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The long-term clinical and esthetic success of implant-supported restorations is determined by osseointegration and optimal remodeling of peri-implant soft tissues. Complications of soft-tissue management are often caused by fibrotic regeneration of oral mucosa after multiple surgical procedures. Knowledge of the proliferative processes in wound healing is necessary to attain adequate soft-tissue conditions. Successful reconstruction of peri-implant soft tissues is feasible even in fibrotic conditions when appropriate surgical techniques are selected. The pleiotropic proliferative cytokine TGF-beta is involved in the regulation of all phases of wound healing and tissue remodeling. The isoform TGF-beta1 is a cytokine associated with the development of fibrotic tissue. Overexpression of TGF-beta1 causes scarring and fibrosis, and results in limited clinical success of intraoral soft-tissue management. Experimental therapeutic approaches with neutralizing antibodies to block TGF-beta1 resulted in less scarring and a reduction of fibrosis. Further molecular biologic research of cell-matrix-cytokine interactions in wound healing will provide highly specific antifibrotic therapeutic approaches in the future. [ABSTRACT FROM AUTHOR]

Abstract Background Human β-defensins (hBD) are antimicrobial peptides that are an integral part of bone innate immunity. Recently, it could be shown that expression of hBD-1, -2 and -3 were upregulated in cases of osteomyelitis of the jaws. In order to gain insight into the possible impairment of hBD metabolism in bisphosphonate-associated osteonecrosis of the jaws (BONJ), the present exploratory study was designed so as to determine the qualitative and quantitative expression of afore mentioned hBDs in BONJ and infected osteoradionecrosis (ORN), both of which represent inflammatory bone diseases. Methods Bone samples were collected from patients with BONJ (n = 20) and ORN (n = 20). Non-infected healthy bone samples (n = 20) were included as controls. Immunohistological staining in an autostainer was carried out by the (Strept-ABC)-method against hBD-1,-2,-3. Specific positive vs. negative cell reaction of osteocytes (labeling index) near the border of bony resection was determined and counted for quantitative analysis. Number of vital osteocytes vs. empty osteocytes lacunae was compared between groups. Results hBD-1,-2 and -3 could be detected in BONJ as well as ORN and healthy bone samples. Immunoreactivity against hBD-2 and -3 was significantly higher in BONJ than in ORN and healthy jaw bone samples. Number of empty osteocyte lacunae was significantly higher in ORN compared with BONJ (P = 0.001). Conclusion Under the condition of BONJ an increased expression of hBD-1,-2,-3 is detectable, similarly to the recently described upregulation of defensins in chronically infected jaw bones. It remains still unclear how these findings may relate to the pathoetiology of these diseases and whether this is contributing to the development of BONJ and ORN or simply an after effect of the disease.

Abstract Background Bisphosphonate associated osteonecrosis of the jaw (BRONJ) implies an impairment in oral hard- and soft tissue repair. An understanding of the signal transduction alterations involved can inform therapeutic strategies. Transforming growth factor β1 (TGFβ1) is a critical regulator of tissue repair; galectin-3 mediates tissue differentiation and specifically modulates periodontopathic bacterial infection. The aim of this study was to compare the expression of TGFβ1-related signaling molecules and Galectin-3 in BRONJ-affected and healthy mucosal tissues. To discriminate between BRONJ-specific impairments in TGFβ1 signaling and secondary inflammatory changes, the results were compared to the expression of TGFβ1 and Galectin-3 in mucosal tissues with osteoradionecrosis. Methods Oral mucosal tissue samples with histologically-confirmed BRONJ (n = 20), osteoradionecrosis (n = 20), and no lesions (normal, n = 20) were processed for immunohistochemistry. Automated staining with an alkaline phosphatase-anti-alkaline phosphatase kit was used to detect TGFβ1, Smad-2/3, Smad-7, and Galectin-3. We semiquantitatively assessed the ratio of stained cells/total number of cells (labeling index, Bonferroni-adjustment). Results TGFβ1 and Smad-2/3 were significantly decreased (p < 0.032 and p(0.028, respectively) in the BRONJ samples and significantly increased (p < 0.04 and p Conclusion Our results showed that disrupted TGFβ1 signaling was associated with delayed periodontal repair in BRONJ samples. The findings also indicated that impairments in TGFβ1-signaling were different in BRONJ compared to osteoradionecrosis. BRONJ appeared to be associated with increased terminal osseous differentiation and decreased soft tissue proliferation. The increase in Galectin-3 reflected the increase in osseous differentiation of mucoperiosteal progenitors, and this might explain the inflammatory anergy observed in BRONJ-affected soft tissues. The results substantiated the clinical success of treating BRONJ with sequestrectomy, followed by strict mucosa closure. BRONJ can be further elucidated by investigating the specific intraoral osteoimmunologic status.

Abstract Background Bone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL); however, osteonecrosis of the jaw (ONJ) is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP)-related and denosumab (anti-RANKL antibody)-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC)-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction (BMP-2/4, RANKL) in ONJ-altered and healthy periodontal tissue. Methods Twenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each) to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels. Results Semi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4 (all p < 0.05) expression in ONJ-adjacent periodontal tissue. ONJ tissue also exhibited decreased relative gene expression for Msx-1 (p < 0.03) and RANKL (p < 0.03) and increased BMP-2/4 expression (p < 0.02) compared to control. Conclusions These results explain the sclerotic and osteopetrotic changes of periodontal tissue following BP application and substantiate clinical findings of BP-related impaired remodeling specific to periodontal tissue. RANKL suppression substantiated the clinical finding of impaired bone remodelling in BP- and aRANKL-induced ONJ-affected bone structures. Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to maxillofacial tissue.

Background: The first-line treatment of oral squamous cell carcinoma (OSCC) involves surgical tumor resection, followed by adjuvant radio(chemo)therapy (R(C)T) in advanced cases. Neoadjuvant radio- and/or chemotherapy has failed to show improved survival in OSCC. Recently, neoadjuvant immunotherapy has shown promising therapeutic efficacy in phase 2 trials. In this context, the addition of radio- and chemotherapy is being reconsidered. Therefore, a better understanding of the tumor-biologic effects of neoadjuvant RCT would be beneficial. The current study was conducted on a retrospective cohort of patients who received neoadjuvant RCT for the treatment of oral cancer. The aim of the study was to evaluate the influence of neoadjuvant RCT on the immunological tumor microenvironment (TME) and hypoxic and glucose metabolisms., Methods: A cohort of 45 OSSC tissue samples from patients were analyzed before and after RCT (total 50.4 Gy; 1.8 Gy 5× weekly; Cisplatin + 5-Fluorouracil). Immunohistochemistry for CD68, CD163, TGF-β, GLUT-1 and HIF-1α was performed using tissue microarrays and automated cell counting. Differences in expression before and after RCT and associations with histomorphological parameters (T-status, N-status) were assessed using the Mann-Whitney U test., Results: Tumor resection specimens after neoadjuvant RCT showed a significant decrease in CD68 infiltration and a significant increase in CD163 cell density. The CD68/CD163 ratio was significantly lower after RCT, indicating a shift toward M2 polarization. The GLUT-1 and HIF-1α expressions were significantly lower after RCT. Larger tumors (T3/T4) showed a lower GLUT-1 expression. Other biomarkers were not associated with the T- and N-status., Conclusions: Neoadjuvant RCT with 50.4 Gy induced a shift toward the M2 polarization of macrophages in the TME. This change in immune composition is not favorable and may be prognostically negative and counteract immunotherapeutic approaches. In addition, the decreased expressions in GLUT-1 and HIF-1α indicate reductions in the glucose metabolism and hypoxic energy metabolism in response to "high dose" neoadjuvant RCT, which may be therapeutically desirable.

