Microalloying Design of Biodegradable Mg-2Zn-0.05Ca Promises Improved Bone-Implant Applications.

Mg and its alloys have been comprehensively studied and show huge potential for clinical orthopedic applications. However, balancing the mechanical strength and corrosion resistance of alloys is still a challenge. In light of this, micro-level contents of Zn and Ca were added to pure Mg to fabricate a Mg-2Zn-0.05Ca microalloy to expectedly enhance the mechanical strength and concurrently improve the corrosion resistance. The characteristics of the rolled Mg-2Zn-0.05Ca microalloy were explored using optical microscopy, X-ray diffraction, and tensile tests. The corrosion behavior and mechanical strength loss were explored using electrochemical and immersion tests. The effects of the microalloy extract on the proliferation, adhesion, and osteogenic differentiation of MC3T3-E1 cells were systematically studied. Moreover, implantations were done in femoral condyles of rabbits to study the degradation properties, osteogenic effect, mechanical strength loss, and biosafety of the microalloy. The ultimate tensile strength and yield strength of the rolled microalloy were found to be significantly elevated to 257 ± 2.74 and 237.6 ± 8.29 MPa, respectively. The microalloy showed a stable and gradual strength loss during degradation, both in vivo and in vitro. Concurrently, the microalloy exhibited improved corrosion resistance ability and especially, in vivo, the rolled microalloy exhibited a comparable degradation rate to that of rolled pure Mg within the initial 12 weeks of implantation. Additionally, the microalloy promoted osteogenesis, both in vitro and in vivo, and no short- and long-term toxicities of the microalloy were observed in rabbits. This study suggested that the rolled Mg-2Zn-0.05Ca microalloy effectively balanced the mechanical strength and corrosion resistance and showed potential application as bone implants.

Synergistic enhancement of tendon-to-bone healing via anti-inflammatory and pro-differentiation effects caused by sustained release of Mg2+/curcumin from injectable self-healing hydrogels.

Poor healing response after rotator cuff reconstruction is multifactorial, with the inflammatory microenvironment and deficiency of stem cell differentiation factors at the lesion site being most relevant. However, there is a lack of effective tissue engineering strategies that can simultaneously exert anti-inflammatory and pro-differentiation effects to promote rotator cuff healing. Methods: In this study, we synthesized and characterized a novel active drug delivery vector that successfully overcame the challenge of simultaneous high-efficiency loading and controlled release of Mg2+ and curcumin. The anti-inflammatory and pro-differentiation effects of the composite hydrogel were evaluated in vitro and in vivo. Moreover, healing of the rotator cuff tendon-to-bone interface was studied by histology, immunofluorescence, and biomechanical tests. Results: The composite hydrogel exhibited excellent biocompatibility and injectability, good adhesiveness, and rapid self-healing. The released curcumin showed obvious anti-inflammatory and antioxidation effects, which protected stem cells and tendon matrix. Furthermore, released Mg2+ promoted stem cell aggregation and chondrogenesis. Moreover, biomechanical tests and histological results of a rat rotator cuff tear model at 8 weeks after surgery indicated that the composite hydrogel significantly enhanced tendon-to-bone healing. Conclusions: The composite hydrogel mediated sustained in situ release of curcumin and Mg2+ to effectively promote rotator cuff tendon-to-bone healing via anti-inflammatory and pro-differentiation effects. Therefore, this composite hydrogel offers significant promise for rotator cuff repair.

Downregulation of Adrenomedullin Leads to the Inhibition of the Tumorigenesis via VEGF Pathway in Human and Nude Mice Osteosarcoma Models.

