Differentiation Of Bone Marrow Mesenchymal Stem Cells Research Articles

Restoring bone-fat equilibrium: Baicalin's impact on P38 MAPK pathway for treating diabetic osteoporosis

Diabetes can lead to a disorder of bone-fat balance, a significant cause of osteoporosis due to changes in environmental factors. Baicalin (Bai), an active ingredient of Scutellaria baicalensis, has been confirmed to possess antioxidant, hypoglycemic, and anti-osteoporotic effects. However, a comprehensive understanding of Bai's influence on diabetic osteoporosis (DOP), including its effects and underlying mechanisms, remains elusive. This study investigated Bai's impact on the bone-fat equilibrium in rats with DOP. The results indicated that Bai alleviated bone damage in DOP by promoting osteogenesis and inhibiting adipogenesis. Concurrently, through bioinformatics analysis, it was suggested that Bai's mechanism of action might involve the P38-MAPK pathway. In vitro, Bai was found to enhance the development of bone marrow mesenchymal stem cells (BMSCs) towards osteogenic lineages while suppressing their differentiation towards adipogenic lineages. It was discovered that Bai's promotion of BMSC osteogenic differentiation depends on the P38-MAPK pathway. Additionally, the synergistic effect mediated by Bai and P38-MAPK inhibitor suppressed BMSC adipogenic differentiation. Our research indicates that the P38-MAPK pathway play a role in Bai's effects on the osteogenic-adipogenic differentiation of BMSCs, showcasing the potential for DOP treatment. This study highlights Bai's ability to regulate the equilibrium between bone and fat, presenting a novel approach to adressing DOP.

Osteogenerative and corrosion-decelerating teriparatide-mediated strontium–zinc phosphate hybrid coating on biodegradable zinc–copper alloy for orthopaedic applications

Zinc–copper (ZnCu) alloys recently showed great potential as biodegradable materials for bone implants thanks to their excellent mechanical strength, biosafety, and promising bioactivities. However, bare ZnCu alloys exhibited poor cytocompatibility and osteointegration. Moreover, the biological functions of ZnCu remain to be corroborated. Herein, the teriparatide (PTH)-mediated strontium–zinc phosphate (SrZnP) organic–inorganic hybrid coating was fabricated on the surface of ZnCu substrate via a chemical conversion method to contain the release of Zn ions and improve biofunctionality in osteogenesis. The characteristics of the hybrid coating were optimised by adjusting the amount of PTH molecules added. A dense and homogenous coating with micellar crystal micro-/nano-structure was obtained. The coated samples showed a significantly decreased corrosion rate and a more uniform corrosion mode, effectively suppressing the release of Zn2+. Additionally, the coated ZnCu enhanced the proliferation of bone marrow mesenchymal stem cells (BMSCs), promoted the expression of alkaline phosphatase and the formation of calcium nodules, and upregulated the levels of osteogenic-related genes. More important, the hybrid coating activated the classic osteogenic signalling pathway of BMP-2/Smad1/Runx2 and Sirt1/Runx2 to induce the osteogenic differentiation of BMSCs. These appealing features can be mainly attributed to the constituents of released Zn2+, Sr2+, and PTH molecules in the control, as well as the unique topographical characteristics of the coatings. Taken together, the PTH&SrZnP hybrid coating could be a promising option for surface modification of biodegradable metallic Zn for orthopaedic applications.

Fabrication of a coral-like barium titanate nano-piezoelectric coating and its effect on promoting osteogenic differentiation of bone marrow mesenchymal stem cell in vitro