Background: The options of (neo-)adjuvant immunotherapy in addition to surgery in the treatment of oral squamous cell carcinoma (OSCC) are steadily increasing, but patients do not always respond to therapy as intended. The objectives of this study were to investigate the systemic perioperative course of the biomarkers CD45RO, TGF-β, and IL-8 in non-tumor-related minor and tumor-related major maxillofacial surgery and to perform association analyses with demographic and histomorphologic parameters. A deeper understanding of surgery-related changes in various of different immune biomarkers could help to better understand the immunologic consequences of surgery which could influence immunotherapeutic protocols., Methods: Peripheral whole blood from 38 patients was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) at five different timepoints before and after maxillofacial surgery to detect changes in mRNA expression of the biomarkers TGF-β, IL-8 and CD45RO. All patients underwent general anesthesia to undergo either resection and free flap reconstruction for OSCC or minor maxillofacial surgery (controls). Statistical analysis was done using Mann-Whitney-U test, Wilcoxon test, and Spearman's correlation., Results: Compared to the preoperative expression, there was a significant postoperative downregulation of CD45RO, TGF-β and IL-8 until the 4th postoperative day (p ≤ 0.003) in OSCC patients. For TGF-β and IL-8, the reduction in expression was significant (p ≤ 0.004) compared to controls. By postoperative day 10, all analyzed parameters converged to baseline levels. Only CD45RO still showed a significant downregulation (p=0.024). Spearman analysis revealed a significant correlation between increased duration of surgery and perioperative reduction in peripheral blood expression of CD45RO, TGF-β and IL-8 (p ≤ 0.004). Perioperative changes in TGF-β and PD-L1 expression were shown to be not correlated. Preoperative TGF-β expression was significantly lower in patients with lymph node metastases (p=0.014)., Conclusion: With regard to the analyzed parameters, major oncologic head-and-neck surgery does not seem to have long-lasting systemic immunologic effects. Reduced CD45RO might be an expression of transient systemic immunosuppression in response to major surgery. The association of duration of surgery with expression changes of immunologic markers supports efforts to keep the duration of surgery as short as possible. As perioperative TGF-β and PD-L1 expression changes are not associated, these results support further investigation of a combined perioperative anti-PD-1 and anti-TGF-β immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Trumet, Ries, Ivenz, Sobl, Wehrhan, Lutz, Kesting and Weber.)

Background: There is increasing evidence of the benefits of adjuvant and neoadjuvant immunotherapy in the treatment of solid malignancies like oral squamous cell carcinoma (OSCC). To optimize (neo-)adjuvant treatment, the systemic immunomodulatory effects of tumor surgery itself need to be considered. Currently, there is little evidence on the immunological effects of major surgery, such as free microvascular flap reconstruction. The current study aims to analyze how and to what extent maxillofacial surgery affects systemic parameters of immune tolerance., Methods: A total of 50 peripheral whole blood samples from patients (Group 1 (G1) = extensive OSCC surgery; Group 2 (G2) = free flap reconstruction without persistent malignant disease; Group 3 (G3) = minor maxillofacial surgery) undergoing surgery were included for real-time quantitative polymerase chain reaction (RT-qPCR) to examine changes in mRNA expression of the biomarkers IL-6, IL-10, FOXP3, and PD-L1. Blood samples were taken immediately before and after surgery as well as on the second, fourth, and tenth postoperative days. Differences in mRNA expression between groups and time points were calculated using statistical tests, including Mann-Whitney U-test and Pearson correlation analysis., Results: Comparing postoperative expression of G1 and G3, there was a significantly higher PD-L1 expression ( p = 0.015) in G1 compared to G3 and a significantly lower IL-6 ( p = 0.001) and FOXP3 ( p = 0.016) expression. Interestingly, IL-10 expression was higher pre- (0.05) and postoperative ( p < 0.001) in G1 compared to G3. Additionally, in G1, there was a significant overexpression of IL-10 post-surgery compared to the preoperative value ( p = 0.03) and a downregulated expression of FOXP3 between pre- and 2 d post-surgery ( p = 0.04). Furthermore, there was a significant correlation between the duration of surgery and the perioperative expression changes of the analyzed biomarkers. As the duration of surgery increased, the expression of IL-10 and PD-L1 increased, and the expression of IL-6 and FOXP3 decreased., Conclusion: Extensive surgery in OSCC patients is associated with a transient shift toward postoperative systemic immune tolerance compared with patients undergoing minor surgery. However, even extensive surgery causes no signs of long-lasting systemic immunosuppression. The degree of immune tolerance that occurred was associated with the duration of surgery. This supports efforts to minimize the duration of surgery.

Purpose: Despite microvascular free tissue transfer being the mainstay of care in the reconstruction of larger maxillofacial defects, a significant number of patients experience postoperative complications due to impaired blood supply of the flap. In this context, the early influence of recipient bed perfusion remains unclear, but there is evidence that it is associated with free flap viability immediately after surgery., Methods: We analyzed flap and recipient bed perfusion within the first 2 weeks after surgery by using the oxygen-to-see device. One hundred ninety-one patients who underwent free flap surgery in our department were included., Results: Flow parameters were higher and postoperative complications were less frequent in radial forearm free flaps compared to any other type of flap. Flow parameters of the recipient bed were higher than transferred tissue at all times, implicating flap autonomization is not completed within 2 weeks. Previous radiotherapy significantly decreased flow parameters of the recipient bed but not of the flaps. Furthermore, irradiated patients with postoperative complications were found to have reduced flow parameters of their recipient bed compared to non-irradiated patients with postoperative complications., Conclusion: We conclude that monitoring of recipient bed perfusion is useful for detecting flap compromise of irradiated patients in the early postoperative period., (© 2022. The Author(s).)

