Mechanical Overload Regulates Osteoblast Proliferation, Differentiation And Mineralization Through Wnt/Β-Catenin Signaling Pathway

Objectives: To study the effect of mechanical overload stimulation on proliferation, differentiation and mineralization of osteoblast and the underlying mechanisms.Methods: MC3T3-E1 cells were divided into overload group and control group. Four-point bending loading device was used to exert mechanical overload stimulation on MC3T3-E1 cells for a certain time. The proliferation of osteoblasts was detected by MTT colorimetric assay. Real-time PCR and Western Blot were used to detect the transcription and expression of osteoblast marker genes and proteins. The specific fluorescent dyes were used to label the actin filament and the nucleus, and the changes of cytoskeleton were observed under laser scanning confocal microscope. The mineralization of osteoblasts was evaluated by the number of calcium nodules formed by alizarin red staining. Results: Compared with the control group, the mechanical overload group significantly inhibited the proliferation of osteoblasts (p <0.05). Real-time PCR and Western Blot showed that the expression of osteoblast differentiation marker gene and protein was inhibited by mechanical overload stimulation. Under laser confocal microscopy, the overload group cell shrinkage deformation was observed, also the microfilament arrangement disorder, the skeleton arrangement loose, the direction difference and the skeleton breakage, but the nucleus does not have obvious change. Alizarin red staining showed that mechanical overload inhibited the formation of calcium nodules in osteoblasts. The expression of β-catenin protein in Wnt signaling pathway was inhibited by overload mechanical stimulation under immunofluorescence microscopy.Conclusion: Mechanical overload stimulation reduces the expression of Runx 2 by affecting the classical Wnt/β-catenin signaling pathway, thus it was inhibited osteoblast proliferation, differentiation and mineralization.

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.

microRNA-136-5p from bone marrow mesenchymal stem cell-derived exosomes facilitates fracture healing by targeting LRP4 to activate the Wnt/β-catenin pathway

Aims Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing. Methods A mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively. Results miR-136-5p promoted fracture healing and osteoblast proliferation and differentiation. BMSC-derived exosomes exhibited an enriched miR-136-5p level, and were internalized by MC3T3-E1 cells. LRP4 was identified as a downstream target gene of miR-136-5p. Moreover, miR-136-5p or exosomes isolated from BMSCs (BMSC-Exos) containing miR-136-5p activated the Wnt/β-catenin pathway through the inhibition of LRP4 expression. Furthermore, BMSC-derived exosomes carrying miR-136-5p promoted osteoblast proliferation and differentiation, thereby promoting fracture healing. Conclusion BMSC-derived exosomes carrying miR-136-5p inhibited LRP4 and activated the Wnt/β-catenin pathway, thus facilitating fracture healing. Cite this article: Bone Joint Res 2021;10(12):744–758.

Linarin, a Glycosylated Flavonoid, with Potential Therapeutic Attributes: A Comprehensive Review

Many flavonoids, as eminent phenolic compounds, have been commercialized and consumed as dietary supplements due to their incredible human health benefits. In the present study, a bioactive flavone glycoside linarin (LN) was designated to comprehensively overview its phytochemical and biological properties. LN has been characterized abundantly in the Cirsium, Micromeria, and Buddleja species belonging to Asteraceae, Lamiaceae, and Scrophulariaceae families, respectively. Biological assessments exhibited promising activities of LN, particularly, the remedial effects on central nervous system (CNS) disorders, whereas the remarkable sleep enhancing and sedative effects as well as AChE (acetylcholinesterase) inhibitory activity were highlighted. Of note, LN has indicated promising anti osteoblast proliferation and differentiation, thus a bone formation effect. Further biological and pharmacological assessments of LN and its optimized semi-synthetic derivatives, specifically its therapeutic characteristics on osteoarthritis and osteoporosis, might lead to uncovering potential drug candidates.

K-Carrageenan Stimulates Pre-Osteoblast Proliferation and Osteogenic Differentiation: A Potential Factor for the Promotion of Bone Regeneration?

Current cell-based bone tissue regeneration strategies cannot cover large bone defects. K-carrageenan is a highly hydrophilic and biocompatible seaweed-derived sulfated polysaccharide, that has been proposed as a promising candidate for tissue engineering applications. Whether κ-carrageenan can be used to enhance bone regeneration is still unclear. In this study, we aimed to investigate whether κ-carrageenan has osteogenic potential by testing its effect on pre-osteoblast proliferation and osteogenic differentiation in vitro. Treatment with κ-carrageenan (0.5 and 2 mg/mL) increased both MC3T3-E1 pre-osteoblast adhesion and spreading at 1 h. K-carrageenan (0.125–2 mg/mL) dose-dependently increased pre-osteoblast proliferation and metabolic activity, with a maximum effect at 2 mg/mL at day three. K-carrageenan (0.5 and 2 mg/mL) increased osteogenic differentiation, as shown by enhanced alkaline phosphatase activity (1.8-fold increase at 2 mg/mL) at day four, and matrix mineralization (6.2-fold increase at 2 mg/mL) at day 21. K-carrageenan enhanced osteogenic gene expression (Opn, Dmp1, and Mepe) at day 14 and 21. In conclusion, κ-carrageenan promoted MC3T3-E1 pre-osteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation, suggesting that κ-carrageenan is a potential osteogenic inductive factor for clinical application to enhance bone regeneration.

Discovery of Thymosin Beta-4 as a Human Exerkine and Growth Factor

Skeletal muscle is an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in inter-organ crosstalk. Using mass spectrometry (MS)-based proteomics, we characterized the secretome and identified thymosin beta-4 (TMSB4X) as the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in plasma of exercising humans irrespective of the insulin resistance condition or exercise mode. Treatment of mice with TMSB4X did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X increased osteoblast proliferation and neurite outgrowth, consistent with its WADA-classification as a prohibited growth factor. Therefore, we report TMSB4X as a human exerkine with a potential role in cellular crosstalk.

Tanshinone IIA Regulates Osteoblast Differentiation and Promotes Fracture Healing via Mammalian Target of Rapamycin Complex 1 Signaling Pathway

This study assessed the effect and potential molecular mechanism of tanshinone IIA on fracture healing. Mice model with fracture were established. Digital radiographic photographic system was used to detect callus formation after treatment with tanshinone II A (Tan IIA) and alkaline phosphatase (ALP) staining analyzed ALP activity. Osteoblast proliferation was also measured. Western blot and Quantitative real-time PCR (qRT-PCR) measured osteogenic markers expression. Compared with control group, Tan IIA treatment could increase callus formation, stimulate osteoblast proliferation, osteogenic proteins and genes expression, and activate mTORC1 signaling pathway. However, Tan IIA’s effects were significantly inhibited after rapamycin treatment. Tan IIA regulates osteoblast differentiation by mTORC1 signaling and promotes intramembranous ossification in the process of callus formation, which accelerates bone healing.