Osteogenic Differentiation Of Mesenchymal Stem Cells Research Articles

Exendin‐4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor‐kappa B and osteoprotegerin signaling pathway

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

Osteogenesis-Related Long Noncoding RNA GAS5 as a Novel Biomarker for Osteonecrosis of Femoral Head.

Background: The lack of effective biomarkers makes it difficult to achieve early diagnosis and intervention for osteonecrosis of the femoral head (ONFH). Hence, we aimed to identify novel long noncoding RNA (lncRNA) biomarkers for ONFH. Methods: High-throughput RNA sequencing was performed to detect lncRNA and mRNA expression levels in subchondral bone samples from three patients with ONFH and three patients with femoral neck fractures. Integrated bioinformatics analyses were conducted to identify lncRNAs associated with ONFH development and their potential functions and signaling pathways. A co-expression network was constructed based on the gene time-series expression data in GSE113253. After selecting lncRNA GAS5 as a novel biomarker for ONFH, bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation assays were performed to verify the association between lncRNA GAS5 and osteogenic differentiation. Alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to measure the osteogenic phenotype and lncRNA GAS5 expression. Finally, for further validation, ONFH rat models were established, and lncRNA GAS5 expression in subchondral bone was detected by RT-qPCR. Results: We identified 126 and 959 differentially expressed lncRNAs and genes, respectively. lncRNA GAS5 expression level was significantly downregulated in patients with ONFH compared to the control group patients. The BMSC osteogenic differentiation assays showed that ALP activity increased gradually from days 3 to 7, while the lncRNA GAS5 expression level was significantly upregulated in the osteogenic differentiation induction groups. Furthermore, in vivo experiments suggested that the bone volume/tissue volume value and trabecular thickness significantly decreased in the ONFH rat model group compared to the control group, whereas the trabecular space significantly increased in the ONFH group compared to the control group. In addition, the lncRNA GAS5 expression level significantly decreased in the ONFH rat model group. Conclusion: The lncRNA GAS5 expression level was highly associated with BMSC osteogenic differentiation and was significantly downregulated in both the subchondral trabecular bone tissue of ONFH patients and ONFH rat models. Therefore, lncRNA GAS5 can serve as an ONFH osteogenic biomarker to provide an effective target for early diagnosis and molecular therapy of ONFH.

Promoting in-vivo bone regeneration using facile engineered load-bearing 3D bioactive scaffold

The worldwide incidence of bone disorders has trended steeply upward and is expected to get doubled by 2030. The biological mechanism of bone repair involves both osteoconductivity and osteoinductivity. Despite the self-healing functionality after injury, bone tissue faces a multitude of pathological challenges. Several innovative approaches have been developed to prepare biomaterial-based bone grafts. To design a suitable bone material, the freeze-drying technique has achieved significant importance among the other conventional methods. However, the functionality of the polymeric freeze-dried scaffold in in-vivo osteogenesis is in a nascent stage. In this study facile, freeze-dried, biomaterial-based load-bearing three-dimensional porous composite scaffolds have been prepared. The biocompatible scaffolds have been made by using chitosan (C), polycaprolactone (P), hydroxyapatite (H), glass ionomer (G), and graphene (gr). Scaffolds of eight different groups (C, P, CP, CPH, CPHG, CPHGgr1, CPHGgr2, CPHGgr3) have been designed and characterized to evaluate their applicability in orthopedics. To evaluate the efficacy of the scaffolds a series of physio-chemical, morphological, and in-vitro and in-vivo biological experiments have been performed. From the obtained results it was observed that the CPHGgr1 is the ideal compatible material for Wharton’s jelly-derived mesenchymal stem cells (MSCs) and the blood cells. The in-vitro bone-specific gene expression study revealed that the scaffold assists MSCs osteogenic differentiation. Additionally, the in-vivo study on the mice model was also performed for a period of four and eight weeks. The subcutaneous implantation of the designed scaffolds did not show any altered physiological condition in the animals, which indicated the in-vivo biocompatibility of the designed material. The histopathological study revealed that after eight weeks of implantation, the CPHGgr1 scaffold supported significantly better collagen deposition and calcification. The facile designing of the CPHGgr1 multicomponent nanocomposite provided an osteo-regenerative biomaterial with desired mechanical strength as an ideal regenerative material for cancellous bone tissue regeneration.

BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models.

Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3% alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.

Stratified-structural hydrogel incorporated with magnesium-ion-modified black phosphorus nanosheets for promoting neuro-vascularized bone regeneration

Angiogenesis and neurogenesis play irreplaceable roles in bone repair. Although biomaterial implantation that mimics native skeletal tissue is extensively studied, the nerve-vascular network reconstruction is neglected in the design of biomaterials. Our goal here is to establish a periosteum-simulating bilayer hydrogel and explore the efficiency of bone repair via enhancement of angiogenesis and neurogenesis. In this contribution, we designed a bilayer hydrogel platform incorporated with magnesium-ion-modified black phosphorus (BP) nanosheets for promoting neuro-vascularized bone regeneration. Specifically, we incorporated magnesium-ion-modified black phosphorus (BP@Mg) nanosheets into gelatin methacryloyl (GelMA) hydrogel to prepare the upper hydrogel, whereas the bottom hydrogel was designed as a double-network hydrogel system, consisting of two interpenetrating polymer networks composed of GelMA, PEGDA, and β-TCP nanocrystals. The magnesium ion modification process was developed to enhance BP nanosheet stability and provide a sustained release platform for bioactive ions. Our results demonstrated that the upper layer of hydrogel provided a bionic periosteal structure, which significantly facilitated angiogenesis via induction of endothelial cell migration and presented multiple advantages for the upregulation of nerve-related protein expression in neural stem cells (NSCs). Moreover, the bottom layer of the hydrogel significantly promoted bone marrow mesenchymal stem cells (BMSCs) activity and osteogenic differentiation. We next employed the bilayer hydrogel structure to correct rat skull defects. Based on our radiological and histological examinations, the bilayer hydrogel scaffolds markedly enhanced early vascularization and neurogenesis, which prompted eventual bone regeneration and remodeling. Our current strategy paves way for designing nerve-vascular network biomaterials for bone regeneration.

Heterotopic ossification vs. fracture healing: Extracellular vesicle cargo proteins shed new light on bone formation

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease in which bone tissue forms in extraskeletal sites, which is known as heterotopic ossification (HO). Extracellular vesicles (EVs) are small phospholipid-enclosed particles released by various cells which have an emerging, but not completely understood role in various (patho)physiological processes. In order to further study the pathophysiology of FOP we conducted a small observational study comparing the proteomic profiles of EV cargo, derived from pooled plasma of four patient groups: FOP patient (N = 1) during active disease phase (flare-up), FOP patients during remission (N = 2), patients after long bone fracture (N = 20) and healthy controls (N = 10). After isolation of EVs – their protein cargo was determined using liquid chromatography / mass spectrometry, after which a functional gene enrichment analysis was performed. Our results show a sizeable difference of the proteomics profiles in which EVs from the bone fracture group show significant activity of integrin interactions, Wnt, VEGF, IGF-1 and PDGF pathways; conversely, FOP patients' EVs indicate that HO occurs via processes of innate immunity and the Ephrin B signaling pathway. We hypothesize that the Ephrin B signaling (expressed in EVs) contributes to HO by aiding in mesenchymal stem cell recruitment and osteogenic differentiation, as well as by contributing to the inflammatory response, including macrophage chemotaxis and activation. This is, to our knowledge, the first published analysis of EV protein cargo in FOP.

Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway.

Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs' vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

Osteostatin improves the Osteogenic differentiation of mesenchymal stem cells and enhances angiogenesis through HIF-1α under hypoxia conditions in vitro

