Compressive force strengthened the pro-inflammatory effect of zoledronic acid on il-1ß stimulated human periodontal fibroblasts.

OBJECTIVES: The number of patients in dentistry taking bisphosphonates (BP) increases every year. There are only little data about the influence of biomechanical stress due to orthodontic treatment and periodontal inflammation in BP patients. This study focused on the effects of the induced inflammation by IL-1ß in compressed human periodontal ligament fibroblasts (HPdLF) exposed to the nitrogen-containing BP zoledronate in vitro. MATERIALS AND METHODS: HPdLF were incubated with 5 µmol/l zoledronate and 10 ng/ml IL-1ß for 48 h. In the last 3 h, cells were exposed to a compressive, centrifugal force of 34.9 g/cm2. Cell viability was analyzed directly after the compressive force by MTT assay. Gene expression of COX-2 and IL-6 was investigated using quantitative qRT-PCR. PGE-2 and IL-6 protein secretion were measured via ELISA. RESULTS: The cell viability of HPdLF was not affected. Without inflammatory pre-stimulation, COX-2 expression was increased by compression and zoledronate. IL-6 expression was increased under compression. On secretion level, the combination of compression and zoledronate induced a slightly increase of IL-6 secretion. In contrast, inflammatory pre-stimulation strengthened the compressive upregulation of COX-2, as well as induced a higher PGE-2 secretion. Further addition of zoledronate to pre-stimulated cells additionally strengthened the compression-induced upregulation of COX-2 and IL-6 expression as well as protein secretion compared to all other groups. CONCLUSIONS: Biomechanical stress might trigger a pro-inflammatory potential of BP further enhanced in the presence of an inflammatory pre-stimulation. CLINICAL RELEVANCE: To prevent excessive host inflammatory responses, occlusal overloading and mechanical stress due to orthodontic treatment should be avoided in BP patients with untreated periodontitis.

Influence of clodronate and compressive force on IL-1ß-stimulated human periodontal ligament fibroblasts.

OBJECTIVES: The aim of this study was to investigate in vitro the effect of clodronate on interleukin-1ß (IL-1ß)-stimulated human periodontal ligament fibroblasts (HPdLFs) with the focus on inflammatory factors of orthodontic tooth movement with and without compressive force. MATERIALS AND METHODS: HPdLFs were incubated with 5 µM clodronate and 10 ng/mL IL-1ß. After 48 h, cells were exposed to 3 h of compressive force using a centrifuge. The gene expression of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), matrix metalloproteinase 8 (MMP-8), and the tissue inhibitor of MMP (TIMP-1) was analyzed using RT-PCR. Prostaglandin E2 (PGE-2), IL-6, and TIMP-1 protein syntheses were quantified via ELISA. RESULTS: Compressive force and IL-1ß induced an overexpression of COX-2 gene expression (61.8-fold; p < 0.05 compared with control), diminished by clodronate (41.1-fold; p < 0.05 compared with control). Clodronate slowed down the compression and IL-1ß induced IL-6 gene expression (161-fold vs. 85.6-fold; p < 0.05 compared with control). TNF-α was only slightly affected without statistical significance. Clodronate reduced IL-1ß-stimulated MMP-8 expression with and without compressive force. TIMP-1 on gene and protein level was downregulated in all groups. Analyzing the MMP-8/TIMP-1 ratio, the highest ratio was detected in IL-1ß-stimulated HPdLFs with compressive force (21.2-fold; p < 0.05 compared with control). Clodronate diminished IL-1ß-induced upregulation of MMP-8/TIMP-1 ratio with (11.5-fold; p < 0.05 compared with control) and without (12.5-fold; p < 0.05 compared with control) compressive force. CONCLUSION: Our study demonstrates a slightly anti-inflammatory effect by clodronate under compressive force in vitro. Additionally, the periodontal remodeling presented by the MMP-8/TIMP-1 ratio seems to be diminished by clodronate. CLINICAL RELEVANCE: Reduction of pro-inflammatory factors and reduction of periodontal remodeling might explain reduced orthodontic tooth movement under clodronate intake.

Comparative effectiveness of delayed-release dimethyl fumarate versus interferon, glatiramer acetate, teriflunomide, or fingolimod: results from the German NeuroTransData registry.

