Bone Marrow-derived Mesenchymal Stem Cells Osteogenesis Research Articles

Carboxypeptidase M modulates BMSCs osteogenesis-adipogenesis via the MAPK/ERK pathway: Anintegrated single-cell and bulk transcriptomic study.

The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. Invitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. Invivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP.

LncRNA SNHG14 activates autophagy via regulating miR-493-5p/Mef2c axis to alleviate osteoporosis progression

Osteoporosis is a progressive bone disease caused by impaired function of endogenous bone marrow-derived mesenchymal stem cells (BMSCs). Herein, we investigated the mechanism of lncRNA SNHG14 in osteoporosis progression. BMSCs were isolated from BALB/c mice. The osteogenic ability of BMSCs was assessed by Alkaline phosphatase (ALP) and Alizarin Red S Staining (ARS) staining. The interaction between miR-493-5p and SNHG14 or myocyte enhancer factor 2 C (Mef2c) was confirmed by dual-luciferase reporter assay. Bone histomorphometry changes were evaluated to analyze SNHG14’roles in osteoporosis in vivo. Our results illustrated SNHG14 and Mef2c levels were increased in a time-dependent manner in BMSCs, and miR-493-5p expression was decreased. SNHG14 knockdown inhibited osteogenic differentiation of BMSCs, and SNHG14 upregulation had the opposite effect. SNHG14 overexpression elevated bone mineral density and bone trabecular number, and alleviated osteoporosis progression in vivo. Mechanically, miR-493-5p was a target of SNHG14, and miR-493-5p targeted the Mef2c gene directly. SNHG14 overexpression reversed the inhibition of miR-493-5p on the osteogenic ability of BMSCs, and miR-493-5p silencing accelerated BMSCs osteogenesis by activating Mef2c-mediated autophagy to accelerate BMSCs osteogenesis. In short, SNHG14 activated autophagy via regulating miR-493-5p/Mef2c axis to alleviate osteoporosis progression, which might provide a new molecular target for osteoporosis treatment.

Stem Cell Self‐Triggered Regulation and Differentiation on Polyvinylidene Fluoride Electrospun Nanofibers

AbstractSmart electroactive materials that can dynamically regulate stem cell fate without external stimuli have fascinated increasing attention. Meanwhile, noninvasive electrical stimulation has emerged as a less restrictive approach, generating considerable interest in its potential biomedical applications. The intricate interplay between cells and materials within complex microenvironments includes encompassing substrate response, ion exchange, and membrane potential alterations. However, the mechanisms by which smart materials influence stem cells have yet to be fully elucidated. Herein, electrospinning technology is utilized to fabricate bone‐mimicking microenvironments comprising disordered and highly oriented polyvinylidene fluoride (PVDF) nanofibers. The aligned annealed PVDF (AA) and random annealed PVDF (RA) membranes present high fractions of β‐phase. By comparing the osteogenic ability, calcium activity, and F‐actin distribution of bone marrow‐derived mesenchymal stem cells (BMSCs) cultured with these PVDF nanofibers, it is proposed that the stem cells autonomously regulate their differentiation by remodeling the cytoskeleton on the electrospun membranes. Electrical stimulation, more adhesion area, and active calcium influx support the greater osteogenesis of BMSCs on RA than AA. This mechanism can provide a basic theory for the design and preparation of bone tissue engineering scaffolds and contribute to the further study of cells and microenvironments.

Geometric Angles and Gene Expression in Cells for Structural Bone Regeneration.

Geometry and angles play crucial roles in cellular processes; however, its mechanisms of regulation remain unclear. In this study, a series of three dimensional (3D)-printed microfibers with different geometries is constructed using a near-field electrostatic printing technique to investigate the regulatory mechanisms of geometry on stem cell function and bone regeneration. The scaffolds precisely mimicked cell dimensions with high porosity and interoperability. Compared with other spatial topography angles, microfibers with a 90° topology can significantly promote the expression of osteogenic gene proteins in bone marrow-derived mesenchymal stem cells (BMSCs). The effects of different spatial structures on the expression profiles of BMSCs differentiation genes are correlated and validated using microRNA sequencing. Enrichment analysis shows that the 90° microfibers promoted osteogenesis in BMSCs by significantly upregulating miR-222-5p/cbfb/Runx2 expression. The ability of the geometric architecture to promote bone regeneration, as assessed using the cranial defect model, demonstrates that the 90° fiber scaffolds significantly promote new bone regeneration and neovascular neural network formation. This study is the first to elucidate the relationship between angular geometry and cellular gene expression, contributing significantly to the understanding of how geometric architecture can promote stem cell differentiation, proliferation, and function for structural bone regeneration.

