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Background Tissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization. Methods We cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a β-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments. Results We found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks. Conclusions This study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.

Although stress can significantly promote atherosclerosis, the underlying mechanisms are still not completely understood. Here we successfully unveiled that high salt-induced nuclear factor of activated T cells 5 (NFAT5) control the endothelial-dependent fibrinolytic activity and the inflammatory adhesion-related molecules expression through regulation of plasminogen activator inhibitor-1 (PAI-1). We first observed that high salt diets instigated the expression of NFAT5 and PAI-1 in the endothelium which brought about the fibrin deposition and macrophage infiltration in the atherosclerotic arteries of ApoE-/- mice. Overexpression of NFAT5 increased PAI-1-mediated antifibrinolytic activity and activated inflammatory adhesion-related genes in endothelial cells. Knockdown of NFAT5 by siRNA inhibited the expression of PAI-1, antifibrinolytic and adhesive molecules. Moreover, chromatin immunoprecipitation assay demonstrated that high salt intake significantly promoted the binding of NFAT5 to PAI-1 promoter (TGGAATTATTT) in endothelial cells. Our study identified that NFAT5 has great potential to activate the PAI-1-mediated fibrinolytic dysfunction and inflammatory cell adhesion, thus promoting high salt-induced atherosclerosis disease.

Vasculogenesis (de novo formation of vessels) induced by endothelial progenitor cells (EPCs) is requisite for vascularized bone regeneration. However, there exist few available options for promoting vasculogenesis within artificial bone grafts except for exogenous EPC transplantation, which suffers from the source of EPC, safety, cost, and time concerns in clinical applications. This study aimed at endogenous EPC recruitment for vascularized bone regeneration by using a bioinspired EPC-induced graft. The EPC-induced graft was created by immobilizing two bioactive peptides, WKYMVm and YIGSR, on the surface of poly(ε-caprolactone) (PCL)/poliglecaprone (PGC) nanofibrous scaffolds via a polyglycolic acid (PGA)-binding peptide sequence. Remarkable immobilization efficacy of WKYMVm and YIGSR peptides and their sustained release (over 14 days) from scaffolds were observed. In vivo and in vitro studies showed robust recruitment of EPCs, which subsequently contributed to early vasculogenesis and ultimate bone regeneration. The dual-peptide-functionalized nanofibrous scaffolds proposed in this study provide a promising therapeutic strategy for vasculogenesis in bone defect repair.

This study aims to investigate the repair of bone defects with prefabricated vascularized bone grafts and double-labeled bone marrow-derived mesenchymal stem cells (BMSCs) in a rat model. BMSCs were separated from rat bone marrow. LTR-CMVpro-RFP and LTR-CMVpro-GFP were transfected into the BMSCs for in vitro and in vivo tracking. BMSCs-RFP and BMSCs-GFP were induced into endothelial progenitor cells (EPCs) and osteoblasts (OBs). Rats were divided into five groups: Group A: in vitro prefabrication with EPCs-RFP + in vivo prefabrication with arteriovenous vascular bundle + secondary OBs-GFP implantation; Group B: in vitro prefabrication with EPCs-RFP + secondary OBs-GFP implantation; Group C: in vivo prefabrication with arteriovenous vascular bundle + secondary OBs-GFP implantation; Group D: implantation of EPCs-RFP + implantation of with arteriovenous vascular bundle + simultaneous OBs-GFP implantation; Group E: demineralized bone matrix (DBM) grafts (blank control). Among five groups, Group A had the fastest bone regeneration and repair, and the regenerated bone highly resembled normal bone tissues; Group D also had fast bone repair, but the repair was slightly slower than Group A. Therefore, in vitro prefabrication with EPCs-RFP plus in vivo prefabrication with arteriovenous vascular bundle and secondary OBs-GFP implantation could be the best treatment for bone defect.

