Mechanisms of vascular invasion after cartilage injury and potential engineering cartilage treatment strategies.

Articular cartilage injury is one of the most common diseases in orthopedic clinics. Following an articular cartilage injury, an inability to resist vascular invasion can result in cartilage calcification by newly formed blood vessels. This process ultimately leads to the loss of joint function, significantly impacting the patient's quality of life. As a result, developing anti-angiogenic methods to repair damaged cartilage has become a popular research topic. Despite this, tissue engineering, as an anti-angiogenic strategy in cartilage injury repair, has not yet been adequately investigated. This exhaustive literature review mainly focused on the process and mechanism of vascular invasion in articular cartilage injury repair and summarized the major regulatory factors and signaling pathways affecting angiogenesis in the process of cartilage injury. We aimed to discuss several potential methods for engineering cartilage repair with anti-angiogenic strategies. Three anti-angiogenic tissue engineering methods were identified, including administering angiogenesis inhibitors, applying scaffolds to manage angiogenesis, and utilizing in vitro bioreactors to enhance the therapeutic properties of cultured chondrocytes. The advantages and disadvantages of each strategy were also analyzed. By exploring these anti-angiogenic tissue engineering methods, we hope to provide guidance for researchers in related fields for future research and development in cartilage repair.

CD62E- and ROS-Responsive ETS Improves Cartilage Repair by Inhibiting Endothelial Cell Activation through OPA1-Mediated Mitochondrial Homeostasis.

Background: In the environment of cartilage injury, the activation of vascular endothelial cell (VEC), marked with excessive CD62E and reactive oxygen species (ROS), can affect the formation of hyaluronic cartilage. Therefore, we developed a CD62E- and ROS-responsive drug delivery system using E-selectin binding peptide, Thioketal, and silk fibroin (ETS) to achieve targeted delivery and controlled release of Clematis triterpenoid saponins (CS) against activated VEC, and thus promote cartilage regeneration. Methods: We prepared and characterized ETS/CS and verified their CD62E- and ROS-responsive properties in vitro. We investigated the effect and underlying mechanism of ETS/CS on inhibiting VEC activation and promoting chondrogenic differentiation of bone marrow stromal cells (BMSCs). We also analyzed the effect of ETS/CS on suppressing the activated VEC-macrophage inflammatory cascade in vitro. Additionally, we constructed a rat knee cartilage defect model and administered ETS/CS combined with BMSC-containing hydrogels. We detected the cartilage differentiation, the level of VEC activation and macrophage in the new tissue, and synovial tissue. Results: ETS/CS was able to interact with VEC and inhibit VEC activation through the carried CS. Coculture experiments verified ETS/CS promoted chondrogenic differentiation of BMSCs by inhibiting the activated VEC-induced inflammatory cascade of macrophages via OPA1-mediated mitochondrial homeostasis. In the rat knee cartilage defect model, ETS/CS reduced VEC activation, migration, angiogenesis in new tissues, inhibited macrophage infiltration and inflammation, promoted chondrogenic differentiation of BMSCs in the defective areas. Conclusions: CD62E- and ROS-responsive ETS/CS promoted cartilage repair by inhibiting VEC activation and macrophage inflammation and promoting BMSC chondrogenesis. Therefore, it is a promising therapeutic strategy to promote articular cartilage repair.

The Mechanism of Action and Experimental Verification of Narenmandula in the Treatment of Postmenopausal Osteoporosis.

BACKGROUND: Narenmandula is a classic ancient remedy in Inner Mongolia, historically used for gastrointestinal diseases. In recent decades, Inner Mongolia Medical University found that it has a significant effect in promoting fracture healing and increasing bone density, and has been used to treat postmenopausal osteoporosis (PMOP), but its mechanism is unclear. OBJECTIVE: Identify the mechanism of action of Narenmandula for PMOP treatment. METHODS: Network pharmacology, molecular docking and ovarian departing rat models were used to verify the relevant mechanism of Narenmandula in the treatment of PMOP. RESULTS: We confirmed that NRMDL prescription can improve OVX-induced bone loss, improve trabecular density, and relieve osteoporosis. Upon screening of network pharmacology, we obtained 238 overlapping genes of Narenmandula and PMOP, and analyzed AKT, IL1B, and IL6 as key genes by network topology. Among the 1143 target genes that interact with PMOP, 107 NRMDL active compounds correspond to 345 target genes and 238 overlapping genes. Network topology analysis showed the top 8 active ingredients, such as quercetin and kaempferol, and the top 20 key genes, such as AKT, IL1B, IL6, INS, JUN, STAT3, TNF, TP53, etc. Enrichment analysis revealed involvement of PI3K-Akt, HIF-1, FoxO, MAPK, and TNF signaling pathways. In addition, we found the most important active compounds bind tightly to core proteins, which were verified by molecular docking analysis. The AKT-related pathway had good binding energy, and the pathway was verified by cell and animal experiments. CONCLUSION: The potential mechanism and efficacy of Narenmandula against PMOP may be related to the PI3K-AKT pathway.