ANKRD1 activates the Wnt signaling pathway by modulating CAV3 expression and thus promotes BMSC osteogenic differentiation and bone formation in ovariectomized mice

Bone marrow-derived mesenchymal stem cells (BMSCs) are considered promising materials for treating bone diseases such as osteoporosis (OP). This research explored the functions and molecular mechanism of ankyrin repeat domain 1 (ANKRD1) in BMSC osteogenesis. An OP model in mice was established by bilateral ovariectomy. Manipulation of ANKRD1 expression in BMSCs or femurs was achieved by lentivirus infection. Increased ANKRD1 expression was observed in BMSCs during osteogenic induction. Silencing of ANKRD1 impaired the osteogenesis of BMSCs, as shown by the decreased alkaline phosphatase (ALP) activity, osteogenic gene (Runx2, Col1a1, Bglap, and Spp1) expression, and mineralized formation. ANKRD1-mediated promotion of osteogenesis was also reproduced in mouse MC3T3-E1 preosteoblastic cells. Activation of Wnt/β-catenin signaling, a well-known osteogenic stimulus, was also impaired in ANKRD1-silenced BMSCs. Overexpression of ANKRD1 resulted in the opposite effects on osteogenesis and Wnt/β-catenin signaling. Mechanistic studies revealed that ANKRD1 modulated caveolin-3 (CAV3) expression by reducing CAV3 ubiquitination, and the knockdown of CAV3 impaired the functions of ANKRD1. Additionally, a very low level of ANKRD1 was observed in the BMSCs from OP mice. Rescue of ANKRD1 significantly restored osteogenic differentiation and Wnt signaling activation in BMSCs from ovariectomized mice. The results of micro-CT, H&E staining, and IHC staining showed that ANKRD1 also promoted bone formation and Wnt activation and ameliorated pathological alterations in the femurs of OP mice. Collectively, this study demonstrated that ANKRD1 plays an important role in regulating the osteogenic differentiation of BMSCs and is a promising target for the treatment of OP and other bone diseases.

Mechanical force induces macrophage-derived exosomal UCHL3 promoting bone marrow mesenchymal stem cell osteogenesis by targeting SMAD1

BackgroundOrthodontic tooth movement (OTM), a process of alveolar bone remodelling, is induced by mechanical force and regulated by local inflammation. Bone marrow-derived mesenchymal stem cells (BMSCs) play a fundamental role in osteogenesis during OTM. Macrophages are mechanosensitive cells that can regulate local inflammatory microenvironment and promote BMSCs osteogenesis by secreting diverse mediators. However, whether and how mechanical force regulates osteogenesis during OTM via macrophage-derived exosomes remains elusive.ResultsMechanical stimulation (MS) promoted bone marrow-derived macrophage (BMDM)-mediated BMSCs osteogenesis. Importantly, when exosomes from mechanically stimulated BMDMs (MS-BMDM-EXOs) were blocked, the pro-osteogenic effect was suppressed. Additionally, compared with exosomes derived from BMDMs (BMDM-EXOs), MS-BMDM-EXOs exhibited a stronger ability to enhance BMSCs osteogenesis. At in vivo, mechanical force-induced alveolar bone formation was impaired during OTM when exosomes were blocked, and MS-BMDM-EXOs were more effective in promoting alveolar bone formation than BMDM-EXOs. Further proteomic analysis revealed that ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) was enriched in MS-BMDM-EXOs compared with BMDM-EXOs. We went on to show that BMSCs osteogenesis and mechanical force-induced bone formation were impaired when UCHL3 was inhibited. Furthermore, mothers against decapentaplegic homologue 1 (SMAD1) was identified as the target protein of UCHL3. At the mechanistic level, we showed that SMAD1 interacted with UCHL3 in BMSCs and was downregulated when UCHL3 was suppressed. Consistently, overexpression of SMAD1 rescued the adverse effect of inhibiting UCHL3 on BMSCs osteogenesis.ConclusionsThis study suggests that mechanical force-induced macrophage-derived exosomal UCHL3 promotes BMSCs osteogenesis by targeting SMAD1, thereby promoting alveolar bone formation during OTM.Graphical

