BMP-2 Levels Research Articles

Electrical Stimulation Therapy and HA/TCP Composite Scaffolds Modulate the Wnt Pathways in Bone Regeneration of Critical-Sized Defects.

Critical bone defects are the most difficult challenges in the area of tissue repair. Polycaprolactone (PCL) scaffolds, associated with hydroxyapatite (HA) and tricalcium phosphate (TCP), are reported to have an enhanced bioactivity. Moreover, the use of electrical stimulation (ES) has overcome the lack of bioelectricity at the bone defect site and compensated the endogenous electrical signals. Such treatments could modulate cells and tissue signaling pathways. However, there is no study investigating the effects of ES and bioceramic composite scaffolds on bone tissue formation, particularly in the view of cell signaling pathway. This study aims to investigate the application of HA/TCP composite scaffolds and ES and their effects on the Wingless-related integration site (Wnt) pathway in critical bone repair. Critical bone defects (25 mm2) were performed in rats, which were divided into four groups: PCL, PCL + ES, HA/TCP and HA/TCP + ES. The scaffolds were grafted at the defect site and applied with the ES application twice a week using 10 µA of current for 5 min. Bone samples were collected for histomorphometry, immunohistochemistry and molecular analysis. At the Wnt canonical pathway, HA/TCP and HA/TCP + ES groups showed higher Wnt1 and β-catenin gene expression levels, especially HA/TCP. Moreover, HA/TCP + ES presented higher Runx2, Osterix and Bmp-2 levels. At the Wnt non-canonical pathway, HA/TCP group showed higher voltage-gated calcium channel (Vgcc), calmodulin-dependent protein kinase II, and Wnt5a genes expression, while HA/TCP + ES presented higher protein expression of VGCC and calmodulin (CaM) at the same period. The decrease in sclerostin and osteopontin genes expressions and the lower bone sialoprotein II in the HA/TCP + ES group may be related to the early bone remodeling. This study shows that the use of ES modulated the Wnt pathways and accelerated the osteogenesis with improved tissue maturation.

INFLUENCE OF CHOLESTEROL ENRICHED DIET ON GENES EXPRESSION ENCODING BONE MORPHOGENETIC PROTEIN-2 AND OSTEOCALCIN IN MOUSE MANDIBLE.

The aim: To evaluate the mRNA expression of the key regulators of osteogenesis - osteocalcin and BMP-2 in the mouse embryos mandible (17th day of pregnancy) which were borne by females on high-cholesterol diet for 30 days before fertilization and throughout pregnancy. Materials and methods: Experimental hypercholesterolemia (2%) was simulated by adding Cholesterol to the diet for 60 days. In experiment were used 40 mature female white mice that were randomly divided to control and experimental groups. The control group were fed with standard chow diet, the experimental group with diet with cholesterol enriched diet (with addition of 2 grams of Cholesterol per 100 grams of standard chow). The mandibles of mouse embryos (E-17) were examined by using molecular genetic methods. Results: In control group the relative level of BMP-2 mRNA / actin mRNA was 27.0«2.82, the relative level of and osteocalcin mRNA / actin mRNA was 30.5«6,28. In the jaws of animals in the experimental group with cholesterol enriched diet, the expression relative level of BMP-2 was 30.9«5.81 that is by 14,4% higher than in control group. Therefore, the expression level of оsteocalcin, on the contrary, decreased by 22.3% and was 23.7+5.31. Conclusions: Our study report influence of the cholesterol enriched diet (2%) on mRNA expression of BMP-2 and osteocalcin encoding genes. The embryos from mouse on cholesterol enriched diet (2%) had increased level of BMP-2 gene expression, however significantly decreased level of osteocalcin gene expression.

Infiltrating circulating monocytes provide an important source of BMP4 at the early stage of spinal cord injury.

