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Introduction: Myasthenia gravis (MG) is an autoimmune disorder. Microvesicle-derived miRNAs have been implicated in autoimmune diseases. However, the role of microvesicle-derived miR-29a-3p in MG remains poorly understood. This study aimed to investigate the therapeutic effect and mechanism of miR-29a-3p derived from stem cell microvesicles (MVs) on experimental autoimmune myasthenia gravis (EAMG) rats. Methods: EAMG was induced in rats by injection of the subunit of the rat nicotinic anti-acetylcholine receptor (AChR) R97–116 peptide.Besides the control group, EAMG rats were randomly allocated into the EAMG model group, MV group, MV-NC-agomir group, and MV- miR-29a-3p-agomir group. Results: Our results found that BMSCs-MV promoted miR-29a-3p expression in gastrocnemius of EAMG rats. Bone marrow mesenchymal stem cells (BMSCs) derived microvesicle miR-29a-3p improved the hanging ability and swimming time of EMGA rats and weakened the degree of muscle fiber atrophy. Furthermore, microvesicles from miR-29a-3p overexpressing BMSCs reduced the content of AchR-Ab in the serum of EAMG rats. BMSC-derived microvesicle miR-29a-3p further suppressed the expression of IFN-γ and enhanced the IL-4 and IL-10 in the serum of EAMG rats by restoring the Th17/Treg cells balance. Discussion: BMSCs-derived microvesicle miR-29a-3p improved the stability of rat myasthenia gravis by regulating Treg/Th17 cells. It may be an effective treatment for MG. [ABSTRACT FROM AUTHOR]

Our previous research revealed a close association between the acetylation of peroxisome proliferator-activated receptor γ (PPARγ) histone H3K27 and the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). We preliminarily explored the epigenetic mechanism of steroid-induced avascular necrosis of the femoral head (SANFH) development, but the specific histone deacetylase (HDAC) involved in this regulatory process remains unknown. In this study, we combined cell, animal, and clinical specimen experiments to screen for specific HDAC genes that could regulate BMSC adipogenic differentiation and to explore their roles. The results showed that dexamethasone (DEX) significantly exacerbated the imbalance between the adipogenic and osteogenic differentiation of BMSCs, and there were differences in HDAC expression in the adipogenic differentiation cell models, with histone deacetylase 10 (HDAC10) showing the most significant decrease in expression. Subsequent use of a chromatin immunoprecipitation assay kit and quantitative polymerase chain reaction (ChIP‒qPCR) revealed a decrease in HDAC10 expression at predicted potential sites within the PPARγ promoter, indicating a significant decrease in HDAC10 enrichment in the PPARγ promoter region of BMSCs, thereby promoting sustained PPARγ expression. Additionally, immunohistochemistry of samples collected from mice and humans with SANFH and normal femoral heads revealed an imbalance between adipogenic and osteogenic differentiation in the necrotic area of femoral heads, with a significant decrease in the relative expression of HDAC10 in the necrotic area of femoral heads with SANFH. In summary, we speculate that HDAC10 affects the progression of SANFH by regulating BMSC adipogenic differentiation, a process possibly related to PPARγ histone acetylation. These findings provide a promising direction for the treatment of SANFH. [ABSTRACT FROM AUTHOR]

Interleukin‐4 (IL‐4)/IL‐4 receptor alpha (IL‐4Rα) signalling pathways play important roles in the complex process of bone formation and bone remodelling. However, whether IL‐4/IL‐4Rα participates in skeletogenesis during embryonic development is not completely understood. We used the anti‐IL‐4Rα monoclonal antibody (anti‐IL‐4Rα mAb) as a powerful investigational tool to evaluate the potential roles of IL‐4/IL‐4Rα in the chondrogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Simultaneously, we explored the effect of IL‐4/IL‐4Rα on bone ossification during rat embryo‐fetal development. In this study, we found that, compared to the control group, IL‐4 can significantly promote the chondrogenic differentiation of BMSCs. Furthermore, following exposure to anti‐IL‐4Rα mAb in pregnant rats, unexpected phenomena were observed in fetal bone development, including non‐ossification of the fetal sternum, an incomplete ossification centre in long bones and a reduced number of ossification points in digit (toe) bones. To further investigate the underlying mechanism of the phenotype, we studied the rat sternum as the target organ, starting from different time points of sternum development in the embryonic stage. The results indicated that the retardation mainly occurred in the middle and late stages of embryonic development. This retardation was characterized by the inhibition of the differentiation process of mesenchymal stem cells into chondrocytes, resulting in reduced angiogenesis near the ossification centre, failure of osteoblasts to invade the centre of the cartilage body with the blood vessels and delayed formation of the primary ossification centre (POC). Overall, our study demonstrated the significant function of IL‐4/IL‐4Rα in chondrogenic differentiation of BMSCs and bone ossification during embryo‐fetal development. [ABSTRACT FROM AUTHOR]

Central nervous system (CNS) diseases are one of the diseases that threaten human health. The delivery of drugs targeting the CNS has always been a significant challenge; the blood–brain barrier (BBB) is the main obstacle that must be overcome. The rise of bone marrow mesenchymal stem cell (BMSC) therapy has brought hope for the treatment of CNS diseases. However, the problems of low homing rate, susceptibility differentiation into astrocytes, immune rejection, and formation of iatrogenic tumors of transplanted BMSCs limit their clinical application. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have become a hot research topic in the treatment of CNS diseases in recent years because of their excellent histocompatibility, low immunogenicity, ease of crossing the BBB, and their ability to serve as natural carriers for treatment. This article reviews the mechanisms of BMSC-Exos in CNS diseases and provides direction for further research. [ABSTRACT FROM AUTHOR]

This study investigated the possible mechanisms of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism. β-Thiol ethanol induced Notch1 mRNA expression, microRNA-124a inhibitor reduced the effects of β-thiol ethanol on Notch1 mRNA expression in BMSCs. Baicalin induced Hes1 mRNA expression, and microRNA-124a inhibitor reduced the effects of baicalin on Hes1 mRNA expression in BMSCs. Si-Notch1 suppressed Hes1 mRNA expression in BMSCs. Baicalin increased the effects of Notch1 on Hes1 mRNA expression in BMSCs. Si-Notch1 increased cell growth of BMSCs. Baicalin reduced the effects of si-Notch1 on cell growth and the differentiation of BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons through Notch/Hes1 signal pathway. [ABSTRACT FROM AUTHOR]

Objective To investigate the impact of inhibiting peroxisome proliferator-activated receptor γ (PPARγ) expression on osteogenic differentiation of BMSCs, so as to provide a theoretical basis for the treatment of primary osteoporosis ( POP) . Methods BMSCs were extracted from patients with normal bone density and categorized into the normal control group (CON group) . BMSCs extracted from patients with primary osteoporosis were categorized into the primary osteoporosis group (POP group). The cells from the POP group were treated with a PPARγ inhibitor and divided into the inhibitor group ( INR group) . Following osteogenic differentiation, the expression of osteopontin (OPN), osteocalcin (OCN), Osterix, and runt-related transcription factor 2 (Runx2) was evaluated in the cells of each group.The alkaline phosphatase ( ALP) staining was employed to assess the osteogenic differentiation of BMSCs in each group. Results The quantity of ALP-positive cells in the POP group was lower than that in the CON and INR groups (P < 0.05). The expression of OCN, OPN, Osterix, and Runx2 was found to be decreased in the POP group when compared to the CON group, with statistically significant differences (P < 0.05). Conversely, the expression of OCN, OPN, Osterix, and Runx2 was elevated in the INR group when compared to the POP group, with statistically significant differences (P < 0.05) . Conclusion Increased expression of osteogenic differentiation-specific genes (ALP, OCN, OPN, Osterix, Runx2) in BMSCs after inhibition of PPARγ expression. [ABSTRACT FROM AUTHOR]

