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BACKGROUND: Inflammation affects the osteogenic differentiation of periodontal ligament stem cells, and the osteogenic ability and autophagy level of periodontal ligament stem cells are closely related. However, there are no relevant reports on whether inflammation affects the osteogenic ability and autophagy level of periodontal ligament stem cells at different stages of osteogenic differentiation. OBJECTIVE: To explore alkaline phosphatase expression and autophagy periodontal ligament stem cells levels in periodontitis and normal conditions. METHODS: Periodontal ligament stem cells from normal and periodontitis patients were isolated and cultured, and underwent Vimentin, pan-CK, and Stro1 fluorescence staining. At 3, 7, and 14 days of osteogenic differentiation, western blot assay was used to detect the protein expression levels of alkaline phosphatase, LC3B, Beclin1, and ATG5 in normal and inflammatory periodontal ligament stem cells. The mRNA expression levels of alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, LC3B, Beclin1, and ATG5 were detected by real-time PCR. RESULTS AND CONCLUSION: (1) Stro-1 was positive, Vimentin was positive, and pan CK was negative in periodontal ligament stem cells. (2) At 3, 7, and 14 days after osteogenic differentiation, compared with normal periodontal ligament stem cells, the mineralization nodules formed by periodontal ligament stem cells from inflammatory sources were significantly reduced (P < 0.01); the expression of alkaline phosphatase protein and mRNA was significantly lower (P < 0.05); the mRNA expression levels of bone sialoprotein, osteocalcin, and Runx2 were significantly decreased (P < 0.05). (3) At 7 and 14 days after osteogenic differentiation, compared with normal periodontal ligament stem cells, the expression levels of ATG5, LC3B, and Beclin1 proteins and mRNA of periodontal ligament stem cells were downregulated (P < 0.05). These findings suggest that inflammation reduces the activity of periodontal ligament stem cells in mineralizing nodule formation and the expression of alkaline phosphatase and weakens the autophagy potential of periodontal ligament stem cells at 7 and 14 days after osteogenic differentiation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Psoralen has a strong anti-osteoporotic activity and may have a restorative effect on chemotherapy-induced osteoporosis. OBJECTIVE: To explore the restorative effect of psoralen on the osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide and its mechanism. METHODS: C57BL/6 mouse bone marrow mesenchymal stem cells were isolated and cultured. Effect of psoralen on viability of bone marrow mesenchymal stem cells was detected by MTT assay. Osteogenic induction combined with alkaline phosphatase staining was used to determine the optimal dose of psoralen to restore the osteogenic differentiation of bone marrow mesenchymal stem cells inhibited by cyclophosphamide. The mRNA expression levels of Runx2, alkaline phosphatase, Osteocalcin, osteoprotegerin, and Wnt/β-catenin signaling pathway-related genes Wnt1, Wnt4, Wnt10b, β-catenin, and c-MYC were measured by RT-qPCR at different time points under the intervention with psoralen. The protein expression of osteogenic specific transcription factor Runx2 and Wnt/β-catenin signaling pathway related genes Active β-catenin, DKK1, c-MYC, and Cyclin D1 was determined by western blot assay at different time points under the intervention with psoralen. RESULTS AND CONCLUSION: (1) There was no significant effect of different concentrations of psoralen on the viability of bone marrow mesenchymal stem cells. The best recovery of the inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells caused by cyclophosphamide was under the intervention of psoralen at a concentration of 200 μmol/L. (2) Psoralen reversed the reduction in osteogenic differentiation marker genes Runx2, alkaline phosphatase, Osteocalcin and osteoprotegerin mRNA expression and Runx2 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium. (3) Psoralen reversed the decrease in Wnt/β-catenin pathway-related genes Wnt4, β-catenin, c-MYC mRNA and Active β-catenin, c-MYC, and Cyclin D1 protein expression and the increase in DKK1 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium. (4) The results showed that cyclophosphamide inhibited osteogenic differentiation of bone marrow mesenchymal stem cells in mice, and psoralen had a restorative effect on it. The best intervention effect was achieved at a concentration of 200 μmol/L psoralen, and this protective effect might be related to the activation of Wnt4/β-catenin signaling pathway by psoralen. [ABSTRACT FROM AUTHOR]