Retinoid X receptor (RXR) and Histone deacetylase (HDAC) are considered important targets for cancer therapy due to their crucial roles in genetic or epigenetic regulations of cancer development and progression. Here, we evaluated the potential of dual targeting of RXR and HDAC using DW22 as a novel therapeutic approach to cancer treatment. We found that the co-expression of RXR-α and HDAC1 was frequently appeared in lung cancer and breast cancer tissues and cell lines. RXR was activated by DW22 in RXRα and HDAC1 overexpressed A549 and MDA-MB-435 cell lines. Meanwhile, DW22 inhibited the activity of HDAC by decreasing its expression in A549 and MDA-MB-435 cell lines, but not in RXRα and HDAC1 deficient cell lines. Moreover, DW22 suppressed cell growth, induced cell differentiation, prompted cell apoptosis and arrested cell cycle in A549, MDA-MB-435 or HL60 cell lines. Treatment human umbilical vascular endothelial cells (HUVECs) with DW22 suppressed migration, invasion and tube formation through decreasing VEGF expression. The up-regulation of Ac-H3 and p21, and down-regulation of VEGF caused by DW22 was markedly attenuated by silencing of HDAC1. Furthermore, knockdown of RXRα by siRNA completely blocked DW22-induced cell differentiation, but partially attenuated DW22-caused inhibition of cell proliferation, induction of cell apoptosis, and suppression of cell migration, invasion and tube formation. Moreover, intravenous administration of DW22 significantly retarded tumor growth of A549 and MDA-MB-435 xenograft mice models, and induced no substantial weight loss and gross toxicity. In addition, DW22 also reduced cell proliferation, angiogenesis, and induced cell apoptosis in vivo. Collectively, our data demonstrates that dual targeting of RXR and HDAC using DW22 possesses pleiotropic antitumor activities both in vitro and in vivo, providing a novel therapeutic approach for cancer treatment.

A novel series of chalcone derivatives were synthesized and their biological activities against HIF-1 were evaluated. Among these compounds, 5d exhibited clearly inhibitory effects on HIF-1 by downregulating the expression of HIF-1α under hypoxic conditions. Meanwhile, it also significantly suppressed VEGF-induced migration and invasion of Hep3B and HUVEC cells in nontoxic concentrations. Additionally, tube formation assay demonstrated its anti-angiogenesis activity. Moreover, the in vivo study indicated that compound 5d could retard tumor growth of Hep3B xenograft models and reduced CD31 and MMP-2 expression in tumor tissues. Finally, in acute intravenous toxicity, 5d was well tolerated and was found to be non-toxic up to 200 mg/kg in Swiss mice. These findings support the further investigation on the anti-invasive and anti-angiogenic potential of this class of compounds as HIF-1 inhibitor.

The demineralized bone matrix (DBM) putty is a traditional bone graft utilized to facilitate the repair and reconstruction of bone. Recent studies indicated the DBM putties with the various carriers were different in bone repairing ability. In order to prepare a kind of DBM putty with a good biocompatibility and bioactivity, the DBM gel was processed from the DBM and the feasibility as a carrier for the DBM putty was evaluated. After the bovine DBM gel was prepared, the BMPs content as well as the ability to promote osteogenic differentiation of MC3T3-E1 cells in vitro were investigated. Then the DBM putty was prepared and filled into the rat calvarial defect model to evaluate the bone repairing ability by micro-CT and histology. The result showed there was 2.953 ± 0.054 ng BMP contained in per gram of the DBM gel. And the ALP production of MC3T3-E1 cells in the DBM gels group increased with prolonged culturing, the mineralized nodules formed in MC3T3-E1 cells on 14th day after co-culture. The putty prepared by DBM gel was easy to handle without loss of DBM particles at room temperature. In the rat calvarial bone defect experiment, histological observation showed more mature bone formed in the DBM putty group than that in the type I collagen group at 12 weeks, which indicated the bone putty prepared by DBM gel exhibited a better bone repair capability.

