Mouse Bone Marrow Mesenchymal Stem Research Articles

Transplanted MSCs promote alveolar bone repair via hypoxia-induced extracellular vesicle secretion.

Mesenchymal stem cell (MSC) therapy is a potential strategy for promoting alveolar bone regeneration. This study evaluated the effects and mechanisms of transplanted MSCs on alveolar bone repair. Mouse alveolar bone defect model was treated using mouse bone marrow mesenchymal stem cell (BMSC) transplantation. The bone repair was evaluated by micro-CT and Masson staining. The conditioned medium of hypoxia-treated BMSCs was co-cultured with normal BMSCs invitro to detect the regulatory effect of transplanted MSCs on the chemotactic and migratory functions of host cells. The mechanisms were investigated using Becn siRNA transfection and western blotting. BMSC transplantation promoted bone defect regeneration. The hypoxic microenvironment induces BMSCs to release multiple extracellular vesicle (EV)-mediated regulatory proteins that promote the migration of host stem cells. Protein array analysis, western blotting, GFP-LC3 detection, and Becn siRNA transfection confirmed that autophagy activation in BMSCs plays a key role during this process. The local hypoxic microenvironment induces transplanted MSCs to secrete a large number of EV-mediated regulatory proteins, thereby upregulating the migration function of the host stem cells and promoting alveolar bone defect regeneration. This process depends on the autophagy-related mechanism of the transplanted MSCs.

Study on the mechanism of magnesium calcium alloys/mineralized collagen composites mediating macrophage polarization to promote bone repair

Magnesium-based composites are a focal point in biomaterials research. However, the rapid degradation rate of magnesium alloys does not align with the healing time of bone tissue. Additionally, the host reaction caused by magnesium implantation hampers its full osteogenic potential. To maintain an appropriate microenvironment, it is important to enhance both corrosion resistance and osteogenic activity of the magnesium matrix. In this study, a composite scaffold composed of mineralized collagen and magnesium alloy was utilized to investigate the regulatory effect of mineralized collagen on mouse macrophages and evaluate its impact on mouse bone marrow mesenchymal stem cells in terms of osteogenesis, immune response, and macrophage-induced osteogenic differentiation. This experiment examined the biocompatibility of mouse bone marrow mesenchymal stem cells and macrophage-induced osteogenic differentiation in vitro, and examined the expression levels of relevant pathways proteins. Magnesium calcium alloys/mineralized collagen exhibited extensive spreading, facilitated by broad and abundant pseudopodia that firmly adhered them to the material surface and promoted growth and pseudopodia formation. The findings revealed that magnesium calcium alloy/mineralized collagen scaffold materials induced osteogenic differentiation mainly through M2 polarization of macrophages. This effect was mainly mediated by promoting the integrin α2β1-FAK-ERK1/2 signaling pathways and inhibiting the RANK signaling pathways.

Bone-Differentiation-Associated Circ-Spen Regulates Death of Mouse Bone Marrow Mesenchymal Stem Cells by Inhibiting Apoptosis and Promoting Autophagy.

The role of estrogen receptor β (ERβ) in bone health is closely associated with its function in vivo, and ERβ-/- mice have been widely utilized to explore the related influences. In this study, ERβ-/- female mice were established to investigate the differential expression of circular RNAs (circRNAs) by RNA-Sequencing (RNA-Seq). Among these circRNAs, mmu_circ_0011379 (named Circ-Spen) exhibited high expression in ERβ-/- female mice. However, the precise mechanism by which Circ-Spen regulates bone health remained unclear. This study identified Circ-Spen as a positive regulator of mouse bone marrow mesenchymal stem cell (mBMSC) viability. The expression of Circ-Spen was markedly increased in ERβ-/- mice femurs tested by RT-qPCR. Moreover, Circ-Spen exhibited an enhanced expression during the bone formation process of mBMSCs. Qualitative experiments also demonstrated that Circ-Spen possessed a circular structure and was localized within the nucleus of mBMSCs. Functionally, it inhibited apoptosis via caspase-3, BCL-2, and BAX, while also promoting autophagy through BECN1 and P62 in mBMSCs tested by MTT assays, transmission electron microscopy (TEM), and Western blotting. These findings reveal the potential of targeting Circ-Spen as a promising therapeutic strategy for rejuvenating senescent mBMSCs and enhancing the efficiency of mBMSC transplantation, which lays the foundation for advancements in the field of bone therapy.