DEPTOR exacerbates bone–fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation

Bone marrow–derived mesenchymal stem cells (BMSCs) tend to differentiate into adipocytes rather than osteoblasts in osteoporosis and other pathological conditions. Understanding the mechanisms underlying the adipo-osteogenic imbalance greatly contributes to the ability to induce specific MSC differentiation for clinical applications. This study aimed to explore whether DEP-domain containing mTOR-interacting protein (DEPTOR) regulated MSC fate and bone–fat switch, which was indicated to be a key player in bone homeostasis. We found that DEPTOR expression decreased during the osteogenesis of BMSCs but increased during adipogenesis and the shift of cell lineage commitment of BMSCs to adipocytes in mice with osteoporosis. DEPTOR facilitated adipogenic differentiation while preventing the osteogenic differentiation of BMSCs. Deptor ablation in BMSCs alleviated bone loss and reduced marrow fat accumulation in mice with osteoporosis. Mechanistically, DEPTOR binds transcriptional coactivator with a PDZ-binding motif (TAZ) and inhibits its transactivation properties, thereby repressing the transcriptional activity of RUNX2 and elevating gene transcription by peroxisome-proliferator-activated receptor-gamma. TAZ knockdown in BMSCs abolished the beneficial role of Deptor ablation in bone–fat balance in mice. Together, our data indicate that DEPTOR is a molecular rheostat that modulates BMSC differentiation and bone–fat balance, and may represent a potential therapeutic target for age-related bone loss.

A regulatory mechanism of a stepwise osteogenesis-mimicking decellularized extracellular matrix on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells.

A cell-derived decellularized extracellular matrix (dECM) plays a vital role in controlling cell functions because of its similarity to the in vivo microenvironment. In the process of stem cell differentiation, the composition of the dECM is not constant but is dynamically remolded. However, there is little information regarding the dynamic regulation by the dECM of the osteogenic differentiation of stem cells. Herein, four types of stepwise dECMs (0, 7, 14, and 21 d-ECM) were prepared from bone marrow-derived mesenchymal stem cells (BMSCs) undergoing osteogenic differentiation for 0, 7, 14, and 21 days after decellularization. In vitro experiments were designed to study the regulation of BMSC osteogenesis by dECMs. The results showed that all the dECMs could support the activity and proliferation of BMSCs but had different effects on their osteogenic differentiation. The 14d-ECM promoted the osteogenesis of BMSCs significantly compared with the other dECMs. Proteomic analysis demonstrated that the composition of dECMs changed over time. The 14d ECM had higher amounts of collagen type IV alpha 2 chain (COL4A2) than the other dECMs. Furthermore, COL4A2 was obviously enriched in the activated focal adhesion kinase (FAK)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Thus, the 14d-ECM could promote the osteogenic differentiation of BMSCs, which might be related to the high content of COL4A2 in the 14d-ECM by activating the FAK/PI3K/AKT signaling pathways.

Fluid shear stress and endothelial cells synergistically promote osteogenesis of mesenchymal stem cells via integrin β1-FAK-ERK1/2 pathway.

