Proliferation Of Bone Mesenchymal Stem Cells Research Articles

A new method regulates bone fracture tissue exosome lncRNA-mRNA to promote mesenchymal stem cell proliferation and migration

Post-injury adaptation (PIA) is a simple and convenient method to promote bone healing, but its mechanism is unclear. This study was to discuss the role of fracture site tissue exosomes lncRNAs-mRNAs networks on PIA promoting bone mesenchymal stem cells (BMSCs) proliferation and migration. Firstly, the effects of PIA accelerating BMSCs proliferation and migration were confirmed by rat fracture model and bone fracture environment in vitro. Besides, the fracture site tissue exosomes were isolated and authenticated. Then the tissue exosomes were the key factor in PIA promoting BMSCs proliferation and migration authenticated by in vitro and in vivo experiments. The high throughput sequencing and RT-PCR were used to analyze the tissue exosomes lncRNAs-mRNAs networks. It was found that PIA treatment upregulated 118 lncRNAs, 295 mRNAs, and downregulated 111 lncRNAs, 2706 mRNAs in tissue exosomes. A total 12,211 genes were the target genes. Akt1, Actb and Uba52 were the hub mRNAs in tissue exosomes. In additions, tissue-derived exosomes of PIA treated rats upregulated 49 genes, 3 lncRNAs and downregulated 28 genes, 1 lncRNA in BMSCs. Kif11 was the hub gene. Overall, PIA promoted BMSCs proliferation and migration in the early stage of fracture healing, which was closely related to the fracture site tissue exosomes. Akt1, Actb and Uba52 were the hub mRNAs in the exosomes. Besides, Kif11 might be the key gene in BMSC regulated by tissue-derived exosomes of PIA treated rats.

Promoted osteogenesis by corona discharge poling induced in electroactive piezoelectric bioceramics

Rapid bone reconstruction and regeneration process is still a problem to be solved in the clinical orthopedic treatment. In vivo electric stimulation (ES) shows great promise in repair and regeneration of damaged tissues, and the piezoelectric characteristic of nature bone tissue make the electrical activity piezoelectricity biomaterials feasible and attractive in bone regeneration. Herein, we prepared a biocompatible lead-free piezoelectric ceramic K0.5Na0.5NbO3 (KNN) via solid phase sintering, which possesses wireless ES capability through corona discharge poling. Compared with the traditional piezoelectric ceramic polarization methods, corona discharge poling is a non-contact and biologically safer electrical poling method without electrodes or insulating liquids. The results of piezoelectricity, the water contact angle, the protein adsorption, surface potential characteristics of KNN ceramics and bone mesenchymal stem cells (BMSCs) behavior on ceramics, showed that after corona discharge poling the surface of KNN piezoelectric ceramic became hydrophilic, and positively charged, and the electrostatic effect formed by these charges promoted protein adsorption and BMSCs attachment and proliferation, which further promoted osteogenic differentiation. In conclusion, we used the corona discharge to polarize KNN piezoelectric ceramics and excellent biocompatibility of the ceramics was maintained after poling. Meanwhile, our work demonstrated the potential application of piezoelectric bio-ceramic materials in bone regeneration.

Hydrogen Promotes the Effectiveness of Bone Mesenchymal Stem Cell Transplantation in Rats with Spinal Cord Injury.