Background/Aims: Osteoarthritis (OA) is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs) possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1) in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

Baicalein has been used for many years as a popular antiviral and antibacterial in China. Recent investigations revealed that baicalein also has anti-inflammatory activities. Our results indicated that baicalein increases ERE-luciferase activity in an estrogen receptor (ER)-dependent manner when either ERα or ERβ were coexpressed in Hela cells. This study examined whether baicalein exerts an anti-inflammatory effect in RAW264.7 cells through an estrogen receptor-dependent pathway and through regulation of NF-ĸB activation. In lipopolysaccharide (LPS)-induced RAW264.7 cells, baicalein exerts anti-inflammatory effects by inhibiting iNOS, COX-2, and TNF-α mRNA expression; NO production; as well as inflammatory cytokine (IL-1β, PGE2, and TNF-α) production through an ER-dependent pathway. These effects are accompanied with the inhibition of the transcription factor NF-ĸB activation and IκBα phosphorylation. We therefore conclude that baicalein inhibits LPS-induced inflammatory cytokine production via regulation of the NF-ĸB pathway and estrogen-like activity, suggesting that it may be useful for preventing inflammation-related diseases.

SL4, a chalcone-based compound, has been shown to retard tumor invasion and angiogenesis by suppressing HIF1 activity and to induce apoptosis by promoting ROS release. Here, we report that SL4 is able to inhibit the proliferation of different types of breast cancer cell in vitro and in vivo by inducing G2/M cell cycle arrest. Our results showed that SL4 exhibited strong anti-proliferative activity in several human breast cancer cell lines, with IC50 values lower than 1.3 μM. Further studies indicated that SL4 induced G2/M arrest in these cell lines. Mechanistically, SL4 reduces the expression of cyclin A2 and cdc25C and decreases the activity of the cdc2/cyclin B1 complex. Notably, SL4 treatment resulted in an obvious increase in p21 mRNA and protein levels through activation of MAPK signaling pathways, but not the TGF-β pathway. SP600125 and PD98059, specific inhibitors of JNK kinase and ERK kinase, significantly blocked the SL4-induced G2/M phase arrest and upregulation of p21. Furthermore, SL4 suppressed the growth of established breast tumors in nude mice through upregulation of p21 and downregulation of cdc25C, and displayed a good safety profile. Taken together, these findings demonstrate the potential value of SL4 as a novel multi-target anti-tumor drug candidate.

Inflammation is a physiological response to infection or injury and involves the innate and adaptive immune system. Tanshinone IIA (Tan IIA) is a well-known flavonoid that elicits an important therapeutic effect by inhibiting inflammatory response. In this study, we examined whether Tan IIA exerts anti-inflammatory activity and investigated the possible mechanisms, including Toll-like receptor 4 (TLR4)-MyD88-nuclear factor kappa B (NF-κB) signaling pathway and microRNA expression in lipopolysaccharide (LPS)-induced RAW264.7 cells. Tan IIA could attenuate the inflammatory reaction via decreasing cytokine, chemokine, and acute-phase protein production, including GM-CSF, sICAM-1, cxcl-1, MIP-1α, and tumor necrosis factor alpha (TNF-α), analyzed by Proteome profile array in LPS-induced RAW264.7 cells. Concurrently, the messenger RNA (mRNA) expressions of IL-1β, TNF-α, and COX-2 were also significantly reduced by Tan IIA. Additionally, Tan IIA decreased LPS-induced NF-κB activation and downregulated TLR4 and MyD88 protein expression levels. We also observed reduced microRNA-155, miR-147, miR-184, miR-29b, and miR-34c expression levels, while LPS-induced microRNA-105, miR-145a, miR-194, miR-383, miR-132, and miR-451a expression levels were upregulated using microRNA (miRNA) qPCR array. Our results indicate that Tan IIA could exert an anti-inflammatory effect on LPS-induced RAW264.7 cells by decreasing TLR4-MyD88-NF-κB signaling pathway and regulating a series of cytokine production and miRNA expression.