Bone morphogenetic protein (BMP)4 plays a critical role in regulating neuronal and glial activity in the course of spinal cord injury (SCI). The underlying cause and cellular source of BMP4 accumulation at the injured spinal cord remain unclear. Here, we observed that plasma BMP4 levels are statistically higher in SCI patients than in healthy donors. When comparing rats in the sham group (T9 laminectomy without SCI) with rats in the SCI group, we found a persistent decline in BBB scores, together with necrosis and mononuclear cell accumulation at the contusion site. Moreover, during 2 weeks after SCI both plasma and cerebrospinal fluid levels of BMP4 displayed notable elevation, and a positive correlation. Importantly, percentages of circulating BMP4-positive (BMP4+) monocytes and infiltrating MDMs were higher in the SCI group than in the sham group. Finally, in the SCI+clodronate liposome group, depletion of monocytes effectively attenuated the accumulation of both BMP4+ MDMs and BMP4 in the injured spinal cord. Our results indicated that, following SCI, infiltrating MDMs provide an important source of BMP4 in the injured spinal cord and, therefore, might serve as a potential therapeutic target.

Serum Levels of Inflammatory and Fibrotic Cytokines in Patients with Carpal Tunnel Syndrome and Hip Osteoarthritis.

A certain percentage of carpal tunnel syndrome (CTS) is associated with inflammatory conditions. Osteoarthritis (OA) increases the risk of CTS, and both diseases are common in the general population. Moreover, OA and CTS are often present in the same patients. Since inflammation and fibrosis are found in both conditions, the question is whether circulating inflammatory cytokines and cytokines involved in fibrosis in OA and CTS patients could serve as indicators of coexisting CTS and OA pathology. This investigation was performed on 31 CTS patients, 29 hip OA patients, and 15 healthy volunteers. Blood samples were collected, and serum levels of TGF-β1, BMP-7, IL-1β, and TNFα were measured using the ELISA method. The statistical analysis was performed to reveal the most significant differences in the serum levels of these cytokines. Statistical significance was set at p-values ≤ 0.05. The serum level of TGF-β1 was the highest in CTS patients (16.36 pg/mL) and significantly different compared to OA and healthy control. Analysis of the cytokine serum level in the subdivided group revealed that serum levels of TGF-β1 and BMP-7 were significantly higher in CTS+/OA+ patients as well as BMP-7 in the OA+/CTS+ group. There was no significant difference in serum levels of the inflammatory cytokines TNFα and IL-1β among all groups. This study showed that in the end stage of CTS and OA, serum levels of inflammatory cytokines (IL1-β and TNFα) were not altered, while the serum levels of TGF-β1 and BMP-7 were significantly higher, especially in patients with coexisting OA and CTS. These findings suggest the possible values of TGF-β1 and BMP-7 as a predictive factor for the comorbidity of CTS and OA.

Scleral Remolding-Related Gene Expression After Scleral Collagen Cross-Linking Using Ultraviolet A and Riboflavin in Myopic Guinea Pig Model

Purpose This study was conducted to evaluate scleral remolding-related gene expression after scleral collagen cross-linking (SCXL) using ultraviolet A (UVA) and riboflavin in lens-induced myopia (LIM) guinea pigs. Methods A total of 100 4-week-old pigmented guinea pigs were randomly divided into five groups (n = 20): SCXL + LIM, LIM, SCXL, Sham, and Control. Refraction, anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were measured using streak retinoscope and A-scan ultrasonography. SCXL was performed using 0.1% riboflavin solution and 365 nm UVA irradiation. Lens-induced myopia was achieved by wearing −10 D concave lenses. Quantitative real-time PCR (qPCR) and western blot were used to measure mRNA and protein levels, respectively. Results Myopia was successfully induced in the LIM group, while myopic refraction was higher and ACD and AL were shorter in SCXL + LIM compared with LIM, suppressing myopia progression. The scleral COL1A1 mRNA levels were significantly decreased and MMP2 and ACTA2 mRNA levels were significantly increased in LIM compared with other groups, while COL1A1 mRNA levels were increased and MMP2 and ACTA2 mRNA levels were decreased in SCXL + LIM compared with LIM. The scleral COL1A1 protein levels were significantly increased at 1 week and 4 weeks and MMP2 protein levels were significantly decreased at 1 week in SCXL compared with SCXL + LIM, LIM and Control. MMP2 protein levels were significantly decreased in SCXL + LIM and SCXL compared with LIM at 4 weeks. The differences in TGFB1, BMP2, CCN2, ITGA2, and ITGB1 mRNA levels and ACTA2 protein levels between the five groups were not significantly different. Conclusion SCXL using UVA and riboflavin could influence the expression of scleral remolding-related genes, including COL1A1, MMP2, TIMP2, and ACTA2, and thus contribute to improving collagen synthesis and reducing collagen degradation and might have an effect on slowing myopia progression.

CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis.

Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease, and so far, there is no specific targeted therapy. Here, we report that CXCL16 is upregulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis and, at the same time, to transcriptionally modulate the levels of BMP4 and hepatocyte growth factor (HGF) in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE and suggest that CXCL16 signaling could be a potential therapeutic target for RE. © 2022 The Pathological Society of Great Britain and Ireland.

Barbaloin Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells: Involvement of Wnt/β-catenin Signaling Pathway.

Barbaloin, found in Aloe vera, exerts broad pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. This study aims to investigate the effects of barbaloin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Osteogenic induction of hBMSCs was performed in the presence or absence of barbaloin. Cell viability was determined by using the CCK-8 assay. The characteristic of hBMSCs was identified by using flow cytometry. Intracellular alkaline phosphatase (ALP) staining was performed to evaluate the ALP activity in hBMSCs. Alizarin Red S staining was performed to evaluate the matrix mineralization. The mRNA and protein levels of target genes were determined using qRT-PCR and western blotting, respectively. Treatment with barbaloin (10 and 20 μg/mL) significantly increased cell viability of hBMSCs after 72 hours. In addition, treatment with barbaloin increased the mRNA expression levels of ALP and its activities. Treatment with barbaloin also increased matrix mineralization and the mRNA and protein levels of late-differentiated osteoblast marker genes BMP2, RUNX2, and SP7 in hBMSCs. The underlying mechanisms revealed that barbaloin increased the protein expressions of Wnt/β-catenin pathway-related biomarkers. Barbaloin promotes osteogenic differentiation of hBMSCs by the regulation of the Wnt/β-catenin signaling pathway.

BMP/Smad Pathway Is Involved in Lithium Carbonate-Induced Neural-Tube Defects in Mice and Neural Stem Cells

Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li2CO3 treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li2CO3 at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li2CO3 or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li2CO3-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li2CO3 exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li2CO3 exposure via inositol deficiency.

High Fatality CBFA2T3-GLIS2 Pediatric Acute Megakaryoblastic Leukemia Is Sensitive to BCL-XL Inhibition