Prevascularization and mechanical stimulation have been reported as effective methods for the construction of functional bone tissue. However, their combined effects on osteogenic differentiation and its mechanism remain to be explored. Here, the effects of fluid shear stress (FSS) on osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (BMSCs) when cocultured with human umbilical vein endothelial cells (HUVECs) were investigated, and underlying signaling mechanisms were further explored. FSS stimulation for 1–4 h/day increased alkaline phosphatase (ALP) activity and calcium deposition in coculture systems and promoted the proliferation of cocultured cells. FSS stimulation for 2 h/day was selected as the optimized protocol according to osteogenesis in the coculture. In this situation, the mRNA levels of ALP, runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and protein levels of OCN and osteopontin (OPN) in BMSCs were upregulated. Furthermore, FSS and coculture with HUVECs synergistically increased integrin β1 expression in BMSCs and further activated focal adhesion kinases (FAKs) and downstream extracellular signal-related kinase (ERK), leading to the enhancement of Runx2 expression. Blocking the phosphorylation of FAK abrogated FSS-induced ERK phosphorylation and inhibited osteogenesis of cocultured BMSCs. These results revealed that FSS and coculture with HUVECs synergistically promotes the osteogenesis of BMSCs, which was mediated by the integrin β1-FAK-ERK signaling pathway.

3D Printed Gelatin/Sodium Alginate Hydrogel Scaffolds Doped with Nano-Attapulgite for Bone Tissue Repair.

IntroductionBone tissue engineering (BTE) is a new strategy for bone defect repair, but the difficulties in the fabrication of scaffolds with personalized structures still limited their clinical applications. The rapid development in three-dimensional (3D) printing endows it capable of controlling the porous structures of scaffolds with high structural complexity and provides flexibility to meet specific needs of bone repair.MethodsIn this study, sodium alginate (SA)/gelatin (Gel) hydrogel scaffolds doped with different contents of nano-attapulgite were fabricated via 3D printing. The surface microstructure, hydrophilicity and mechanical properties were fully evaluated. Furthermore, mouse bone marrow-derived mesenchymal stem cells (BMSCs) were cultured with the composite hydrogels in vitro, and proliferation and osteoblastic differentiation were assessed. A rabbit tibia plateau defect model was used to evaluate the osteogenic potential of the composite hydrogel in vivo.ResultsWhen increasing nano-ATP content, the Gel/SA/nano-ATP composite hydrogels showed better mechanical property and printability. Moreover, Gel/SA/nano-ATP composite hydrogels showed excellent bioactivity, and a significant mineralization effect was observed on the surface after being incubated in simulated body fluid (SBF) for 14 days. The Gel/SA/nano-ATP composite hydrogel also showed good biocompatibility and promoted the osteogenesis of BMSCs. Finally, histological analysis demonstrates that the Gel/SA/nano-ATP composite hydrogels could effectively enhance bone regeneration in vivo.ConclusionThese properties render the Gel/SA/nano-ATP composite hydrogel scaffolds an ideal bone tissue engineering material for the repair of bone defects.

Long noncoding RNA ZFAS1 suppresses osteogenic differentiation of bone marrow-derived mesenchymal stem cells by upregulating miR-499-EPHA5 axis

Emerging evidence suggests that the shift between osteogenic and adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) determines bone mass. Our study was aimed at testing whether a long noncoding RNA called zinc finger antisense 1 (ZFAS1) participates in the differentiation commitment of BMSCs during osteoporosis. We found that ZFAS1 expression was downregulated during osteogenic differentiation and upregulated during adipogenic differentiation. ZFAS1 knockdown facilitated osteogenic differentiation and suppressed adipogenic differentiation. Furthermore, ZFAS1 knockdown suppressed cell senescence and promoted autophagy. Ovariectomized mice injected with a ZFAS1 knockdown construct showed increased bone mass. Mechanismly, ZFAS1 affected the osteogenic and adipogenic differentiation of BMSCs through sponging miR-499 thereby upregulating ephrin type-A receptor 5 (EPHA5). Taken together, our results revealed that the ZFAS1–miR-499–EPHA5 axis may be important for the osteoporosis-related switch between the osteogenesis and adipogenesis of BMSCs, indicating that ZFAS1 represents a plausible therapeutic target for reversing osteoporotic bone loss.

The let-7f-5p-Nme4 pathway mediates tumor necrosis factor α-induced impairment in osteogenesis of bone marrow-derived mesenchymal stem cells.

