In vitro study on proliferation kinetics of oral mucosal keratinocytes.

OBJECTIVE: The limited availability of autogenous oral mucosa in oral and maxillofacial surgery for intraoral grafting after trauma or tumor resection can be balanced by the use of tissue-engineered oral mucosa. However, the use of tissue engineering in autologous grafts is still subject to further research. The aim of this study was to evaluate conditions that lead to a rapid proliferation of vital and highly proliferative oral keratinocytes, which can be used in tissue engineering and consequently help improve surgical management of intraoral mucosal defects. MATERIAL AND METHODS: Human oral keratinocytes were obtained from oral mucosal specimens and cultivated. According to their affinity to ß1-integrin, epidermal stem cell populations were isolated by using collagen type IV and laminin-coated dishes. Cell proliferation and cell viability were measured by using the CASY cell counter, WST-1 assays, and real-time cell analysis (xCELLigence). RESULTS: Measurements on cell proliferation (CASY cell counter) and cell viability (WST-1 assay) showed the characteristic proliferation stages of in vitro-cultivated cells. No statistically significant differences could be monitored (P > .05). Real-time cell analysis, as a more direct and precise technique, revealed a steeper growth curve of adherent cells and therefore generally higher proliferation kinetics compared with cells derived from the supernate. CONCLUSION: Data from real-time cell analysis showed an increased proliferation of adherent cells compared with those derived from the supernate. These results demonstrate the increase of the proliferation capacity by cultivation of keratinocytes derived by adhesion to extracellular matrix proteins.

Histomorphologic characteristics of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a known side effect of the therapy with bisphosphonates. No specific pathologic aspects or histological features are included in the most current definition. This study investigates characteristics of BRONJ with a special emphasis on histomorphologic aspects, evaluating the role of Actinomyces spp. as well as other disease-promoting factors in a formal pathogenetic context. METHODS: We investigated 23 patients (14 female, nine male; mean age: 66 ± 11.8 years) who received bisphosphonates with a gender- /age-matched control group. Tissue specimens were treated according to local standards and analyzed histologically. RESULTS: In 18 (78.3%) BRONJ cases, we found Actinomyces spp. colonies. Bone remodeling could be found in three specimens (13%). Eight specimens (34.8%) showed signs of epithelial proliferation. Analysis of dental treatment before the onset of BRONJ did not reveal significant differences (P > 0.20). In 10 patients (83%; P > 0.05) of the reported cases a relationship between dental treatment and the occurrence of a purulent bone necrosis could be observed. Statistically significant differences in thickness of trabeculae were detected between the two study groups (P = 0.04). CONCLUSIONS: This study demonstrates the important influence of the osteoblast-osteoclast balance in a histomorphologic analysis. Together with cofactors, which are able to trigger the onset of BRONJ, a new pathogenesis model was developed.

Role of oxidative and nitrosative stress in autogenous bone grafts to the mandible using guided bone regeneration and a deproteinized bovine bone material.

UNLABELLED: The aim of this study was to evaluate the role of oxidative and nitrosative stress in autogenous bone grafts to the mandible based on immunohistochemical analysis. MATERIAL AND METHODS: Using a well-established sheep model autogenous bone grafts were harvested form the iliac bone. A combination of a Collagen Membrane (CM) and Deproteinized Bovine Bone Material (DBBM) was used to cover the bone graft (Experiment 2). This modification was compared with simple onlay bone grafts (Experiment 1). Immunohistochemically, the expression of specific stable degradation products of oxidative and nitrosative stress was compared between the two experimental groups. RESULTS: Specific markers for oxidative and nitrosative stress showed statistically significant differences in expression in the different experimental groups. The influence of oxidative and nitrosative stress on osteoblasts (OB), osteoclasts (OC), and osteocytes (OCy) was analysed. Experiment 2 showed increased expression of markers in OB and decreased expression in OC. CONCLUSIONS: Taking the result of this study and reports from the literature into consideration grafts in Experiment 2 showed less resorption and atrophy, higher activity of OB and inhibition of OC, and less expression of Reactive Oxygen and Nitrogen Species (RONS) as markers of oxidative stress within the graft. These data illustrate the improved remodelling processes in grafts using CM and DBBM.

