Christoph Klingelhöffer

Positive frozen section margins predict local recurrence in R0-resected squamous cell carcinoma of the head and neck

OBJECTIVES The purpose of this study was to analyse the impact of surgical margins on tumour recurrence and survival of patients with carcinomas of the head and neck. MATERIAL AND METHODS A cohort of 156 patients with primary squamous cell carcinoma of the head and neck treated by local resection with negative margins and neck dissection between 2004 and 2012 was investigated. Margin status in frozen sections and permanent paraffin tissues were analysed and compared to clinical and histopathological parameters as well as to tumour recurrence (local, regional and distant) and disease-specific survival (DSS). RESULTS Close margins (<5mm) on permanent sections were correlated to high-grade differentiation (p=0.070), lymphangiosis (p=0.009) and positive neck nodes (p=0.025) implicating regional and distant recurrence (p=0.001) as well as unfavorable DSS (p=0.002). Positive margins on initial frozen section analysis revised into negative margins during further surgery were the strongest predictor for local recurrence in uni- and multivariate analysis (p<0.001, hazard ratio 3.34). However, positive frozen section margins were not significantly predictive for DSS (p=0.150). Significant predictors for DSS in univariate analysis were local recurrence (p=0.026), T-stage (p=0.02), N-stage (p<0.001), grading (p=0.02) and lymphangiosis (p=0.001). Multivariate DSS analysis revealed lymph node metastasis (p=0.005) and local recurrence (p=0.017) as significant negative predictors. CONCLUSION Close margins on permanent sections are associated with aggressive tumour characteristics, regional and distant metastasis implicating worse DSS. The accuracy of frozen section analysis seems limited as positive frozen section margins revised into negative margins bear a high risk of local recurrence.

The influence of the donor on dental apical papilla stem cell properties

Stem cells from the human dental apical papilla (SCAP) can be obtained from almost all extracted wisdom teeth with an immature tooth root. Although different stem cell lines are used for studies, it remains elusive whether specific characteristics of the dental stem cell cultures such as proliferation rates or the cell differentiation potential are related to the cell source, e.g. the donor tissue of the dental apical papilla. To answer this question, we compared two independent SCAP cell lines from the same donor and compared them with a third cell line from another donor. We investigated the expression of stem cell markers, the efficiency of colony forming units, cell proliferation and the differentiation potential. Results showed particular differences for typical stem cell attributes such as stem cell marker expression, cell proliferation and the adipogenic differentiation. These differences were regardless of the donor of the cell lines. In conclusion, we suppose that stem cell characteristics of SCAP cell cultures are independent from the donor.

Can dental panoramic radiographic findings serve as indicators for the development of medication-related osteonecrosis of the jaw?

OBJECTIVES The purpose of this case-control study was to find a correlation between certain imaging findings in dental panoramic radiographs and the risk for developing a medication-related osteonecrosis of the jaw (MRONJ) in patients taking antiresorptive therapy (AT). METHODS Randomized and blinded dental panoramic radiographs of 60 patients undergoing antiresorptive drug treatment (36 patients with MRONJ, 24 patients without MRONJ) and of 60 patients without AT were analyzed by 3 experts for the following signs: sequestrum, osteosclerosis, difference in sclerosing of alveolar process and body of mandible, visible alveolar socket, enhancement and loss of lamina dura, enhancement of the oblique ridge, enhancement of the mandibular canal, proliferative periostitis and osteolytic processes at the cortex. RESULTS Signs were seen significantly more often in patients undergoing AT than in the control group (CG) (osteosclerosis p-value = 0.019, visible alveolar socket p-value = 0.001, enhancement of lamina dura p-value < 0.001, enhancement of the mandibular canal p-value = 0.025, proliferative periostitis p-value = 0.05 and osteolytic processes at the cortex p-value < 0.001). While there is no significant difference between the CG and the group of patients with AT without manifest MRONJ for any sign, the significance increases when taking the group of patients under AT with manifest MRONJ into consideration. In addition, if medication was administered for malignant reasons, the signs visible alveolar socket, enhancement of the lamina dura and the enhancement of the mandibular canal were seen significantly more often. CONCLUSIONS The radiographic findings mentioned above are not indicators for the development of MRONJ, as they are seen only in patients with manifest osteonecrosis. However, these findings could be important to assess the dimension and potency of a MRONJ.