Aim: The present study examined the leaching and cytotoxicity of bismuth from ProRoot MTA and aimed to identify whether bismuth leaching was affected by the cement base and the immersion regime used. Methodology: The leaching profile of bismuth was examined from ProRoot MTA and compared with hydroxyapatite containing 20% bismuth oxide as well as hydroxyapatite and tricalcium silicate to investigate whether bismuth release changed depending on the cement base. Bismuth leaching was determined after 30 and 180 days of ageing immersed in Dulbecco's modified Eagle's medium (DMEM) using mass spectroscopy (ICP‐MS). The media were either unchanged or regularly replenished. The pH, surface microstructure and phase changes of aged materials were assessed. Wistar rat femoral bone marrow stromal cells (BMSCs) and cutaneous fibroblasts were isolated, cultured and seeded for cell counting (trypan blue live/dead) after exposure to non‐aged, 30‐ and 180‐days‐aged samples in regularly replenished DMEM. Aged DMEM in contact with materials was also used to culture BMSCs to investigate the effect of material leachates on the cells. Gene expression analysis was also carried out after direct exposure of cells to non‐aged materials. Differences between groups were statistically tested at a significance level of 5%. Results: All materials exhibited alterations after immersion in DMEM and this increased with longer exposure times. The bismuth leached from ProRoot MTA as detected by ICP‐MS. Aged ProRoot MTA samples exhibited a black discolouration and surface calcium carbonate deposition. ProRoot MTA influenced cell counts after direct exposure and its 180‐days leachates reduced BMSC viability. After direct BMSC contact with non‐aged ProRoot MTA an upregulation of metallothionein (MT1 and MT2A) expression and down‐regulation of collagen‐1a (Col‐1a) and bone sialoprotein (BSP) expression was identified. Conclusions: Bismuth leaching was observed throughout 180‐days observation period from all materials containing bismuth oxide. This negatively influenced cell viability and gene expression associated with bismuth exposure. This is the first study to report that metallothionein gene expression was influenced by exposure to ProRoot MTA. [ABSTRACT FROM AUTHOR]

Aim: Impaired wound healing in patients with diabetes can develop into nonhealing ulcerations. Because bone marrow mesenchymal stem cells (BMSCs) exosomes can promote wound healing, this study aims to investigate the mechanism of BMSCs‐isolated exosomal miR‐221‐3p in angiogenesis and diabetic wound healing. Methods: To mimic diabetes in vitro, human umbilical vein endothelial cells (HUVECs) were subjected to high glucose (HG). Exosomes were derived from BMSCs and identified by transmission electron microscopy (TEM), western blot analysis and dynamic light scattering (DLS). The ability to differentiate BMSCs was assessed via Oil red O staining, alkaline phosphatase (ALP) staining and alizarin red staining. The ability to internalise PKH26‐labelled exosomes was assessed using confocal microscopy. Migration, cell viability and angiogenesis were tested by scratch, MTT and tube formation assays separately. The miRNA and protein levels were analysed by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) or western blotting. The relationship among miR‐221‐3p, FOXP1 and SPRY1 was determined using the dual‐luciferase reporter, ChIP and RIP assays. Results: Exosomal miR‐221‐3p was successfully isolated from BMSCs and delivered into HUVECs. HG was found to suppress the angiogenesis, cell viability and migration of HUVECs and exosomal miR‐221‐3p separated from BMSCs inhibited the above phenomenon. FOXP1 could transcriptionally upregulate SPRY1, and the silencing of FOXP1 reversed the HG‐stimulated angiogenesis inhibition, cell viability and migration in HUVECs via the downregulation of SPRY1. Meanwhile, miR‐221‐3p directly targeted FOXP1 and the overexpression of FOXP1 reversed the positive effect of exosomal miR‐221‐3p on HUVEC angiogenesis. Conclusion: Exosomal miR‐221‐3p isolated from BMSCs promoted angiogenesis in diabetic wounds through the mediation of the FOXP1/SPRY1 axis. Furthermore, the findings of this study can provide new insights into probing strategies against diabetes. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. Methods: A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process. Results: Mean callus volume was larger in the elastic fixation group (1,755 mm3 (standard error of the mean (SEM) 297)) than in the stiff fixation group (258 mm3 (SEM 65)). Pathological observation found that the expression levels of osterix (OSX), collagen, type I, alpha 1 (COL1α1), and alkaline phosphatase (ALP) in the callus of the elastic fixation group were higher than those of the stiff fixation group. The protein sequence of the callus revealed 199 DEPs, 124 of which were highly expressed in the elastic fixation group. In the in vitro study, it was observed that a stress of 200 g led to upregulation of thrombospondin 1 (THBS1) and osteoglycin (OGN) expression in bone marrow mesenchymal stem cells (BMSCs). Additionally, these genes were found to be upregulated during the osteogenic differentiation process of the BMSCs. Conclusion: Elastic fixation can promote fracture healing and osteoblast differentiation in callus, and the ability of elastic fixation to promote osteogenic differentiation of BMSCs may be achieved by upregulating genes such as THBS1 and OGN. Cite this article: Bone Joint Res 2024;13(10):559–572.

Abstract Our previous research revealed a close association between the acetylation of peroxisome proliferator-activated receptor γ (PPARγ) histone H3K27 and the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). We preliminarily explored the epigenetic mechanism of steroid-induced avascular necrosis of the femoral head (SANFH) development, but the specific histone deacetylase (HDAC) involved in this regulatory process remains unknown. In this study, we combined cell, animal, and clinical specimen experiments to screen for specific HDAC genes that could regulate BMSC adipogenic differentiation and to explore their roles. The results showed that dexamethasone (DEX) significantly exacerbated the imbalance between the adipogenic and osteogenic differentiation of BMSCs, and there were differences in HDAC expression in the adipogenic differentiation cell models, with histone deacetylase 10 (HDAC10) showing the most significant decrease in expression. Subsequent use of a chromatin immunoprecipitation assay kit and quantitative polymerase chain reaction (ChIP‒qPCR) revealed a decrease in HDAC10 expression at predicted potential sites within the PPARγ promoter, indicating a significant decrease in HDAC10 enrichment in the PPARγ promoter region of BMSCs, thereby promoting sustained PPARγ expression. Additionally, immunohistochemistry of samples collected from mice and humans with SANFH and normal femoral heads revealed an imbalance between adipogenic and osteogenic differentiation in the necrotic area of femoral heads, with a significant decrease in the relative expression of HDAC10 in the necrotic area of femoral heads with SANFH. In summary, we speculate that HDAC10 affects the progression of SANFH by regulating BMSC adipogenic differentiation, a process possibly related to PPARγ histone acetylation. These findings provide a promising direction for the treatment of SANFH. Graphical abstract

Abstract This study investigated the possible mechanisms of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism. β-Thiol ethanol induced Notch1 mRNA expression, microRNA-124a inhibitor reduced the effects of β-thiol ethanol on Notch1 mRNA expression in BMSCs. Baicalin induced Hes1 mRNA expression, and microRNA-124a inhibitor reduced the effects of baicalin on Hes1 mRNA expression in BMSCs. Si-Notch1 suppressed Hes1 mRNA expression in BMSCs. Baicalin increased the effects of Notch1 on Hes1 mRNA expression in BMSCs. Si-Notch1 increased cell growth of BMSCs. Baicalin reduced the effects of si-Notch1 on cell growth and the differentiation of BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons through Notch/Hes1 signal pathway.

