scholarly journals IGF-2 Modified by the m6A Demethylation Enzyme ALKBH5 in the Ossification of the Ligamentum Flavum

2020 ◽  
Author(s):  
Ming-jie Kuang ◽  
Hai-Feng Wang ◽  
Jie Qiu ◽  
An-bang Wang ◽  
Feng Wang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Site Selection ◽  
Methylation Level ◽  
Ligamentum Flavum ◽  
Control Group ◽  
Biological Functions ◽  
Methylation Site ◽  
Methylation Analysis ◽  
Alizarin Red ◽  
Distinct Features

Abstract Background: Ossification of the ligamentum flavum (OLF) is a pathological heterotopic ossification of the paravertebral ligament. However, the specific pathophysiology mechanism of this disease is still unknown. The m6A methylation and its potential functions in OLF remain to be unexplored. Method: In this study, we performed a transcriptome-wide methylation analysis using the OLF and normal ligaments to explore the mechanism of OLF. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. We screened out the methylase-ALKBH5, and by promoting or inhibiting its expression, observing the content of m6A, and measuring the ossification of ligaments, and then measuring IGF expression situation by alizarin red staining, alkaline phosphatase, immunofluorescence, immunohistochemistry, etc. Result: MeRIP-seq and qPCR showed that the m6A methylation level of OLF group was usually higher than that of control group. In addition, we found that ALKBH5 is an important demethyltransferase in OLF, which promotes the expression of m6A. ALKBH5 promotes the expression of IGF-2, which in turn promotes osteogenesis in OLF.Conclusion: Overall, we provided a region-specific map of m6A methylation and characterized the distinct features of specific and common methylation in OLF, and we proved that IGF-2 can be regulated by ALKBH5 to promote the process of OLF.

scholarly journals IGF-2 modified by the m6A demethylation enzyme ALKBH5 in the ossification of the ligamentum flavum

2021 ◽  
Author(s):  
Ming-jie Kuang ◽  
Hai-feng Wang ◽  
Jie Qiu ◽  
An-bang Wang ◽  
Feng Wang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Site Selection ◽  
Methylation Level ◽  
Ligamentum Flavum ◽  
Control Group ◽  
Biological Functions ◽  
Methylation Site ◽  
Methylation Analysis ◽  
Alizarin Red ◽  
Distinct Features

Abstract Abstract Background: Ossification of the ligamentum flavum (OLF) is a pathological heterotopic ossification of the paravertebral ligament. However, the specific pathophysiology mechanism of this disease is still unknown. The m 6 A methylation and its potential functions in OLF remain to be unexplored. Method: In this study, we performed a transcriptome-wide methylation analysis using the OLF and normal ligaments to explore the mechanism of OLF. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. We screened out the methylase-ALKBH5, and by promoting or inhibiting its expression, observing the content of m 6 A, and measuring the ossification of ligaments, and then measuring IGF expression situation by alizarin red staining, alkaline phosphatase, immunofluorescence, immunohistochemistry, etc. Result: MeRIP-seq and qPCR showed that the m6A methylation level of OLF group was usually higher than that of control group. In addition, we found that ALKBH5 is an important demethyltransferase in OLF, which promotes the expression of m 6 A. ALKBH5 promotes the expression of IGF-2, which in turn promotes osteogenesis in OLF. Conclusion: Overall, we provided a region-specific map of m 6 A methylation and characterized the distinct features of specific and common methylation in OLF, and we proved that IGF-2 can be regulated by ALKBH5 to promote the process of OLF.

scholarly journals Evaluation of the Effects of Cuminum cyminum on Cellular Viability, Osteogenic Differentiation and Mineralization of Human Bone Marrow-Derived Stem Cells

Medicina ◽  
2021 ◽  
Vol 57 (1) ◽  
pp. 38
Author(s):  
Hyunjin Lee ◽  
Youngmin Song ◽  
Yoon-Hee Park ◽  
Md. Salah Uddin ◽  
Jun-Beom Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Alizarin Red S ◽  
Control Group ◽  
Cellular Viability ◽  
Alizarin Red

