Alveolar Repair Using Cancellous Bone and Beta Tricalcium Phosphate Seeded With Adipose-Derived Stem Cell.

INTRODUCTION: Adipose-derived stem cells (ADSCs) have been subject of several studies due to their abundance, ease of preparation, and application in bone regeneration. We aim to compare effectiveness of alveolar reconstruction utilizing human cancellous freeze-dried graft (HCG) and beta tricalcium phosphate (BTP), both seeded with human ADSC (hADSC) and autologous bone graft (ABG). MATERIAL AND METHODS: A 5 × 5 mm alveolar defect in 36 male Wistar rats were treated using: ABG (C), HCG-hADSC (H1), and BTP-hADSC (H2). At 1 and 8 weeks after surgery, runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osterix (OSX), and bone morphogenetic protein 2 (BMP2; g/mL) were quantified using immunohistochemistry, while bone tissue volume (BV, mm3), bone tissue volume fraction (BF, percentage), and trabecular thickness of bone (TT, mm) were assessed using micro-computed tomography (CT). RESULTS: One week after surgery, H2 was higher in RUNX2, OSX, ALP, and BMP2 than C (P < .05). Only RUNX2 and OSX were found to be higher in H1 than C, while ALP and BMP2 were higher in H2 than H1. Micro-CT revealed that H2 had a higher TT than C and C had a higher TT than H1 (P < .05). Eight weeks after surgery, both H2 and H1 was higher in RUNX2, OSX, ALP, and BMP2 than C (P < .05). RUNX2 and BMP2 were found to be higher in H1 than H2. Micro-CT revealed that H2 had higher BV and TT than C and H1 (P < .05). CONCLUSIONS: Exogenous hADSC strengthened the effectiveness of HCG and BTP to accelerate osteogenesis, osteoconduction, and osteoinduction. The latter was the most successful in bone formation, followed by HCG and ABG.

Remineralization and antibacterial/antibiofilm effects of toothpaste containing nanohydroxyapatite and Curcuma aeruginosa extract.

The aim of this study was to evaluate the effect of nanohydroxyapatite (nanoHAP) and Curcuma aeruginosa (C. aeruginosa) toothpastes on tooth remineralization and antibacterial/antibiofilm activity. Remineralization was evaluated by the morphological changes in extracted human premolar teeth following toothpaste application. The antibacterial and antibiofilm activities were evaluated by agar diffusion and microdilution methods, respectively, against S. mutans. Statistical approach was utilized to formulate 20 toothpastes with different concentration of nanoHAP and C. aeruginosa. We observed that the interaction among toothpaste ingredients determined the remineralization and antibacterial/antibiofilm activities. The optimum toothpaste formula (OF1) suggested by the prediction model was shown to induce remineralization and have comparable antibacterial activity to that of the control (chlorhexidine gluconate). Furthermore, the antibiofilm activity of this formula was higher to that of the control. The result obtained indicate that these novel toothpastes have potential in decreasing caries prevalence.

Decreased Inhibition of Proliferation and Induction of Apoptosis in Breast Cancer Cell Lines (T47D and MCF7) from Treatment with Conditioned Medium Derived from Hypoxia-Treated Wharton's Jelly MSCs Compared with Normoxia-Treated MSCs.

