OBJECTIVE: To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of Plastrum Testudinis (PT) in the treatment of osteoporosis (OP). METHODS: Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method, including sham group, ovariectomized group (OVX), ovariectomized groups treated with high-, medium-, and low-dose PT (160, 80, 40 mg/kg per day, respectively), with 6 rats in each group. Except for the sham group, the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment, bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry, Western blot, and quantitative polymerase chain reaction. In addition, Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently, PT extract was used to rescue the effects of Dkk-1 and miR-214, and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated. RESULTS: PT-M and PT-L significantly reduced the weight gain in OVX rats (P<0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats, and increased the expressions of bone formation-related factors including alkaline phosphatase, bone morphogenetic protein type 2, collagen type I alpha 1, and runt-related transcription factor 2 when compared with the OVX group (P<0.05 or P<0.01). Meanwhile, different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats, and increased the mRNA or protein expressions of Wnt3a, β-catenin, glycogen synthase kinase-3β, and low-density lipoprotein receptor-related protein 5 (P<0.05 or P<0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition, the expression of miR-214 was decreased in OVX rats (P<0.01), and it was negatively correlated with the osteogenic differentiation of BMSCs (P<0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (P<0.05 or P<0.01). Conversely, PT effectively counteracted the effect of miR-214 mimic, thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (P<0.05 or P<0.01). CONCLUSION PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway, which may be related to inhibiting miR-214 in BMSCs.
Animals ; MicroRNAs/genetics* ; Female ; Rats, Sprague-Dawley ; Wnt Signaling Pathway/genetics* ; Osteogenesis/genetics* ; Mesenchymal Stem Cells/cytology* ; Cell Differentiation/drug effects* ; Bone Density/drug effects* ; Ovariectomy ; Osteoporosis/drug therapy* ; beta Catenin/metabolism* ; Rats ; Intercellular Signaling Peptides and Proteins/metabolism* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVES: The core pathology of osteoporosis lies in bone resorption exceeding bone formation; thus, promoting osteogenesis is a key therapeutic strategy. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) forms the biological basis of bone formation. Astragaloside IV (A-IV), a major active component of Astragalus membranaceus, is known to enhance osteogenesis, but its precise molecular mechanisms remain unclear. This study aims to investigate the effects of A-IV on the proliferation and osteogenic differentiation of BMSCs from osteoporotic rats and to elucidate its molecular mechanism through the regulation of microRNA-21 (miR-21) and Notch2 expression. METHODS: After 1 week of adaptive feeding, mature female SD rats were randomly divided into a sham-operated (Sham) group (n=4) and an ovariectomized (OVX) group (n=8) to establish an osteoporosis model. Twelve weeks after surgery, BMSCs were isolated from femoral bone marrow and cultured. Cells were divided into a S-BMSCs group (from Sham), an O-BMSCs group (from OVX), and an A-BMSCs group (from OVX-derived BMSCs treated with A-IV). S-BMSCs and O-BMSCs were induced for osteogenic differentiation using osteogenic induction medium, whereas A-BMSCs were treated with A-IV before induction. Flow cytometry was used to identify mesenchymal stem cell surface markers (CD29) and hematopoietic stem cell marker (CD34) to confirm BMSC characteristics. Cell proliferation was assessed using the methyl thiazolyl tetrazolium (MTT) assay. Alizarin red staining was performed to quantify calcium nodule formation, and alkaline phosphatase (ALP) activity assays were used to evaluate osteogenic differentiation. Real-time reverse transcription PCR (real-time RT-PCR) was used to detect changes in osteogenic-related genes, runt-related transcription factor 2 (Runx2) and osteopontin (OPN), as well as miR-21 expression. Western blotting was performed to assess Runx2, OPN, and Notch2 protein expression. RESULTS: Flow cytometry confirmed that O-BMSCs retained the phenotypic characteristics of mesenchymal stem cells. A-IV significantly enhanced the proliferation of BMSCs from osteoporotic rats (P<0.05), increased ALP activity, and upregulated the mRNA and protein expression of Runx2 and OPN (P<0.05). Bioinformatic and experimental analyses demonstrated that miR-21 directly targeted Notch2. A-IV treatment increased miR-21 expression while suppressing Notch2 protein expression and inhibiting activation of the Notch signaling pathway (P<0.05). CONCLUSIONS Astragaloside IV promotes the osteogenic differentiation of BMSCs derived from osteoporotic rats by upregulating miR-21 expression and inhibiting the key Notch signaling protein Notch2, thereby relieving the Notch2-mediated suppression of osteogenesis.