Objective: To investigate the biocompatibility of coral-like barium titanate nano-piezoelectric coatings and the influence of ultrasound-excited piezoelectric effect on the early osteogenic differentiation. Methods: The barium titanate nano-piezoelectric coating (the coating group) was prepared on the surface of titanium metal by anodic oxidation, hydrothermal reaction and high-temperature annealing, and polished titanium specimens were used as control group. The surface morphology, composition, and crystal phase and hydrophilicity of the two groups of titanium specimens were characterized using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy and contact angle meter. The piezoelectric properties of the materials were characterized by piezoresponse force microscopy (PFM). Rat bone marrow mesenchymal stem cells (BMSC) were cultured and identified and seeded the surface of titanium specimens in two groups. The cells seeded on blank culture plates were used as blank group. After low intensity pulsed ultrasound intervention, cell proliferation and live/dead staining were detected to evaluate cytocompatibility of the coatings. Alkaline phosphatase (ALP) activity of each group was detected by ALP staining kit, and the expression of osteogenesis-related genes [integrin, bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX2)] was detected by real-time fluorescent quantitative PCR (RT-qPCR) to evaluate the effect of the coating on promoting the early osteogenic differentiation of BMSC. Results: The surface of titanium specimens in the coating group showed a uniform coral-like morphology, and the diameter of the coral tentacles was 70-100 nm. The main component was tetragonal barium titanate. The surface hydrophilicity of the coating group (water contact angle 10.12°±0.93°) was significantly better than that of the control group (water contact angle 78.32°±0.71°) (F=10 165.91, P<0.001). The coating has a stable piezoelectric property with a piezoelectric constant of about 5 pC/N. Cell experiments showed that, with or without ultrasound, the cell proliferation activity of the coating group was significantly lower than that of the blank group and the control group on the third day (P<0.05). On the fifth day, with or without ultrasound, there was no significant difference in cell proliferation activity between the three groups (P>0.05). After 7 days of culture, the ALP activity of the coating group was significantly higher than that of the blank group and the control group (P<0.05). The results of RT-qPCR showed that the mRNA expression of integrin and BMP-2 in the coating group with ultrasound was significantly higher than that in the other groups with ultrasound, and was higher than that of the coating group without ultrasound (P<0.05). The expression of integrin mRNA in the control group with ultrasound was significantly higher than that in the control group without ultrasound (P<0.05). The expression of RUNX2 mRNA in the coating group with ultrasound was significantly higher than that in the coating group without ultrasound (P<0.05). Conclusions: The coral-like barium titanate nano-piezoelectric coating exhibits favorable biocompatibility and stable piezoelectric property, and facilitates the early osteogenic differentiation of BMSC under the excitation of low-intensity pulsed ultrasound.

Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved. Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process. AIM To assess the influence of interleukin-10 (IL-10) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) following their interaction with macrophages in an inflammatory environment. METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment. In this study, we investigated its impact on the proliferation, migration, and osteogenesis of BMSCs. The expression levels of signal transducer and activator of transcription 3 (STAT3) and its activated form, phosphorylated-STAT3, were examined in IL-10-stimulated macrophages. Subsequently, a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling. RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution, and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs. Mechanistically, STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages. Specifically, IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response, as evidenced by its diminished impact on the osteogenic differentiation of BMSCs. CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs. The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’ osteogenic differentiation.

Transplantation of Wnt5a-modified Bone Marrow Mesenchymal Stem Cells Promotes Recovery After Spinal Cord Injury via the PI3K/AKT Pathway.

Spinal cord injury (SCI) is a severe neurological condition that can lead to paralysis or even death. This study explored the potential benefits of bone marrow mesenchymal stem cell (BMSC) transplantation for repairing SCI. BMSCs also differentiate into astrocytes within damaged spinal cord tissues hindering the cell transplantation efficacy, therefore it is crucial to enhance their neuronal differentiation rate to facilitate spinal cord repair. Wnt5a, an upstream protein in the non-classical Wnt signaling pathway, has been implicated in stem cell migration, differentiation, and neurite formation but its role in the neuronal differentiation of BMSCs remains unclear. Thus, this study investigated the role and underlying mechanisms of Wnt5a in promoting neuronal differentiation of BMSCs both in vivo and in vitro. Wnt5a enhanced neuronal differentiation of BMSCs in vitro while reducing astrocyte differentiation. Additionally, high-throughput RNA sequencing revealed a correlation between Wnt5a and phosphoinositide 3-kinase (PI3K)/protein kinase B(AKT) signaling, which was confirmed by the use of the PI3K inhibitor LY294002 to reverse the effects of Wnt5a on BMSC neuronal differentiation. Furthermore, transplantation of Wnt5a-modified BMSCs into SCI rats effectively improved the histomorphology (Hematoxylin and eosin [H&E], Nissl and Luxol Fast Blue [LFB] staining), motor function scores (Footprint test and Basso-Beattie-Bresnahan [BBB]scores)and promoted neuron production, axonal formation, and remodeling of myelin sheaths (microtubule associated protein-2 [MAP-2], growth-associated protein 43 [GAP43], myelin basic protein [MBP]), while reducing astrocyte production (glial fibrillary acidic protein [GFAP]). Therefore, targeting the Wnt5a/PI3K/AKT pathway could enhance BMSC transplantation for SCI treatment.