Although bone mesenchymal stem cell (BMSC) transplantation has been applied to the treatment of spinal cord injury (SCI), the effect is unsatisfactory due to the specific microenvironment (inflammation and oxidative stress) in the SCI area, which leads to the low survival rate of transplanted cells. Thus, additional strategies are required to improve the efficacy of transplanted cells in the treatment of SCI. Hydrogen possesses antioxidant and anti-inflammatory properties. However, whether hydrogen can enhance the effect of BMSC transplantation in the treatment of SCI has not yet been reported. This study was aimed at investigating whether hydrogen promotes the therapeutic effect of BMSC transplantation in the treatment of SCI in rats. In vitro, BMSCs were cultured in a normal medium and a hydrogen-rich medium to study the effect of hydrogen on the proliferation and migration of BMSCs. BMSCs were treated with a serum-deprived medium (SDM), and the effects of hydrogen on the apoptosis of BMSCs were studied. In vivo, BMSCs were injected into the rat model of SCI. Hydrogen-rich saline (5 ml/kg) and saline (5 ml/kg) were given once a day via intraperitoneal injection. Neurological function was evaluated using the Basso, Beattie, and Bresnahan (BBB) and CatWalk gait analyses. Histopathological analysis, oxidative stress, inflammatory factors (TNF-α, IL-1β, and IL-6), and transplanted cell viability were detected at 3 and 28 days after SCI. Hydrogen can significantly enhance BMSC proliferation and migration and tolerance to SDM. Hydrogen and BMSC codelivery can significantly enhance neurological function recovery by improving the transplant cell survival rate and migration. Hydrogen can enhance the migration and proliferation capacity of BMSCs to repair SCI by reducing the inflammatory response and oxidative stress in the injured area. Hydrogen and BMSC codelivery is an effective method to improve BMSC transplantation in the treatment of SCI.

3D printed porous titanium filled with mineralized UV-responsive chitosan hydrogel promotes cell proliferation and osteogenesis in vitro

The modification of 3D printed porous titanium (Ti), especially for the internal pore structure, is critical and has received more attention to promoting its osteogenesis for clinical use. Ultra-violet (UV) responsive chitosan (CSMA), as an injectable filling material, was firstly incorporated into porous Ti, and then CSMA was in-situ mineralized by carbon oxide (CO2) diffusion (CSMA/CaCO3). Their physical-chemical and biological properties were investigated in vitro. CaCO3 crystals within CSMA hydrogels were successfully deposited into pores of porous Ti, which exhibited favorable biocompatibility. Ti implants filled with CSMA/CaCO3 promoted adhesion and proliferation of bone mesenchymal stem cells (BMSCs). Moreover, Ti implant filled CSMA/CaCO3 hydrogels could increase alkaline phosphatase (ALP) activities, up-regulate osteopontin (OPN) and osteocalcin (OCN) expression levels, and enhance extracellular mineralization. 3D printed porous Ti filled with mineralized UV-responsive chitosan hydrogel could promote proliferation and osteogenesis of BMSCs, and have great potential for the modification of porous Ti implants in bone tissue engineering.

Novel Peptides from Sea Cucumber Intestinal Enzyme Hydrolysates Promote Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Phosphorylation of PPARγ at Serine 112.

Osteo-adipogenic differentiation imbalance of bone mesenchymal stem cells (BMSCs) has been linked to a variety of pathophysiological processes such as obesity and osteoporosis. Recent studies report that the phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ) Ser112 affects the fate decision of BMSCs. Novel peptides from the sea cucumber intestinal peptide (SCIP) have been proved to promote the growth of longitudinal bone. However, it is unclear the effect of SCIP on BMSCs differentiation fate. BMSCs in vitro and glucocorticoid induced mice are employed to investigate the effects of SCIP on osteo-adipogenic differentiation of BMSCs. In vitro results show that SCIP supplement significantly promotes the proliferation and osteogenic differentiation of BMSCs, upregulates the expression of osteogenic marker. In vivo results show that SCIP supplement ameliorates the osteo-adipogenic differentiation imbalance in glucocorticoid-treated mice, decreases bone marrow fat, and elevates bone mineral density. Mechanistically, SCIP supplement promotes and maintains the phosphorylation of PPARγ Ser112 through AMPK/ERK and TAZ signals, thereby inducing the osteogenic differentiation of BMSCs. Supplement with SCIP promotes BMSCs to differentiate into osteoblasts. These results suggest that SCIP has potential as a functional food to improve obesity and osteoporosis.

Combined computational analysis and cytology show limited depth osteogenic effect on bone defects in negative pressure wound therapy.