Natural fracture healing is most efficient when the fine-tuned mechanical force and proper micromotion are applied. To mimick this micromotion at the fracture gap, a near-infrared-II (NIR–II)–activated hydrogel was fabricated by integrating two-dimensional (2D) monolayer Nb2C nanosheets into a thermally responsive poly(N-isopropylacrylamide) (NIPAM) hydrogel system. NIR–II–triggered deformation of the NIPAM/Nb2C hydrogel was designed to generate precise micromotion for co-culturing cells. It was validated that micromotion at 1/300 Hz, triggering a 2.37-fold change in the cell length/diameter ratio, is the most favorable condition for the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Moreover, mRNA sequencing and verification revealed that micromotion-induced augmentation was mediated by Piezo1 activation. Suppression of Piezo1 interrupts the mechano-sensitivity and abrogates osteogenic differentiation. Calvarial and femoral shaft defect models were established to explore the biocompatibility and osteoinductivity of the Micromotion Biomaterial. A series of research methods, including radiography, micro-CT scanning, and immunohistochemical staining have been performed to evaluate biosafety and osteogenic efficacy. The in vivo results revealed that tunable micromotion strengthens the natural fracture healing process through the sequential activation of endochondral ossification, promotion of neovascularization, initiation of mineral deposition, and combinatory acceleration of full-thickness osseous regeneration. This study demonstrated that Micromotion Biomaterials with controllable mechanophysical characteristics could promote the osteogenic differentiation of BMSCs and facilitate full osseous regeneration. The design of NIPAM/Nb2C hydrogel with highly efficient photothermal conversion, specific features of precisely controlled micromotion, and bionic-mimicking bone-repair capabilities could spark a new era in the field of regenerative medicine.

Delayed or failed bone healing is a significant clinical challenge worldwide. Bone marrow mesenchymal stem cells (BMSCs) offer a promising approach for improving fracture healing. Isomangiferin, a xanthone C-glucoside, is known for its pharmacological activities, but its role in fracture healing remains unclear. In this study, we investigated the effects of isomangiferin on BMSCs under oxidative stress conditions induced by hydrogen peroxide (H2O2). Our results showed that isomangiferin promotes osteogenic differentiation and migration of H2O2-treated BMSCs, reduces apoptosis and reactive oxygen species production, and activates the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC) pathway. These findings suggest that isomangiferin may be a potential therapeutic agent for enhancing bone healing by modulating BMSC function.

Abstract Background The progression of osteoporosis (OP) can dramatically increase the risk of fractures, which seriously disturb the life of elderly individuals. Specific protein 1 (SP1) is involved in OP progression. However, the mechanism by which SP1 regulates OP progression remains unclear. Objective This study investigated the mechanism underlying the function of SP1 in OP. Methods SAMP6 mice were used to establish an in vivo model of age-dependent OP, and BALB/c mice were used as controls. BMSCs were extracted from two subtypes of mice. Hematoxylin and eosin staining were performed to mark the intramedullary trabecular bone structure to evaluate histological changes. ChIP assay was used to assess the targeted regulation between SP1 and miR-133a-3p. The binding sites between MAPK3 and miR-133a-3p were verified using a dual-luciferase reporter assay. The mRNA levels of miR-133a-3p and MAPK3 were detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The protein expression of SP1, MAPK3, Colla1, OCN, and Runx2 was examined using Western blotting. Alkaline phosphatase (ALP) kit and Alizarin Red S staining were used to investigate ALP activity and mineralized nodules, respectively. Results The levels of SP1 and miR-133a-3p were upregulated, whereas the expression of MAPK3 was downregulated in BMSCs from SAMP6 mice, and miR-133a-3p inhibitor accelerated osteogenic differentiation in BMSCs. SP1 directly targeted miR-133a-3p, and MAPK3 was the downstream mRNA of miR-133a-3p. Mechanically, SP1 accelerated osteogenic differentiation in BMSCs via transcriptional mediation of the miR-133a-3p/MAPK3 axis. Conclusion SP1 regulates osteogenic differentiation by mediating the miR-133a-3p/MAPK3 axis, which would shed new light on strategies for treating senile OP.

Abstract Background Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. Methods The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague–Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. Results The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. Conclusion NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.

Abstract Osteoporosis is a metabolic bone disease that involves gradual loss of bone density and mass, thus resulting in increased fragility and risk of fracture. Inflammatory cytokines, such as tumour necrosis factor α (TNF‐α), inhibit osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and several microRNAs are implicated in osteoporosis development. This study aimed to explore the correlation between TNF‐α treatment and miR‐27a‐3p expression in BMSC osteogenesis and further understand their roles in osteoporosis. An osteoporosis animal model was established using ovariectomized (OVX) mice. Compared with Sham mice, the OVX mice had a significantly elevated level of serum TNF‐α and decreased level of bone miR‐27a‐3p, and in vitro TNF‐α treatment inhibited miR‐27a‐3p expression in BMSCs. In addition, miR‐27a‐3p promoted osteogenic differentiation of mouse BMSCs in vitro, as evidenced by alkaline phosphatase staining and Alizarin Red‐S staining, as well as enhanced expression of the osteogenic markers Runx2 and Osterix. Subsequent bioinformatics analysis combined with experimental validation identified secreted frizzled‐related protein 1 (Sfrp1) as a downstream target of miR‐27a‐3p. Sfrp1 overexpression significantly inhibited the osteogenic differentiation of BMSCs in vitro and additional TNF‐α treatment augmented this inhibition. Moreover, Sfrp1 overexpression abrogated the promotive effect of miR‐27a‐3p on the osteogenic differentiation of BMSCs. Furthermore, the miR‐27a‐3p–Sfrp1 axis was found to exert its regulatory function in BMSC osteogenic differentiation via regulating Wnt3a–β‐catenin signalling. In summary, this study revealed that TNF‐α regulated a novel miR‐27a‐3p–Sfrp1 axis in osteogenic differentiation of BMSCs. The data provide new insights into the development of novel therapeutic strategies for osteoporosis.

Abstract Suitable biomaterials with seed cells have promising potential to repair bone defects. However, bone marrow mesenchymal stem cells (BMSCs), one of the most common seed cells used in tissue engineering, cannot differentiate efficiently and accurately into functional osteoblasts. In view of this, a new tissue engineering technique combined with BMSCs and scaffolds is a major task for bone defect repair. Lentiviruses interfering with miR‐136‐5p or Smurf1 expression were transfected into BMSCs. The effects of miR‐136‐5p or Smurf1 on the osteogenic differentiation (OD) of BMSCs were evaluated by measuring alkaline phosphatase activity and calcium deposition. Then, the targeting relationship between miR‐136‐5p and Smurf1 was verified by bioinformatics website analysis and dual luciferase reporter assay. Then, a rabbit femoral condyle bone defect model was established. miR‐136‐5p/BMSCs/β‐TCP scaffold was implanted into the defect, and the repair of the bone defect was detected by Micro‐CT and HE staining. Elevating miR‐136‐5p‐3p or suppressing Smurf1 could stimulate OD of BMSCs. miR‐136‐5p negatively regulated Smurf1 expression. Overexpressing Smurf1 reduced the promoting effect of miR‐136‐5p on the OD of BMSCs. miR‐136‐5p/BMSCs/β‐TCP could strengthen bone density in the defected area and accelerate bone repair. SmurF1‐targeting miR‐136‐5p‐modified BMSCs combined with 3D‐printed β‐TCP scaffolds can strengthen osteogenic activity and alleviate bone defects.

