Expression Of BMP-2 Research Articles

BMAL1 improves assisted reproductive technology outcomes in patients with polycystic ovary syndrome by targeting BMP6 and regulating ovarian granulosa cell apoptosis.

To investigate BMAL1 and BMP6 expressive differences in ovarian granulosa cells (OGCs) of patients with polycystic ovary syndrome (PCOS), explore regulatory relationship, assess their impacts on OGC proliferation and apoptosis, and analyze their correlations with ART outcomes of patients. A clinical study selected 40 PCOS patients who underwent IVF/ICSI in our hospital from January to October 2022 and 39 controls with male or tubal factor infertility. RT-qPCR and Western blot assessed BMAL1 and BMP6 mRNA/protein levels. The number of oocytes retrieved, 2PN fertilized oocytes, available embryos, and high-quality embryos were compared between groups and analyzed their correlations with BMAL1 and BMP6 expression levels. Cellular experiments were performed by overexpressing or knocking down BMAL1 in KGN cells by plasmid transfection. The dual-luciferase reporter assay was used to identify BMAL1/BMP6 regulatory relationship. CCK-8 and flow cytometry assessed cellular proliferation and apoptosis. BMAL1 mRNA/protein expression (P < 0.001) in the PCOS group was significantly lower than that in controls, as was the number of high-quality embryos (P = 0.001). Contrastingly, BMP6 (P < 0.001) was significantly higher in the PCOS group. BMAL1 expression levels were negatively correlated with BMP6 (r = - 0.684, P = 0.002) and positively correlated with the number of 2PN fertilized oocytes, available embryos, and high-quality embryos (r = 0.659, P = 0.003; r = 0.623, P = 0.006; and r = 0.738, P < 0.001). Cellular experiments showed that overexpression of BMAL1 significantly decreased relative luciferase activity (P < 0.01). Overexpression of BMAL1 significantly decreased KGN cell apoptosis (P < 0.01) and enhanced proliferation (P < 0.01). BMAL1 regulates OGCs proliferation and apoptosis by targeting BMP6, thereby influencing ART outcomes in patients with PCOS. This study might provide molecular factors that indicate ART outcomes and therapeutic targets for PCOS. Registration number: ChiCTR2100052331; registration date: 2021-10-24.

Bone Marrow Stromal Cells Generate a Pro-Healing Inflammasome When Cultured on Titanium-Aluminum-Vanadium Surfaces with Microscale/Nanoscale Structural Features.

The surface topography and chemistry of titanium-aluminum-vanadium (Ti6Al4V) implants play critical roles in the osteoblast differentiation of human bone marrow stromal cells (MSCs) and the creation of an osteogenic microenvironment. To assess the effects of a microscale/nanoscale (MN) topography, this study compared the effects of MN-modified, anodized, and smooth Ti6Al4V surfaces on MSC response, and for the first time, directly contrasted MN-induced osteoblast differentiation with culture on tissue culture polystyrene (TCPS) in osteogenic medium (OM). Surface characterization revealed distinct differences in microroughness, composition, and topography among the Ti6Al4V substrates. MSCs on MN surfaces exhibited enhanced osteoblastic differentiation, evidenced by increased expression of RUNX2, SP7, BGLAP, BMP2, and BMPR1A (fold increases: 3.2, 1.8, 1.4, 1.3, and 1.2). The MN surface also induced a pro-healing inflammasome with upregulation of anti-inflammatory mediators (170-200% increase) and downregulation of pro-inflammatory factors (40-82% reduction). Integrin expression shifted towards osteoblast-associated integrins on MN surfaces. RNA-seq analysis revealed distinct gene expression profiles between MSCs on MN surfaces and those in OM, with only 199 shared genes out of over 1000 differentially expressed genes. Pathway analysis showed that MN surfaces promoted bone formation, maturation, and remodeling through non-canonical Wnt signaling, while OM stimulated endochondral bone development and mineralization via canonical Wnt3a signaling. These findings highlight the importance of Ti6Al4V surface properties in directing MSC differentiation and indicate that MN-modified surfaces act via signaling pathways that differ from OM culture methods, more accurately mimicking peri-implant osteogenesis in vivo.