BACKGROUND: Comparative effectiveness (CE) research allows real-world treatment comparisons using outcome measurements important to physicians/patients. This German NeuroTransData registry-based analysis compared delayed-release dimethyl fumarate (DMF) effectiveness with interferons (IFN), glatiramer acetate (GA), teriflunomide (TERI), or fingolimod (FTY) in patients with relapsing-remitting multiple sclerosis (RRMS) using propensity score matching (PSM). METHODS: Data from registry patients aged ≥ 18 years with RRMS, ≥ 1 relapse, and Expanded Disability Status Scale (EDSS) assessment(s) after index therapy initiation underwent 1:1 PSM to match DMF with comparator populations baseline characteristics. Primary outcome measurement was time to first relapse (TTFR). Secondary outcome measurements included annualised relapse rate (ARR), proportion of patients relapse free at 12 and 24 months, time to index therapy discontinuation (TTD), and reasons for discontinuation. Exploratory analyses included time to 3- and 6-month EDSS confirmed disability progression (CDP). Non-pairwise censoring was the primary analysis method; pairwise censoring was the main sensitivity analysis method. FINDINGS: Post-matched cohorts were well-balanced. By non-pairwise censoring, TTFR and ARR were significantly lower in DMF populations versus matched IFN, GA, and TERI, but there was no evidence of difference between DMF and FTY. TTD was similar between DMF and IFN, GA, and TERI, but significantly shorter versus FTY. Time to CDP generally showed no evidence of difference between DMF and comparator populations. Pairwise censored analysis results confirmed the non-pairwise censoring results. INTERPRETATION: These results support previous CE studies in demonstrating relative improvement in real-world effectiveness with DMF versus first-line agents IFN, GA, and TERI, and similar effectiveness versus FTY.

Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology.

A challenge for circulating tumor cell (CTC)-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1) their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2) their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers.

Comparison of Safety Margin Generation Concepts in Image Guided Radiotherapy to Account for Daily Head and Neck Pose Variations.

PURPOSE: Intensity modulated radiation therapy (IMRT) of head and neck tumors allows a precise conformation of the high-dose region to clinical target volumes (CTVs) while respecting dose limits to organs a risk (OARs). Accurate patient setup reduces translational and rotational deviations between therapy planning and therapy delivery days. However, uncertainties in the shape of the CTV and OARs due to e.g. small pose variations in the highly deformable anatomy of the head and neck region can still compromise the dose conformation. Routinely applied safety margins around the CTV cause higher dose deposition in adjacent healthy tissue and should be kept as small as possible. MATERIALS AND METHODS: In this work we evaluate and compare three approaches for margin generation 1) a clinically used approach with a constant isotropic 3 mm margin, 2) a previously proposed approach adopting a spatial model of the patient and 3) a newly developed approach adopting a biomechanical model of the patient. All approaches are retrospectively evaluated using a large patient cohort of over 500 fraction control CT images with heterogeneous pose changes. Automatic methods for finding landmark positions in the control CT images are combined with a patient specific biomechanical finite element model to evaluate the CTV deformation. RESULTS: The applied methods for deformation modeling show that the pose changes cause deformations in the target region with a mean motion magnitude of 1.80 mm. We found that the CTV size can be reduced by both variable margin approaches by 15.6% and 13.3% respectively, while maintaining the CTV coverage. With approach 3 an increase of target coverage was obtained. CONCLUSION: Variable margins increase target coverage, reduce risk to OARs and improve healthy tissue sparing at the same time.

Effect of compressive loading and incubation with clodronate on the RANKL/OPG system of human osteoblasts.

OBJECTIVES: In vivo studies have shown that bisphosphonates result in slow rates of orthodontic tooth movement. This study investigated whether clodronate modifies the impact of mechanical loading on the RANKL/OPG system of human osteoblasts. METHODS: Osteoblasts were cultured in vitro with 0.5 or 5.0 µM clodronate for 48 h and/or subjected to 3 h of compressive loading at 34.9 g/cm(2). Cell viability was determined by MTT assay. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemical staining were used to analyze the cells for their production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) at the transcriptional and protein levels. RESULTS: Compressive loading did not affect osteoblast viability in a significant way. Clodronate (5.0 µM) mildly reduced the viability of both compressed and uncompressed cells. Compressive loading induced a 4.2-fold increase in RANKL gene expression, while clodronate led to a concentration-dependent inhibition of this effect (1.8-fold increase at 5.0 µM). OPG gene expression was decreased by compressive loading both in the presence of 0.5 µM clodronate and in the absence of clodronate, and OPG protein synthesis in the compressed cells was significantly decreased in the presence of clodronate. Immunocytochemical staining revealed an increase of RANKL protein synthesis in compressed cells, while clodronate and cell compression reduced this increase. CONCLUSION: This study demonstrates that clodronate decreases the compression-induced RANKL/OPG ratio expressed by human osteoblasts. Reported in vivo findings of reduced osteoclast numbers on the compression side of orthodontic tooth movement under the action of clodronate-and the associated slow rate of tooth movement-might be attributable not only to a direct impact on osteoclasts but also to changes in osteoblast-osteoclast interaction resulting from the presence of clodronate.

Mechanical loading influences the effects of bisphosphonates on human periodontal ligament fibroblasts.