Tumour necrosis factor α regulates the miR-27a-3p-Sfrp1 axis in a mouse model of osteoporosis.

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.

Biodegradable Zn-2Cu-0.5Zr alloy promotes the bone repair of senile osteoporotic fractures via the immune-modulation of macrophages

Delayed bone-healing of senile osteoporotic fractures remains a clinical challenge due to the alterations caused by aging in bone and immune systems. The novel biomaterials that address the deficiencies in both skeletal cells and immune systems are required to effectively treat the bone injuries of older patients. Zinc (Zn) has shown promise as a biodegradable material for use in orthopedic implants. To address the bone-healing deficiencies in elderly patients with bone injuries, we developed a biodegradable Zn-based alloy (Zn–2Cu-0.5Zr) with enhanced mechanical properties, including a yield strength of 198.7 MPa and ultimate tensile strength of 217.6 MPa, surpassing those of pure Zn and Zn–2Cu alloys. Cytotoxicity tests conducted on bone marrow mesenchymal stem cells (BMSCs) and MC3T3-E1 cells demonstrated that the extracts from Zn–2Cu-0.5Zr alloy exhibited no observable cytotoxic effects. Furthermore, the extracts of Zn–2Cu-0.5Zr alloy exhibited significant anti-inflammatory effects through regulation of inflammation-related cytokine production and modulation of macrophage polarization. The improved immune-osteo microenvironment subsequently contributed to osteogenic differentiation of BMSCs. The potential therapeutic application of Zn–2Cu-0.5Zr in senile osteoporotic fracture was tested using a rat model of age-related osteoporosis. The Zn–2Cu-0.5Zr alloy met the requirements for load-bearing applications and accelerated the healing process in a tibial fracture in aged rats. The imaging and histological analyses showed that it could accelerate the bone-repair process and promote the fracture healing in senile osteoporotic rats. These findings suggest that the novel Zn–2Cu-0.5Zr alloy holds potential for influencing the immunomodulatory function of macrophages and facilitating bone repair in elderly individuals with osteoporosis.

YTHDF2-Mediated m6A methylation inhibition by miR27a as a protective mechanism against hormonal osteonecrosis in BMSCs

BackgroundWith the increasing incidence of steroid-induced necrosis of the femoral head (SNFH), numerous scholars have investigated its pathogenesis. Current evidence suggests that the imbalance between lipogenesis and osteoblast differentiation in bone marrow mesenchymal stem cells (BMSCs) is a key pathological feature of SNFH. MicroRNAs (miRNAs) have strong gene regulatory effects and can influence the direction of cell differentiation. N6-methyladenosine (m6A) is a prevalent epigenetic modification involved in diverse pathophysiological processes. However, knowledge of how miRNAs regulate m6A-related factors that affect BMSC differentiation is limited.ObjectiveWe aimed to investigate the role of miR27a in regulating the expression of YTHDF2 in BMSCs.MethodsWe compared miR27a, YTHDF2, and total m6A mRNA levels in SNFH-affected and control BMSCs. CCK-8 and TUNEL assays were used to assess BMSC proliferation and apoptosis. Western blotting and qRT‒PCR were used to measure the expression of osteogenic (ALP, RUNX2, and OCN) and lipogenic (PPARγ and C/EBPα) markers. Alizarin Red and Oil Red O staining were used to quantify osteogenic and lipogenic differentiation, respectively. miR27a was knocked down or overexpressed to evaluate its impact on BMSC differentiation and its relationship with YTHDF2. Bioinformatics analyses identified YTHDF2 as a differentially expressed gene in SNFH (ROC analysis) and revealed potential signaling pathways through GSEA. The effects of YTHDF2 silencing on the lipogenic and osteogenic functions of BMSCs were assessed.ResultsmiR27a downregulation and YTHDF2 upregulation were observed in the SNFH BMSCs. miR27a knockdown/overexpression modulated YTHDF2 expression, impacting BMSC differentiation. miR27a silencing decreased m6A methylation and promoted osteogenic differentiation, while YTHDF2 silencing exerted similar effects. GSEA suggested potential signaling pathways associated with YTHDF2 in SNFH.ConclusionmiR27a regulates BMSC differentiation through YTHDF2, affecting m6A methylation and promoting osteogenesis. This finding suggests a potential therapeutic target for SNFH.