Background: The treatment of bone defects remains a clinical challenge. The effect of negative pressure wound therapy (NPWT) on osteogenesis in bone defects has been recognized; however, bone marrow fluid dynamics under negative pressure (NP) remain unknown. In this study, we aimed to examine the marrow fluid mechanics within trabeculae by computational fluid dynamics (CFD), and to verify osteogenic gene expression, osteogenic differentiation to investigate the osteogenic depth under NP. Methods: The human femoral head is scanned using micro-CT to segment the volume of interest (VOI) trabeculae. The VOI trabeculae CFD model simulating the bone marrow cavity is developed by combining the Hypermesh and ANSYS software. The effect of trabecular anisotropy is investigated, and bone regeneration effects are simulated under NP scales of -80, -120, -160, and -200mmHg. The working distance (WD) is proposed to describe the suction depth of the NP. Finally, gene sequence analysis, cytological experiments including bone mesenchymal stem cells (BMSCs) proliferation and osteogenic differentiation are conducted after the BMSCs are cultured under the same NP scale. Results: The pressure, shear stress on trabeculae, and marrow fluid velocity decrease exponentially with an increase in WD. The hydromechanics of fluid at any WD inside the marrow cavity can be theoretically quantified. The NP scale significantly affects the fluid properties, especially those fluid close to the NP source; however, the effect of the NP scale become marginal as WD deepens. Anisotropy of trabecular structure coupled with the anisotropic hydrodynamic behavior of bone marrow; An NP of -120mmHg demonstrates the majority of bone formation-related genes, as well as the most effective proliferation and osteogenic differentiation of BMSCs compared to the other NP scales. Conclusion: An NP of -120mmHg may have the optimal activated ability to promote osteogenesis, but the effective WD may be limited to a certain depth. These findings help improve the understanding of fluid mechanisms behind NPWT in treating bone defects.

Photobiomodulation (800 nm Light-Emitting Diode) Treatment Promotes Bone Mesenchymal Stem Cell Proliferation Via Long Noncoding RNA MEG3-MicroRNA-217-5P Pathway.

Background: Patients with osteoporosis (OP) have a high risk of bone fracture. Abnormal bone mesenchymal stem cell (BMSC) differentiation is an essential process of OP development. In recent years, photobiomodulation has been shown to effectively promote BMSC proliferation. However, the mechanism by which photobiomodulation promotes BMSC proliferation is unclear. Long noncoding RNAs (lncRNAs) are essential mediators in multiple biological processes. The lncRNA maternally expressed gene 3 (MEG3) is a novel lncRNA gene and is related to cell proliferation. Studies have indicated that MEG3 serves as a promotor in BMSC proliferation. Objective: To investigate the effects and mechanisms of 800 nm light-emitting diode (LED) photobiomodulation in BMSC proliferation. Materials and methods: The BMSCs collected from mouse tibias and femurs were irradiated by 800 nm LED for 180 sec. CCK-8 assay was used to detect the cell viability. A dual-luciferase reporter assay was used to determine IncRNA MEG3 acted as a miR-217-5p sponge. We used reverse transcription-polymerase chain reaction (RT-PCR) and western blot to detect the mRNA and protein levels of MEG3, miR-217-5p, Notch2, Hes1, Hey2. Results: In the present study, we revealed that photobiomodulation (800 nm LED) could increase the mRNA level of MEG3, and protein levels of Notch2, Hes1, and Hey2. Moreover, we also identified that upregulated MEG3 could act as a miR-217-5p sponge to activate the Notch signaling pathway. Conclusions: The current study revealed the MEG3-related mechanism of photobiomodulation treatment in OP and identified potential gene therapies for OP.

Screening and analysis of key genes in the biological behavior of bone mesenchymal stem cells seeded on gradient nanostructured titanium compared with native pure Ti.