Whole-Transcriptome Analysis Reveals the RNA Profiles in Mouse Bone Marrow Mesenchymal Stem Cells or Zebrafish Embryos After Exposure to Environmental Level of Tributyltin.

To understand the underlying molecular mechanisms, mouse bone marrow mesenchymal stem cells (BMSCs) and zebrafish embryos were exposed to the control group and Tributyltin (TBT) group (10 ng/L, environmental concentration) for 48h, respectively. The expression profiles of RNAs were investigated using whole-transcriptome analysis in mouse BMSCs or zebrafish embryos after TBT exposure. For mouse BMSCs, the results showed 2,449 differentially expressed (DE) mRNAs, 59 DE miRNAs, 317 DE lncRNAs, and 15 circRNAs. Similarly, for zebrafish embryos, the results showed 1,511 DE mRNAs, 4 DE miRNAs, 272 DE lncRNAs, and 28 circRNAs. According to KEGG pathway analysis showed that DE RNAs were mainly associated with immune responses, signaling, and cellular interactions. Competing endogenous RNA (ceRNA) network analysis revealed that the regulatory network of miRNA-circRNA constructed in zebrafish embryos was more complex compared to that of mouse BMSCs.

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

PH-Responsive nanoplatform synergistic gas/photothermal therapy to eliminate biofilms in poly(L-lactic acid) scaffolds.

To date, implant-associated infection is still a significant clinical challenge, which cannot be effectively eliminated by single therapies due to the formation of microbial biofilms. Herein, a pH-responsive nanoplatform was constructed via the in situ growth of zinc sulfide (ZnS) nanoparticles on the surface of Ti3C2 MXene nanosheets, which was subsequently introduced in poly(L-lactic acid) (PLLA) to prepare a composite bone scaffold via selective laser sintering technology. In the acidic biofilm microenvironment, the degradation of ZnS released hydrogen sulfide (H2S) gas to eliminate the biofilm extracellular DNA (eDNA), thus destroying the compactness of the biofilm. Then, the bacterial biofilm became sensitive to hyperthermia, which could be further destroyed under near-infrared light irradiation due to the excellent photothermal property of MXene, finally achieving gas/photothermal synergistic antibiofilm and efficient sterilization. The results showed that the synergistic gas/photothermal therapy for the composite scaffold not only evidently inhibited the formation of biofilms, but also effectively eradicated the eDNA of the already-formed biofilms and killed 90.4% of E. coli and 84.2% of S. aureus under near infrared light irradiation compared with single gas or photothermal therapy. In addition, the composite scaffold promoted the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Thus, the designed scaffold with excellent biofilm elimination and osteogenesis ability has great potential as an alternative treatment for implant-associated bone infections.

MiR-155-5p/Bmal1 Modulates the Senescence and Osteogenic Differentiation of Mouse BMSCs through the Hippo Signaling Pathway.