Purpose: Medication-related osteonecrosis occurs exclusively in the jaw bones. However, the exact pathogenesis of medication-related osteonecrosis of the jaw (MRONJ) and the unique predisposition of the jaw bones have not been elucidated, making its treatment a challenge. Recent evidence indicates that macrophages might play a pivotal role in MRONJ pathogenesis. The aim of the present study was to compare the macrophage populations between the craniofacial and extracranial skeleton and to investigate the changes induced by zoledronate (Zol) application and surgical interventions., Materials and Methods: An in vivo experiment was performed. 120 wistar rats were randomized to 4 groups (G1, G2, G3, G4). G1 served as an untreated control group. G2 and G4 received Zol injections for 8 weeks. Afterwards, the right lower molar of the animals from G3 and G4 was extracted and the right tibia osteotomized followed by osteosynthesis. Tissue samples were taken from the extraction socket and the tibia fracture at fixed time points. Immunohistochemistry was conducted to determine the labeling indexes of CD68 + and CD163 + macrophages., Results: Comparing the mandible and the tibia, we observed a significantly higher number of macrophages and a heightened pro-inflammatory environment in the mandible compared to the tibia. Tooth extraction caused an increase of the overall number of macrophages and a shift toward a more pro-inflammatory microenvironment in the mandible. Zol application amplified this effect., Conclusion: Our results indicate fundamental immunological differences between the jaw bone and the tibia, which might be a reason for the unique predisposition for MRONJ in the jaw bones. The more pro-inflammatory environment after Zol application and tooth extraction might contribute to the pathogenesis of MRONJ. Targeting macrophages might represent an attractive strategy to prevent MRONJ and improve therapy. In addition, our results support the hypothesis of an anti-tumoral and anti-metastatic effect induced by BPs. However, further studies are needed to delineate the mechanisms and specify the contributions of the various macrophage phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Struckmeier, Wehrhan, Preidl, Mike, Mönch, Eilers, Ries, Trumet, Lutz, Geppert, Kesting and Weber.)

Expression of signaling proteins in bone cells depends on their embryological mesoderm-derived (e.g. tibia) or cranial neural crest (CNC)-derived (e.g. jaw) origin. Connexin 43 (Cx43) is a gap junction protein that plays an essential role in the mode of action of bisphosphonates (BP). This study aimed to investigate Cx43 expression and the influence of BP application on mesoderm- and CNC-derived bone. Using a rat model, molar extraction and tibia osteotomy with (Group 4) or without (Group 3) previous BP application was performed. Untreated (Group 1) and animals selectively treated with BPs (Group 2) served as controls. Cx43 expression was immunohistochemically determined 12 and 16 weeks postoperatively via a labeling index. Cx43 expression in CNC-derived bone was significantly higher compared with mesodermal bone. BP application decreased Cx43 expression; however, detected expression levels were still higher in jawbone (Group 2 tibia vs jaw: 5.83 ± 5.06 vs 23.52 ± 6.42; p = 0.007). During bone healing after surgical intervention (Group 3) there were no expression differences between tibia and jawbone. BP treatment prior to surgery resulted in significantly lower Cx43 expression in CNC-derived compared with tibia bone (Group 4 tibia vs jaw: 56.84 ± 15.57 vs 16.40 ± 5.66; p < 0.01). Increased Cx43 expression in jaw compared with tibia bone is in line with their embryological origins. A significant Cx43 suppression in jawbone after BP application and surgery might contribute to the selectively altered osseous turnover and development of MRONJ in CNC-derived bone., Competing Interests: Declaration of Competing Interest The authors of this manuscript have no conflicts of interest to disclose., (Copyright © 2021 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate intraluminal vessel diameters and endothelial expression levels of pro-inflammatory and -thrombotic mediators in patent and non-patent microvascular anastomoses. Endothelial expression of CD31, VCAM-1, E- and P-Selectin, eNOS, iNOS and PAI-1 was evaluated by immunohistochemistry and compared to non-anastomosed arteries as controls. Intraluminal diameters were determined via H.E.-staining. In 20 human anastomoses (8 patent, 12 non-patent) neither the analysis of endoluminal de-endothelialization (p = 0.966) nor luminal narrowing (p = 0.750) revealed any significant differences between patent and non-patent microanastomoses. Expressions of pro-inflammatory mediators were significantly higher in patent anastomoses compared to controls but did not show any difference compared to non-patent anastomoses (p > 0.050). iNOS was higher in non-patent compared to patent anastomoses (p = 0.030) and controls (p = 0.001), whereas eNOS did not reveal any differences between these groups (p = 0.611 and p = 0.130). In non-patent anastomoses PAI-1 was expressed higher compared to patent anastomoses and controls (p = 0.021 and p < 0.001). Irrespective of their patency, anastomoses are characterized by endothelial dysfunction with a pro-inflammatory and pro-thrombotic milieu. Avoiding endothelial trauma during suturing is essential in order not to aggravate existing endothelial dysfunction in microanastomoses. Additionally, the influence of medication-related changes on anastomoses should be investigated as this is still an indistinctive topic., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflict of interest to disclose. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2021 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Purpose: Vestibuloplasty with free gingival grafting is a frequently performed surgical procedure to generate sufficient keratinized mucosa (KM) around dental implants. Avascular porcine collagen matrices (CM) have been proclaimed to be sufficient substitutes as alternatives to free gingival grafts (FGGs). However, the process of graft integration and vascularization is still incompletely understood., Methods: In 18 patients a vestibuloplasty in the lower edentulous jaw situation was performed during implant exposure, either with FGGs from the palate or a porcine CM (mucoderm). Tissue perfusion of the soft tissue grafts was measured using laser-doppler-spectrophotometer intraoperatively and on postoperative days 2, 5, 10, 30 and between days 60 and 90. With graft perfusion expressed by oxygen saturation [SO2%], the relative amount of hemoglobin [rHb], blood flow, and velocity [AU] was detected and compared between groups and the surrounding mucosa., Results: Healing was uneventful in both groups, with mature KM around dental implants after healing. Blood flow and velocity significantly increased until postoperative day 10, comparable to perfusion values of the surrounded mucosa. Intergroup comparisons revelated no significant differences concerning the flow between CM and FGGs. Oxygen saturation also significantly increased within the first 5 postoperative days in both groups. Hemoglobin content did not show any differences during the investigated period., Conclusions: The perfusion mainly progresses within the first postoperative week with only minimal further detectable alterations until the final investigation, comparable in both groups. Although integration of FGGs (revascularized) and the CM (new tissue formation) is biologically different, both transplants show comparable perfusion patterns, leading to sufficient KM., (Copyright © 2020 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The programmed cell death ligand 1/programmed cell death receptor 1 (PD-L1/PD-1) Immune Checkpoint is an important modulator of the immune response. Overexpression of the receptor and its ligands is involved in immunosuppression and the failure of an immune response against tumor cells. PD-1/PD-L1 overexpression in oral squamous cell carcinoma (OSCC) compared to healthy oral mucosa (NOM) has already been demonstrated. However, little is known about its expression in oral precancerous lesions like oral leukoplakia (OLP). The aim of the study was to investigate whether an increased expression of PD-1/PD-L1 already exists in OLP and whether it is associated with malignant transformation., Material and Methods: PD-1 and PD-L1 expression was immunohistologically analyzed separately in the epithelium (E) and the subepithelium (S) of OLP that had undergone malignant transformation within 5 years (T-OLP), in OLP without malignant transformation (N-OLP), in corresponding OSCC and in NOM. Additionally, RT-qPCR analysis for PD-L1 expression was done in the entire tissues. Additionally, the association between overexpression and malignant transformation, dysplasia and inflammation were examined., Results: Compared to N-OLP, there were increased levels of PD-1 protein in the epithelial and subepithelial layers of T-OLP (p E = 0.001; p S = 0.005). There was no significant difference in PD-L1 mRNA expression between T-OLP and N-OLP ( p = 0.128), but the fold-change increase between these groups was significant (Relative Quantification (RQ) = 3.1). In contrast to N-OLP, the PD-L1 protein levels were significantly increased in the epithelial layers of T-OLP ( p = 0.007), but not in its subepithelial layers ( p = 0.25). Importantly, increased PD-L1 levels were significantly associated to malignant transformation within 5 years., Conclusion: Increased levels of PD-1 and PD-L1 are related to malignant transformation in OLP and may represent a promising prognostic indicator to determine the risk of malignant progression of OLP. Increased PD-L1 levels might establish an immunosuppressive microenvironment, which could favor immune escape and thereby contribute to malignant transformation. Hence, checkpoint inhibitors could counteract tumor development in OLP and may serve as efficient therapeutic strategy in patients with high-risk precancerous lesions.