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of pediatric acute myeloid leukemia (AML) with dismal survival prognosis in children below 3 years of age (<40% of cure rates). In two thirds of cases, recurrent and mutually exclusive oncogenic fusions are detected, such as CBFA2T3-GLIS2 (CG2) or NUP98 rearrangements (e.g. NUP98-KDM5A), which are considered the transformative event in this disease. The development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material, which is among other things attributed to myelofibrosis and the rarity of primary patients. Therefore, our research group has engineered synthetic human models of high-fatality pediatric AMKL with different oncogenic fusions that faithfully phenocopy the disease in a patho-physiological context. In recent years, inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, entered the therapeutic arena in adult AML and pediatric leukemia with promising results. The molecules emphasized on are Venetoclax (a specific inhibitor of BCL2) and Navitoclax (broader affinity to BCL2, BCL-XL and BCL-W). Even though we see promising results in other leukemia, these agents were so far not investigated as potential therapies in pediatric high-fatality AMKL. To first overcome the paucity of patient material for research, we generated synthetic models of high-risk AMKL by transducing cord-blood derived hematopoietic stem and progenitor cells (HSPC) with lentiviral particles carrying the CBFA2T3-GLIS2 oncogenic fusion, followed by xenotransplantation into NSG mice. This approach generated leukemia with a latency between 9.9 and 36.1 weeks in primary mice which are able to sustain serial rounds of transplantation in recipient mice (up to 5 rounds tested). Furthermore, leukemic blasts immunophenotypically recapitulate AMKL, demonstrating expression of megakaryopoietic markers such as CD41, CD61 and CD56 that are even maintained after in vitro culture of 6 days and subsequent transplantation. Furthermore, we derived a CG2 specific gene expression signature from transcriptomic profiling of our models as well as patient samples that showed high expression of NCAM1, BMP2, ERG and low GATA1 levels, as reported for CG2 leukemia. Remarkedly, one of the specific up regulated genes in our signature for CG2 was pro-survival factor BCL2. In addition, our models are located in the megakaryocytic-erythroid differentiation space, as assessed by single cell RNAseq, and similar to their healthy counterparts of the megakaryopoietic lineage they demonstrated high expression of BCL-XL on RNA and protein level, in contrast to AML samples. Based on the aberrant expression of BCL2 in our synthetic leukemia models and lineage related expression of BCL-XL, we set out to test Venetoclax and Navitoclax in the context of AMKL. We identified Navitoclax (inhibitor of BCL2, BCL-XL and BCL-W) as a therapeutic vulnerability in our models of AMKL (CG2 and NUP98 rearranged), whereas our cells demonstrated resistance to treatment with Venetoclax. We further investigated the molecular mechanism and showed that shRNA mediated knock-down or inhibition of BCL-XL, by either Navitoclax or the BCL-XL specific proteasomal degrader DT2216, results in significant induction of apoptosis in our models of AMKL. On the contrary, genetic or pharmacological inhibition of BCL2 did not induce apoptosis in our synthetic models of AMKL, even at doses as high as 10µM. Furthermore, a genotype matched leukemic model of NUP98-KDM5A, which was presenting as monocytic (CD68+LYZ+) AML instead of AMKL, was resistant to either treatment with Venetoclax or Navitoclax whereas ML2, a MLL rearranged AML cell line, showed increased sensitivity to both Venetoclax and Navitoclax treatment. Moreover, in vivo treatment of a xenografted CG2 AMKL model with Navitoclax reduced leukemic burden in mice in bone-marrow and spleen in comparison to vehicle treated controls. In conclusion, we generated a model of rare CG2 pediatric high-fatality leukemia that faithfully mimics the patient situation and allows biomass generation for large scale multi-omic approaches. Furthermore, we demonstrate a lineage- and genotype specific targetable dependency of pediatric AMKL towards inhibition of BCL-XL but not BCL2, that could be transferred to the clinic as a novel therapeutic option in high-risk infant leukemia.

Biological, Functional and Genetic Characterization of Bone Marrow Mesenchymal Stromal Cells from Adult Ph+ Acute Lymphoblastic Leukemia

