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Background Dendritic Cell Cytokine-induced killer cell (DC- CIK) coculture treatment in cancer immunotherapy has been shown to be effective. However, the cost of DC- CIK therapy is prohibitive for many patients, and the lack of standard manufacturing processes and treatment strategies are major limitations. Our study used tumor lysate as a tumor-associated antigen source and DCs and CIK cells in coculture. We developed an efficient method to obtain autologous DCs- and CIK cells from peripheral blood. We used flow cytometry to assess DC activation and the cytometric bead array assay to quantify cytokines secreted by CIK cells. Results We evaluated the antitumor activity of DC- CIK coculture in vitro with the K562 cell line. We demonstrated that a manufacturing process employing frozen immature DCs can yield the lowest loss with the highest economic benefits. DC-CIK coculture can effectively upgrade CIK cells’ immunological specificity to tumors in the presence of tumor-associated antigens. Conclusion In vitro experiments revealed that when the DC- CIK cell ratio was 1: 20 in the coculture, CIK cells secreted the highest number of cytokines on the 14th day and the antitumor immune effect showed the highest potency. CIK cells’ cytotoxicity to K562 cells was highest when the CIK: K562 cell ratio was 25: 1. We developed an efficient manufacturing process for DC- CIK coculture, while also establishing the optimal DC- CIK cell ratio for immunological activity and the best cytotoxic CIK: K562 cell ratio.

Background Chitosan (Chi) is a natural material which has been widely used in neural applications due to possessing better biocompatibility. In this research study, a novel of nanocomposites film based on Chitosan (Chi) with Hyaluronic Acid (HA), combined with varying amounts of gold nanoparticles (AuNPs) was created resulting in pure Chi, Chi-HA, Chi-HA-AuNPs (25 ppm) and Chi-HA-AuNPs (50 ppm). Methods This study focused on evaluating their effects on Mesenchymal Stem Cell (MSC) viability, colony formation and biocompatibility. The surface morphology and chemical position was characterized through UV-Visible spectroscopy (UV-Vis), Fourier-transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and contact-angle assessment. Results When seeding MSCs on Chi-HA-AuNPs (50 ppm), the results showed high cell viability, biocompatibility, and the highest colony formation ability. Meanwhile, the evidence showed that Chi-HA-Au nano film was able to inhibit Nestin and β-Tubulin expression of MSCs, as well as inhibit the ability of neurogenic differentiation. Furthermore, the results of Matrix Metalloproteinase 2/9 (MMP2/9) expression in MSCs were also significantly higher in the Chi-HA-AuNP (50 ppm) group, guiding with angiogenesis and wound healing abilities. In addition, in our rat model, both capsule thickness and collagen deposition were the lowest in Chi-HA-AuNPs (50 ppm). Conclusion Thus, in view of the in vitro and in vivo results, Chi-HA-AuNPs (50 ppm) could not only maintain the greatest stemness properties and regulate the neurogenic differentiation ability of MSCs, but was able to also induce the least immune response. Herein, Chi-HA-Au 50 ppm nano film holds promise as a suitable material for nerve regeneration engineering.

Cardiovascular Diseases (CVDs) such as atherosclerosis, where inflammation occurs in the blood vessel wall, are one of the major causes of death worldwide. Mesenchymal Stem Cells (MSCs)-based treatment coupled with nanoparticles is considered to be a potential and promising therapeutic strategy for vascular regeneration. Thus, angiogenesis enhanced by nanoparticles is of critical concern. In this study, Polyethylene Glycol (PEG) incorporated with 43.5 ppm of gold (Au) nanoparticles was prepared for the evaluation of biological effects through in vitro and in vivo assessments. The physicochemical properties of PEG and PEG–Au nanocomposites were first characterized by UV-Vis spectrophotometry (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), and Atomic Force Microscopy (AFMs). Furthermore, the reactive oxygen species scavenger ability as well as the hydrophilic property of the nanocomposites were also investigated. Afterwards, the biocompatibility and biological functions of the PEG–Au nanocomposites were evaluated through in vitro assays. The thin coating of PEG containing 43.5 ppm of Au nanoparticles induced the least platelet and monocyte activation. Additionally, the cell behavior of MSCs on PEG–Au 43.5 ppm coating demonstrated better cell proliferation, low ROS generation, and enhancement of cell migration, as well as protein expression of the endothelialization marker CD31, which is associated with angiogenesis capacity. Furthermore, anti-inflammatory and endothelial differentiation ability were both evaluated through in vivo assessments. The evidence demonstrated that PEG–Au 43.5 ppm implantation inhibited capsule formation and facilitated the expression of CD31 in rat models. TUNEL assay also indicated that PEG–Au nanocomposites would not induce significant cell apoptosis. The above results elucidate that the surface modification of PEG–Au nanomaterials may enable them to serve as efficient tools for vascular regeneration grafts.

Gold nanoparticles (AuNPs) are well known to interact with cells, leading to different cell behaviors such as cell proliferation and differentiation capacity. Biocompatibility and biological functions enhanced by nanomedicine are the most concerning factors in clinical approaches. In the present research, AuNP solutions were prepared at concentrations of 1.25, 2.5, 5 and 10 ppm for biocompatibility investigations. Ultraviolet–visible spectroscopy was applied to identify the presence of AuNPs under the various concentrations. Dynamic Light Scattering assay was used for the characterization of the size of the AuNPs. The shape of the AuNPs was observed through a Scanning Electron Microscope. Afterward, the mesenchymal stem cells (MSCs) were treated with a differentiation concentration of AuNP solutions in order to measure the biocompatibility of the nanoparticles. Our results demonstrate that AuNPs at 1.25 and 2.5 ppm could significantly enhance MSC proliferation, decrease reactive oxygen species (ROS) generation and attenuate platelet/monocyte activation. Furthermore, the MSC morphology was observed in the presence of filopodia and lamellipodia while being incubated with 1.25 and 2.5 ppm AuNPs, indicating that the adhesion ability was enhanced by the nanoparticles. The expression of matrix metalloproteinase (MMP-2/9) in MSCs was found to be more highly expressed under 1.25 and 2.5 ppm AuNP treatment, relating to better cell migrating ability. Additionally, the cell apoptosis of MSCs investigated with Annexin-V/PI double staining assay and the Fluorescence Activated Cell Sorting (FACS) method demonstrated the lower population of apoptotic cells in 1.25 and 2.5 ppm AuNP treatments, as compared to high concentrations of AuNPs. Additionally, results from a Western blotting assay explored the possibility that the anti-apoptotic proteins Cyclin-D1 and Bcl-2 were remarkably expressed. Meanwhile, real-time PCR analysis demonstrated that the 1.25 and 2.5 ppm AuNP solutions induced a lower expression of inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6 and IL-8). According to the tests performed on an animal model, AuNP 1.25 and 2.5 ppm treatments exhibited the better biocompatibility performance, including anti-inflammation and endothelialization. In brief, 1.25 and 2.5 ppm of AuNP solution was verified to strengthen the biological functions of MSCs, and thus suggests that AuNPs become the biocompatibility nanomedicine for regeneration research.