Inhibition of HDAC6 promotes microvascular endothelial cells to phagocytize myelin debris and reduces inflammatory response to accelerate the repair of spinal cord injury.

AIMS: To identify an effective strategy for promoting microvascular endothelial cells (MECs) to phagocytize myelin debris and reduce secretion of inflammatory factors following spinal cord injury (SCI). METHODS: We established a coculture model of myelin debris and vascular-like structures. The efficiency with which MECs phagocytize myelin debris under different conditions was examined via ELISA, flow cytometry, and immunofluorescence. Tubastatin-A was used to interfere with the coculture model. The anti-inflammatory effects of Tubastatin-A were observed by HE staining, flow cytometry, immunofluorescence, and ELISA. RESULTS: MECs phagocytized myelin debris via IgM opsonization, and phagocytosis promoted the secretion of inflammatory factors, whereas IgG-opsonized myelin debris had no effect on inflammatory factors. Application of the HDAC6 inhibitor Tubastatin-A increased the IgG levels and decreased the IgM levels by regulating the proliferation and differentiation of B cells. Tubastatin-A exerted a regulatory effect on the HDAC6-mediated autophagy-lysosome pathway, promoting MECs to phagocytize myelin debris, reducing the secretion of inflammatory factors, and accelerating the repair of SCI. CONCLUSIONS: Inhibition of HDAC6 to regulate the immune-inflammatory response and promote MECs to phagocytize myelin debris may represent a novel strategy in the treatment of SCI.

Morroniside Inhibits Inflammatory Bone Loss through the TRAF6-Mediated NF-κB/MAPK Signalling Pathway.

Osteoporosis is a chronic inflammatory disease that severely affects quality of life. Cornus officinalis is a Chinese herbal medicine with various bioactive ingredients, among which morroniside is its signature ingredient. Although anti-bone resorption drugs are the main treatment for bone loss, promoting bone anabolism is more suitable for increasing bone mass. Therefore, identifying changes in bone formation induced by morroniside may be conducive to developing effective intervention methods. In this study, morroniside was found to promote the osteogenic differentiation of bone marrow stem cells (BMSCs) and inhibit inflammation-induced bone loss in an in vivo mouse model of inflammatory bone loss. Morroniside enhanced bone density and bone microstructure, and inhibited the expression of IL6, IL1ß, and ALP in serum (p < 0.05). Furthermore, in in vitro experiments, BMSCs exposed to 0-256 µM morroniside did not show cytotoxicity. Morroniside inhibited the expression of IL6 and IL1ß and promoted the expression of the osteogenic transcription factors Runx2 and OCN. Furthermore, morroniside promoted osteocalcin and Runx2 expression and inhibited TRAF6-mediated NF-κB and MAPK signaling, as well as osteoblast growth and NF-κB nuclear transposition. Thus, morroniside promoted osteogenic differentiation of BMSCs, slowed the occurrence of the inflammatory response, and inhibited bone loss in mice with inflammatory bone loss.

Iron induces B cell pyroptosis through Tom20-Bax-caspase-gasdermin E signaling to promote inflammation post-spinal cord injury.