Titanium (Ti) and Ti-based alloy materials are ideal brackets that restore bone defect, and the mechanism of related genes inducing bone mesenchymal stem cells (BMSCs) to osteogenic differentiation is currently a hot research topic. In order to screen key genes of BMSCs during the osteogenic expression process, we acquired data sets (GSE37237 and GSE84500) which were in the database Gene Expression Omnibus (GEO). Investigations on differentially expressed genes (DEGs) and their enrichment of functions were conducted. We constructed relative protein-protein interaction (PPI) network by using Search Tool for the Retrieval of Interacting Genes (STRING) and visualized the expression of DEGs with Cytoscape. A total of 279 DEGs were discerned, which could be divided into 177 down regulated genes and 102 up regulated genes. In addition, the DEGs' enrichment and pathways included regulation of actin cytoskeleton, inflammatory mediator regulation of transient receptor potential (TRP) channels, peroxisome proliferator-activated receptors (PPAR) pathway, cell cycle, Rheumatoid arthritis, mitogen-activated protein kinases (MAPK) signaling pathway and Ras signaling pathway ect. It showed that 10 notable up regulated genes were mainly in AMP-activated protein kinase (AMPK) pathway. Then we used a technology named surface mechanical attrition treatment (SMAT) to prepare gradient nanostructured (GNS) surface Ti and seeded well-growing BMSCs on the surface of SMAT Ti and native pure Ti. Cell Counting Kits-8 (CCK-8), apoptosis experiment, immunofluorescence technology and staining experiments for alka-line phosphatase (ALP) and alizarin red staining (ARS) were used to research the proliferation, adhesion and differentiation ability of BMSCs seeded on SMAT Ti compared with native pure Ti. We used quantitative real-time PCR (qRT-PCR) technology so as to verify the expression of the most significant 5 genes. In summary, these results indicated novel point of views into candidate genes and potential mechanism for the further study of BMSCs' behaviors seeded on SMAT Ti.

The effect of BMP2/Smads pathway mediating platelet-rich fibrin on rat bone mesenchymal stem cells.

We explored the influences on platelet-rich fibrin (PRF) to rat Bone Mesenchymal Stem Cells (BMSCs), as well as the role of bone morphogenetic protein 2 (BMP2)/maternal signal protein homolog (Smads) pathway. The proposed research is approved by the ethics board of the Second Affiliated Hospital of Harbin Medical University. The BMSCs were isolated and purified. The BMSCs were assigned to a control group arbitrarily, PRF group, BMP activator group and BMP inhibitor group (hereinafter referred to as activator group and inhibitor group). Each group of BMSCs in the logarithmic growth phase was detected for the alkaline phosphatase (ALP) activity since 3 days and 14 days of culture; CCK-8 assay was conducted for detection of the proliferation of BMSCs; Real time PCR was conducted for detection of the osteogenic differentiation marker collagen I (COL-I), BMP2, Runt-related transcription factor 2(RUNX2), osteocalcin (OCN) mRNA relative expression levels; Western-Blot detection of BMP2, OCN, P-SMAD1/5/8, relative expression level of RUNX2 protein. In contrast to the control group, BMSCs' the ALP activity of the PRF group, activator group, as well as inhibitor group increased for 3 days and 14 days, and the activator group>PRF group>inhibitor group (p≤0.05). ALP activity in each group was elevated with the increase in culture time, the ALP activity of the control group, PRF group, activator group and inhibitor group increased (p≤0.05). In comparison to the control group, the relevant expression levels of COL-I, BMP-2, RUNX2 and OCN in the PRF group, activator group, and inhibitor group increased, and the activator group>PRF group>inhibitor group (p≤0.05). The relative expression levels of BMP2, OCN, p-SMAD1/5/8 and RUNX2 protein in each group were statistically different, the activator group>PRF group>control group>inhibitor group (p≤0.05). PRF can promote the proliferation and osteogenic differentiation of BMSCs by activating the BMP2/Smads signaling pathway.

Enhanced anti-microbial activity and osseointegration of Ta/Cu co-implanted polyetheretherketone

Polyetheretherketone (PEEK) has been widely applied for orthopedic and oral implants due to its excellent mechanical properties, biocompatibility, and radiolucency. However, its bioinert and the lack of anti-microbial activity limit its application. We modified the PEEK surface with Ta/Cu co-implantation using plasma immersion ion-implantation technology. After implantation of Ta/Cu ions, the morphology and roughness of the PEEK surface were not significantly changed at micron level. We estimated the cytocompatibility, anti-microbial ability, and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) of the modified surfaces in vitro. Compared to the untreated surfaces, the Ta ion-treated surface showed improved adhesion, proliferation, ALP activity, ECM mineralization, and osteogenic gene expression of BMSCs. Further, the Cu ion-treated surface showed reduced initial adhesion and proliferation of Escherichia coli and Staphylococcus aureus in vitro and proliferation of Staphylococcus aureus in the mouse subcutaneous implant-associated infection model. According to a rat bone repair model, all Ta ion-implanted groups demonstrated improved new bone formation. In summary, Ta/Cu ion co-impanation improved anti-microbial activity and promoted osseointegration of the PEEK surface.

