Local hemostasis with collagen fleece, local flap, and acrylic splint seems to be sufficient to prevent postoperative bleeding.

Influence of lip closure on alveolar cleft width in patients with cleft lip and palate.

BACKGROUND: The influence of surgery on growth and stability after treatment in patients with cleft lip and palate are topics still under discussion. The aim of the present study was to investigate the influence of early lip closure on the width of the alveolar cleft using dental casts. METHODS: A total of 44 clefts were investigated using plaster casts, 30 unilateral and 7 bilateral clefts. All infants received a passive molding plate a few days after birth. The age at the time of closure of the lip was 2.1 month in average (range 1-6 months). Plaster casts were obtained at the following stages: shortly after birth, prior to lip closure, prior to soft palate closure. We determined the width of the alveolar cleft before lip closure and prior to soft palate closure measuring the alveolar cleft width from the most lateral point of the premaxilla/anterior segment to the most medial point of the smaller segment. RESULTS: After lip closure 15 clefts presented with a width of 0 mm, meaning that the mucosa of the segments was almost touching one another. 19 clefts showed a width of up to 2 mm and 10 clefts were still over 2 mm wide. This means a reduction of 0% in 5 clefts, of 1-50% in 6 clefts, of 51-99% in 19 clefts, and of 100% in 14 clefts. CONCLUSIONS: Early lip closure reduces alveolar cleft width. In most cases our aim of a remaining cleft width of 2 mm or less can be achieved. These are promising conditions for primary alveolar bone grafting to restore the dental bony arch.

Metastases in odontogenic cysts: literature review and case presentation.

Malignant tumors in the oral cavity are relatively rare. About 5% of all malignant growths in the body are localized in the oral cavity. The vast majority of oral malignancies are primary tumors with squamous cell carcinoma being the most frequent and sarcomas occurring very seldom. Secondary tumors caused by hematogenous spread arising from a tumor localized elsewhere in the body are extremely rare. About 1% of all oral cancers are metastases to the jawbones and the surrounding soft tissues. Metastases to the jaws are mainly caused by malignant tumors of the breast, lung, kidney, bone, and colon. They occur in the late state of the disease and are regularly detected by staging examinations including scintigraphy. Even more rare are metastases into odontogenic cysts. Odontogenic cysts include dentigerous cysts, periapical or radicular cysts, and the keratocysts-nowadays declared as keratocystic odontogenic tumor. The incidence of odontogenic cysts is about 10% to 15% of all oral biopsies and therefore general dentists are frequently faced with these types of lesions. The aim of this study was to review the literature regarding metastasis into odontogenic cysts and to further highlight this rare entity with the help of a clinical case.