Extracellular Vesicles Derived from Neutrophils Accelerate Bone Regeneration by Promoting Osteogenic Differentiation of BMSCs.

The reconstruction of bone defects has been associated with severe challenges worldwide. Nowadays, bone marrow mesenchymal stem cell (BMSC)-based cell sheets have rendered this approach a promising way to facilitate osteogenic regeneration in vivo. Extracellular vesicles (EVs) play an essential role in intercellular communication and execution of various biological functions and are often employed as an ideal natural endogenous nanomedicine for restoring the structure and functions of damaged tissues. The perception of polymorphonuclear leukocytes (neutrophils, PMNs) as indiscriminate killer cells is gradually changing, with new evidence suggesting a role for these cells in tissue repair and regeneration, particularly in the context of bone healing. However, the role of EVs derived from PMNs (PMN-EVs) in bone regeneration remains largely unknown, with limited research being conducted on this aspect. In the current study, we investigated the effects of PMN-EVs on BMSCs and the underlying molecular mechanisms as well as the potential application of PMN-EVs in bone regeneration. Toward this end, BMSC-based cell sheets with integrated PMN-EVs (BS@PMN-EVs) were developed for bone defect regeneration. PMN-EVs were found to significantly enhance the proliferation and osteogenic differentiation of BMSCs in vitro. Furthermore, BS@PMN-EVs were found to significantly accelerate bone regeneration in vivo by enhancing the maturation of the newly formed bone in rat calvarial defects; this is likely attributable to the effect of PMN-EVs in promoting the expression of key osteogenic proteins such as SOD2 and GJA1 in BMSCs. In conclusion, our findings demonstrate the crucial role of PMN-EVs in promoting the osteogenic differentiation of BMSCs during bone regeneration. Furthermore, this study proposes a novel strategy for enhancing bone repair and regeneration via the integration of PMN-EVs with BMSC-based cell sheets.

Preliminary study on the mechanism by which exosomes derived from human exfoliated deciduous teeth improve the proliferation and osteogenic inhibitory effect of glucocorticoid-induced BMSCs

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a disease characterized by a collapsed femoral head caused by the overuse of glucocorticoids. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is an important pathological feature of SONFH. In this study, we investigated whether exosomes from SHEDs (stem cells from human exfoliated deciduous teeth) have a therapeutic effect on glucocorticoid-induced inhibition of proliferation and osteogenesis in BMSCs, and elucidated the underlying mechanisms involved. MethodsPrimary dental pulp cells were isolated and cultured from human deciduous tooth pulp, SHEDs were isolated and purified by the limiting dilution method and exosomes were isolated from the supernatants of SHEDs by ultracentrifugation. The cell surface markers CD31, CD34, CD45, CD73, CD90 and CD105 were detected by flow cytometry. A Cell-Counting-Kit-8 assay was used to detect cell activity. ALP and Alizarin Red staining were used to identify osteogenic differentiation ability, and exosomes were identified using transmission electron microscopy, NanoFCM and Western blotting. PKH67 fluorescence was used to track the uptake of exosomes by BMSCs. Transcriptome analysis combined with quantitative real-time PCR was used to explore the underlying mechanism involved. ResultsExosomes secreted by SHEDs can be endocytosed by BMSCs, and can partially reverse the inhibitory effects of glucocorticoids on the viability and osteogenic differentiation of BMSCs. Transcriptome sequencing analysis revealed that the differentially expressed mRNAs regulated by SHED-derived exosomes were enriched mainly in signaling pathways such as the apoptosis pathway, the PI3K-Akt signaling pathway, the Hippo signaling pathway and the p53 signaling pathway. qPCR showed that SHED-derived exosomes reversed the dexamethasone-induced upregulation of HGF and ITGB8 expression and the inhibition of EFNA1 expression, but further increased the dexamethasone-induced downregulation of IL7 expression. In conclusion, SHED-derived exosomes partially reversed the inhibitory effects of glucocorticoids on BMSC proliferation and osteogenesis by inhibiting the expression of HGF, ITGB8 and IL7, and upregulating the expression of EFNA1.