Background : Mesenchymal stem cells (MSCs) are an appealing source of adult stem cells for cell therapy due to the high rate of proliferation, self-renewal capability, and applicable therapy. Wharton's jelly (WJ), the main component of the umbilical cord extracellular matrix, comprises multipotent stem cells with a high proliferation rate and self-renewal capability and has anti-cancer properties. MSCs have been reported to secrete a variety of cytokines that have a cytotoxic effect in various cancers. Oxygen tension affects MSCs proliferation, cytokines level but no in surface markers expression, MSCs' differentiation. We explored the cytotoxic effect and inducing apoptosis of Wharton's jelly derived mesenchymal stem cells (WJMSCs) secretions from normoxic WJMSCs (WJMSCs-norCM) (CM: conditioned medium) and hypoxic WJMSCs (WJMSCs-hypoCM) in breast cancer cell lines (T47D and MCF7). Materials and Methods: Cytotoxic activity was determined using the MTS assay. RT-PCR was performed to measure the expression of apoptosis-inducing genes, specifically P53, BAX, and CASP9, and the antiapoptotic gene BCL-2. Results: WJMSCs-norCM and WJMSCs-hypoCM were potent inhibitors of the proliferation in both cell lines. WJMSCs-norCM had more anticancer activity in T47D and MCF7. The IC50 value of WJMSCs-norCM on MCF7 was 42.34%, and on T47D was 42.36%. WJMSCs-norCM significantly induced the gene expression of apoptotic P53, BAX, and CASP9 and insignificantly decreased the antiapoptotic gene BCL-2 in both MCF7 and T47D cells. WJMSCs-CM has anticancer activity by inducing P53, BAX, and CASP9 apoptotic genes. Conclusion: WJMSCs-norCM has more anticancer activity than WJMSCs-hypoCM.

Effect of interleukins (IL-2, IL-15, IL-18) on receptors activation and cytotoxic activity of natural killer cells in breast cancer cell.

INTRODUCTION: Breast cancer is one of the leading cause of cancer deaths in women. Metastasis in BC is caused by immunosurveillance deficiency, such NK cell maturation, low NK activity and decreasing cytotoxicity. This study was performed to improve activating receptors and cytotoxicity of NK cells using interleukins (ILs). METHODS: Human recombinant IL-2, -15, and -18 were used to induce NK cells. We measured the activating and inhibiting receptors, proliferation activity of NK cells, and the cytotoxicity of NK cells on BC cells (MCF7). The effects of ILs were tested on the NK cell receptors CD314, CD158a and CD107a with flowcytometry, proliferation at various incubation times with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and concentrations of TNF-α and IFN-γ by NK cells with ELISA. RESULTS: ILs increased NK cell receptor levels (CD314, CD158a, and CD107a) at 24 hours of incubation. ILs increased NK cell viability, which increased with longer incubation. Moreover, ILs-induced NK cells inhibited proliferation in MCF7 cells, as well as increased TNF-α, IFN-γ, PRF1 and GzmB secretion. CONCLUSION: IL-2, IL-15, and IL-18 improved activating receptors and proliferation of NK cells. IL-induced NK cells increased TNF-α, IFN-γ, PRF1 and GzmB secretion and cytotoxic activity on BC cells. High NK cell numbers increased BC cell growth inhibition.

Effect of Conditioned Medium from IGF1-Induced Human Wharton's Jelly Mesenchymal Stem Cells (IGF1-hWJMSCs-CM) on Osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a chronic disease that attacks joints and bones which can be caused by trauma or other joint diseases. Stem cell and Conditioned Medium (CM) of stem cells are developed for OA therapy, which is minimally invasive. It can decrease inflammation and be a replacement for knee surgery. This study aimed to utilize human Wharton's Jelly-Mesenchymal Stem Cells (hWJMSCs) as an alternative OA therapy. METHODS: CM from hWJMSCs induced by IGF1 was collected. The OA cells model (IL1ß-CHON002) culture was treated as follows: 1) with hWJMSCs-CM 15% (v/v); 2) with hWJMSCs-CM 30% (v/v); 3) with IGF1-hWJMSCs (IGF1-hWJMSCs-CM) 15% (v/v); 4) with IGF1-hWJMSCs-CM 30% (v/v). Parameters including inflammatory cytokines (IL10 and TNFα), extracellular matrix degradation (MMP3 expression), and chondrogenic marker (COL2 expression) were determined. RESULTS: The most significant increase in COL2 chondrogenic markers was found in IL1ß-CHON002 treatment using 15% CM of hWJMSCs induced with IGF1. CM of hWJMSCs can reduce inflammatory cytokines (TNFα and IL10) and matrix degradation mediator MMP3. Better result was gained from IGF1-induced hWJMSCs-CM. CONCLUSION: CM of IGF1-hWJMSCs reduce inflammation while repairing injured joint in the human chondrocyte OA model.