A nanocomposite composed of polyethylene glycol (PEG) incorporated with various concentrations (~17.4, ~43.5, ~174 ppm) of gold nanoparticles (Au) was created to investigate its biocompatibility and biological performance in vitro and in vivo. First, surface topography and chemical composition was determined through UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), free radical scavenging ability, and water contact angle measurement. Additionally, the diameters of the PEG-Au nanocomposites were also evaluated through dynamic light scattering (DLS) assay. According to the results, PEG containing 43.5 ppm of Au demonstrated superior biocompatibility and biological properties for mesenchymal stem cells (MSCs), as well as superior osteogenic differentiation, adipocyte differentiation, and, particularly, neuronal differentiation. Indeed, PEG-Au 43.5 ppm induced better cell adhesion, proliferation and migration in MSCs. The higher expression of the SDF-1α/CXCR4 axis may be associated with MMPs activation and may have also promoted the differentiation capacity of MSCs. Moreover, it also prevented MSCs from apoptosis and inhibited macrophage and platelet activation, as well as reactive oxygen species (ROS) generation. Furthermore, the anti-inflammatory, biocompatibility, and endothelialization capacity of PEG-Au was measured in a rat model. After implanting the nanocomposites into rats subcutaneously for 4 weeks, PEG-Au 43.5 ppm was able to enhance the anti-immune response through inhibiting CD86 expression (M1 polarization), while also reducing leukocyte infiltration (CD45). Moreover, PEG-Au 43.5 ppm facilitated CD31 expression and anti-fibrosis ability. Above all, the PEG-Au nanocomposite was evidenced to strengthen the differentiation of MSCs into various cells, including fat, vessel, and bone tissue and, particularly, nerve cells. This research has elucidated that PEG combined with the appropriate amount of Au nanoparticles could become a potential biomaterial able to cooperate with MSCs for tissue regeneration engineering.

TIMP metallopeptidase inhibitor 1 (TIMP-1) has been identified as a multifunctional molecule with divergent functions. It participates in wound healing and regeneration, cell morphology and survival, tumor metastasis, angiogenesis, and inflammatory responses. An imbalance of Matrix Metalloproteinase/TIMP regulation has been implicated in several inflammatory diseases. TIMP-1 could be considered an important regulator in the process of liver fibrosis and bile duct degeneration. Thus, we aimed to determine the role of TIMP-1 in a rat model of Common Bile Duct Ligation (CBDL). Male Sprague-Dawley rats were divided into several groups, including those with/ without CBDL surgery and those with/without amiodarone or simvastatin administration. Amiodarone/simvastatin treatment was given at a daily dose of 15 mg/kg and 18 mg/kg by means of intergalactic gavage, which began 7 days prior to CBDL induction. Two weeks after surgery, the animals in each group were sacrificed and hepatocyte degeneration severity was examined using histological morphologies. Large-scale array for secretory factors is intended for the purpose of finding key functional protein after CBDL. The hepatic level of miR-21 was determined through Taqman miRNA analysis. Furthermore, the TIMP-1 level in liver tissue was also visualized by histological stain. Liver injury and fibrosis were founded in CBDL rats based upon histopathological examination and serum biochemical analysis. Hepatic miR-21 and TIMP-1 were significantly up-regulated in CBDL rats, while being slightly rescued in response to amiodarone or simvastatin treatment. Up-regulation of miR-21 and TIMP-1 may result in the progression of hepatic cirrhosis after bile duct obstruction. Drug intervention for cirrhosis, like the use of statin, may function via similar mechanisms.

Background: Dynamin-related protein 1 (DRP1) is a GTPase involved in mitochondrial fission, mitochondrial protein import, and drug sensitivity, suggesting an association with cancer progression. This study was conducted to evaluate the prognostic significance of DRP1 in glioblastoma multiforme (GBM). Methods: DRP1 expression was measured by immunohistochemistry and Western blotting. Correlations between DRP1 expression and clinicopathological parameters were determined by statistical analysis. Differences in survival were compared using the log-rank test. DRP1 expression was detected in 87.2% (41/47) of the investigated patients with GBM. Results: The patients with higher DRP1 levels had worse survival (p = 0.0398). In vitro, the silencing of DRP1 reduced cell proliferation, invasive potential, and radiation resistance. The addition of shikonin inhibited DRP1 expression and increased drug uptake. Moreover, shikonin reduced the nuclear entry of DNA repair-associated enzymes and increased radiation sensitivity, suggesting that reducing DRP1 expression could inhibit DNA repair and increase the radiation sensitivity of GBM cells. Conclusion: Our results indicate that DRP1 overexpression is a prospective radio-resistant phenotype in GBM. Therefore, DRP1 could be a potential target for improving the effectiveness of radiation therapy.