The core clock gene brain and muscle ARNT like-1 (Bmal1) is involved in the regulation of bone tissue aging. However, current studies are mostly limited to the establishment of the association between Bmal1 and bone senescence, without in-depth exploration of its main upstream and downstream regulatory mechanisms. The luciferase reporter assay, RT-qPCR and Western blotting were performed to detect the interaction between miR-155-5p and Bmal1. The effects of miR-155-5p and Bmal1 on the aging and osteogenic differentiation ability of mouse bone marrow mesenchymal stem cells (BMSCs) were investigated by cell counting kit-8 (CCK-8) assay, flow cytometry, β-gal staining, alkaline phosphatase quantitative assay and alizarin red staining in vitro. The potential molecular mechanism was identified by ChIP-Seq, RNA-seq database analysis and immunofluorescence staining. The expression of Bmal1 declined with age, while the miR-155-5p was increased. miR-155-5p and Bmal1 repressed each other's expression, and miR-155-5p targeted the Bmal1. Besides, miR-155-5p inhibited the proliferation and osteogenic differentiation of BMSCs, promoted cell apoptosis and senescence, inhibited the expression and nuclear translocation of YAP and TAZ. However, Bmal1 facilitated the osteogenic differentiation and suppressed the aging of BMSCs, meanwhile inactivated the Hippo pathway. Moreover, YAP inhibitors abrogated the positive regulation of aging and osteogenic differentiation in BMSCs by miR-155-5p and Bmal1. In mouse BMSCs, miR-155-5p and Bmal1 regulated the aging and osteogenic differentiation ability of BMSCs mainly through the Hippo signaling pathway. Our findings provide new insights for the interventions in bone aging.

Biomimetic Hydroxyapatite on 3D-Printed Nanoattapulgite/Polycaprolactone Scaffolds for Bone Regeneration of Rat Cranium Defects.

Nanoattapulgite (nano-ATP), a magnesium-aluminum silicate clay, can absorb substances and is a suitable material for bone repair and regeneration. In this study, using three-dimensional printing technology, a nano-ATP/polycaprolactone (PCL) scaffold was fabricated and modified using NaOH to form a rough surface. Biomimetic hydroxyapatite (HA) on nano-ATP/PCL scaffolds was fabricated using a biomineralized approach. The scaffold provided structural support through PCL and was modified with ATP and HA to improve hydrophilicity and promote the delivery of nutrients. The biocompatibility and osteogenic induction of scaffolds were assessed in vitro using mouse bone marrow mesenchymal stem cells. According to the in vitro study results, the nano-ATP/PCL/HA composite scaffold significantly boosted the expression levels of genes related to osteogenesis (p < 0.05), attributed to its superior alkaline phosphatase activity and calcium deposition capabilities. The outcomes of in vivo experimentation demonstrated an augmentation in bone growth at the rat cranial defect site when treated with the ATP/PCL/HA composite scaffold. It can be inferred from the results that the implementation of ATP and HA for the bone tissue engineering repair material displays encouraging prospects.

Enhancing mechanical property, osteogenesis and angiogenesis of 3D-plotted β-tricalcium phosphate bioceramic scaffolds incorporated with magnesium silicate: In Vitro and In Vivo study

The balance between porosity and compressive strength, as well as the pro-osteogenic and pro-angiogenesis effects of β-tricalcium phosphate (β-TCP) are not satisfactory in the clinical application even though it has good biocompatibility, osseointegration and osteoconductivity. Herein, different contents (10, 20 and 30 wt%) of magnesium silicate (Mg2SiO4, MS) and β-TCP composite bioceramic scaffolds (MS/β-TCP) were fabricated by three-dimensional fiber deposition (3DF) technology. The physicochemical, in vitro osteogenic and angiogenic properties, the early (6 weeks) cranium defect repair effects in rats and the underlying molecular mechanism on accelerating osteogenic differentiation of MS/β-TCP were systematically investigated. The results showed that MS compounding not only promoted the sintering of β-TCP and significantly enhanced the compressive strength, but also greatly improved its osteogenic and angiogenic performances, such as the viability, adhesion, and proliferation of mouse bone marrow mesenchymal stem cells (mBMSCs) and human umbilical vein endothelial cells (HUVECs), ALP activity and the expression of adhesion-related, osteogenesis-related and angiogenesis-related genes. The in vivo experiments presented that 10MS/β-TCP had more new bone formation and better angiogenesis compared to β-TCP and Blank. Analysis of the transcriptome sequencing results confirmed that the Wnt signaling pathway was involved in the regulation of osteogenic differentiation in mBMSCs by the MS/β-TCP composite bioceramic scaffold. The MS/β-TCP holds great potential in bone repair and regeneration.

