CD24+ tumor-initiating cells from oral squamous cell carcinoma induce initial angiogenesis in vivo.

BACKGROUND: In oral squamous cell carcinoma (OSCC), a minor subset of cancer stem cells has been identified using the surface marker CD24. The CD24+ cell population is involved in initiating, maintaining, and expanding tumor growth, but has not been reported to be involved in angiogenesis to date. METHODS: NOD/SCID mice were equipped with dorsal skinfold chambers and gelatin sponges seeded with CD24+, CD24-, and unsorted cancer cells suspended in Matrigel® were implanted. Following intravital fluorescence microscopy, specimens were examined by immunohistology. RESULTS: Sponges seeded with CD24+ cells showed a significantly higher functional capillary density than those seeded with CD24- cells. The presence of endothelial cells was confirmed by immunohistochemistry for CD31. CONCLUSION: For the first time, CD24+ tumorigenic cells with angiogenic potential, which were isolated from OSCC, were characterized. Our findings provide a promising in vivo model to facilitate the development of therapeutic agents against cancer stem cells and their angiogenic pathways.

Putative CD133+ melanoma cancer stem cells induce initial angiogenesis in vivo.

Tumor angiogenesis is essential for tumor growth and metastasis, and is regulated by a complex network of various types of cells, chemokines, and stimulating factors. In contrast to sprouting angiogenesis, tumor angiogenesis is also influenced by hypoxia, inflammation, and the attraction of bone-marrow-derived cells. Recently, cancer stem cells have been reported to mimic vascularization by differentiating into endothelial cells and inducing vessel formation. In this study, the influence of cancer stem cells on initial angiogenesis was evaluated for the metastatic melanoma cell line D10. Following flow cytometry, CD133+ and CD133- cells were isolated using magnetic cell separation and different cell fractions were transferred to porcine gelatin sponges, which were implanted into the dorsal skinfold chamber of immunocompromised mice. Angiogenesis was analyzed based on microvessel density over a 10-day period using in vivo fluorescence microscopy, and the results were verified using immunohistology. CD133+ D10 cells showed a significant induction of early angiogenesis in vivo, contrary to CD133- D10 cells, unsorted D10 cells, and negative control. Neovascularization was confirmed by visualizing endothelial cells by immunohistology using an anti-CD31 antibody. Because CD133+ cells are rare in clinical specimens and hardly amenable to functional assays, the D10 cell line provides a suitable model to study the angiogenic potential of putative cancer stem cells and the leukocyte-endothelial cell interaction in the dorsal skinfold chamber in vivo. This cancer stem cell model might be useful in the development and evaluation of therapeutic agents targeting tumors.

hMSC-Derived VEGF Release Triggers the Chemoattraction of Alveolar Osteoblasts.

Human mesenchymal stem cells (hMSCs) are promising candidates for regenerative periodontal strategies, due to the broad spectrum of supportive effects on cells and tissues at the site of application. Although positive effects are visible, the understanding of their underlying mechanisms still requires further elucidation. Recently, we have shown that hMSCs are capable to prompt osteogenic differentiation of alveolar osteoblasts, thereby presumably contributing to alveolar bone regeneration. Another issue that is critical in this context is the attraction of hard tissue-forming cells to regeneration sites, but it is an open question whether hMSCs can afford this. In the present manuscript, we show by life cell imaging that in interactive cocultures, hMSCs successfully trigger osteoblast chemotaxis. Gene expression analysis for hMSC-innate chemoattractive biomolecules, orchestrating this process, revealed vascular endothelial growth factor (VEGF), PgE synthase, osteoprotegerin (OPG), monocyte colony-stimulating factor, and transforming growth factor ß1, which was confirmed for VEGF and OPG on the protein level. Noteworthy, we showed that only corresponding levels of VEGF but not OPG attracted alveolar osteoblasts similar to hMSC coculture, while VEGF inhibitor abolished both the VEGF and the hMSC-triggered chemoattraction. In summary, we have identified secreted OPG and VEGF proteins as potential chemoattractants, of which further characterization yielded VEGF as a causative for hMSC-directed osteoblast chemotaxis. With respect to the better understanding of potential hMSC-based periodontal regeneration strategies, we propose hMSC-derived VEGF release as a mechanism in the recruitment of hard tissue-forming cells to alveolar bone sites in need of regeneration.

Orbital reconstruction: prefabricated implants, data transfer, and revision surgery.