Background and Objectives: Cuminum cyminum L. has long been used in the treatment of various diseases in multiple geographical regions. This study was performed to determine the effects of C. cyminum methanolic extract (CCT) on the cellular viability, alkaline phosphatase activity and mineralization of human mesenchymal stem cells. Materials and Methods: Bone marrow-derived stem cells were cultured in the presence of CCT at concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/mL. Evaluations of cell morphology were performed on days 1, 3, 7 and 14. Cellular viability was evaluated on days 1, 3, 5 and 7. On the 7th and 14th day, alkaline phosphatase activity measurements and Alizarin red S staining were conducted to assess the osteogenic differentiation of stem cells. A real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, COL2A1 and β-catenin mRNAs. Results: Stem cells in the control group showed fibroblast-like morphology and the addition of CCT at 0.001, 0.01, 0.1 and 1 μg/mL did not generate noticeable changes in morphology compared with the untreated control group. The application of CCT did not produce significant changes in cellular viability or alkaline phosphatase activity compared with controls. Alizarin Red S staining was significantly increased with the application of CCT. Treatment with CCT increased the expressions of RUNX2, BSP and OCN. Conclusions: These results indicate that CCT enhanced the osteogenic differentiation of stem cells derived from bone marrow by regulating the expressions of RUNX2, BSP and OCN. Thus, the use of CCT may be applied to achieve beneficial effects on the mineralization of stem cells.

scholarly journals Comparison of osteopromoting ability of human tooth powder with the demineralized freeze-dried bone allograft, a bovine xenograft, and a synthetic graft: An in vitro study

2020 ◽  
Vol 12 (1) ◽  
pp. 19-23
Author(s):  
Mahdi Kadkhodazadeh ◽  
Alireza Fathiazar ◽  
Zahra Yadegari ◽  
Reza Amid
Keyword(s):  
Cell Proliferation ◽  
Alkaline Phosphatase ◽  
Alkaline Phosphatase Level ◽  
In Vitro Study ◽  
Human Tooth ◽  
Control Group ◽  
Synthetic Graft ◽  
Alizarin Red ◽  
Bovine Xenograft

Background. The present study aimed to evaluate the osteopromoting ability of human tooth powder and compare it to a bovine xenograft, a synthetic material, and the DFDBA allograft. Methods. In this in vitro study, 30 teeth without caries, inflammation, and infection, which had been extracted for orthodontic reasons, were collected. The crowns were removed, pulpectomy was carried out, and the samples were ground to a powder with particles <500 µm. Osteoblast-like cells of MG-63 were cultured with the tooth powder, Cerabone, DFDBA, and Osteon II. Cell proliferation was assessed by the MTT assay at 24- and 72-hour intervals. The alizarin red test was carried out after three and five days. The alkaline phosphatase level was measured after 24, 48, and 72 hours to assess the osteoblastic activity. The results were analyzed with one-way ANOVA. Results. According to the MTT assay, all the materials exhibited a higher proliferation rate than the control group in 24 hours. In 72 hours, DFDBA had the lowest cell proliferation rate at concentrations of 40 and 80 mg/mL. DFDBA and the positive control group were able to create calcified nodules by the alizarin red test. At the 48- and 72-hour intervals, DFDBA had the lowest alkaline phosphatase activity at a concentration of 40 mg/mL. At the 72-hour interval, bovine xenograft had the highest alkaline phosphatase level, followed by the synthetic material and tooth powder. Conclusion. The tooth powder was able to increase cell proliferation in comparison with the bovine xenograft, the synthetic graft, and the DFDBA. However, its osteopromoting ability was less than that of the osteogenic materials.