Animals ; Triterpenes/pharmacology* ; Saponins/pharmacology* ; Osteogenesis/drug effects* ; MicroRNAs/metabolism* ; Rats, Sprague-Dawley ; Female ; Cell Differentiation/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Signal Transduction/drug effects* ; Osteoporosis/pathology* ; Rats ; Cells, Cultured ; Receptor, Notch2/metabolism* ; Receptors, Notch/metabolism* ; Ovariectomy ; Cell Proliferation/drug effects*

Bone is a highly calcified and vascularized tissue. The vascular system plays a vital role in supporting bone growth and repair, such as the provision of nutrients, growth factors, and metabolic waste transfer. Moreover, the additional functions of the bone vasculature, such as the secretion of various factors and the regulation of bone-related signaling pathways, are essential for maintaining bone health. In the bone microenvironment, bone tissue cells play a critical role in regulating angiogenesis, including osteoblasts, bone marrow mesenchymal stem cells (BMSCs), and osteoclasts. Osteogenesis and bone angiogenesis are closely linked. The decrease in osteogenesis and bone angiogenesis caused by aging leads to osteoporosis. Long noncoding RNAs (lncRNAs) are involved in various physiological processes, including osteogenesis and angiogenesis. Recent studies have shown that lncRNAs could mediate the crosstalk between angiogenesis and osteogenesis. However, the mechanism by which lncRNAs regulate angiogenesis‒osteogenesis crosstalk remains unclear. In this review, we describe in detail the ways in which lncRNAs regulate the crosstalk between osteogenesis and angiogenesis to promote bone health, aiming to provide new directions for the study of the mechanism by which lncRNAs regulate bone metabolism.
RNA, Long Noncoding/physiology* ; Osteogenesis/physiology* ; Humans ; Neovascularization, Physiologic/genetics* ; Bone and Bones/metabolism* ; Animals ; Mesenchymal Stem Cells ; Signal Transduction ; Osteoblasts ; Osteoclasts ; Angiogenesis

Periodontitis is a common oral disease caused by bacteria coupled with an excessive host immune response. Stem cell therapy can be a promising treatment strategy for periodontitis, but the relevant mechanism is complicated. This study aimed to explore the therapeutic potential of mitochondria from human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) for the treatment of periodontitis. The gingival tissues of periodontitis patients are characterized by abnormal mitochondrial structure. Human gingival fibroblasts (HGFs) were exposed to 5 μg/mL lipopolysaccharide (LPS) for 24 h to establish a cell injury model. When treated with hESC-MSCs or mitochondria derived from hESC-MSCs, HGFs showed reduced expression of inflammatory genes, increased adenosine triphosphate (ATP) level, decreased reactive oxygen species (ROS) production, and enhanced mitochondrial function compared to the control. The average efficiency of isolated mitochondrial transfer by hESC-MSCs was determined to be 8.93%. Besides, a therapy of local mitochondrial injection in mice with LPS-induced periodontitis showed a reduction in inflammatory gene expression, as well as an increase in both the mitochondrial number and the aspect ratio in gingival tissues. In conclusion, our results indicate that mitochondria derived from hESC-MSCs can reduce the inflammatory response and improve mitochondrial function in HGFs, suggesting that the transfer of mitochondria between hESC-MSCs and HGFs serves as a potential mechanism underlying the therapeutic effect of stem cells.