Controlled and Sequential Delivery of Stromal Derived Factor-1 α (SDF-1α) and Magnesium Ions from Bifunctional Hydrogel for Bone Regeneration.

Bone healing is a complex process that requires the participation of cells and bioactive factors. Stromal derived factor-1 α (SDF-1α) and magnesium ions (Mg2+) both are significant bioactive factors for cell recruitment and osteogenesis during bone regeneration. Thus, a bifunctional hydrogel containing a sequential delivery system is fabricated to improve osteogenesis. During sequential delivery of the hydrogel, SDF-1α is predominantly released at the early stage of bone mesenchymal stem cells (BMSCs) recruitment, while Mg2+ are constantly delivered at a later stage to improve osteogenic differentiation of recruited cells. In addition, due to the early release of SDF-1α, the hydrogel showed strong BMSCs recruitment and proliferation activity. Mg2+ can not only induce up-regulation of osteogenic gene expression in vitro, but also promote bone tissue and angiogenesis in vivo. Taken together, the injection of xanthan gum-polydopamine crosslinked hydrogel co-loading SDF-1α and Mg2+ (XPMS hydrogel) provides a novel strategy to repair bone defects.

Regulation of mesenchymal stem cell osteogenic potential via microfluidic manipulation of microcarrier surface curvature

Microsphere-carriers have gained great interests as three-dimensional substrates for cultivation/expansion of tissue cells, required by cell-based therapy. However, how the microsphere curvature affects the cell proliferation/differentiation as well as the underlying signaling pathway of stem cells, and how to consequently regulate those cellular functionalities via manipulating microcarrier surface curvature, still remain to be explored. The current study was thus designed to develop a microfluidic manipulation technology to precisely control poly (lactic-co-glycolic) acid (PLGA) microsphere surface curvature, and subsequently to investigate the cellular responses and responding pathways of rat bone mesenchymal stem cells (BMSCs) cultured on these microspheres of predetermined curvature. A microfluidic device was developed to produce mono-distributed PLGA microspheres of diameters ranging from 52 µm to 250 µm, corresponding to curvatures (κ) from 1/26 µm−1 to 1/125 µm−1. BMSCs attachment and proliferation was evaluated on them and the one of κ = 1/82.5 µm−1 was shown to provide the most suitable microenvironment for cells to grow and undergo osteogenic differentiation. It was even found that F-actin cytoskeletal organization, nuclear distortion and expression of Lamin A were significantly enhanced by cells on the microcarriers of κ = 1/82.5 µm−1. Furthermore, a long non-coding RNA named lnc-LMNA, was found in this study to be the key factor associated with Lamin A to regulate osteogenic differentiation of BMSCs on spherical substrates. The current study thus provides a smart manipulation technology via microfluidic-manufacturing microcarriers to regulate cell functionalities, thereby enhancing desired therapeutic outcomes of cell-based regeneration or repair.

Differentiation of Bone Mesenchymal Stem Cells Into Vascular Endothelial Cell-Like Cells Using Functionalized Single-Walled Carbon Nanotubes.

Carbon nanotubes (CNTs) are a promising bioactive scaffold for bone regeneration because of their superior mechanical and biological properties. Vascularization is crucial in bone tissue engineering, and insufficient vascularization is a long-standing problem in tissue-engineered scaffolds. However, the effect of CNTs on vascularization is still minimal. In the current study, pristine single-walled carbon nanotubes (SWNTs) were purified to prepare different ratios of SWNTs/EDC composites, and their surface morphology and physicochemical properties of SWNTs/EDC were studied. Furthermore, the effect of SWNTs/EDC on vascularization was investigated by inducing the differentiation of bone mesenchymal stem cells (BMSCs) into vascular endothelial cell-like cells (VEC-like cells). Results showed that SWNTs/EDC composite was successfully prepared, and EDC was embedded in the SWNTs matrix and uniformly distributed throughout the composites. The AFM, FTIR spectra, and Raman results confirmed the formation of SWNTs/EDC composites. Besides, the surface topography of the SWNTs/EDC composites presents a rough surface, which may positively affect cell function. In vitro cell culture revealed that SWNTs and SWNTs/EDC composites exhibited excellent biocompatibility and bioactivity. The SWNTs/EDC composite at mass/volume ratios 1:10 had the best enhancement of proliferation and differentiation of BMSCs. Moreover, after culture with SWNTs/EDC composite, approximately 78.3% ± 4.2% of cultured cells are double-positive for FITC-UEA-1 and DiI-Ac-LDL double staining. Additionally, the RNA expression of representative endothelial cell markers VEGF, VEGF-R2, CD31, and vWF in the SWNTs/EDC composite group was significantly higher than those in the control and SWNTs group. With the limitation of our study, the results suggested that SWNTs/EDC composite can promote BMSCs differentiation into VEC-like cells and positively affect angiogenesis and bone regeneration.