AIM Metalloproteinase 2 (MMP2) is a multi-functional protein which has been shown to be up-regulated in patients with oral cancer, especially those with lymph node metastasis. However, the association of MMP2 genotype with oral cancer risk or metastatic behavior is unknown. This study aimed to evaluate the role of MMP2 promoter 1306 and -735 genotypes in the risk of oral cancer and metastasis. MATERIALS AND METHODS In this case-control study, MMP2 promoter 1306 (rs243865) and -735 (rs2285053) genotypes and their interaction with consumption of areca, cigarettes, and alcohol in determining oral cancer risk were investigated among 788 patients with oral cancer and 956 gender-matched healthy controls. In addition, their role in oral cancer metastasis were also examined. RESULTS The distribution of CC, CT and TT for MMP2 promoter 1306 genotype was 79.0, 20.1 and 0.9% in the oral cancer group and 68.7, 29.2 and 2.1% in the non-cancer control group, respectively (p for trend=4.3E-6). The allelic frequency distributions showed that the variant T allele of MMP2 promoter 1306 conferred lower oral cancer susceptibility than the wild-type C allele (odds ratio=0.61, 95% confidence interval=0.50-0.75, p=1.1E-6). As for the MMP2 -735 genotypes, there was no differential distribution in genotypic or allelic frequencies. The variant CT and TT genotypes were also associated with lower metastasis rates within 5 years among the patients with oral cancer (odds ratio=0.34, 95% confidence interval=0.15-0.80, p=0.0102). CONCLUSION The CT and TT genotypes of MMP2 promoter 1306 may have a protective effect on oral cancer susceptibility and metastasis risk within 5 years for Taiwanese. They may serve as predictive markers for oral cancer in precise medical practice.

In this report, a novel nanocomposite based on type I collagen (Col) was prepared, which contained varying amounts (15.1, 30.2, and 75.5 ppm) of silver nanoparticles (AgNPs). The surface morphology and chemical composition pure Col and Col-AgNP nanocomposites (Col-Ag) was characterized by UV–Vis spectroscopy (UV–Vis), atomic force microscope (AFM) and Fourier transform IR spectrometer (FTIR). The biocompatibility effect and biological activity of Col-Ag culturing with mesenchymal stem cells (MSCs), as well as guiding for angiogenesis differentiation, were evaluated via in vitro assay. The Col-Ag in 30.2 ppm demonstrated better biological properties and compatibility culturing with MSCs. The biological properties could be associated with cell adhesion, proliferation, migration and differentiation. Afterwards, the induced angiogenesis and differentiation of MSCs by the expression of von Willebrand Factor (vWF) and CD31 were also investigated. Furthermore, both anti-inflammatory and endothelialization ability were also investigated in vivo assay. It was observed that Col-Ag nanocomposites not only inhibited CD86 expression, but also facilitated endothelialization capacity, the expression of CD31 when implanting Col-Ag into rats subcutaneously after 4 weeks. This current research indicates that Col-Ag nanocomposites has potential of being employed as a surface modification approach, and is better in clinical treatments with MSCs for vascular regeneration applications.

BackgroundDynamin-related protein 1 (DRP1) is a GTPase involved in mitochondrial fission, mitochondrial protein imports, and drug sensitivity, suggesting an association with cancer progression. This study is to evaluate the prognostic significance of DRP1 in glioblastoma multiforme (GBM).Material and MethodsDRP1 expression was measured by immunohistochemistry and Western blotting. Correlations between DRP1 expression and clinicopathological parameters were analyzed by statistical analysis. Differences in survival were compared by a log-rank test.ResultsDRP1 expression was detected in 87.2% (41/47) patients with GBM. Patients with higher DRP1 levels had worse survival (p = 0.0398). In vitro, silencing of DRP1 reduced cell proliferation, metastatic potential, and radiation resistance. The addition of shikonin inhibited DRP1 expression and increased drug uptake. Moreover, shikonin reduced the nuclear entry of DNA repair-associated enzymes and increased radiation sensitivity, suggesting that to reduce DRP1 expression could inhibit DNA repair and increase the radiation sensitivity of GBM cells.ConclusionOur results indicate that DRP1 overexpression is a prospective radio-resistant phenotype in GBM. Therefore, DRP1 could be a potential target for improving the effectiveness of radiation therapy.Abbreviations used are: ATAD3A, the ATPase family, AAA domain containing 3A; CIM, confocal immunofluorescence microscopy; DRP1, dynamin-related protein 1; ER, endoplasmic reticulum; GBM, glioblastoma multiforme; hHR23A, human homolog of yeast Rad23 protein A; IDH1, isocitrate dehydrogenase 1; MAM, mitochondria-associated membrane; MGMT, O6-methylguanine-DNA-methyltransferase; SAHA, suberoylanilide hydroxamic acid (vorinostat); TMZ, temozolomideTranslational RelevanceThis study shows that dynamin-related protein 1 (DRP1), an essential 80-kDa GTPase, which is involved in mitochondrial fission, and mitochondrial protein imports, is highly expressed in glioblastoma multiforme (GBM). Moreover, we demonstrate that DRP1 expression is closely associated with radiation sensitivity, cancer progression, and patients’ cumulative survival. In vitro, inhibition of DRP1 expression reduced the nuclear entry of DNA repair-associated enzymes, such as ATM, but increased radiation sensitivity and nuclear drug uptakes of glioblastoma cells. More importantly, the silencing of DRP1 induced cellular autophagy. These results indicate that DRP1 overexpression could be a prospective radio-resistant phenotype in GBM and a clinically important target for improving the effectiveness of radiation therapy.

