Paradox: Curcumin, a Natural Antioxidant, Suppresses Osteosarcoma Cells via Excessive Reactive Oxygen Species.

Osteosarcoma (OS) is an aggressive tumor with a rare incidence. Extended surgical resections are the prevalent treatment for OS, which may cause critical-size bone defects. These bone defects lead to dysfunction, weakening the post-surgical quality of patients' life. Hence, an ideal therapeutic agent for OS should simultaneously possess anti-cancer and bone repair capacities. Curcumin (CUR) has been reported in OS therapy and bone regeneration. However, it is not clear how CUR suppresses OS development. Conventionally, CUR is considered a natural antioxidant in line with its capacity to promote the nuclear translocation of a nuclear transcription factor, nuclear factor erythroid 2 (NRF2). After nuclear translocation, NRF2 can activate the transcription of some antioxidases, thereby circumventing excess reactive oxygen species (ROS) that are deleterious to cells. Intriguingly, this research demonstrated that, in vitro, 10 and 20 µM CUR increased the intracellular ROS in MG-63 cells, damaged cells' DNA, and finally caused apoptosis of MG-63 cells, although increased NRF2 protein level and the expression of NRF2-regulated antioxidase genes were identified in those two groups.

Compressed Prostate Cancer Cells Decrease Osteoclast Activity While Enhancing Osteoblast Activity In Vitro.

Once prostate cancer cells metastasize to bone, they perceive approximately 2 kPa compression. We hypothesize that 2 kPa compression stimulates the epithelial-to-mesenchymal transition (EMT) of prostate cancer cells and alters their production of paracrine signals to affect osteoclast and osteoblast behavior. Human DU145 prostate cancer cells were subjected to 2 kPa compression for 2 days. Compression decreased expression of 2 epithelial genes, 5 out of 13 mesenchymal genes, and increased 2 mesenchymal genes by DU145 cells, as quantified by qPCR. Conditioned medium (CM) of DU145 cells was added to human monocytes that were stimulated to differentiate into osteoclasts for 21 days. CM from compressed DU145 cells decreased osteoclast resorptive activity by 38% but did not affect osteoclast size and number compared to CM from non-compressed cells. CM was also added to human adipose stromal cells, grown in osteogenic medium. CM of compressed DU145 cells increased bone nodule production (Alizarin Red) by osteoblasts from four out of six donors. Compression did not affect IL6 or TNF-α production by PC DU145 cells. Our data suggest that compression affects EMT-related gene expression in DU145 cells, and alters their production of paracrine signals to decrease osteoclast resorptive activity while increasing mineralization by osteoblasts is donor dependent. This observation gives further insight in the altered behavior of PC cells upon mechanical stimuli, which could provide novel leads for therapies, preventing bone metastases.

K-Carrageenan Stimulates Pre-Osteoblast Proliferation and Osteogenic Differentiation: A Potential Factor for the Promotion of Bone Regeneration?

Current cell-based bone tissue regeneration strategies cannot cover large bone defects. K-carrageenan is a highly hydrophilic and biocompatible seaweed-derived sulfated polysaccharide, that has been proposed as a promising candidate for tissue engineering applications. Whether κ-carrageenan can be used to enhance bone regeneration is still unclear. In this study, we aimed to investigate whether κ-carrageenan has osteogenic potential by testing its effect on pre-osteoblast proliferation and osteogenic differentiation in vitro. Treatment with κ-carrageenan (0.5 and 2 mg/mL) increased both MC3T3-E1 pre-osteoblast adhesion and spreading at 1 h. K-carrageenan (0.125-2 mg/mL) dose-dependently increased pre-osteoblast proliferation and metabolic activity, with a maximum effect at 2 mg/mL at day three. K-carrageenan (0.5 and 2 mg/mL) increased osteogenic differentiation, as shown by enhanced alkaline phosphatase activity (1.8-fold increase at 2 mg/mL) at day four, and matrix mineralization (6.2-fold increase at 2 mg/mL) at day 21. K-carrageenan enhanced osteogenic gene expression (Opn, Dmp1, and Mepe) at day 14 and 21. In conclusion, κ-carrageenan promoted MC3T3-E1 pre-osteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation, suggesting that κ-carrageenan is a potential osteogenic inductive factor for clinical application to enhance bone regeneration.