Objectives: This study analyzed the expression of the PD1 receptor in tumor tissue and peripheral blood of oral squamous cell carcinoma (OSCC) patients, and correlated it with the PD1 ligands PD-L1 and PD-L2. The currently low response rates of checkpoint inhibitor treatment in OSCC could be increased by a better understanding of immune checkpoint biology. Despite evidence in the literature for upregulation of PD1 checkpoint ligands in OSCC tissue, there has been no correlation analysis of the PD1 receptor with its ligands in tissue specimens and peripheral blood of OSCC patients., Materials and Methods: An RT-qPCR analysis of PD1 mRNA expression was performed in oral cancer specimens, healthy mucosa, and corresponding blood samples. A cut-off point (COP) was determined and a chi-square (χ 2 ) test was carried out. PD1 expression was correlated with previously reported PD-L1 and PD-L2 expression values using the Spearman test., Results: Tissue and blood specimens of 48 OSCC patients and 26 healthy individuals were analyzed. PD1 expression in OSCC specimens was significantly increased (p = 0.006) compared with healthy oral mucosa. PD1 overexpression in tissue samples showed a significant association with the presence of malignancy (p = 0.006). PD1 expression in tissue samples showed a significant positive correlation (p < 0.001) with the ligands PD-L1 and PD-L2. In contrast, there was no correlation between PD1 and its ligands in blood samples. However, there was a significant positive correlation (p < 0.001) between the ligands PD-L1 and PD-L2, both in tissue and blood samples., Conclusions: Increased PD1 expression might be a manifestation of T-cell exhaustion in OSCC specimens, leading to immune tolerance. PD-L1/PD-L2-PD1 interaction may be a major mediator of local immunosuppression in OSCC, requiring advanced multimodal treatment protocols., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2020 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Maxillofacial hard tissues have several differences compared to bones of other localizations of the human body. These could be due to the different embryological development of the jaw bones compared to the extracranial skeleton. In particular, the immigration of neuroectodermally differentiated cells of the cranial neural crest (CNC) plays an important role. These cells differ from the mesenchymal structures of the extracranial skeleton. In the ontogenesis of the jaw bones, the development via the intermediate stage of the pharyngeal arches is another special developmental feature. The aim of this review was to illustrate how the development of maxillofacial hard tissues occurs via the cranial neural crest and pharyngeal arches, and what significance this could have for relevant pathologies in maxillofacial surgery, dentistry and orthodontic therapy. The pathogenesis of various growth anomalies and certain syndromes will also be discussed.

Background: Immunomodulatory properties of bisphosphonates (BP) are suggested to contribute to the development of medication-associated osteonecrosis of the jaw (MRONJ). Furthermore, bisphosphonate-derived immune modulation might contribute to the anti-metastatic effect observed in breast cancer patients. Macrophages are potential candidates for the mediation of immunomodulatory effects of bisphosphonates. The study aimed to investigate the influence of bisphosphonates alone and in combination with surgical trauma on systemic macrophage polarization (M1 vs. M2) using an in vivo rat model., Methods: A total of 120 animals were divided into four groups. Groups 2 and 4 were treated with 8 × 40 μg/kg body weight of the BP Zoledronate i.p. (week 0-7). Groups 3 and 4 were exposed to surgical trauma (week 8, tooth extraction + tibia fracture), whereas in Group 1 neither medication nor surgical trauma was applied. After 8, 10, 12 and 16 weeks, skin, lung and spleen were immunohistochemically examined for macrophage polarization via expression analysis of CD68, CD163 and iNOS using a tissue microarray (TMA)., Results: A significant shift of macrophage polarization towards M1 was observed in skin, spleen and lung tissue of animals, with and without surgical trauma, treated with BP when compared to those without BP application. Surgical trauma did not cause a significant increase towards M1 polarization., Conclusions: BP application leads to a systemic pro-inflammatory situation in vivo, independent of surgical trauma, as evidenced by the shift in macrophage polarization towards M1 in various somatic tissues. This provides a possible explanation for the clinically observed anti-tumor effect of bisphosphonates and might also contribute to pathogenesis of MRONJ., Competing Interests: The authors declare that they have no competing interests.

Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.

Background: Craniofacial osteosarcomas (COS) and extracranial osteosarcomas (EOS) show distinct clinical differences. COS show a remarkably lower incidence of metastases and a better survival. However, in contrast to EOS, they show a poor response to neoadjuvant chemotherapy. Tumor-associated macrophages and their polarization as well as developmental biological signaling pathways are possible candidates for explaining the clinical differences between COS and EOS. The aim of the study was to analyze differential expression of macrophage markers and important regulators of these pathways. Methods: Twenty osteosarcoma cases (10 COS and 10 EOS) were immunohistochemically stained to assess CD68, CD11c, CD163, MRC1, Gli1, and Gli2 expression. Statistical differences between COS and EOS were tested using the Mann-Whitney U test. Additionally, the paper describes an example of multidisciplinary treatment of a patient suffering from COS and discusses the surgical challenges in treatment and rehabilitation of COS. Results: COS showed a significantly ( p < 0.05) increased infiltration of CD11c-positive M1 macrophages and a shift toward M1 polarization compared to EOS. Additionally, COS revealed a significantly ( p < 0.05) lower Gli1 expression than EOS. Conclusion: The reduced Gli1 expression in COS can be interpreted as reduced activation of the Hedgehog (Hh) signaling pathway. The increased M1 polarization and reduced Hh activation in COS could explain the low incidence of metastases in these osteosarcomas., (Copyright © 2020 Weber, Söder, Sander, Ries, Geppert, Kesting and Wehrhan.)

Correction to: Strahlenther Onkol 2018 https://doi.org/10.1007/s00066-018-1382-3 The original version of this article unfortunately contained a mistake. The correct version of the Acknowledgements is given ….