Preparation, characterization, and in vitro biological evaluation of salicylic acid‐modified polyphenylene sulfide/quartz fiber/hydroxyapatite composite with high mechanical property for bone replacement

AbstractThe surface inertness of pure polyphenylene sulfide (PPS) limits its application in orthopedic implant materials. On that basis, modified PPS containing hydroxyl and carboxyl groups, that is, SAPPS was synthesized by nucleophilic substitution of dihalide monomers and sodium sulfide under high pressure. And consequently, a kind of ternary composite consisting of polyphenylene sulfide salicylate/quartz fiber/hydroxyapatite (SAPPS/QF/HA) was developed. The structure, composition, cross section morphology, degradation behavior, mechanical property, and biological property of all the composites were characterized by Fourier transform infrared spectroscopy, x‐ray diffraction, differential scanning calorimetry, thermogravimetric analyzer, scanning electron microscope, energy dispersive spectrometer, and in vitro cytocompatibility tests. The composites exhibited good thermal property, which achieved a high melting point (277.8°C) and decomposition temperature (493.9–502.7°C at 5% weight loss). When the mass fraction of 20 wt% QF and 20 wt% HA was added in the pure SAPPS, the bending yield strength and bending modulus of the composite (SAPPS/20QF/20HA) were 177.4 MPa and 8.03 GPa, respectively, which increased by 61.6% and 158.2% compared with that of the pure SAPPS. Observing the considerable proliferation and adhesion of mouse bone marrow mesenchymal stem cells (mBMSCs) in the experiment, we demonstrated that the SAPPS and its composites were non‐toxic, and the SAPPS/QF/20HA composites had the most prominent effect in promoting the proliferation and adhesion of mBMSCs.Highlights Salicylic acid‐modified polyphenylene sulfide was studied as a biomaterial. The composite material has mechanics matching with human cortical bone. Cell experiments showed that all materials were nontoxic.

RIOK3 potentially regulates osteogenesis-related pathways in ankylosing spondylitis and the differentiation of bone marrow mesenchymal stem cells

RNA-binding proteins (RBPs), which are key effectors of gene expression, play critical roles in inflammation and immune regulation. However, the potential biological function of RBPs in ankylosing spondylitis (AS) remains unclear. We identified differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) of five patients with AS and three healthy persons by RNA-seq, obtained differentially expressed RBPs by overlapping DEGs and RBPs summary table. RIOK3 was selected as a target RBP and knocked down in mouse bone marrow mesenchymal stem cells (mBMSCs), and transcriptomic studies of siRIOK3 mBMSCs were performed again using RNA-seq. Results showed that RIOK3 knockdown inhibited the expression of genes related to osteogenic differentiation, ribosome function, and β-interferon pathways in mBMSCs. In vitro experiments have shown that RIOK3 knockdown reduced the osteogenic differentiation ability of mBMSCs. Collectively, RIOK3 may affect the differentiation of mBMSCs and participate in the pathogenesis of AS, especially pathological bone formation.

Isoliquiritin treatment of osteoporosis by promoting osteogenic differentiation and autophagy of bone marrow mesenchymal stem cells.

Osteoporosis is a chronic progressive bone disease characterized by the decreased osteogenic ability of osteoblasts coupled with increased osteoclast activity. Natural products showing promising therapeutic potential for postmenopausal osteoporosis remain underexplored. In this study, we aimed to analyze the therapeutic effects of isoliquiritin (ISL) on osteoporosis in mice and its possible mechanism of action. An ovariectomy-induced osteoporosis mouse model and bone marrow mesenchymal stem cells (BMSCs) were used to analyze the effects of ISL on bone regeneration in vivo and in vitro, respectively. Mitogen-activated protein kinase (MAPK) and autophagy inhibitors were used, to investigate whether the MAPK signaling pathway and autophagy affect the osteogenic differentiation of BMSCs. ISL significantly improved bone formation and reduced bone resorption in mouse femurs without inducing any detectable toxicity in critical organs such as the liver, kidney, brain, heart, and spleen. In vitro experiments showed that ISL enhanced the proliferation and osteogenic differentiation of BMSCs and that its osteogenic effect was attenuated by p38/extracellular regulated protein kinase (ERK) and autophagy inhibitors. Further studies showed that the inhibition of phosphorylated p38/ERK blocked ISL autophagy in BMSCs. ISL promoted the osteogenic differentiation of BMSCs through the p38/ERK-autophagy pathway and was therapeutically effective in treating osteoporosis in ovariectomized mice without any observed toxicity to vital organs. These results strongly suggest the promising potential of ISL as a safe and efficacious candidate drug for the treatment of osteoporosis.