Background: Low-intensity pulsed ultrasound (LIPUS) has been used as an effective noninvasive method for treating fractures and osteoarthrosis, but the application in the field of oral implantation is in its infancy. This study aimed to clarify the effect and mechanism of LIPUS on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and implant osseointegration, and to provide an experimental basis for future clinical applications. Methods: Dental implants were inserted into Wistar rat femurs, and LIPUS was performed for 4 weeks. Micro-CT and toluidine blue staining were used to assess implant osseointegration. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to identify enriched functional terms and signalling pathways for differentially expressed genes from LIPUS-treated rat BMSC RNAseq data obtained from the GEO database. The random forest method was used to identify key risk genes according to the mean decrease Gini (MDG) coefficient. Then, LIPUS was applied to treat rat BMSCs, and alkaline phosphatase (ALP) staining, alizarin red staining, RT-PCR and western blotting were used to determine whether LIPUS could promote BMSC osteogenic differentiation via integrin α11 (ITGA11) and the focal adhesion pathway. Results: Our in vivo experimentations verified that LIPUS significantly increased new bone formation and osseointegration around the implant in rats. Bioinformatics analysis of RNA-seq data revealed that the upregulated genes in BMSCs after LIPUS treatment were significantly enriched in osteoblast differentiation-related functions and focal adhesion-related pathways. Random forest analysis revealed that ITGA11 was the most significant factor affecting BMSC osteogenic differentiation among the differentially expressed genes. In addition, LIPUS significantly increased ALP expression and mineralized nodule formation in rat BMSCs by upregulating ITGA11 and increasing the activity of FAK/PI3K/AKT/GSK3β/β-catenin pathway. Conclusions: LIPUS can effectively promote implant osseointegration in rats and improve rat BMSC osteogenic differentiation by upregulating ITGA11 and increasing the activity of the downstream focal adhesion pathway. [ABSTRACT FROM AUTHOR]

An improved understanding of the molecular actions underpinning bone marrow mesenchymal stem cell (BMSC) differentiation could highlight new therapeutics for osteoporosis (OP). Current evidence indicates that microRNAs (miRNAs) exert critical roles in many biological systems, including osteoblast differentiation. In this study, we examined miR-468-3p effects on osteogenic differentiation (OD). Distinct miR-468-3p reductions were identified during OD. MiR-468-3p also suppressed BMSC OD in gain- and loss-of-function assays, while it negatively regulated Runx2 as shown by molecular, protein, and bioinformatics approaches. When Runx2 was inhibited by small-interfering RNA (siRNA), the inhibitory effects of miR-468-3p toward BMSC osteogenesis were considerably reversed. Also, silenced miR-468-3p in ovariectomized (OVX) and sham mice augmented bone mass (BM) and bone formation (BF) and improved trabecular (Tb) microarchitecture. Therefore, miR-468-3p is a novel Runx2 regulator with key physiological action in BF and OD. [ABSTRACT FROM AUTHOR]

Calcific aortic valve disease (CAVD), a common heart valve disease, is increasingly prevalent worldwide and causes high morbidity and mortality. Here, we aimed to investigate a possible role for miR-34c in the development of osteogenic differentiation during CAVD and to find out the underlying mechanisms. Valvular interstitial cells (VICs) were isolated from the clinical aortic valve tissue samples of CAVD patients and patients with acute aortic dissection and collected. Then, RT-qPCR was performed to determine miR-34c expression and western blot analysis was applied to confirm the relevant protein expression in these VICs. Dual luciferase reporter gene assay was applied to confirm the relation between miR-34c and STC1. Alkaline phosphatase (ALP) staining and alizarin red staining was performed to further confirm the degree of calcification in these samples. MiR-34c was lowly expressed and STC1 was highly expressed in the CAVD tissues. Furthermore, STC1 was the target of miR-34c and was negatively regulated by miR-34c. Overexpression of miR-34c in VICs was concomitant with suppression of both STC1 expression and phosphorylation level of c-Jun N-terminal kinase (JNK). In addition, significant decrease of bone morphogenetic protein-2 (BMP2) and osteocalcin, as well as the decrease of calcification degree were also observed in VICs with miR-34c overexpressed. Taken together, miR-34c could inhibit osteogenic differentiation and calcification of VICs by suppressing the STC1/JNK signaling pathway in CAVD, making miR-34c a novel therapeutic target for the treatment of CAVD. [ABSTRACT FROM AUTHOR]

Background: Cellular senescence is a key driver of decreased bone formation and osteoporosis. Leptin (LEP) has been implicated in cellular senescence and osteogenic differentiation. The aim of this study was to investigate the mechanisms by which LEP mediates cellular senescence and osteogenic differentiation. Methods: C3H10T1/2 cells were treated with etoposide to induce cellular senescence, which was assessed by β-galactosidase staining. Quantitative real-time PCR and western blotting were used to measure the levels of senescence markers p21 and p16, as well as osteogenic differentiation-related genes ALP, COL1A1, and RUNX2. Alkaline phosphatase (ALP) staining and alizarin red S staining were performed to evaluate osteogenic differentiation. The NF-κB pathway and O-GlcNAcylation were assessed by western blotting. Results: Etoposide treatment increased the number of senescent cells and the levels of p21 and p16, along with elevated LEP expression. These effects were reversed by LEP knockdown. Additionally, LEP knockdown increased ALP staining density and osteoblast mineralization nodules, as well as the mRNA and protein levels of ALP, COL1A1, and RUNX2, indicating that LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells. Mechanistically, LEP knockdown inactivated the NF-κB pathway by inhibiting the nuclear translocation of p65. Furthermore, OGT was found to promote O-GlcNAcylation of LEP at the S50 site. Conclusion: Our findings demonstrated that O-GlcNAcylation of LEP inactivated the NF-κB pathway by reducing LEP protein levels, thereby inhibiting cellular senescence and promoting osteogenic differentiation in C3H10T1/2 cells. This study may provide a novel therapeutic target for the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Osteoporosis is characterized by increased resorption and decreased bone formation; it is predominantly influenced by genetic factors. G-protein coupled receptors (GPCRs) play a vital role in bone homeostasis, and mutations in these genes are associated with osteoporosis. This study aimed to investigate the impact of single nucleotide polymorphism (SNP) rs1042713 in the ADRB2 gene, encoding the beta-2-adrenergic receptor, on osteoblastogenesis. Herein, using quantitative polymerase chain reaction, western immunoblotting, immunofluorescence assays, and flow cytometry, we examined the expression of ADRB2 and markers of bone matrix synthesis in mesenchymal stem cells (MSCs) derived from osteoporosis patient (OP-MSCs) carrying ADRB2 SNP in comparison with MSCs from healthy donor (HD-MSCs). The results showed significantly reduced ADRB2 expression in OP-MSCs at both the mRNA and protein levels, alongside decreased type 1 collagen expression, a key bone matrix component. Notably, OP-MSCs exhibited increased ERK kinase expression during differentiation, indicating sustained cell cycle progression, unlike that going to HD-MSC. These results provide novel insights into the association of ADRB2 gene polymorphisms with osteogenic differentiation. The preserved proliferative activity of OP-MSCs with rs1042713 in ADRB2 contributes to their inability to undergo effective osteogenic differentiation. This research suggests that targeting genetic factors may offer new therapeutic strategies to mitigate osteoporosis progression. [ABSTRACT FROM AUTHOR]