Psoralen mediates BMSCs osteogenic differentiation via cross-talk between Wnt/β-catenin and BMP/smad signaling pathways

Background: To explore the mechanism of psoralen mediated Wnt/β-catenin and bone morphogenetic protein (BMP) signaling pathway to induce osteogenesis of BMSC. Methods: Bone marrow mesenchymal stem cells (BMSCs) were treated with psoralen to detect its osteogenic differentiation. In addition, lentivirus and siRNA were used to construct cell models of β-catenin or BMP2 overexpression and knockdown, separately. They may help to clarify the role of β-catenin and BMP2 crosstalk in osteogenic differentiation of BMSCs. What’s more, C57BL/6 mice were selected to be treated psoralen with psoralen to further verify the osteogenic effect. Results: Various in vitro studies on BMSCs showed that psoralen could promote the osteogenic differentiation of BMSCs. Overexpression of β-catenin could promote the expression of BMP2 in BMSCs, and psoralen can enhance the effect of bone differentiation. Knockdown β-catenin decreased the expression of BMP2 and inhibited psoralen in promoting bone differentiation. In addition, it was found that the effect of psoralen on β-catenin level did not change significantly after overexpression or knockdown of BMP2, but the effect of psoralen on promoting bone differentiation was inhibited by knockdown of BMP2. In mice, psoralen intervention regulated the crosstalk of Wnt/β-catenin and BMP signaling pathway to reached to promote osteogenic differentiation of bone tissue. Conclusions: Psoralen can activate β-catenin signaling pathway and up-regulate the expression of BMP signaling pathway to increase the cross talk between β-catenin and BMP, to eventually reach to promote osteogenic differentiation of BMSCs. This trial was funded by the Program of National Natural Science Foundation of China (81973889 to FY), the Program of Shaanxi Province Natural Science Foundation (2023-YBSF-210 to FY &amp; 2023-JC-QN-0978 to WL), the Program of Shaanxi Education Department (19JC013 to FY &amp; 22JK0346 to WL), the Program of Shaanxi Administration Bureau of Traditional Chinese Medicine (2021-01-22-009 to FY &amp;2022-QCYZH-029 to WL), Youth Innovation Team in Shaanxi universities, and the Program of Health Commission of Shaanxi Province (2022B002 to WL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Beta-adrenergic receptor antagonist propranolol prevents bisphosphonate-related osteonecrosis of the jaw by promoting osteogenesis

Background/purposeBisphosphonate-related osteonecrosis of the jaw (BRONJ), a complication arising from the use of bisphosphonates (BPs), inflicts long-term suffering on patients. Currently, there is still a lack of effective treatments. This study aimed to explore the preventive effects of propranolol (PRO) on BRONJ in vitro and in vivo, given PRO's potential in bone health enhancement. Materials and methodsIn vitro, effect of PRO on zoledronic acid (ZA)-pretreated bone marrow mesenchymal stem cells (BMSCs) was detected by cell counting kit-8, alkaline phosphatase (ALP) staining, alizarin red staining, real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In vivo, forty mice were divided into four groups: control, ZA, PRO, and ZA-PRO. The maxillary extraction sockets sides were analyzed with micro-CT and histomorphometry. Hematoxylin-eosin (H&E), Masson staining, immunofluorescence staining of ALP, bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2) and TUNEL staining were performed. ResultsPRO increased proliferation and osteogenic differentiation of BMSCs. PRO stimulated bone formation and facilitated the healing process in zoledronic acid-induced osteonecrosis of jaw in mouse model. Compared with ZA group, control and PRO group showed more BMP2+, RUNX2+, and ALP+ cells (P < 0.05). However, PRO rescued the decreased expression of ALP, RUNX2, BMP2 due to ZA and decreased the expression of TUNEL (P < 0.05). ConclusionThe findings suggest that propranolol may offer a promising preventive strategy against BRONJ by enhancing bone regeneration. This research contributes to the understanding of the pathogenesis of BRONJ and opens avenues for potential treatments of BRONJ focusing on β-adrenergic signaling.