Expression of growth factors during the healing process of alveolar ridge augmentation procedures using autogenous bone grafts in combination with GTR and an anorganic bovine bone substitute: an immunohistochemical study in the sheep.

OBJECTIVES: This study aims to evaluate the expression of various immunohistochemical growth factors and vascularization markers in augmentation on the mandible comparing onlay bone grafts and Guided Bone Regeneration (GBR). MATERIALS AND METHODS: Using a sheep in vivo model, autogenous bone grafts were harvested from the iliac crest. A combination of a resorbable collagen membrane (CM) and a Deproteinized Bovine Bone Material (DBBM) was performed. This modification of the host side was compared with an onlay bone graft control group. Expression of different vascularization markers was compared between these groups. RESULTS: The expression of revascularization markers was significantly higher within the modification of the host side using GBR and DBBM. Regarding different graft regions, a significantly higher expression within the bone graft using GBR and DBBM could be observed in staining on bone morphogenetic protein-2 (BMP-2) (5.75 vs. 3.55), vascular endothelial growth factor (VEGF) (3.08 vs. 1.64), VEGF Receptor 1 (VEGFR-1) and VEGF Receptor 2 (VEGFR-2) (4.88 vs. 2.24 and 5.06 vs. 2.74), and endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) (5.29 vs. 3.28 and 5.22 vs. 3.09; p = 0.000, all others p < 0.05), whereas the control group showed a higher rate of resorption during the surveillance period until euthanasia of sheep after 16 weeks. CONCLUSION: The use of GBR and DBBM in the transplantation process of autogenous bone grafts compared with the therapeutical use of certain growth factors may enhance vascularization and lower atrophy and resorption. CLINICAL RELEVANCE: The use of a combination of GBR and DBBM in augmentation procedures on the mandible shows less resorption than simple onlay bone grafts and seems to be superior in a clinical use.

Immunohistochemial study on the expression of von Willebrand factor (vWF) after onlay autogenous iliac grafts for lateral alveolar ridge augmentation.

INTRODUCTION: The main problems of autogenous bone transplants are their unpredictable atrophy and their loss of structure. One key factor lies in the poor revascularization of simple onlay grafts. The the aim of this study was to evaluate the revascularization processes in autogenous bone grafts from the iliac crest to the alveolar ridge. METHODS: In a sheep model, autogenous bone grafts were harvested from the iliac crest. A combination of a resorbable collagen membrane (CM) and deproteinized bovine bone material (DBBM) was used to modify the bone graft (experiment 2). This was compared with a simple onlay bone graft (control group, experiment 1). The amount of vessels in bone and connective tissue (CT), and the amount of CT were analyzed. The expression of von Willebrand factor (vWF) was compared between the two experimental groups using immunohistochemical analysis. RESULTS: The ratio of the amount of vessels in bone and CT changed over time, and more vessels could be detected in bone at 12-16 weeks of graft healing. The number of vessels were significantly higher in experiment 2 than in experiment 1. More CT was found in experiment 1, whereas the amount of CT in both experiments decreased over time. CONCLUSION: This study shows a more intensive and extensive revascularization in experiment 2, as significantly more vessels were detected. The decreased amount of CT in experiment 2 clarifies its clinical superiority.

Tissue engineering of human oral mucosa on different scaffolds: in vitro experiments as a basis for clinical applications.