Objective.: This study was aimed to investigate the potential roles of circTMTC1 in the underlying pathophysiological mechanisms of ossification of the ligamentum flavum (OLF). Summary of Background Data.: OLF is the primary contributor to thoracic spinal stenosis, which may cause severe neurological symptoms. There is a lack of effective medical therapy for OLF available so far because the exact underlying mechanism of OLF has not been fully elucidated. CircRNAs are a special class of noncoding RNAs and have attracted a growing interest of research in various human diseases recently. Therefore, we explored the potential roles of circRNAs in the underlying pathophysiological mechanisms of OLF. Materials and Methods.: We performed RNA-seq analysis to investigate the differentially expression profile of circRNAs in osteogenic differentiation of human LF cells, and identified a key circular RNA circTMTC1 functioned in OLF. Subsequently, we performed a series of experiments to investigate the exact molecular and cellular mechanisms in osteogenic differentiation of human ligamentum flavum cells. Results.: CircTMTC1 is significantly upregulated during osteogenic differentiation of human LF cells. Mechanistically, we found that circTMTC1 could interact with the RNA binding protein DDX3X and enhance its nucleocytoplasmic translocation. An increased cytoplasmic level of DDX3X activated the NLRP3 inflammasome pathway and thus promoted osteogenic differentiation of human ligamentum flavum cells. Conclusion.: Our findings suggested the circTMTC1-DDX3X-NLRP3 inflammasome signaling plays a pivotal role in osteogenic differentiation of human ligamentum flavum cells, which may provide novel diagnostic and therapeutic strategies for OLF. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recent studies have shown that research on naringin anti-osteoporosis mostly stays in in vitro and in vivo experiments. Understanding the mechanism of related signaling pathways and the expression of related proteins and some specific genes is an important way to deeply understand naringin anti-osteoporosis. At present, traditional Chinese medicine has been confirmed to have a significant role in anti-osteoporosis. Naringin is one of the main active ingredients in Rhizoma Drynariae. Its effectiveness and mechanism of action against osteoporosis have been gradually recognized by scholars, and its clinical and basic research has been gradually emphasized. OBJECTIVE: To analyze and summarize the research progress of naringin in anti-osteoporosis in vitro and in vivo, thereby providing some ideas for the next step to study its related mechanism of action. METHODS: The relevant literatures included in CNKI and PubMed database were searched with the Chinese search terms of “naringin, osteoporosis, traditional Chinese medicine compound, pathogenesis, signaling pathway, bone marrow mesenchymal stem cells, osteoblasts, osteoclasts” in Chinese and English, respectively. The corresponding criteria were established according to the research needs, and finally 69 articles were included for review. RESULTS AND CONCLUSION: Naringin blocks the increase in the number of osteoclasts and adipocytes, the decrease in the number of osteocytes and osteocalcin (+) cells induced by fructose-rich diet, and promotes the secretion of Sema3A from osteoblasts and osteocytes, thereby enhancing local bone formation and inhibiting osteoclast production by activating the Wnt/β-catenin pathway. Naringin is an important way to induce autophagy of osteoblasts, but autophagy-related proteins participate in osteoblast differentiation and bone formation. Lack of autophagy in osteoblasts reduces mineralization and leads to an imbalance in the number of osteoblasts and osteoclasts, which results in bone loss and decreased bone density. The composite scaffold loaded with naringin can be used as a necessary carrier for bone defect repair and has excellent bone repair properties. Naringin can also accelerate the growth of new bone tissue by increasing the local contents of bone morphogenetic protein 2 and vascular endothelial growth factor. Naringin can regulate bone metabolism and inhibit oxidative stress via ERK, PI3K/Akt and Wnt signaling pathways to improve osteoporosis, which can play a good role in preventing and controlling the disease. However, the depth and breadth of the relevant research is insufficient. Based on the mechanism of the current study, we should investigate the specific mechanisms by which naringin regulates different pathways and inter-pathway interactions in the future, which will be beneficial to the multifaceted development of naringin used in the treatment of osteoporosis.. [ABSTRACT FROM AUTHOR]