Polyhedral Oligomeric Silsesquioxane /Platelets Rich Plasma/Gelrite-Based Hydrogel Scaffold for Bone Tissue Engineering

2020 ◽  
Vol 26 (26) ◽  
pp. 3147-3160
Author(s):  
Saeedeh Ahmadipour ◽  
Jaleh Varshosaz ◽  
Batool Hashemibeni ◽  
Leila Safaeian ◽  
Maziar Manshaei
Keyword(s):  
Bone Fractures ◽  
Compressive Strain ◽  
Gelation Time ◽  
Polyhedral Oligomeric Silsesquioxane ◽  
Local Drug Delivery ◽  
Gelling Agent ◽  
Control Group ◽  
Hydrogel Scaffold ◽  
Alizarin Red ◽  
Oligomeric Silsesquioxane

Background: Polyhedral oligomeric silsesquioxane (POSS) is a monomer with silicon structure and an internal nanometric cage. Objective: The purpose of this study was to provide an injectable hydrogel that could be easily located in open or closed bone fractures and injuries, and also to reduce the possible risks of infections caused by bone graft either as an allograft or an autograft. Methods: Various formulations of temperature sensitive hydrogels containing hydroxyapatite, Gelrite, POSS and platelets rich plasma (PRP), such as the co-gelling agent and cell growth enhancer, were prepared. The hydrogels were characterized for their injectability, gelation time, phase transition temperature and viscosity. Other physical properties of the optimized formulation including compressive stress, compressive strain and Young’s modulus as mechanical properties, as well as storage and loss modulus, swelling ratio, biodegradation behavior and cell toxicity as rheometrical parameters were studied on human osteoblast MG-63 cells. Alizarin red tests were conducted to study the qualitative and quantitative osteogenic capability of the designed scaffold, and the cell adhesion to the scaffold was visualized by scanning electron microscopy. Results: The results demonstrated that the hydrogel scaffold mechanical force and injectability were 3.34±0.44 Mpa and 12.57 N, respectively. Moreover, the scaffold showed higher calcium granules production in alizarin red staining compared to the control group. The proliferation of the cells in G4.5H1P0.03PRP10 formulation was significantly higher than in other formulations (p<0.05). Conclusion: The optimized Gelrite/Hydroxyapatite/POSS/PRP hydrogel scaffold has useful impacts on osteoblasts activity, and may be beneficial for local drug delivery in complications including a break or bone loss.

scholarly journals Replacing Maize Grain with Ancient Wheat Lines By-Products in Organic Laying Hens’ Diet Affects Intestinal Morphology and Enzymatic Activity

2021 ◽  
Vol 13 (12) ◽  
pp. 6554
Author(s):  
Nicola Francesco Addeo ◽  
Basilio Randazzo ◽  
Ike Olivotto ◽  
Maria Messina ◽  
Francesca Tulli ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Enzymatic Activity ◽  
Laying Hens ◽  
Triticum Aestivum L ◽  
Treated Group ◽  
Intestinal Morphology ◽  
Control Group ◽  
Positive Effects ◽  
Maize Grain ◽  
By Products

The effects of replacement of maize grain with ancient wheat by-products on intestinal morphometry and enzymatic activity in laying hens was studied. Eighty hens were divided into two groups (40 each, 8 replicates, 5 hens/replicate) fed two isoproteic and isoenergetic diets. In the treated group, part of the maize was replaced by a mix of ancient grains (AGs) middling, in a 50:50 ratio of Triticum aestivum L. var. spelta (spelt) and Triticum durum dicoccum L. (emmer wheat). The AG diet affected the weight of all the large intestine tracts, decreasing the weight of caeca (p < 0.01) and increasing those of colon (p < 0.01), rectum and cloaca (p < 0.05). Villus height in the AG group was higher (p < 0.01) than the control for the duodenum and jejunum, while for the ileum, the control group showed the highest values (p < 0.01). The submucosa thickness was higher (p < 0.01) in the control group for the duodenum and ileum, while the jejunum for the AG group showed the highest (p < 0.05) submucosa thickness. The crypts depth was higher (p < 0.01) in the control group for the duodenum and ileum. Enzyme activity was enhanced by AGs (p < 0.01) in the duodenum. Regarding the jejunum, sucrase-isomaltase and alkaline phosphatase had higher activity (p < 0.05 and p < 0.01, respectively) in the AG group. In the ileum, sucrase-isomaltase showed higher activity (p < 0.01) in the control group, while alkaline phosphatase showed the highest values (p < 0.05) in the AG group. Overall, results suggested that the dietary inclusion of AGs exerted positive effects in hens, showing an improved intestinal function.

scholarly journals Biological characterization of implant surfaces - in vitro study

2015 ◽  
Vol 44 (4) ◽  
pp. 195-199 ◽  
Author(s):  
Priscilla Barbosa Ferreira Soares ◽  
Camilla Christian Gomes Moura ◽  
Huberth Alexandre da Rocha Júnior ◽  
Paula Dechichi ◽  
Darceny Zanetta-Barbosa
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Viability ◽  
Total Protein ◽  
Cell Attachment ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Serum Total Protein ◽  
Trypan Blue Exclusion ◽  
Experimental Surface

<title>Abstract</title><sec><title>Objective</title><p>Evaluate the biological performance of titanium alloys grade IV under different surface treatments: sandblasting and double etching (Experimental surface 1; Exp1, NEODENT); surface with wettability increase (Experimental surface 2; Exp2, NEODENT) on response of preliminary differentiation and cell maturation.</p></sec><sec><title>Material and method</title><p>Immortalized osteoblast cells were plated on Exp1 and Exp2 titanium discs. The polystyrene plate surface without disc was used as control group (C). Cell viability was assessed by measuring mitochondrial activity (MTT) at 4 and 24 h (n = 5), cell attachment was performed using trypan blue exclusion within 4 hours (n = 5), serum total protein and alkaline phosphatase normalization was performed at 4, 7 and 14 days (n = 5). Data were analyzed using one-way ANOVA and Tukey test.</p></sec><sec><title>Result</title><p>The values of cell viability were: 4h: C– 0.32±0.01<sup>A</sup>; Exp1– 0.34±0.08<sup>A</sup>; Exp2– 0.29±0.03<sup>A</sup>. 24h: C– 0.43±0.02<sup>A</sup>; Exp1– 0.39±0.01<sup>A</sup>; Exp2– 0.37±0.03<sup>A</sup>. The cell adhesion counting was: C– 85±10<sup>A</sup>; Exp1- 35±5<sup>B</sup>; Exp2– 20±2<sup>B</sup>. The amounts of serum total protein were 4d: C– 40±2<sup>B</sup>; Exp1– 120±10<sup>A</sup>; Exp2– 130±20<sup>A</sup>. 7d: C– 38±2<sup>B</sup>; Exp1– 75±4<sup>A</sup>; Exp2– 70±6<sup>A</sup>. 14 d: C– 100±3<sup>A</sup>; Exp1– 130±5<sup>A</sup>; Exp2– 137±9<sup>A</sup>. The values of alkaline phosphatase normalization were: 4d: C– 2.0±0.1<sup>C</sup>; Exp1– 5.1±0.8<sup>B</sup>; Exp2– 9.8±2.0<sup>A</sup>. 7d: C– 1.0±0.01<sup>C</sup>; Exp1– 5.3±0.5<sup>A</sup>; Exp2– 3.0±0.3<sup>B</sup>. 14 d: C– 4.1±0.3<sup>A</sup>; Exp1– 4.4±0.8<sup>A</sup>; Exp2– 2.2±0.2<sup>B</sup>. Different letters related to statistical differences.</p></sec><sec><title>Conclusion</title><p>The surfaces tested exhibit different behavior at dosage of alkaline phosphatase normalization showing that the Exp2 is more associated with induction of cell differentiation process and that Exp1 is more related to the mineralization process.</p></sec>

ALKALINE PHOSPHATASE AS A PROGNOSTIC MARKER IN CANCER BREAST PATIENTS IN A TERTIARY CARE HOSPITAL

2021 ◽  
pp. 14-16
Author(s):  
Asha Premlata Omega Oraon ◽  
Bela Rose Ekka
Keyword(s):  
Alkaline Phosphatase ◽  
Cancer Patients ◽  
Prognostic Marker ◽  
Prognostic Markers ◽  
Tertiary Care Hospital ◽  
Serum Alkaline Phosphatase ◽  
Tertiary Care ◽  
Control Group ◽  
Care Hospital ◽  
Cancer Breast

OBJECTIVE: To estimate the value of Alkaline Phosphatase in cancer breast patients in a tertiary care hospital. MATERIAL AND METHODS: The study was conducted to estimate the value of Serum Alkaline Phosphatase in 50 cancer breast patients and 50 normal patients of same age as a control group. RESULTS: The level of serum Alkaline Phosphatase was signicantly increased (p<0.05)in cancer patients compared to the control group. CONCLUSION: There is an increase in serum Alkaline Phosphatase in cancer patients compared to the control group and can be a prognostic markers for the progress of the disease.

The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

2021 ◽  
pp. 1-5
Author(s):  
Kari Hanson ◽  
Carly Isder ◽  
Kristen Shogren ◽  
Anthony L. Mikula ◽  
Lichun Lu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
High Dose ◽  
Inhibitory Effects ◽  
Alizarin Red ◽  
Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

scholarly journals Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-α

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Tingting Meng ◽  
Ying Zhou ◽  
Jingkun Li ◽  
Meilin Hu ◽  
Xiaomeng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Alkaline Phosphatase Activity ◽  
Periodontal Ligament ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Induced Apoptosis

Background and Objective. This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-α in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-α had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-α and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-κB signaling pathway proteins p65, phosphorylated p65, IκB-α, phosphorylated IκB-α, and β-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis. Results. TNF-α stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-α. Western blot analysis showed that β-catenin, phosphorated p65, and phosphorylated IκB-α protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10 μg/ml, 20 μg/ml) prevented TNF-α-induced apoptosis by decreasing caspase-8 and caspase-3 expression. Conclusions. Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-κB signaling pathways and by suppressing TNF-α-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

scholarly journals Bone metabolism biomarkers in walking children with cerebral palsy

2020 ◽  
Vol 7 (4) ◽  
pp. 79-86
Author(s):  
Vladimir M. Kenis ◽  
Svetlana L. Bogdanova ◽  
Tatyana N. Prokopenko ◽  
Andrei V. Sapogovskiy ◽  
Tatyana I. Kiseleva
Keyword(s):  
Vitamin D ◽  
Alkaline Phosphatase ◽  
Cerebral Palsy ◽  
Bone Metabolism ◽  
Control Group ◽  
Study Group ◽  
Flat Feet ◽  
Children With Cerebral Palsy ◽  
Calcium Phosphorus ◽  
Orthopedic Patients

Backgrоund. Osteoporosis is an important factor in the pathogenesis of orthopedic manifestations in children with cerebral palsy. It was previously demonstrated that children with cerebral palsy have specific changes in bone metabolism, which can cause changes in laboratory parameters compared with other orthopedic patients without neurological backgrounds. Aim. The aim of this study was to assess bone metabolism biomarkers in children with cerebral palsy, identifying distinguishing characteristic patterns in comparison with patients with orthopedic pathology without neurological backgrounds. Materials and methods. This study evaluated the concentrations of calcium, phosphorus, -cross laps, osteocalcin, vitamin D, CICP, and alkaline phosphatase in the blood serum of 50 children with cerebral palsy aged between 6 to 12 years with GMFCS levels IIII. The control group consisted of 50 patients with plano-valgus deformities of the feet. Results. The alkaline phosphatase activity in the group of children with cerebral palsy was 170.25 59.35 u/L, while in the control group it was 145.58 46.29 u/L; the CICP concentration in the study group was higher than in the control group (324.01 174.10 and 269.68 240.98, respectively). The concentration of -cross laps, osteocalcin, calcium, and vitamin D in the study group was lower than in children with flat feet. Conclusions. This study demonstrated multidirectional changes in the biomarkers of bone metabolism that are characteristic of walking children with cerebral palsy. These changes are characterized by a corresponding increase in the activity of osteoresorption and osteoreparation. This makes it possible to justify the combined use of metabolites and metabolic activators (calcium and vitamin D) and drugs that suppress osteoresorption (bisphosphonates) for the prevention and treatment of osteoporosis in children with cerebral palsy.

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