Objective: One of the severe side effects of bisphosphonate (BP) therapy is bisphosphonate-associated osteonecrosis of the jaw (BONJ). However, there is no information available about its pathogenesis. Hence, the aim of this observational study was to contribute to discerning this pathogenesis by comparing salivary quantity and quality in patients with BONJ and undergoing BP treatment., Materials and Methods: This study included 60 patients divided into three groups. The first group consisted of 20 patients with established BONJ, the second group had 20 patients undergoing BP treatment, and the third group comprised 20 healthy individuals. These groups were analysed for the flow rate of stimulated saliva, buffer capacity, and salivary pH level., Results: Reduced salivation was observed in a significantly high number of patients with established BONJ (n = 8) and those undergoing BP treatment (n = 9) in comparison with the healthy control group (n = 4; p = 0.039). Though the distribution of the mean value of stimulated saliva flow rates in patients undergoing BP treatment was lower than in the control group, the difference was not significant. Moreover, there were no significant differences in the salivary pH level and buffer capacity in patients undergoing BP treatment as compared with the healthy control group., Conclusions: It is possible that the quantity of human saliva is affected by BP treatment. This reduction in saliva production could have a negative effect on mucosal health and is perhaps a cofactor in the pathogenesis of BONJ., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Most oral squamous cell carcinomas (OSCC) occur on the basis of oral leukoplakias (OLP). The histologic degree of dysplasia is insufficient for the prediction of OLP malignant transformation. Immunologic parameters are gaining importance for prognostic assessment and therapy of cancer. M2 polarized macrophages were shown to be associated with OSCC progression and inferior prognosis. The current study aims to answer the question if OLP with malignant transformation into OSCC within 5 years differ from OLP without transformation regarding macrophage infiltration and polarization., Methods: 201 specimens (50 transforming OLP, 53 non-transforming OLP, 49 corresponding OSCC and 49 healthy oral mucosa controls) were processed for immunohistochemistry. Samples were stained for CD68, CD163 and CD11c expression, completely digitalized and computer-assisted cell counting was performed. Epithelial and subepithelial compartments were differentially assessed. Groups were statistically compared using the Mann-Whitney U-test. A cut-off point for the discrimination of transforming and non-transforming OLP was determined and the association between macrophage infiltration and malignant transformation was calculated using the Chi-square test (χ 2 test)., Results: Macrophage infiltration and M2 polarization in OLP with malignant transformation within 5 years was significantly increased compared to OLP without malignant transformation (p < 0.05). OSCC samples showed the highest macrophage infiltration and strongest M2 polarization (p < 0.05). Additionally, transforming OLP revealed a significant shift of macrophage infiltration towards the epithelial compartment (p < 0.05). χ 2 test revealed a significant association of increased macrophage infiltration with malignant transformation (p < 0.05)., Conclusion: Immunological changes precede malignant transformation of OLP. Increased macrophage infiltration and M2 polarization was associated with the development of oral cancer in OLP. Macrophage infiltration could serve as predictive marker for malignant transformation.

Purpose: Surgical treatment of medication-related osteonecrosis of the jaw (MRONJ) consists of necrotic bone removal followed by dense mucosal closure. Fluorescence-guided surgery has become a promising tool to intraoperatively distinguish between healthy and necrotic bone. Until now, there has been a lack of histopathological studies correlating the intraoperative fluorescence situation to histopathological analyses of the respective bone areas in order to further validate this method., Materials and Methods: Histopathological sections from intraoperatively detected fluorescence- and non-fluorescence-labeled bone were analyzed detecting osteocyte and collagen content, RANK(L) and TRAP expression as well as proportion of immature bone regeneration. Samples were compared with viable-looking bone areas according to the intraoperative clinical situation., Results: Staining revealed a significant decrease of osteocytes and collagen type-I fibers in necrotic, non-fluorescing areas compared to fluorescing bone (R/RGB [%]: 0.56 ± 0.38 (fluorescence positive) vs. 3.18 ± 2.22 (fluorescence negative), p = 0.041). Furthermore, the number of osteocytes was higher in fluorescing, clinically viable bone samples (cell/mm 2 : 151.26 ± 95.77 (fluorescence positive) vs. 0.56 ± 0.38 (fluorescence negative), p = 0.028). Additionally, the amount of immature bone was substantially increased in luminescent jaw bone (proportion of red [%]: 6.78 ± 7.00 (fluorescence positive) vs. 2.24 ± 1.36 (fluorescence negative), p = 0.442). RANK(L) and TRAP expression did not differ between the investigated areas, resembling a generalized decrease in osteocyte-osteoclast function all over the jaw (RANK(L) -positive cells per mm 2 : 8.97 ± 7.85 (fluorescence positive) vs. 7.76 ± 6.41 (fluorescence negative), p = 0.793; TRAP-positive cells per mm 2 : 0.36 ± 0.38 (fluorescence positive) vs. 0.33 ± 0.41 (fluorescence negative), p = 0.887)., Conclusion: Intraoperative fluorescence-guided surgery might be more precise in identifying and resecting the necrotic bone compared to previous indicators like bone bleeding, which could be useful to further improve surgical therapy in MRONJ patients., (Copyright © 2019 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Introduction: Dental cysts can be of inflammatory (radicular cysts) or noninflammatory (dentigerous cysts) origin. Apical periodontitis is a necrosis of the pulp and infection of the root canal causing the development of apical granulomas or radicular cysts. The immunology of granuloma and cyst formation is important because modern root filling materials are immunologically active and can contribute to the resolution of apical granulomas. In contrast, radicular cysts often require apicectomy. A better understanding of the pathophysiology of inflammation and bone resorption in apical periodontitis could be the basis for developing new root filling materials with superior immunomodulatory properties., Methods: Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray of the 87 consecutive specimens was created, and human leukocyte antigen-DR isotype (HLA-DR)-, CD83-, receptor activator of nuclear factor kappa B ligand-, macrophage colony-stimulating factor (MCSF)-, galectin-3 (Gal3)-, CD4-, and CD8-positive cells were detected by immunohistochemistry. Tissue microarrays were digitized, and the expression of markers was quantitatively assessed., Results: HLA-DR, CD83, MCSF, and Gal3 expression was significantly (P < .05) higher in radicular cysts compared with apical granulomas. HLA-DR, CD83, MCSF, receptor activator of nuclear factor kappa B ligand, and Gal3 expression in dentigerous cysts was significantly (P < .05) lower than in both periapical lesions (apical granulomas and radicular cysts). CD4 and CD8 infiltration was not statistically different between apical granulomas and radicular cysts. Dentigerous cysts showed a significantly (P < .05) lower T-cell infiltration than apical periodontitis. The CD4/CD8 ratio was not significantly different between the analyzed groups., Conclusions: The development of radicular cysts in apical periodontitis is associated with an increased expression of myeloid inflammatory markers and bone resorption parameters. Antigen-presenting cells and myeloid cells might be more relevant for the pathogenesis of apical periodontitis than T cells. Increased inflammation might promote the formation of radicular cysts and more pronounced bone resorption., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Purpose: Vestibuloplasty is a frequently performed surgical procedure to create or increase soft tissue mucosal sealing around dental restorations. Collagen matrices have exhibited comparable clinical results as free gingival grafts in the context of intraoral tissue augmentation. However, the process of matrix vascularization, the basic requirement for local healing, is incompletely understood. Therefore, this study investigated collagen matrix perfusion in a clinical intraoral setting., Materials and Methods: In a prospective cohort study, vestibuloplasty was performed during implant exposure using prefabricated collagen matrices. Matric perfusion was determined intraoperatively and at days 2, 5, 7, 14, 30, and 90 using a laser Doppler spectrophotometer measuring oxygen saturation, relative amount of hemoglobin, blood flow, and blood velocity as primary outcome variables. These parameters were compared with perfusion of the oral mucosa surrounding the matrices. Statistical analysis was performed by applying variance and regression models., Results: In 10 patients (average age, 60.9 yr), vestibuloplasty was performed exclusively in the anterior mandible. Blood flow and tissue oxygen saturation in the augmented zones markedly increased until postoperative day 5 and approximated perfusion values of the adjacent mucosa at the following 2 time points. Likewise, matrix oxygen saturation markedly increased until day 7 and subsequently converged to perfusion parameters of the surrounding mucosa at the following time points., Conclusion: Flow signals in incorporated collagen matrices occurred on day 2 after vestibuloplasty and further increased until days 5 to 7. Therefore, matrix perfusion mainly occurs within the first postoperative week, converging to perfusion levels of the surrounding mucosa with minimal alterations during the following course., (Copyright © 2019 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Leukoplakia is a potential precursor of oral as well as laryngeal squamous cell carcinoma. Risk assessment of malignant transformation based on the grade of dysplasia of leukoplakia often does not lead to reliable results. However, oral squamous cell carcinoma, laryngeal squamous cell carcinoma, and leukoplakia express single or multiple members of the melanoma-associated antigens A (MAGE-A) family, while MAGE-A are absent in healthy mucosal tissue. The present study aimed at determining if there is an association between the expression of MAGE-A in leukoplakia and malignant transformation to oral or laryngeal squamous cell carcinoma. Paraffin-embedded tissues of 205 oral and laryngeal leukoplakia, 90 corresponding tumors, and 40 healthy oral mucosal samples were included in the study. The grade of dysplasia of the leukoplakia samples was determined histopathologically. The leukoplakia samples were divided into lesions that transformed to oral and laryngeal squamous cell carcinoma (n = 91) and lesions that did not (n = 114) during a 5 years follow-up. The expression of MAGE-A3/6 and MAGE-A4 was analyzed by real-time RT-PCR. The expression of MAGE-A 1-4, 6, and 12 was determined by immunohistochemistry. A total of 59.3% of the transforming leukoplakia expressed at least one of the examined antigens as opposed to an expression rate of 3.5% of all non-transforming leukoplakia. There was no MAGE-A expression in healthy oral mucosa. The risk of malignant transformation was statistically significantly associated with MAGE-A expression in immunohistochemistry (p < 0.001) and real-time RT-PCR (MAGE-A3/6, p = 0.001; MAGE-A4, p = 0.002) analyses. There was no significant association between MAGE-A expression and the grade of dysplasia ("low-grade", D0/D1; "high-grade", D2/D3) in immunohistochemistry (p = 0.412) and real-time RT-PCR (MAGE-A3/6, p = 0.667; MAGE-A4, p = 0.756). It seems that the analysis of the MAGE-A expression profile may support the identification of leukoplakia at risk for malignant transformation. Therefore, efforts should be made to establish this analysis as a routine procedure in addition to conventional histopathology.