The effect of TLR-4 on the proliferation and differentiation of bone mesenchymal stem cells and its relationship with the Wnt signal transduction pathway during bone nonunion.

BackgroundBone nonunion is a special fracture complication that occurs in about 5% to 10% of cases. This type of fracture is difficult to heal, and causes great pain to patients and affects their quality of life. The mechanism of bone nonunion is not clear. In our study, we investigated the influence of Toll-like receptor (TLR)-3, TLR-4, and Wnt signaling pathways on the occurrence of bone nonunion.MethodsFirstly, we established a Sprague Dawley (SD) rat model of femoral nonunion, and detected the expression levels of TLR-3, TLR-4, β-catenin, nemo-like kinase (NLK), c-Jun N-terminal kinase (JNK), and other proteins during model construction. For in vitro experiments, primary cultured bone mesenchymal stem cells (BMSCs) were divided into 4 groups: lipopolysaccharide (LPS, agonist of TLR-4) group, LPS + CLI095 (inhibitor of TLR-4) group, control group, and LPS + substance P (SP) group. The expression of β-catenin, NLK, JNK, and ALP and the osteogenic differentiation ability of cells were detected during culture.ResultsX-ray and hematoxylin and eosin (HE) staining results confirmed the successful modeling of bone nonunion. During the formation of the bone nonunion model, the expression of TLR-4 showed an upward trend. In vitro experiment results showed that inhibition of TLR-4 expression could enhance the proliferation and differentiation ability of BMSCs. The expression of β-catenin, the core protein of the canonical Wnt signaling pathway, increased rapidly in the first 2 weeks of bone nonunion construction, and decreased after 2 weeks. Non-canonical Wnt signaling pathway proteins NLK and JNK had no change in the first 2 weeks, and showed an upward trend after 2 weeks. In vitro experiment results showed that the expression of β-catenin was dominant in BMSCs with strong proliferation and differentiation ability, while the expression of NLK and JNK was dominant in BMSCs with weak proliferation and differentiation ability. These results suggest that the Wnt signaling pathway may regulate the occurrence of bone nonunion.ConclusionsTLR-4 inhibits the proliferation and differentiation of BMSCs, and the transformation of the canonical Wnt signaling pathway to the non-canonical Wnt signaling pathway may lead to bone nonunion. Our study may provide new insights into the treatment of bone nonunion.

CircRNA-011235 Counteracts The Deleterious Effect of Irradiation Treatment on Bone Mesenchymal Stem Cells by Regulating The miR-741-3p/CDK6 Pathway.