BACKGROUND AND AIMS: Osteosarcoma is one of the most pernicious primary bone tumor characterized by high malignancy and metastasis, however its pathogenesis remain largely unknown. Our previous study showed elevated expression of adrenomedullin (ADM) is correlated with prognosis and disease severity in osteosarcoma patients. In this research, we continue to study the mechanisms of ADM-induced osteosarcoma cells proliferation in vitro and in vivo osteosarcoma models. METHODS: The Radioimmunoassay was used to test the correlations of ADM and vascular endothelial growth factor (VEGF) expressions in plasma of osteosarcoma patients. The MTT and flow cytometry analysis were accepted to monitor the proliferation of osteosarcoma cells, meanwhile the ADM and VEGF expression were detected by real-time PCR and western blot. Moreover, the relationship among ADM and VEGF expression was also assessed in the osteosarcoma nude mice models. RESULTS: In this study, a significant correlation was found between ADM and VEGF expression in the plasma of osteosarcoma patients. As important stimuli in osteosarcoma proliferation, hypoxia could stimulate ADM and VEGF expression in MG-63 cells. The expressions of ADM and VEGF increased with the duration of hypoxia, which also identify the positive correlations between the expression of ADM and VEGF. The proliferation of MG-63 cells decreased when ADM was inhibited. And the proliferation increased when adding exogenous ADM, while VEGF inhibition attenuated this effect. Furthermore, ADM reduction also inhibited VEGF, CD31 expression and tumor cells growth in osteosarcoma nude mice models. CONCLUSION: These results suggested inhibition of osteosarcoma cells proliferation might be influenced by ADM through VEGF pathway, which implied ADM-VEGF signal was a potential target for impeding the proliferation of malignant cells in osteosarcoma.

pH-responsive injectable hydrogels with mucosal adhesiveness based on chitosan-grafted-dihydrocaffeic acid and oxidized pullulan for localized drug delivery.

Injectable hydrogels with multifunctional properties, including tissue adhesiveness and pH-sensitivity are highly desired for localized drug delivery in disease treatment, and their design is still challenging. We developed a series of multifunctional injectable mucoadhesive and pH-responsive hydrogels based on chitosan-grafted-dihydrocaffeic acid (CS-DA) and oxidized pullulan (OP) via a Schiff base reaction. These hydrogels exhibited good injectability, suitable gelation time, in vitro pH-dependent equilibrated swelling ratios, morphologies, and rheological characteristics. The desirable in vitro pH-sensitive drug release behavior of these hydrogels was demonstrated by a drug release test with anti-cancer drug doxorubicin (DOX) loaded hydrogels at different pH values. The hydrogels showed good DOX release, effectively killing colon tumor cells (HCT116 cells) and good antibacterial properties against E. coli and S. aureus in vitro when the antibacterial model drug amoxicillin was encapsulated in the hydrogels. A lap-shear test was also carried out with these hydrogels. The hydrogels exhibited good mucosal adhesion, indicating their potential use in mucosa-localized drug delivery systems. All these results suggest that these injectable pH-responsive adhesive hydrogels are ideal candidates for development of colon cancer drug delivery carriers or mucoadhesive drug delivery systems.

Using urinary bFGF and TIMP3 levels to predict the presence of juvenile pilocytic astrocytoma and establish a distinct biomarker signature.

OBJECTIVE The authors report the use of urinary biomarkers as a novel, noninvasive technique to detect juvenile pilocytic astrocytomas (JPAs), capable of distinguishing JPAs from other CNS diseases, including other brain tumors. Preliminary screening of an array of tumors implicated proteases (including matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinase [TIMPs]) as well as growth factors (including basic fibroblast growth factor [bFGF]) as candidate biomarkers. These data led the authors to hypothesize that tissue inhibitor of metalloproteinase 3 (TIMP3) and bFGF would represent high-probability candidates as JPA-specific biomarkers. METHODS Urine was collected from 107 patients, which included children with JPA (n = 21), medulloblastoma (n = 17), glioblastoma (n = 9), arteriovenous malformations (n = 25), moyamoya (n = 14), and age- and sex-matched controls (n = 21). Biomarker levels were quantified with enzyme-linked immunosorbent assay, tumor tissue expression was confirmed with immunohistochemical analysis, and longitudinal biomarker expression was correlated with imaging. Results were subjected to univariate and multivariate statistical analyses. RESULTS Using optimal urinary cutoff values of bFGF > 1.0 pg/µg and TIMP3 > 3.5 pg/µg, multiplexing bFGF and TIMP3 predicts JPA presence with 98% accuracy. Multiplexing bFGF and MMP13 distinguishes JPA from other brain tumor subtypes with up to 98% accuracy. Urinary biomarker expression correlated with both tumor immunohistochemistry and in vitro tumor levels. Urinary bFGF and TIMP3 decrease following successful tumor treatment and correlate with changes in tumor size. CONCLUSIONS This study identifies 2 urinary biomarkers-bFGF and TIMP3-that successfully detect one of the most common pediatric brain tumors with high accuracy. These data highlight potential benefits of urinary biomarkers and support their utility as diagnostic tools in the treatment of children with JPA.