Although tumor necrosis factor α (TNF-α)-mediated inflammation significantly impacts osteoporosis, the mechanisms underlying the osteogenic differentiation defects of bone marrow-derived mesenchymal stem cells (BM-MSCs) caused by TNF-α remain poorly understood. We found that TNF-α stimulation of murine BM-MSCs significantly upregulated the expression levels of several microRNAs (miRNAs), including let-7f-5p, but this increase was significantly reversed by treatment with the kinase inhibitor BAY 11-7082. To study gain- or loss of function, we transfected cells with an miRNA inhibitor or miRNA mimic. We then demonstrated that let-7f-5p impaired osteogenic differentiation of BM-MSCs in the absence and presence of TNF-α, as evidenced by alkaline phosphatase and alizarin red staining as well as quantitative assays of the mRNA levels of bone formation marker genes in differentiated BM-MSCs. Moreover, let-7f-5p targets the 3' untranslated region of Nucleoside diphosphate kinase 4 (Nme4) mRNA and negatively regulates Nme4 expression in mouse BM-MSCs. Ectopic expression of Nme4 completely reversed the inhibitory effects of the let-7f-5p mimic on osteogenic differentiation of mouse BM-MSCs. Furthermore, inhibition of let-7f-5p or overexpression of Nme4 in BM-MSCs restored in-vivo bone formation in an ovariectomized animal model. Collectively, our work indicates that let-7f-5p is involved in TNF-α-mediated reduction of BM-MSC osteogenesis via targeting Nme4.

Ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss

N-acetyltransferase 10 (NAT10) is the key enzyme for N4-acetylcytidine (ac4C) modification of mRNA, which participates in various cellular processes and is related to many diseases. Here, we explore the relationships among osteoblast differentiation, NAT10, and ac4C, and we found that NAT0 expression and the ac4C level of total RNA were decreased in the bone tissues of bilateral ovariectomized (OVX) mice and osteoporosis patients. Adenoviruses overexpressing NAT10 reversed bone loss, and Remodelin, an NAT10 inhibitor, enhanced the loss of bone mass in OVX mice. Moreover, bone marrow-derived mesenchymal stem cells (BMSCs) with low-level ac4C modification formed fewer calcium nodules in vitro with NAT10 silencing, whereas BMSCs with high-level ac4C modification formed more calcium nodules with NAT10 overexpression. Moreover, we demonstrated that the ac4C level of runt-related transcription factor 2 (RUNX2) mRNA was increased after BMSCs were cultured in osteogenic medium (OM) and decreased after NAT10 silencing. The RUNX2 mRNA half-life and protein expression decreased after silencing NAT10 in BMSCs. Therefore, NAT10-based ac4C modification promotes the osteogenic differentiation of BMSCs by regulating the RUNX2 ac4C level. Because abnormal levels of NAT10 are probably one of the mechanisms responsible for osteoporosis, NAT10 is a new potential therapeutic target for this disease.

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

IRF2-mediated upregulation of lncRNA HHAS1 facilitates the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by acting as a competing endogenous RNA.

BackgroundMesenchymal stem cells (MSCs) are the major source of osteoblasts. Long noncoding RNAs (lncRNAs) are abundantly expressed RNAs that lack protein‐coding potential and play an extensive regulatory role in cellular biological activities. However, the regulatory network of lncRNAs in MSC osteogenesis needs further investigation.MethodsQRT‐PCR, western blot, immunofluorescence, and immunohistochemistry assays were used to determine the levels of relevant genes. The osteogenic differentiation capability was evaluated by using Alizarin Red S (ARS) staining, alkaline phosphatase activity assays, hematoxylin & eosin staining or micro‐CT. RNA fluorescence in situ hybridization (FISH) and RNAscope were used to detect HHAS1 expression in cells and bone tissue. A microarray assay was performed to identify differentially expressed microRNAs. RNA immunoprecipitation and RNA pull‐down were used to explore the interactions between related proteins and nucleic acids.ResultsThe level of lncRNA HHAS1 increased during bone marrow‐derived MSC (BMSC) osteogenesis and was positively related to the levels of osteogenic genes and ARS intensity. HHAS1 was located in both the cytoplasm and the nucleus and was expressed in human bone tissue. HHAS1 facilitated BMSC osteogenic differentiation by downregulating miR‐204‐5p expression and enhancing the level of RUNX family transcription factor 2 (RUNX2). In addition, interferon regulatory factor 2 (IRF2) was increased during BMSC osteogenic differentiation and interacted with the promoter of HHAS1, which resulted in the transcriptional activation of HHAS1. Furthermore, IRF2 and HHAS1 helped improve bone defect repair in vivo.ConclusionsOur study identified a novel lncRNA, HHAS1, that facilitates BMSC osteogenic differentiation and proposed a role for the IRF2/HHAS1/miR‐204‐5p/RUNX2 axis in BMSC osteogenesis regulation. These findings help elucidate the regulatory network of BMSC osteogenesis and provide potential targets for clinical application.