External impact to the orbit may cause a blowout or zygomatico-maxillary fractures. Diagnosis and treatment of orbital wall fractures are based on both physical examination and computed tomography scan of the orbit. Injuries of the orbit often require a reconstruction of its orbital walls. Using computer-assisted techniques, anatomically preformed orbital implants, and intraoperative imaging offers precise and predictable results of orbital reconstructions. Secondary reconstruction of the orbital cavity is challenging due to fractures healed in malposition, defects, scarring, and lack of anatomic landmarks, and should be avoided by precise primary reconstruction. The development of preformed orbital implants based on topographical analysis of the orbital cavity was a milestone for the improvement of primary orbital reconstruction.

Is there more to the clinical outcome in posttraumatic reconstruction of the inferior and medial orbital walls than accuracy of implant placement and implant surface contouring? A prospective multicenter study to identify predictors of clinical outcome.

PURPOSE: Reconstruction of orbital wall fractures is demanding and has improved dramatically with the implementation of new technologies. True-to-original accuracy of reconstruction has been deemed essential for good clinical outcome, and reasons for unfavorable clinical outcome have been researched extensively. However, no detailed analysis on the influence of plate position and surface contour on clinical outcome has yet been published. MATERIALS AND METHODS: Data from a previous study were used for an ad-hoc analysis to identify predictors for unfavorable outcome, defined as diplopia or differences in globe height and/or globe projection of >2 mm. Presumed predictors were implant surface contour, aberrant implant dimension or position, accuracy of reconstructed orbital volume, and anatomical fracture topography according to the current AO classification. RESULTS: Neither in univariable nor in multivariable regression models were unfavorable clinical outcomes associated with any of the presumed radiological predictors, and no association of the type of implant, i.e., standard preformed, CAD-based individualized and non-CAD-based individualized with its surface contour could be shown. CONCLUSION: These data suggest that the influence of accurate mechanical reconstruction on clinical outcomes may be less predictable than previously believed, while the role of soft-tissue-related factors may have been underestimated.

Sinusitis and oroantral fistula in patients with bisphosphonate-associated necrosis of the maxilla.

BACKGROUND: The management of bisphosphonate related necrosis of the jaw has become clinical routine. While approximately two thirds of the lesions are in the mandible, one third is located in the maxilla. In 40-50 % of maxillary necrosis the maxillary sinus is involved, leading to maxillary sinusitis and oro-antral communications. METHODS: This retrospective single center study includes all patients with diagnosis of BP-ONJ of the maxilla and concomitant maxillary sinusitis. The information collected includes age, gender, primary disease, bisphosphonate intake, involving type of bisphosphonate, route of administration and duration of BP treatment previous to surgical treatment and treatment outcome. RESULTS: A total of 12 patients fulfill the criteria of the diagnosis of maxillary sinusitis associated to maxillary necrosis, of which 6 Patients showed purulent sinusitis. All patients underwent surgical treatment with complete resection of the affected bone and a multilayer wound closure. A recurrence appeared in one patient with open bone and no sign of sinusitis and was treated conservatively. CONCLUSIONS: Purulent maxillary Sinusitis is a common complication of bisphosphonate-related necrosis of the maxilla. The surgical technique described can be suggested for the treatment of these patients.

A prospective multicenter study to compare the precision of posttraumatic internal orbital reconstruction with standard preformed and individualized orbital implants.

PURPOSE: A variety of implants are available for orbital reconstruction. Titanium orbital mesh plates are available either as standard preformed implants or able to be individualized for the patient. The aim of this study was to analyze whether individualized orbital implants allow a more precise reconstruction of the orbit than standard preformed implants. MATERIALS AND METHODS: A total of 195 patients treated between 2010 and 2014 were followed up to 12 weeks after surgery. Of the patients, 100 had received standardized preformed and 95 individualized implants. The precision of orbital reconstruction with the different implants was determined by comparing the variances in the volume difference between the reconstructed and the contralateral orbit on the postoperative computed tomographic scans. Clinical volume-related parameters including globe position, vision, motility, and diplopia and surgical details including approach, timing and technique of implant modification, use of navigation, duration of surgery, as well as adverse events were documented. RESULTS: Orbital reconstruction was significantly more precise when individualized implants were used. The same was seen with intraoperative navigation. An overlap in the use of individualized implants and navigation makes it difficult to attribute the improved precision to a single factor. CONCLUSION: This study demonstrated that individualization and navigation provide clinical benefit.

Moving the mandible in orthognathic surgery - A multicenter analysis.