Generation of human induced pluripotent stem cell line from Alzheimer's disease patient with PSEN2 N141I mutation using integration-free non-viral method.

Induced pluripotent stem cell (iPSC) line HUi001-A was reprogrammed from skin fibroblasts via non-integrating, virus free self-replicating RNA. Skin fibroblasts from an 81-year-old female Caucasian familial Alzheimer's disease patient of Volga German family carrying N141I mutation in the PSEN2 gene (familial AD4, clinical summary confirmed Alzheimer's disease) was obtained from the Coriell Institute (AG09908). Generated iPSCs were characterized and pluripotency was confirmed.

Establishment human induced pluripotent stem cell line from idiopathic non-familial Parkinson's disease patient using self-replicating RNA vector.

Induced pluripotent stem cell (iPSC) line HUi002-A was reprogrammed from skin fibroblasts via non-integrating, virus free self-replicating RNA. Skin fibroblasts from a 53-year-old male Caucasian, non-familial Parkinson's disease patient, idiopathic (clinical summary confirmed Parkinson's disease) was obtained from the Coriell Institute (AG20442). Generated iPSCs were characterized and pluripotency was confirmed.

Effects of insulin-like growth factor-induced Wharton jelly mesenchymal stem cells toward chondrogenesis in an osteoarthritis model.

OBJECTIVES: This study aimed to determine the collagen type II (COL2) and SOX9 expression in interleukin growth factor (IGF-1)-induced Wharton's Jelly mesenchymal stem cells (WJMSCs) and the level of chondrogenic markers in co-culture IGF1-WJMSCs and IL1ß-CHON002 as osteoarthritis (OA) cells model. MATERIALS AND METHODS: WJMSCs were induced with IGF1 (75, 150, and 300 ng/ml) to enhance their chondrogenesis capability. The gene expression of SOX9 and COL2 was evaluated with quantitative RT-PCR. Furthermore, IGF1-WJMSCs were co-cultured with IL1ß-CHON002 cells in varied ratios (1:2, 1:1, 2:1). Chondrogenic markers ADAMTS1, ADAMTS5, MMP3, MMP1, and RANKL were measured with ELISA. RESULTS: The IGF1-WJMSCs had an increased expression of COL2 and SOX9. ADAMTS1, ADAMTS5, MMP1, MMP3, and RANKL levels were decreased in the co-culture IGF1-WJMSCs and IL1ß-CHON002. CONCLUSION: The IGF1-induced WJMSCs were capable to enhance chondrogenesis, indicated by increased expression of SOX9 and COL2 and decreased expression of ADAMTS1, ADAMTS5, MMP3, MMP1, and RANKL. These findings can be further used in the osteoarthritis treatment.

Biocompatibility of Yttria-Tetragonal Zirconia Polycrystal Seeded with Human Adipose Derived Mesenchymal Stem Cell.

INTRODUCTION: The scaffold is a place for regeneration of new bone and bone tissue growths in tissue engineering applications. hADMSC is a multipotent cell which can differentiate into osteogenic, chondrogenic and adipogenic. Y-TZP has been shown to have several advantages over other ceramics because of its hard nature, namely fracture toughness and high flexural strength. AIM: This study aimed to analyze the biocompatibility of Y-TZP as a scaffold seeded with hADMSCs by in vitro analysis. MATERIAL AND METHODS: This research involved several processes, namely Y-TZPS manufacture process, XRD examination, differentiation and characterization of hADMSC, SEM observation, and then TT. RESULTS: The results of the XRD examination showed that Y-TZPSs had sharp peaks. It suggests that they had high crystal purity. The marked expression of the characterization of hADMSC is the positive expression of Cluster of differentiation (CD), namely CD 90, CD 73 and CD 105 above NMT and negative expressions of CD 14, CD 19, CD 34, CD 45 and also HLA-DR below NLT. The analysis of observations on the Y-TZPSs with SEM, subsequently, indicated the porosity of Y-TZPSs, as a result, the adhesion of HADMSCs occurred and grew in the porosity in the Y-TZPSs. CONCLUSIONS: Y-TZPSs with low porosity and toxicity can be able to proliferate and differentiate if seeded with hADMSC. Y-TZPSs are expected to be used as implantable biomaterials using hADMSCs with high biocompatibility.