Humans ; Gingiva/cytology* ; Fibroblasts/metabolism* ; Mitochondria/physiology* ; Mesenchymal Stem Cells/cytology* ; Animals ; Periodontitis/therapy* ; Mice ; Reactive Oxygen Species/metabolism* ; Inflammation ; Lipopolysaccharides ; Human Embryonic Stem Cells/cytology* ; Cells, Cultured ; Adenosine Triphosphate/metabolism* ; Male

OBJECTIVES: To explore the mechanism by which Tougu Xiaotong Capsule (TXC) promotes chondrogenic differentiation and cartilage repair in mice with osteoarthritis (OA). METHODS: Fifty 8-week-old male C57BL mice were randomly divided into normal control group, cartilage damage (induced by subchondral ring-shaped drilling) model group and TXC treatment groups at low, moderate and high doses (184, 368 and 736 mg/kg, respectively). Saline (in normal control and model groups) and TXC were administered after modeling by daily gavage for 6 consecutive weeks. The changes of cartilage damage in the mice were assessed by measuring thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) and using micro-CT, modified safranine O and fast green staining, HE staining, and qPCR. Primary cultures of mouse synovial mesenchymal stem cells (SMSCs) with lentivirus vector transfection for interfering CXCL12, TXC treatment, or both for 24 h were examined for chondrogenic differentiation using immunofluorescence staining, scratch assay, immunocytochemistry, and Western blotting. RESULTS: In mouse models with cartilage damage, TXC treatment at the moderate dose significantly alleviated joint pain, promoted cartilage repair, and upregulated the mRNA expression levels of CXCL12, GDF5, collagen II, aggrecan, Comp and Sox9 in the cartilage tissue. In primary mouse SMSCs, CXCL12 knockdown resulted in significant reduction of GDF5 protein expression, migration ability and Sox9 protein expression, and these changes were obviously reversed by TXC treatment. CONCLUSIONS TXC promotes chondrogenic differentiation of mouse SMSCs to promote repair of cartilage damage in mice by activating the CXCL12/GDF5 pathway.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Osteoarthritis/metabolism* ; Male ; Growth Differentiation Factor 5/metabolism* ; Mice, Inbred C57BL ; Mice ; Chemokine CXCL12/metabolism* ; Signal Transduction/drug effects* ; Cell Differentiation/drug effects* ; Cartilage, Articular/drug effects* ; Mesenchymal Stem Cells/cytology*

The ambiguity of etiology makes temporomandibular joint osteoarthritis (TMJOA) "difficult-to-treat". Emerging evidence underscores the therapeutic promise of exosomes in osteoarthritis management. Nonetheless, challenges such as low yields and insignificant efficacy of current exosome therapies necessitate significant advances. Addressing lower strontium (Sr) levels in arthritic synovial microenvironment, we studied the effect of Sr element on exosomes and miRNA selectively loading in synovial mesenchymal stem cells (SMSCs). Here, we developed an optimized system that boosts the yield of SMSC-derived exosomes (SMSC-EXOs) and improves their miRNA profiles with an elevated proportion of beneficial miRNAs, while reducing harmful ones by pretreating SMSCs with Sr. Compared to untreated SMSC-EXOs, Sr-pretreated SMSC-derived exosomes (Sr-SMSC-EXOs) demonstrated superior therapeutic efficacy by mitigating chondrocyte ferroptosis and reducing osteoclast-mediated joint pain in TMJOA. Our results illustrate Alix's crucial role in Sr-triggered miRNA loading, identifying miR-143-3p as a key anti-TMJOA exosomal component. Interestingly, this system is specifically oriented towards synovium-derived stem cells. The insight into trace element-driven, site-specific miRNA selectively loading in SMSC-EXOs proposes a promising therapeutic enhancement strategy for TMJOA.
