Effects of Selective Alveolar Decortication on the Periodontal Complex

Background Modification of bone turnover has been reported following selective alveolar decortication but the molecular signals in the periodontal ligament space (PDL) remain unanswered. The objective of this study was to understand how selective alveolar decortication affects the biological reactions in the periodontal ligament. Methods Selective alveolar decortication in wild-type mice (n=25) was performed on mandibular right buccal cortical plate adjacent to the mandibular right third molar and euthanized at 3, 7, 14 and 28 days. We also performed selective alveolar decortication in Lrp5ACT (n=5) mice and Ad-Dkk1 treated mice (n=5), and euthanized at 7 days. The periodontium around the mandibular third molars were examined using histology, immunohistochemical analyses for osteogenic markers, TGF-β, RANKL, TRAP and alkaline phosphatase activity. Results The expression of osteogenic markers in the wild-type PDL was maintained during healing time period after selective alveolar decortication. Increased osteoclast activity in the wild-type mice was observed at 3 and 7 days after selective alveolar decortication. The PDL in Lrp5G171V (Lrp5ACT) mice and adenovirus Dkk1 (Ad-Dkk1) treated mice also showed insignificant changes in the expression of osteogenic markers following selective alveolar decortication. In Lrp5ACT mice where there was a reduction of bone resorption, selective alveolar decortication caused a dramatic increase in osteoclast activity. Conclusions Selective alveolar decortication affects only bone turnover, but not the expression of osteogenic markers in the PDL.

Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

Nanovibrational stimulation inhibits osteoclastogenesis and enhances osteogenesis in co-cultures

Models of bone remodelling could be useful in drug discovery, particularly if the model is one that replicates bone regeneration with reduction in osteoclast activity. Here we use nanovibrational stimulation to achieve this in a 3D co-culture of primary human osteoprogenitor and osteoclast progenitor cells. We show that 1000 Hz frequency, 40 nm amplitude vibration reduces osteoclast formation and activity in human mononuclear CD14+ blood cells. Additionally, this nanoscale vibration both enhances osteogenesis and reduces osteoclastogenesis in a co-culture of primary human bone marrow stromal cells and bone marrow hematopoietic cells. Further, we use metabolomics to identify Akt (protein kinase C) as a potential mediator. Akt is known to be involved in bone differentiation via transforming growth factor beta 1 (TGFβ1) and bone morphogenetic protein 2 (BMP2) and it has been implicated in reduced osteoclast activity via Guanine nucleotide-binding protein subunit α13 (Gα13). With further validation, our nanovibrational bioreactor could be used to help provide humanised 3D models for drug screening.

Porous 3D Scaffolds Enhance MSC Vitality and Reduce Osteoclast Activity

In the context of an aging population, unhealthy Western lifestyle, and the lack of an optimal surgical treatment, deep osteochondral defects pose a great challenge for the public health system. Biodegradable, biomimetic scaffolds seem to be a promising solution. In this study we investigated the biocompatibility of porous poly-((D,L)-lactide-ε-caprolactone)dimethacrylate (LCM) scaffolds in contrast to compact LCM scaffolds and blank cell culture plastic. Thus, morphology, cytotoxicity and metabolic activity of human mesenchymal stromal cells (MSC) seeded directly on the materials were analyzed after three and six days of culturing. Further, osteoclastogenesis and osteoclastic activity were assessed using reverse-transcriptase real-time PCR of osteoclast-specific genes, EIA and morphologic aspects after four, eight, and twelve days. LCM scaffolds did not display cytotoxic effects on MSC. After three days, metabolic activity of MSC was enhanced on 3D porous scaffolds (PS) compared to 2D compact scaffolds (CS). Osteoclast activity seemed to be reduced at PS compared to cell culture plastic at all time points, while no differences in osteoclastogenesis were detectable between the materials. These results indicate a good cytocompatibility of LCM scaffolds. Interestingly, porous 3D structure induced higher metabolic activity of MSC as well as reduced osteoclast activity.