siRNA targeting stathmin inhibits invasion and enhances chemotherapy sensitivity of stem cells derived from glioma cell lines.

Glioma is one of the most highly angiogenic tumors, and glioma stem cells (GSCs) are responsible for resistance to chemotherapy and radiotherapy, as well as recurrence after operation. Stathmin is substantial for mitosis and plays an important role in proliferation and migration of glioma-derived endothelial cells. However, the relationship between stathmin and GSCs is incompletely understood. Here we isolated GSCs from glioma cell lines U87MG and U251, and then used siRNA targeting stathmin for silencing. We showed that silencing of stathmin suppressed the proliferation, increased the apoptosis rate, and arrested the cell cycle at G2/M phase in GSCs. Silencing of stathmin in GSCs also resulted in inhibited the migration/invasion as well as the capability of vasculogenic mimicry. The susceptibility of GSCs to temozolomide was also enhanced by stathmin silencing. Our findings suggest stathmin as a potential target in GSCs for glioma treatment.

Netrin-1 promotes medulloblastoma cell invasiveness and angiogenesis, and demonstrates elevated expression in tumor tissue and urine of patients with pediatric medulloblastoma.

Invasion and dissemination of medulloblastoma within the central nervous system is the principal factor predicting medulloblastoma treatment failure and death. Netrin-1 is an axon guidance factor implicated in tumor and vascular biology, including in invasive behaviors. We found that exogenous netrin-1 stimulated invasion of human medulloblastoma cells and endothelial cells in contrast to VEGF-A, which promoted invasion of endothelial cells but not medulloblastoma cells. Furthermore, medulloblastoma cells expressed endogenous netrin-1 along with its receptors, neogenin and UNC5B. Blockades in endogenous netrin-1, neogenin, or UNC5B reduced medulloblastoma invasiveness. Neogenin blockade inhibited netrin-1-induced endothelial cells tube formation and recruitment of endothelial cells into Matrigel plugs, two hallmarks of angiogenesis. In patients with pediatric medulloblastoma, netrin-1 mRNA levels were increased 1.7-fold in medulloblastoma tumor specimens compared with control specimens from the same patient. Immunohistochemical analyses showed that netrin-1 was elevated in medulloblastoma tumors versus cerebellum controls. Notably, urinary levels of netrin-1 were 9-fold higher in patients with medulloblastoma compared with control individuals. Moreover, urinary netrin-1 levels were higher in patients with invasive medulloblastoma compared with patients with noninvasive medulloblastoma. Finally, we noted that urinary netrin-1 levels diminished after medulloblastoma resection in patients. Our results suggest netrin-1 is a candidate biomarker capable of detecting an invasive, disseminated phenotype in patients with medulloblastoma and predicting their disease status.

MicroRNA-9 inhibits vasculogenic mimicry of glioma cell lines by suppressing Stathmin expression.