CGRP Regulates the Age-Related Switch Between Osteoblast and Adipocyte Differentiation.

Osteoporosis is a chronic age-related disease. During aging, bone marrow-derived mesenchymal stem cells (BMSCs) display increased adipogenic, along with decreased osteogenic, differentiation capacity. The aim of the present study was to investigate the effect of calcitonin gene-related peptide (CGRP) on the osteogenic and adipogenic differentiation potential of BMSC-derived osteoblasts. Here, we found that the level of CGRP was markedly lower in bone marrow supernatant from aged mice compared with that in young mice. In vitro experiments indicated that CGRP promoted the osteogenic differentiation of BMSCs while inhibiting their adipogenic differentiation. Compared with vehicle-treated controls, aged mice treated with CGRP showed a substantial promotion of bone formation and a reduction in fat accumulation in the bone marrow. Similarly, we found that CGRP could significantly enhance bone formation in ovariectomized (OVX) mice in vivo. Together, our results suggested that CGRP may be a key regulator of the age-related switch between osteogenesis and adipogenesis in BMSCs and may represent a potential therapeutic strategy for the treatment of age-related bone loss.

Approaches to promoting bone marrow mesenchymal stem cell osteogenesis on orthopedic implant surface.

Bone marrow-derived mesenchymal stem cells (BMSCs) play a critical role in the osseointegration of bone and orthopedic implant. However, osseointegration between the Ti-based implants and the surrounding bone tissue must be improved due to titanium’s inherent defects. Surface modification stands out as a versatile technique to create instructive biomaterials that can actively direct stem cell fate. Here, we summarize the current approaches to promoting BMSC osteogenesis on the surface of titanium and its alloys. We will highlight the utilization of the unique properties of titanium and its alloys in promoting tissue regeneration, and discuss recent advances in understanding their role in regenerative medicine. We aim to provide a systematic and comprehensive review of approaches to promoting BMSC osteogenesis on the orthopedic implant surface.

Cordycepin promotes osteogenesis of bone marrow-derived mesenchymal stem cells and accelerates fracture healing via hypoxia in a rat model of closed femur fracture

Fracture is the most frequently encountered traumatic large-organ injury observed in human patients. Cordycepin possesses beneficial effects in osteogenesis of mesenchymal stem cells (MSCs), but its effect on fracture healing is largely unknown. A rat model of closed femur fracture was established, and treated with therapy using bone marrow-derived MSCs (BMMSCs). The effect of cordycepin on the osteogenic process of BMMSCs in vitro was evaluated by Alizarin Red S (ARS) staining and expressions of osteogenic marker genes. Radiographic evaluations and four-point bending mechanical testing were performed on model rats after BMMSC treatment, to assess the effect of cordycepin on fracture healing. Cordycepin promoted osteogenesis of BMMSCs in vitro, and enhanced radiographic parameters and mechanical properties in rat closed femur fracture model using BMMSC therapy in vivo. A hypoxia inhibitor echinomycin could negate the above-mentioned therapeutic effects of cordycepin, indicating that the beneficial effects of cordycepin were mediated via hypoxic response pathway. This study demonstrates that cordycepin promotes osteogenesis of BMMSCs and accelerates fracture healing via hypoxia in a rat model of closed femur fracture, and proposes the clinical potential of cordycepin in bone fracture treatments.

