Three-Dimensionally Cultured Jaw Periosteal Cells Attenuate Macrophage Activation of CD4+ T Cells and Inhibit Osteoclastogenesis.

The implementation of a successful therapeutic approach that includes tissue-engineered grafts requires detailed analyses of graft-immune cell interactions in order to predict possible immune reactions after implantation. The phenotypic plasticity of macrophages plays a central role in immune cell chemotaxis, inflammatory regulation and bone regeneration. The present study addresses effects emanating from JPC-seeded ß-TCP constructs (3DJPCs) co-cultivated with THP-1 derived M1/M2 macrophages within a horizontal co-culture system. After five days of co-culture, macrophage phenotype and chemokine secretion were analyzed by flow cytometry, quantitative PCR and proteome arrays. The results showed that pro-inflammatory factors in M1 macrophages were inhibited by 3DJPCs, while anti-inflammatory factors were activated, possibly affected by the multiple chemokines secreted by 3D-cultured JPCs. In addition, osteoclast markers of polarized macrophages were inhibited by osteogenically induced 3DJPCs. Functional assays revealed a significantly lower percentage of proliferating CD4+ T cells in the groups treated with secretomes from M1/M2 macrophages previously co-cultured with 3DJPCs compared to controls without secretomes. Quantifications of pit area resorption assays showed evidence that supernatants from 3DJPCs co-cultured with M1/M2 macrophages were able to completely suppress osteoclast maturation, compared to the control group without secretomes. These findings demonstrate the ability of 3D cultured JPCs to modulate macrophage plasticity.

Differences in analysis and treatment of upper airway obstruction in Robin sequence across different countries in Europe.

The goal of this study was to explore the availability of diagnostic and treatment options for managing upper airway obstruction (UAO) in infants with Robin Sequence (RS) in Europe. Countries were divided in lower- (LHECs, i.e., PPP per capita < $4000) and higher-health expenditure countries (HHECs, i.e., PPP per capita ≥ $4000). An online survey was sent to European healthcare professionals who treat RS. The survey was designed to determine the availability of diagnostic tools such as arterial blood gas analysis (ABG), pulse oximetry, CO2 analysis, polysomnography (PSG), and sleep questionnaires, as well as to identify the used treatment options in a specific center. Responses were received from professionals of 85 centers, originating from 31 different countries. It was equally challenging to provide care for infants with RS in both LHECs and HHECs (3.67/10 versus 2.65/10, p = 0.45). Furthermore, in the LHECs, there was less access to ABG (85% versus 98%, p = 0.03), CO2 analysis (45% versus 70%, p = 0.03), and PSG (54% versus 93%, p < 0.01). There were no significant differences in the accessibility concerning pulse oximetry, sleep questionnaires, home saturation monitoring, nasopharyngeal tubes, Tuebingen plates, and mandibular distraction.    Conclusion: This study demonstrates a large difference in available care for infants with RS throughout Europe. LHECs have less access to diagnostic tools in RS when compared to HHECs. There is, however, no difference in the availability of treatment modalities between LHECs and HHECs. What is Known: • Patients with Robin sequence (RS) require complex and multidisciplinary care. They can present with moderate to severe upper airway obstruction (UAO). There exists a large variety in the use of diagnostics for both UAO treatment indications and evaluations. In most cases, conservative management of UAO in RS is sufficient. Patients with UAO that persist despite conservative management ultimately need surgical intervention. To determine which intervention is best suitable for the individual RS patient, the level of UAO needs to be determined through diagnostic testing. • There is a substantial variation among institutions across Europe for both diagnostics and treatment options in UAO. A standardized, internationally accepted protocol for the assessment and management of UAO in RS could guide healthcare professionals in the timing of assessment and indications to prevent escalation of UAO. Creating such a protocol might be a challenge, as there are large financial differences between countries in Europe (e.g., health expenditure per capita in purchasing power parity in international dollars ranges from $600 to over $8500). What is New: • There is a substantial variation in the availability of objective diagnostic tools between European countries. Arterial blood gas analysis, CO2 analysis and polysomnography are not equally accessible for lower-healthcare expenditure countries (LHECs) compared to higher-healthcare expenditure countries (HHECs). These differences are not only limited to availability; there is also a difference in quality of these diagnostic tools. Surprisingly, there is no difference in access to treatment tools between LHECs and HHECs. • There is national heterogeneity in access to tools for diagnosis and treatment of RS, which suggests centralization of health care, showing that specialized care is only available in tertiary centers. By centralization of care for RS infants, diagnostics and treatment can be optimized in the best possible way to create a uniform European protocol and ultimately equal care across Europe. Learning what is necessary for adequate monitoring could lead to better allocation of resources, which is especially important in a low-resource setting.