Background: Evidence has confirmed that forkhead box protein A1 (FOXA1) inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells. However, whether FOXA1 regulates the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to participate in periodontitis process is unclear. Methods: Lipopolysaccharide (LPS) was used to treat hPDLSCs to mimic inflammation environments. FOXA1 expression was examined by quantitative real-time PCR and western blot. The levels of IL-6 and TNF-α were evaluated by quantitative real-time PCR, ELISA and immunohistochemistry staining. hPDLSCs osteogenic differentiation was assessed by measuring alkaline phosphatase activity, alizarin red S intensity and the levels of osteogenic differentiation-related markers. Besides, the expression of signal transducer and activator of transcription 3 (STAT3) pathway-related markers were examined by western blot and immunofluorescence staining. Results: FOXA1 was upregulated in the periodontal ligament tissues of periodontitis patients, and its knockdown enhanced osteogenic differentiation of hPDLSCs. Besides, downregulation of FOXA1 suppressed inflammation levels in LPS-induced hPDLSCs. Also, FOXA1 silencing promoted the osteogenic differentiation of LPS-induced hPDLSCs by the inactivation of STAT3 pathway. Conclusion: Our data confirmed that knockdown of FOXA1 attenuated inflammation and enhanced osteogenic differentiation of LPS-induced hPDLSCs by regulating STAT3 pathway, indicating that FOXA1 might be a target for periodontitis treatment. [ABSTRACT FROM AUTHOR]

Objective: To investigate the impact of polydatin (PD) on osteogenesis and differentiation in ankylosing spondylitis (AS) mice and its regulation on Wnt-Smad pathway. Methods: Thirty-six SPF C57BL/6 male mice were randomly divided into 6 groups: control group, model group, positive group (sulfasalazine group), PD low, medium and high doses groups, with 6 mice in each group. HE staining was used to detect the pathological morphology of the synovium of the sacroiliac joint in mice; the contents of TNF-α, MMP-3 and OPG in serum of mice were detected by ELISA. Osteoblasts were isolated and cultured, the proliferation activity of osteoblasts was detected by CCK-8; ALP activity was measured with the kit; Alizarin red staining was used to detect the mineralization ability; Western blot was used to detect the expressions of Wnt1, β-Catenin, DKK-1, BMP-2, p-Smad1 and Runx2 proteins. Results: Compared with control group, the number of capillaries in the synovial tissue of the model group was increased, the surface of the tissue was swollen and congested, and the synovial cells were irregularly arranged; the contents of TNF-α, MMP-3 and OPG in serum were increased; the survival rate, ALP activity, mineralization ability and the expression of Wnt1, β-Catenin, BMP-2, p-Smad1, Runx2 proteins of osteoblasts in AS mice were increased, while the expression of DKK-1 protein was decreased (P<0.05); compared with model group, the proliferation of capillaries in the synovial tissue of the positive group and PD low, medium and high dose groups were decreased, the swelling of the tissue surface was decreased, and the synovial cells were translucent and arranged neatly; the contents of TNF-α, MMP-3 and OPG in serum were decreased; the survival rate, ALP activity, mineralization ability and the expressions of Wnt1, β-Catenin, BMP-2, p-Smad1, Runx2 proteins of osteoblasts in AS mice were decreased, while the expression of DKK-1 protein was increased (P<0.05). Conclusion: PD may inhibit the osteogenic differentiation of AS mice by inhibiting the activation of Wnt-Smad pathway. [ABSTRACT FROM AUTHOR]

Non-union fractures pose a significant clinical challenge, often leading to prolonged pain and disability. Understanding the molecular mechanisms underlying non-union fractures is crucial for developing effective therapeutic interventions. This study integrates bioinformatics analysis and experimental validation to unravel key genes and pathways associated with non-union fractures. We identified differentially expressed genes (DEGs) between non-union and fracture healing tissues using bioinformatics techniques. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to elucidate the biological processes and pathways involved. Common DEGs were identified, and a protein–protein interaction (PPI) network was constructed. Fibronectin-1 (FN1), Thrombospondin-1 (THBS1), and Biglycan (BGN) were pinpointed as critical target genes for non-union fracture treatment. Experimental validation involved alkaline phosphatase (ALP) and Alizarin Red staining to confirm osteogenic differentiation. Our analysis revealed significant alterations in pathways related to cell behavior, tissue regeneration, wound healing, infection, and immune responses in non-union fracture tissues. FN1, THBS1, and BGN were identified as key genes, with their upregulation indicating potential disruptions in the bone remodeling process. Experimental validation confirmed the induction of osteogenic differentiation. The study provides comprehensive insights into the molecular mechanisms of non-union fractures, emphasizing the pivotal roles of FN1, THBS1, and BGN in extracellular matrix dynamics and bone regeneration. The findings highlight potential therapeutic targets and pathways for further investigation. Future research should explore interactions between these genes, validate results using in vivo fracture models, and develop tailored treatment strategies for non-union fractures, promising significant advances in clinical management. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) is a common systemic bone metabolic disease clinically, mainly induced by the imbalance or deviation of bone homeostasis caused by various factors. Abundant research has confirmed that long non-coding RNA (IncRNA), such as IncRNA XIST, plays a crucial role in the metabolic imbalance processes of OP, such as bone homeostasis imbalance or deviation, proliferation and apoptosis of chondrocytes, and interactions between miRNA and proteins. This may provide a new perspective for in-depth exploration of the pathogenesis of OP. This article aims to review the relevant studies of IncRNA XIST in OP, in order to reveal its key mechanisms in the occurrence and development of OP, and provide theoretical support for the clinical treatment of bone metabolic diseases using IncRNA XIST. [ABSTRACT FROM AUTHOR]