BMP6, a potential biomarker of inflammatory fibrosis and promising protective factor for dilated cardiomyopathy

BackgroundDilated cardiomyopathy (DCM) stands as one of the most prevalent and severe causes of heart failure. Inflammation plays a pivotal role throughout the progression of DCM to heart failure, while age acts as a natural predisposing factor for all cardiovascular diseases. These two factors often interact, contributing to cardiac fibrosis, which is both a common manifestation and a pathogenic driver of adverse remodeling in DCM-induced heart failure.MethodBulk RNA-seq, single-cell RNA-seq, Mendelian randomization analysis, animal model construction, and BMP6 knockdown were utilized to identify and validate potential specific markers and targets for intervention in DCM heart failure.ResultsWe found that DCM hearts exhibit pronounced inflammatory cell infiltration and fibrosis. Both bulk RNA-seq and single-cell RNA-seq analyses revealed aberrant BMP6 expression specifically in fibroblasts. The ROC curve underscores the high specificity of BMP6 in relation to DCM, while Mendelian randomization analysis further confirms BMP6 as a protective factor against DCM. Notably, BMP6 knockdown led to a decrease in SMAD6 expression and a marked elevation in COL1A1 expression levels, indicating its antifibrotic role.ConclusionBMP6 emerges as a promising biomarker for DCM, and its functional role in exerting an antifibrotic effect underscores its potential as a therapeutic target.

Implications of Mineralization Biomarkers in Breast Cancer Outcomes Beyond Calcifications.

The aim of this work was to explore the biomarkers associated with epithelial to mesenchymal transition (EMT) and mineralization processes as new prognostic factors across different breast cancer phenotypes. To this end, 133 breast biopsies, including benign and malignant lesions, with or without microcalcifications, were retrospectively collected. Immunohistochemical analysis was performed to evaluate the expression of vimentin, BMP-2, BMP-4, RANKL, Runx2, OPN, PTX3, and SDF-1, while Kaplan-Meier plots were used to assess their prognostic impact on overall survival in a dataset of 2976 breast cancer patients. The expression of vimentin, BMP-2, BMP-4, and SDF-1 was significantly higher in malignant lesions compared to benign ones, regardless of the presence of microcalcifications. Notably, these markers showed no correlation with traditional prognostic factors, such as tumor grade or hormone receptor status. The bioinformatics analysis provided valuable insights into the possible prognostic and therapeutic significance of BMP-2, BMP-4, SDF-1, and vimentin in breast cancer. In fact, all these biomarkers impact on the overall survival in specific molecular breast cancer types. In addition, high expression of SDF-1 and vimentin is able to predict the response to chemotherapy. The findings here reported suggest that vimentin, BMP-2, BMP-4, and SDF-1 could be independent prognostic biomarkers in breast cancer, providing insights beyond traditional clinical factors.

Hypothyroidism Alters Uterine Kisspeptin System and Activity Modulators in Cyclic Rats.

Hypothyroidism causes ovarian dysfunction and infertility in women and animals and impairs the hypothalamic expression of kisspeptin (Kp). However, kisspeptin is also expressed in the genital system, and the lack of the Kp receptor (Kiss1r) in the uterus is linked to reduced implantation rates. This study investigated the impact of hypothyroidism on the uterine expression of Kp and Kiss1r in female rats throughout the estrous cycle and the associated changes in uterine activity modulators. Hypothyroidism was induced through daily administration of propylthiouracil (PTU) over a period of 14 days. Plasma levels of LH, E2, and P4, cyclicity, body and uterine weight, uterine histomorphometry, and the gene and/or protein expression of Kiss1, Kiss1r, estrogen receptor α (ERα), progesterone receptor (PR), and thyroid hormone receptor α (TRα) were assessed. Additionally, proliferative activity (CDC-47) and the gene expression of uterine receptivity mediators (SMO, WNT4, BMP2, HAND2, MUC1, and LIF) were evaluated. Hypothyroidism prolonged the diestrus and increased progesterone levels during this phase, while decreasing luteinizing hormone and estradiol on proestrus. In the uterus, hypothyroidism reduced Kp immunostaining on diestrus and KISS1R mRNA levels on proestrus. These changes were accompanied by reduced endometrial glands, reduced uterine proliferative activity, and reduced ERα gene and protein expression. Additionally, hypothyroidism led to reduced uterine gene expression of LIF, BMP2, WNT4, and HAND2. On the other hand, thyroid hypofunction increased uterine PR and TRα immunostaining, while it reduced PGR gene expression on diestrus. These findings demonstrate that hypothyroidism reduces the expression of Kiss1/Kiss1r system in the uterus, which is associated with disrupted uterine estrogen and progesterone signaling and reduced expression of uterine receptivity mediators across the rat estrous cycle.

Small but mighty: nanoemulsion particle size dictates bone regeneration potential of FTY720.