Autologous Tooth Transplantation in Craniofacial Malformations.

OBJECTIVE: To evaluate the applicability of transplanted teeth in young patients with craniofacial anomalies. DESIGN: Observational study. SETTING: Comprehensive Centre for Cleft Palate and Craniofacial Malformations. PATIENTS/PARTICIPANTS: Patients with craniofacial anomalies who underwent tooth transplantation. Only children with complete clinical and radiological documentation and a follow-up period of at least 1.5 years were included. INTERVENTIONS: Tooth transplantation. MAIN OUTCOME MEASURE(S): Retrospective evaluation of clinical records, pre- and postoperative radiographs, and operative charts. Clinical characteristics of patients, preoperative parameters and postoperative outcome parameters were collected. RESULTS: A total of 17 patients with 23 tooth transplantations were included. The median follow-up period was 6.7 years. The pooled survival and success rates were 91%. Notably, one out of two teeth that were transplanted into the bone grafted alveolar cleft site had to be extracted, which might indicating a higher risk for this procedure. In total, two transplanted teeth had to be extracted during the follow-up period, one due to external resorption and the other one due to perio-endo lesion. One patient needed endodontic treatment due to pulp necrosis. CONCLUSION: We consider tooth transplantation to be a reliable and suitable procedure in the dental rehabilitation of young patients with craniofacial anomalies and fitting concomitant circumstances. We encourage craniofacial teams to reconsider this option more frequently in appropriate cases.

Neurocognitive development in isolated Robin sequence treated with the Tuebingen palatal plate.

OBJECTIVES: We aimed to determine the neurocognitive development of cleft palate patients with and without Robin sequence (RS). MATERIALS AND METHODS: Children with isolated RS with cleft palate and children with cleft palate only (CPO) were contacted at the age of 5-6 years. All RS children had undergone initial polygraphic sleep study (PG) with a mixed-obstructive apnea index (MOAI) of ≥ 3/h and were consequently treated with the Tuebingen palatal plate. A standardized clinical examination as well as a neuropediatric and neuropsychological examination included the Wechsler Pre-school and Primary Scale of Intelligence (WPPSI-III), Kaufman Assessment Battery for Children (K-ABC), and an assessment of developmental milestones. RESULTS: In total, 44 children (22RS, 22CPO) were included. RS children were younger at study (70.5 ± 7.3 and 75.2 ± 7.5 months; P = .035). Both groups achieved the evaluated milestones within the normed time frame. WPPSI-III and K-ABC results showed no group differences. Mean values for Verbal IQ (101.8 ± 11.1 vs. 97.1 ± 15.7), Performance IQ (102.9 ± 12.1 vs. 99.6 ± 14.5), Processing Speed Quotient (98.9 ± 15.6 vs. 94.5 ± 15.7), Full-Scale IQ (103.2 ± 12.1 vs. 98.4 ± 15.3), and Sequential Processing Scale (102.1 ± 13.1 vs. 94.2 ± 17.3) were within the reference range (IQ 85-115) for RS and CPO children, respectively, indicating average performance of both groups. CONCLUSION: No neurocognitive, physical, or mental impairments were detected suggesting that RS children having upper airway obstruction (UAO) treated early and effectively may use their potential for an age-appropriate neurocognitive development. CLINICAL RELEVANCE: Tuebingen palatal plate treatment successfully releases UAO. Thus, isolated RS does not necessarily result in developmental delay or an impaired neurocognitive outcome. TRIAL REGISTRATION: Deutsches Register Klinischer Studien, DRKS00006831, https://www.drks.de/drks_web/.

Impact of Fluid Dynamics on the Viability and Differentiation Capacity of 3D-Cultured Jaw Periosteal Cells.

Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (ß-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration.