OBJECTIVES: There is increasing evidence that bisphosphonates affect orthodontic tooth movement. The object of the study was to investigate the changes produced by tensile strain on human periodontal ligament fibroblasts (HPdLFs) treated with clodronate or zoledronate. MATERIALS AND METHODS: HPdLF were cultured with 5 and 50 µM clodronate or zoledronate for 48 h and applied to tensile strain (TS) (5 and 10 %) for 12 h in vitro. Viability was verified by MTT assay and apoptosis rate via caspase 3/7 assay. Gene expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) was investigated using real-time PCR. OPG was also analyzed by ELISA and RANKL by immunocytochemical staining. RESULTS: Zoledronate (50 µM) reduced the viability of HPdLF (76 vs 100 %) and combined with 5 % TS to 53 %. TS of 10 % and clodronate reduced viability to 79 % with increased caspase 3/7 activity. Clodronate (5 µM) led to a slight increase of OPG gene expression, zoledronate (5 µM) to a slight decrease. Combined with 5 % TS, both increased OPG gene expression (2-3-fold) and OPG synthesis. Zoledronate increased gene expression of RANKL (4-fold). Combined with 5 % of TS, this increase was abolished. TS of 10 % in combination amplified increase of RANKL ending up with a 9-fold gene expression by clodronate and high RANKL protein synthesis. CONCLUSIONS: This study shows for the first time that mechanical loading alters the effects of bisphosphonates on viability, apoptosis rate, and OPG/RANKL system of HPdLF dependent on the applied strength. Low forces and bisphosphonates increase factors for bone apposition, whereas high forces combined with bisphosphonates stimulate osteoclastogenesis. CLINICAL RELEVANCE: Mechanical loading of periodontal ligament with high strengths should be avoided during bisphosphonate therapy.

Induction of IL-6 and MMP-8 in human periodontal fibroblasts by static tensile strain.

OBJECTIVES: Mechanical loading is a potential activator of inflammation and able to stimulate factors for periodontal and alveolar bone destruction. Aim of this study was to investigate the inflammatory response and synthesis of proteinases by human periodontal ligament fibroblast (HPdLF) dependent on different strengths of static tensile strain (STS). MATERIALS AND METHODS: HPdLFs were loaded with different STS strengths (1, 5, and 10 %) in vitro. Gene expressions of cyclooxygenase (COX)-2 and interleukin (IL)-6 were analyzed by quantitative real-time polymerase chain reaction. Production of IL-6, prostaglandin E2 (PGE2), matrix metalloproteinase (MMP)-8, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were measured by enzyme-linked immunosorbent assay. Receptor activator of nuclear factor-kappa ligand (RANKL) synthesis was detected by immunocytochemical staining. RESULTS: Ten percent STS led to an increased gene expression of IL-6 and COX-2 (34.4-fold) in HPdLF, and 1 and 5 % STS slightly reduced the gene expression of IL-6. Synthesis of IL-6 was significantly reduced by 1 % STS and stimulated by 10 % STS. Ten percent STS significantly induced PGE2 production. RANKL was not detectable at any strength of STS. MMP-8 synthesis showed significantly higher values only at 10 % STS, but TIMP-1 was stimulated by 5 and 10 % STS, resulting into highest TIMP-1/MMP-8 ratio at 5 % STS. CONCLUSIONS: High-strength STS is a potent inducer of periodontal inflammation and MMP-8, whereas low-strength STS shows an anti-inflammatory effect. Moderate-strength STS causes the highest TIMP-1/MMP-8 ratio, leading to appropriate conditions for reformation of the extracellular matrix. CLINICAL RELEVANCE: Furthermore, this study points out that the strength of force plays a pivotal role to achieve orthodontic tooth movement without inducing periodontal inflammation and to activate extracellular matrix regeneration.

Osteogenic differentiation of periodontal fibroblasts is dependent on the strength of mechanical strain.

OBJECTIVE: During orthodontic therapy the correct strength of mechanical strain plays a key role for bone remodelling during tooth movement. Aim of this study was to investigate the osteogenic differentiation of human periodontal ligament fibroblasts (HPdLF) depending on the applied strength of mechanical strain compared to osteoblasts (HOB). DESIGN: HPdLF and HOB were loaded with different strengths (1%, 5% and 10%) of static mechanical strain (SMS) for 12h in vitro. Viability was verified by MTT and apoptosis by TUNEL assay. Gene expression of cyclin D1, collagen type-1 (COL-I), alkaline phosphatase (ALP), osteocalcin, osteoprotegerin (OPG) and receptor activator of the NF-κB ligand (RANKL) were investigated using RT-PCR. OPG and RANKL synthesis was measured by ELISA and ALP activity by colorimetric assay. RESULTS: 10% of SMS led to a decrease in cell viability of both cells lines, but no increased rate of apoptosis. RT-PCR showed the highest increase of cyclin D1 expression for HPdLF and HOB when applied to 5% of SMS, and HOB showed a doubling of COL-I gene expression. HPdLF and HOB showed a strength-dependent synthesis of OPG and ALP activity, whereas HOB demonstrated a decrease in OPG synthesis and ALP activity when applied to 10% of SMS. CONCLUSION: Osteogenic differentiation of HPdLF correlates with increasing strength of SMS. HOB show decreased activity when applied to high SMS, demonstrating potential damage to the bone remodelling due to strain of high strength. SMS up to 5% provides the best conditions for bone formation at the tension site of tooth movement.