BACKGROUND: Immune inflammatory responses play an important role in spinal cord injury (SCI); however, the beneficial and detrimental effects remain controversial. Many studies have described the role of neutrophils, macrophages, and T lymphocytes in immune inflammatory responses after SCI, although little is known about the role of B lymphocytes, and immunosuppression can easily occur after SCI. METHODS: A mouse model of SCI was established, and HE staining and Nissl staining were performed to observe the pathological changes. The size and morphology of the spleen were examined, and the effects of SCI on spleen function and B cell levels were detected by flow cytometry and ELISA. To explore the specific mechanism of immunosuppression after SCI, B cells from the spleens of SCI model mice were isolated using magnetic beads and analyzed by 4D label-free quantitative proteomics. The level of inflammatory cytokines and iron ions were measured, and the expression of proteins related to the Tom20 pathway was quantified by western blotting. To clarify the relationship between iron ions and B cell pyroptosis after SCI, we used FeSO4 and CCCP, which induce oxidative stress to stimulate SCI, to interfere with B cell processes. siRNA transfection to knock down Tom20 (Tom20-KD) in B cells and human B lymphocytoma cell was used to verify the key role of Tom20. To further explore the effect of iron ions on SCI, we used deferoxamine (DFO) and iron dextran (ID) to interfere with SCI processes in mice. The level of iron ions in splenic B cells and the expression of proteins related to the Tom20-Bax-caspase-gasdermin E (GSDME) pathway were analyzed. RESULTS: SCI could damage spleen function and lead to a decrease in B cell levels; SCI upregulated the expression of Tom20 protein in the mitochondria of B cells; SCI could regulate the concentration of iron ions and activate the Tom20-Bax-caspase-GSDME pathway to induce B cell pyroptosis. Iron ions aggravated CCCP-induced B cell pyroptosis and human B lymphocytoma pyroptosis by activating the Tom20-Bax-caspase-GSDME pathway. DFO could reduce inflammation and promote repair after SCI by inhibiting Tom20-Bax-caspase-GSDME-induced B cell pyroptosis. CONCLUSIONS: Iron overload activates the Tom20-Bax-caspase-GSDME pathway after SCI, induces B cell pyroptosis, promotes inflammation, and aggravates the changes caused by SCI. This may represent a novel mechanism through which the immune inflammatory response is induced after SCI and may provide a new key target for the treatment of SCI.

Construction and optimization of a coculture system of mouse brain microvascular endothelial cells and myelin debris.

Microvascular endothelial cells are a newly discovered cell type involved in the phagocytosis of myelin debris, which play a key role in the repair of spinal cord injuries. Several methods for the preparation of myelin debris and parameters for constructing a coculture system of microvascular endothelial cells and myelin debris are available, but no systematic studies have yet been conducted, which hinders further exploration of the mechanisms of demyelinating disease repair. Herein, we aimed to develop a standardized method for this process. Myelin debris of different sizes was obtained from the brains of C57BL/6 mice by stripping the brains under aseptic conditions, multiple grinding, gradient centrifugation, etc. Transmission electron microscopy and nanoparticle size analysis were used to characterize myelin debris. Microvascular endothelial cells were cultured on a matrix gel, and myelin debris of different sizes (fluorescently labeled using CFSE) was placed in coculture after forming a vascular-like structure. Subsequently, myelin debris of different concentrations was cocultured in the vascular-like structure, and phagocytosis of myelin debris by microvascular endothelial cells was detected using immunofluorescence staining and flow cytometry. We found that myelin debris could be successfuly obtained from the mouse brain with secondary grinding and other steps and cocultured with microvascular endothelial cells at a concentration of 2 mg/mL, which promoted the phagocytosis of microvascular endothelial cells. In conclusion, we provide a reference for the protocol of a coculture system of microvascular endothelial cells and myelin debris.

Discovering the Potential Value of Coenzyme Q10 in Oxidative Stress: Enlightenment From a Synthesis of Clinical Evidence Based on Various Population.

Background: Oxidative stress (OS) is associated with ferroptosis. Coenzyme Q10 (CoQ10), as an adjuvant treatment, has shown to be beneficial against OS. However, the efficacy of CoQ10 as a therapeutic agent against OS has not been promptly updated and systematically investigated. Methods: A systematic literature search was performed using the Medline, EMBASE, Web of science, Cochrane Central Register of Controlled Trials, CNKI, CBM, Science direct and clinical trial. gov to identify randomized clinical trials evaluating the efficacy of CoQ10 supplementation on OS parameters. Standard mean differences and 95% confidence intervals were calculated for net changes in OS parameters using a random-effects model. Results: Twenty-one randomized clinical studies met the eligibility criteria to be included in the meta-analysis. Overall, CoQ10 supplementation increased the levels of antioxidant enzymes [including superoxide dismutase (SOD) (SMD = 0.63; 95% CI: 0.38 to 0.88; p < 0.001), catalase (CAT) (SMD = 0.44; 95% CI:0.16 to 0.72; p = 0.002)] significantly and the levels of malondialdehyde (MDA) (SMD = -0.68; 95% CI: 0.93 to -0.43; p < 0.001) was decreased considerably. However, significant associations were not observed between this supplement and total antioxidant capacity (TAC), glutathione peroxidase (GPx) activity. Conclusion: CoQ10 can improve OS as indicated by statistical significance in CAT and MDA concentrations, as well as SOD activity. Future studies focusing on long-term results and specific valuation of OS parameters are required to confirm the efficacy of CoQ10 on OS. We also believe that with the further research on ferroptosis, CoQ10 will gain more attention. Systematic Review Registration: [https://inplasy.com/], identifier [INPLASY2021120123].