Intraoral scanning of neonates and infants with craniofacial disorders: feasibility, scanning duration, and clinical experience.

OBJECTIVE: The aim of this study was to evaluate intraoral scanning (IOS) in infants, neonates, and small children with craniofacial anomalies for its feasibility, scanning duration, and success rate. Impression taking in vulnerable patients can be potentially life-threatening, with the risk of airway obstruction and aspiration of impression material. The advantage of increasingly digitalized dentistry is demonstrated. MATERIALS AND METHODS: IOS was captured with the Trios 3® (3Shape, Copenhagen, Denmark) intraoral scanner. The underlying disorders were divided into cleft lip and palate (CLP), Trisomy 21 (T21), Robin Sequence (RS), Treacher Collins syndrome (TC), and isolated mandibular retrognathia (MR). Scan data were analysed by scanning duration, number of images, possible correlations of these factors with the different craniofacial disorders, patient age, and relationship between first and subsequent scans. Clinical experiences with the repeated digital impressions are described. RESULTS: Patient data of 141 scans in 83 patients were analysed within an 11-month period. Patients had a median age of 137 days. Median scanning duration was 138 seconds, resulting in a median of 352 images. There was a statistically significant difference in scanning duration (P = 0.001) between infants and neonates. IOS took longest in patients with CLP (537 seconds) and shortest in T21 patients (21 seconds), although there was no statistically significant difference between aetiologies. There was no statistically significant difference between first and subsequent scans in scanning duration. In four cases the IOS had to be repeated, and one patient ultimately required conventional impression taking (all CLP patients; success rate 94%). No severe adverse events occurred. CONCLUSION: IOS is a fast, safe, and feasible procedure for neonates, small children, and infants with craniofacial malformations. One special challenge for both technician and user was identified in patients with CLP, though implementing this new approach of digital impression taking was otherwise found to be highly successful in everyday clinical routine.

Routine panendoscopy in oral squamous cell cancer patients: mandatory or facultative?

OBJECTIVES: This study investigated benefits of routine panendoscopy in staging of oral squamous cell cancer patients. MATERIALS AND METHODS: From 2013 to 2017, 194 oral squamous cell cancer patients were staged. Reports of routine flexible panendoscopy including oropharyngolaryngoscopy, bronchoscopy, and esophagogastroduodenoscopy were retrospectively analyzed for diagnoses of inflammation and second primary malignancies (carcinoma in situ or cancer) and compared to results of computed tomography. The effects of alcohol and tobacco history of 142 patients were assessed. RESULTS: Overall, a second primary malignancy was detected in seven patients. In four patients this discovery was only found by panendoscopy. One invasive carcinoma (esophagus) was detected as well as three carcinoma in situ. The second primary malignancies were located in the lung (3), esophagus (3), and stomach (1). In one patient index tumor therapy was modified after panendoscopy. Upper gastrointestinal inflammation was present in 73.2% of patients and 61.9% required treatment. About 91.8% of bronchoscopies and 34.5% of panendoscopies were without therapeutic consequences. Patients with higher risk from smoking were more likely to benefit from panendoscopy and to have a Helicobacter pylori infection. CONCLUSION: We do not recommend routine panendoscopy for all oral squamous cell cancer patients. Esophagogastroduodenoscopy benefitted smoking patients primarily concerning the secondary diagnosis of inflammation of the upper digestive tract. Selective bronchoscopy, esophagogastroduodenoscopy, and oropharyngolaryngoscopy should be performed if clinical examination or medical history indicates risks for additional malignancies of the upper aerodigestive tract. CLINICAL RELEVANCE: Routine panendoscopy is not recommended in all, especially not in low-risk oral cancer patients like non-smokers and non-drinkers.

Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization.

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.

Influence of Human Jaw Periosteal Cells Seeded ß-Tricalcium Phosphate Scaffolds on Blood Coagulation.

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.

iPSC-Derived MSCs Versus Originating Jaw Periosteal Cells: Comparison of Resulting Phenotype and Stem Cell Potential.

Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-ß-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.

Surgical suture course for dental students with the Peyton-4-step approach versus the PDCA cycle using video assisted self-monitoring.