Potential mechanism of Ziyin Tongluo Formula in the treatment of postmenopausal osteoporosis: based on network pharmacology and ovariectomized rat model

Background Amending from ancient classic, Ziyin Tongluo Formula (ZYTLF) has been prescribed to treat postmenopausal osteoporosis (PMOP) for decades with good curative effect. However, the possible mechanisms of it are still unknown. Methods Ovariectomized rat model was established to validate the therapeutic effect of ZYTLF on PMOP by Micro-CT bone analysis and pathological observation. Subsequently, active ingredients of ZYTLF and corresponding putative targets were identified by online databases. Overlapping genes were first obtained from mining genes associated with PMOP and then overlapped them with the putative targets. Key genes were selected from the multiple constructed and analyzed networks. GO and KEGG pathway enrichment analysis were performed by importing the key genes to the DAVID database. Moreover, validation of the binding association between key targets and their corresponding active compounds were accomplished by AutoDock Tools and other software. Lastly, Enzyme linked immunosorbent assay (Elisa) detection and Western blot analysis were utilized to further explore the possible mechanism of ZYTLF on PMOP. Results With 129 target genes interacting with PMOP, 92 active compounds of ZYTLF corresponded to 243 targets, and 50 key genes were chosen. Network analysis revealed the top 10 active ingredients, such as quercetin and kaempferol and the top 50 key genes, such as ERα, p38 MAPK, p-AKT and TGF-β1. Enrichment analysis uncovered multiple signaling pathways, including estrogen signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. Furthermore, our finding of the foremost active compounds was tightly bound to the core proteins, which were verified by molecular docking analysis. Through experimental studies, we confirmed that the prescription of ZYTLF could ameliorate the OVX-induced bone loss, suppress the osteoclast activity and boost osteoblast ability through experimental studies. Conclusion The potential mechanisms and therapeutic effects of ZYTLF against PMOP may be ascribed to inhibition of osteoclast activity, boost of osteoblast activity and enhancement of the expression of ERα.

Potential Mechanism of Ziyin Tongluo Formula in the Treatment of Postmenopausal Osteoporosis：based on Network Pharmacology and Ovariectomized Rat Model

BackgroundAmending from ancient classic, Ziyin Tongluo Formula (ZYTLF) has been prescribed to treat Postmenopausal osteoporosis (PMOP) for decades and obtained beneficial effect. However, the possible mechanisms of it are still unknown. MethodsOvariectomized rat model were established to validate the therapeutic effect of ZYTLF on PMOP by Micro-CT bone analysis and pathological observation. Subsequently, active ingredients of ZYTLF and corresponding putative targets were identified by online databases. Overlapping genes were obtained by mining genes associated with PMOP and then overlapping them with the putative targets. Key genes were selected from the multiple constructed and analyzed networks. GO and KEGG pathway enrichment analysis were performed by importing the key genes to the DAVID database. Moreover, validation of the binding association between key targets and their corresponding active compounds were accomplished by AutoDock Tools and other softwares. Lastly, Enzyme linked immunosorbent assay (Elisa) detection and Western blot analysis were utilized to further explore the possible mechanism of ZYTLF on PMOP. ResultsWith 129 target genes interacting with PMOP, 92 active compounds of ZYTLF corresponded to 243 targets, and 50 key genes were chosen. Network analysis revealed the top 10 active ingredients, such as quercetin, kaempferol and the top 50 key genes, such as ERα, p38 MAPK , p-AKT, TGF-β1. Enrichment analysis uncovered multiple signaling pathways, including estrogen signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway. Furthermore, our finding of the foremost active compounds were tightly bound to the core proteins was verified by molecular docking analysis. We confirmed that the prescription of ZYTLF could ameliorate the OVX-induced bone loss, suppress the osteoclast activity and boost osteoblastg ability through experimental studies. ConclusionThe potential mechanisms and therapeutic effects of ZYTLF against PMOP may be ascribed to inhabit osteoclast activity, boost osteoblast activity and enhance the expression of ERα .