The purpose of this study was to investigate the functions of microRNA-9, which is a tissue-specific microRNA in central nervous system, in the vasculogenic mimicry (VM) of glioma cell lines in vitro and in vivo. Glioma cell lines U87MG, U251 and SHG44 were transfected with microRNA-9 mimic, microRNA-9 inhibitor or scramble sequences. The amount of microRNA-9 and Stathmin (STMN1) mRNA was determined by quantitative real-time PCR, and the protein expression of STMN1 was determined by western blot. Cell proliferation and apoptosis were assessed. The interactions between the 3'UTR of STMN1 and miR-9 was determined by luciferase reporter assay. The VM capacity in vitro was evaluated using VM formation assay, and the rescue experiment of STMN1 was carried out in U251 cells. The in vivo experiment was applied with animal models implanted with U87MG cells.MicroRNA-9 mimic transfection reduced proliferation and increased apoptosis in glioma cell lines (p < 0.05). MicroRNA-9 mimic up-regulated STMN1 mRNA levels but reduced its protein levels (p < 0.05), and luciferase activity of STMN1 was suppressed by microRNA-9 mimic transfection (p < 0.05). Furthermore, microRNA-9 mimic transfection suppressed tumor volume growth, as well as VM both in vitro and in vivo. The cell viability and microtube density were upregulated in U251 cells after STMN1 up-regulation (p < 0.05). STMN1 is a target of microRNA-9, and microRNA-9 could modulate cell proliferation, VM and tumor volume growth through controlling STMN1 expression. MicroRNA-9 and its targets may represent a novel panel of molecules for the development of glioma treatment.

Cyclosporin A and sanglifehrin A enhance chemotherapeutic effect of cisplatin in C6 glioma cells.

Glioma is the most common type of brain tumors in adults, and treatment of high-grade gliomas is still palliative. Studies to date have revealed only modest effect in attenuating growth of these tumors with single agent therapy, but combination treatment appears to be more effective. Cyclophilin A (CypA), a target of immunosuppressive drugs cyclosporin A (CsA) and sanglifehrin A (SFA), is an intracellular protein that has peptidyl-prolyl cis-trans isomerase (PPIase) enzymatic activity. Previously, we showed that overexpressed CypA induced chemoresistance in cancer cells. Here we provide evidence that combination of cisplatin with either CsA or SFA synergistically enhances apoptotic cell death in C6 glioma cells, compared with single agent treatment. Enhanced apoptotic cell death is a result of an increase in ROS generation and a decrease in intracellular glutathione levels. Consistently, CypA knockdown by siRNA also enhances cisplatin-induced apoptosis. Immunohistochemical analysis showed increased expression of CypA in human glioblastoma multiforme, but not in normal human astrocytes. CypA was also shown to be up-regulated in C6 glioma cells during hypoxia. In conclusion, CsA or SFA in combination with cisplatin synergistically enhances cisplatin-induced apoptosis in C6 glioma cells via inhibition of PPIase activity of CypA, indicating that development of new drugs that selectively inhibit the CypA PPIase activity without immune suppression may facilitate alleviation of chemoresistance in treatment of high-grade glioma.

[Study on the chemical constituents of the leaves of Ipomoea batatas].

OBJECTIVE: To study the chemical constituents of the leaves of Ipomoea batatas. METHODS: The constituents were isolated and purified by silica gel and TLC, and their structures were elucidated by spectroscopy. RESULTS: Six compounds were isolated from 90% ethanol extract and identified as tetracosane (I ), myristic acid (II), beta-sitosterol (II), beta-carotene (IV), daucosterol (V) and quercetin (VI). CONCLUSION: Compounds I, II, IV, V are isolated from this plant for the first time.

Molecular cloning and expression of MyD88 in large yellow croaker, Pseudosciaena crocea.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). In this report, the full-length cDNA of MyD88 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1574 bp, including a 5'-terminal untranslated region (UTR) of 89 bp, a 3'-terminal UTR of 621bp and an open reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids. It contained a typical death domain at the N-terminal and a conservative Toll/IL-1R (TIR) domain structure at the C-terminal. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of MyD88 with the highest expression in the spleen and the weakest expression in the muscle. The expression of MyD88 after challenge with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. It suggested that the highest expression was in the spleen (p<0.05) with 1.9 times (at 48 h) as much as that in the control and the lowest expression of MyD88 was in the liver (p<0.05) with 0.29 times (at 3h) of that in the control. These results indicated that as a universal key adaptor in the Toll-like receptor pathway in mammals, MyD88 might play an important role in large yellow croaker defense against pathogenic infection.

Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker, Pseudosciaena crocea.

Toll-like receptors (TLRs) are the archetypal pattern-recognition receptors in sensing foreign pathogens. In this report, two alternative splicing isoforms of TLR9 (named PcTLR9A and PcTLR9B) cDNA were cloned from the large yellow croaker, Pseudosciaena crocea. The full-length cDNA of PcTLR9A was of 3637bp, including a 5'-terminal untranslated region (UTR) of 111bp, 3'-terminal UTR of 355bp and an open reading frame (ORF) of 3171bp encoding a polypeptide of 1056 amino acids. However, the full-length cDNA of PcTLR9B was 119bp longer than that of PcTLR9A from the position of 3079-3197bp, which encoded a polypeptide of 1006 amino acids. Both of the PcTLR9A and PcTLR9B contained 12 typical structures of leucine-rich repeats (LRRs), an LRRTYP and an LRRCT in the extracellular region and a conservative Toll/IL-1R (TIR) domain in the intracellular region. However, compared to PcTLR9A, conservative Box 3 was absent in PcTLR9B TIR domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR9A and PcTLR9B with the highest expression in spleen and the weakest expression in muscle. Expression of PcTLR9A and PcTLR9B after stimulation with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. This indicated that the highest expression was 3.3 times (at 24h) as much as that in the control in the spleen (p<0.05) and the lowest expression of PcTLR9A was 1/4 times (at 3h) of that in the control in the liver (p<0.05). PcTLR9B showed a similar expression pattern to PcTLR9A post-injection. These results suggested that both PcTLR9A and PcTLR9B might play an important role in large yellow croaker defense against pathogenic infection.

Endogenous hydrogen peroxide regulates glutathione redox via nuclear factor erythroid 2-related factor 2 downstream of phosphatidylinositol 3-kinase during muscle differentiation.

We reported previously that endogenous reactive oxygen species (ROS) function as myogenic signaling molecules. It has also been determined that excess ROS induce electrophile-response element (EpRE)-driven gene expression via activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Nonetheless, the relationship between the metabolism of ROS (eg, H(2)O(2)) through glutathione (GSH) up-regulation, GSH-dependent reduction of H(2)O(2), and Nrf2-dependent gene regulation is not well established. Therefore, we attempted to determine whether H(2)O(2) controls the intracellular GSH redox state via the Nrf2-glutamate-cysteine ligase (GCL)/glutathione reductase (GR)-GSH signaling pathway. In our experiments, enhanced H(2)O(2) generation was accompanied by an increase in both total GSH levels and the GSH/GSSG ratio during muscle differentiation. Both GCL and GR transcriptional expression levels were markedly increased during muscle differentiation but reduced by catalase treatment. Nrf2 protein expression and nuclear translocation increased during myogenesis. The inhibition of GCL, GR, and Nrf2 both by inhibitors and by RNA interference blocked muscle differentiation. Phosphatidylinositol 3-kinase regulated the expression of the GCL C (a catalytic subunit) and GR genes via the induction of Nrf2 nuclear translocation and expression. In conclusion, endogenous H(2)O(2) generated during muscle differentiation not only functions as a signaling molecule, but also regulates the GSH redox state via activation of the Nrf2-GCL/GR-GSH signaling pathway downstream of phosphatidylinositol 3-kinase.

A new method for purification of functional recombinant GST-cyclophilin A protein from E. coli.

The expression of glutathione-S-transferase (GST) fusion protein is extensively utilized in the study of protein-protein interactions. In the commonly used purification method, the overexpressed GST fusion protein is bound to the glutathione (GSH)-coupled resins via affinity chromatography, and then eluted by an excessive quantity of reduced GSH. However, this technique has certain limitations, such as low product purity, retention of GSH in the sample, as well as relatively high cost. To overcome these limitations, in this study, elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer. By using this method, highly purified GST-cyclophilin A (CypA) fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities. Moreover, the procedure is also cost-effective, due to the low cost of formic acid as compared with GSH.