• Mmp9 expression was specifically elevated during fusion of posterior frontal (PF) suture compared with other patent sutures in mice. • MMP9 depletion disturbed PF suture fusion ex vivo and in vivo. • Mmp9 knockout reduced suture cells proliferation, chondrogenesis and osteogenesis during PF suture closure. • Mmp9 knockout disturbed PF suture fusion via upregulating ribosome biogenesis in mice. Cranial sutures function as growth centers for calvarial bones. Abnormal suture closure will cause permanent cranium deformities. MMP9 is a member of the gelatinases that degrades components of the extracellular matrix. MMP9 has been reported to regulate bone development and remodeling. However, the function of MMP9 in cranial suture development is still unknown. Here, we identified that the expression of Mmp9 was specifically elevated during fusion of posterior frontal (PF) suture compared with other patent sutures in mice. Interestingly, inhibition of MMP9 ex vivo or knockout of Mmp9 in mice (Mmp9-/-) disturbed the fusion of PF suture. Histological analysis showed that knockout of Mmp9 resulted in wider distance between osteogenic fronts, suppressed cell condensation and endocranial bone formation in PF suture. Proliferation, chondrogenesis and osteogenesis of suture cells were decreased in Mmp9-/- mice, leading to the PF suture defects. Moreover, transcriptome analysis of PF suture revealed upregulated ribosome biogenesis and downregulated IGF signaling associated with abnormal closure of PF suture in Mmp9-/- mice. Inhibition of the ribosome biogenesis partially rescued PF suture defects caused by Mmp9 knockout. Altogether, these results indicate that MMP9 is critical for the fusion of cranial sutures, thus suggesting MMP9 as a potential therapeutic target for cranial suture diseases. [ABSTRACT FROM AUTHOR]

Objectives: Dental pulp stem cells (DPSCs) have been extensively used for tissue regeneration owing to their notable capabilities. Insulin‐like growth factor‐binding protein 5 (IGFBP5) regulates osteogenic differentiation of mesenchymal stem cells (MSCs); however, the underlying regulatory mechanisms require further investigation. Materials and Methods: Carboxyfluorescein succinimidyl ester, an alkaline phosphatase (ALP) activity assay and Alizarin Red staining were used to reveal the role of IGFBP5 in DPSCs. Protein expression levels were determined using western blotting. Immunofluorescence was used to observe cell sub‐localisation. Subcutaneous transplantation in nude mice was used to observe the osteogenesis of DPSCs in vivo. Results: IGFBP5 enhanced the proliferation and osteogenic differentiation of DPSCs. Deletion of the nuclear localisation sequence (NLS) of IGFBP5 prevented its nuclear import and abolished all its promoting effects on DPSCs; ivermectin stimulation attenuated the enhancement of ALP activity by IGBFP5. Bone‐like tissue formation promoted by IGFBP5 in vivo vanishes when the NLS is deleted. Inhibition of IGFBP5 nuclear import attenuated the IGFBP5‐induced phosphorylation of JNK (p‐JNK) and phosphorylated ERK (p‐ERK) in DPSCs. Conclusion: Our findings suggest that cell proliferation and osteogenic differentiation effects exerted by IGFBP5 on DPSCs are closely associated with their entry into the nucleus, thereby providing a novel potential target for tissue regeneration. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) plays an essential role during many biological processes including development from early embryonic stages until the terminal differentiation of specialized cells. This review summarizes the current knowledge about the involvement of PKC in molecular processes during the differentiation of stem/precursor cells into tissue cells with a particular focus on osteogenic, adipogenic, chondrogenic and neuronal differentiation by using a comprehensive approach. Interestingly, studies examining the overall role of PKC, or one of its three isoform groups (classical, novel and atypical PKCs), often showed controversial results. A discrete observation of distinct isoforms demonstrated that the impact on differentiation differs highly between the isoforms, and that during a certain process, the influence of only some isoforms is crucial, while others are less important. In particular, PKCβ inhibits, and PKCδ strongly supports osteogenesis, whereas it is the other way around for adipogenesis. PKCε is another isoform that overwhelmingly supports adipogenic differentiation. In addition, PKCα plays an important role in chondrogenesis, while neuronal differentiation has been positively associated with numerous isoforms including classical, novel and atypical PKCs. In a cellular context, various upstream mediators, like the canonical and non-canonical Wnt pathways, endogenously control PKC activity and thus, their activity interferes with the influence of PKC on differentiation. Downstream of PKC, several proteins and pathways build the molecular bridge between the enzyme and the control of differentiation, of which only a few have been well characterized so far. In this context, PKC also cooperates with other kinases like Akt or protein kinase A (PKA). Furthermore, PKC is capable of directly phosphorylating transcription factors with pivotal function for a certain developmental process. Ultimately, profound knowledge about the role of distinct PKC isoforms and the involved signaling pathways during differentiation constitutes a promising tool to improve the use of stem cells in regenerative therapies by precisely manipulating the activity of PKC or downstream effectors. [ABSTRACT FROM AUTHOR]