Electrospun sandwich mesh structures loaded with naringenin and vitamin K2 polycaprolactone/gelatin nanofibers synergistically promote bone regeneration

Osteoblasts and osteoclasts play a crucial role in the dynamically coupled balance during bone regeneration and remodeling. They complement and restrict each other in the human body. Decreased osteoblasts lead to insufficient bone formation or excessive formation of osteoclasts, leading to increased bone resorption, which will destroy the structure of the bone tissue. This will greatly increase the risk of diseases such as osteoporosis and nonunions caused by bone defects.Herein, gelatin and polycaprolactone were used as substrates, and biomaterial membranes with mesh and sandwich structures were constructed using the electrospinning technology. Naringenin was loaded into the shell, and vitamin K2 was loaded into the core layer of the nanofibrous membrane. The biocompatibility and osteogenic capacity of the membranes were assessed in vitro using mouse bone marrow mesenchymal stem cells (BMSCs). During osteoclast induction, the receptor activator of nuclear factor kappa-Β ligand (RANKL) was used to coculture RAW264.7 cells with various materials. The regulatory effect of various membranes on osteoclast growth was evaluated by detecting the expression levels of osteoclast-related genes and proteins in the cells.Subsequently, we constructed a model of a rat skull defect and implanted different membranes into the defect. Then, we evaluated the new bone formation in the defect using histological staining and micro-computed tomography after 4 and 8 weeks. The results of in vitro experiments confirmed that the incorporation of naringenin and vitamin K2 stimulated the expression of osteogenesis-related genes and the secretion of osteogenesis-related proteins. Simultaneously, the results showed that naringenin and vitamin K2 inhibited the formation and growth of osteoclasts. Therefore, naringenin and vitamin K2 have a synergistic effect in promoting bone growth and regulating osteoclast growth.

Effects of Zinc and Strontium Doping on In Vitro Osteogenesis and Angiogenesis of Calcium Silicate/Calcium Phosphate Cement.

Based on multiple biological functions (mainly osteogenesis and angiogenesis) of bioactive ions, Zn/Sr-doped calcium silicate/calcium phosphate cements (Zn/Sr-CS/CPCs, including 10Zn-CS/CPC, 20Sr-CS/CPC, and 10Zn/20Sr-CS/CPC) were prepared by the addition of Zn and Sr dual active ions into CS/CPC to further accelerate its bone regeneration in this study. The physicochemical and biological properties of the Zn/Sr-CS/CPCs were systematically investigated. The results showed that the setting time was slightly prolonged, the compressive strength and porosity did not change much, and all groups maintained good injectability after the doping of Zn and Sr. Besides, the doping of Zn and Sr had little effect on the phase and microstructure of hydrated products of CS/CPC. The degradation rate of Zn/Sr-CS/CPCs decreased after doping with Zn and Sr. In mouse bone marrow mesenchymal stem cells (mBMSC) experiments, all Zn/Sr-CS/CPCs stimulated the viability, adhesion, proliferation, and alkaline phosphatase (ALP) activity together with osteogenesis-related genes (ALP, Runx2, Col-I, OCN, and OPN). The further addition of Zn and Sr played better and synergistic roles in in vitro osteogenesis. Thereinto, 10Zn/20Sr-CS/CPC manifested the optimum in vitro osteogenic performance. As for human umbilical vein endothelial cell (HUVEC) experiments, the incorporation of CS doped with Zn and Sr into CPC possessed good vascularization properties of proliferation, NO secretion, tube formation, and the expression of angiogenesis-related genes (VEGF, bFGF, and eNOS). In conclusion, the doping of Zn and Sr into CS/CPC could exhibit excellent osteogenesis and good angiogenesis potentials and 10Zn/20Sr-CS/CPC could be considered as a promising candidate in bone repair.