Mechanical Stretch Promotes Macrophage Polarization and Inflammation via the RhoA-ROCK/NF-κB Pathway.

Macrophages play an essential role in the pathogenesis of most inflammatory diseases. Recent studies have shown that mechanical load can influence macrophage function, leading to excessive and uncontrolled inflammation and even systemic damage, including cardiovascular disease and knee osteoarthritis. However, the molecular mechanism remains unclear. In this study, murine RAW264.7 cells were treated with mechanical stretch (MS) using the Flexcell-5000T Tension System. The expression of inflammatory factors and cytokine release were measured by RT-qPCR, ELISA, and Western blotting. The protein expression of NF-κB p65, Iκb-α, p-Iκb-α, RhoA, ROCK1, and ROCK2 was also detected by Western blotting. Then, Flow cytometry was used to detect the proportion of macrophage subsets. Meanwhile, Y-27632 dihydrochloride, a ROCK inhibitor, was added to knockdown ROCK signal transduction in cells. Our results demonstrated that MS upregulated mRNA expression and increased the secretion levels of proinflammatory factors iNOS, IL-1ß, TNF-α, and IL-6. Additionally, MS significantly increased the proportion of CD11b+CD86+ and CD11b+CD206+ subsets in RAW264.7 macrophages. Furthermore, the protein expression of RhoA, ROCK1, ROCK2, NF-κB p65, and IκB-α increased in MS-treated RAW264.7 cells, as well as the IL-6 and iNOS. In contrast, ROCK inhibitor significantly blocked the activation of RhoA-ROCK and NF-κB pathway, decreased the protein expression of IL-6 and iNOS, reduced the proportion of CD11b+CD86+ cells subpopulation, and increased the proportion of CD11b+CD206+ cell subpopulation after MS. These data indicate that mechanical stretch can regulate the RAW264.7 macrophage polarization and enhance inflammatory responses in vitro, which may contribute to activation the RhoA-ROCK/NF-κB pathway.

Quercetin Attenuates Osteoporosis in Orchiectomy Mice by Regulating Glucose and Lipid Metabolism via the GPRC6A/AMPK/mTOR Signaling Pathway.

Quercetin, a flavonoid found in natural medicines, has shown a role in disease prevention and health promotion. Moreover, because of its recently identified contribution in regulating bone homeostasis, quercetin may be considered a promising agent for improving bone health. This study aimed to elucidate the role of quercetin in androgen deprivation therapy-induced osteoporosis in mice. C57BL/6 mice were subjected to orchiectomy, followed by quercetin treatment (75 and 150 mg/kg/d) for 8 weeks. Bone microstructure was then assessed by micro-computed tomography, and a three-point bending test was used to evaluate the biomechanical parameters. Hematoxylin and eosin (H&E) staining was used to examine the shape of the distal femur, gastrocnemius muscle, and liver. The balance motion ability in mice was evaluated by gait analysis, and changes in the gastrocnemius muscle were observed via Oil red O and Masson's staining. ELISA and biochemical analyses were used to assess markers of the bone, glucose, and lipid metabolism. Western blotting analyses of glucose and lipid metabolism-related protein expression was performed, and expression of the GPCR6A/AMPK/mTOR signaling pathway-related proteins was also assessed. After 8 weeks of quercetin intervention, quercetin-treated mice showed increased bone mass, bone strength, and improved bone microstructure. Additionally, gait analysis, including stride length and frequency, were significantly increased, whereas a reduction of the stride length and gait symmetry was observed. H&E staining of the gastrocnemius muscle showed that the cross-sectional area of the myofibers had increased significantly, suggesting that quercetin improves balance, motion ability, and muscle mass. Bone metabolism improvement was defined by a reduction of serum levels of insulin, triglycerides, total cholesterol, and low-density lipoprotein, whereas levels of insulin-like growth factor-1 and high-density lipoprotein were increased after quercetin treatment. Expression of proteins involved in glucose uptake was increased, whereas that of proteins involved in lipid production was decreased. Moreover, the GPRC6A and the phospho-AMPK/AMPK expression ratio was elevated in the liver and tibia tissues. In contrast, the phospho-mTOR/mTOR ratio was reduced in the quercetin group. Our findings indicate that quercetin can reduce the osteoporosis induced by testosterone deficiency, and its beneficial effects might be associated with the regulation of glucose metabolism and inhibition of lipid metabolism via the GPCR6A/AMPK/mTOR signaling pathway.

[Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair].

Objective: To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods: CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups ( n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results: The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 µm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes ( P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers ( P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B ( P<0.05), and the level of TIMP-1 in group C was significantly higher ( P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B ( P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B ( P<0.05). Conclusion: CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.

A new method for primary culture of microglia in rats with spinal cord injury.

At present, the primary culture method of microglia is complicated, and the culture of spinal cord microglia is rare, so we will explore to establish a new and efficient primary culture method of microglia in rats with spinal cord injury (SCI). The SCI model of SD rats was established by modified A11en's method, and the model of SCI was performed on 1 d, 3 d, 7 d and 14 d respectively. Then the injured spinal cord was removed, mechanically separated and filtered. The morphology of microglia was observed the next day and its purity was identified by CD11b and Iba1 immunofluorescence labeling. According to the above results, the morphological changes of microglia after 3 d of SCI were observed at 1 d, 2 d and 4 d. The results showed that the purity of microglia was 98%. The number of microglia after 3 d of SCI was the most. After SCI, the migration ability of microglia was enhanced, the number of microglia in the injured area increased, and the number was the highest at 3 d, then gradually decreased. In addition, the microglia after SCI would gradually change from active state to resting state with the passage of time. Therefore, we can use a simple and efficient mechanical separation method to extract primary microglia, which provides the basis for the study of microglia.

Cartilage Repair Using Clematis Triterpenoid Saponin Delivery Microcarrier, Cultured in a Microgravity Bioreactor Prior to Application in Rabbit Model.

Cartilage tissue engineering provides a promising method for the repair of articular cartilage defects, requiring appropriate biological scaffolds and necessary growth factors to enhance the efficiency of cartilage regeneration. Here, a silk fibroin (SF) microcarrier and a clematis triterpenoid saponin delivery SF (CTS-SF) microcarrier were prepared by the high-voltage electrostatic differentiation and lyophilization method, and chondrocytes were carried under the simulated microgravity condition by a rotating cell culture system. SF and CTS-SF microspheres were relatively uniform in size and had a porous structure with good swelling and cytocompatibility. Further, CTS-SF microcarriers could sustainably release CTSs in the monitored 10 days. Compared with the monolayer culture, chondrocytes under the microgravity condition maintained a better chondrogenic phenotype and showed better proliferation ability after culture on microcarriers. Moreover, the sustained release of CTS from CTS-SF microcarriers upregulated transforming growth factor-ß, Smad2, and Smad3 signals, contributing to promote chondrogenesis. Hence, the biophysical effects of microgravity and bioactivities of CTS-ST were used for chondrocyte expansion and phenotype maintenance in vitro. With prolonged expansion, SF- and CTS-SF-based microcarrier-cell composites were directly implanted in vivo to repair rabbit articular defects. Gross evaluations, histopathological examinations, and biochemical analysis indicated that SF- and CTS-SF-based composites exhibited cartilage-like tissue repair compared with the nontreated group. Further, CTS-SF-based composites displayed superior hyaline cartilage-like repair that integrated with the surrounding cartilage better and higher cartilage extracellular matrix content. In conclusion, these results provide an alternative preparation method for drug-delivered SF microcarrier and a culture method for maintaining the chondrogenic phenotype of seed cells based on the microgravity environment. CTS showed its bioactive function, and the application of CTS-SF microcarriers can help repair and regenerate cartilage defects.

Current status and development trend of miRNAs in osteoporosis-related research: A bibliometric analysis.