Background: Artemisia absinthium (A. absinthium), commonly known as absinthe, is a perennial plant with distinctive broad ovate pointed leaves of a silvery-gray color, reaching a height of 1.5 m. The utilization of this herb as a source of natural compounds and as the primary ingredient in the alcoholic beverage absinthe has recently seen a resurgence following a period of prohibition. This study investigates the biological effects of A. absinthium extract on healthy human periodontal ligament stem cells (hPDLSCs) and the human tongue squamous carcinoma cell line (HSC-3). Methods: A. absinthium element characterization was performed using High-Performance Liquid Chromatography (HPLC) and the Folin method. Alizarin assays evaluated the osteogenic capacity of human periodontal ligament cells (hPDLSCs) while CCK-8 and MTT determined the cytotoxicity of the extract against HSC-3 and hPDLSCs. Results: High artemisinin levels were detected, revealing a concentration of 89 μM (25 μg/mL). The total phenolic concentration of the extract was 1.07 mM +/− 0.11. The in vitro cytotoxicity assays revealed the biocompatible profile of the Artemisia extract in hPDLSCs without exhibiting any osteogenic potential. After 24 h of incubation with HSC-3, Artemisia extract (10 µM) decreased cancer cell viability by 99% and artemisinin by 64%, and increased the expression of Caspase 3 and 9 almost six and two times, respectively. Conclusions: In summary, our preliminary findings suggest that A. absinthium extract exhibits a toxic effect against carcinoma cell lines without affecting healthy human periodontal ligament stem cells. [ABSTRACT FROM AUTHOR]

Periodontitis is one of the most common complications of diabetes, which seriously affects patients' life quality. It is important to find the key factors and mechanisms to improve the treatment of periodontitis. In our study, high glucose (HG) and lipopolysaccharide (LPS) treated human periodontal ligament cells (hPDLCs) and LPS treated diabetic mice was used to establish the diabetic periodontitis model in vitro and in vivo. O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) and O-linked N-acetylglucosamine transferase (OGT) protein levels were detected by western blot assay. Cell counting kit-8, alkaline phosphatase (ALP), and alizarin red staining (ARS) assays were used to observe the O-GlcNAcylation and OGT effects on cell viability and osteoblast differentiation. Co-immunoprecipitation (Co-IP) assay was used to detect the relationship between OGT and ALP. The results showed that the levels of OGT and O-GlcNAcylation were significantly increased in both cell and mouse models. ALP and ARS staining results showed that silencing of OGT or inhibition of O-glycosylation notably improved osteogenic differentiation, increased the osteoprotegerin (OPG) protein levels and decreased the receptor activator for nuclear factor-κB Ligand (RANKL) protein levels of the HG and LPS treated hPDLCs. In diabetic periodontitis mice, knockdown of OGT relieved the injury of gingival tissue, increased the ALP and OPG levels and decreased the RANKL levels. Besides, ALP interacted with OGT protein, and OGT protein was found to act on ALP serine 513 glycosylation. In conclusion, our study demonstrated that excessive O-GlcNAcylation could restrain osteoblast differentiation by O-glycosylation in ALP. [ABSTRACT FROM AUTHOR]

Many tissues exhibit structural anisotropy, which imparts orientation-specific properties and functions. However, recapitulating the cellular patterns found in anisotropic tissues presents a remarkable challenge, particularly when using soft and wet hydrogels. Herein, we develop self-assembled anisotropic magnetic Fe3O4 micropatterns on polyethylene glycol hydrogels utilizing dipole–dipole interactions. Under the influence of a static magnetic field, Fe3O4 nanoparticles align into highly ordered structures with a height of 400–600 nm and a width of 8–10 μm. Furthermore, our layer-by-layer assembly technique enables the creation of oriented micropatterns with varying densities and heights, which can be further manipulated to form three-dimensional structures by adjusting the angle of the magnetic field. These anisotropic magnetic Fe3O4 micropatterns can be applied to various substrates, including treated glass slides, standard glass slides, silicon wafers, and polydimethylsiloxane. The patterned Fe3O4 scaffolds, modified with gold coating, effectively enhance cellular adhesion, orientation, and osteogenic differentiation of bone marrow-derived stem cells, which is crucial for effective tissue repair. Overall, this study presents an efficient strategy for constructing anisotropic Fe3O4 micropattern hydrogels, providing a bioactive platform that significantly enhances cellular functions. [ABSTRACT FROM AUTHOR]