The engineering of vascular regeneration still involves barriers that need to be conquered. In the current study, a novel nanocomposite comprising of fibronectin (denoted as FN) and a small amount of silver nanoparticles (AgNP, ~15.1, ~30.2 or ~75.5 ppm) was developed and its biological function and biocompatibility in Wharton’s jelly-derived mesenchymal stem cells (MSCs) and rat models was investigated. The surface morphology as well as chemical composition for pure FN and the FN-AgNP nanocomposites incorporating various amounts of AgNP were firstly characterized by atomic force microscopy (AFM), UV-Visible spectroscopy (UV-Vis), and Fourier-transform infrared spectroscopy (FTIR). Among the nanocomposites, FN-AgNP with 30.2 ppm silver nanoparticles demonstrated the best biocompatibility as assessed through intracellular ROS production, proliferation of MSCs, and monocytes activation. The expression levels of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, were also examined. FN-AgNP 30.2 ppm significantly inhibited pro-inflammatory cytokine expression compared to other materials, indicating superior performance of anti-immune response. Mechanistically, FN-AgNP 30.2 ppm significantly induced greater expression of vascular endothelial growth factor (VEGF) and stromal-cell derived factor-1 alpha (SDF-1α) and promoted the migration of MSCs through matrix metalloproteinase (MMP) signaling pathway. Besides, in vitro and in vivo studies indicated that FN-AgNP 30.2 ppm stimulated greater protein expressions of CD31 and von Willebrand Factor (vWF) as well as facilitated better endothelialization capacity than other materials. Furthermore, the histological tissue examination revealed the lowest capsule formation and collagen deposition in rat subcutaneous implantation of FN-AgNP 30.2 ppm. In conclusion, FN-AgNP nanocomposites may facilitate the migration and proliferation of MSCs, induce endothelial cell differentiation, and attenuate immune response. These finding also suggests that FN-AgNP may be a potential anti-inflammatory surface modification strategy for vascular biomaterials.

The ideal characteristics of surface modification on the vascular graft for clinical application would be with excellent hemocompatibility, endothelialization capacity, and antirestenosis ability. Here, Fourier transform infrared spectroscopy (FTIR), surface enhanced Raman spectroscopy (SERS), atomic force microscopy (AFM), contact angle (θ) measurement, and thermogravimetric analysis (TGA) were used to evaluate the chemical and mechanical properties of collagen-gold nanocomposites (collagen+Au) with 17.4, 43.5, and 174 ppm of Au and suggested that the collagen+Au with 43.5 ppm of Au had better biomechanical properties and thermal stability than pure collagen. Besides, stromal-derived factor-1α (SDF-1α) at 50 ng/mL promoted the migration of mesenchymal stem cells (MSCs) on collagen+Au material through the α5β3 integrin/endothelial oxide synthase (eNOS)/metalloproteinase (MMP) signaling pathway which can be abolished by the knockdown of vascular endothelial growth factor (VEGF). The potentiality of collagen+Au with MSCs for vascular regeneration was evaluated by our in vivo rat model system. Artery tissues isolated from an implanted collagen+Au-coated catheter with MSCs expressed substantial CD-31 and α-SMA, displayed higher antifibrotic ability, antithrombotic activity, as well as anti-inflammatory response than all other materials. Our results indicated that the implantation of collagen+Au-coated catheters with MSCs could be a promising strategy for vascular regeneration.

Aim Arsenic trioxide (As2O3), known as pi-shuang and the most toxic compound in traditional Chinese medicine, has been used as an antitumor agent for thousands of years. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phenol that has significant anti-bacterial, anti-fungaI and antiaging activities. Our study aimed to examine the combined anticancer effects of As2O3 and resveratrol against human neuroblastoma SK-N-SH cells, and elucidate the underlying intracellular signaling. Materials and methods SK-N-SH cells were treated with an extremely low-dose (2-4 μM) of As2O3 alone or combined with 75 μg/ml resveratrol for further comparisons. Cell viability, apoptotic signaling as well as synergistic cytotoxic effects were estimated using the MTT assay, microscopy observation, flow cytometric analysis for loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and typical quantitative western blotting analysis. Student's t-test, and one- and two-way analysis of variance (ANOVA) were used for examination of significant differences. Results The combined treatment was more effective than single treatment of As2O3 or resveratrol alone in suppressing cell viability, which correlated with the elevation of ROS levels. The intracellular mechanisms of cytotoxicity of As2O3 plus resveratrol were revealed as ROS accumulation and relative decrease of MMP, leading to activation of caspase-3 and -9, but not of caspase-1, -7 and-8. Combination treatment reduced the expression of B-cell lymphoma 2 (BCL2), BH3 interacting domain death agonist (BID), and BCL-x/L. Conclusion Combined treatment at extremely low concentration of two agents from natural products, As2O3 and resveratrol, has high potential as a cocktail of anticancer drugs for neuroblastoma.

Metalloproteinase 2 (MMP2) is a multi-functional protein which has been shown to be up-regulated in patients with oral cancer, especially those with lymph node metastasis. However, the association of MMP2 genotype with oral cancer risk or metastatic behavior is unknown. This study aimed to evaluate the role of MMP2 promoter 1306 and -735 genotypes in the risk of oral cancer and metastasis.In this case-control study, MMP2 promoter 1306 (rs243865) and -735 (rs2285053) genotypes and their interaction with consumption of areca, cigarettes, and alcohol in determining oral cancer risk were investigated among 788 patients with oral cancer and 956 gender-matched healthy controls. In addition, their role in oral cancer metastasis were also examined.The distribution of CC, CT and TT for MMP2 promoter 1306 genotype was 79.0, 20.1 and 0.9% in the oral cancer group and 68.7, 29.2 and 2.1% in the non-cancer control group, respectively (p for trend=4.3E-6). The allelic frequency distributions showed that the variant T allele of MMP2 promoter 1306 conferred lower oral cancer susceptibility than the wild-type C allele (odds ratio=0.61, 95% confidence interval=0.50-0.75, p=1.1E-6). As for the MMP2 -735 genotypes, there was no differential distribution in genotypic or allelic frequencies. The variant CT and TT genotypes were also associated with lower metastasis rates within 5 years among the patients with oral cancer (odds ratio=0.34, 95% confidence interval=0.15-0.80, p=0.0102).The CT and TT genotypes of MMP2 promoter 1306 may have a protective effect on oral cancer susceptibility and metastasis risk within 5 years for Taiwanese. They may serve as predictive markers for oral cancer in precise medical practice.