Yu Li,1 Guangren Yue,1 Shuying Yu,1 Zheng Liu,1 Yilin Cao,1– 3 Ximei Wang1 1Department of Plastic and Reconstructive Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 2Shanghai Key Laboratory of Tissue Engineering, Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3National Tissue Engineering Center of China, Shanghai, People’s Republic of ChinaCorrespondence: Ximei Wang, Department of Plastic and Reconstructive Surgery, The First Affiliated Hospital of Zhengzhou University, 1st Jianshe Eastern Road, Zhengzhou, 450052, People’s Republic of China, Tel +86 371-66278102, Email tracywang@zzu.edu.cnIntroduction: Autologous stem cell transplantation has emerged as a promising strategy for bone repair. However, the osteogenic potential of mesenchymal stem cells derived from diabetic patients is compromised, possibly due to hyperglycemia-induced senescence. The objective of this study was to assess the preconditioning effects of extracellular vesicles derived from H2O2-stimulated adipose-derived stem cells (ADSCs) and non-modified ADSCs on the osteogenic potential of diabetic bone marrow mesenchymal stem cells (BMSCs).Methods: Sprague-Dawley (SD) rats were experimentally induced into a diabetic state through a high-fat diet followed by an injection of streptozotocin, and diabetic BMSCs were collected from the bone marrow of these rats. Extracellular vesicles (EVs) were isolated from the conditioned media of ADSCs, with or without hydrogen peroxide (H2O2) preconditioning, using density gradient centrifugation. The effects of H2O2 preconditioning on the morphology, marker expression, and particle size of the EVs were analyzed. Furthermore, the impact of EV-pretreatment on the viability, survivability, migration ability, osteogenesis, cellular senescence, and oxidative stress of diabetic BMSCs was examined. Moreover, the expression of the Nrf2/HO-1 pathway was also assessed to explore the underlying mechanism. Additionally, we transplanted EV-pretreated BMSCs into calvarial defects in diabetic rats to assess their in vivo bone formation and anti-senescence capabilities.Results: Our study demonstrated that pretreatment with EVs from ADSCs significantly improved the viability, senescence, and osteogenic differentiation potential of diabetic BMSCs. Moreover, in-vitro experiments revealed that diabetic BMSCs treated with H2O2-activated EVs exhibited increased viability, reduced senescence, and enhanced osteogenic differentiation compared to those treated with non-modified EVs. Furthermore, when transplanted into rat bone defects, diabetic BMSCs treated with H2O2-activated EVs showed improved bone regeneration potential and enhanced anti-senescence function t compared to those treated with non-modified EVs. Both H2O2-activated EVs and non-modified EVs upregulated the expression of the Nrf2/HO-1 pathway in diabetic BMSCs, however, the promoting effect of H2O2-activated EVs was more pronounced than that of non-modified EVs.Conclusion: Extracellular vesicles derived from H2O2-preconditioned ADSCs mitigated senescence in diabetic BMSCs and enhanced their bone regenerative functions via the activation of the Nrf2/HO-1 pathway.Keywords: extracellular vesicles, ADSCs, BMSCs, diabetes mellitus, bone regeneration

Objective Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis‐derived BMSCs (OP‐BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6‐methyladenosine RNA binding protein 3) on the osteogenic traits of OP‐BMSCs and identify potential therapeutic targets to boost their bone formation ability. Methods We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single‐cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. Results Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single‐cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP‐BMSCs substantially impedes their proliferation and differentiation. Conclusion YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP‐BMSCs.

Ovarian aging reduced the quality of oocytes, resulting in age-related female infertility. It is reported that mesenchymal stem cells (MSCs) therapy can improve age-related ovarian function decline and the success rate of in vitro maturation (IVM) in assisted reproductive therapy. In order to investigate the effectiveness and mechanisms of MSCs to enhance oocyte quality of cumulus oocyte complexes (COCs) in advanced age, this study focus on the respective functional improvement of oocytes and granulosa cells (GCs) from aging mice and further to explore and verify the possible mechanisms. Here, we studied a popular but significant protein of follicular development, Forkhead box O-3a (FOXO3a), which is a transcription factor that mediates a variety of cellular processes, but the functions of which in regulating oocyte quality in MSCs therapy still remain inconclusive. In this study, the RNA-seq data of metaphase II (MII) oocytes and GCs isolated from COCs confirmed that, GCs of immature follicles show the most potential to be the targeted cells of bone marrow mesenchymal stem cells (BMSCs) by FOXO3a signaling pathway. Furthermore, we demonstrated the effectiveness of BMSCs co-culture with aging COCs to enhance oocyte quality and found its mechanism to function via ameliorating the biological function of GCs by alleviating FOXO3a levels. These results provide significant fundamental research on MSCs therapy on ovarian aging, as well as offering guidance for raising the success rate of assisted reproductive technology such IVM in clinical and non-clinical settings. [ABSTRACT FROM AUTHOR]

Aim: RADA16-PLGA composite scaffolds constructed with simultaneous loading of BMSCs and TGF-β3 and explored their ability for chondrogenic differentiation in vitro. Methods: The performance of the composite scaffolds is assessed by rheometer assay, electron microscopic structural observation and ELISA release assay. The biosafety of the composite scaffolds is assessed by cytocompatibility assay and cell migration ability. The chondrogenic differentiation ability of composite scaffolds is evaluated by Alisin blue staining, PCR and immunofluorescence staining. Results: The composite scaffold has a good ECM-like structure, the ability to control the release of TGF-β3 and good biocompatibility. More importantly, the composite scaffolds can induce the differentiation of BMSCs to chondrocytes. Conclusion: Composite scaffolds are expected to enhance the endogenous NP repair process. Graphical Abstract RADA16 aqueous solution, PLGA microspheres and BMSCs are mixed to form a composite aqueous solution. The aqueous solution is injected into the body and exposed to the acidic environment of the NP. The nucleus pulposus pH induces gelation of the aqueous solution to form a composite scaffold. The composite scaffold can function in vivo. It induces the differentiation of BMSCs into chondrocytes and subsequently secretes a large amount of ECM such as Collagen II, GAGAs and SOX-9. In addition, it also enhances the self-repair of endogenous BMSCs, which is to replenish the reduction of nucleus pulposus cells and the loss of ECM. Article highlights There is an endogenous repair process during myeloid degeneration, but there is an insufficient number of stem cells and a lack of sustained release of growth factors. BMSCs can differentiate into chondrocytes and secrete extracellular matrix in response to TGFβ3. The physico-mechanical properties and ultrastructure of the RADA16 hydrogel meet the requirements of the target material. The suitable conditions for RADA16 are 1% w/v and pH = 7.0. The RADA16-PLGA hydrogel-microsphere composite scaffold structure can encapsulate TGF-β3 to form a good controlled release system. The suitable conditions for the composite scaffold are 1% w/v and pH = 7.0. Composite scaffolds have good cytocompatibility. Composite scaffolds can recruit BMSCs for migration to the composite scaffold. The composite scaffold can adapt to the acidic environment of the degenerated disc, inducing the differentiation of BMSCs to chondrocyte-like cells while secreting large amounts of ECM. Composite scaffolds are expected to enhance the endogenous NP repair process and are promising tissue-engineered scaffolds for IVDD repair. [ABSTRACT FROM AUTHOR]