Green tea extract protects endothelial progenitor cells from oxidative insult through reduction of intracellular reactive oxygen species activity.

OBJECTIVES: Many studies have reported that tea consumption decreases cardiovascular risk, but the mechanisms remain unclear. Green tea is known to have potent antioxidant and free radical scavenging activities. This study aimed to investigate whether green tea extract (GTE) can protect endothelial progenitors cells (EPCs) against oxidative stress through antioxidant mechanisms. MATERIALS AND METHODS: Mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation with Ficoll. The cells were then plated on fibronectin-coated culture dishes. After 7 days of culture, EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptake and lectin binding. EPCs were further identified by assessing the expression of CD34/45, CD133, and KDR. EPCs were then treated with hydrogen peroxide (H2O2) at doses of 50, 100, 200 µM and incubated with or without GTE (25 µg/ml). The intracellular reactive oxygen species (ROS) levels were detected by flow cytometry using a 2',7'-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. RESULTS: GTE ameliorated the cell viability of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 25.47, 22.52, and 11.96% higher than controls, respectively. GTE also decreased the intracellular ROS levels of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 84.24, 92.27, and 93.72% compared to controls, respectively. CONCLUSION: GTE improves cell viability by reducing the intracellular ROS accumulation in H2O2-induced EPCs.

Immunoregulatory effects of AFP domains on monocyte-derived dendritic cell function.

BACKGROUND: Alpha-fetoprotein (AFP) is a tumor-associated glycoprotein that functions in regulation of both ontogenic and oncogenic growth. Recent study showed that AFP can induce apoptosis or impair monocyte-derived dendritic cell (MDDC) function. However, it is still unclear which AFP domain (D-AFP) plays major role in this function. RESULTS: As expected monocytes cultured in the presence of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Interleukin-4 (IL-4) developed into MDDC. Up-regulation of HLA-DR and CD11c as well as loss of CD14 molecules could be observed. Full length AFP (FL-AFP), domain 2 AFP (D2-AFP) and D3-AFP, but not D1-AFP, significantly inhibited the expression of HLA-DR high/CD11c high and CD80+/CD86 high molecules. In contrast, CD83 expression was substantially down-regulated in all samples. Expression of CD40 was significantly suppressed by FL-AFP but not by any D-AFPs. Finally, both FL-AFP and D-AFP impaired the MDDC ability to secrete IL-12 (p70). CONCLUSIONS: D2- and D3- but not D1-AFP extensively suppresses the MDDC function. All the recombinant AFP proteins impaired the ability of MDDC to secrete IL-12.

Alpha-1-acid glycoprotein as potential biomarker for alpha-fetoprotein-low hepatocellular carcinoma.