Vascularization of engineered tissues remains one of the key problems. Here, we described a novel approach to promote vascularization of engineered tissues using fibronectin (FN) incorporated gold nanoparticles (AuNP) coated onto catheters with mesenchymal stem cells (MSCs) for tissue engineering. We found that the FN-AuNP composite with 43.5 ppm of AuNP exhibited better biomechanical properties and thermal stability than pure FN. FN-AuNP composites promoted MSC proliferation and increased the biocompatibility. Mechanistically, vascular endothelial growth factor (VEGF) promoted MSC migration on FN-AuNP through the endothelial oxide synthase (eNOS)/metalloproteinase (MMP) signaling pathway. Vascular femoral artery tissues isolated from the implanted FN-AuNP-coated catheters with MSCs expressed substantial CD31 and alpha-smooth muscle actin (α-SMA), displayed higher antithrombotic activity, as well as better endothelialization ability than those coated with all other materials. These data suggested that the implantation of FN-AuNP-coated catheter with MSCs could be a novel strategy for vascular biomaterials applications.

Three-dimensional (3-D) tissue engineered constructs provide a platform for examining how the local extracellular matrix contributes to the malignancy of various cancers, including human glioblastoma multiforme. Here, we describe a simple and innovative 3-D culture environment and assess its potential for use with glioblastoma stem cells (GSCs) to examine the diversification inside the cell mass in the 3-D culture system. The dissociated human GSCs were cultured using gelatin foam. These cells were subsequently identified by immunohistochemical staining, reverse transcriptase-polymerase chain reaction, and Western blot assay. We demonstrate that the gelatin foam provides a suitable microenvironment, as a 3-D culture system, for GSCs to maintain their stemness. The gelatin foam culture system contributes a simplified assessment of cell blocks for immunohistochemistry assay. We show that the significant transcription activity of hypoxia and the protein expression of inflammatory responses are detected at the inside of the cell mass in vitro, while robust expression of PROM1/CD133 and hypoxia-induced factor-1 alpha are detected at the xenografted tumor in vivo. We also examine the common clinical trials under this culture platform and characterized a significant difference of drug resistance. The 3-D gelatin foam culture system can provide a more realistic microenvironment through which to study the in vivo behavior of GSCs to evaluate the role that biophysical factors play in the hypoxia, inflammatory responses and subsequent drug resistance.

Although suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been used in clinical trials for cancer therapies, its pharmacological effects occur through a poorly understood mechanism. Here, we report that SAHA specifically triggers autophagy and reduces cell viability via promotion of apoptosis in the late phase of glioblastoma stem cells (GSCs). Using a cell line cultured from a glioblastoma biopsy, we investigated the properties and effects of GSCs under SAHA treatment in vitro. In vivo xenograft assays revealed that SAHA effectively caused tumor growth slowdown and the induction of autophagy. SAHA was sufficient to increase formation of intracellular acidic vesicle organelles, recruitment of LC3-II to the autophagosomes, potentiation of BECN1 protein levels and reduced SQSTM1 levels. We determined that SAHA triggered autophagy through the downregulation of AKT-MTOR signaling, a major suppressive cascade of autophagy. Interestingly, upon depletion or pharmacological inhibition of autophagy, SAHA facilitates apoptosis and results in cell death at the early phase, suggesting that SAHA-induced autophagy functions probably act as a prosurvival mechanism. Furthermore, our results also indicated that the inhibition of SAHA-induced autophagy using chloroquine has synergistic effects that further increase apoptosis. Moreover, we found that a reduced dose of SAHA functioned as a potent modulator of differentiation and senescence. Taken together, our results provide a new perspective on the treatment of GSCs, indicating that SAHA is a promising agent for targeting GSCs through the induction of autophagy.

Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a common flavonoid in many types of plants and has several beneficial biological effects, including anti-inflammation, anti-oxidant, and anti-cancer properties. However, the detail mechanisms of luteolin in suppressing tumor invasion and metastasis are poorly understood. Here, we investigated the effects of luteolin on suppressing glioblastoma tumor cell invasion and migration activity. Under the non-cytotoxic doses (15 and 30 μM), luteolin exhibited an inhibitory effect on migration and invasion in U-87 MG and T98G glioblastoma cells. Additionally, filopodia assembly in U-87 MG cells was markedly suppressed after luteolin treatment. The treatment of luteolin also showed a decrease of Cdc42 (cell division cycle 42) protein levels and reduced PI3K/AKT activation, whereas there was no association between this decrease and phosphorylated ERK or altered transcription levels of Cdc42. Over expression of constitutive Cdc42 (Q61L) using transient transfection in U-87 MG cells induced a partial cell migration, but did not affected the degradation of the protein levels of Cdc42 after luteolin treatment. Moreover, inhibition of the proteaosome pathway by MG132 caused a significant recovery in the migration ability of U-87 MG cells and augmented the Cdc42 protein levels after luteolin treatment, suggesting that pharmacological inhibition of migration via luteolin treatment is likely to preferentially facilitate the protein degradation of Cdc42. Taken together, the study demonstrated that flavonoids of luteolin prevent the migration of glioblastoma cells by affecting PI3K/AKT activation, modulating the protein expression of Cdc42 and facilitating their degradation via the proteaosome pathway.

This paper proposes a GA-SVM classification method which is applied to the dynamic evaluation of taekwondo. For classifying a dynamic action, we converted a dynamic action signal to a frequency spectrum signal for analysis. However, the useful features were concentrated in a part of the frequency spectrum, and the useless features led to a decline in accuracy, operation speed, and efficiency of the classifier. Therefore, we propose a classification method that involves using a support vector machine (SVM) with a genetic algorithm (GA) for the dynamic evaluation of taekwondo. The GA determines the useful features, and the useless features are eliminated, thus reducing the dimensionality and improving the accuracy of the classifier. The SVM can solve problems of small, nonlinear, and high-dimension samples efficiently, exhibiting superior performance in the classification of a dynamic action.