BACKGROUND: In this prospective study the Peyton 4-step approach of demonstration-deconstruction-comprehension (verbalization by the learner), and performance by the learner was compared to the PDCA cycle/Deming-Circle (Plan-Do-Check (video assisted self-monitoring)-Act) as a teaching method for surgical suturing and nodes with end performance as the primary objective. METHODS: Students of the third clinical semester in dental medicine were randomly selected to one of the two teaching methods. They completed a first course during the third clinical semester and a subsequent course during the fourth clinical semester. The focus was on learning surgical suturing techniques. Before the course started a questionnaire was handed out to both groups to evaluate their initial level of performance. Each course ended with a practical test to review the content of the course. The evaluation followed standardized parameters. Some of the test tasks in test one were repeated in test two to measure a horizontal as well as vertical difference in performance level. RESULTS: 53 students (Peyton: n = 28/18 female, 10 male; PDCA: n = 25/14 female, 11 male) have completed both courses. The evaluation of the subjective questionnaires showed that the members of the PDCA-groups achieved a higher subjective increase in performance. The objective results also indicated higher learning success in the PDCA-groups compared to the Peyton-Group. DISCUSSION/CONCLUSION: This study demonstrated significant learning success for both groups in their own self-assessment as well as in the results of the practical exercises. Subsequently, the superiority of the PDCA cycle could be shown for almost all criteria for surgical suturing techniques. Several studies prioritize the teaching of practical skills according to Peyton and consider step 3 ("comprehension") to be the essential factor. The PDCA cycle, which has its origins in industrial quality management, and its success can be understood from the perspective of learning theory in terms of Jean Piaget's model of equilibration. The necessity of active reflection on the learning content through practice constitutes the key element for transfer into long-term memory.

Fungus Ball of the Maxillary Sinus-Modern Treatment by Osteoplastic Approach and Functional Endoscopic Sinus Surgery.

PURPOSE: Functional endoscopic sinus surgery (FESS) is considered standard surgical therapy for fungus ball of the maxillary sinus. However, recent findings have indicated an odontogenic etiology, which requires simultaneous treatment of the dental origin. This study presents the authors' treatment results of fungus ball of the maxillary sinus using a combination of FESS and an endoscopically assisted osteoplastic approach through the anterior wall of the maxillary sinus, enabling simultaneous treatment of the dental origin. MATERIALS AND METHODS: A cohort of 22 patients with histopathologically confirmed fungus ball of the maxillary sinus was retrospectively analyzed. Clinical records and medical imaging data were reviewed to evaluate the etiology, clinical and radiologic findings, and postoperative outcome. RESULTS: Only 15 patients presented nonspecific clinical symptoms compatible with chronic unilateral maxillary sinusitis. Computed tomography visualized complete opacity of the maxillary sinus in 11 patients and intralesional hyperdensity in 12 patients. An odontogenic association was verified in 18 patients. Twenty-one patients underwent endoscopically assisted osteoplastic surgery through the anterior maxillary sinus wall. In 12 cases, the assumed persistent odontogenic source was treated simultaneously. Depending on the patency of the ostiomeatal complex, the accompanying chronic sinusitis was treated by FESS. CONCLUSIONS: The present data support the assumption of an odontogenic etiology of fungus ball of the maxillary sinus. Hence, surgical management requires simultaneous treatment of the fungal mass, the odontogenic origin of the disease, and the accompanying chronic sinusitis. To properly treat fungus ball, the authors present a modern treatment concept, using a minimally invasive endoscopically assisted osteoplastic approach through the anterior maxillary wall, for sufficient and necessary surgical treatment.

Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions.

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.

Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA.

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

Comparative Study of MSCA-1 and CD146 Isolated Periosteal Cell Subpopulations.