Manganese-Implanted Titanium Modulates the Crosstalk between Bone Marrow Mesenchymal Stem Cells and Macrophages to Improve Osteogenesis.

Manganese (Mn) is an essential micronutrient in various physiological processes, but its functions in bone metabolism remain undefined. This is partly due to the interplay between immune and bone cells because Mn plays a central role in the immune system. In this study, we utilized the plasma immersion ion implantation and deposition (PIII&D) technique to introduce Mn onto the titanium surface. The results demonstrated that Mn-implanted surfaces stimulated the shift of macrophages toward the M1 phenotype and had minimal effects on the osteogenic differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs) under mono-culture conditions. However, they promoted the M2 polarization of macrophages and improved the osteogenic activities of mBMSCs under co-culture conditions, indicating the importance of the crosstalk between mBMSCs and macrophages mediated by Mn in osteogenic activities. This study provides a positive incentive for the application of Mn in the field of osteoimmunology.

Ursolic acid alleviates steroid-induced avascular necrosis of the femoral head in mouse by inhibiting apoptosis and rescuing osteogenic differentiation

Steroid-induced avascular necrosis of femoral head (SANFH) is a common disorder worldwide with high disability. Overdose of glucocorticoid (GC) is the most common non-traumatic cause of SANFH. Up until now, there are limited therapeutic strategies for curing SANFH, and the mechanisms underlying SANFH progression remain unclear. Nevertheless, Osteogenic dysfunction is considered to be one of the crucial pathobiological mechanisms in the development of SANFH, which involves mouse bone marrow mesenchymal stem cells (BMSCs) apoptosis and osteogenic differentiation disorder. Ursolic acid (UA), an important component of the Chinese medicine formula Yougui Yin, has a wide range of pharmacological properties such as anti-tumor, anti-inflammatory and bone remodeling. Due to the positive effect of Yougui Yin on bone remodeling, the purpose of this study was to investigate the effects of UA on dexamethasone (DEX)-induced SANFH in vitro and vivo. In vitro, we demonstrated that UA can promote mouse BMSCs proliferation and resist DEX-induced apoptosis by CCK8, Western blotting, TUNEL and so on. In addition, vitro experiments such as ALP and Alizarin red staining assay showed that UA had a beneficial effect on the osteogenic differentiation of mouse BMSCs. In vivo, the results of H&E staining, immunohistochemistry staining, Elisa and micro-CT analysis showed that UA had a bone repair-promoting effect in SANFH model. Moreover, the results of Western blot and TUNEL experiments showed that UA could delay the disease progression of SANFH in mice by inhibiting apoptosis. Overall, our study suggests that UA is a potential compound for the treatment of SANFH.

Eicosapentaenoic acid supplementation modulates the osteoblast/osteoclast balance in inflammatory environments and protects against estrogen deficiency-induced bone loss in mice