Objectives: To clarify the biological characteristics of bone marrow mesenchymal stromal cells (MSCs) in Ph+ acute lymphoblastic leukemia (Ph+ ALL) patients in different disease states, and compare protective effects of MSCs in different disease states on leukemia cells and explore the possible mechanisms. Methods: Bone marrow specimens from Ph+ ALL patients in various disease status (newly diagnosis, complete remission, relapse/ refractory) and healthy donors were collected. Bone marrow mononuclear cells (BMNCs) were isolated and cultured for primary MSCs. (1) CCK-8 method was used to compare proliferation ability of MSCs in vitro. (2) IC50 concentrations of dasatinib (Dasa), imatinib (Ima) and dexamethasone (Dex) in SUP-B15 were determined. SUP-B15 were co-cultured with HS-5 (a human stromal cell line) and MSCs respectively, then the apoptosis ratio of SUP-B15 cells after drug treatment was detected. (3) Transcriptome sequence (RNA-seq) of MSCs was performed, and differentially-expressed genes (DEGs) and signal pathway enrichment analysis among MSCs and BMNCs groups were performed. (5) qRT-PCR was used to detect the expression levels of candidate genes in MSCs. Results: (1) The proliferation ability of DR-MSCs in vitro was stronger than ALL-MSCs (P<0.01, Figure 1A). Proliferation ability of MSCs from refractory and relapsed group (RR-MSCs) were stronger than that from complete remission group (CR-MSCs) and newly diagnosed group (ND-MSCs)(P<0.05, P<0.05, Figure 1A). There was no significant difference between newly diagnosed-complete remission (ND-CR) paired MSCs (P>0.05). (2) The ratios of apoptotic SUP-B15 cells were significant decreased when co-cultured with HS-5 with and without the presence of Dasa, Ima, and Dex (P＜0.05, P＜0.0001, P＜0.001, P＜0.01, Figure 1B). (3) Ratios of apoptotic SUP-B15 cells were significantly reduced when co-cultured with different MSCs under the treatment of blank control, Dasa, Ima, and Dex. The abilities to prevent SUP-B15 cell apoptosis of DR-MSCs and ND-MSCs was stronger than that of CR-MSCs (P<0.001, P<0.05, P<0.01, P<0.0001, P<0.05, P<0.05, P<0.05, P<0.001) (Figure 1B). (4) The RNA-seq results showed that the gene expression thermogram among DR-MSCs, ND-MCSs and CR-MCSs were quite different (Figure 1C-D). Expression of BMP4 in ND-MSCs was significantly up-regulated (FC=3.311, P. adj=0.024) comparing with DR-MSCs. RR-MSCs expressed higher level of BMP4 than CR-MSCs (P＜0.05). The expression level of BMP6 in CR-MSCs was lower than that in ND-MSCs (P<0.05). DEGs were mainly enriched in BMP/SMADs signaling pathway by GO enrichment analysis (P. adj＜0.0001). (5) Comparing with DR-MSCs, DEGs in ND-MSCs were enriched in cytokine receptors and immune gene-related pathways by Reactome enrichment analysis (P. adj=0.023). (6) TIDE scores of CR-MSCs and RR-MSCs were higher than those of ND-MSCs (P=0.045 and P =0.06, respectively). Conclusion: Compared with DR-MSCs, Ph+ ALL-MSCs had a weaker proliferative capacity in vitro. MSCs derived from different disease status have protective effects on the apoptosis of SUP-B15 induced by drugs, the effects of DR-MSCs and ND-MSCs were stronger than that of CR-MSCs. The protective effect may be achieved through the activity of BMP/SMADs signaling pathway. Figure Legend: A. In Vitro proliferation of MSCs;B.The effect of MSCs cells on apoptosis of SUP-B15;C.Differential gene expression thermogram of DR-MSCs and ND-MCSs;D.Differential gene expression thermogram of ND-CR compared samples. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Early pregnancy exposure to beta-cypermethrin compromises endometrial decidualisation in mice via downregulation of cyclin D3, CDK4/6, and p21.

Beta-cypermethrin (β-CYP) is a highly effective broad-spectrum insecticide that can potentially affect female reproduction. However, little is known about the effect of β-CYP on uterine decidualisation, which is a vital process by which the uterus provides a suitable microenvironment for pregnancy maintenance. Therefore, we focused on the effect and mechanism of β-CYP on endometrial decidualisation during early pregnancy in mice. The results indicated that the expression levels of HOXA10, BMP2, and IGFBP1 was significantly downregulated in the decidual tissue and primary endometrial stromal cells of pregnant and pseudopregnant mice following β-CYP treatment. Serum E2 concentration was significantly increased, whereas P4 concentration and oestrogen receptor (ERα) and progesterone receptor (PRA) expression were significantly downregulated following β-CYP exposure. The number of polyploid decidual cells was lower in the β-CYP-treated group. Furthermore, β-CYP significantly downregulated the protein expression levels of CDK4 and CDK6, and the mRNA expression levels of cyclin D3 and p21. The number of foetuses per female in the first litter was markedly reduced following exposure to β-CYP. In summary, early pregnancy exposure to β-CYP may result in defective endometrial decidualisation via compromised proliferation of uterine stromal cells and reduced expressions of cyclin D3, CDK4/6, and p21 in mice.