The burgeoning field of nano-bone regeneration is yet to establish a definitive optimal particle size for nanocarriers. This study investigated the impacts of nanocarrier's particle size on the bone regeneration efficacy of fingolimod (FTY720)-loaded nanoemulsions. Two distinct particle sizes (60 and 190 nm, designated as NF60 and NF190, respectively) were produced using low-energy and high-energy emulsion techniques, maintaining a consistent surfactant, co-surfactant, and oil. In vitro studies using rat mesenchymal stem cells revealed that both NF60 and NF190 exhibited cell viability and reduced lactate dehydrogenase. Interestingly, NF60 demonstrated superior antioxidant properties, significantly reducing nitric oxide and intracellular reactive oxygen species (ROS) levels compared to NF190. Furthermore, NF60 significantly enhanced ALP activity and calcium deposition during osteogenic differentiation, indicating its potential to promote the early stages of bone formation. In vivo studies using a rat calvarial bone defect model demonstrated that both NF60 and NF190 significantly upregulated the expression of key osteogenic genes, including Runx2, Col, ALP, OCN, and BMP2. Notably, NF60 induced significantly higher expression of Runx2 and BMP2. X-ray and histological investigations revealed significantly improved bone regeneration in the NF60 group, highlighting the superior bone healing potential of smaller FTY720 nanoemulsions, without infiltration of inflammatory cells. The smaller particle size demonstrated superior antioxidant properties, enhanced osteogenic differentiation, and improved bone regeneration, suggesting smaller nanoparticles, with their larger surface area, accelerated drug release rate, and lower viscosity, interact more effectively with cells, leading to increased and effective drug delivery and cellular uptake. Findings highlight the importance of nanocarrier size in optimizing drug delivery for bone tissue engineering applications.

Discovery of potent antiosteoporotic cyclic depsipeptides with an unusual nitrile hydroxy acid from Microascus croci.

Two cyclic octadepsipeptides, microascusins A and B (1 and 2), were identified from the marine sponge-associated Microascus croci IMB19-064 co-cultivated with Escherichia coli. Their structures and conformations in solution were determined by comprehensive spectroscopic data analysis. The absolute configurations of amino and hydroxy acids were determined by the advanced Marfey's and O-Marfey's methods, respectively, as well as chiral-phase HPLC analysis. Microascusins A (1) and B (2) represent a new type of cyclic octadepsipeptides characterized by the presence of the unique hydroxy acid l-2-hydroxy-4-cyanobutyric acid. Compound 1 exhibited potent in vitro antiosteoporotic activity with dual regulation effects of promoting the osteoblast-mediated bone formation as well as inhibiting the RANKL-induced osteoclast differentiation at the concentrations of 0.1-10nM. Further in vivo study showed that compound 1 could improve bone mineralization after dexamethasone-induced bone damage in zebrafish through up-regulating the expression of the osteoblast-specific genes Bmp2 and Oc.

Therapeutic potential of targeting BMP/ALK pathway in pathological retinal neovascularization

Aims/Purpose: Pathological retinal neovascularization (PRNV) in ischemic retinopathies presents a significant clinical challenge. This study investigates the role of bone morphogenetic protein (BMP) signaling via ALK receptors in PRNV, with a focus on epigenetic modifications.Methods: The oxygen‐induced retinopathy (OIR) mouse model was utilized to investigate BMP signaling. Experimental groups included OIR mice treated with Noggin (a BMP inhibitor) or vehicle via ip from postnatal day 12‐16. Additional groups included a combined ALK2 and ALK3 endothelial conditional knockout mice (ALK2/3eKO), subjected to OIR. Capillary degeneration and neovascularization were assessed at postnatal day 17 in flat‐mount retinas using isolectin‐B4 staining. Immunostaining of retinal sections evaluated BMP2, BMP4, and ALK3 localization. In vitro experiments involved human retinal endothelial cells (HRECs) treated with BMP2 (50 ng/ml) or exposed to hypoxia (1% O2) for 48 hours. The impact of BMP2 on vascular endothelial growth factor (VEGF) was assessed through ELISA. MicroRNA (miRNA) expression was analyzed using the GeneChip miRNA 3.0 array. DNA methylation of HRECs was quantified using a colorimetric kit.Results: Immunostaining revealed an upregulation of BMP2, BMP4, and ALK3 expression in retinal vessels. Noggin‐treated and ALK2/3eKO OIR mice exhibited a significant reduction in capillary dropout and neovascularization (p &lt; 0.01). BMP2 treatment of HRECs elevated VEGF production. MiRNA array analysis indicated significant alterations by BMP2 (4%) and hypoxia (5%), including miRNAs linked to VEGF signaling and angiogenesis. Specifically, BMP2 downregulated miR‐4521, miR‐769‐3p, miR‐23b‐5p, miR‐3195, and miR‐6781‐5p, while upregulating miR‐126‐3p, miR‐921, miR‐7515, miR‐29c, and miR‐20b‐5p. Interestingly, BMP2 and hypoxia induced similar effects on certain miRNAs, such as miR‐769‐3p, miR‐23b, and miR‐20b. The DNA methylation assay demonstrated a statistically significant reduction induced by BMP2.Conclusions: BMP/ALK signaling plays a potential role in the pathogenesis of PRNV, possibly mediated through epigenetic modifications. Thereby, targeting BMP/ALK signaling holds a promise to mitigate PRNV in ischemic retinopathies