IL-1ß Damages Fibrocartilage and Upregulates MMP-13 Expression in Fibrochondrocytes in the Condyle of the Temporomandibular Joint.

The temporomandibular joint (TMJ), which differs anatomically and biochemically from hyaline cartilage-covered joints, is an under-recognized joint in arthritic disease, even though TMJ damage can have deleterious effects on physical appearance, pain and function. Here, we analyzed the effect of IL-1ß, a cytokine highly expressed in arthritic joints, on TMJ fibrocartilage-derived cells, and we investigated the modulatory effect of mechanical loading on IL-1ß-induced expression of catabolic enzymes. TMJ cartilage degradation was analyzed in 8-11-week-old mice deficient for IL-1 receptor antagonist (IL-1RA-/-) and wild-type controls. Cells were isolated from the juvenile porcine condyle, fossa, and disc, grown in agarose gels, and subjected to IL-1ß (0.1-10 ng/mL) for 6 or 24 h. Expression of catabolic enzymes (ADAMTS and MMPs) was quantified by RT-qPCR and immunohistochemistry. Porcine condylar cells were stimulated with IL-1ß for 12 h with IL-1ß, followed by 8 h of 6% dynamic mechanical (tensile) strain, and gene expression of MMPs was quantified. Early signs of condylar cartilage damage were apparent in IL-1RA-/- mice. In porcine cells, IL-1ß strongly increased expression of the aggrecanases ADAMTS4 and ADAMTS5 by fibrochondrocytes from the fossa (13-fold and 7-fold) and enhanced the number of MMP-13 protein-expressing condylar cells (8-fold). Mechanical loading significantly lowered (3-fold) IL-1ß-induced MMP-13 gene expression by condylar fibrochondrocytes. IL-1ß induces TMJ condylar cartilage damage, possibly by enhancing MMP-13 production. Mechanical loading reduces IL-1ß-induced MMP-13 gene expression, suggesting that mechanical stimuli may prevent cartilage damage of the TMJ in arthritic patients.

Transdifferentiation of Human Dermal Fibroblasts to Smooth Muscle-Like Cells to Study the Effect of MYH11 and ACTA2 Mutations in Aortic Aneurysms.

Mutations in genes encoding proteins of the smooth muscle cell (SMC) contractile apparatus contribute to familial aortic aneurysms. To investigate the pathogenicity of these mutations, SMC are required. We demonstrate a novel method to generate SMC-like cells from human dermal fibroblasts by transdifferentiation to study the effect of variants in genes encoding proteins of the SMC contractile apparatus (ACTA2 and MYH11) in patients with aortic aneurysms. Dermal fibroblasts from seven healthy donors and cells from seven patients with MYH11 or ACTA2 variants were transdifferentiated into SMC-like cells within a 2-week duration using 5 ng/ml TGFß1 on a scaffold containing collagen and elastin. The induced SMC were comparable to primary human aortic SMC in mRNA expression of SMC markers which was confirmed on the protein level by immunofluorescence quantification analysis and Western blotting. In patients with MYH11 or ACTA2 variants, the effect of intronic variants on splicing was demonstrated on the mRNA level in the induced SMC, allowing classification into pathogenic or nonpathogenic variants. In conclusion, direct conversion of human dermal fibroblasts into SMC-like cells is a highly efficient method to investigate the pathogenicity of variants in proteins of the SMC contractile apparatus.

EphA2 Targeted Doxorubicin-Nanoliposomes for Osteosarcoma Treatment.