BACKGROUND/AIMS: Periosteal tissue is a valuable source of multipotent stem cells for bone tissue engineering. To characterize these cells in detail, we generated an immortalized human cranial periosteal cell line and observed an increased MSCA-1 and CD146 expression, as well as an earlier and stronger mineralization compared to the parental cells. Further, we detected a higher osteogenic potential of MSCA-1high compared to MSCA-1low cranial periosteal cell (CPC) fractions. In the present study, a possible synergism of MSCA-1 and CD146 for periosteal cell mineralization was investigated. METHODS: MSCA-1/CD146 positive and negative CPCs were magnetically isolated (MACS) or sorted by flow cytometry (FACS) and subjected to osteogenic differentiation. The expression of osteogenic marker genes in the four subpopulations was analyzed by quantitative real-time PCR. Furthermore, the co-expression of osteogenic markers/antigens was analyzed by multispectral imaging flow cytometry (ImageStream, AMNIS). The mineralization potential was assessed by the quantification of alizarin stainings. RESULTS: While the total cell yield after separation was higher using MACS compared to the FACS approach, the isolation of MSCA-1+/- and CD146+/- subpopulations was more efficient with the FACS separation. The accuracy of the FACS separation of the four distinguished cell subpopulations was confirmed by multispectral imaging flow cytometry. Further, we detected increasing levels of MSCA-1 and CD146 during in vitro differentiation in all subpopulations. However, MSCA-1 expression was significantly higher in the MSCA-1+/CD146+ and MSCA-1+/ CD146- subpopulations, while CD146 expression remained clearly lower in these fractions. Significantly higher gene expression levels of osteogenic markers, ALP and RUNX2, were detected in MSCA-1+ compared to MSCA-1- CPCs at different time points during in vitro differentiation. Staining and quantification of calcium phosphate precipitates revealed a significantly higher mineralization potential of MACS separated MSCA-1+ and CD146- CPCs, compared to their respective counterparts. FACS sorted CPCs displayed earlier mineralization in both MSCA-1+ fractions (d13), while later (d28) only the CD146+/MSCA-1- fraction had a significantly lower calcium phosphate concentration compared to all other fractions. CONCLUSION: Our results demonstrate, that MSCA-1+ cells isolated from CPCs represent a subpopulation with a higher osteogenic potential. In contrast, we found a lower osteogenic potential in CD146+ CPCs. In conclusion, only MSCA-1, but not CD146, is a suitable marker for the isolation of osteoprogenitors from CPCs.

Immunophenotyping of patients with oral squamous cell carcinoma in peripheral blood and associated tumor tissue.

The immune system is important for elimination of cancer cells. Tumors including oral squamous cell carcinoma (OSCC) are capable of escaping detection by host immune cells through apoptotic depletion of tumor-infiltrating lymphocytes (TILs). Circulating peripheral blood lymphocytes (PBLs) and corresponding TILs of tumor specimen were evaluated before and after curative tumor resection (n = 30) compared with PBLs of controls (n = 87). PBLs were characterized for the total number of T cells (CD3(+)), T helper cells (Th, CD3(+)/CD4(+)), regulatory T cells (Treg, CD4(+)/CD25(+)/CD127(low)), cytotoxic T cells (Tc, CD3(+)/CD8(+)), activated T cells (CD3(+)/HLA-DR(+)), and natural killer (NK) cells (CD3(-)/CD16(+)/CD56(+)). In tumor tissue, the prevalence of CD3(+), CD4(+), and CD8(+) TILs was assessed using immunohistochemistry, whereas the incidence of apoptosis was assessed using terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labeling (TUNEL) assay. In PBLs of pretreated OSCC patients, a highly significant decrease in total number of T cells (p = 0.0001), Th cells (p < 0.0001), Treg cells (p < 0.0001), Tc cells (p < 0.0001), and NK cells (p = 0.0037) were found compared with controls. Decreased PBLs of OSCC patients were correlated with decreased numbers of corresponding TILs, which were associated with increased detection of apoptosis in the tumor tissue. Compared with the controls, the total number of T cells remained unchanged after surgery but the total number of NK cells significantly increased. Standardized immunophenotyping of OSCC may help to identify patients likely to benefit from cancer immunotherapy strategies and/or chemoradiation. Finally, future attempts to enhance an effective tumor-reactive immune response by immunotherapy or vaccination should be made by promoting tumor-specific Th and/or Tc cell/NK cell responses.

Glutaminolysis and carcinogenesis of oral squamous cell carcinoma.