Bovine lactopontin promotes bone development in growing rats by altering the composition of intestinal flora and bile acid metabolism.

Lactopontin (LPN) is an important milk protein with the potential to improve bone health; however, its specific effects have not been determined. This study aims to investigate the effects of LPN on early bone growth and development. 3 week-old SD rats (n = 32) were assigned to the control group, whey protein concentration (WPC) group, LPN-L (low-dose LPN) group, and LPN-H (high-dose LPN) group, with intragastric administration of deionized water, 65.8 mg kg-1 WPC, and 5 and 45 mg kg-1 LPN for 30 days, respectively. LPN-H supplementation increased body length and femur length, improved femoral bone volume and microarchitecture, and upregulated osteoblast activity-related mRNA (Bmp2, Smad8, and Runx-2) expression in the femur. The content of secondary bile acid glycolithocholic acid (GLCA) in stool and serum was significantly increased after LPN-H intervention and positively correlated with the increased abundance of Parabacteroides in feces. In addition, the bile acid receptor FXR in femur was also activated in the LPN-H group, suggesting that LPN may regulate bone health through the microbiota-metabolite-bone axis. The results of this study suggest that LPN has potential application value in regulating bone metabolism during growth.

Investigation of the molecular mechanism of quercetin in inhibiting ankylosing spondylitis ossification via the bone morphogenetic protein/smad signaling pathway.

Ankylosing spondylitis (AS) is a chronic inflammatory disease involving the spine and bone joints, which is characterized by hyperosteogeny, ossification of ligaments, and ankylosis. Quercetin is a natural polyphenolic compound with various biological activities such as antioxidant, anti-inflammatory, and anti-tumor. It was to explore the effect of quercetin on AS ossification and its molecular mechanism. In vitro culture of AS mesenchymal stem cells was conducted. Cells were treated with 0, 10, 30, 60, and 80μM quercetin, divided into control, 10μM, 30μM, 60μM, and 80μM groups. Alkaline phosphatase (ALP) staining, Alizarin Red staining, real-time quantitative polymerase chain reaction (qRT-PCR), and Western blot (WB) were employed to investigate the effect of quercetin on the expression of osteogenic-related genes and proteins. Additionally, bone morphogenetic protein (BMP) and Smad genes were knocked out to explore quercetin's regulation of BMP/Smad. In vivo experiments were conducted using 50 mice, including 10 in the normal group. An AS model was established in 36 mice, divided into negative control (n = 18, 0.9% saline) and quercetin groups (n = 18, quercetin). Safranin O-fast green (HE) staining and MicroCT scanning were performed before and 4weeks after injection. In the 60μM and 80μM quercetin groups, ALP activity, Ca2+ deposition area, and relative protein/mRNA levels of BMP-1, BMP-2, Smad1, Smad4, and Smad5 in AS mesenchymal stem cells were significantly lower compared to the control, 10μM, and 30μM groups (P < 0.05). The 80μM group exhibited lower levels than the 60μM group (P < 0.05). In the siRNA + 80μM group, the reduction in mRNA expression of BMP1, BMP2, Smad1, Smad4, and Smad5 was significantly greater compared to the siRNA group and the 80μM group (P < 0.05). At 4weeks post-injection, mice in the quercetin group showed significantly reduced severity of articular cartilage lesions, lymphocyte infiltration, and tissue edema, with no significant increase in sacroiliac joint fusion. Quercetin downregulates the expression of BMP and Smad-related proteins, inhibiting osteogenic differentiation of AS mesenchymal stem cells and effectively reducing ALP activity and Ca2+ deposition levels. These findings suggest that quercetin holds potential application value in the control and treatment of AS disease.

Exploring Bone Morphogenetic Protein-2 and -4 mRNA Expression and Their Receptor Assessment in a Dynamic In Vitro Model of Vascular Calcification.

