Effects of the Highly COX-2-Selective Analgesic NSAID Etoricoxib on Human Periodontal Ligament Fibroblasts during Compressive Orthodontic Mechanical Strain

Human periodontal ligament (hPDL) fibroblasts play a major role during periodontitis and orthodontic tooth movement, mediating periodontal inflammation, osteoclastogenesis, and collagen synthesis. The highly COX-2-selective NSAID etoricoxib has a favorable systemic side effect profile and high analgesic efficacy, particularly for orthodontic pain. In this in vitro study, we investigated possible side effects of two clinically relevant etoricoxib concentrations on the expression pattern of mechanically strained hPDL fibroblasts and associated osteoclastogenesis in a model of simulated orthodontic compressive strain occurring during orthodontic tooth movement. hPDL fibroblasts were incubated for 72h under physiological conditions with etoricoxib at 0M, 3.29M, and 5.49M, corresponding to clinically normal and subtoxic dosages, with and without mechanical strain by compression (2g/cm(2)) for the final 48h, simulating conditions during orthodontic tooth movement in compressive areas of the periodontal ligament. We then determined gene and/or protein expression of COX-2, IL-6, PG-E-2, RANK-L, OPG, ALPL, VEGF-A, P4HA1, COL1A2, and FN1 via RT-qPCR, ELISA, and Western blot analyses as well as apoptosis, necrosis, cell viability, and cytotoxicity via FACS, MTT, and LDH assays. In addition, hPDL fibroblast-mediated osteoclastogenesis was assessed by TRAP staining in coculture with RAW267.4 cells for another 72h. Gene and protein expression of all evaluated factors was significantly induced by the mechanical compressive strain applied. Etoricoxib at 3.29M and 5.49M significantly inhibited PG-E-2 synthesis, but not COX-2 and IL-6 gene expression nor RANK-L-/OPG-mediated osteoclastogenesis or angiogenesis (VEGF-A). Extracellular matrix remodeling (COL1A2, FN1) and bone anabolism (ALPL), by contrast, were significantly stimulated particularly at 5.49M. In general, no adverse etoricoxib effects on hPDL fibroblasts regarding apoptosis, necrosis, cell viability, or cytotoxicity were detected. Clinically dosed etoricoxib, that is, a highly selective COX-2 inhibition, did not have substantial effects on hPDL fibroblast-mediated periodontal inflammation, extracellular matrix remodeling, RANK-L/OPG expression, and osteoclastogenesis during simulated orthodontic compressive strain

Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force

Objective: Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. Methodology: Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients’ genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). Results: The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). Conclusions: Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force

An Evaluation of Different 3D Cultivation Models on Expression Profiles of Human Periodontal Ligament Fibroblasts with Compressive Strain

The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT–qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain

Alternatives to free flap surgery for maxillofacial reconstruction: focus on the submental island flap and the pectoralis major myocutaneous flap

Background Microvascular tissue transfer (MTT) has been established as the gold standard in oral- and maxillofacial reconstruction. However, free flap surgery may be critical in multimorbid elderly patients and after surgery or radiotherapy, which aggravate microsurgery. This study evaluates indications and outcome of the submental island flap (SMIF) and the pectoralis major myocutaneous flap (PMMF) as alternatives to the free radial forearm flap (RFF). Methods This retrospective study included 134 patients who had undergone resection and reconstruction with SMIF, PMMF, or RFF at our department between 2005 and 2020. The level of comorbidity was measured with the Age-adjusted Charlson comorbidity index (ACCI). Primary outcome variables were flap success, complications, wound dehiscence, surgery duration, as well as time at the ICU and the ward (hospitalization). Chi-square tests, t-tests, and ANOVA were performed for statistics. Results 24 SMIFs, 52 RFFs, and 58 PMMFs were included in this study. The flap types did not significantly differ in terms of flap success, complications, and healing disorders. The SMIF presented a success rate of 95.8% and was significantly more often used in elderly patients (mean age = 70.2 years; p < 0.001) with increased comorbidities than the PMMF (p < 0.01) and RFF (p < 0.001). SMIF reconstruction reduced surgery duration (p < 0.001) and time at the ICU (p = 0.009) and the ward (p < 0.001) more than PMMF and RFF reconstructions. PMMF reconstruction was successful in 91.4% of patients and was more frequently used after head and neck surgery (p < 0.001) and radiotherapy (p < 0.001) than SMIF and RFF reconstructions. Patients undergoing PMMF reconstruction more frequently required segmental jaw resection and had presented with advanced tumor stages (both p < 0.001). Nicotine and alcohol abuse was more frequent in the RFF and PMMF groups (both p < 0.001) than in the SMIF group. Conclusions The pedicled SMIF represents a valuable reconstructive option for elderly patients with increased comorbidity because of the shorter duration of surgery and hospitalization. On the other hand, the PMMF serves as a solid backup solution after head and neck surgery or radiotherapy. The rates of flap success, complications, and healing disorders of both pedicled flaps are comparable to those of free flap reconstruction

The role of 25-hydroxyvitamin-D3 and vitamin D receptor gene in human periodontal ligament fibroblasts as response to orthodontic compressive strain: an in vitro study

Background: This study aimed to investigate, if diferent physiological concentrations of vitamin D (25(OH)D3) and single nucleotide polymorphisms in vitamin D receptor (VDR) gene have an impact on gene expression in human periodontal ligament (hPDL) fbroblasts induced by simulated orthodontic compressive strain. Methods: A pool of hPDL fbroblasts was treated in absence or presence of 25(OH)D3 in 3 diferent concentra�tions (10, 40 and 60 ng/ml). In order to evaluate the role of single nucleotide polymorphisms in the VDR gene, hPDL fbroblasts from 9 patients were used and treated in absence or presence of 40 ng/ml 25(OH)D3. Each experiment was performed with and without simulated orthodontic compressive strain. Real-time PCR was used for gene expression and allelic discrimination analysis. Relative expression of dehydrocholesterol reductase (DHCR7), Sec23 homolog A, amidohydrolase domain containing 1 (AMDHD1), vitamin D 25-hydroxylase (CYP2R1), Hydroxyvitamin D-1-α hydroxy�lase, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2) and interleukin-6 (IL6) was assessed. Three single nucleotide polymorphisms in VDR were genotyped. Parametric or non�parametric tests were used with an alpha of 5%. Results: RANKL, RANKL:OPG ratio, COX-2, IL-6, DHCR7, CYP2R1 and AMDHD1 were diferentially expressed during simulated orthodontic compressive strain (p<0.05). The RANKL:OPG ratio was downregulated by all concentrations (10 ng/ml, 40 ng/ml and 60 ng/ml) of 25(OH)D3 (mean=0.96±0.68, mean=1.61±0.66 and mean=1.86±0.78, respectively) in comparison to the control (mean 2.58±1.16) (p<0.05). CYP2R1 gene expression was statistically modulated by the diferent 25(OH)D3 concentrations applied (p=0.008). Samples from individuals carrying the GG genotype in rs739837 presented lower VDR mRNA expression and samples from individuals carrying the CC genotype in rs7975232 presented higher VDR mRNA expression (p<0.05). Conclusions: Simulated orthodontic compressive strain and physiological concentrations of 25(OH)D3 seem to regu�late the expression of orthodontic tooth movement and vitamin-D-related genes in periodontal ligament fbroblast

Significance of site‐specific radiation dose and technique for success of implant‐based prosthetic rehabilitation in irradiated head and neck cancer patients—A cohort study

Background Radiotherapy aggravates implant‐based prosthetic rehabilitation in patients with head and neck cancer. Purpose To evaluate the impact of radiation dose at implant and parotid gland site for prosthetic rehabilitation. Material and methods The retrospective study includes 121 irradiated head and neck cancer patients with 751 inserted implants. Radiation doses on implant bed and parotid gland site were recorded by 3‐dimensional modulated radiation plans. Implant success was clinically and radiographically evaluated according to modified Albrektsson criteria and compared to treatment‐ and patient‐specific data. Results Implant overall survival after 5 years was 92.4% with an implant success rate of 74.9%. Main reasons for implant failure were marginal bone resorption (20.9%), implant not in situ or unloaded (9.6%) and peri‐implantitis (7.5%). A mean radiation dose of 62.6 Gy was applied with a mean parotid dose of 35 Gy. Modulating radiation techniques went along with lower grades of xerostomia (p 50 Gy (HR 7.9), parotid dose >30 Gy (HR 2.3), bone (HR 14.5) and soft tissue (HR 4.5) transplants, bad oral hygiene (HR 3.8), nonmodulated radiation treatment planning (HR 14.5), and nontelescopic prosthetics (HR 5.2). Conclusion Radiotherapy impedes implant success in a dose‐dependent manner at implant site. Modern radiation techniques effectively reduce xerostomia favoring implant‐based prosthetic rehabilitation. Implantation in bone grafts is more critical and telescopic‐retained overdentures should be preferred

A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common tumors within the oral cavity. Early diagnosis and prognosis tools are urgently needed. This study aimed to investigate the activation of the complement system in OSCC patients as potential biomarker. Therefore, an innovative complement activation array was developed. Characterized antibodies detecting the complement activation specific epitopes C3a, C5a and sC5b-9 along with control antibodies were implemented into a suspension bead array. Human serum from a healthy (n = 46) and OSCC patient (n = 57) cohort were used to investigate the role of complement activation in oral tumor progression. The novel multiplex assay detected C3a, C5a and sC5b-9 from a minimal sample volume of human tears, aqueous humor and blood samples. Limits of detection were 0.04 ng/mL for C3a, 0.03 ng/mL for C5a and 18.9 ng/mL for sC5b-9, respectively. Biological cut-off levels guaranteed specific detections from serum. The mean serum concentration of a healthy control cohort was 680 ng/mL C3a, 70 ng/mL C5a and 2247 ng/mL sC5b-9, respectively. The assay showed an intra-assay precision of 2.9-6.4% and an inter-assay precision of 9.2-18.2%. Increased systemic C5a (p < 0.0001) and sC5b-9 (p = 0.01) concentrations in OSCC patients were determined using the validated multiplex complement assay. Higher C5a concentrations correlated with tumor differentiation and OSCC extension state. Systemic sC5b-9 determination provided a novel biomarker for infiltrating tumor growth and C3a levels were associated with local tumor spreading. Our study suggests that systemic complement activation levels in OSCC patients may be useful to assess disease progression

Impact of Spatially Heterogeneous Trop-2 Expression on Prognosis in Oral Squamous Cell Carcinoma

Oral cancer often presents with aggressive behavior and a high risk of recurrence and metastasis. For oral squamous cell carcinoma (OSCC), which is the most frequent histological subtype, therapy strategies include surgery, radiation therapy, chemotherapy, immune checkpoint inhibitors, and EGFR inhibitors. Recently, a Trop-2 antibody-drug conjugate (ADC) has been approved in the United States of America for the treatment of advanced triple-negative breast cancer. However, this ADC has also been tested in other solid tumors including head & neck squamous cell carcinoma. The prognostic impact of Trop-2 has already been reported for several cancers. We studied the prognostic influence of Trop-2 protein expression on OSCC patients’ survival. The cohort comprised n = 229 OSCC patients with available archived tumor tissue and corresponding non-neoplastic oral mucosa tissue. Using immunohistochemistry, we investigated Trop-2 expression in both the central and peripheral regions of each tumor and in corresponding non-neoplastic oral mucosa. In patients suffering from OSCC with combined high central and low peripheral Trop-2 expression, five-year overall survival (OS) was 41.2%, whereas 55.6% of OSCC patients who presented lower central and/or higher peripheral tumoral Trop-2 expression were alive after five years (p = 0.075). In multivariate Cox regression, the expression pattern of high central tumoral and lower peripheral Trop-2 expression was significantly correlated with impaired OS (HR = 1.802, 95%-CI: 1.134–2.864; p = 0.013) and recurrence-free survival (RFS) (HR = 1.633, 95%-CI: 1.042–2.560; p = 0.033), respectively, when adjusting for co-variables. Hence, Trop-2 may serve as an independent prognostic biomarker in OSCC. In subsequent studies, the pathophysiological meaning of downregulated Trop-2 expression in the OSCC periphery has to be analyzed

Loss of MMP-27 Predicts Mandibular Bone Invasion in Oral Squamous Cell Carcinoma

Invasion of the mandibular bone is frequent in oral squamous cell carcinoma (OSCC), which often results in extensive ablative and reconstructive procedures for the patient. The purpose of this single-center, retrospective study was to identify and evaluate potential biomarkers and risk factors for bone invasion in OSCC. Initially, in silico gene expression analysis was performed for different HNSCC tumor T-stages to find factors associated with invasive (T4a) tumor growth. Afterwards, the protein expression of bone-metabolizing MMP-27, TNFRSF11B (Osteoprotegerin, OPG), and TNFSF11 (RANKL) was investigated via Tissue Microarrays (TMAs) for their impact on mandibular bone invasion. TMAs were assembled from the bone–tumor interface of primary OSCCs of the floor of the mouth and gingiva from 119 patients. Sixty-four carcinomas with patho-histological jaw invasion (pT4a) were compared to 55 carcinomas growing along the mandible without invasion (pT2, pT3). Tissue samples were additionally evaluated for patterns of invasion using the WPOI grading system. Statistical analysis of in silico data revealed decreased MMP-27 mRNA expression to be strongly associated with the pT4a-stage in OSCC, indicating invasive tumor growth with infiltration of adjacent anatomical structures. Our own clinico-pathological data on OSCCs presented a significant decrease of MMP-27 in tumors invading the nearby mandible (pT4a), compared to pT2 and pT3 tumors without bone invasion. Loss of MMP27 evolved as the strongest predictor of mandibular bone invasion in binary logistic regression analysis. To our knowledge, this is the first study investigating the role of MMP-27 expression in OSCC and demonstrating the importance of the loss of MMP-27 in mandibular bone invasion

Influence of Single-Nucleotide Polymorphisms on Vitamin D Receptor Expression in Periodontal Ligament Fibroblasts as a Response to Orthodontic Compression

This study aimed to evaluate if single-nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene are associated with gene expression in human periodontal ligament (hPDL) fibroblasts under simulated orthodontic compressive force. hPDL samples from 57 patients were used. A physiological compressive strain was performed to simulate orthodontic tooth movement in pressure areas under cell culture conditions. The RNA from hPDL fibroblasts was isolated to determine the relative gene expression (mRNA) of the VDR. The DNA was also isolated for the genotyping analysis of five SNPs in the VDR gene: BglI (rs739837, G/T), BsmI (rs1544410, T/C), ApaI (rs7975232, A/C), FokI (rs2228570, A/G), and TaqI (rs731236, A/G). Real-time polymerase chain reaction was used for both analyses. Kruskal–Wallis tests were used to compare VDR expression among genotypes of each SNP. A linear regression analysis was performed to evaluate SNP–SNP interaction. An established alpha of 5% was used. The relative mRNA VDR expression according to the genotypes in the SNPs BglI, BsmI, ApaI, FokI, and TaqI was not statistically significantly different (p > 0.05). The SNP–SNP interaction evaluated by regression analysis did not demonstrate any statistically significant association. No association was observed (p > 0.05). In conclusion, the SNPs BglI (rs739837), BsmI (rs1544410), ApaI (rs7975232), FokI (rs2228570), and TaqI (rs731236) did not show an impact on VDR gene expression in hPDL fibroblasts under simulated orthodontic compressive force