Tobacco toxins trigger bone marrow mesenchymal stem cells aging by inhibiting mitophagy

Smoking disrupts bone homeostasis and serves as an independent risk factor for the development and progression of osteoporosis. Tobacco toxins inhibit the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), promote BMSCs aging and exhaustion, but the specific mechanisms are not yet fully understood. Herein, we successfully established a smoking-related osteoporosis (SROP) model in rats and mice through intraperitoneal injection of cigarette smoke extract (CSE), which significantly reduced bone density and induced aging and inhibited osteogenic differentiation of BMSCs both in vivo and in vitro. Bioinformatics analysis and in vitro experiments confirmed that CSE disrupts mitochondrial homeostasis through oxidative stress and inhibition of mitophagy. Furthermore, we discovered that CSE induced BMSCs aging by upregulating phosphorylated AKT, which in turn inhibited the expression of FOXO3a and the Pink1/Parkin pathway, leading to the suppression of mitophagy and the accumulation of damaged mitochondria. MitoQ, a mitochondrial-targeted antioxidant and mitophagy agonist, was effective in reducing CSE-induced mitochondrial oxidative stress, promoting mitophagy, significantly downregulating the expression of aging markers in BMSCs, restoring osteogenic differentiation, and alleviating bone loss and autophagy levels in CSE-exposed mice. In summary, our results suggest that BMSCs aging caused by the inhibition of mitophagy through the AKT/FOXO3a/Pink1/Parkin axis is a key mechanism in smoking-related osteoporosis.

Mogroside V Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells from Diabetic Mice by Altering MicroRNA Profiles.

Mogroside V (MV), a triterpene glycoside, exhibits diverse biological functions. However, its ability to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under diabetic conditions is yet to be elucidated. To study the regulation of osteogenic differentiation of BMSCs in diabetic mice by MV and determine the potential mechanism. BMSCs were isolated from both normal (referred to as N-BMSCs) and diabetic (referred to as DM-BMSCs) C57BL/6 mice. DM-BMSCs were treated with different concentrations of MV for varying durations, and cell viability was detected using the cell counting kit-8 assay. Following 2 weeks of osteogenic induction, osteogenic differentiation capability was evaluated using alizarin red S staining, alkaline phosphatase (ALP) activity analysis, and quantitative real-time reverse transcription polymerase chain reaction. Furthermore, the microRNA (miRNA) expression profiles of N-BMSCs, DM-BMSCs, and DM-BMSCs treated with MV were tested using high-throughput sequencing. Treatment with MV enhanced the viability of DM-BMSCs and mitigated the reduction of calcium nodule deposition, ALP activity, and mRNA expression of ALP, osteocalcin, and runt-related transcription factor 2. Of the analyzed miRNAs, miR-10b-5p was the only one that exhibited differential expression in N-BMSCs, DM-BMSCs, and DM-BMSCs treated with MV. An analysis of the top four protein clusters based on KEGG suggested that the target genes of differentially expressed miRNAs were closely linked to the PI3K/AKT pathway. MV significantly enhances the viability and osteogenic differentiation of BMSCs under diabetic conditions. The alteration of miRNA profiles provides a foundation for further research into the regulatory role of miRNAs and MV in this process.

Acetyl-CoA-dependent ac4C acetylation promotes the osteogenic differentiation of LPS-stimulated BMSCs