Vascular calcification (VC) is a dynamic, tightly regulated process driven by cellular activity and resembling the mechanisms of bone formation, with specific molecules playing pivotal roles in its progression. We aimed to investigate the involvement of the bone morphogenic proteins (BMP-2, BMP-4, BMPR-1a/1b, and BMPR-2) system in this process. Our study used an advanced in vitro model that simulates the biological environment of the vascular wall, assessing the ability of a phosphate mixture to induce the osteoblastic switch in human coronary artery smooth muscle cells (HCASMCs). HCASMCs were grown in mono- and co-culture with human coronary artery endothelial cells (HCAECs) in a double-flow bioreactor (LiveBox2 and IVTech), allowing static and dynamic conditions through a peristaltic pump. The VC was stimulated by incubation in a calcifying medium for 7 days. A BMP system Real-Time PCR was performed at the end of each experiment. In monocultures, BMP-2 expression increased in calcified HCASMCs in static (p = 0.01) and dynamic conditions. BMP-4 and the biological receptors were expressed in all the experimental settings, increasing mainly in dynamic flow conditions. In co-cultures, we observed a marked increase in BMP-2 and BMP-4, BMPR-1a (p = 0.04 and p = 0.01, respectively), and BMPR-2 (p = 0.001) in the calcifying setting mostly in dynamic conditions. The increase in BMP-2/4 in co-culture suggests that these genes might promote the switch towards an osteogenic-like phenotype, data also supported by the rise of both BMPR-1a and BMPR-2. Thus, our findings provide insights into the mechanisms by which dynamic co-culture modulates the BMP system activation in an environment mimicking in vivo VC's cellular and mechanical characteristics.

Equol promotes osteogenic differentiation of hPDLSCs by inhibiting oxidative stress via IL1B/NF-κB/CXCL1 signaling axis.

Oxidative stress (OS) inhibits the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Equol (Eq), a phytoestrogen, exhibits notable antioxidant properties and potential for preventing osteoporosis. However, the research on the regulatory effects of Eq on stem cell osteogenesis remains limited. This investigation aimed to identify whether Eq could protect the osteogenic potential of hPDLSCs under H2O2-induced oxidative microenvironment. We employed a series of assays, including CCK-8, DCFH-DA, ALP staining, ARS, RT-qPCR, and Western Blotting, to assess the changes in cell viability, antioxidant capacity, and osteogenic potential following H2O2 and Eq treatments. Our findings indicated that low concentrations of Eq had no cytotoxic effects on hPDLSCs and promoted their proliferation. Eq pre-treatment (0.5μmol/L) partially counteracted the inhibitory effect of H2O2, reduced the generation of reactive oxygen species, and increased glutathione levels, thereby inhibiting oxidative damage. Eq suppressed the H2O2-induced inhibition of osteogenic differentiation, presenting as restoring the alkaline phosphatase levels and calcium nodule formation, as well as by upregulating the expression of BMP2 and RUNX2. Furthermore, bioinformatics analysis in this study suggested that the IL1B/NF-κB/CXCL1 signaling pathway might be a key pathway for Eq's enhancement of osteogenic differentiation potential of hPDLSCs under OS conditions. The activation of this axis by H2O2, which Eq can alleviate, was confirmed by validation experiments. This study provides new insights into the potential therapeutic application of Eq in alveolar bone resorption and bone regeneration research.

Discovery of potential ferroptosis and osteoporosis biomarkers through TMT proteomics and bioinformatics analysis

BackgroundPrimary osteoporosis has increasingly emerged as a major issue affecting human health, with a complex specific pathogenic mechanism. As a research hotspot, ferroptosis plays a vital role in the pathogenesis of primary osteoporosis, aiming to explore the link and specific target genes between ferroptosis and primary osteoporosis.MethodsBy utilizing TMT proteomics and bioinformatics analyses, we elucidated the linkages and key targets of the ferroptosis pathway in an ovariectomized osteoporotic rat model. Forty 12-week-old SD female rats were employed in the study, of which 20 female SD rats were ovariectomized as the OVX group and 20 female SD rats were employed as the SHAM group. At the end of the experiments, the femurs of the rats were excised for computed tomography tests and used for hematoxylin and eosin staining. Finally, we extracted bone tissue proteins for TMT proteomics analysis and protein blotting verification.ResultsThe proteomics results of the VX and SHAM groups showed that 133 proteins were significantly changed, of which 91 proteins were upregulated and 42 proteins were downregulated, including TXN, TMSB4X, TFRC, TF, RELA, PARP14, CP, CAPG, and ADIPOQ. The expression of key proteins in the bone tissues was detected by protein blotting. The expression of TFR1, TFRC and TF was upregulated, whereas the expression of Cp, TXN and BMP-2 was downregulated.ConclusionsTMT proteomics and functional enrichment analyses in our study substantiated that in osteoporosis, disturbances in lipid metabolism lead to the emergence of oxidative stress with iron homeostasis imbalance.