This paper proposes a novel biodegradable nerve conduit comprising 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linked gelatin, annexed with β-tricalcium phosphate (TCP) ceramic particles (EDC-Gelatin-TCP, EGT). In this study, the EGT-implant site in rats was irradiated using a large-area 660 nm AlGaInP diode laser (50 mW) to investigate the feasibility of laser stimulation in the regeneration of a 15-mm transected sciatic nerve. The animals were divided into three groups: a sham-irradiated group (EGT/sham); an experimental group undergoing low-level laser (LLL) therapy (EGT/laser); a control group undergoing autologous nerve grafts (autografts). Twelve weeks after implantation, walking track analysis showed a significantly higher sciatic functional index (p < 0.05) and improved toe spreading development in the EGT/laser and autograft groups than in the EGT/sham group. In electrophysiological measurement, both the mean peak amplitude and the area under the compound muscle action potential curves in the EGT/laser and autograft groups showed significantly improved functional recovery than the EGT/sham group (p < 0.05). Compared with the EGT/sham group, the EGT/laser and autograft groups displayed a reduction in muscular atrophy. Histomorphometric assessments revealed that the EGT/laser group had undergone more rapid nerve regeneration than the EGT/sham group. The laser-treated group also presented greater neural tissue area as well as larger axon diameter and thicker myelin sheath than the tube group without the laser treatment, indicating improved nerve regeneration. Thus, these assessments demonstrate that LLL therapy can accelerate the repair of a transected peripheral nerve in rats after being bridged with EGT conduit. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:239–252, 2013.

This study proposed a biodegradable GGT nerve conduit containing genipin crosslinked gelatin annexed with tricalcium phosphate (TCP) ceramic particles for the regeneration of peripheral nerves. Cytotoxicity tests revealed that GGT-extracts were non-toxic and promoted proliferation and neuronal differentiation in the induction of stem cells (i-ASCs) derived from adipose tissue. Furthermore, the study confirmed the effectiveness of a GGT/i-ASCs nerve conduit as a guidance channel in the repair of a 10-mm gap in the sciatic nerve of rats. At eight weeks post-implantation, walking track analysis showed a significantly higher sciatic function index (SFI) (P

This study used a biodegradable composite containing genipin-cross-linked gelatin annexed with β-tricalcium phosphate ceramic particles (genipin-gelatin-tricalcium phosphate, GGT), developed in a previous study, as a nerve guide conduit. The aim of this study was to analyse the influence of a large-area irradiated aluminium-gallium-indium phosphide (AlGaInP) diode laser (660 nm) on the neural regeneration of the transected sciatic nerve after bridging the GGT nerve guide conduit in rats. The animals were divided into two groups: group 1 comprised sham-irradiated controls and group 2 rats underwent low-level laser (LLL) therapy. A compact multi-cluster laser system with 20 AlGaInP laser diodes (output power, 50mW) was applied transcutaneously to the injured peripheral nerve immediately after closing the wound, which was repeated daily for 5 min for 21 consecutive days. Eight weeks after implantation, walking track analysis showed a significantly higher sciatic function index (SFI) score (P

Stroke represents one of the leading causes of death and disability in humans, but despite intense research, only a few options exist for the treatment of stroke-related infarction of brain tissue. Thus far, in experimental strokes, cell therapy appears to partly reverse some behavioral deficits. However, the mechanisms of action remain uncertain as most studies reveal only little, if any, evidence for neuronal replacement and observed behavioral improvements. This present study was performed to test rodent fetus forebrain derived neural stem cells (NSCs) implantation into rats subjected to suture-induced middle cerebral artery occlusion (MCAO). Efficacy of cell therapy was studied regarding behavior recovery, infarct volume, and protection possibility of related molecular mechanisms. Here, we show that grafted cells can home in on damaged regions by MCAO and significantly improve behavior of ischemic rats. Infarct volumes and brain atrophy were diminished after grafted NSCs treatment. Furthermore, we detected inflammation related molecules such as COX-2 and IL-1beta and found that grafted NSCs treatment after ischemic stroke could repress expression of inflammation molecular protein levels. We also detected protein levels of heat shock protein 27 (HSP27) as a protective protein against apoptosis. The results showed that grafted NSCs treatment induced the protein level of HSP27 and down-regulated activity of caspase-3 compared with the vehicle control. Our results demonstrate that transplanted NSCs provide benefits in behavioral function recovery after MCAO and increase neuroprotection whilst repressing inflammatory destruction. These data reveal another essential explanation of cellular transplantation therapy in damage recovery from ischemic stroke and offer new therapeutic possibilities.

Neural progenitors cells are capable of promoting neurogenesis after ischemic stroke in the adult mammalian brain; however the function of these cells and their fate is still not clear. Therefore the purpose of this study investigated the relationship between neural progenitors and reactive astrocytes after middle cerebral artery occlusion (MCAO). Brain infarction was induced by occlusion of a right cerebral artery in male Wistar rats. The fate of progenitor cells and the surrounding cells was investigated by immunochemical staining for nestin, vimentin and glial fibrillary acidic protein (GFAP) positive cells at several locations. Vimentin and nestin positive cells were observed in the ipsilateral subventricular zone (SVZ), striatum, and cortex at 3 and 7 days after MCAO, but those cells were not found at 28 days after ischemia. In contrast, reactive astrocyte positive cells increased following MCAO. These reactive astrocytes induced astrocytes differentiation of progenitor cells and formed dense astroglioses surrounding the ischemic lesion. Reactive astrocytes are thought to protect the penumbra during brain ischemia. We examined which brain cell expressed nestin and GFAP in the ipsilateral co-expression at 7 days after MCAO, especially at the core of injury. These results suggest that robust reactive astrocytes after MCAO were possibly differentiation from the induced nestin-positive cells after early ischemia.