BackgroundHypoxia conditions induced by bone defects would prolong the duration of bone regeneration. The effect of osteostatin (OST) on the osteogenic differentiation of mesenchymal stem cells (MSCs) and angiogenesis under hypoxia conditions remain unexplored. MethodsSPF mice were obtained, and MSCs were isolated from bone marrow. MSCs were treated with 1% oxygen for hypoxia induction, and 200 nM of OST was used to treat cells under nomorxia or hypoxia conditions. Cell proliferation was evaluated using CCK8 assay, and trypan blue staining was implemented for determining cell death ratio. Alkaline phosphatase activity and alizarin redS staining was conducted to histologically evaluated osteogenic differentiation. Flow cytometry was used for the detection of CD31hiEmcnhi cells (Type H ECs), whose migration was detected by Transwell assay and angiogenesis was measured by tube formation assay. Protein level was measured by western blotting and mRNA level was monitored via RT-qPCR. ResultsThe MSC proliferation was enhanced by OST under hypoxia conditions. The osteogenic differentiation of MSCs was decreased under hypoxia conditions, and treatment of OST significantly reversed its inhibitory effect. The hypoxia treated culture medium of MSCs promoted the proliferation, migration, and angiogenesis of type H ECs, while the effects were further strengthened by OST addition. HIF-1α was found to be upregulated in hypoxia treated MSCs, whereas silencing of HIF-1α had reversed effects on the angiogenic capacity of Type H ECs. ConclusionOST improved the proliferation and osteogenic differentiation of MSCs and further promoted angiogenesis of type H ECs through upregulating HIF-1α expression.

Silicon-Gold Nanoparticles Affect Wharton's Jelly Phenotype and Secretome during Tri-Lineage Differentiation.

Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton’s Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon–gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4–9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.

Osteogenic differentiation of mesenchymal stem cells promotes c-Jun-dependent secretion of interleukin 8 and mediates the migration and differentiation of CD4+ T cells

BackgroundThe immune system and the skeletal system have complex interactions in the bone marrow and even in the joints, which has promoted the development of the concept of osteoimmunology. Some evidence has indicated that T cells and B cells contribute to the balance between the resorption and formation of bone. However, there has been little discussion on the regulation of CD4+ T lymphocytes by cells involved in bone metabolism. Mesenchymal stem cells (MSCs), which exert core functions related to immunoregulation and osteogenic differentiation, are crucial cells linked to both bone metabolism and the immune system. Previous studies have shown that the immunoregulatory capacity of MSCs changes following differentiation. However, it is still unclear whether the osteogenic differentiation of MSCs affects the migration and differentiation of CD4+ T cells.MethodsMSCs were cultured in growth medium or osteogenic medium for 10 days and then cocultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of specific cytokines were analyzed by quantitative real-time PCR and enzyme-linked immunosorbent assays. A Proteome Profiler Human XL Cytokine Array Kit was used to analyze supernatants collected from MSCs. Alizarin red S staining and Alkaline phosphatase assay were used to detect the osteogenic differentiation of MSCs.ResultsHere, we found that the migration of CD4+ T cells was elevated, and the capacity to induce the differentiation of regulatory T (Treg) cells was weakened during MSC osteogenic differentiation, while the differentiation of T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells was not affected. Further studies revealed that interleukin (IL)-8 was significantly upregulated during MSC osteogenic differentiation. Both a neutralizing antibody and IL-8-specific siRNA significantly inhibited the migration of CD4+ T cells and promoted the differentiation of Treg cells. Finally, we found that the transcription factor c-Jun was involved in regulating the expression of IL-8 and affected the osteogenic differentiation of MSCs, thereby mediating the migration and differentiation of CD4+ T cells.ConclusionThis study demonstrated that MSC osteogenic differentiation promoted c-Jun-dependent secretion of IL-8 and mediated the migration and differentiation of CD4+ T cells. These results provide a further understanding of the crosstalk between bone and the immune system and reveal information about the relationship between osteogenesis and inflammation in the field of osteoimmunology.

The effects of cigarette smoking and nicotine on the therapeutic potential of mesenchymal stem cells.

Due to their immunoregulatory properties and capacity for multi-lineage differentiation, mesenchymal stem cells (MSCs) have been used as new therapeutic agents in regenerative medicine. Numerous lifestyle habits and behavioral risk factors may modulate metabolic and cell growth signaling pathways in MSCs, affecting their phenotype and function. Accordingly, identification of these factors and minimization of their influence on viability and function of transplanted MSCs may greatly contribute to their better therapeutic efficacy. A large number of experimental and clinical studies have demonstrated the detrimental effects of cigarette smoke and nicotine on proliferation, homing, chondrogenic and osteogenic differentiation of MSCs. Cigarette smoke down-regulates expression of chemokine receptors and modulates activity of anti-oxidative enzymes in MSCs, while nicotine impairs synthesis of transcriptional factors that regulate the cell cycle, metabolism, migration, chondrogenesis and osteogenesis. In this review article, we summarize current knowledge about molecular mechanisms that are responsible for cigarette smoke and nicotine-dependent modulation of MSCs' therapeutic potential.