Orthognathic surgery has always been a classical focus of maxillofacial surgery. Since more than 100 years, various surgical techniques for mandibular repositioning have been developed and clinically tested. Since the establishment of plate and screw osteosynthesis, orthognathic surgery became more stable and safe. Nowadays, different surgical methods for mobilising the mandible are existing. This international multicenter analysis (n = 51 hospitals) is providing first evidence based data for the current use of different surgical methods. The dominating techniques were Obwegeser/dal Pont (61%) followed by Hunsuck/Epker (37%) and Perthes/Schlössmann (29%). The main osteosynthesis materials were plates (82%), bicortical screws (23.5%), or a combination of both (5.9%). 47% of all centers reported to use several surgical methods at the same time, depending on the anatomical problem and the surgeon's preference. This shows that different surgical methods seem to work as comparable, safe, and reliable procedures in everydays clinical practise. On this basis, further prospective studies could evaluate possible advantages for our patients.

Microvascular transplants in head and neck reconstruction: 3D evaluation of volume loss.

BACKGROUND: Despite oversized latissimus dorsi free flap reconstruction in the head and neck area, esthetic and functional problems continue to exist due to the well-known occurrence of transplant shrinkage. The purpose of this study was to acquire an estimation of the volume and time of the shrinkage process. MATERIALS AND METHODS: The assessment of volume loss was performed using a 3D evaluation of two postoperative CT scans. A retrospective review was conducted on all latissimus dorsi free flap reconstructions performed between 2004 and 2013. Inclusion criteria for the assessment were: resection of an oral carcinoma and microsurgical defect coverage with latissimus dorsi free flap; a first postoperative CT (CT1) performed between 3 weeks and a maximum of 3 months after reconstruction surgery; and an additional CT scan (CT2) performed at least one year postoperatively. The exclusion criterion was surgical intervention in the local area between the acquisition of CT1 and CT2. The effect of adjuvant radiation therapy was considered. Volume determination of the transplant was carried out in CT1 and CT2 by manual segmentation of the graft. RESULTS: Fifteen patients were recruited. 3D evaluation showed an average volume loss of 34.4%. In the consideration of postoperative radiotherapy the volume reduction was 39.2% in patients with radiotherapy and 31.3% in patients without radiotherapy. CONCLUSION: The reconstruction flap volume required for overcorrection of the surgical defect was investigated. This study indicates that a volume loss of more than 30% could be expected one or more years after latissimus dorsi free flap reconstruction. Clinical trial number DRKS00007534.

The validity of surgical clips as radiographic markers for the tumour resection cavity in head and neck cancer treatment.

BACKGROUND: A prerequisite of irradiation after advanced head and neck tumour resection is the accurate localization of the tumour resection margin. The purpose of the following study is to evaluate the use of surgical clips placed in the tumour resection margins for use as radiographic markers to facilitate focussed adjuvant radiation therapy. MATERIALS: To evaluate whether the clips remain predictive for the resection margin, we analysed the deviation of each clip in two postoperative CT scans on different days. Bone registration points were used to fuse the two CT scans in the region of the primary tumour and the distances between corresponding clips were measured. RESULTS: The tumour resection margins were labelled with an average of 18 titanium clips. In total 282 clips were evaluated. Metric analysis of clip deviation between the two postoperative CT scans found a mean distance of 4.5 mm ± 2.5 mm with a range of 0.5-11.8 mm. No significant statistical relationship of the clip differences as a function of time, the method of reconstruction or administered radiotherapy could be demonstrated. CONCLUSION: Placement of surgical clips in the cavity walls after complete tumour resection provides an easy and inexpensive approach for defining resection margins and allows for increased accuracy of adjuvant treatment. Clinical trial number DRKS00007534.

Marking of tumor resection borders for improved radiation planning facilitates reduction of radiation dose to free flap reconstruction in head and neck cancer surgery.

Accurate localization of tumor resection borders is crucial for adjuvant radiotherapy. An improvement to adjuvant radiotherapy with the reduction of radiation doses to free flap reconstruction by virtual navigation procedures and titanium clips was evaluated. Thirty-three patients with oral cancer were prospectively included in the study. Following complete local excision of the primary tumor, resection borders were marked virtually using a navigation pointer and with titanium ligature clips. Postoperative delineation of tumor resection borders was examined. In five patients with microvascular free flap reconstruction a reduction of the radiation dose to the free flap reconstruction was achieved. The tumor resection borders in 30 patients were marked with titanium ligature clips. Surgical clip insertion was successful in 91%. We demonstrate a significant relationship between the reconstruction volume and the part of the target volume which will receive a reduced radiation dose. A cumulative dose of 60 Gy was administered to the target volume and a significant reduction of the administered radiation dose to the center of the flap could be demonstrated. We demonstrate an accurate delineation of the tumor resection margins. These improvements in tumor resection margin delineation allow for increased accuracy in adjuvant treatment and a reduction of radiation dose to the vascular free flap reconstruction.