This study employed a rat model of sciatic nerve injury to investigate the effects of postoperative low-power far-infrared (FIR) radiation therapy on nerve repair following end-to-end neurorrhaphy. The rat models were divided into the following 3 groups: (1) nerve injury without FIR biostimulation (NI/sham group); (2) nerve injury with FIR biostimulation (NI/FIR group); and (3) noninjured controls (normal group). Walking-track analysis results showed that the NI/FIR group exhibited significantly higher sciatic functional indices at 8 weeks after surgery (P<0.05) compared with the NI/sham group. The decreased expression of CD4 and CD8 in the NI/FIR group indicated that FIR irradiation modulated the inflammatory process during recovery. Compared with the NI/sham group, the NI/FIR group exhibited a significant reduction in muscle atrophy (P<0.05). Furthermore, histomorphometric assessment indicated that the nerves regenerated more rapidly in the NI/FIR group than in the NI/sham group; furthermore, the NI/FIR group regenerated neural tissue over a larger area, as well as nerve fibers of greater diameter and with thicker myelin sheaths. Functional recovery, inflammatory response, muscular reinnervation, and histomorphometric assessment all indicated that FIR radiation therapy can accelerate nerve repair following end-to-end neurorrhaphy of the sciatic nerve.

This study proposed a novel combination of neural regeneration techniques for the repair of damaged peripheral nerves. A biodegradable nerve conduit containing genipin-cross-linked gelatin was annexed using beta-tricalcium phosphate (TCP) ceramic particles (genipin-gelatin-TCP, GGT) to bridge the transection of a 15 mm sciatic nerve in rats. Two trigger points were irradiated transcutaneously using 660 nm of gallium-aluminum arsenide phosphide (GaAlAsP) via laser diodes for 2 min daily over 10 consecutive days. Walking track analysis showed a significant improvement in sciatic functional index (SFI)(P<0.01)and pronounced improvement in the toe spreading ability of rats undergoing laser stimulation. Electrophysiological measurements (peak amplitude and area) illustrated by compound muscle action potential (CMAP) curves demonstrated that laser stimulation significantly improved nerve function and reduced muscular atrophy. Histomorphometric assessments revealed that laser stimulation accelerated nerve regeneration over a larger area of neural tissue, resulting in axons of greater diameter and myelin sheaths of greater thickness than that observed in rats treated with nerve conduits alone. Motor function, electrophysiological reactions, muscular reinnervation, and histomorphometric assessments all demonstrate that the proposed therapy accelerated the repair of transected peripheral nerves bridged using a GGT nerve conduit.

This study proposes a biodegradable nerve conduit comprising 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linked gelatin annexed with β-tricalcium phosphate (β-TCP) ceramic particles (EDC-gelatin-TCP, EGT). For this study, the EGT-implant site in rats was irradiated using 660-nm GaAlAsP laser diodes (50 mW) for trigger point therapy to investigate the use of low-level laser (LLL) stimulation in the regeneration of a 15-mm transected sciatic nerve. Animals were divided into three groups: a control group undergoing autologous nerve graft (autograft); a sham-irradiated group (EGT), and an experimental group undergoing laser stimulation (EGT/LS). Two trigger points on the surgical incision along the sciatic nerve were irradiated transcutaneously for 2 min daily for 10 consecutive days. Twelve weeks after implantation, walking track analysis showed a significantly higher sciatic functional index (SFI; p0.05) and improved toe spreading development in the autograft and EGT/LS groups, compared to the EGT group. In the electrophysiological measurement, the mean recovery index (peak amplitude and area) of the compound muscle action potential curves in the autograft and EGT/LS groups showed significantly improved functional recovery than in the EGT group (p0.05). Compared with the EGT group, the autograft and EGT/LS groups showed a reduction in muscular atrophy. Histomorphometric assessments showed that the EGT/LS group had undergone more rapid nerve regeneration than the EGT group. Therefore, motor function, electrophysiological reaction, muscular reinnervation, and histomorphometric assessments demonstrate that LLL therapy can accelerate the repair of a 15-mm transected peripheral nerve in rats after being bridged with the EGT nerve conduit.

This study proposed a biodegradable GGT nerve conduit containing genipin crosslinked gelatin annexed with tricalcium phosphate (TCP) ceramic particles for the regeneration of peripheral nerves. Cytotoxicity tests revealed that GGT-extracts were non-toxic and promoted proliferation and neuronal differentiation in the induction of stem cells (i-ASCs) derived from adipose tissue. Furthermore, the study confirmed the effectiveness of a GGT/i-ASCs nerve conduit as a guidance channel in the repair of a 10-mm gap in the sciatic nerve of rats. At eight weeks post-implantation, walking track analysis showed a significantly higher sciatic function index (SFI) (P 0.05) in the GGT/i-ASC group than in the autograft group. Furthermore, the mean recovery index of compound muscle action potential (CMAP) differed significantly between GGT/i-ASCs and autograft groups (P 0.05), both of which were significantly superior to the GGT group (P 0.05). No severe inflammatory reaction in the peripheral nerve tissue at the site of implantation was observed in either group. Histological observation and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/i-ASCs nerve conduits were similar to those of the autografts. These promising results achieved through a combination of regenerative cells and GGT nerve conduits suggest the potential value in the future development of clinical applications for the treatment of peripheral nerve injury.

This study proposes a biodegradable nerve conduit containing genipin-cross-linked gelatin annexed with tricalcium phosphate ceramic particles (genipin-gelatin-tricalcium phosphate, GGT) in peripheral nerve regeneration. Firstly, cytotoxicity tests revealed that the GGT-extracts were not toxic, and promoted the proliferation and neuronal differentiation of adipose tissue-derived stem cells (ADSCs). Secondly, the GGT composite film effectively supported ADSCs attachment and growth. Additionally, the GGT substrate was biocompatible with the neonatal rat sciatic nerve and produced a beneficial effect on peripheral nerve repair through in vitro tissue culture. Finally, the experiments in this study confirmed the effectiveness of a GGT/ADSCs nerve conduit as a guidance channel for repairing a 10-mm gap in a rat sciatic nerve. Eight weeks after implantation, the mean recovery index of compound muscle action potentials (CMAPs) was significantly different between the GGT/ADSCs and autografts groups (p < 0.05), both of which were significantly superior to the GGT group (p < 0.05). Furthermore, walking track analysis also showed a significantly higher sciatic function index (SFI) score (p < 0.05) and better toe spreading development in the GGT/ADSCs group than in the autograft group. Histological observations and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/ADSCs nerve conduits were similar to those of the autografts. The GGT nerve conduit offers a better scaffold for the incorporation of seeding undifferentiated ADSCs, and opens a new avenue to replace autologous nerve grafts for the rapid regeneration of damaged peripheral nerve tissues and an improved approach to patient care. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