OBJECTIVES: The lack of oral mucosa in oral and maxillofacial surgery for intraoral grafting after trauma or tumor resection can be balanced by tissue-engineered oral mucosa. The aim of this study was to generate a tissue-engineered oral mucosa equivalent (OME). STUDY DESIGN: First, primary oral fibroblasts were cultured for 7 days on different materials: Tissufoil E (TFE), dermal regeneration template (DRT), and Vicryl. Then, cocultures were established by seeding of primary oral keratinocytes and culturing for another 7-14 days. Immunohistochemical staining for CD90, cytokeratin 14 and collagen IV as well as gene expression analysis using reverse-transcription quantitative polymerase chain reaction were used to get information about cell architecture and basal membrane formation. RESULTS: Vicryl showed good mechanical stability but mixed cell growth. TFE provided the best cell growth with good cell architecture and basal membrane formation but showed degradation. The best results for the above-mentioned criteria were seen with DRT. CONCLUSIONS: It was possible to create OMEs on all 3 scaffolds. The arrangement of the cells strongly depends on the texture of the material.

Hemimandibulectomy after bisphosphonate treatment for complex regional pain syndrome: a case report and review on the prevention and treatment of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: The increase in reported cases of osteonecrosis of the jaw has increased the clinical significance of bisphosphonate therapeutic agents in the dentistry field. METHODS: We present a rare and severe case of bisphosphonate-related osteonecrosis of the jaw caused by medicamentous treatment of complex regional pain syndrome. This article reviews the current international prevention and treatment guidelines with regard to bisphosphonate treatment. RESULTS: Even rare indications for bisphosphonate treatment may lead to devastating effects on the patient. CONCLUSIONS: Dentists and physicians who prescribe bisphosphonates should be familiar with the side effects of these drugs and the management of these side effects. To prevent negative outcomes, it is important that there be a close collaboration among the doctors involved and that a thorough medical history is obtained; this is especially true because the range of indications for bisphosphonate treatment increases every year.

Mucormycosis of the head and neck.

INTRODUCTION: Mucormycosis of the head and neck is a rare disease increasingly occurring in immunocompromised patients. We report on two cases with different outcomes. CASE REPORTS: A 63-year-old female presented with a recently developed deformation of her right cheek and nose combined with a loosening of the teeth. Further examination revealed mucormycosis of the maxilla. Hemimaxillectomy and secondary bony reconstruction with oral rehabilitation were performed. The second patient was a 54-year-old male who suffered from multiple myeloma. After receiving an allogeneic haematopoietic stem cell transplant, he developed a necrotizing infection of the right midface. Histopathological investigation confirmed the diagnosis of mucormycosis. The patient died one day after radical surgical resection. DISCUSSION: These two cases demonstrate the variability of mucormycosis. Although slow progression of the disease is possible, a high level of attentiveness and expedient treatment are necessary due to the high risk of a devastating course.

In vivo degradation of low temperature calcium and magnesium phosphate ceramics in a heterotopic model.

Bone replacement using synthetic and degradable materials is desirable in various clinical conditions. Most applied commercial materials are based on hydroxyapatite, which is not chemically degradable under physiological conditions. Here we report the effect of a long-term intramuscular implantation regime on the dissolution of various low temperature calcium and magnesium phosphate ceramics in vivo. The specimens were analysed by consecutive radiographs, micro-computed tomography scans, compressive strength testing, scanning electron microscopy and X-ray diffractometry. After 15months in vivo, the investigated materials brushite (CaHPO(4)·2H(2)O), newberyite (MgHPO(4)·3H(2)O), struvite (MgNH(4)PO(4)·6H(2)O) and hydroxyapatite (Ca(9)(PO(4))(5)HPO(4)OH) showed significant differences regarding changes of their characteristics. Struvite presented the highest loss of mechanical performance (95%), followed by newberyite (67%) and brushite (41%). This was accompanied by both a distinct extent of cement dissolution as well as changes of the phase composition of the retrieved cement implants. While the secondary phosphate phases (brushite, newberyite, struvite) completely dissolved, re-precipitates of whitlockite and octacalcium phosphate were formed in either particulate or whisker-like morphology. Furthermore, for the first time the possibility of a macropore-free volume degradation mechanism of bioceramics was demonstrated.