Background: The progression of osteoporosis (OP) can dramatically increase the risk of fractures, which seriously disturb the life of elderly individuals. Specific protein 1 (SP1) is involved in OP progression. However, the mechanism by which SP1 regulates OP progression remains unclear. Objective: This study investigated the mechanism underlying the function of SP1 in OP. Methods: SAMP6 mice were used to establish an in vivo model of age-dependent OP, and BALB/c mice were used as controls. BMSCs were extracted from two subtypes of mice. Hematoxylin and eosin staining were performed to mark the intramedullary trabecular bone structure to evaluate histological changes. ChIP assay was used to assess the targeted regulation between SP1 and miR-133a-3p. The binding sites between MAPK3 and miR-133a-3p were verified using a dual-luciferase reporter assay. The mRNA levels of miR-133a-3p and MAPK3 were detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The protein expression of SP1, MAPK3, Colla1, OCN, and Runx2 was examined using Western blotting. Alkaline phosphatase (ALP) kit and Alizarin Red S staining were used to investigate ALP activity and mineralized nodules, respectively. Results: The levels of SP1 and miR-133a-3p were upregulated, whereas the expression of MAPK3 was downregulated in BMSCs from SAMP6 mice, and miR-133a-3p inhibitor accelerated osteogenic differentiation in BMSCs. SP1 directly targeted miR-133a-3p, and MAPK3 was the downstream mRNA of miR-133a-3p. Mechanically, SP1 accelerated osteogenic differentiation in BMSCs via transcriptional mediation of the miR-133a-3p/MAPK3 axis. Conclusion: SP1 regulates osteogenic differentiation by mediating the miR-133a-3p/MAPK3 axis, which would shed new light on strategies for treating senile OP. [ABSTRACT FROM AUTHOR]

Background: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. Methods: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague–Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. Results: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. Conclusion: NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs. [ABSTRACT FROM AUTHOR]

Background: Cellular senescence is an important process related to the pathogenic mechanism of different disorders, especially bone loss. During senescence, bone marrow stromal cells (BMSCs) lose their self-renewal and functional differentiation abilities. Therefore, finding signals opposing the osteogenic differentiation of BMSCs within bone marrow microenvironment is the important for elucidating these above-mentioned mechanisms. Inflammatory cytokines affect bone physiology and remodeling. However, the function of interleukin-19 (IL-19) in skeletal system remains unclear. Methods: The mouse model of IL-19 knockout was established through embryonic stem cell injection for analyzing how IL-19 affected bone formation. Micro-CT examinations were performed to evaluate bone microstructures. We performed a three-point bending test to measure bone stiffness and the ultimate force. Antibody arrays were performed to detect interleukin family members in bone marrow aspirates. BMSCs were cultured and induced for osteogenic differentiation. Results: According to our findings, there was increased IL-19 accumulation within bone marrow in old mice relative to that in their young counterparts, resulting in bone loss via the inhibition of BMSCs osteogenic differentiation. Among Wnt/β-catenin pathway members, IL-19 strongly upregulated sFRP1 via STAT3 phosphorylation. The inhibition of STAT3 and sFRP1 abolished IL-19's inhibition against the BMSCs osteogenic differentiation. Conclusion: To sum up, IL-19 inhibited BMSCs osteogenic differentiation in old mice. Our findings shed novel lights on pathogenic mechanism underlying age-related bone loss and laid a foundation for further research on identifying novel targets to treat senile osteoporosis. [ABSTRACT FROM AUTHOR]

Osteoporosis is a metabolic bone disease that involves gradual loss of bone density and mass, thus resulting in increased fragility and risk of fracture. Inflammatory cytokines, such as tumour necrosis factor α (TNF‐α), inhibit osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and several microRNAs are implicated in osteoporosis development. This study aimed to explore the correlation between TNF‐α treatment and miR‐27a‐3p expression in BMSC osteogenesis and further understand their roles in osteoporosis. An osteoporosis animal model was established using ovariectomized (OVX) mice. Compared with Sham mice, the OVX mice had a significantly elevated level of serum TNF‐α and decreased level of bone miR‐27a‐3p, and in vitro TNF‐α treatment inhibited miR‐27a‐3p expression in BMSCs. In addition, miR‐27a‐3p promoted osteogenic differentiation of mouse BMSCs in vitro, as evidenced by alkaline phosphatase staining and Alizarin Red‐S staining, as well as enhanced expression of the osteogenic markers Runx2 and Osterix. Subsequent bioinformatics analysis combined with experimental validation identified secreted frizzled‐related protein 1 (Sfrp1) as a downstream target of miR‐27a‐3p. Sfrp1 overexpression significantly inhibited the osteogenic differentiation of BMSCs in vitro and additional TNF‐α treatment augmented this inhibition. Moreover, Sfrp1 overexpression abrogated the promotive effect of miR‐27a‐3p on the osteogenic differentiation of BMSCs. Furthermore, the miR‐27a‐3p–Sfrp1 axis was found to exert its regulatory function in BMSC osteogenic differentiation via regulating Wnt3a–β‐catenin signalling. In summary, this study revealed that TNF‐α regulated a novel miR‐27a‐3p–Sfrp1 axis in osteogenic differentiation of BMSCs. The data provide new insights into the development of novel therapeutic strategies for osteoporosis. What is the central question of this study?What is the mechanism by which TNF‐α plays its critical role in osteogenesis via the modulation of bone loss and formation?What is the main finding and its importance?TNF‐α regulates a novel miR‐27a‐3p–Sfrp1 axis in osteogenic differentiation of bone marrow mesenchymal stem cells. [ABSTRACT FROM AUTHOR]

This study determines the role and mechanism of APS in cyclophosphamide-induced myelosuppression in mice and bone mesenchymal stem cells (BMSCs) cell model. Cy-induced myelosuppression mice and BMSCs cell model were established. Fifty C57BL/6 mice (weighing 20 ± 2 g) were randomly divided into five groups. Femur and tibia samples, bone marrow samples, and blood samples were collected 3 days after the last injection of Cy. Histopathology changes and cell apoptosis were detected. Cell viability, apoptosis, cycle distribution, reactive oxygen species activity, osteogenesis ability, and protein levels were detected. γ-H2AX and senescence-associated β-galactosidase activity expression was detected by immunofluorescence. Cy-induced senescence and Wnt/β-catenin related protein levels were detected using western blotting. The results showed that APS effectively induced Cy-induced histological injury and cell apoptosis rate. After treated with APS, ROS and ALP levels were significantly increased. In BMSCs, cell viability, apoptosis, and cell cycle distribution were also influenced by APS treatment. Compared with the control group, cell viability was significantly increased, the cell apoptosis rate was decreased while the number of cells remained in the G0-G1 phase was increased. Meanwhile, ROS levels were significantly increased in APS group. Cell senescence and Wnt/β-catenin related protein (γ-H2AX, SA-β-gal, p21, p16, p-β-catenin/ β-catenin, c-Myc, and AXIN2) levels were also altered both in vivo and in vitro. Interestingly, the effects of APS were reversed by BML-284. Our results indicate that APS protected Cy-induced myelosuppression through the Wnt/β-catenin pathway and APS is a potential therapeutic drug for Cy-induced myelosuppression. • By treated with APS, cell senescence and Wnt/β-catenin related protein levels were also altered both in vivo and in vitro. • APS protected Cy-induced myelosuppression through the Wnt/β-catenin pathway. • APS is a potential therapeutic drug for Cy-induced myelosuppression. [ABSTRACT FROM AUTHOR]

Suitable biomaterials with seed cells have promising potential to repair bone defects. However, bone marrow mesenchymal stem cells (BMSCs), one of the most common seed cells used in tissue engineering, cannot differentiate efficiently and accurately into functional osteoblasts. In view of this, a new tissue engineering technique combined with BMSCs and scaffolds is a major task for bone defect repair. Lentiviruses interfering with miR‐136‐5p or Smurf1 expression were transfected into BMSCs. The effects of miR‐136‐5p or Smurf1 on the osteogenic differentiation (OD) of BMSCs were evaluated by measuring alkaline phosphatase activity and calcium deposition. Then, the targeting relationship between miR‐136‐5p and Smurf1 was verified by bioinformatics website analysis and dual luciferase reporter assay. Then, a rabbit femoral condyle bone defect model was established. miR‐136‐5p/BMSCs/β‐TCP scaffold was implanted into the defect, and the repair of the bone defect was detected by Micro‐CT and HE staining. Elevating miR‐136‐5p‐3p or suppressing Smurf1 could stimulate OD of BMSCs. miR‐136‐5p negatively regulated Smurf1 expression. Overexpressing Smurf1 reduced the promoting effect of miR‐136‐5p on the OD of BMSCs. miR‐136‐5p/BMSCs/β‐TCP could strengthen bone density in the defected area and accelerate bone repair. SmurF1‐targeting miR‐136‐5p‐modified BMSCs combined with 3D‐printed β‐TCP scaffolds can strengthen osteogenic activity and alleviate bone defects. [ABSTRACT FROM AUTHOR]