Changes in glycocalyx-related biochemical parameters during lung resection in non-small cell carcinoma cases : A pilot study

Objective: Non-small cell lung cancer (NSCLC) is one of the most common neoplasms with high mortality rates, and new studies are needed to understand its characteristics better. This study aimed to determine the changes in the glycocalyx structure related to surgery regarding histopathologic subtypes and to evaluate the correlation of these changes on the development of metastasis and mortality. Material and methods: Serum levels of hyaluronan, VEGF-A, FGF-10, BMP-2, and BMP-4 were measured before and after surgery in 42 patients with NSCLC. The alterations in serum levels of studied markers were evaluated as related to metastasis status and mortality in post-operative 18-24 months. Results: Our study included 15 adenocarcinoma and 27 squamous cell carcinoma cases. Pre- and post-operative values of serum hyaluronan, VEGF-A, FGF-10, and BMP-2 showed significant differences for the whole group (p=0.006, p=0.001, p=0.002, and p=0.004, respectively). Post-operative BMP-2 values also correlated with hyaluronan and VEGF-A values. Post-operative values of hyaluronan and VEGF-A values found incorrelation with metastasis and mortality, while BMP-2 with metastasis and FGF-10 with mortality. Serum values of hyaluronan, VEGF-A, FGF-10 and BMP-2 differed significantly in-between the pre- and post-operative measurements in adenocarcinoma cases (p=0.020, p=0.009, p=0.003, and p=0.011, respectively) , but not that of squamous cell carcinoma. Conclusion: Pre- and post-operative changes in serum hyaluronan, VEGF-A, FGF-10 and BMP-2 values may be associated with metastasis and/or mortality in NSCLC. These findings were also more prominent in adenocarcinoma cases, though further extended studies are needed for a better conclusion.

The Effect of Angiogenesis-Based Scaffold of MesoporousBioactive Glass Nanofiber on Osteogenesis.

There is still an urgent need for more efficient biological scaffolds to promote the healing of bone defects. Vessels can accelerate bone growth and regeneration by transporting nutrients, which is an excellent method to jointly increase osteogenesis and angiogenesis in bone regeneration. Therefore, we aimed to prepare a composite scaffold that could promote osteogenesis with angiogenesis to enhance bone defect repair. Here, we report that scaffolds were prepared by coaxial electrospinning with mesoporous bioactive glass modified with amino (MBG-NH2) adsorbing insulin-like growth factor-1 (IGF-1) as the core and silk fibroin (SF) adsorbing vascular endothelial growth factor (VEGF) as the shell. These scaffolds were named MBG-NH2/IGF@SF/VEGF and might be used as repair materials to promote bone defect repair. Interestingly, we found that the MBG-NH2/IGF@SF/VEGF scaffolds had nano-scale morphology and high porosity, as well as enough mechanical strength to support the tissue. Moreover, MBG-NH2 could sustain the release of IGF-1 to achieve long-term repair. Additionally, the MBG-NH2/IGF@SF/VEGF scaffolds could significantly promote the mRNA expression levels of osteogenic marker genes and the protein expression levels of Bmp2 and Runx2 in bone marrow mesenchymal stem cells (BMSCs). Meanwhile, the MBG-NH2/IGF@SF/VEGF scaffolds promoted osteogenesis by simulating Runx2 transcription activity through the phosphorylated Erk1/2-activated pathway. Intriguingly, the MBG-NH2/IGF@SF/VEGF scaffolds could also significantly promote the mRNA expression level of angiogenesis marker genes and the protein expression level of CD31. Furthermore, RNA sequencing verified that the MBG-NH2/IGF@SF/VEGF scaffolds had excellent performance in promoting bone defect repair and angiogenesis. Consistent with these observations, we found that the MBG-NH2/IGF@SF/VEGF scaffolds demonstrated a good repair effect on a critical skull defect in mice in vivo, which not only promoted the formation of blood vessels in the haversian canal but also accelerated the bone repair process. We concluded that these MBG-NH2/IGF@SF/VEGF scaffolds could promote bone defect repair under accelerating angiogenesis. Our finding provides a new potential biomaterial for bone tissue engineering.

SIRT1/Notch1 signal axis involves in the promoting effect of Segetalin B on bone formation.