Bone tissue engineering is a technique that simulates the bone tissue microenvironment by utilizing cells, tissue scaffolds, and growth factors. The collagen hydrogel is a three-dimensional network bionic material that has properties and structures comparable to those of the extracellular matrix (ECM), making it an ideal scaffold and drug delivery system for tissue engineering. The clinical applications of this material are restricted due to its low mechanical strength. In this investigation, a collagen-based gel (atelocollagen/glycerol/pullulan [Col/Gly/Pul] gel) that is moldable and injectable with high adhesive qualities was created by employing a straightforward technique that involved the introduction of Gly and Pul. This study aimed to characterize the internal morphology and chemical composition of the Col/Gly/Pul gel, as well as to verify its osteogenic properties through in vivo and in vitro experiments. When compared to a standard pure Col hydrogel, this material is more adaptable to the complexity of the local environment of bone defects and the apposition of irregularly shaped flaws due to its greater mechanical strength, injectability, and moldability. Overall, the Col/Gly/Pul gel is an implant that shows great potential for the treatment of complex bone defects and the enhancement of bone regeneration. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) is an epidemic bone remodeling disorder of growing relevance with the aging population. Considering that isorhamnetin (ISO), a flavonoid derived from plant, has been newly reckoned as an active ingredient in treating OP, our paper was conducted to investigate the regulatory role and mechanism of ISO in OP. CCK-8 method detected cell activity. Alkaline phosphatase (ALP) assay kit, ALP staining and alizarin red S staining measured osteogenic differentiation. RT-qPCR and Western blot examined the expressions of osteoblast-related proteins. Wound healing and cell adhesion assays severally detected cell migration and adhesion. Also, Western blot tested the expressions of extracellular signal-regulated kinase (ERK) signaling-associated proteins. As illustrated, after MC3T3-E1 pre-osteoblasts were stimulated to differentiate to osteoblasts, ISO markedly promoted the differentiation, mineralization, migration and adhesion of MC3T3-E1 osteoblasts in a concentration-dependent manner. In addition, administration of ISO functioned as an activator of ERK-dependent BMP2-Smad signaling in MC3T3-E1 osteoblasts and pretreatment with ERK inhibitor PD98059 partially compensated the impacts of ISO on MC3T3-E1 osteoblasts differentiation, mineralization, migration as well as adhesion. To be summarized, ISO might activate ERK-dependent BMP2-Smad signaling to facilitate the differentiation, mineralization, migration and adhesion of MC3T3-E1 osteoblasts, suggesting the protective potential of ISO in OP. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell's metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including KLF1, NFATC3, ZNF148, ASCL1, FOXI1, and KLF5. These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation. [ABSTRACT FROM AUTHOR]

Ossification of the posterior longitudinal ligament (OPLL) is a condition comprising ectopic bone formation from spinal ligaments. This disease is a leading cause of myelopathy in the Asian population. However, the molecular mechanism underlying OPLL and efficient preventive interventions remain unclear. Here, we performed single-cell RNA sequencing and revealed that type I interferon (IFN) signaling was activated in the ossified ligament of patients with OPLL. We also observed that IFN-β stimulation promoted the osteogenic differentiation of preosteoblasts in vitro and activated the ossification-related gene SPP1, thereby confirming the single-cell RNA sequencing findings. Further, blocking the IFN-α/β subunit 1 receptor (IFNAR1) using an anti-IFNAR1 neutralizing antibody markedly suppressed osteogenic differentiation. Together, these results demonstrated that the type I IFN signaling pathway facilitated ligament ossification, and the blockade of this signaling might provide a foundation for the prevention of OPLL. [ABSTRACT FROM AUTHOR]

A general decline in the osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMSCs) in the elderly is a clinical consensus, with diverse opinions on the mechanisms. Many studies have demonstrated that metformin (MF) significantly protects against osteoporosis and reduces fracture risk. However, the exact mechanism of this effect remains unclear. In this study, we found that the decreased miR-181a-5p expression triggered by MF treatment plays a critical role in recovering the osteogenic ability of aging hBMSCs (derived from elderly individuals). Notably, the miR-181a-5p expression in hBMSCs was significantly decreased with prolonged MF (1000 μM) treatment. Further investigation revealed that miR-181a-5p overexpression markedly impairs the osteogenic ability of hBMSCs, while miR-181a-5p inhibition reveals the opposite result. We also found that miR-181a-5p could suppress the protein translation process of plasminogen activator inhibitor-1 (PAI-1), as evidenced by luciferase assays and Western blots. Additionally, low PAI-1 levels were associated with diminished osteogenic ability, whereas high levels promoted it. These findings were further validated in human umbilical cord mesenchymal stem cells (hUCMSCs). Finally, our in vivo experiment with a bone defects rat model confirmed that the agomiR-181a-5p (long-lasting miR-181a-5p mimic) undermined bone defects recovery, while the antagomiR-181a-5p (long-lasting miR-181a-5p inhibitor) significantly promoted the bone defects recovery. In conclusion, we found that MF promotes bone tissue regeneration through the miR-181a-5p/PAI-1 axis by affecting MSC osteogenic ability, providing new strategies for the treatment of age-related bone regeneration disorders. [ABSTRACT FROM AUTHOR]