Objective: Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis‐derived BMSCs (OP‐BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6‐methyladenosine RNA binding protein 3) on the osteogenic traits of OP‐BMSCs and identify potential therapeutic targets to boost their bone formation ability. Methods: We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single‐cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. Results: Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single‐cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP‐BMSCs substantially impedes their proliferation and differentiation. Conclusion: YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP‐BMSCs. [ABSTRACT FROM AUTHOR]

In our previous study, IRX5 has been revealed a significant role in adipogenesis of hBMSCs. Considering the expansion of adipose tissue in bone marrow in aged and ovariectomy‐related osteoporosis, the effect of IRX5 on the osteogenesis of BMSCs still needs to be elucidated. In vivo, models of aging‐induced and ovariectomy‐induced osteoporotic mice, and in vitro studies of IRX5 gene gain‐ and loss‐of‐function in hBMSCs were employed. Histology, immunofluorescence, qRT‐PCR, and Western blot analysis were performed to detect the functions of IRX5 in hBMSCs osteogenic differentiation. RNA‐seq, transmission electron microscopy, Seahorse mito‐stress assay, and Surface Sensing of Translation assay were conducted to explore the effect of mammalian/mechanistic target of rapamycin (mTOR)‐mediated ribosomal translation and mitochondrial functions in the regulation of hBMSCs differentiation by IRX5. As a result, elevated IRX5 protein expression levels were observed in the bone marrow of osteoporotic mice compared to normal mice. IRX5 overexpression attenuated osteogenic processes, whereas IRX5 knockdown resulted in enhanced osteogenesis in hBMSCs. RNA‐seq and enrichment analysis unveiled that IRX5 overexpression exerted inhibitory effects on ribosomal translation and mitochondrial functions. Furthermore, the application of the mTOR activator, MHY1485, effectively reversed the inhibitory impact of IRX5 on osteogenesis and mitochondrial functions in hBMSCs. In summary, our findings suggest that IRX5 restricts mTOR‐mediated ribosomal translation, consequently impairing mitochondrial OxPhos, which in turn results in osteogenic dysfunction of hBMSCs. [ABSTRACT FROM AUTHOR]

To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene transfected bone marrow mesenchymal stem cells (BMSCs) on vascular endothelial injury, coagulation function and inflammatory factors in septic mice. 60 SD mice were divided into Sham group, model (Model) group, BMSCs group and GM-CSF-BMSCs group, with 15 mice in each group, sepsis model of mice was prepared by cecal ligation and puncture. The expression of GM-CSF gene was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The tail bleeding time of 24h mice was recorded. The levels of plasma prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (Fib) were detected. The levels of serum interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1 and IL-10 were detected by ELISA. The morphology of vascular tissue was observed by hematoxylin-eosin (HE) staining. Apoptosis of vascular endothelial cells was detected by TUNEL method. The expression of vascular tissue factor (TF), tissue factor pathway inhibitor (TFPI), antithrombin (ATIII), vascular cell adhesion molecule 1 (VCAM1) and protease-activated receptor 1 (PAR1) were detected by Western blot. Compared with BMSCs in CK group, the expression of GM-CSF mRNA in BMSCs in Transfection group was significantly increased (P<0.05). Compared with Sham group, the vascular morphology of the mice in Model group was significantly damaged, the tail bleeding time, Fib content, serum IL-10 level and ATIII protein expression in vascular endothelial tissue of the mice in Model group were significantly decreased, PT, APTT, TT time, serum IL-6, TNF-α, IL-1 levels, apoptosis rate, TF, TFPI, VCAM1, PAR1 protein expression in vascular endothelial tissue were significantly increased (P<0.05). Compared with Model group, the damage of vascular morphology and structure in BMSCs group and GM-CSF-BMSCs group was significantly reduced, the tail bleeding time, Fib content, serum IL-10 level and ATIII protein expression in vascular endothelial tissue were significantly increased, PT, APTT time, serum IL-6, TNF-α, IL-1 level, apoptosis rate, TF, TFPI, VCAM1 and PAR1 protein expression in vascular endothelial tissue were significantly decreased (P<0.05). The detection indexes in GM-CSF-BMSCs group were better than those in BMSCs group (P<0.05). GM-CSF transfected BMSCs can reduce the inflammatory response and vascular endothelial cell injury in septic mice, and improve coagulation function. [ABSTRACT FROM AUTHOR]

Recently, epidermal growth factor-like domain protein 6 (EGFL6) was proposed as a candidate gene for coupling angiogenesis to osteogenesis during bone repair; however, the exact role and underlying mechanism are largely unknown. Here, using immunohistochemical and Western blotting analyses, we found that EGFL6 was downregulated in the femoral head tissue of patients with steroid-induced osteonecrosis of the femoral head (SONFH) compared to patients with traumatic femoral neck fracture (FNF), accompanied by significantly downregulation of osteogenic and angiogenic marker genes. Then, bone marrow mesenchymal stem cells (BMSCs) were isolated from the FNF and the SONFH patients, respectively, and after identification by immunofluorescence staining surface markers, the effect of EGFL6 on their abilities of osteogenic differentiation and angiogenesis was evaluated. Our results of alizarin red staining and tubular formation experiment revealed that BMSCs from the SONFH patients (SONFH-BMSCs) displayed an obviously weaker ability of osteogenesis than FNF-BMSCs, and EGFL6 overexpression improved the abilities of osteogenic differentiation and angiogenesis of SONFH-BMSCs. Moreover, EGFL6 overexpression activated extracellular signal-regulated kinases 1/2 (ERK1/2). ERK1/2 inhibitor U0126 reversed the promoting effect of EGFL6 overexpression on the expression of osteogenesis and angiogenesis-related genes in the SONFH femoral head. In conclusion, EGFL6 plays a protective role in SONFH, it promotes osteogenesis and angiogenesis of BMSCs, and its effect is likely to be related to ERK1/2 activation. [ABSTRACT FROM AUTHOR]

Urethral stricture (US) is a challenging problem in urology and its pathogenesis of US is closely related to the fibrotic process. Previous evidence has indicated the downregulation of microRNA (miR)‐486 in injured urethral specimens of rats. This study aimed to explore the effects of miR‐486‐overexpressed bone marrow mesenchymal stem cells (BMSCs) on US. BMSCs were identified by detecting their multipotency and surface antigens. Lentivirus virus expressing miR‐486 was transduced into rat BMSCs to overexpress miR‐486. Transforming growth factor (TGF)‐β1 induced fibrotic phenotypes in urethral fibroblasts (UFs) and rat models. Western blotting showed protein levels of collagen I/III and collagen type XIII alpha 1 chain (Col13a1). Real time quantitative polymerase chain reaction was utilized for messenger RNA level evaluation. Hematoxylin‐eosin, Masson's trichrome, and Von Willebrand Factor staining were conducted for histopathological analysis. Immunofluorescence staining was employed for detecting alpha smooth muscle actin (α‐SMA) expression. Luciferase reporter assay verified the interaction between miR‐486 and Col13a1. The results showed that miR‐486‐overexpressed BMSCs suppressed collagen I/III and α‐SMA expression in TGF‐β1‐stimulated UFs. miR‐486‐overexpressed BMSCs alleviated urethral fibrosis, collagen deposition, and epithelial injury in the urethral tissue of US rats. miR‐486 targeted and negatively regulated Col13a1 in US rats. In conclusion, overexpression of miR‐486 in BMSCs targets Col13a1 and attenuates urethral fibrosis in TGF‐β1‐triggered UFs and US rats. [ABSTRACT FROM AUTHOR]

Existing studies have shown that Astragalus membranaceus (AM) and its active ingredients astragalus polysaccharides, oninon, and astragalus methyl glycosides can attenuate X-ray radiation-induced injury. However, there are no studies on how isoliquiritigenin (ISL) attenuate the bystander effect of bone marrow mesenchymal stem cells (BMSCs) induced by carbon ion radiation therapy for lung cancer. This study aimed to investigate the AM-derived small molecule ISL to enhance radiotherapy sensitivity by attenuating the carbon ion radiation-induced bystander effect (RIBE) in BMSCs to elucidate its mechanism of action. In this study, we established a C57BL/6 mouse lung cancer transplantation tumor model in vivo and a co-culture model of A549 cells and BMSCs in vitro, and the models were successfully treated with carbon ions. In further work, we used flow cytometry, immunofluorescence, Western blot, enzyme-linked immunosorbent assay (ELISA), inhibitor, short hairpin RNA (shRNA), Cell Counting Kit-8 (CCK-8), and other methods to illustrate the mechanism. In the next experiments, we found that ISL combined with carbon ion radiotherapy had a significant anti-tumor effect and protected BMSCs from radiation damage. The aim of this study was to investigate the potential of ISL in enhancing the sensitivity of lung cancer cells to radiotherapy and attenuating RIBE in both in vitro and in vivo settings. Traditional Chinese medicine combined with radiation therapy is a promising and innovative treatment for non-small cell lung cancer. These results establish a theoretical foundation for further clinical development of ISL as a potential radiosensitizer option.

Various biological effects of ionizing radiation, especially continuous exposure to low-dose radiation (LDR), have attracted considerable attention. Impaired bone structure caused by LDR has been reported, but little is known about the mechanism involved in the disruption of bone metabolism. In this study, given that LDR was found to (at a cumulative dose of 0.10 Gy) disturb the serum Mg2+ level and Notch1 signal in the mouse femur tissues, the effects of LDR on osteogenesis and the underlying molecular mechanisms were investigated based on an in vitro culture system for bone marrow stromal cells (BMSCs). Our data showed that cumulative LDR suppressed the osteogenic potential in BMSCs as a result of upregulation of Notch1 signaling. Further analyses indicated that the upregulation of NICD1 (Notch1 intracellular domain), the key intracellular domain for Notch1 signaling, under LDR was a consequence of enhanced protein stabilization caused by SUMOylation (small ubiquitin-like modification). Specifically, the downregulation of SENP1 (sentrin/SUMO-specific protease 1) expression induced by LDR enhanced the SUMOylation of NICD1, causing the accumulation of Notch1 signaling, which eventually inhibited the osteogenic potential of BMSCs. In conclusion, this work expounded on the mechanisms underlying the impacts of LDR on bone metabolism and shed light on the research on bone regeneration under radiation.

Abstract The accumulation of reactive oxygen species (ROS) within the bone marrow microenvironment leads to diminished osteogenic differentiation and heightened lipogenic differentiation of mesenchymal stem cells residing in the bone marrow, ultimately playing a role in the development of osteoporosis (OP). Mitigating ROS levels is a promising approach to counteracting OP. In this study, a nanozyme composed of magnesium-based zeolitic imidazolate frameworks (Mg-ZIF) was engineered to effectively scavenge ROS and alleviate OP. The results of this study indicate that Mg-ZIF exhibits significant potential in scavenging ROS and effectively promoting osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Additionally, Mg-ZIF was found to inhibit the differentiation of BMSCs into adipose cells. In vivo experiments further confirmed the ability of Mg-ZIF to mitigate OP by reducing ROS levels. Mechanistically, Mg-ZIF enhances the differentiation of BMSCs into osteoblasts by upregulating lipid metabolic pathways through ROS scavenging. The results indicate that Mg-ZIF has potential as an effective therapeutic approach for the treatment of osteoporosis.

Abstract Background Fractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing. Methods Rat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed. Results The results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins. Conclusion Silencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing.

Abstract Treatment of bone defects remains crucial challenge for successful bone healing, which arouses great interests in designing and fabricating ideal biomaterials. In this regard, the present study focuses on developing a novel fluffy scaffold of poly Lactide-co-glycolide (PLGA) composites with hydroxyapatite (HA) scaffold used in bone defect repair in rabbits. This fluffy PLGA/HA composite scaffold was fabricated by using multi-electro-spinning combined with biomineralization technology. In vitro analysis of human bone marrow mesenchymal stem cells (BMSCs) seeded onto fluffy PLGA/HA composite scaffold showed their ability to adhere, proliferate and cell viability. Transplant of fluffy PLGA/HA composite scaffold in a rabbit model showed a significant increase in mineralized tissue production compared to conventional and fluffy PLGA/HA composite scaffold. These findings are promising for fluffy PLGA/HA composite scaffolds used in bone defects. Graphical Abstract

Multiple regulatory mechanisms are in place to ensure the normal processes of bone metabolism, encompassing both bone formation and absorption. This study has identified chaperone‐mediated autophagy (CMA) as a critical regulator that safeguards bone formation from the detrimental effects of excessive inflammation. By silencing LAMP2A or HSCA8, we observed a hindrance in the osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. To further elucidate the role of LAMP2A, we generated LAMP2A gene knockdown and overexpression of mouse BMSCs (mBMSCs) using adenovirus. Our results showed that LAMP2A knockdown led to a decrease in osteogenic‐specific proteins, while LAMP2A overexpression favored the osteogenesis of mBMSCs. Notably, active‐β‐catenin levels were upregulated by LAMP2A overexpression. Furthermore, we found that LAMP2A overexpression effectively protected the osteogenesis of mBMSCs from TNF‐α, through the PI3K/AKT/GSK3β/β‐catenin pathway. Additionally, LAMP2A overexpression significantly inhibited osteoclast hyperactivity induced by TNF‐α. Finally, in a murine bone defect model, we demonstrated that controlled release of LAMP2A overexpression adenovirus by alginate sodium capsule efficiently protected bone healing from inflammation, as confirmed by imaging and histological analyses. Collectively, our findings suggest that enhancing CMA has the potential to safeguard bone formation while mitigating hyperactivity in bone absorption. [ABSTRACT FROM AUTHOR]

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. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus‐mediated CPM overexpression had the reverse effects. In vivo, 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. [ABSTRACT FROM AUTHOR]

Recent interest in preventing the development of osteoporosis has focused on the regulation of redox homeostasis. However, the action of lycopene (LYC), a strong natural antioxidant compound, on osteoporotic bone loss remains largely unknown. Here, we show that oral administration of LYC to OVX rats for 12 weeks reduced body weight gain, improved lipid metabolism, and preserved bone quality. In addition, LYC treatment inhibited ROS overgeneration in serum and bone marrow in OVX rats, and in BMSCs upon H2O2 stimulation, leading to inhibiting adipogenesis and promoting osteogenesis during bone remodeling. At the molecular level, LYC improved bone quality via an increase in the expressions of FoxO1 and Runx2 and a decrease in the expressions of PPARγ and C/EBPα in OVX rats and BMSCs. Collectively, these findings suggest that LYC attenuates osteoporotic bone loss through promoting osteogenesis and inhibiting adipogenesis via regulation of the FoxO1/PPARγ pathway driven by oxidative stress, presenting a novel strategy for osteoporosis management. [ABSTRACT FROM AUTHOR]