Glioblastoma is one of the most angiogenic malignancy, the neoplastic vessels of which are likely to arise by angiogenesis and vasculogenesis. An alternative mechanism of tumor vasculature is described, termed vasculogenic mimicry, by which highly aggressive tumor cells can form vessel-like structures themselves, by virtue of their high cellular plasticity. Evidence suggests that cancer stem cells acquire a multi-potent plastic phenotype and show vasculogenic potential. In this study, we report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133(+)-GSC) and CD133- (CD133(-)-GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with CD133(-)-GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133(+)-GSC-derived tumor xenografts. Subsets of CD133(+)-GSC but not CD133(-)-GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133(-)-GSC and exclusively dispersed within the tumor tissues. Although the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells.

The neurological functional disabilities caused by cerebral infarction significantly deteriorate life quality and increase the medical and socio-economic costs. Although some molecular agents show potential in acting against the pathological mechanisms in animal studies, none has been proven effective for cerebral ischemia treatment in human patients. New treatment strategy needs to be developed. Stem cell therapy is promising for neural regeneration and thus become one of the current trends. More evidence has shown stem cells, such as embryonic stem cells (ESCs), skeletal muscle satellite cells and mesenchymal stem cells, to be useful in tissue repair and regeneration. However all these stem cells mentioned above have limitations. Adipose tissue-derived stem cells (ADSCs) are an alternative autologous stem cell source for the characters as abundant, easy to obtain, immunological and ethic problem free. So far, this treatment strategy has been rarely adopted on ischemic brain injury. In this study, we investigated the transplantation effects of rat ADSCs for the treatment of cerebral ischemia in rats. ADSCs were isolated from rat adipose tissue and then induced to initiate neural differentiation. Following neural induction, ADSCs developed neural morphology and displayed molecular expression of Nestin, MAP2 and GFAP. We evaluate the neurobehavioral function, infarct volume and cell properties as apoptosis, survival, migration, proliferation, differentiation and immunogenicity. Treatment with i-ADSCs (induction from ADSCs) results in better functional recovery and more reduction in hemispheric atrophy then without i-ADSCs in other groups. Our study demonstrates that i-ADSCs therapy is promising in stroke treatment and finally leads to an efficacious therapeutic modalities for much better outcome in clinical patients.

To modulate the mechanical properties of nerve guide conduit for surgical manipulation, this study develops a biodegradable composite containing genipin cross-linked gelatin annexed with β-tricalcium phosphate ceramic particles as a nerve guide material. The conduit was dark bluish and round with a rough and compact outer surface compared to the genipin cross-linked gelatin conduit (without β-tricalcium phosphate). Water uptake and swelling tests indicate that the conduit noticeably increases the stability in water, and the hydrated conduit does not collapse and stenose. The conduit has a sufficiently high level of mechanical properties to serve as a nerve guide. After subcutaneous implantation on the dorsal side of a rat, the degraded conduit only evokes a mild tissue response, and the formation of a very thin fibrous capsule surrounds the conduit. This paper assesses the effectiveness of the conduit as a guidance channel when we use it to repair a 10 mm gap in the rat's sciatic nerve. The experimental results show no gross inflammatory reactions of the peripheral nerve tissues at the implantation site in either group. In overall gross examination, the diameter of the intratubular and newly formed nerve fibers in the conduits exceeds that of the silicone tubes during the implantation period. The quantitative results indicate the superiority of the conduits over the silicone tubes. This study microscopically observes the nerve regeneration in the tissue section at the middle region of all implanted conduits. Therefore, the histomorphometric assessment demonstrates that the conduit could be a candidate for peripheral nerve repair.

This study proposes a biodegradable GGT composite nerve guide conduit containing genipin-cross-linked gelatin and tricalcium phosphate (TCP) ceramic particles in peripheral nerve regeneration. The proposed genipin-cross-linked gelatin annexed with TCP ceramic particles (GGT) conduit was dark bluish and round with a rough and compact surface. Water uptake and swelling tests indicated that the hydrated GGT conduit exhibited increased stability with not collapsing or stenosis. The GGT conduit had higher mechanical properties than the genipin-cross-linked gelatin without TCP ceramic particles (GG) conduit and served as a better nerve guide conduit. Cytotoxicity tests revealed that the GGT conduit was not toxic and that it promoted the viability and growth of neural stem cells. The experiments in this study confirmed the effectiveness of the GGT conduit as a guidance channel for repairing a 10-mm gap in rat sciatic nerve. Walking track analysis showed a significantly higher sciatic function index score and better toe spreading development in the GGT group than in the silicone group 8 weeks after implantation. Gross examination revealed that the diameter of the intratubular newly formed nerve fibers in GGT conduits exceeded those in silicone tubes after the implantation period. Histological observations revealed that the morphology and distribution patterns of nerve fibers in the GGT conduits at 8 weeks after implantation were similar to those of normal nerves. The quantitative results indicated the superiority of the conduits over the silicone tubes. Motor functional and histomorphometric assessments demonstrate that the proposed GGT conduit is a suitable candidate for peripheral nerve repair.

We have a clarification to make in our paper “Low-Level Laser Stimulation on Adipose-Tissue-Derived Stem Cell Treatments for Focal Cerebral Ischemia in Rats” [1]. Because the GAPDH gene is often stably and constitutively expressed at high levels in most cells and tissues, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot. GAPDH was just used as internal control for gene expression analysis as it was supposed to have a uniform expression in both Figures 5(a) and 9(a).