Phytoestrogens are a class of potential natural medicines for treating postmenopausal osteoporosis (PMOP). Segetalin B (SB) is a cyclic peptide compound showing estrogenic activity. This study reports the effect of SB on bone formation among ovariectomized (OVX) rats. The bone marrow mesenchymal stem cells (BMSCs) from OVX rats were cultured in vitro. Alizarin Red staining was utilized to observe the effect of SB on the mineralization of BMSCs. The levels of alkaline phosphatase (ALP), osteocalcin, bone morphogenetic protein (BMP-2), and Sirtuin 1 (SIRT1) activities were detected. The OVX rats were treated with SB in vivo. Micro-CTwas utilized for imaging analysis. Urine calcium and phosphorus, and ALP activity in bone marrow were assayed. Western blot analysis and immunofluorescence were incorporated to detect protein expressions in vitro and in vivo. The results showed that SB dose-dependently promoted mineralization of OVX rat-derived BMSCs in vitro increased the level of Osteocalcin, BMP-2, ALP, and SIRT1 activity. Moreover, it upregulated expressions of Runx2, Osterix, and SIRT1, downregulated expressions of Notch intracellular domain (NICD), acetyl-NICD, and hairy and enhancer of split 1 (Hes1). In addition, SB treatment significantly decreased bone loss, inhibited calcium and phosphorus loss, elevated ALP activity, upregulated Runx2, Osterix, and SIRT1, and downregulated NICD and Hes1 in OVX rats in vivo. However, EX527, a SIRT1-selective inhibitor, could reverse the above effects of SB in vitro or in vivo. These results indicate that SB is a potential natural medicine to improve PMOP. Thus, its mechanism of promoting bone formation involves the SIRT1/Notch1 signaling axis.

Iron-dependent BMP6 Regulation in Liver Sinusoidal Endothelial Cells Is Instructed by Hepatocyte-derived Secretory Signals.

Iron-dependent BMP6 Regulation in Liver Sinusoidal Endothelial Cells Is Instructed by Hepatocyte-derived Secretory Signals.

Ameliorative effect of different mesoporous bioactive glass materials in experimental tibial defects in rats.

Enhancing the bone healing procedure would resultantly improve the post-recovery life quality, as well as the speed with which the patient returns to their former life quality. Porous structures can provide a large surface area and abundant channels to facilitate mass transfer. To evaluate the application of mesoporous materials in the bone healing of surgically created defects on the tibiae of male adult Wistar rats. The defect areas were evaluated after implantation of 4 types of bioactive glass histopathologically and immunohistochemically. Fifty adult rats were divided into 5 groups including a control group without material. The used products were mesoporous bioactive glass (MBG), Cu-MBG, Zn-MBG, and Cu-Zn-MBG. Unicortical bone defects with a 3 mm diameter were performed in both tibiae of the animals and filled with 4 types of glass particles. The rats were then euthanized at 15 d and 30 d. Tibial samples were collected and the tissues forwarded for histological processing, and examined using light microscopy. Additionally, bone healing was evaluated by assessing the levels of bone morphogenetic protein BMP2, collagen 1, osteocalcin (OST), and vascular endothelial growth factor (VEGF) using immunohistochemical methods. Within the 15th day, all groups presented connective tissue septa; at the 30th day, the new bone formation was more intense in the Cu-Zn-MBG group. Additionally, BMP2, collagen 1, OST, and VEGF immune expression were more prominent in the Cu-Zn-MBG group. The study results indicated that MBG may be used for the repairing of bone defects. Cu-Zn-MBG may be the best choice for this purpose.

Bone Healing in Rat Segmental Femur Defects with Graphene-PCL-Coated Borate-Based Bioactive Glass Scaffolds.

Bone is a continually regenerating tissue with the ability to heal after fractures, though healing significant damage requires intensive surgical treatment. In this study, borate-based 13-93B3 bioactive glass scaffolds were prepared though polymer foam replication and coated with a graphene-containing poly (ε-caprolactone) (PCL) layer to support bone repair and regeneration. The effects of graphene concentration (1, 3, 5, 10 wt%) on the healing of rat segmental femur defects were investigated in vivo using male Sprague–Dawley rats. Radiographic imaging, histopathological and immuno-histochemical (bone morphogenetic protein (BMP-2), smooth muscle actin (SMA), and alkaline phosphatase (ALP) examinations were performed 4 and 8 weeks after implantation. Results showed that after 8 weeks, both cartilage and bone formation were observed in all animal groups. Bone growth was significant starting from the 1 wt% graphene-coated bioactive glass-implanted group, and the highest amount of bone formation was seen in the group containing 10 wt% graphene (p < 0.001). Additionally, the presence of graphene nanoplatelets enhanced BMP-2, SMA and ALP levels compared to bare bioactive glass scaffolds. It was concluded that pristine graphene-coated bioactive glass scaffolds improve bone formation in rat femur defects.