Objectives SL4, a chalcone-based compound, exhibits clearly inhibitory effects on HIF-1 and has been shown to effectively suppress tumour invasion and angiogenesis in vitro and in vivo. Here, studies were conducted to determine SL4's anti-apoptotic effects and its underlying mechanisms, in human cancer cells. Materials and methods Cytotoxicity, apoptotic induction and its involved mechanisms of SL4 were investigated using normal cells, cancer cells and mouse xenograft models. The role of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signalling in SL4-induced apoptosis was explored by manipulating specific scavenger or signalling inhibitors, in cultured cells. Results SL4 significantly inhibited cell population growth of human cancer cell lines but exhibited lower cytotoxicity against normal cells. In addition, SL4 effectively induced apoptosis of Hep3B and MDA-MB-435 cells by activating procaspase-8, -9 and -3, and down-regulating expression levels of XIAP, but did not affect HIF-1 apoptosis-related targets, Survivin and Bcl-XL. Further study showed that SL4 also reduced mitochondrial membrane potential and promoted generation of ROS. ROS generation and apoptotic induction by SL4 were blocked by NAC, a scavenger of ROS, suggesting SL4-induced apoptosis via ROS accumulation. We also found that MAPKs, JNK and p38, but not ERK1/2, to be critical mediators in SL4-induced apoptosis. SP600125 and SB203580, specific inhibitors of JNK kinase and p38 kinase, significantly retarded apoptosis induced by SL4. Moreover, anti-oxidant NAC blocked activation of JNK and p38 induced by SL4, indicating that ROS may act as upstream signalling of JNK and p38 activation. It is noteworthy that animal studies revealed dramatic reduction (49%) in tumour volume after 11 days SL4 treatment. Conclusions These data demonstrate that SL4 induced apoptosis in human cancer cells through activation of the ROS/MAPK signalling pathway, suggesting that it may be a novel lead compound, as a cancer drug candidate, with polypharmacological characteristics.

To study the effect of Schwann cells (SCs) promoting the function of nitric oxide (NO) secretion of bone marrow mesenchymal stem cells (BMSCs) derived endothelial cells so as to lay the experimental foundation for research of the effect of nerves on vessels during the process of tissue engineering bone formation.SCs were collected from 1-day-old Sprague Dawley (SD) rats, and identified through S100 immunohistochemistry (IHC). BMSCs were collected from 2-week-old SD rats and induced into endothelial cells (IECs), which were identified through von Willebrand factor (vWF) and CD31 immunofluorescence (IF). Transwell system was used for co-culture of SCs and IECs without contact as the experimental group, and simple culture of IECs served as the control group. The NO concentration in the medium was measured at 1, 3, 5, and 7 days after culture; the mRNA expressions of nitric oxide synthetase 2 (NOS2) and NOS3 were detected by real-time fluorescence quantitative PCR (RT -qPCR) at 1, 3, 7, and 10 days.SCs and IECs were identified through morphology and immunology indexes of S100 IHC, vWF and CD31 IF. Significant differences were found in the NO concentration among different time points in 2 groups (P0.05); the NO concentration of the experimental group was significantly higher than that of the control group at the other time points (P0.05) except at 3 days. NOS2 mRNA expression of the experimental group was significantly higher than that of the control group (P0.05); difference was significant in the NOS2 mRNA expression among different time points in 2 groups (P0.05). NOS3 mRNA expression of the experimental group was significantly higher than that of the control group at the other time points (P0.05) except at 10 days. No significant difference was found in NOS3 mRNA expression among different time points in the experimental group (F = 6.673, P = 0.062), but it showed significant differences in the control group (F = 36.581, P = 0.000).SCs can promote NO secretion of BMSCs derived endothelial cells, which is due to promoting the activity of NOS.