Background: With an increasing indication spectrum of antiresorptive drugs, the medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy [MRONJ (BP)] is continuously gaining clinical relevance. Impaired osteoclast function, accompanied by altered cell morphology and expression of osteoclastic effector proteins, contributes to the pathogenesis of MRONJ (BP). However, the underlying regulatory mechanisms at a transcriptional level are unaddressed so far. These mechanisms are crucial to the development of disease-characteristic osteoclastic anomalies, that contribute to the pathogenesis of MRONJ (BP). NFATc1 is considered a master upstream osteoclastic activator, whereas BCL6 acts as osteoclastic suppressor. The present study aimed to elucidate the NFATc1 and BCL6 mediated osteoclastic regulation and activity in MRONJ (BP) compared to osteoradionecrosis (ORN) and osteomyelitis (OM) and normal jaw bone., Methods: Formalin-fixed jaw bone specimens from 70 patients [MRONJ (BP) n = 30; OM: n = 15, ORN: n = 15, control: n = 10] were analyzed retrospectively for osteoclast expression of NFATc1 and BCL6. The specimens were processed for H&E staining and immunohistochemistry. The histological sections were digitalized and analyzed by virtual microscopy., Results: Osteoclastic expression of NFATc1 and BCL6 was significantly higher in MRONJ (BP) specimens compared to OM and control specimens. NFATc1 and BCL6 labeling indices revealed no significant differences between MRONJ (BP) and ORN. The ratio of nuclear BCL6+ osteoclasts to cytoplasmic BCL6+ osteoclasts revealed significantly higher values for MRONJ (BP) specimens compared to OM and controls., Conclusion: This study displays that osteoclasts in MRONJ (BP) tissues feature increased expression of the higher-level regulators, paradoxically of both NFATc1 and BCL6. These observations can help to explain the genesis of morphologically altered and resorptive inactive osteoclasts in MRONJ (BP) tissues by outlining the transcriptional regulation of the pathomechanically relevant osteoclastic effector proteins. Furthermore, they strengthen the etiological delineation of MRONJ (BP) from OM and extend the osteoclast profiles of MRONJ (BP), OM and ORN and thus could lead to a better histopathological differentiation that can improve treatment decision and motivate new therapeutic concepts.

Background: Despite the observed association of increased PD-L1 expression in peripheral blood of oral squamous cell carcinoma (OSCC) patients with histomorphologic parameters, the role of the PD1 ligands-PD-L1 and PD-L2-is insufficiently understood. Aim of the study was to investigate whether the alterations of PD-L1 and PD-L2 expression in blood are associated with survival and could serve as immune monitoring parameter. Moreover, it should be analyzed if PD-L2 is differentially expressed in tissue and blood samples of OSCC patients compared to healthy controls and if there is an association of PD-L2 expression with histomorphologic and prognostic tumor parameters., Methods: PD-L2 mRNA expression was analyzed in tumors and healthy oral mucosa specimens and in corresponding peripheral blood samples of 48 OSCC patients and 26 healthy controls using RT-qPCR. A cutoff point (COP) was determined and a chi-square test (χ 2 test) was carried out. Survival analysis of PD-L2 and previously reported PD-L1 expression data was performed using Kaplan-Meier analysis (Log-rank test)., Results: PD-L2 expression in tissue samples was significantly (P < 0.001) higher in OSCC patients compared to healthy controls. A significant association of PD-L2 expression above the COP (positive) with malignancy was ascertained (P < 0.001). A significant (P = 0.01) association of previously reported PD-L1 expression rates in peripheral blood with survival could be shown., Conclusion: Peripheral blood PD-L1 expression might be a prognostic marker for OSCC patients and a possible parameter to monitor immune dysfunction in malign diseases. In the peripheral blood, PD-L1 might be more relevant for immune tolerance than PD-L2. Local PD-L2 expression in tissue samples might be useful as a diagnostic parameter for malignancy and could contribute to the immunosuppressive local microenvironment in OSCC., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Microvascular free flap reconstruction has become a standard technique in head and neck reconstructive surgery. Pre-operative radiotherapy is associated with a higher incidence of free flap malperfusion and the need for operative revision. Irradiated vessels present characteristic histomorphological and structural changes. Alterations in endothelial cells of irradiated arteries remain incompletely investigated especially with regard to long-term changes in endothelial dysfunction supporting an intraluminal pro-thrombotic and pro-inflammatory milieu., Methods: Endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E‑ and P‑selectin, endothelial NO-synthase (eNOS), thrombomodulin and plasminogen activator inhibitor-1 (PAI-1) in irradiated and non-irradiated arteries was analysed using immunohistochemistry and Remmele scale grading. The average radiation dose was 58.7 ± 7.0 Gy; the time interval between end of radiation and tissue sampling was 106.0 ± 86.8 months., Results: Endothelial expression of ICAM-1, VCAM-1, E‑ and P‑selectin as well as PAI-1 was significantly increased in previously irradiated arteries compared with non-irradiated controls, whereas thrombomodulin and eNOS expression did not show any differences. However, when comparing non-irradiated free flap arteries with irradiated arteries from the head and neck area in respective individuals, eNOS expression was significantly lower in irradiated vessels whereas ICAM-1, VCAM-1, E‑/p-Selectin and PAI-1 showed significantly higher expression levels., Conclusion: There is ongoing endothelial dysfunction in terms of increased expression of pro-thrombotic and pro-inflammatory markers in irradiated arteries even years after radiotherapy. Treating this endothelial dysfunction might reduce the complication rates associated with microvascular free flap reconstructions in irradiated patients.