Kimura's disease in a white man.

BACKGROUND: Kimura's disease is a rare inflammatory disease that mainly affects Asians and most often occurs in the deep lymph nodes of the head and neck. We report on a rare case of Kimura's disease in the hard palate of a white man. METHOD: A 56-year-old white man was seen with a rapidly growing mass in the upper jaw. A complete tumor resection with hemimaxillectomy was performed. The tumor, which showed signs of inflammation, was located within the bone and the soft tissue. RESULTS: Kimura's disease was diagnosed by histopathologic examination of the resected tumor. CONCLUSION: This case demonstrates that Kimura's disease, though rare, is not limited to the Asian population. We present a case of a tumor in a white man. This adds another possibility for uncertain differential diagnoses of rapidly growing tumor masses.

MAGE-A antigens in lesions of the oral mucosa.

Oral squamous cell carcinoma develops continuously out of predamaged oral mucosa. For the physician and pathologist, difficulties arise in distinguishing precancerous from cancerous lesions. MAGE-A antigens are tumor antigens that are found solely in malignant transformed cells. These antigens might be useful in distinguishing precancerous from cancerous lesions. The aim of this study was to verify this assumption by comparing MAGE-A expression in benign, precancerous, and cancerous lesions of the oral mucosa. Retrospectively, biopsies of different oral lesions were randomly selected. The lesions that were included are 64 benign oral lesions (25 traumatic lesions (oral ulcers), 13 dental follicles, and 26 epulis), 26 oral lichen planus, 123 epithelial precursor lesions (32 epithelial hyperplasia found in leukoplakias, 24 epithelial dysplasia found in leukoplakias, 26 erythroplasia with oral epithelial dysplasia, and 41 carcinomas in situ in erythroleukoplakias). The lesions were immunohistochemically stained with the poly-MAGE-A antibody 57B, and the results were compared. Biopsies of oral lichen planus, oral ulcers, dental follicles, epulis, and leukoplakia without dysplasia showed no positive staining for MAGE-A antigens. Leukoplakia with dysplasia, dysplasia, and carcinomata in situ displayed positive staining in 33%, 65%, and 56% of the cases, respectively. MAGE-A antigens were not detectable via immunohistochemistry in benign lesions of the oral mucosa. The staining rate of dysplastic precancerous lesions or malignant lesions ranged from 33% to 65%. The MAGE-A antigens might facilitate better differentiation between precancerous and cancerous lesions of the oral mucosa.

Low temperature fabrication of magnesium phosphate cement scaffolds by 3D powder printing.

Synthetic bone replacement materials are of great interest because they offer certain advantages compared with organic bone grafts. Biodegradability and preoperative manufacturing of patient specific implants are further desirable features in various clinical situations. Both can be realised by 3D powder printing. In this study, we introduce powder-printed magnesium ammonium phosphate (struvite) structures, accompanied by a neutral setting reaction by printing farringtonite (Mg(3)(PO(4))(2)) powder with ammonium phosphate solution as binder. Suitable powders were obtained after sintering at 1100°C for 5 h following 20-40 min dry grinding in a ball mill. Depending on the post-treatment of the samples, compressive strengths were found to be in the range 2-7 MPa. Cytocompatibility was demonstrated in vitro using the human osteoblastic cell line MG63.

Cryopreservation of autologous bone grafts: an experimental study on a sheep animal model.