Objective The present work was aimed at uncovering the effect of circRNA-011235 (circ-011235) on irradiation-induced bone mesenchymal stem cells (BMSCs) injury and its regulatory mechanism, with a view to establish a scientific basis for its possible medical applications. Materials and Methods In this experimental study, after irradiation with different doses (0, 2, 4, 6 GY), the relative expression levels of circ-011235, miR-741-3p, and cyclin-dependent kinases 6 (CDK6) were detected in the BMSCs, using the real time-quantitative polymerase chain reaction (RT-qPCR). The overexpression effects of circ-011235 and CDK6 on the cell proliferation in irradiation-treated BMSCs were measured by the Cell Counting Kit-8 (CCK8) assay. And also, their effects on the cell cycle were evaluated by flow cytometry. RT-qPCR and immunoblotting were performed to detect the effects of pcDNA-circ-011235 and pcDNA-CDK6 on the expression of cyclin D1 and cyclin- dependent kinases 4 (CDK4) at the gene and protein levels, respectively. Results Irradiation treatment elevated the expression of circ-011235 and CDK6, but reduced miR-741-3p expression in the BMSCs with a dose-dependent effect. The proliferation of BMSCs was significantly inhibited in the irradiation treatment group, while the overexpression of circ-011235 and CDK6 effectively attenuated this inhibition. Also, overexpression of circ-011235 and CDK6 elevated the expression of cyclin D1 in irradiation-treated BMSCs, but had no significant effect on the CDK4 expression. ConclusionOur results demonstrated that circ-011235 up-regulated the expression of cyclin D1 via miR-741-3p/ CDK6 signal pathway, thereby promoting cell cycle progression and proliferation of irradiation-treated BMSCs. This finding suggested circ-011235/ miR-741-3p/CDK6 pathway exerted a protective role in the response to irradiation and will be a potential new target for future research on the mechanism involved in the resistance of BMSCs to radiation.

Naringin promotes osteogenesis and ameliorates osteoporosis development by targeting JAK2/STAT3 signalling.

Osteoporosis is a systemic bone metabolism disorder, which increases the risk of fractures, and in severe cases it may cause disability or even death. An important factor contributing to osteoporosis is the imbalance between bone formation and resorption. Naringin was reported to promote osteoblast differentiation, thus enhancing bone formation and alleviating osteoporosis development. However, the signalling pathways related to the regulatory mechanism of naringin in osteoporosis development are not clear. Proliferation of bone mesenchymal stem cells (BMSCs) treated with naringin in vitro was detected by CCK-8. An osteogenesis differentiation medium supplemented with naringin was applied to explore the effects of naringin on BMSC osteogenic differentiation, as detected by Alizarin red staining. Ovariectomy (OVX)-induced postmenopausal osteoporosis (PMOP) rats were orally administered with naringin. Dual-energy X-ray absorptiometry (DEXA) and micro-CT were applied to measure bone mineral density (BMD), bone volume/total volume (BV/TV), trabecula thickness (Tb.Th), trabecula number (Tb.N), trabecular separation (Tb.Sp) and bone surface/bone volume (BS/BV). H&E staining was performed to show pathological changes of the femur in PMOP rats after naringin treatment. Bone metabolism indicators were assessed by ELISA. We found that naringin suppressed the activation of the JAK2/STAT3 pathway. Naringin promoted BMSC proliferation and osteogenic differentiation. Furthermore, naringin alleviates bone loss and improves abnormal bone metabolism of PMOP rats. Collectively, naringin promotes BMSC osteogenic differentiation to ameliorate osteoporosis development by targeting JAK2/STAT3signalling.

Osteogenic Induction of Low-dose Ipriflavone on Bone Marrow Mesenchymal Stem Cells Extracted from Osteoporosis Rats.

To investigate the effects of ipriflavone (IPF), a synthetic isoflavone plant oestrogen with a structure similar to that of oestradiol, on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). BMSCs were derived from ovariectomised rats (rBMSCs-OVX) and then induced with or without IPF. Cell cytotoxicity, mineralisation in vitro and osteoblast-specific gene expression of BMSCs were studied. IPF at a concentration of 10-8, 10-7 and 10-6 mol/l exhibited no cytotoxic effect on the proliferation of BMSCs but increased alkaline phosphatase activity and osteoblast-specific gene expression. IPF enhances osteogenic differentiation of rBMSCs-OVX partly in vitro, thus its use offers a potential strategy for the treatment of osteoporosis.

MiR-140-3p regulates the osteogenic differentiation ability of bone marrow mesenchymal stem cells by targeting spred2-mediated autophagy.