To study the feasibility of preparation of the poly-D, L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide linkage formation.PDLLA scaffolds (8 mm diameter, 1 mm thickness) were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma. The consequent scaffolds were labeled as aminated PDLLA (A/PDLLA). The pore size, porosity, and surface water contact angle of groups 0 (un-treated control), 5, 10, and 20 minutes A/PDLLA were measured. A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solution which contain 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC-HCl) and N-hydroxysuccinimide (NHS), the molar ratio of peptides/EDC*HCL/NHS was 1.5 : 1.5 : 1.0, then brachytely slashed for 24 hours in room temperature. The consequent scaffolds were labelled as peptides conjugated A/PDLLA (PA/PDLLA). The scaffolds in groups 0, 5, 10, and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy (XPS). The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance liquid chromatography (HPLC), un-treated and un-conjugated scaffolds employed as control. Bone marrow mesenchymal stem cells (BMSCs) from SD rats were isolated and cultured by whole bone marrow adherent culture method. BMSCs at the 3rd-6th passages were seeded to the scaffolds as follows: 20 minutes ammonia plasma treatment (group A/PDLLA), 20 minutes ammonia plasma treatment and conjugation of GRGDS (group PA/PDLLA), and untreated PDLLA control (group PDLLA). After 16 hours of culture, the adhesive cells on scaffolds and the adhesive rate were calculated. After 4 and 8 days of culture, the BMSCs/scaffold composites was observed by scanning electron microscope (SEM).No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments (P0.05). With increased time of ammonia plasma treatment, the water contact angle of A/PDLLA scaffolds surface was decreased, and the hydrophilicity in the treated scaffolds was improved gradually, showing significant differences when these groups were compared with each other (P0.001). XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma. With time passing, the peak N1s became more visible, and the ratio of N/C increased more obviously. After PDLLA scaffolds treated for 0, 5, 10, and 20 minutes with ammonia plasma and subsequent conjugation of peptides, the ratio of N/C increased and the peak of S2p appeared on the surface. The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time. The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully, showing significant differences between 10 minutes and 20 minutes groups (P0.001). However, the amount of peptides conjugated in un-treated control and 0, 5 minutes PA/PDLLA scaffolds was too small to detect. After 16 hours co-culture of BMSCs/scaffolds, the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds, showing significant difference between every 2 groups (P0.01). Also, SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group, especially in PA/PDLLA group.Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide linkage formation. This new type of biomimetic bone has stabilized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.

OBJECTIVE To explore the cytotoxicity, labeled time, marking rate, and effect on adhesion of quantum dot 655 (QD655) labeled rat bone marrow mesenchymal stem cells (BMSCs) in vitro, and to confirm its feasibility for stem cell labeling and tracer means for rat.BMSCs were collected from the femur and tibia bone marrow cavity of a 2-week-old SD rat, cultured and identified. The 3rd passage of BMSCs were incubated with QD655 as the experimental group according to the recommended concentration of the markers. The cells were not labeled by QD655 as control group. The cell survival rate after QD655 labeling was detected by trypan-blue exclusion. The effect of QD655 on cell proliferation was observed by MTT. The osteogenic differentiation potential was identified by Alizarin red staining, alkaline phosphatase (ALP) staining, and real-time fluorogenic quantitative PCR. At immediately, 1, 2, 4, and 6 weeks, fluorescent microscopy was used to observe the labeled rate and scanning electron microscope was used to observe the cell adhesion to scaffold (bioglass/collagen composite).The cell survival rates were more than 90% in both experimental group and control group, showing no significant difference (P0.05). There was no significant difference in the cell proliferation between 2 groups (P0.05). Alizarin red staining and ALP staining showed positive results. Real-time fluorogenic quantitative PCR result showed that the mRNA expression levels of osteopontin, osteocalcin, collagen type I, ALP, and BMP-2 in the experimental group was significantly higher than those in the control group. The labeled rates were 96.50% +/- 1.59%, 93.30% +/- 1.51%, 72.40% +/- 2.90%, 40.10% + 3.60%, and 10.00% +/- 1.70% immediately, 1, 2, 4, and 6 weeks after labeling, respectively. The labeled rate in the control group was 0. Scanning electron microscope showed a good distribution of fusiform or polygonal cells in the pores of scaffold.QD655 can be used as a labeling marker for BMSCs. Rat BMSCs labeled with QD655 is of high efficiency and safety.