The impaired osteogenic capability of bone marrow mesenchymal stem cells (BMSCs) caused by persistent inflammation is the main pathogenesis of inflammatory bone diseases. Recent studies show that metabolism is disturbed in osteogenically differentiated BMSCs in response to Lipopolysaccharide (LPS) treatment, while the mechanism involved remains incompletely revealed. Herein, we demonstrated that BMSCs adapted their metabolism to regulate acetyl-coenzyme A (acetyl-CoA) availability and RNA acetylation level, ultimately affecting osteogenic differentiation. The mitochondrial dysfunction and impaired osteogenic potential upon inflammatory conditions accompanied by the reduced acetyl-CoA content, which in turn suppressed N4-acetylation (ac4C) level. Supplying acetyl-CoA by sodium citrate (SC) addition rescued ac4C level and promoted the osteogenic capacity of LPS-treated cells through the ATP citrate lyase (ACLY) pathway. N-acetyltransferase 10 (NAT10) inhibitor remodelin reduced ac4C level and consequently impeded osteogenic capacity. Meanwhile, the osteo-promotive effect of acetyl-CoA-dependent ac4C might be attributed to fatty acid oxidation (FAO), as evidenced by activating FAO by L-carnitine supplementation counteracted remodelin-induced inhibition of osteogenesis. Further in vivo experiments confirmed the promotive role of acetyl-CoA in the endogenous bone regeneration in rat inflammatory mandibular defects. Our study uncovered a metabolic-epigenetic axis comprising acetyl-CoA and ac4C modification in the process of inflammatory osteogenesis of BMSCs and suggested a new target for bone tissue repair in the context of inflammatory bone diseases.

The IFT80/Hedgehog Pathway Regulates the Osteogenic-adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Steroid-induced avascular necrosis of the femoral head (SANFH) is a typical refractory disease that often progresses irreversibly and has a high disability rate. Numerous studies have confirmed that abnormal osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) is one of the major factors of SANFH. However, the mechanism remains to be elucidated. This study aimed to investigate the mechanism and effect of the IFT80/Hedgehog-mediated osteogenic-adipogenic differentiation of BM-MSCs in SANFH. Femoral head specimens of SANFH patients and femoral neck fractures (FNF) patients were collected to detect the expression of IFT80, Shh and osteogenic-adipogenic differentiation-related genes by immunohistochemistry (IHC), western blot (WB) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). Based on the rabbit SANFH model, the mRNA expression and protein level of IFT80 and Shh were detected by RT-qPCR and WB. After the osteogenic/adipogenic differentiation based on rabbit BM-MSCs, the IFT80, Gli1, PPAR-γ, and Runx2 expression were detected. Differences in alkaline phosphodiesterase activity, calcium nodule, quantification/distribution of lipid droplets, expression of IFT80/Hedgehog axis, and the level of osteogenic- adipogenic associated factors were determined after IFT80 overexpression. RT-qPCR, WB and IHC revealed that IFT80 and Shh lowly expressed in the osteoblasts and intra-trabecular osteocytes at the edge of trabeculae and in the intercellular matrix of the bone marrow lumen in the SANFH specimens. The Runx2 expression was low, while the PPAR-γ expression was high in both human specimens and animal models of SANFH, suggesting that the balance of osteogenic-adipogenic differentiation was dysregulated. Rabbit BM-MSCs with stable overexpression of IFT80 showed increased alkaline phosphatase activity after induction of osteogenic differentiation, increased calcium nodule production, and decreased adipogenesis after induction of adipogenic differentiation. There is a dysregulation of the balance of osteogenic-adipogenic differentiation in SANFH. IFT80 may inhibit adipogenic differentiation while promoting osteogenic differentiation in rabbit BM-MSCs by activating the Hedgehog pathway.

Highly biologically functional magnesium silicate-coated 3D printed round pore-shaped titanium scaffold alters exosomal miRNA expression to promote osteogenic differentiation for bone defect repair

Bone defects are a serious condition that has a significant impact on the function and quality of life of the patient. In severe bone defects, restoring the structure and function of bone tissue is a complex task, and titanium (Ti) implants designed with superior performance can be an alternative. In this study, in order to develop a Ti implant that effectively promotes osseointegration and new bone ingrowth, 3D printing-based magnesium silicate (MgSiO3)-coated round pore-shaped Ti scaffold (S-R/MgSi) was fabricated by standardizing pore size and porosity and designing different pore shapes and selecting the optimal pore shape (round) as well as subsequent hydrothermal synthesis to finally form MgSiO3 coating. The experimental results of Ti scaffolds with different pore shapes showed that S-R is optimal for promoting osteogenic differentiation. After S-R was coated with MgSiO3, S-R/MgSi enhanced the adhesion, proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as regulated angiogenesis. In vivo studies, S-R/MgSi significantly promoted new bone ingrowth and was effective in repairing bone defects. Following the mechanism exploration, it was found that exosomes secreted by BMSCs co-cultured with S-R/MgSi (Exo_S-R/MgSi) were more able to promote the osteogenic differentiation of BMSCs. In conclusion, this study not only successfully prepared a new type of Ti implant with superior ability to promote new bone ingrowth, but also provided a new explanation for the mechanism of scaffold surface chemistry affecting osteogenesis from the viewpoint of scaffold surface coating affecting the amount and function of cellular exosome secretion.