Bone marrow mesenchymal stem cells (BMSC) are promising candidates for the treatment of trans-territory perforator flap necrosis. However, the low retention and survival rate of engrafted BMSCs limit their therapeutic efficacy. Strategies either modifying BMSCs or alleviating the inflammatory environment may solve this problem. Thus, we aimed to explore the therapeutic efficacy of sequential transplantation of exosomes and hypoxia pretreated BMSCs on flap necrosis. After the perforator flap model was created, the exosomes derived from BMSCs were injected immediately into choke zone II followed by transplantation of hypoxia pretreated BMSCs on Day 2. Gross view was performed to assess the flap survival, enzyme-linked immunosorbent assay was performed to evaluate the inflammatory factor level, microvessel number was assessed and quantitative polymerase chain reaction (qPCR) was performed to assess angiogenesis. We found that exosome delivery significantly reduced inflammatory cytokines levels on Day 1 and Day 3 and promoted the engrafted BMSCs' survival on Day 7. After combining with transplantation of hypoxia pretreated BMSCs, the flap survival rate and the angiogenesis-related gene expression were significantly higher than in the other three groups; the von Willebrand factor (vWF) vascular diameter and vWF vascular count were significantly higher than in the phosphate buffered saline (PBS) group. Thus, we concluded that sequential transplantation of exosomes and BMSCs combinatorially pretreated with hypoxia further facilitated flap survival. This sequential transplantation approach provides novel insights into the clinical treatment of flap necrosis. [ABSTRACT FROM AUTHOR]

Current myocardial infarction (MI) treatments are suboptimal, necessitating deeper pathogenesis understanding of MI. This research explored how exosomes (Exo) derived from bone marrow mesenchymal stem cells (BMSCs) contribute to MI mitigation and their therapeutic potential. Isolated BMSCs was identified by microscope, flow cytometry, alizarin red and oil red O staining. Exo were identified by TEM, NTA and western blot. HE staining, masson staining, and cardiac function parameters were used to assess the cardiac function in MI mice. TUNEL staining, western blot and qRT-PCR were used to detect apoptosis, inflammatory factors and M1/M2 markers. The NF-κB pathway activation was detected through western blot assays. Immunofluorescence, qRT-PCR, western blot, and flow cytometry were employed to evaluate macrophage polarization. MI mice showed cardiac injury, increased apoptosis and inflammation, while BMSCs-Exo treatment alleviated these effects. In MI mice, the macrophage M1 polarization was increased and the NF-κB pathway was activated, whereas BMSCs-Exo treatment reversed these changes. Furthermore, CISH expression was reduced in MI mice, but was elevated with BMSCs-Exo treatment. In vitro, LPS shifted RAW264.7 cells to M1 phenotype and activated the NF-κB pathway, yet BMSCs-Exo shifted them to M2 phenotype and inhibited the NF-κB pathway. Mechanistically, BMSCs-Exo induced macrophage M2 polarization by transmitting CISH to inhibit NF-κB activation. BMSCs-Exo mitigates MI by transmitting CISH to inhibit the NF-κB pathway, promoting macrophages to M2 type. This implies BMSCs-Exo could be a useful treatment for MI, and CISH could be a potential therapy target. [ABSTRACT FROM AUTHOR]

The accumulation of reactive oxygen species (ROS) within the bone marrow microenvironment leads to diminished osteogenic differentiation and heightened lipogenic differentiation of mesenchymal stem cells residing in the bone marrow, ultimately playing a role in the development of osteoporosis (OP). Mitigating ROS levels is a promising approach to counteracting OP. In this study, a nanozyme composed of magnesium-based zeolitic imidazolate frameworks (Mg-ZIF) was engineered to effectively scavenge ROS and alleviate OP. The results of this study indicate that Mg-ZIF exhibits significant potential in scavenging ROS and effectively promoting osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Additionally, Mg-ZIF was found to inhibit the differentiation of BMSCs into adipose cells. In vivo experiments further confirmed the ability of Mg-ZIF to mitigate OP by reducing ROS levels. Mechanistically, Mg-ZIF enhances the differentiation of BMSCs into osteoblasts by upregulating lipid metabolic pathways through ROS scavenging. The results indicate that Mg-ZIF has potential as an effective therapeutic approach for the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Background: Fractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing. Methods: Rat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed. Results: The results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins. Conclusion: Silencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing. [ABSTRACT FROM AUTHOR]

This study aims to investigate the induction effect of LncRNA-CIR6 on MSC differentiation into cardiogenic cells in vitro and in vivo. In addition to pretreatment with Ro-3306 (a CDK1 inhibitor), LncRNA-CIR6 was transfected into BMSCs and hUCMSCs using jetPRIME. LncRNA-CIR6 was further transfected into the hearts of C57BL/6 mice via 100 μL of AAV9-cTnT-LncRNA-CIR6-ZsGreen intravenous injection. After three weeks of transfection followed by AMI surgery, hUCMSCs (5 × 105/100 μL) were injected intravenously one week later. Cardiac function was evaluated using VEVO 2100 and electric mapping nine days after cell injection. Immunofluorescence, Evans blue-TTC, Masson staining, FACS, and Western blotting were employed to determine relevant indicators. LncRNA-CIR6 induced a significant percentage of differentiation in BMSCs (83.00 ± 0.58)% and hUCMSCs (95.43 ± 2.13)% into cardiogenic cells, as determined by the expression of cTnT using immunofluorescence and FACS. High cTNT expression was observed in MSCs after transfection with LncRNA-CIR6 by Western blotting. Compared with the MI group, cardiac contraction and conduction function in MI hearts treated with LncRNA-CIR6 or combined with MSCs injection groups were significantly increased, and the areas of MI and fibrosis were significantly lower. The transcriptional expression region of LncRNA-CIR6 was on Chr17 from 80209290 to 80209536. The functional region of LncRNA-CIR6 was located at nucleotides 0–50/190–255 in the sequence. CDK1, a protein found to be related to the proliferation and differentiation of cardiomyocytes, was located in the functional region of the LncRNA-CIR6 secondary structure (from 0 to 17). Ro-3306 impeded the differentiation of MSCs into cardiogenic cells, while MSCs transfected with LncRNA-CIR6 showed a high expression of CDK1. LncRNA-CIR6 mediates the repair of infarcted hearts by inducing MSC differentiation into cardiogenic cells through CDK1. [ABSTRACT FROM AUTHOR]

[Display omitted] Angiogenesis plays a pivotal role in tissue development and regeneration, but few safe and efficient pro-angiogenic factors are available for long-term clinical therapy. Although stem cell-derived exosomes exhibit excellent biocompatibility and considerable pro-angiogenic capacity, their low yield extremely limits their clinical potential. Exosome-mimetics (EMs) employ similar features to exosomes and can be produced in a higher yield. In this study, the HY-EMs were produced to enhance in vitro and in vivo pro-angiogenic ability from hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) using a serial extrusion method. The yield of EMs was over 10 times greater than that of exosomes. Furthermore, compared with the EMs group, the tube number of human umbilical vein endothelial cells (HUVECs) in vitro doubled in the HY-EMs treated group, and the number of blood vessels increased by 186% in subcutaneously implanted scaffold with HY-EMs. More importantly, the HY-EMs effectively promoted vascularized osteogenesis in a critical-sized cranial defect model, with the new bone volume and vessel number in regenerated bone increased to 120% and 175%, respectively. Consequently, our current work provides a promising strategy to customize and mass-produce functional EMs for vascularized osteogenesis. [ABSTRACT FROM AUTHOR]