Endothelial Progenitor Cell Fraction Contained in Bone Marrow-Derived Mesenchymal Stem Cell Populations Impairs Osteogenic Differentiation.

In bone tissue engineering (TE) endothelial cell-osteoblast cocultures are known to induce synergies of cell differentiation and activity. Bone marrow mononucleated cells (BMCs) are a rich source of mesenchymal stem cells (MSCs) able to develop an osteogenic phenotype. Endothelial progenitor cells (EPCs) are also present within BMC. In this study we investigate the effect of EPCs present in the BMC population on MSCs osteogenic differentiation. Human BMCs were isolated and separated into two populations. The MSC population was selected through plastic adhesion capacity. EPCs (CD34(+) and CD133(+)) were removed from the BMC population and the resulting population was named depleted MSCs. Both populations were cultured over 28 days in osteogenic medium (Dex(+)) or medium containing platelet lysate (PL). MSC population grew faster than depleted MSCs in both media, and PL containing medium accelerated the proliferation for both populations. Cell differentiation was much higher in Dex(+) medium in both cases. Real-time RT-PCR revealed upregulation of osteogenic marker genes in depleted MSCs. Higher values of ALP activity and matrix mineralization analyses confirmed these results. Our study advocates that absence of EPCs in the MSC population enables higher osteogenic gene expression and matrix mineralization and therefore may lead to advanced bone neoformation necessary for TE constructs.

Zoledronate induces osteonecrosis of the jaw in sheep.

INTRODUCTION: The treatment of bisphosphonate-related osteonecrosis of the jaw has become routine in maxillofacial hospitals. However, the etiopathology has not yet been fully understood. The aim of this study was to develop a large animal model for medication-related osteonecrosis of the jaw (MRONJ). MATERIAL AND METHODS: Eight Swiss mountain sheep were randomly assigned into two groups. Group I received 0.075 mg/kg zoledronate (ZOL) intravenously every third week for 16 weeks. After 16 weeks, extraction of the first and second lower left premolar was performed. Group II underwent surgery and no ZOL was administered. After surgery, Group I continued to receive ZOL infusions; after 16 weeks, all animals were euthanized. The jaw bones were investigated macroscopically, radiographically (computed tomography) and histologically. RESULTS: Osteonecrosis of the jaw was observed at all extraction sites in all the animals receiving ZOL, and at none of the sites in animals without ZOL. All ZOL-treated animals spontaneously developed exposed bone lesions in the oral cavity at sites where no surgical intervention was performed. CT imaging shows persistent alveolar extraction sockets 16 weeks after surgery in all animals of the ZOL-group, and healed alveolar extraction sockets in non-ZOL-treated animals. CONCLUSION: Sheep treated with ZOL reproducibly demonstrated osteonecrosis of the jaw after tooth extraction, and spontaneous development of exposed bone in the oral cavity at sites where no manipulation was performed. This animal model can be used for further research in the fields of BP-ONJ etiopathology, oral implantology, bone and fracture healing and periodontology.

Clipping of tumour resection margins allows accurate target volume delineation in head and neck cancer adjuvant radiation therapy.

BACKGROUND: Accurate tumour bed localisation is a key requirement for adjuvant radiotherapy. A new procedure is described for head and neck cancer treatment that improves tumour bed localisation using titanium clips. MATERIALS AND METHODS: Following complete local excision of the primary tumour, the tumour bed was marked with titanium clips. Preoperative gross target volume (GTV) and postoperative tumour bed were examined and the distances between the centres of gravity were evaluated. RESULTS: 49 patients with squamous cell carcinoma of the oral cavity were prospectively enrolled in this study. All patients underwent tumour resection, neck lymph node dissection and defect reconstruction in one stage. During surgery, 7-49 clips were placed in the resection cavity. Surgical clip insertion was successful in 88% (n=43). Clip identification and tumour bed delineation was successful in all 43 patients. The overall distance between the centres of gravity of the preoperative tumour extension to the tumour bed was 0.9cm. A significant relationship between the preoperative tumour extension and the postoperative tumour bed volume could be demonstrated. CONCLUSION: We demonstrate a precise delineation of the former tumour cavity. Improvements in tumour bed delineation allow an increase of accuracy for adjuvant treatment.

Description of a method: computer generated virtual model for accurate localisation of tumour margins, standardised resection, and planning of radiation treatment in head & neck cancer surgery.

Communication between the surgeon, the pathologist and the radiation oncologist is improved by a virtual model of the final resection combining 3D imaging with computer aided navigation. The pathologist localises any questionable margins and the oncologist plans focussed delivery of radiation to native tissue in an area of complex anatomy.