Mesenchymal Stem Cell-Immune Cell Interaction and Related Modulations for Bone Tissue Engineering.

Critical bone defects and related delayed union and nonunion are still worldwide problems to be solved. Bone tissue engineering is mainly aimed at achieving satisfactory bone reconstruction. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells that can differentiate into bone cells and can be used as one of the key pillars of bone tissue engineering. In recent decades, immune responses play an important role in bone regeneration. Innate immune responses provide a suitable inflammatory microenvironment for bone regeneration and initiate bone regeneration in the early stage of fracture repair. Adaptive immune responses maintain bone regeneration and bone remodeling. MSCs and immune cells regulate each other. All kinds of immune cells and secreted cytokines can regulate the migration, proliferation, and osteogenic differentiation of MSCs, which have a strong immunomodulatory ability to these immune cells. This review mainly introduces the interaction between MSCs and immune cells on bone regeneration and its potential mechanism, and discusses the practical application in bone tissue engineering by modulating this kind of cell-to-cell crosstalk. Thus, an in-depth understanding of these principles of bone immunology can provide a new way for bone tissue engineering.

NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells.

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD+); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

The cannabinoid receptor I (CB1) enhanced the osteogenic differentiation of BMSCs by rescue impaired mitochondrial metabolism function under inflammatory condition

BackgroundPeriodontitis is a chronic infectious disease leading to bone resorption and periodontal tissue disruption under inflammatory stimulation. The osteogenic differentiation ability of mesenchymal stem cells (MSCs) is impaired under the inflammatory environment, which limits the effect of treatment. The cannabinoid receptor I (CB1) is the main effector of the endogenous cannabinoid system (ECS), and our previous study verified that CB1 could enhance the osteo/dentinogenic differentiation of dental MSCs, which might be a target for alveolar bone regeneration. However, the effect of CB1 on the osteogenic differentiation of MSCs derived from bone remains unknown. In present study, we investigated the role and mechanism of CB1 on mitochondrial function and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) under inflammatory environment.MethodsAlkaline phosphatase (ALP) activity, alizarin red staining, quantitative calcium analysis, and osteogenic markers were used to detect the osteogenic differentiation ability of BMSCs. Real-time RT-PCR and Western blot were used to detect the gene expression. Seahorse Cell Mito Stress Test was used to detect the oxygen consumption rate (OCR). JC-10 assay was used to determine the mitochondrial membrane potential (MMP).ResultsCB1 increased osteogenic differentiation potential and mitochondrial energy metabolism, including the OCR, MMP, and enhanced the expressions of Nrf1 and Nrf2 in hBMSCs without or with TNF-α or INF-γ stimulation. Then, the inhibitor of mitochondrial electron transport chain (ETC), rotenone (ROT), inhibited the osteogenic differentiation in hBMSCs, and CB1 could rescue ROT impaired osteogenic differentiation potentials of hBMSCs without or with TNF-α or INF-γ stimulation. Activation of ETC by Coenzyme Q10 (CoQ10) could restore the impaired osteogenic differentiation of hBMSCs by depletion of CB1 without or with TNF-α or INF-γ stimulation. Mechanismly, CB1 could activate the JNK signaling pathway, p38 MAPK signaling pathway, and inhibit the Erk1/2 signaling pathway.ConclusionsThe activating of CB1 enhanced the osteogenic differentiation by rescuing the mitochondrial metabolism function in hBMSCs under the inflammatory environment, suggesting that CB1 is a potential target for enhancing bone regeneration under the inflammatory environment.