An imbalance of osteoblasts (OBs) and osteoclasts (OCs) in a chronic inflammatory microenvironment is an important pathological factor leading to osteoporosis. Eicosapentaenoic acid (EPA) has been shown to suppress inflammation in macrophages and adipocytes. However, the effect of EPA on OBs and OCs has yet to be fully elucidated. We explored the roles of EPA in the differentiation of OBs and OCs, as well as the coupling between OBs and OCs in an inflammatory microenvironment. The effects of EPA on estrogen deficiency-induced osteoporosis were also evaluated. Mouse bone marrow mesenchymal stem cells (mBMSCs) and mouse bone marrow-derived macrophages (mBMMs) were used for invitro OBs and OCs differentiation. TNF-α was used to create an inflammatory microenvironment. We examined the effects of EPA on osteoblastogenesis in the absence or presence of TNF-α and collect OBs' culture medium as the conditioned medium (CM). Then we examined the effects of EPA and CM on RANKL-induced osteoclastogenesis. The invivo effects of EPA were determined using an ovariectomized (OVX) mouse model treated with EPA or vehicle. High-dose EPA was shown to promote osteoblastogenesis in an inflammatory environment invitro, as well as upregulate expression of OBs-specific proteins and genes. ARS and ALP staining also showed that high-dose EPA-treated groups restored mBMSCs' impaired osteogenic capacity caused by TNFa. Mechanistically, EPA suppressed the NF-κB pathway activated by TNF-α in mBMSCs and rescued TNF-α-mediated inhibition of osteoblastogenesis. EPA was also shown to inhibit expression of RANKL and decrease the RANKL/OPG ratio in OBs in an inflammatory environment. CM from TNF-α-stimulated OBs promoted osteoclastogenesis of mBMMs; EPA-treated CM prevented this. In the OVX mouse model, EPA supplementation prevented bone loss in an estrogen deficiency-induced inflammatory environment. EPA was demonstrated for the first time to restore mBMSCs' impaired osteogenic capacity caused by TNFa-induced inflammation and rescue the OBs/OCs balance via regulation of RANKL and OPG expression in OBs. EPA showed a remarkable ability to prevent bone loss in OVX mice, suggesting a potential application of EPA in postmenopausal osteoporosis.

Aquaporin-7 Facilitates Proliferation and Adipogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells by Regulating Hydrogen Peroxide Transport.

Hydrogen peroxide (H2O2) is a major form of reactive oxygen species that play an important role in the survival, proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). The regulatory mechanisms of H2O2 homeostasis in BMSCs are not fully understood. Here we demonstrate for the first time that aquaglyceroporin AQP7 is a functional peroxiporin expressed in BMSCs and remarkably upregulated upon adipodenic induction. The proliferation ability of BMSCs from AQP7-/- mice was significantly decreased, as indicated by fewer clonal formation and cell cycle arrest compared with wildtype BMSCs. AQP7 deficiency caused accumulation of intracellular generated H2O2 during BMSCs proliferation, leading to oxidative stress and inhibition of PI3K/AKT and STAT3 signaling pathways. After adipogenic induction, however, the AQP7-/- BMSCs exhibited greatly reduced adipogenic differentiation with fewer lipid droplets formation and lower cellular triglycerides content than wildtype BMSCs. In such case AQP7 deficiency was found to diminish import of extracellular H2O2 produced by plasma membrane NADPH Oxidases, resulting in altered AMPK and MAPK signaling pathways and reduced expression of lipogenic genes C/EBPα and PPARγ. Our data revealed a novel regulatory mechanism of BMSCs function through AQP7-mediated H2O2 transport across plasma membrane. AQP7 is a peroxiporin mediating H2O2 transport across the plasma membrane of BMSCs. During proliferation, AQP7 deficiency results in accumulation of intracellular generated H2O2 due to reduced export, which inhibited STAT3 and PI3K/AKT/insulin receptor signaling pathways and cell proliferation. During adipogenic differentiation, however, AQP7 deficiency blocked the uptake of extracelluar H2O2 generated through plasma membrane NOX enzymes. The reduced intracellular H2O2 level causes decreased expression of lipogenic genes C/EBPα and PPARγ due to altered AMPK and MAPK signaling pathways, leading to impaired adipogenic differentiation.

Chitosan nanoparticles for sustained release of metformin and its derived synthetic biopolymer for bone regeneration.

