Alterations in macrophage polarization in the craniofacial and extracranial skeleton after zoledronate application and surgical interventions - an in vivo experiment.

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.

Pedicled buccal flaps as a backup procedure for intraoral reconstruction.

BACKGROUND: Intraoral soft tissue deficiency and impaired wound beds are common problems after cleft and tumour surgery or after dental trauma. Frequently, limited defects are overtreated with extensive microvascular reconstruction procedures, but pedicled flaps remain useful, as they are simple to harvest, and they provide a reliable outcome. The buccal flap, first described in the 1970s, has been used for palatine lengthening in cleft patients over decades. In the following, we present an expanded indication in cases of palatal fistula, complex vestibulum, exposed bone in orthognathic surgery, and osteoradionecrosis. METHODS: We conducted a retrospective chart review and report on all buccal flaps harvested in our department within the last 3 years with a follow-up period of at least half a year after flap surgery. Patients of all age groups and treatment indications in which a buccal flap was used were implicated in the evaluation. RESULTS: Sixteen buccal flaps were performed in 10 patients. The median age at the time of surgery was 42 years, reaching from 12 up to 66 years. Fourteen buccal flaps were used for upper jaw or palatal coverage; two buccal flaps were used in the mandible. In terms of complications (four flaps; 25%), there were two partial flap failures, one wound dehiscence and one wound dehiscence. There were no failures of the remaining mucosal flap islands after pedicle dissection. CONCLUSION: The buccal flap is a reliable and straightforward approach to challenging intraoral wound beds with soft tissue deficiency. We thoroughly discuss the additional indications for buccal flap surgery, describe the harvest technique, and provide strategies to prevent intra- and postoperative complications.

Recipient bed perfusion as a predictor for postoperative complications in irradiated patients with microvascular free tissue transfer of the head and neck area: a clinical analysis of 191 microvascular free flaps.

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.

DICER1-Mutated Botryoid Fibroepithelial Polyp of the Parotid Duct: Report of the First Case.

DICER1, a member of the ribonuclease III family, is involved in the biogenesis of microRNAs and, hence, it influences gene expression regulation. DICER1 germline (associated with the inherited DICER1 syndrome) or somatic mutations have been linked to tumorigenesis in histogenetically diverse benign and malignant neoplasms in different organs including pleuropulmonary blastoma, cystic nephroma, embryonal rhabdomyosarcoma, nasal chondromesenchymal hamartoma, poorly differentiated thyroid carcinoma, thyroblastoma, intracranial sarcoma and gonadal Sertoli-Leydig cell tumors in addition to others. Moreover, rare botryoid (giant) fibroepithelial polyps may harbor this mutation. Herein, we describe the first reported case of a DICER1-mutated botryoid fibroepithelial polyp occurring within the parotid duct of a 65-year-old female who has no other features or family history of the DICER1 syndrome. Based on its distinctive morphology, we tested this lesion specifically for DICER1 mutations and confirmed the presence of a pathogenic DICER1 variant with a low allele frequency, consistent with a somatic mutation.

Lineage-associated connexin 43 expression in bisphosphonate-exposed rat bones.

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.

Zoledronate Causes a Systemic Shift of Macrophage Polarization towards M1 In Vivo.

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.

Endothelial inflammatory and thrombogenic expression changes in microvascular anastomoses - An immunohistochemical analysis.

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.

Free Gingival Graft and Collagen Matrix Revascularization in an Enoral Open Wound Situation.

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.

Correction to: Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

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 ….

Perioperative Management in Patients With Cleft Lip and Palate.

In cleft care, perioperative treatment strategies like ear nose and throat (ENT) diagnostics as well as postoperative antibiotics, feeding, and duration of inpatient stay are nonstandardized procedures varying between different centers. Likewise, intraoperative choice of suture materials and time of suture removal are performed inconsistently. Therefore, we wanted to collect information on protocols focusing on these topics to summarize and subsume currently approved treatment strategies of centers around the world. We ask members of international cleft centers for their respective treatment strategies and performed descriptive statistics.Absorbable suture material is used for reconstruction of the outer lip skin in 20 of 70 centers. Removal of skin sutures is conducted after 7.0 ±â€Š1.5 days. Suturing of the orbicularis oris muscle, the enoral and nasal mucosa, as well as the palatal musculature is predominantly performed with absorbable suture materials. Intraoperative antibiotic prophylaxis is applied in 82.9% of the participating centers. In contrast, 31.9% of the departments do not apply any antibiotic postoperatively. Postoperative feeding is performed in 27 centers via a nasogastric tube for 4.6 ±â€Š2.3 days on average. Mean length of postoperative inpatient stay is 4.1 ±â€Š2.6 days in children after cleft lip surgery and 4.5 ±â€Š2.7 days after cleft palate surgery. ENT consultation before surgery is routinely conducted in 52.8% of the centers and 82.9% of ENT colleagues investigate middle ear pathologies in the same operation in which cleft repair is performed.Closure of the lip skin is predominantly performed with nonabsorbable suture material followed by a suture removal after 1 week. Intraoperative antibiotic prophylaxis as well as inpatient hospital stay of 4 to 5 days in combination with oral feeding and a preoperative consultation and intraoperative cooperation with the ENT department seems to be well-proven concepts in cleft lip palate patient care. However, this analysis illustrated the variations and differing approaches in perioperative care emphasizing the need to verify perioperative management concepts in cleft surgery-preferably in the context of multicenter studies.

Fluorescence-guided bone resection: A histological analysis in medication-related osteonecrosis of the jaw.

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/mm2: 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 mm2: 8.97 ± 7.85 (fluorescence positive) vs. 7.76 ± 6.41 (fluorescence negative), p = 0.793; TRAP-positive cells per mm2: 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.

Collagen Matrix Vascularization in a Peri-Implant Vestibuloplasty Situation Proceeds Within the First Postoperative Week.

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.

Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

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 P­selectin, 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 P­selectin 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.

Galectin 3 expression in regional lymph nodes and lymph node metastases of oral squamous cell carcinomas.

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.

Macrophage polarization differs between apical granulomas, radicular cysts, and dentigerous cysts.

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.

Galectin 3 expression in primary oral squamous cell carcinomas.

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.

Re-expression of pro-fibrotic, embryonic preserved mediators in irradiated arterial vessels of the head and neck region.

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 Y­box 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.

Osteoclast profile of medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy: a comparison with osteoradionecrosis and osteomyelitis.

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.

Macrophage and osteoclast polarization in bisphosphonate associated necrosis and osteoradionecrosis.

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.

PD-L1 expression in tumor tissue and peripheral blood of patients with oral squamous cell carcinoma.

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.