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway

BackgroundBone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms.MethodshDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria.ResultsThe calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture.ConclusionsThe stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

In animal models, the administration of ciliary neurotrophic factor (CNTF) was demonstrated to reduce bone mass and to participate in bone remodeling. Cementoblasts, a cell type embedded in the cementum, are the main cells to produce and mineralize the extracellular matrix. The effect of CNTF on cementoblasts has not yet been addressed. Thus, the goal of this in vitro study was to investigate possible influences of exogenous CNTF on cementogenesis, as well as autophagy regulation and subsequent mechanisms in cementoblasts. Cementoblasts (OCCM-30) were stimulated with exogenous CNTF. Alizarin Red staining was performed to analyze the functional differentiation (mineralization) of OCCM-30 cells. The release of OPG was quantified by ELISA. The expression of cementogenesis markers (RUNX-2, OCN, BMP-7, BSP, and SPON-2) was evaluated by RT-qPCR. Western blotting (WB) was performed for the protein expression of STAT3, COX-2, SHP-2, cPLAα, cPLAβ; ERK1/2, P38, and JNK. The autophagic flux was assessed using WB and RT-qPCR analysis of LC3A/B, Beclin-1, and Atg-5, and the autophagosome was investigated by immunofluorescence staining (IF). The ERK1/2 (FR180204) or STAT3 (sc-202818) antagonist was added, and the cellular response was analyzed using flow cytometry. Exogenous CNTF significantly attenuated mineralized nodule formation, impaired OPG release, and downregulated the mRNA levels of RUNX-2, OCN, BMP-7, and BSP. Moreover, CNTF induced the phosphorylation of STAT3 and activated a transient activation of SHP-2, cPLAβ, ERK1/2, P38, and JNK protein. CNTF also induced autophagosome formation and promoted autophagy-associated gene and protein expressions. Additionally, the inhibition of ERK1/2 or STAT3 reversed a CNTF-induced mineralization impairment and had regulatory effects on CNTF-induced autophagosome formation. Our data revealed that CNTF acts as a potent inhibitor of cementogenesis, and it can trigger autophagy, in part by ERK1/2 and STAT3 commitment in the cementoblasts. Thus, it may play an important role in inducing or facilitating inflammatory root resorption during orthodontic tooth movement.

Effects of cell seeding technique and cell density on BMP-2 production in transduced human mesenchymal stem cells.

Small animal models have demonstrated the efficacy of ex vivo regional gene therapy using scaffolds loaded with BMP-2-expressing mesenchymal stem cells (MSCs). Prior to clinical translation, optimization of seeding techniques of the transduced cells will be important to minimize time and resource expenditure, while maximizing cell delivery and BMP-2 production. No prior studies have investigated cell-seeding techniques in the setting of transduced cells for gene therapy applications. Using BMP-2-expressing transduced adipose-derived MSCs and a porous ceramic scaffold, this study compared previously described static and dynamic seeding techniques with respect to cell seeding efficiency, uniformity of cell distribution, and in vitro BMP-2 production. Static and negative pressure seeding techniques demonstrated the highest seeding efficiency, while orbital shaking was associated with the greatest increases in BMP-2 production per cell. Low density cell suspensions were associated with the highest seeding efficiency and uniformity of cell distribution, and the greatest increases in BMP-2 production from 2 to 7 days after seeding. Our results highlight the potential for development of an optimized cell density and seeding technique that could greatly reduce the number of MSCs needed to produce therapeutic BMP-2 levels in clinical situations. Further studies are needed to investigate in vivo effects of cell seeding techniques on bone healing.