INTRODUCTION: The occurrence of osteoporosis (OP) has drawn considerable attention from scholars around the world due to the significant impacts thereof on the social economy and the quality of human life. OP research has been rapidly expanding since the inclusion of microRNAs (miRNAs) as critical regulators of gene-expression. However, despite the ability to evaluate miRNA gene therapy in OP being enhanced, there has been a scarcity of updated citation analyses that reflect such developments. In the present study, through bibliometric analysis, the global research activity and trends in regard to the relationship between OP and miRNAs were reviewed. METHODS: Publications related to miRNA and OP from 2000 to 2021 were retrieved via Web of Science (WoS). The data included publication years, countries, journals, institutions, authors and keywords, and were sorted and summarized by bibliometrics, before being visually analyzed through VOS Viewer. RESULTS: In the past five years, 599 articles have been published, with said studies accounting for 79.11% of all relevant documents, indicating the increased interest in the present research topic. The country with the highest contribution rate was China, and the publication rate of Journal of Bone and Mineral Research was the highest, followed by Bone. The institutions with the highest contribution rate were Nanjing Medical University. The most frequently occurring keywords were clustered into five groups. The research area of the first group described that circulating miRNA would be a potential biomarker for postmenopausal osteoporosis (PMOP). The remaining four groups involved the influences of miRNAs and exosomes on the osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs), and the interactions of lncRNA and miRNA with OP. CONCLUSIONS: The results of the present study will expand the research on miRNAs and OP. The research direction with the highest frequency was the miRNAs acting on osteoblasts and osteoclasts. The influence of miRNAs carried by exosomes on the differentiation of MSCs might become an effective method for OP cell-free treatment.

Trans effects of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models.

BACKGROUND: Trisomy 21 causes Down syndrome (DS), but the mechanisms by which the extra chromosome leads to deficient intellectual and immune function are not well understood. RESULTS: Here, we profile CpG methylation in DS and control cerebral and cerebellar cortex of adults and cerebrum of fetuses. We purify neuronal and non-neuronal nuclei and T lymphocytes and find biologically relevant genes with DS-specific methylation (DS-DM) in each of these cell types. Some genes show brain-specific DS-DM, while others show stronger DS-DM in T cells. Both 5-methyl-cytosine and 5-hydroxy-methyl-cytosine contribute to the DS-DM. Thirty percent of genes with DS-DM in adult brain cells also show DS-DM in fetal brains, indicating early onset of these epigenetic changes, and we find early maturation of methylation patterns in DS brain and lymphocytes. Some, but not all, of the DS-DM genes show differential expression. DS-DM preferentially affected CpGs in or near specific transcription factor binding sites (TFBSs), implicating a mechanism involving altered TFBS occupancy. Methyl-seq of brain DNA from mouse models with sub-chromosomal duplications mimicking DS reveals partial but significant overlaps with human DS-DM and shows that multiple chromosome 21 genes contribute to the downstream epigenetic effects. CONCLUSIONS: These data point to novel biological mechanisms in DS and have general implications for trans effects of chromosomal duplications and aneuploidies on epigenetic patterning.

Genome-wide divergence of DNA methylation marks in cerebral and cerebellar cortices.

BACKGROUND: Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function. METHODOLOGY/SIGNIFICANT FINDINGS: Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum. CONCLUSIONS: These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions.

Identification of coding single nucleotide polymorphisms and mutations by combination of genome tiling arrays and enrichment/depletion of mismatch cDNAs.

Genome tiling array technology combined with a method for both enrichment and depletion of mismatch-containing cDNA fragments offers a useful approach for detecting coding single nucleotide polymorphisms (cSNPs) and mutations in pooled cDNA samples. Enriched mismatch and perfect match cDNA samples from human primary melanoma cells and normal melanocytes were obtained by selection using mismatch repair thymine DNA glycosylase-bound beads. These cDNA samples were then labeled and hybridized to Encyclopedia of DNA Elements genome tiling arrays. The results revealed that the hybridization intensity values of potential cDNA variation regions of the enriched mismatch samples increased, whereas the hybridization intensity values of corresponding regions of the enriched perfect match samples decreased. Six potential mutations were confirmed by polymerase chain reaction product sequencing, including two novel heterozygous mutations in melanoma cells. We suggest that this strategy should increase the efficiency of both cSNP and mutation detection throughout the entire human genome and decrease the cost and complexity of genomewide analysis of cDNA variations.

Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression.

Induction of cyclin D1 gene transcription by estrogen receptor alpha (ERalpha) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERalpha has been mapped to an alternative response element, a cyclic AMP-response element at -57, with possible participation of an activating protein-1 site at -954. The action of ERbeta at the cyclin D1 promoter is unknown, although evidence suggests that ERbeta may inhibit the proliferative action of ERalpha. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERalpha, ERalphaK206A (a derivative that is superactive at alternative response elements), or ERbeta. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERbeta occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERalpha, inhibit expression with ERbeta. Strikingly, the presence of ERbeta completely inhibits cyclin D1 gene activation by estrogen and ERalpha or even by estrogen and the superactive ERalphaK206A. The observation of the opposing action and dominance of ERbeta over ERalpha in activation of cyclin D1 gene expression has implications for the postulated role of ERbeta as a modulator of the proliferative effects of estrogen.