Background: Altered expressions of miR-186, miR-494 and miR-3651 in whole blood of OSCC patients have already been demonstrated. However, nothing is known about their expression in tumor tissues. This study aimed at evaluating differences in miRNA expression in OSCC tissues compared to healthy oral mucosa and at checking if the altered expression is reflected in corresponding blood., Methods: In 53 OSCC and 40 healthy mucosal tissues the expression of miR-186, -494 and -3651 was determined by RT-qPCR and expression levels were compared between the groups. The altered expression in tissues was compared with that determined for corresponding blood. MiR-3651 target genes were identified using TargetScan software., Results: Differential expression of miR-186 and miR-494 could not be observed in tumor tissues compared to normal mucosa (pmiR-186= 0.13; pmiR-494= 0.35). Contrary to the detected upregulation of the miR-3651 in the blood of OSCC patients, a significant downregulation was observed in OSCC tissues. A significant correlation between underexpression and diagnosis was ascertained (p= 0.0001). 1710 possible target genes of miR-3651 were identified., Conclusions: Decreased expression of miR-3651 in OSCC tissues may be a diagnostic biomarker. The opposite change in expression level in the blood might indicate different functions of this miRNA in circulation and tissue.

Background: Neck dissection is standard in surgical management of oral squamous cell carcinomas (oscc). However, the immunologic link between primary tumor and lymph nodes is insufficiently understood. Galectin 3 (Gal3) promotes M2 polarization of macrophages and contributes to immunosuppression. The current study analyzes the association between Gal3 expression in regional lymph nodes of oscc with histomorphologic parameters (T-, N-, L- Pn-stage, grading) of the primary tumor. Additionally, Gal3 expression is correlated with markers of macrophage polarization (M1 vs. M2)., Methods: Preoperative diagnostic biopsies (n = 26), tumor resection specimens (n = 34), tumor-free lymph nodes (n = 28) and lymph node metastases (n = 10) of T1/T2 oscc patients were immunohistochemically analyzed for Gal3 and macrophage marker (CD68, CD11c, CD163 and MRC1) expression. The number of positive cells and the expression ratios were quantitatively assessed., Results: High Gal3 expression in tumor-free regional lymph nodes was significantly (p < 0.05) associated with increased tumor size. The epithelial compartment of lymph node metastases showed a significantly (p < 0.05) increased Gal3 expression compared to biopsies and tumor resection specimens. Cell density of M2 macrophages was significantly (p < 0.05) and positively correlated with the number of Gal3 expressing cells in lymph nodes and tumor specimens., Conclusion: Gal3 expression in regional lymph nodes might be associated with oscc progression. The increased Gal3 expression in regional lymph nodes of larger tumors underlines the need of immunomodulatory treatment concepts in early-stage oscc. Blocking of Gal3 might be a therapeutic option in oral cancer.

Objectives: Apical periodontitis can appear clinically as apical granulomas or radicular cysts. There is evidence that immunologic factors are involved in the pathogenesis of both pathologies. In contrast to radicular cysts, the dentigerous cysts have a developmental origin. Macrophage polarization (M1 vs M2) is a main regulator of tissue homeostasis and differentiation. There are no studies comparing macrophage polarization in apical granulomas, radicular cysts, and dentigerous cysts., Materials and Methods: Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray (TMA) of the 87 consecutive specimens was created, and CD68-, CD11c-, CD163-, and MRC1-positive macrophages were detected by immunohistochemical methods. TMAs were digitized, and the expression of macrophage markers was quantitatively assessed., Results: Radicular cysts are characterized by M1 polarization of macrophages while apical granulomas show a significantly higher degree of M2 polarization. Dentigerous cysts have a significantly lower M1 polarization than both analyzed periapical lesions (apical granulomas and radicular cysts) and accordingly, a significantly higher M2 polarization than radicular cysts. Macrophage cell density in dentigerous cysts is significantly lower than in the periapical lesions., Conclusions: The development of apical periodontitis towards apical granulomas or radicular cysts might be directed by macrophage polarization. Radicular cyst formation is associated with an increased M1 polarization of infiltrating macrophages. In contrast to radicular cysts, dentigerous cysts are characterized by a low macrophage infiltration and a high degree of M2 polarization, possibly reflecting their developmental rather than inflammatory origin., Clinical Relevance: As M1 polarization of macrophages is triggered by bacterial antigens, these results underline the need for sufficient bacterial clearance during endodontic treatment to prevent a possible M1 macrophage-derived stimulus for radicular cyst formation.

Background: Immunologic factors can promote the progression of oral squamous cell carcinomas (oscc). The phylogenetic highly conserved protein Galectin 3 (Gal3) contributes to cell differentiation and immune homeostasis. There is evidence that Gal3 is involved in the progression of oscc and influences the regulation of macrophage polarization. Macrophage polarization (M1 vs. M2) in solid malignancies like oscc contributes to tumor immune-escape. However, the relationship between macrophage polarization and Gal3 expression in oscc is not yet understood. The current study analyzes the association between histomorphologic parameters (T-, N-, L- Pn-status, grading) and Gal3 expression resp. the ratio between Gal3 expressing cells and CD68 positive macrophages in oscc specimens., Methods: Preoperative diagnostic biopsies (n = 26) and tumor resection specimens (n = 34) of T1/T2 oscc patients were immunohistochemically analyzed for Gal3 and CD68 expression. The number of Gal3 expressing cells and the ratio between CD68 and Gal3 expressing cells was quantitatively assessed., Results: In biopsy and tumor resection specimens, the number of Gal3 positive cells as well as the Gal3/CD68 ratio were significantly (p < 0.05) higher in T2 oscc compared to T1 cases. In biopsy specimens, a significantly (p < 0.05) increased Gal3 expression and Gal3/CD68 ratio was associated with the progression marker lymph vessel infiltration (L1). Tumor resection specimens of cases with lymph node metastases (N+) had a significantly (p < 0.05) increased Gal3 expression. Additionally, a high Gal3/CD68 ratio correlated significantly (p < 0.05) with higher grading (G3) in tumor resection specimens., Conclusion: High Gal3 expression in oscc is associated with tumor size (T-status) and parameters of malignancy (N-, L-status, grading). Gal3 might contribute to M2 macrophage mediated local immune tolerance. Gal3 expression shows association with prognosis in oscc and represent a potential therapeutic target.