A sheep animal model was used to investigate the clinical behavior of autologous bone transplants after cryopreservation. The aim of the present study was to compare fresh, cryopreserved and deep-frozen bone transplants in terms of their osseointegration. We used a serum-free cryopreservation protocol with DMSO as cryoprotectant for the bone transplants, which were harvested from the iliac crest of the sheep. The bicortical iliac bone grafts were either cryopreserved or immediately frozen to -80 degrees C for 4 weeks. Four, 8, 12 and 16 weeks after the autologous transplantation of the cryopreserved, fresh or deep-frozen bone transplants to the contralateral iliac crest, the animals were sacrificed and the bone specimens were evaluated clinically, by staining for hematoxylin/eosin and for tartrate-resistant acid phosphatase, by quantified computed tomography, immunohistochemistry (Ki67) and polychrome sequential labeling. The best results were obtained for the fresh specimens with 83% bone healing compared with 75% (cryopreserved bone) and 50% (deep frozen bone). All parameters indicate that bone formation and remodeling processes take place in fresh and cryopreserved transplants. The deep-frozen specimens displayed no fluorochrome uptake in the sequential labeling. These findings indicate that osseointegration of the fresh transplants was the most successful and that osteogenic effects in fresh and cryopreserved transplants are located in the surface area, whereas only the osteoconductive effects are important in the center of the transplants. Thus, cryopreservation is a useful method for the clinical routine because it keeps the osteogenic cells viable, making it superior to deep freezing of abundant bone.

Bridging of the sella turcica in skeletal Class III subjects.

Several investigations have analysed the frequency of sella turcica anomalies in patients with severe craniofacial deviations. Until now, there have been no studies concerning the prevalence of sella turcica bridging in homogenous groups of patients. Therefore, the aims of this controlled study were to analyse the prevalence of sella turcica bridging and measure the size of the sella turcica in two well-defined groups of Caucasian individuals. In a multicentre retrospective study, 400 pre-treatment lateral cephalograms of adult patients (over 17 years of age) with a skeletal Class III (n = 250, 132 females and 118 males) or a skeletal Class I (n = 150, 94 females and 56 males) malocclusion were analysed. The morphology, length, depth, and diameter of the sella turcica were investigated. For statistical analysis, chi-square and t-tests were used. Skeletal Class III patients presented a significantly higher rate of sella turcica bridging, 16.8 per cent (P = 0.031), in comparison with skeletal Class I patients, whose rate was 9.4 per cent. No differences between females and males were detected for the length, depth, and diameter of the sella turcica. Bridging of the sella turcica could be seen radiographically in skeletal Class III subjects.

Expression of cyclooxygenase-2 (COX-2) in an advanced metastasized hypopharyngeal carcinoma and cultured tumor cells.

PURPOSE: The inducible enzyme cyclooxygenase-2 (COX-2) catalyzes PGE(2) production and plays an important role in the progression of many solid cancers. However, the role of COX-2 expression in cervical lymph node metastases of head and neck cancer has not been clarified yet. PATIENT AND METHODS: We comment on a male patient aged 53 who was admitted to an ENT-department with acute bleeding from an advanced hypopharyngeal carcinoma and a frontotemporal mass. Prior to palliative intended radiotherapy, the metastasis was resected. During the procedure, a small amount of tumor tissue was harvested for primary tumor cell culture. RESULTS: COX-2 overexpression was demonstrated in the primary tumor tissue, the metastasis, in the cultured tumor cells by standard immunohistochemistry, as well as cytochemistry. CONCLUSIONS: A simultaneous expression of COX-2 in head and neck carcinoma was presented for the first time. Besides the prognostic impact in oral carcinogenesis, this COX-2 role of biomarker for aggressive head and neck squamous cell carcinomas should be further evaluated. Additionally, treatment of hypopharyngeal carcinomas with selective COX-2 inhibitors could be beneficial when administered in combination with radiochemotherapy.

Phase composition, mechanical performance and in vitro biocompatibility of hydraulic setting calcium magnesium phosphate cement.