Understanding the function and regulatory mechanism of miR-140-3p on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Alizarin Red staining, Alkaline phosphatase (ALP) staining, and ALP activity were used to detect the ability osteogenic differentiation. miR-140-3p or Spred2 overexpression into BMSCs using lentiviral vectors and the result were analyzed by Reverse transcription quantitative polymerase chain reaction (RT-qPCR). The relation between miR-140-3p and Spred2 was examined by luciferase reporter assay. CCK8 assay was used to detect the proliferation of BMSCs. RT-qPCR and Western blot analysis were both used to detect altered gene and protein in osteogenic differentiation of BMSCs, respectively. The BMSCs which were induced for 21days were analyzed by Alizarin Red staining, (ALP) staining and ALP activity. RT-qPCR analysis showed that overexpressed miR-140-3p promotes osteogenic differentiation. Western blots results indicated that the overexpression of Spred2 suppressed miR-140-3p. Luciferase reporter assay indicated that Spred2 can integrate with miR-140-3p directly. Meanwhile, the protein level of ALP, OCN, and Runx2, the markers of chondrogenesis, was increased when miR-140-3p increased or Spred2 overexpressed in the osteoinductive medium applied to the BMSCs. Our study demonstrated the association between miR-140-3p and Spred2 in osteogenic differentiation of BMSCs for the first time. Furthermore, our detections also revealed that Spred2-induced autophagic signaling accelerates the progress of osteogenic differentiation ability of BMSCs.

Hsa_circ_0066523 promotes the proliferation and osteogenic differentiation of bone mesenchymal stem cells by repressing PTEN.

AimsCircular RNAs (circRNAs) are a novel type of non-coding RNA that plays major roles in the development of diverse diseases including osteonecrosis of the femoral head (ONFH). Here, we explored the impact of hsa_circ_0066523 derived from forkhead box P1 (FOXP1) (also called circFOXP1) on bone mesenchymal stem cells (BMSCs), which is important for ONFH development.MethodsRNA or protein expression in BMSCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell Counting Kit 8 (CCK8) and 5-ethynyl-2’-deoxyuridine (EdU) were used to analyze cell proliferation. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red S staining were employed to evaluate the osteoblastic differentiation. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pull down, and RNA immunoprecipitation (RIP) assays were combined for exploring molecular associations.ResultsCirc_0066523 was upregulated in osteogenic induction process of BMSCs. Silencing circ_0066523 restrained the proliferation and osteogenic differentiation of BMSCs. Mechanistically, circ_0066523 activated phosphatidylinositol-4,5-bisphosphate 3-kinase / AKT serine/threonine kinase 1 (PI3K/AKT) pathway via recruiting lysine demethylase 5B (KDM5B) to epigenetically repress the transcription of phosphatase and tensin homolog (PTEN). Functionally, AKT signalling pathway agonist or PTEN knockdown counteracted the effects of silenced circ_0066523 on BMSC proliferation and differentiation.ConclusionCirc_0066523 promotes the proliferation and differentiation of BMSCs by epigenetically repressing PTEN and therefore activating AKT pathway. This finding might open new avenues for the identification of therapeutic targets for osteoblast differentiation related diseases such as ONFH.Cite this article: Bone Joint Res 2021;10(8):526–535.

Cornuside I promoted osteogenic differentiation of bone mesenchymal stem cells through PI3K/Akt signaling pathway

BackgroundOsteoporosis is a common disease closely associated with aging. In this study, we aimed to investigate the role of Cornuside I in promoting osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and the potential mechanism.MethodsBMSCs were isolated and treated with different concentrations of Cornuside I (0, 10, 30, 60 μM). Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. RNA sequencing was performed on the isolated BMSCs with control and Cornuside I treatment. Differentially expressed genes were obtained by the R software. Alkaline phosphatase (ALP) staining and Alizarin Red Staining (ARS) were performed to assess the osteogenic capacity of the NEO. qRT-PCR and western blot were used to detect the expression of osteoblast markers.ResultsCornuside I treatment significantly improved BMSC proliferation. The optimal dose of Cornuside I was 30 μM (P < 0.05). Cornuside I dose dependently increased the ALP activity and calcium deposition than control group (P < 0.05). A total of 704 differentially expressed genes were identified between Cornuside I and normal BMSCs. Cornuside I significantly increased the PI3K and Akt expression. Moreover, the promotion effects of Cornuside I on osteogenic differentiation of BMSCs were partially blocked by PI3K/Akt inhibitor, LY294002.ConclusionCornuside I plays a positive role in promoting osteogenic differentiation of BMSCs, which was related with activation of PI3K/Akt signaling pathway.