MicroRNAs/metabolism* ; Mesenchymal Stem Cells/drug effects* ; Osteoarthritis/drug therapy* ; Exosomes/drug effects* ; Strontium/pharmacology* ; Synovial Membrane/cytology* ; Humans ; Animals ; Temporomandibular Joint Disorders/therapy* ; Temporomandibular Joint

Dental mesenchymal stem cells (DMSCs) are pivotal for tooth development and periodontal tissue health and play an important role in tissue engineering and regenerative medicine because of their multidirectional differentiation potential and self-renewal ability. The cellular microenvironment regulates the fate of stem cells and can be modified using various optimization techniques. These methods can influence the cellular microenvironment, activate disparate signaling pathways, and induce different biological effects. "Epigenetic regulation" refers to the process of influencing gene expression and regulating cell fate without altering DNA sequences, such as histone methylation. Histone methylation modifications regulate pivotal transcription factors governing DMSCs differentiation into osteo-/odontogenic lineages. The most important sites of histone methylation in tooth organization were found to be H3K4, H3K9, and H3K27. Histone methylation affects gene expression and regulates stem cell differentiation by maintaining a delicate balance between major trimethylation sites, generating distinct chromatin structures associated with specific downstream transcriptional states. Several crucial signaling pathways associated with osteogenic differentiation are susceptible to modulation via histone methylation modifications. A deeper understanding of the regulatory mechanisms governing histone methylation modifications in osteo-/odontogenic differentiation and immune-inflammatory responses of DMSCs will facilitate further investigation of the epigenetic regulation of histone methylation in DMSC-mediated tissue regeneration and inflammation. Here is a concise overview of the pivotal functions of epigenetic histone methylation at H3K4, H3K9, and H3K27 in the regulation of osteo-/odontogenic differentiation and renewal of DMSCs in both non-inflammatory and inflammatory microenvironments. This review summarizes the current research on these processes in the context of tissue regeneration and therapeutic interventions.
Mesenchymal Stem Cells/physiology* ; Humans ; Osteogenesis/genetics* ; Histones/metabolism* ; Cell Differentiation/physiology* ; Methylation ; Odontogenesis/genetics* ; Epigenesis, Genetic

Periodontal bone defects, primarily caused by periodontitis, are highly prevalent in clinical settings and manifest as bone fenestration, dehiscence, or attachment loss, presenting a significant challenge to oral health. In regenerative medicine, harnessing developmental principles for tissue repair offers promising therapeutic potential. Of particular interest is the condensation of progenitor cells, an essential event in organogenesis that has inspired clinically effective cell aggregation approaches in dental regeneration. However, the precise cellular coordination mechanisms during condensation and regeneration remain elusive. Here, taking the tooth as a model organ, we employed single-cell RNA sequencing to dissect the cellular composition and heterogeneity of human dental follicle and dental papilla, revealing a distinct Platelet-derived growth factor receptor alpha (PDGFRA) mesenchymal stem/stromal cell (MSC) population with remarkable odontogenic potential. Interestingly, a reciprocal paracrine interaction between PDGFRA⁺ dental follicle stem cells (DFSCs) and CD31⁺ Endomucin⁺ endothelial cells (ECs) was mediated by Vascular endothelial growth factor A (VEGFA) and Platelet-derived growth factor subunit BB (PDGFBB). This crosstalk not only maintains the functionality of PDGFRA⁺ DFSCs but also drives specialized angiogenesis. In vivo periodontal bone regeneration experiments further reveal that communication between PDGFRA⁺ DFSC aggregates and recipient ECs is essential for effective angiogenic-osteogenic coupling and rapid tissue repair. Collectively, our results unravel the importance of MSC-EC crosstalk mediated by the VEGFA and PDGFBB-PDGFRA reciprocal signaling in orchestrating angiogenesis and osteogenesis. These findings not only establish a framework for deciphering and promoting periodontal bone regeneration in potential clinical applications but also offer insights for future therapeutic strategies in dental or broader regenerative medicine.