Brushite (CaHPO(4) x 2H(2)O)-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. However, their short setting times and low mechanical strengths limit broad clinical application. In this study, we showed that a significant improvement of these properties of brushite cement could be achieved by the use of magnesium-substituted beta-tricalcium phosphate with the general formula Mg(x)Ca((3-x))((PO(4))(2) with 0 < x < 3 as cement reactants. The incorporation of magnesium ions increased the setting times of cements from 2 min for a magnesium-free matrix to 8-11 min for Mg(2.25)Ca(0.75)(PO(4))(2) as reactant. At the same time, the compressive strength of set cements was doubled from 19 MPa to more than 40 MPa after 24h wet storage. Magnesium ions were not only retarding the setting reaction to brushite but were also forming newberyite (MgHPO(4) x 3H(2)O) as a second setting product. The biocompatibility of the material was investigated in vitro using the osteoblast-like cell line MC3T3-E1. A considerable increase of cell proliferation and expression of alkaline phosphatase, indicating an osteoblastic differentiation, could be noticed. Scanning electron microscopy analysis revealed an obvious cell growth on the surface of the scaffolds. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium, magnesium and phosphate ions were altered markedly due to the chemical solubility of the scaffolds. We conclude that the calcium magnesium phosphate (newberyite) cements have a promising potential for their use as bone replacement material since they provide a suitable biocompatibility, an extended workability and improved mechanical performance compared with brushite cements.

Impact of MAGE-A antigens on taxane response in oral squamous cell carcinoma.

MAGE-A antigens are a subgroup of cancer/testis antigens that are exclusively expressed in malignant cells. Only scarce information on the function of MAGE-A antigens is available. There is some evidence that they may influence the response to chemotherapeutic drugs. This study aimed to evaluate the impact of the MAGE-A antigen subgroups MAGE-A2, -A3, -A4 and -A6 on oral squamous cell carcinoma cell lines treated with docetaxel and paclitaxel. Five oral squamous cell carcinoma cell lines were characterized for their quantitative expression of MAGE-A2, -A3, -A4 and -A6. The cell lines were treated with concentrations ranging from 0.025 to 0.8 µM of docetaxel and paclitaxel. The amount of viable cells after 24 and 48 h was measured. The measurements were statistically correlated with MAGE-A expression. All cell lines responded to docetaxel and paclitaxel. One cell line showed a statistically significant weaker response to the taxane treatment. This cell line was the only one that expressed MAGE-A4. MAGE-A4 has a statistically significant impact on the tumour response to docetaxel and paclitaxel in oral squamous cell carcinoma. This may influence treatment options and the course of the disease. Therefore, patients should be evaluated for MAGE-A4 expression before treatment with taxanes.

MAGE-A antigens in patients with primary oral squamous cell carcinoma.

MAGE-A antigens are only expressed on tumor cells. The aim of this study was to identify their expression in patients with oral squamous cell carcinoma (OSCC). Forty-seven patients with primary OSCC was selected retrospectively. Histo-pathological sections were stained immunohistochemically with MAGE-A antibody 57B. The results were evaluated regarding tumor size (T), lymph-node metastasis (N), blood vessel infiltration (V), lymph vessel infiltration (L), grading (G), and sex. MAGE-A antigens were expressed in 55% of all patients. Expression increased with tumor size (T1 = 56%; T2 = 44%; T3 = 67%; T4 = 71%). Lymph-node metastasis had no influence (N0 and N1 about 50%). Tumors with blood and lymph vessel infiltration had higher expression (V0 = 50%; V1 = 100%; L0 = 46%; L1 = 71%). Less-differentiated tumors showed higher rates (G1 = 50%; G2 = 45%; G3 = 83%). OSCC in men were positive in 62% and in women in 38%. MAGE-A antigens are frequently expressed in OSCC. Their expression seems to increase with tumor dedifferentiation.

Analysis of expression profiles of MAGE-A antigens in oral squamous cell carcinoma cell lines.

BACKGROUND: The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs. METHODS: The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B) and compared with those of an adult keratinocyte cell line (NHEK). RESULTS: All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference. CONCLUSION: MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy.