Exosome Secreted from Mesenchymal Stem Cells (MSCs) of Osteoporosis Inhibits the Osteogenic Differentiation

This study analyze the effect of exosome secreted from MSCs on osteogenic differentiation in OP rats. The exosome was obtained from cultivated MSCs isolated from OP rats with ultracentrifugation. OP rats were treated with exosome secreted from MSCs of normal rats, exosome secreted from MSCs of OP rats and exosome secreted from MSCs of OP rats with overexpression of ALP followed by analysis of the osteogenic differentiation, the expression of ALP, Bglap and Runx2 and the targeted correlation between miR-351 and ALP. The MSCs in normal rats and OP rats were able to adhere to wall. There was elongated. The level of miR-351 in OP rats was significantly higher than normal rats. The Runx2 expression and ALP activity in rats treated with exosome secreted from MSCs of OP rats was declined significantly compared to that from MSCs of normal rats. ALP was a target gene of miR-351. In conclusion, the exosome secreted from MSCs of OP rats inhibits the osteogenic differentiation possibly through restraining miR-351-ALP.

miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

Impacts of Angelica Polysaccharide on Proliferation and Differentiation of Mesenchymal Stem Cells of Rat Bone Marrow

In literature, antiosteoporotic effects of Angelica sinensis root have been confirmed, but the impact of Angelica sinensis polysaccharide (ASP) on osteoblastic or adipogenic distinction of BMSCs is limited. This paper aimed to explore the role of ASP on proliferation and differentiation of rat BMSCs. Rat BMSCs were subjected to isolation and identification through flow cytometry. The proliferation of rat BMSCs under ASP was performed by CCK-8 kit. Measures of osteogenesis under different concentrations of ASP were detected by using alizarin red staining for mesenchymal cells differentiation and ALP activity assay to identify ALP activity. Quantitative RT-PCR was selected to identify osteoblastic or adipogenic biomarkers from a genetic perspective. Likewise, we have evaluated measures of indicators of Wnt/β-catenin signal. ASP significantly promoted the proliferation, increased osteogenesis, and decreased adipogenesis of rat BMSCs within the limit of 20–60 mg/L in a dose-dependent manner but was suppressed at 80 mg/L. The expression of cyclin D1 and ß-catenin showed a considerable rise over the course of ASP induced osteogenesis. Dickkopf 1 (DKK1) suppressed the regulation of rat BMSCs differentiation through the mediation of ASP. We have observed that ASP upregulated the osteogenic but downregulated adipogenic differentiation of BMSCs, and our findings help to contribute to effective solutions for treating bone disorders.

LncRNA-KCNQ1OT1 Promotes the Odontoblastic Differentiation of Dental Pulp Stem Cells via Regulating miR-153-3p/RUNX2 Axis

Background and Objective: Long non-coding RNAs (LncRNAs) play a key role in the odontoblastic differentiation. This study aimed to explore the role of LncRNA-KCNQ1OT1 in the odontoblastic differentiation of human dental pulp stem cells (DPSCs) and its possible mechanism. Methods: The expression of LncRNA-KCNQ1OT1, miR-153-3p, RUNX2 in the odontoblastic differentiation was detected by qRT-PCR. Interaction between LncRNA-KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and RUNX2 were detected by dual-luciferase assay. The cell viability of DPSCs was detected by cell counting kit-8 (CCK-8), and the effect of LncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs was observed by alizarin red staining, alkaline phosphatase (ALP) activity assay and Western blot for RUNX2, DSPP, DMP-1. Results: During odontoblastic differentiation of DPSCs, the expression of LncRNA-KCNQ1OT1 increased, miR-153-3p expression decreased, and RUNX2 expression increased. Dual-luciferase assay showed that LncRNA-KCNQ1OT1 sponges miR-153-3p and miR-153-3p targets on RUNX2. After LncRNA-KCNQ1OT1 and miR-153-3p expressions of DPSCs were changed, the cell viability was not notably changed, but the odontoblastic differentiation was notably changed which was confirmed with alizarin red staining, ALP activity and Western blot for RUNX2, DSPP, DMP-1. Conclusion: LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provide a therapeutic clue for odontogenesis.

Melatonin-Medicated Neural JNK3 Up-Regulation Promotes Ameloblastic Mineralization

Introduction: Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied.Objective: To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis.Method: ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and β-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression.Results: In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.

Gingival crevicular fluid alkaline phosphatase activity as growth marker: A systematic review

Background: This review synthesizes the available evidence about the individual skeletal maturity with biological maturity indicators and compares it with the levels of gingival crevicular fluid (GCF) alkaline phosphatase (ALP) activity in growing children. Aims: This systematic review aimed to clarify the question: Is GCF ALP a reliable biomarker to assess skeletal maturity during growth? Objectives: The objective of this systematic review is to collect, compile, and review the existing evidence on the levels of GCF ALP activity in growing children and comparing its reliability with contemporary growth indicators. Materials and Methods: A literature appraisal executed using Entres PubMed, www.ncbi.nim.nih.gov, Scupose, Hinary, Ebsco, Embass, Cochrane; Google Scholar Electronic database search engines were used. The MeSH term used “growth markers in gingival crevicular fluid” or “growth markers in growing children.” Studies published till October, 2020, were included in this study. Data Extraction and Quality Assessment: The data have extracted from the selected articles based on year of publication, study design, age of subjects, instruments used, and author’s conclusions. The quality assessment was executed using BIOCROSS Scale. This scale is exclusive for cross-sectional studies with biomarkers. Results: Literature search identified 731 records from electronic databases and from the partial grey literature (Google scholar) search. Finally, six articles fulfilled eligibility criteria included in the review. Conclusions: All the six studies concluded that GCF ALP activity is a reliable method in determining a skeletal maturity indicator in growing children.

Nutraceuticals Synergistically Promote Osteogenesis in Cultured 7F2 Osteoblasts and Mitigate Inhibition of Differentiation and Maturation in Simulated Microgravity

Microgravity is known to impact bone health, similar to mechanical unloading on Earth. In the absence of countermeasures, bone formation and mineral deposition are strongly inhibited in Space. There is an unmet need to identify nutritional countermeasures. Curcumin and carnosic acid are phytonutrients with anticancer, anti-inflammatory, and antioxidative effects and may exhibit osteogenic properties. Zinc is a trace element essential for bone formation. We hypothesized that these nutraceuticals could counteract the microgravity-induced inhibition of osteogenic differentiation and function. To test this hypothesis, we cultured 7F2 murine osteoblasts in simulated microgravity (SMG) in a Random Positioning Machine in the presence and absence of curcumin, carnosic acid, and zinc and evaluated cell proliferation, function, and differentiation. SMG enhanced cell proliferation in osteogenic medium. The nutraceuticals partially reversed the inhibitory effects of SMG on alkaline phosphatase (ALP) activity and did not alter the SMG-induced reduction in the expression of osteogenic marker genes in osteogenic medium, while they promoted osteoblast proliferation and ALP activity in the absence of traditional osteogenic media. We further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.

Isolation and identification of peptides from Pinctada martensii with osteogenic activity

Marine organisms have attracted considerable attention in recent years. In this study, peptides with osteogenic activity from Pinctada martensii were isolated and identified. Additionally, the effects of the hydrolysates on MC3T3-E1 cell proliferation and differentiation were evaluated using the MTT and alkaline phosphatase (ALP) assays, respectively. First, trypsin, pancreatin, and neutral protease were used to hydrolyse the intact shellfish. The hydrolysates with the greatest effects on osteoblast proliferation and ALP activity were separated and purified. Second, fraction WP2 was isolated and purified using a Sephadex G-25 column. WP2, which had the highest osteogenic activity, increased cell growth by 48.57 ± 0.05% and ALP activity by 6.27 ± 0.07 mU. Finally, four novel peptides were identified in WP2 (FDNEGKGKLPEEY, IVLDSGDGVTH, IVLDSGDGVSH, and SSENSDLQRQ) by Orbitrap Fusion Lumos Tribrid orbital liquid chromatography-mass spectrometry. Our findings revealed that P. martensii contains peptides with potential osteogenic activity.

Icariin and Icariside II Reciprocally Stimulate Osteogenesis and Inhibit Adipogenesis of Multipotential Stromal Cells through ERK Signaling

Herba Epimedii is a famous Chinese herbal medicine for treating bone diseases. Icariin and icariside II, the main chemical constituents, have attracted great attention from scientists for their potential as antiosteoporosis agents. Our study aimed to evaluate their effects on the lineage commitment of multipotential stromal cells (MSCs). The osteogenesis and adipogenesis of MSCs were assessed by ALP activity, calcium deposition, and adipocyte formation. The expression profiles and levels of osteogenic and adipogenic specific genes were evaluated by cDNA microarray and quantitative real-time PCR. The involvement of extracellular signal-regulated kinase (ERK) signaling was studied by enzyme-linked immunosorbent assay. Icariin and icariside II significantly increased ALP activity and mineralization during osteogenic differentiation of MSCs. Runx2, Col1, and Bmp2 were upregulated in the presence of icariin and icariside II. Meanwhile, they downregulated Pparg, Adipsin, and Cebpb expression during adipogenic differentiation. cDNA microarray revealed 57 differentially expressed genes during lineage commitment of MSCs. In addition, icariin and icariside II enhanced the phosphorylation of ERK, and the above biological effects were blocked by ERK inhibitor U0126. Icariin and icariside II may drive the final lineage commitment of MSCs towards osteogenesis and inhibit adipogenesis through the ERK signaling pathway. Both of them exert multiple osteoprotective effects and deserve more attention for their medicinal and healthcare prospects.

Effects of Various Mineral Trioxide Aggregates on Viability and Mineralization Potential of 3-Dimensional Cultured Dental Pulp Stem Cells

Three-dimensionally (3D) cultured dental pulp stem cells (DPSCs) reportedly exhibit superior multi-lineage differentiation capacities and have a higher expression in regeneration-related gene categories compared to conventionally cultured DPSCs. This study aimed to evaluate the effects of various mineral trioxide aggregates (MTAs) on DPSCs cultured in 3D, assessing their cell viability and tissue mineralization properties. We examined the morphology, cell viability, alkaline phosphate (ALP) activity and qualitative alizarin red S staining assay of the DPSCs that reacted with various MTAs, which included ProRoot (PRM), Biodentine (BIO), and Well-Root PT (WRP), in two different culture plates, an ultra-low attachment plate (ULA) and a conventional monolayer plate (2D). As a control, MTA-free and IRM samples were prepared. None of the MTA groups affected the microsphere-forming characteristics of DPSCs that had been cultured in ULA. The DPSCs that were cultured in ULA showed high cell viability in all MTA groups compared to IRM. The mineralization potential was favorable in all MTA groups, with a significantly higher ALP activity among the DPSCs that were cultured in ULA. Among MTAs, the PRM group showed substantially higher ALP activity than the other MTA groups. In conclusion, our results indicate that 3D-cultured DPSCs with various MTAs showed comparable viability and mineralization capacity similar to those cultured without reacting with MTA cement.