The WNT inhibitor APCDD1 sustains the expression of β-catenin during the osteogenic differentiation of human dental follicle cells.

In hair follicle cells APCDD1 inhibits the canonical WNT/β-Catenin pathway and its inactivation is associated with an autosomal dominant form of hair loss. We analyzed the role of APCDD1 for the osteogenic differentiation in dental follicle cells (DFCs) and identified a new and surprising function. Contrarily to hair follicle cells APCDD1 was crucial for the expression of β-Catenin and for the activity of the TCF/LEF reporter assay in DFCs. In addition, a depletion of APCDD1 inhibits the expression of osteogenic markers such as RUNX2 and decreased the matrix mineralization. However, similar to hair follicle cells in previous studies a control cell culture with oral squamous carcinoma cells showed that APCDD1 inhibits the expression of β-Catenin and of typical target genes of the canonical WNT/β-Catenin pathway. In conclusion, our data disclosed an unusual role of APCDD1 in DFCs during the osteogenic differentiation. APCDD1 sustains the expression and activation of β-Catenin.

Contrast-enhanced ultrasonography as a new method for assessing autonomization of pedicled and microvascular free flaps in head and neck reconstructive surgery

OBJECTIVE Evaluating vascular autonomization of pedicled and microvascular free flaps for soft tissue reconstruction in the head and neck area by means of postoperative quantitative measurement of dynamic contrast values obtained with contrast-enhanced ultrasound. METHODS 8/18 patients underwent lip reconstruction with a pedicle flap, 10 patients reconstruction of other parts of the head with a microvascular free transplant. Ultrasound examinations were conducted within the 1st postoperative week and 4 weeks after surgery. After the intravenous bolus of the ultrasound contrast agent, examinations were carried out for 30 sec without compression followed by 30 sec with compression of the vascular pedicle in bolus and flash kinetics. Digital cine loops were analyzed off-line with a quantification software (VueBox™) to determine the Rise Time (RT) between flap tissue with and without compression. RESULTS Measurements showed increasing autonomous perfusion, independent of the vascular pedicle. No transplant was lost, but 4/10 patients with a microvascular flap and 1/8 patients with a pedicle flap developed postoperative complications. RT values for the pedicled and microvascular flaps obtained under compression differed significantly between the 1st and the 4th week (p = 0.025). CONCLUSIONS Reliable neovascularization was achieved 4 weeks postoperatively. CEUS showed to be a useful method for assessing the degree of autonomization of pedicle and microvascular free flaps.

The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3

The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Students t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs.

miRNA-101 supports the osteogenic differentiation in human dental follicle cells.

OBJECTIVE Human dental follicle cells (DFCs) are genuine precursor cells of cementoblasts and alveolar bone osteoblasts. MicroRNAs (miRNAs) represent a class of non-coding endogenous RNAs that silence gene expression post-transcriptionally. miRNA101 actively regulates the osteogenic differentiation of periodontal ligament cells. Therefore the aim of this study was to investigate the role of miRNA101 during the osteogenic differentiation in DFCs. MATERIALS AND METHODS DFCs were isolated, cultivated and osteogenic differentiated in differentiation medium. Total RNA including miRNAs was isolated and the expression of miRNA101 was examined by real-time RT-PCRs. The expression of miRNA101 was induced by miRNA101-mimic transfection and the gene expression of osteogenic transcription factors was obtained by real-time RT-PCRs. Moreover the induction of the osteogenic differentiation was evaluated by the activity of alkaline phosphatase. RESULTS miRNA101 was regulated in DFCs during the osteogenic differentiation. After miRNA101-mimic transfection the alkaline phosphatase was increased and the gene expression of typical osteogenic transcription factors such as SP7 (osterix) was up-regulated. CONCLUSION Our results suggest that miRNA101 sustains the osteogenic differentiation of DFCs.