Targeted silencing of miRNA-132-3p expression rescues disuse osteopenia by promoting mesenchymal stem cell osteogenic differentiation and osteogenesis in mice

BackgroundSkeletal unloading can induce severe disuse osteopenia that often occurs in spaceflight astronauts or in patients subjected to prolonged bed-rest or immobility. Previously, we revealed a mechano-sensitive factor, miRNA-132-3p, that is closely related to the osteoblast function. The aim of this study was to investigate whether miRNA-132-3p could be an effective target for treating disuse osteopenia.MethodsThe 2D-clinostat device and the hindlimb-unloaded (HU) model were used to copy the mechanical unloading condition at the cellular and animal levels, respectively. Mimics or inhibitors of miRNA-132-3p were used to interfere with the expression of miRNA-132-3p in bone marrow-derived mesenchymal stem cells (BMSCs) in vitro for analyzing the effects on osteogenic differentiation. The special in vivo antagonists of miRNA-132-3p was delivered to the bone formation regions of HU mice for treating disuse osteopenia by a bone-targeted (AspSerSer)6-cationic liposome system. The bone mass, microstructure, and strength of the hindlimb bone tissue were analyzed for evaluating the therapeutic effect in vivo.ResultsmiRNA-132-3p expression was declined under normal conditions and increased under gravitational mechanical unloading conditions during osteogenic differentiation of BMSCs in vitro. The upregulation of miRNA-132-3p expression resulted in the inhibition of osteogenic differentiation, whereas the downregulation of miRNA-132-3p expression enhanced osteogenic differentiation. The inhibition of miRNA-132-3p expression was able to attenuate the negative effects of mechanical unloading on BMSC osteogenic differentiation. Most importantly, the targeted silencing of miRNA-132-3p expression in the bone tissues could effectively preserve bone mass, microstructure, and strength by promoting osteogenic differentiation and osteogenesis in HU mice.ConclusionThe overexpression of miRNA-132-3p induced by mechanical unloading is disadvantageous for BMSC osteogenic differentiation and osteogenesis. Targeted silencing of miRNA-132-3p expression presents a potential therapeutic target for the prevention and treatment of disuse osteoporosis.

Foxf1 knockdown promotes BMSC osteogenesis in part by activating the Wnt/β-catenin signalling pathway and prevents ovariectomy-induced bone loss.

Forkhead box protein f1 (Foxf1) is associated with cell differentiation, and may be a key player in bone homoeostasis. However, the effect of Foxf1 on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) and ovariectomy-induced bone loss, as well as its clinical implications, is unknown. By quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, we assayed Foxf1 expression in bone tissue, BMSCs, and bone marrow-derived macrophages (BMMs), derived from ovariectomised (OVX) mice, and during osteogenic differentiation and osteoclast differentiation. Using a loss-of-function approach (small interfering RNA [siRNA]-mediated knockdown) in vitro, we examined whether Foxf1 regulates osteoblast differentiation of BMSCs via the Wnt/β-catenin signalling pathway. Furthermore, we assessed the anabolic effect of Foxf1 knockdown (siFoxf1) in OVX mice in vivo. We also assayed the expression of Foxf1 in bone tissue derived from postmenopausal osteoporosis (PMOP) patients and its link with bone mineral density (BMD). Finally, we examined the effect of Foxf1 knockdown on the osteoblastic differentiation of human BMSCs. Foxf1 expression was significantly increased in bone extract and BMSCs from OVX mice and gradually decreased during osteoblastic differentiation of BMSCs but did not differ significantly in OVX mouse-derived BMMs or during osteoclast differentiation. In vitro, Foxf1 knockdown markedly increased the expression of osteoblast specific genes, alkaline phosphatase (ALP) activity, and mineralisation. Moreover, siFoxf1 activated the Wnt/β-catenin signalling pathway. The siFoxf1-induced increase in osteogenic differentiation was partly rescued by inhibitor of Wnt signalling (DKK1). In OVX mice, Foxf1 siRNA significantly reduced bone loss by enhancing bone formation. Foxf1 expression levels negatively correlated with reduced bone mass and bone formation in bone tissue from PMOP patients. Finally, Foxf1 knockdown significantly promoted osteogenesis by human BMSCs. Our findings indicate that Foxf1 knockdown promotes BMSC osteogenesis and prevents OVX-induced bone loss. Therefore, Foxf1 has potential as a biomarker of osteogenesis and may be a therapeutic target for PMOP.