PPARγ inhibition promotes osteogenic differentiation of bone marrow mesenchymal stem cells and fracture healing.

This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 μM, 10 μM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.

Mijiao formula regulates NAT10-mediated Runx2 mRNA ac4C modification to promote bone marrow mesenchymal stem cell osteogenic differentiation and improve osteoporosis in ovariectomized rats

Ethnopharmacological relevanceThe Mijiao (MJ) formula, a traditional herbal remedy, incorporates antlers as its primary constituent. It can effectively treat osteoporosis (OP), anti-aging, enhance immune activity, and change depression-like behavior. In this study, we investigated that MJ formula is a comprehensive treatment strategy, and may provide a potential approach for the clinical treatment of postmenopausal osteoporosis. Aim of the studyThe purpose of this study was to determine whether MJ formula promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and improved osteoporosis in ovariectomized rats by regulating the NAT10-mediated Runx2 mRNA ac4C modification. Materials and methodsFemale Sprague-Dawley (SD) rats were used to investigate the potential therapeutic effect of MJ formula on OP by creating an ovariectomized (OVX) rat model. The expression of osteogenic differentiation related proteins in BMSCs was detected in vivo, indicating their role in promoting bone formation. In addition, the potential mechanism of its bone protective effect was explored via in vitro experiments. ResultsOur study showed that MJ formula significantly mitigated bone mass loss in the OVX rat model, highlighting its potential as an OP therapeutic agent. We found that the possible mechanism of action was the ability of this formulation to stabilize Runx2 mRNA through NAT10-mediated ac4C acetylation, which promoted osteogenic differentiation of BMSCs and contributed to the enhancement of bone formation. ConclusionsMJ formula can treat estrogen deficiency OP by stabilizing Runx2 mRNA, promoting osteogenic differentiation and protecting bone mass. Conceivably, MJ formulation could be a safe and promising strategy for the treatment of osteoporosis.

Ganoderma spore lipid ameliorates docetaxel, cisplatin, and 5-fluorouracil chemotherapy-induced damage to bone marrow mesenchymal stem cells and hematopoiesis

BackgroundA triplet chemotherapy regimen of docetaxel, cisplatin, and 5-fluorouracil (TPF) is used to treat head and neck squamous cell carcinoma; however, it is toxic to bone marrow mesenchymal stem cells (BMSCs). We previously demonstrated that Ganoderma spore lipid (GSL) protect BMSCs against cyclophosphamide toxicity. In this study, we investigated the protective effects of GSL against TPF-induced BMSCs and hematopoietic damage.MethodsBMSCs and C57BL/6 mice were divided into control, TPF, co-treatment (simultaneously treated with GSL and TPF for 2 days), and pre-treatment (treated with GSL for 7 days before 2 days of TPF treatment) groups. In vitro, morphology, phenotype, proliferation, senescence, apoptosis, reactive oxygen species (ROS), and differentiation of BMSCs were evaluated. In vivo, peripheral platelets (PLTs) and white blood cells (WBCs) from mouse venous blood were quantified. Bone marrow cells were isolated for hematopoietic colony-forming examination.ResultsIn vitro, GSL significantly alleviated TPF-induced damage to BMSCs compared with the TPF group, recovering their morphology, phenotype, proliferation, and differentiation capacity (p < 0.05). Annexin V/PI and senescence-associated β-galactosidase staining showed that GSL inhibited apoptosis and delayed senescence in TPF-treated BMSCs (p < 0.05). GSL downregulated the expression of caspase-3 and reduced ROS formation (p < 0.05). In vivo, GSL restored the number of peripheral PLTs and WBCs and protected the colony-forming capacity of bone marrow cells (p < 0.05).ConclusionsGSL efficiently protected BMSCs from damage caused by TPF and recovered hematopoiesis.