BACKGROUND: The outcome of patients with hepatocellular carcinoma (HCC) remains poor because of late diagnosis. We determined the performances of α -1-acid glycoprotein (AAG) and des-γ-carboxy prothrombin (DCP) for the diagnosis of HCC, especially for α-fetoprotein (AFP)-low HCC. METHODS: Of the 220 patients included in this retrospective study, 124 had HCC, and 61 (49%) of these were AFP-low HCC (AFP ≤ 20 ng/mL). The remaining 96 patients, including 49 with chronic hepatitis B or C and 47 with cirrhosis, were considered as control. Plasma AAG was analyzed using high performance liquid chromatography (HPLC) and confirmed using Western blot technique. RESULTS: When all patients with HCC were evaluated, the area under receiver operating characteristic (ROC) curves for AAG (0.94, 95% CI: 0.91-0.97) and DCP (0.92, 95% CI: 0.88-0.95) were similar (P = 0.40). AAG had better area under ROC curve (0.96, 95% CI: 0.94-0.99) than DCP (0.87, 95% CI: 0.81-0.93) for AFP-low HCC (P < 0.05). At the specificity 95%, the sensitivity of AAG was higher in AFP-low HCC than in AFP-high HCC (82% and 62%, respectively). In contrast, higher sensitivity was obtained from DCP in discriminating HCC patients with low AFP than that in high AFP (57% and 90%, respectively). CONCLUSION: Our cross-sectional study showed that AAG was better performance in diagnosing HCC patients with low AFP, while DCP did better in those with high AFP.

Combination of alpha-1-acid glycoprotein and alpha-fetoprotein as an improved diagnostic tool for hepatocellular carcinoma.

BACKGROUND: To evaluate the diagnostic value of alpha-1-acid glycoprotein (AAG) and the combination with alpha-fetoprotein (AFP) in hepatocellular carcinoma (HCC) patients. METHODS: AAG was measured in serum of 65 HCC patients and 54 chronic liver diseases (CLD) patients by using proteomic approach. Sensitivity and specificity of AAG and its combination with AFP were determined and compared with AFP alone for the diagnosis of HCC. RESULTS: The expression concentration of AAG was significantly higher in HCC patients than chronic liver disease with sensitivity (77%) and accuracy (83%). Receiver operating characteristic analysis yielded the following AUC: AFP 0.750 (CI 95% 0.663-0.837), AAG 0.907 (CI 95% 0.855-0.960) and AFP+AAG 0.943 (CI 95% 0.897-0.988). At a specificity of 90%, the combination of AFP+AAG had sensitivity 89% and accuracy 90%, which was higher than sensitivity (52.3%) and accuracy (70%) when using AFP alone. CONCLUSION: The combination of AAG and AFP shows high sensitivity and improves the accuracy of HCC diagnosis.

Solution NMR characterizations of oligomerization and dynamics of equine infectious anemia virus matrix protein and its interaction with PIP2.

Budding of retroviruses requires the structural precursor polyprotein, Gag, to target the plasma membrane through its N-terminal matrix (MA) domain. For HIV-1, the interaction between membrane signaling molecule phosphatidylinositol 4,5-diphosphate (PIP2) and MA induces the exposure of myristate and promotes membrane binding. Here we studied oligomerization of the naturally unmyristylated equine infectious anemia virus (EIAV) MA and its interaction with PIP2-C4 primarily using solution NMR spectroscopy. The measured 1H-15N residual dipolar coupling agrees with the atomic coordinates from the EIAV MA crystal structure. The analytical ultracentrifugation results show a dominant population of monomeric EIAV MA at a concentration of 63 microM and 20 degrees C, along with a small trimer and a broad distribution of other oligomers. The monomer-trimer equilibrium model and the quaternary packing of the trimer were further established by the concentration-dependent 15N spin relaxation rates and chemical shifts. Binding of MA to PIP2-C4 was detected by chemical shift mapping (CSM) with an apparent Kd of 182 +/- 56 microM, a value similar to that reported for HIV-1 MA. The PIP2 binding site includes the Loop region between Helix2 and Helix3 in the EIAV MA. CSM and spin relaxation dispersion reveal a coupling of conformational change and submillisecond dynamics, respectively, between the Loop and trimeric Interface Residues due to PIP2 binding. We infer that PIP2 participates in the initial trimer formation of EIAV MA, but more importantly, the concentration effect is dominant in shifting the equilibrium toward trimer, in line with the entropic switch mechanism proposed for myristylated HIV-1 MA.