Prenatal glucocorticoid exposure and congenital abdominal wall defects: Involvement of CXCR4 – SDF-1 signaling

Developmental defects of the ventral abdominal wall, such as gastroschisis, have been associated with prenatal stress exposure. To investigate this further, dexamethasone (DEX), a synthetic glucocorticoid, was administered to fertilized chicken eggs on day 1 of incubation to simulate stress, and embryonic development was subsequently analyzed through in-situ hybridization, immunohistochemistry, and histological methods. Significant developmental abnormalities were displayed by DEX-treated embryos, including open abdomens, reduced MYOG expression in the abdominal wall, and disrupted muscle fiber formation, as indicated by altered Myosin heavy chain patterns. Additionally, early markers of muscle development, such as Pax3, and the CXCR4-SDF-1 signaling axis, crucial for the migration of myogenic precursors of the dermomyotome, were markedly affected. Significant alterations in the expression of mesenchymal markers, including Vimentin and Fibronectin in the lateral plate mesoderm, were observed, alongside alterations in Pitx2, BMP4 and TFAP2A expression. Importantly, a downregulation of Glucocorticoid Receptors was identified, emphasizing the chronic stress exposure. These results provide critical insights into how DEX interferes with key developmental pathways, particularly those involving chemokines like CXCR4 and SDF-1, and other markers of mesodermal differentiation. An advancement in the understanding of the mechanisms underlying ventral abdominal wall defects in the context of prenatal stress is provided by this research, with potential implications for preventing these congenital anomalies.

Peripheral nerve-derived CSF1 induces BMP2 expression in macrophages to promote nerve regeneration and wound healing

The precise mechanisms regulating inflammatory and prorepair macrophages have not been fully elucidated, despite the pivotal role played by innate immunity in wound healing. We first employed a denervation wound model to validate the crosstalk between neurons and macrophages. Compared to normal wound healing, the denervation wound healing process involved fewer macrophages, decreased angiogenesis, and delayed wound healing. Consistent with the results of the scRNA-seq libraries, the number of early-phase wound proinflammatory and late-phase wound prorepair macrophages were decreased during the denervation wound healing process. We profiled early-phase and late-phase skin wounds in mice at the transcriptional and functional levels and compared them to those of normal wounds. We revealed a neuroimmune regulatory pathway driven by peripheral nerve-derived CSF1 that induces BMP2 expression in prorepair macrophages and enhances nerve regeneration. Crosstalk between neurons and macrophages facilitates the healing process of wounds and provides a potential strategy for wound healing therapy.

Bioactive Porous Composite Implant Guides Mesenchymal Stem Cell Differentiation and Migration to Accelerate Bone Reconstruction.

Delayed healing and non-healing of bone defects pose significant challenges in clinical practice, with metal materials increasingly recognized for their significance in addressing these issues. Among these materials, Strontium (Sr) and Zinc (Zn) have emerged as promising agents for promoting bone repair. Building upon this insight, this research evaluates the impact of a porous Sr@Zn@SiO2 nanocomposite implant on bone regeneration, aiming to advance the field of bone repair. The preparation of the Sr@Zn@SiO2 composite implant involves various techniques such as roasting, centrifugation, and washing. The material's composition is examined, and its microstructure and element distribution are analyzed using TEM and elemental scanning technology. In vitro experiments entail the isolation and characterization of BMSCs followed by safety assessments of the implant material, evaluation of cell migration capabilities, and relevant proliferation markers. Mechanistically, this study delves into key targets associated with significant changes in the osteogenic process. In vivo experiments involve establishing a rat femur bone defect model, followed by assessment of the osteogenic potential of Sr@Zn@SiO2 using Micro-CT imaging and tissue section staining. Through in vivo and in vitro investigations, we validate the osteogenic efficacy of the Sr@Zn@SiO2 composite implant. In vitro analyses demonstrate that porous Sr@Zn@SiO2 nanocomposite materials upregulate BMP-2 expression, leading to the activation of Smad1/5/9 phosphorylation and subsequent activation of downstream osteogenic genes, culminating in BMSCs osteogenic differentiation and bone proliferation. And the migration of BMSCs is closely related to the high expression of CXCL12/CXCR4, which will also provide the conditions for osteogenesis. In vivo, the osteogenic ability of Sr@Zn@SiO2 was also confirmed in rats. In our research, the porous Sr@Zn@SiO2 composite implant displays prominent osteogenic effect and promotes the migration and differentiation of BMSCs to promote bone defect healing. This bioactive implant has surgical application potential in the future.