PURPOSE: To employ Doxorubicin-loaded liposomes, modified with YSA-peptide to target EphA2, to reduce adverse effects against primary bone cells and maximize toxicity against Saos-2 osteosarcoma cells. METHODS: PEGylated liposomes were prepared by thin film method using Dipalmitoylphosphatidylcholine (DPPC), cholesterol and distearylphosphatidylethanolamine-polyethyleneglycol conjugate (DSPE-mPEG) in 67.9:29.1:3 M ratios, and loaded with DOX (L-DOX) by pH-gradient method. Targeted liposomes (YSA-L-DOX), were prepared by conjugating YSA-peptide to DSPE-mPEG. Liposomes were physicochemically characterized and tested in cellular toxicity assays. RESULTS: YSA conjugation efficiency was >98%. Size and polydispersity index of both L-DOX and YSA-L-DOX were around 88 nm and 0.188, respectively. Both had similar zeta potential, and 85% DOX loading efficiencies. DOX release kinetics followed the Korsmeyer-Peppa model, and showed comparable release for both formulations from 1-8 h, and a plateau of 29% after 48 h. Both formulations could be stably stored for ≥6 months at 4°C in the dark. Toxicity assays showed a significant 1.91-fold higher cytotoxicity compared to free DOX in the Saos-2 cells, and 2-fold lesser toxicity in primary bone cells compared to the Saos-2 cells. Cellular uptake studies showed higher and more nuclear uptake in YSA-L-DOX compared to L-DOX treated cells. CONCLUSIONS: YSA-L-DOX vesicles might be effective for targeted treatment of osteosarcoma.

Toward a new generation of pelvic floor implants with electrospun nanofibrous matrices: A feasibility study.

OBJECTIVE: The use of knitted, polypropylene meshes for the surgical treatment of pelvic organ prolapse (POP) is frequently accompanied by severe complications. Looking for alternatives, we studied the potential of three different electrospun matrices in supporting the adhesion, proliferation, and matrix deposition of POP and non-POP fibroblasts, the most important cells to produce extracellular matrix (ECM), in vitro. STUDY DESIGN: We electrospun three commonly used medical materials: nylon; poly (lactide-co-glycolide) blended with poly-caprolactone (PLGA/PCL); and poly-caprolactone blended with gelatin (PCL/Gelatin). The matrices were characterized for their microstructure, hydrophilicity, and mechanical properties. We seeded POP and non-POP fibroblasts from patients with POP and we determined cellular responses and ECM deposition. RESULTS: All matrices had >65% porosity, homogenous microstructures, and close to sufficient tensile strength for pelvic floor repair: 15.4 ± 3.3 MPa for Nylon; 12.4 ± 1.6 MPa for PLGA/PCL; and 3.5 ± 0.9 MPa for PCL/Gelatin. Both the POP and non-POP cells adhered to the electrospun matrices; they proliferated well and produced ample ECM. Overall, the best in vitro performance appeared to be on nylon, presumably because this was the most hydrophilic material with the thinnest fibers. CONCLUSION: Electrospun nanofibrous matrices show feasible mechanical strength and great biocompatibility for POP and non-POP fibroblasts to produce their ECM in vitro and, thus, may be candidates for a new generation of implants for pelvic floor repair. Further studies on electrospun nanofibrous matrices should focus on mechanical and immunological conditions that would be presented in vivo. Neurourol. Urodynam. 36:565-573, 2017. © 2016 Wiley Periodicals, Inc.

Flow Preconditioning of Endothelial Cells on Collagen-Immobilized Silicone Fibers Enhances Cell Retention and Antithrombotic Function.

Stability and antithrombotic functionality of endothelial cells on silicone hollow fibers (SiHFs) are critical in the development of biohybrid artificial lungs. Here we aimed to enhance endothelial cell retention and anti-thrombotic function by low (12 dyn/cm2 , 24 h) fluid shear stress ("flow") preconditioning of endothelial cells seeded on collagen-immobilized SiHFs. The response of endothelial cells without preconditioning (48 h static culture) and with preconditioning (24 h static culture followed by 24 h flow preconditioning) on hollow fibers to high fluid shear stress (30 dyn/cm2 , 1 h) was assessed in a parallel-plate flow chamber. Finite element (FE) modeling was used to simulate shear stress within the flow chamber. We found that collagen immobilization on hollow fibers using carbodiimide bonds provided sufficient stability to high shear stress. Flow preconditioning for 24 h before treatment with high shear stress for 1 h on collagen-immobilized hollow fibers increased cell retention (1.3-fold). The FE model showed that cell flattening due to flow preconditioning reduced maximum shear stress on cells by 32%. Flow preconditioning prior to exposure to high fluid shear stress enhanced the production of nitric oxide (1.3-fold) and prostaglandin I2 (1.2-fold). In conclusion, flow preconditioning of endothelial cells on collagen-immobilized SiHFs enhanced cell retention and antithrombotic function, which could significantly improve current biohybrid artificial lungs.