Background: Immune checkpoints like programmed cell death-1 (PD-1) and its ligand PD-L1 are involved in immune escape mechanisms of solid tumors including oral squamous cell carcinoma (OSCC). Inhibitors of the pathway are successfully used for treating especially advanced disease. However, the physiological relevance of PD-1/PD-L1-signaling in OSCC is insufficiently understood. The aim of the study was to analyze if PD-L1 expression in tumor tissue and peripheral blood samples of OSCC patients is associated with histomorphological tumor parameters and if PD-L1 expression in patients is different from controls., Results: OSCC tumor specimens showed a significantly higher PD-L1 expression than oral mucosa controls ( p < 0.001; upregulation more than 3-fold). Cross-tabulation revealed an association of increased expression of PD-L1 mRNA in tissue specimens with malignancy ( p < 0.001).OSCC cases with higher tumor grade and cases with lymph node metastases (N+) were significantly ( p < 0.05) associated with increased PD-L1 expression in peripheral blood. Cross-tabulation revealed an significant association with lymph node metastases (N+) ( p ≤ 0.002)., Materials and Methods: PD-L1 mRNA expression was analyzed in tumor specimens and corresponding samples of healthy oral mucosa and peripheral blood of 45 OSCC patients and 36 healthy control persons using RT-qPCR analysis. A Mann-Whitney U-test , a cut-off point analysis and a Chi-square test were carried out., Conclusions: PD-L1 expression in OSCC could contribute to the immunosuppressive local tumor microenvironment. Increased malignant behavior (higher tumor grade, positive nodal status) might be associated with PD-L1 mediated systemic immune tolerance. Thus, PD-L1 expression in peripheral blood might be an indicator of the existence of metastatic disease (N+) in OSCC., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

Purpose: Surgical treatment of head and neck malignancies frequently includes microvascular free tissue transfer. Preoperative radiotherapy increases postoperative fibrosis-related complications up to transplant loss. Fibrogenesis is associated with re-expression of embryonic preserved tissue developmental mediators: osteopontin (OPN), regulated by sex-determining region Y‑box 9 (Sox9), and homeobox A9 (HoxA9) play important roles in pathologic tissue remodeling and are upregulated in atherosclerotic vascular lesions; dickkopf-1 (DKK1) inhibits pro-fibrotic and atherogenic Wnt signaling. We evaluated the influence of irradiation on expression of these mediators in arteries of the head and neck region., Materials and Methods: DKK1, HoxA9, OPN, and Sox9 expression was examined immunohistochemically in 24 irradiated and 24 nonirradiated arteries of the lower head and neck region. The ratio of positive cells to total cell number (labeling index) in the investigated vessel walls was assessed semiquantitatively., Results: DKK1 expression was significantly decreased, whereas HoxA9, OPN, and Sox9 expression were significantly increased in irradiated compared to nonirradiated arterial vessels., Conclusion: Preoperative radiotherapy induces re-expression of embryonic preserved mediators in arterial vessels and may thus contribute to enhanced activation of pro-fibrotic downstream signaling leading to media hypertrophy and intima degeneration comparable to fibrotic development steps in atherosclerosis. These histopathological changes may be promoted by HoxA9-, OPN-, and Sox9-related inflammation and vascular remodeling, supported by downregulation of anti-fibrotic DKK1. Future pharmaceutical strategies targeting these vessel alterations, e. g., bisphosphonates, might reduce postoperative complications in free tissue transfer.

Background: The medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy [MRONJ (BP)] is characterized by non-healing exposed bone in the maxillofacial region. The pathogenesis of MRONJ (BP) is not fully understood. Giant, hypernucleated, inactive osteoclasts were found in MRONJ (BP) tissues, which indicated that accelerated cell-cell fusion might play a role. Dendritic cell-specific transmembrane protein (DC-STAMP) is associated with the cell-cell fusion of osteoclasts and precursor cells. Tartrate-resistant acid phosphatase (TRAP) is essential for osteoclastic bone resorption. The cell-cell fusion, as part of the osteoclastogenesis, and the resorptive activity can determine the morphology of osteoclasts. This study analyzed jaw bone from patients with MRONJ (BP), osteomyelitis (OM) and osteoradionecrosis (ORN) because a comparison with the osteoclast profiles of OM and ORN is essential for characterizing the osteoclast profile of MRONJ (BP)., Methods: Formalin-fixed routine jaw bone specimens from 70 patients [MRONJ (BP) n = 30; OM: n = 15, ORN: n = 15, control: n = 10] were analyzed retrospectively for osteoclast quantity, morphology and the expression of TRAP and DC-STAMP. The specimens were processed for hematoxylin and eosin staining (H&E), histochemistry (TRAP) and immunohistochemistry (anti-DC-STAMP) and were analyzed via virtual microscopy., Results: The quantity, diameter and nuclearity of osteoclasts were significantly higher in MRONJ (BP) specimens than in OM, ORN and control specimens. Giant, hypernucleated osteoclasts were detected in MRONJ (BP) specimens only. Osteoclastic TRAP expression was lower in MRONJ (BP) and ORN specimens than in OM and control specimens. The DC-STAMP expression of osteoclasts and mononuclear cells was significantly higher in MRONJ (BP) and ORN specimens than in OM and control specimens., Conclusions: This study indicates that the osteoclast profile of MRONJ (BP) is characterized by osteoclast inactivation and a high cell-cell fusion rate; however, the presence of giant, hypernucleated osteoclasts cannot be attributed to increased DC-STAMP-triggered cell-cell fusion alone. The incidental characterization of the osteoclast profiles of OM and ORN revealed differences that might facilitate the histopathological differentiation of these diseases from MRONJ (BP), which is essential because their therapies are somewhat different.

Purpose: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a complication of antiresorptive therapy with nitrogen-containing bisphosphonates (BP). With various suggestions as to pathogenesis, the etiology of BRONJ is not sufficiently understood. Osteoclasts and their precursors, that is, macrophages, are the main target cells of BP. BP can repolarize regeneration- and healing-associated M2 macrophages towards the tissue destructive M1-type. The current study aims to elucidate differences in macrophage and osteoclast polarization in BRONJ, osteoradionecrosis (ORN) and healthy control specimens., Materials and Methods: A total of 39 jaw bone samples (18 BRONJ, 8 ORN and 13 healthy controls) were processed for immunohistochemistry to detect CD68-, CD11c- and CD163-positive cells. Macrophages and osteoclasts were distinguished on the basis of morphological differences. Samples were digitized, and the macrophage and osteoclast cell counts were quantitatively analyzed., Results: In jaw bone affected by BRONJ, a significantly increased macrophage infiltration and M1 polarization of macrophages can be seen. The density of CD68-expressing osteoclasts is significantly increased in BRONJ specimens compared to ORN and to healthy controls., Conclusions: A bisphosphonate-derived shift of macrophage polarization towards M1-polarized macrophages might impair bone tissue homeostasis and thus contribute to the pathogenesis of BRONJ. The observed increase in osteoclast density might be caused by BP-induced prolonged osteoclast survival., (Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)