Calcific aortic valve disease-associated long noncoding RNA H19 promotes the osteogenic transition of valvular interstitial cells via the JAG1/NOTCH1 axis

Abstract Background Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease causing tremendous suffering globally. The osteogenic differentiation of the valvular interstitial cells (VICs) is a key process in the pathogenesis of CAVD leading to calcification and stiffening. Here, we focus on the contribution of long noncoding RNA H19 in the osteogenic differentiation of VICs promoting CAVD. Methods and results A human lncRNA array performed in AVS patients with coronary artery disease (CAD) revealed the differential expression of lncRNA H19. For the experiments in this study, loss of function (LOF) was performed in primary VICs isolated from the aortic valves of patients with CAVD, and commercially-available VICs. Therefore, H19 expression was effectively downregulated via RNAi. Subsequent gene expression analysis via RT-qPCR revealed that the expression of calcification markers RUNX2 and BMP2, as well as the expression of the H19 downstream targets JAG1 and NOTCH1 were unchanged after the H19 knockdown alone. Then, the VICs were additionally treated with osteogenic medium (OM) and procalcifying medium (PCM) for 7 d to induce the calcification of the cells. In the OM, inorganic phosphate is generated in an alkaline phosphatase (ALPL)-dependent manner and integrated into newly formed hydroxyapatite crystals, thus promoting cellular calcification. PCM contains inorganic phosphate to form hydroxyapatite crystals and is therefore not ALPL-dependent. Gene expression analysis via RT-qPCR and protein analysis via immunofluorescence revealed that the calcification markers BMP2 and RUNX2 were upregulated after H19 knockdown and calcification induction. This indicates that H19 regulates the expression of calcification makers promoting cellular calcification. Furthermore, RT-qPCR and immunoblotting revealed the differential expression of several potential H19 downstream targets, namely JAG1, NOTCH1, and STAT3. The analysis showed that the potential downstream targets were upregulated after H19 knockdown and calcification treatment hinting at the involvement of the NOTCH1 or STAT3 pathway in the pathogenesis of CAVD. Moreover, an Alizarin Red S staining of calcified nodules revealed reduced calcification after H19 knockdown and calcification treatment for 21 d. This demonstrates that H19 also regulates a late-stage event in the cellular calcification of VICs. Conclusion Our study showed that H19 regulates calcification marker expression and the expression of potential downstream targets JAG1, NOTCH1, and STAT3 both in healthy and AVS patient VICs. This hints at the involvement of the H19/JAG1/NOTCH1 pathway in the calcification of VICs, which might play a critical role in the pathogenesis of CAVD.

CircCSPP1 Competitively Binds miR-10a to Regulate BMP7 Expression and Affects the Proliferation of Dermal Papilla Cells.

A series of differentially expressed circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were identified through sequencing in the hair follicle tissues of Hu sheep with small-waved and straight wool patterns. Based on these findings, the circCSPP1-miR-10a-BMP7 (Bone Morphogenetic Protein 7) regulatory network was constructed. The preliminary study highlighted that miR-10a and the BMP7 gene exhibited not only significant differential expression across hair follicle tissues with different patterns in Hu sheep but also had an impact on the proliferation of hair papilla cells. The proliferation of hair papilla cells is intricately linked to hair follicle development and growth. Consequently, we selected the circCSPP1-miR-10a-BMP7 regulatory network to validate its role in promoting hair papilla cell proliferation in Hu sheep. Firstly, the authenticity of circCSPP1 was successfully confirmed through RNase R digestion and reverse primer amplification. Additionally, nucleoplasmic localization analysis determined that circCSPP1 was predominantly distributed in the cytoplasm. Using the dual-luciferase gene reporter system, we verified the targeting relationship between circCSPP1 and miR-10a, building upon our previous validation of the miR-10a-BMP7 interaction. This clarified the competing endogenous RNA (ceRNA) mechanism within the circCSPP1-miR-10a-BMP7. Furthermore, rescue experiments confirmed that circCSPP1 competitively binds to miR-10a, thereby regulating BMP7 expression and influencing the proliferation of hair papilla cells in Hu sheep. This discovery provides a solid foundation for future investigations into the mechanisms underlying wool curvature and the formation of lambskin patterns, offering insights into the complex regulatory networks that govern these phenotypic traits in Hu sheep.