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator-deficient mice.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.

Functional characteristics of vaginal fibroblastic cells from premenopausal women with pelvic organ prolapse.

Pelvic organ prolapse (POP) remains a great therapeutic challenge with no optimal treatment available. Tissue maintenance and remodelling are performed by fibroblasts, therefore altered cellular functionality may influence tissue quality. In this study, we evaluated functional characteristics of fibroblastic cells from tissues involved in POP. To rule out normal ageing tissue degeneration, biopsies from 18 premenopausal women were collected from the precervical region (non-POP site) after hysterectomy of 8 healthy and 10 POP cystocele cases (POP-Q stage ≥ II). Extra tissues from the prolapsed sites were taken in the POP cases to distinguish between intrinsic and acquired cellular defects. Twenty-eight primary fibroblastic cultures were studied in vitro. A contractility assay was used to test fibroblast-mediated collagen contraction. Cellular mechanoresponses on collagen-coated or uncoated substrates were evaluated by measuring matrix remodelling factors at protein or gene expression levels. No differences were found between fibroblasts from the controls and the non-POP site of the case group. Fibroblastic cells from the prolapsed site showed delayed fibroblast-mediated collagen contraction and lower production of matrix metalloproteinase-2 (MMP-2) on collagen-coated plates. On uncoated surfaces the gene MMP-2 and its tissue inhibitor of metalloproteinases-2 were up-regulated in POP site fibroblastic cells. In conclusion, fibroblastic cells derived from prolapsed tissues of patients with cystocele, display altered in vitro functional characteristics depending on the surface substrate and compared with non-prolapsed site. This implies an acquired rather than an intrinsic defect for most patients with cystocele, and should be taken into account when trying to improve treatments for POP.

Notch signaling is involved in human articular chondrocytes de-differentiation during osteoarthritis.

CONTEXT: During osteoarthritis (OA), chondrocytes undergo de-differentiation, resulting in the acquisition of a fibroblast-like morphology, decreased expression of collagen type II (colII) and aggrecan, and increased expression of collagen type I (colI), metalloproteinase 13 (MMP13) and nitric oxide synthase (eNOS). Notch signaling plays a crucial role during embryogenesis. Several studies showed that Notch is expressed in adulthood. OBJECTIVE: The aim of our study was to confirm the involvement of Notch signaling in human OA at in vitro and ex vivo levels. MATERIALS AND METHODS: Normal human articular chondrocytes were cultured during four passages either treated or not with a Notch inhibitor: DAPT. Human OA cartilage was cultured with DAPT for five days. Chondrocytes secreted markers and some Notch pathway components were analyzed using Western blotting and qPCR. RESULTS: Passaging chondrocytes induced a decrease in the cartilage markers: colII and aggrecan. DAPT-treated chondrocytes and OA cartilage showed a significant increase in healthy cartilage markers. De-differentiation markers, colI, MMP13 and eNOS, were significantly reduced in DAPT-treated chondrocytes and OA cartilage. Notch1 expression was proportional to colI, MMP13 and eNOS expression and inversely proportional to colII and aggrecan expression in nontreated cultured chondrocytes. Notch ligand: Jagged1 increased in chondrocytes culture. DAPT treatment resulted in reduced Jagged1 expression. Notch target gene HES1 increased during chondrocyte culture and was reduced when treated with DAPT. CONCLUSION: Targeting Notch signaling during OA might lead to the restitution of the typical chondrocyte phenotype and even to chondrocyte redifferentiation during the pathology.

Applying shear stress to endothelial cells in a new perfusion chamber: hydrodynamic analysis.