Circ_0019693 promotes osteogenic differentiation of bone marrow mesenchymal stem cell and enhances osteogenesis-coupled angiogenesis via regulating microRNA-942-5p-targeted purkinje cell protein 4 in the development of osteoporosis

ABSTRACT Circular RNA (circRNA) is a crucial regulator in multiple human diseases, including osteoporosis (OP). However, the function of numerous circRNAs remains unclear. This study aimed to explore the role and mechanism of circ_0019693 in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteogenesis-coupled angiogenesis. The expression of circ_0019693, miR-942-5p and purkinje cell protein 4 (PCP4) was measured using quantitative real-time PCR (qPCR) or Western blot. Osteogenic differentiation was monitored according to the protein levels of RUNX family transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) by Western blot analysis, and the activity of alkaline phosphatase (ALP). Angiogenesis was evaluated by tube formation assay. The targeting relationship between miR-942-5p and circ_0019693 or PCP4 was identified using pull-down, dual-luciferase reporter, and RNA immunoprecipitation assays. Circ_0019693 was downregulated in serum samples and bone tissues from OP patients relative to normal subjects. Circ_0019693 expression was enhanced in the stages of BMSC osteogenic differentiation. Circ_0019693 overexpression enhanced the activity of ALP and the expression of RUNX2, OPN and OCN, and its overexpression also promoted angiogenesis. However, circ_0019693 knockdown played the opposite effects. MiR-942-5p was ensured to be a target of circ_0019693, and miR-942-5p enrichment reversed the effects of circ_0019693. In addition, PCP4 was a target of miR-942-5p, and miR-942-5p inhibitor-promoted BMSC osteogenic differentiation and angiogenesis were partly repressed by PCP4 knockdown. In conclusion, circ_0019693 promotes BMSC osteogenic differentiation osteogenesis-coupled angiogenesis via regulating miR-942-5p-targeted PCP4, thus blocking the development of OP.

MicroRNA-149 suppresses osteogenic differentiation of mesenchymal stem cells via inhibition of AKT1-dependent Twist1 phosphorylation

Osteogenic differentiation is a vital process for growth, repair, and remodeling of bones. Accumulating evidence have suggested that microRNAs (miRNAs or miRs) play a crucial role in osteogenic differentiation of mesenchymal stem cells (MSCs). Hence, the current study set out to elucidate the role of miR-149 in osteogenic differentiation of MSCs and the underlying mechanism. First, rat models of bone differentiation were established using the Masquelet-induced membrane technique, and MSCs were isolated. The expression of miR-149 and AKT1 in the rats and cells was detected with RT-qPCR and western blot analysis. The relationships among miR-149, AKT1, and Twist1 were further predicted by online bioinformatics prediction and verified using dual luciferase reporter gene assay. Alteration of miR-149, AKT1, or Twist1 was performed to further explore their effect on osteogenic differentiation of MSCs. miR-149 was poorly expressed in the process of osteogenic differentiation of MSCs, while AKT1 was highly expressed. miR-149 negatively regulated the expression of AKT1, which in turn diminished the protein levels of Twist1 and promoted the phosphorylation levels of Twist1. Lastly, miR-149 acted as an inhibitor of osteogenic differentiation of MSCs, which could be reversed by AKT1. To sum up, miR-149 silencing promoted osteogenic differentiation of MSCs by enhancing Twist1 degradation through AKT1 upregulation, representing a new method for bone repair treatment.

YTHDC2 Inhibits Rat Bone Mesenchymal Stem Cells Osteogenic Differentiation by Accelerating RUNX2 mRNA Degradation Via m6A Methylation

YTHDC2 Inhibits Rat Bone Mesenchymal Stem Cells Osteogenic Differentiation by Accelerating RUNX2 mRNA Degradation Via m6A Methylation

Bone Regeneration and Angiogenesis by Co-transplantation of Angiotensin II–Pretreated Mesenchymal Stem Cells and Endothelial Cells in Early Steroid-Induced Osteonecrosis of the Femoral Head

Mesenchymal stem cells (MSCs) have been shown to exert a positive impact on osteonecrosis of the femoral head (ONFH) in preclinical experiments and clinical trials. After the femoral head suffers avascular necrosis, the transplanted MSCs undergo a great deal of stress-induced apoptosis and senescence in this microenvironment. So, survival and differentiation of MSCs in osteonecrotic areas are especially important in ONFH. Although MSCs and endothelial cells (ECs) co-culture enhancing proliferation and osteogenic differentiation of MSCs and form more mature vasculature in vivo, it remains unknown whether the co-culture cells are able to repair ONFH. In this study, we explored the roles and mechanisms of co-transplantation of angiotensin II (Ang II)-MSCs and ECs in repairing early ONFH. In vitro, when MSCs and ECs were co-cultured in a ratio of 5:1, both types of cells managed to proliferate and induce both osteogenesis and angiogenesis. Then, we established a rabbit model of steroid-induced ONFH and co-transplantation of Ang II-MSCs and ECs through the tunnel of core decompression. Four weeks later, histological and Western blot analyses revealed that ONFH treated with Ang II-MSCs and ECs may promote ossification and revascularization by increasing the expression of collagen type I, runt-related transcription factor 2, osteocalcin, and vascular endothelial growth factor in the femoral head. Our data suggest that co-transplantation of Ang II-MSCs and ECs was able to rescue the early steroid-induced ONFH via promoting osteogenesis and angiogenesis, which may be regarded as a novel therapy for the treatment of ONFH in a clinical setting.