Zinc is the second most abundant trace element in the human body, stored mainly in the bones. Zinc is required for bone growth and homeostasis and is also a crucial cofactor for numerous proteins that play key roles in maintaining microstructural integrity and bone remodeling. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent progenitors found in the bone marrow stroma and can differentiate along multiple lineage pathways. In this study, we investigated the effect of zinc on the osteogenic differentiation of BMSCs. We stimulated the osteogenic differentiation of BMSCs with high phosphate and Ca-containing osteogenic medium (PiCa) in the presence or absence of zinc. We followed calcification by measuring ECM mineralization, the Ca content of the ECM, mRNA, and the protein expression of the osteo-chondrogenic transcription factor RUNX2 and SOX9 and its targets OCN and ALP. Zinc dose-dependently abolished PiCa-induced ECM mineralization and decreased the expression of RUNX2, SOX9, OCN, and ALP. Serum albumin did not alter the inhibitory effect of zinc on BMSC mineralization. Our further analysis with the zinc-chelator TPEN and ZnCl2 confirmed the specific inhibitory effect of free zinc ions on BMSC mineralization. Zinc inhibited phosphate uptake and PiCa-induced upregulation of the sodium-dependent phosphate cotransporters (PiT-1 and PiT-2). Zinc attenuated the PiCa-induced increase in ROS production. Taken together, these data suggest that zinc inhibits PiCa-induced BMSC calcification by regulating phosphate uptake and ROS production. [ABSTRACT FROM AUTHOR]

A biodegradable, shape memory polymer (SMP) scaffold based on poly(ε‐caprolactone) (PCL) represents an attractive alternative therapy for the repair of critically sized bone defects given its ability to press‐fit within irregular defects. Clinical translation of SMP scaffolds requires successful movement beyond proof‐of‐concept rodent studies through a relevant large‐animal model and into the clinical setting. In addition to representing a clinical veterinary population, the canine species is a strong translational model for humans due to similarities in orthopedic disorders, biomechanics, and bone healing. The present study was performed to assess in vitro cytocompatibility and osteogenic differentiation of canine multipotent stromal cells (cMSCs) cultured on SMP scaffolds in preparation for future canine in vivo studies. Two different SMP scaffold compositions were utilized: a "PCL‐only" scaffold prepared from PCL‐diacrylate (PCL‐DA) and a semi‐interpenetrating network (semi‐IPN) formed from PCL‐DA and poly(L‐lactic acid) (PCL:PLLA). The PCL:PLLA scaffolds degrade faster and are more mechanically rigid versus the PCL scaffolds. Canine bone marrow–derived MSCs (cMSCs) were evaluated in terms of attachment, proliferation, and osteogenic differentiation. cMSCs exhibited excellent cytocompatibility, attachment, and proliferation on both SMP scaffold compositions. PCL scaffolds were more conducive to both early‐ and late‐stage in vitro osteogenesis of cMSCs versus PCL:PLLA scaffolds. However, cMSCs deposited mineralized extracellular matrix over 21 days when cultured on both SMP scaffold compositions. These results demonstrate that the SMP scaffolds are suitable for in vitro cMSC attachment, proliferation, and osteogenic differentiation, representing a significant step toward canine in vivo studies and potential translation to human patients. [ABSTRACT FROM AUTHOR]

Degradable phosphate glasses have shown favorable properties for tissue engineering. By changing the composition of the glasses, the degradation rate, and ion release are controllable. Zinc oxide can function as a glass network modifier and has been shown to play a positive role in bone formation. Also, phosphate glasses can easily be processed into microspheres, which can be used as microcarriers. This study aims to develop zinc phosphate glasses microspheres and explore the optimized size and composition for applications in bone tissue engineering. Zinc–titanium–calcium–sodium phosphate glasses with 0, 1, 3, 5, or 10 mol % zinc oxide were prepared and processed into microspheres. The smaller microspheres ranged in size from 50 to 106 μm, while the larger ones ranged from 106 to 150 μm. The characteristics of glasses were examined. The osteoblastic cell line MC3T3‐E1 was cultured on the surface of microspheres and the cell viability was examined. To evaluate osteogenic differentiation, Alizarin Red S staining, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed after 14 days. Different sizes of zinc phosphate glass microspheres were successfully made. The glass microspheres with <10 mol % zinc oxide were able to support the adhesion and proliferation of MC3T3‐E1 cell lines. The relative gene expression of BMP2 was significantly upregulated in the smaller glass microspheres containing 3 mol % zinc oxide (26‐fold, p <.001) and both sizes of microspheres containing 5 mol % zinc oxide (smaller: 27‐fold, p <.001; larger: 35‐fold, p <.001). Additionally, cluster formation was observed in glass microspheres after 14 days, and the mineralization of MC3T3‐E1 cell lines was promoted. Based on these findings, the glass microspheres containing 3–5 mol % of zinc oxide can promote osteogenic differentiation for MC3T3‐E1 cells. [ABSTRACT FROM AUTHOR]