Glutaminolysis is a crucial factor for tumor metabolism in the carcinogenesis of several tumors but has not been clarified for oral squamous cell carcinoma (OSCC) yet. Expression of glutaminolysis-related solute carrier family 1, member 5 (SLC1A5)/neutral amino acid transporter (ASCT2), glutaminase (GLS), and glutamate dehydrogenase (GLDH) was analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. SLC1A5/ASCT2 and GLS were significantly overexpressed in the carcinogenesis of OSCC compared with normal tissue, while GLDH was weakly detected. Compared with SIN I-III SLC1A5/ASCT2 and GLS expression were significantly increased in OSCC. GLDH expression did not significantly differ from SIN I-III compared with OSCC. This study shows the first evidence of glutaminolysis-related SLC1A5/ASCT2, GLS, and GLDH expression in OSCC. The very weak GLDH expression indicates that glutamine metabolism is rather related to nucleotide or protein/hexosamine biosynthesis or to the function as an antioxidant (glutathione) than to energy production or generation of lactate through entering the tricarboxylic acid cycle. Overcoming glutaminolysis by targeting c-Myc oncogene (e.g. by natural compounds) and thereby cross-activation of mammalian target of rapamycin complex 1 or SLC1A5/ASCT2, GLS inhibitors may be a useful strategy to sensitize cancer cells to common OSCC cancer therapies.

Standardized pretreatment inflammatory laboratory markers and calculated ratios in patients with oral squamous cell carcinoma.

Analyzing the inflammatory microenvironment has become an important issue in the management of oral squamous cell carcinoma (OSCC). Pretreatment C-reactive protein (CRP) levels, leucocytes, monocytes, lymphocytes, neutrophils, basophils, eosinophils, platelets, neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) derived from the peripheral blood were analyzed. Receiver operating characteristic (ROC) curves determined a cut-off value for each parameter in 146 patients with OSCC compared with 93 controls and the results were associated with clinicopathological characteristics. CRP expression of tumors was measured by immunohistochemistry. ROC analysis determined cut-off values for CRP levels, leucocytes, monocytes, lymphocytes, neutrophils, NLR, dNLR, LMR, PLR and showed significant differences between the OSCC and control group. Compared with single laboratory tests calculated ratios were superior in measuring sensitivity and specificity of OSCC disease. NLR was significant directly associated and correlated with PLR. LMR was significant inversely associated and correlated with NLR and PLR. Immunohistochemical analysis did not show CRP expression of OSCCs. This study highlights the first analysis for cut-off values of pretreatment single laboratory tests and calculated ratios, which are strongly needed for a follow-up of cancer patients. Additionally, the calculated baselines can be used as a goal for successful immunotherapies in the future. The links between NLR, LMR, and PLR might be helpful for the clinical course (monitoring) of cancer patients and have been first described for OSCC in this study. Taken together, analyzing these data provides an additional practical guideline of further postoperative OSCC management.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. MATERIALS AND METHODS: THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50 µM) and clodronate (125, 250 and 500 µM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72 h with zoledronate, ibandronate, alendronate and clodronate. RESULTS: Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5 µM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. CONCLUSIONS: All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. CLINICAL RELEVANCE: These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Malocclusion in the primary dentition in children with and without deformational plagiocephaly.

OBJECTIVES: Asymmetries of the jaw and orthodontic abnormalities are suspected as long-term consequences of positional cranial deformity. But only few data exist on this issue. As plagiocephaly is a common problem in infancy, potential functional impairments should be investigated to initiate appropriate measures if necessary. The aim of our study was to compare the orthodontic situation in primary dentition of children with positional plagiocephaly and children without cranial deformities. MATERIAL AND METHODS: Fifty children treated by helmet therapy for plagiocephaly and 50 non-affected children (age 1.98-5.69 years) were examined in a cross-sectional study. Orthodontic parameters of all dimensions were assessed and analyzed. RESULTS: Children of the plagiocephalic group showed more often orthodontic alterations compared to the others. Especially the frequencies of a class II malocclusion (36 vs. 14 %), an edge-to edge bite (28 vs. 12 %), and deviations of the midline (38 vs. 16 %) were conspicuous. However, none of the differences was significant (p > 0.003). Of all observed mandibular asymmetries, 69 % appeared as a shift to the contralateral side of the former flattened occipital region. CONCLUSION: Positional head deformity might be associated in some cases with a higher prevalence of occlusal abnormalities in primary dentition. CLINICAL RELEVANCE: Positional plagiocephaly interfaces medicine and dentistry. As it is a common disorder, this etiology has to be considered in the prevention and therapy of malocclusion.