Enhancing Bone Formation Through bFGF-Loaded Mesenchymal Stromal Cell Spheroids During Fracture Healing in Mice.

This study aimed to evaluate the osteogenic potential of mesenchymal stromal cell (MSC) spheroids combined with the basic fibroblast growth factor (bFGF) in a mouse femur fracture model. To begin, MSC spheroids were generated, and the expression of key trophic factors (bFGF Bmp2, and Vegfa) was assessed using quantitative PCR (qPCR). A binding assay confirmed the interaction between the bFGF and the spheroids' extracellular matrix. The spheroid cultures significantly upregulated bFGF, Bmp2, and Vegfa expression compared to the monolayers (p < 0.001), and the binding assay demonstrated effective bFGF binding to the MSC spheroids. Following these in vitro assessments, the mice were divided into five groups for the in vivo study: (1) no treatment (control), (2) spheroids alone, (3) bFGF alone, (4) bFGF-loaded spheroids (bFGF-spheroids), and (5) non-viable (frozen) bFGF-loaded spheroids (bFGF-dSpheroids). Bone formation was analyzed by a micro-CT, measuring the bone volume (BV) and bone mineral content (BMC) of the mice four weeks post-fracture. A high dose of the bFGF (10 µg) significantly promoted bone formation regardless of the presence of spheroids, as evidenced by the increases in BV (bFGF, p = 0.010; bFGF-spheroids, p = 0.006; bFGF-dSpheroids, p = 0.032) and BMC (bFGF, p = 0.023; bFGF-spheroids, p = 0.004; bFGF-dSpheroids, p = 0.014), compared to the controls. In contrast, a low dose of the bFGF (1 µg) combined with the MSC spheroids significantly increased BV and BMC compared to the control (BV, p = 0.012; BMC, p = 0.015), bFGF alone (BV, p = 0.012; BMC, p = 0.008), and spheroid (BV, p < 0.001; BMC, p < 0.001) groups. A low dose of the bFGF alone did not significantly promote bone formation (p > 0.05). The non-viable (frozen) spheroids loaded with a low dose of the bFGF resulted in a higher BV and BMC compared to the spheroids alone (BV, p = 0.003; BMC, p = 0.017), though the effect was less pronounced than in the viable spheroids. These findings demonstrate the synergistic effect of the bFGF and MSC spheroids on bone regeneration. The increased expression of the BMP-2 and VEGF observed in the initial experiments, coupled with the enhanced bone formation in vivo, highlight the therapeutic potential of this combination. Future studies will aim to elucidate the underlying molecular mechanisms and assess the long-term outcomes for bone repair strategies.

Voluntary exercise in mice triggers an anti-osteogenic and pro-tenogenic response in the ankle joint without affecting long bones

Biomechanical stimulation is proposed to occupy a central place in joint homeostasis, but the precise contribution of exercise remains elusive. We aimed to characterize in vivo the impact of mechanical stimulation on the cell-controlled regulation of ossification within the ankles of healthy mice undergoing mild physical activity. DBA/1 male mice were subjected to voluntary running exercise for two weeks, and compared to mice housed in standard conditions (n = 20 per group). Free access to activity wheels resulted in a running exercise of 5.5 ± 0.8 km/day at 14.5 ± 0.5 m/min. Serum levels of alkaline phosphatase, IL-6, IL-8/Kc, IL-17a, and TNF-α were measured. No change in systemic inflammation was detected. The bone architecture of the femur and the calcaneus was unchanged, as revealed by μCT and histology of the enthesis of the Achilles tendon. mRNAs were extracted from femurs, tibias, and ankle joints before RT-qPCR analysis. The expression of the mechanosensitive genes Sclerostin (Sost) and Periostin (Postn) was not impacted by the exercise in long bones. Oppositely, Sost and Postn levels were modulated by exercise in joints, and osteogenic markers (Col10a1, Runx2, Osx, and Dmp1) were downregulated in the exercise group. In addition, the tenogenic markers Scx, Mkx, and Tnmd were upregulated by exercise. Thus, voluntary exercise affected the phenotype of joint cells without impacting long bones. As gene expression of Bmp2, Bmp4, and Id1 was also reduced in these cells, an off-regulation of BMP signaling could be partly responsible for their mechanosensitive response. Running exercise seemed to preserve the tendon from its progressive ossification, as seen in numerous enthesopathies. This study paves the way to future experiments for investigating the effects of mechanical stimulation in various mouse models.