Three-dimensional Printing of Biomimetic Titanium Mimicking Trabecular Bone Induces Human Mesenchymal Stem Cell Proliferation: An In-vitro Analysis.

In vitro analysis. The aim of this study was to assess the effect of three-dimensional (3D) printing of porous titanium on human mesenchymal stem cell (hMSC) adhesion, proliferation, and osteogenic differentiation. A proprietary implant using three-dimensional porous titanium (3D-pTi) that mimics trabecu-lar bone structure, roughness, porosity, and modulus of elasticity was created (Ti-LIFE technology™, Spineart SA Switzerland). Such implants may possess osteoinductive properties augmenting fusion in addition to their structural advantages. However, the ability of 3D-pTi to affect in vitro cellular proliferation and osteogenic differentiation remains undefined. Disks of 3D-pTi with a porosity of 70% to 75% and pore size of 0.9 mm were produced using additive manufacturing technology. 2D Ti6Al4V (2D-Ti) and 2D polyetheretherketone (2D-PEEK) disks were prepared using standard manufacturing process. Tissue culture plastic (TCP) served as the control surface. All discs were characterized using 2D-micros-copy, scanning electron microscopy (SEM), and x-ray micro-computed tomography. Forty thousand hMSCs were seeded on the disks and TCP and cultured for 42 days. hMSC morphology was assessed using environmental SEM and confocal imaging following phalloidin staining. hMSC proliferation was evaluated using DNA fluorescent assay. hMSC differentiation was assessed using RT-qPCR for genes involved in hMSC osteogenic differentiation and biochemical assays were performed for alkaline phosphatase activity (ALP) and calcium content. 3D-pTi lead to a higher cell number as compared to 2D-Ti and 2D-PEEK at D21, D28 and D42. ALP activity of hMSCs seeded into 3D-pTi scaffolds was as high as or higher than that of hMSCs seeded onto TCP controls over all time points and consistently higher than that of hMSCs seeded onto 2D-Ti scaffolds. However, when ALP activity was normalized to protein content, no statistical differences were found between all scaffolds tested and TCP controls. 3D-pTi provides a scaffold for bone formation that structurally mimics cancellous bone and improves hMSC adhesion and proliferation compared to 2D-Ti and PEEK.

Bioprinted Constructs that Mimic the Ossification Center Microenvironment for Targeted Innervation in Bone Regeneration

AbstractAlthough great progress has been made in engineered bone tissues, delayed or ineffective bone regeneration remains an issue due to the lack of neural network reconstruction in their design. Therefore, an engineered bone tissue construct that mimics the ossification center microenvironment to promote innervation is proposed. Based on this, the NGF@Lap constructs are constructed through bioprinting technology, which can release nerve growth factor (NGF) for a long time and simulate the ossification center's microenvironment with high expression NGF. In vitro, NGF@Lap‐GA can promote axonal extension. Meanwhile, the NGF and Laponite from the constructs can respectively promote the expression and secretion of calcitonin gene‐related peptide (CGRP) in sensory neurons. Further, the constructs show a CGRP‐dependent osteogenic and inhibition of adipogenesis, which is mainly regulated by AMP‐activated protein kinase‐peroxisome proliferator activated receptor pathway. In vivo, the constructs increased neurovascular network density in the tissue surrounding the implant, promoted bone marrow mesenchymal stem cells osteogenic differentiation, and effectively improved bone regeneration in the cranial defect model. In conclusion, the novel tissue‐engineered bone simulates the ossification center microenvironment, promotes innervation, and has promising potential for future application in bone regeneration.