The AKT signaling pathway sustains the osteogenic differentiation in human dental follicle cells

Signaling transduction pathways are established by interactions between growth factors, protein kinases, and transcription factors, and they play a crucial role in tooth development. Precursor cells of the dental follicle (DFCs) are used for in vitro studies about molecular mechanisms during periodontal development. Previous studies have already shown that the growth factor BMP2 and the transcription factor EGR1 are involved in the osteogenic differentiation in DFCs while interactions with protein kinase-based pathways remain elusive. In this current study, we investigated the role of the AKT kinase signaling pathway for the osteogenic differentiation in DFCs. The AKT signaling pathway was activated in DFCs after the induction of the osteogenic differentiation by BMP2. The inhibition of AKT in DFCs repressed the differentiation and the expression of the transcription factor EGR1. Interestingly, EGR1 bound to the phosphorylated form of SMAD1/5 (pSMAD). The binding of pSMAD to EGR1 was increased after the induction with BMP2. Moreover, the overexpression EGR1 increased the osteogenic differentiation of DFCs. Our results suggest that the AKT signaling pathway submits the BMP2-dependent osteogenic differentiation in DFCs via the expression of the transcription factor EGR1.

Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions.

BackgroundDental stem cells in combination with implant materials may become an alternative to autologous bone transplants. For tissue engineering different types of soft and rigid implant materials are available, but little is known about the viability and the osteogenic differentiation of dental stem cells on these different types of materials. According to previous studies we proposed that rigid bone substitute materials are superior to soft materials for dental tissue engineering.MethodsWe evaluated the proliferation, the induction of apoptosis and the osteogenic differentiation of dental stem/progenitor cells on a synthetic bone-like material and on an allograft product. The soft materials silicone and polyacrylamide (PA) were used for comparison. Precursor cells from the dental follicle (DFCs) and progenitor cells from the dental apical papilla of retained third molar tooth (dNC-PCs) were applied as dental stem cells in our study.ResultsBoth dental cell types attached and grew on rigid bone substitute materials, but they did not grow on soft materials. Moreover, rigid bone substitute materials only sustained the osteogenic differentiation of dental stem cells, although the allograft product induced apoptosis in both dental cell types. Remarkably, PA, silicone and the synthetic bone substitute material did not induce the apoptosis in dental cells.ConclusionsOur work supports the hypothesis that bone substitute materials are suitable for dental stem cell tissue engineering. Furthermore, we also suggest that the induction of apoptosis by bone substitute materials may not impair the proliferation and the differentiation of dental stem cells.

Accuracy in orthognathic surgery─comparison of preoperative plan and postoperative outcome using computer-assisted two-dimensional cephalometry by the Onyx Ceph® system

PURPOSE This retrospective study analyzes deviations between preoperative planning and postoperative outcome in orthognathic surgery using 2D Onyx Ceph®-cephalometric analyzing and planning system. MATERIALS AND METHODS A total of 100 patients with a mean age 25.1 of years were included in this study. In 33 patients a bilateral sagittal split osteotomy and in seven patients a Le Fort I osteotomy was performed. A total of 60 patients were treated by a bimaxillary approach. Onyx Ceph® was used as cephalometric planning software (Onyx Ceph®), followed by mock operations. Postoperative cephalograms were obtained after 3.3 days and compared to preoperative planning cephalograms for sagittal (SNA, SNB, ANB) and vertical (ArGoMe, ML-NSL, NL-NSL) angle measurements. Real and absolute mean deviation were documented. RESULTS Absolute mean deviation (degrees) between postoperative and planned jaw movement was lower for the sagittal parameters SNA (0.58), SNB (1.15) and ANB (1.05) compared to the vertical parameters NL-NSL (1.47), ML-NSL (1.96) and ArGoMe (3.20). SNA, SNB and ANB showed constant deviations independent from the extent of jaw movement. With regard to the vertical parameters ML-NSL, ArGoMe and NL-NSL the extent of the postoperative rotational jaw movement was not as much as planned, particularly for vertical shifts of more than 4°. CONCLUSION By using the 2D Onyx Ceph® cephalometric software for orthognathic surgery, the deviations between planned and actual movements are within an acceptable and predictable range. Planning of extensive vertical alterations may result in greater deviations after surgery.