Receptor, Platelet-Derived Growth Factor alpha/metabolism* ; Humans ; Neovascularization, Physiologic/physiology* ; Dental Sac/cytology* ; Single-Cell Analysis ; Transcriptome ; Mesenchymal Stem Cells/metabolism* ; Bone Regeneration ; Animals ; Dental Papilla/cytology* ; Periodontium/physiology* ; Stem Cells/metabolism* ; Regeneration ; Angiogenesis

Pulmonary diseases, as a prevalent category of respiratory system disorders, have become a significant global public health concern. The increasing incidence of these diseases, caused by environmental pollution and occupational hazards, poses a substantial threat to human health and the overall quality of life. Mesenchymal stem cells (MSCs) are known for their remarkable immunomodulatory, anti-bacterial, and anti-apoptotic capabilities. Exosomes derived from MSCs, carrying a diverse array of proteins, lipids, nucleic acids, and other bio-active molecules, have demonstrated considerable therapeutic potential in treating pulmonary diseases, and have come to the forefront of medical research. This review summarized the therapeutic role of exosomes derived from various sources of mesenchymal stem cells in the context of pulmonary diseases, aiming to provide a robust foundation for their clinical application in diagnosis and treatment.
Exosomes/transplantation* ; Humans ; Mesenchymal Stem Cells/metabolism* ; Lung Diseases/therapy* ; Animals

Objective To investigate the effects and underlying mechanisms of human placental chorionic plate-derived mesenchymal stem cells (hpcMSCs) on cognitive dysfunction, neuroinflammation, neuronal damage and synaptic plasticity in a mouse model of stroke. Methods A mouse model of middle cerebral artery occlusion (MCAO) was adopted. The mice were randomly divided into three groups: sham operation group, MCAO group and hpcMSCs treatment group, with seven mice in each group. The hpcMSCs treatment group received hpcMSCs transplantation on the 1st, 3rd and 10th day after MCAO. One month after MCAO, the cognitive ability of the mice was evaluated by Morris water maze and Y maze behavioral tests; the morphological changes and synaptic functions of hippocampal neurons were analyzed by HE staining, Nissl staining, Golgi staining and immunofluorescence staining techniques; the density and activation status of microglia was analyzed by Fluorescent labeling method; the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6 in brain tissue were analyzed by ELISA; the expressions of phosphorylated-mitogen-activated protein kinase kinase 1 (p-MEK1), phosphorylated-extracellular regulated protein kinase (p-ERK) and phosphorylated-cAMP-response element binding protein (p-CREB) and other proteins related to neuroprotection in the signal pathways were detected by Western blotting; and electrophysiological detection was performed using hippocampal slices in vitro. Results Compared with the MCAO group, mice in the hpcMSCs treatment group showed significant improvements, including improved cognitive ability, alleviated neuroinflammation (demonstrated by reduced microglial activation and decreased levels of inflammatory factors TNF-α, IL-1β and IL-6), and increased neuronal density with normalized morphology of neurons in the hippocampal CA1 region. The treatment group also demonstrated a significantly increased number of Nissl-positive cells and density of dendritic spines of hippocampal neurons, along with restored frequency of miniature excitatory postsynaptic potential (mEPSP). Moreover, hpcMSCs treatment significantly increased the expression levels of p-MEK1, p-ERK and p-CREB in the hippocampus. Conclusion Transplantation of hpcMSCs ameliorates cognitive dysfunction and hippocampal neuronal injury in stroke mice through the reduction of neuroinflammation, restoration of hippocampal neuronal function, promotion of synaptic plasticity and activation of the MEK/ERK/CREB signaling pathway. These findings suggest a new potential therapeutic approach for post-stroke neural repair.
Animals ; Hippocampus/physiopathology* ; Mice ; Cognitive Dysfunction/etiology* ; Mesenchymal Stem Cell Transplantation ; Male ; Neurons/metabolism* ; Stroke/metabolism* ; Humans ; Neuroinflammatory Diseases/therapy* ; Female ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Disease Models, Animal ; Mesenchymal Stem Cells/cytology* ; Mice, Inbred C57BL