Background: There are considerable socioeconomic costs associated with bone defects, making regenerative medicine an increasingly attractive option for treating them. Chitosan is a natural biopolymer; it is used in approaches for sustained slow release and osteogenesis, and metformin has osteoinductivity. Our study aimed to synthesize chitosan and human serum albumin (HSA) with a metformin nanoformulation to evaluate the therapeutic effects of this nanoformulation on bone defects in vitro. Methods: A pluripotent differentiation assay was performed in vitro on mouse bone marrow mesenchymal stem cells (BMSCs). Cell Counting Kit-8 was used to detect whether metformin was toxic to BMSCs. The osteogenesis-related gene expression of osteocalcin (OCN) and osteoprotegerin (OPG) from BMSCs was tested by real-time polymerase chain reaction (PCR). HSA, metformin hydrochloride, and chitosan mixtures were magnetically stirred to finish the assembly of metformin/HSA/chitosan nanoparticles (MHC NPs). The MHC NPs were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FT-IR). To test the expression of OCN and OPG, western blot were used. MHC NPs were evaluated in vitro for their osteoinductivity using alkaline phosphatase (ALP). Results: BMSCs successfully differentiated into osteogenic and adipogenic lineages in vitro. According to real-time PCR, a 50µM concentration of metformin promoted osteogenesis in BMSCs most effectively by upregulating the osteogenic markers OCN and OPG. The microstructure of MHC NPs was spherical with an average nanosize of 20 ± 4.7nm and zeta potential of -8.3mV. A blueshift and redshift were observed in MHC NPs following exposure to wavelengths of 1,600-1,900 and 2,000-3,700nm, respectively. The encapsulation (%) of metformin was more than 90%. The simulation study showed that MHC NPs have good stability and it could release metformin slowly in vitro at room temperature. Upon treatment with the studied MHC NPs for 3days, ALP was significantly elevated in BMSCs. In addition, the MHC NPs-treated BMSCs upregulated the expression of OPG and OCN, as shown by real-time PCR and western blot. Conclusion: MHC NPs have a stable metformin release effect and osteogenic ability. Therefore, as a derived synthetic biopolymer, it is expected to play a role in bone tissue regeneration.

3D Bioprinting of a Bioactive Composite Scaffold for Cell Delivery in Periodontal Tissue Regeneration.

Hydrogels have been widely applied to the fabrication of tissue engineering scaffolds via three-dimensional (3D) bioprinting because of their extracellular matrix-like properties, capacity for living cell encapsulation, and shapeable customization depending on the defect shape. However, the current hydrogel scaffolds show limited regeneration activity, especially in the application of periodontal tissue regeneration. In this study, we attempted to develop a novel multi-component hydrogel that possesses good biological activity, can wrap living cells for 3D bioprinting and can regenerate periodontal soft and hard tissue. The multi-component hydrogel consisted of gelatin methacryloyl (GelMA), sodium alginate (SA) and bioactive glass microsphere (BGM), which was first processed into hydrogel scaffolds by cell-free 3D printing to evaluate its printability and in vitro biological performances. The cell-free 3D-printed scaffolds showed uniform porous structures and good swelling capability. The BGM-loaded scaffold exhibited good biocompatibility, enhanced osteogenic differentiation, apatite formation abilities and desired mechanical strength. The composite hydrogel was further applied as a bio-ink to load with mouse bone marrow mesenchymal stem cells (mBMSCs) and growth factors (BMP2 and PDGF) for the fabrication of a scaffold for periodontal tissue regeneration. The cell wrapped in the hydrogel still maintained good cellular vitality after 3D bioprinting and showed enhanced osteogenic differentiation and soft tissue repair capabilities in BMP2- and PDGF-loaded scaffolds. It was noted that after transplantation of the cell- and growth factor-laden scaffolds in Beagle dog periodontal defects, significant regeneration of gingival tissue, periodontal ligament, and alveolar bone was detected. Importantly, a reconstructed periodontal structure was established in the treatment group eight weeks post-transplantation of the scaffolds containing the cell and growth factors. In conclusion, we developed a bioactive composite bio-ink for the fabrication of scaffolds applicable for the reconstruction and regeneration of periodontal tissue defects.