Perfusion bioreactors have been proved to be an impartible part of vascular tissue engineering due to its broad range of applications as a means to distribute nutrients within porous scaffold along with providing appropriate physical and mechanical stimuli. To better understand the mechanical phenomena inside a bioreactor, computational fluid dynamics (CFD) was adopted followed by a validation technique. The fluid dynamics of the media inside the bioreactor was modeled using the Navier-Stokes equation for incompressible fluids while convection through the scaffold was described by Brinkman's extension of Darcy's law for porous media. Flow within the reactor determined the orientation of endothelial cells on the scaffold. To validate flow patterns, streamlines and shear stresses, colorimetry technique was used following attained results from CFD. Our bioreactor was modeled to simulate the optimum condition and flow patterns over scaffold to culture ECs for in vitro experimentation. In such experiments, cells were attached firmly without significant detachment and more noticeably elongation process was triggered even shortly after start up.

Growth and mineralization of fetal mouse long bones under microgravity and daily 1 g gravity exposure.

In a previous Space Shuttle/Spacelab experiment (STS-42), we observed direct responses of isolated fetal mouse long bones to near weightlessness. This paper aimed to verify those results and study the effects of daily 1×g exposure during microgravity on the growth and mineralization of these bones. Two experiments were conducted: one on an American Space Shuttle mission (IML-2 on STS-65) and another on a Russian Bio-Cosmos flight (Bion-10 on Cosmos-2229). Despite differences in hardware, both used 17-day-old fetal mouse metatarsals cultured for 4 days. Results showed reduced proteoglycan content under microgravity compared to 1×g conditions, with no main differences in other cellular structures. While the overall metatarsal length was unaffected, the length increase of the mineralized diaphysis was significantly reduced under microgravity. Daily 1×g exposure for at least 6 h abolished the microgravity-induced reduction in cartilage mineralization, indicating the need for long-duration exposure to 1×g as an in-flight countermeasure using artificial gravity.

Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates.

INTRODUCTION AND HYPOTHESIS: Little is known about dynamic cell-matrix interactions in the context of pathophysiology and treatments for pelvic organ prolapse (POP). This study sought to identify differences between fibroblasts from women with varying degrees of prolapse in reaction to mechanical stimuli and matrix substrates in vitro. METHODS: Fibroblasts from the vaginal wall of three patients with POP Quantification (POP-Q) system stages 0, II, and IV were stretched on artificial polymer substrates either coated or not coated with collagen I. Changes in morphology and anabolic/catabolic compounds that affect matrix remodelling were evaluated at protein- and gene-expression levels. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey-Kramer's post hoc test. RESULTS: POP fibroblasts show delayed cell alignment and lower responses to extracellular matrix remodelling factors at both enzymatic- and gene-expression levels compared with healthy fibroblasts. CONCLUSION: POP fibroblasts, when compared with healthy cells, show differential mechanoresponses on two artificial polymer substrates. This should be taken into account when designing or improving implants for treating POP.

Osteogenic Activity on NaOH-Etched Three-Dimensional-Printed Poly-ɛ-Caprolactone Scaffolds in Perfusion or Spinner Flask Bioreactor.

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

Radiation modulates expression and related activities of c-Met protein in oral tongue squamous cell carcinoma cell lines.

OBJECTIVES: c-Met, a receptor tyrosine kinase, is involved in the growth, invasion and metastasis of a variety of cancers. In a set of cell lines from several solid tumors, a five-fold increase in c-Met expression after irradiation has been reported. This study aimed to assess if c-Met is likewise abundantly expressed in oral tongue squamous cell carcinoma (OTSCC) upon exposure to irradiation, followed by a Met-induced biological response. MATERIALS AND METHODS: Six OTSCC cell lines were exposed to gamma radiation doses of 2, 4, and 6 Gray. The changes in c-Met protein levels were assessed by western blot and flow cytometry. c-Met gene expression, cell migration, proliferation and cell cycle assays were performed as phenotypic readouts. RESULTS: Irradiation resulted in upregulation of c.Met in all cell lines with different time kinetics. On average the cells displayed minimal c-Met expression on their surface ranging from 5 to 30% of total protein. Abrupt downregulation of c-Met surface expression occurred one hour after radiation but recovered 48 h post-radiation. Intracellularly, the highest level of expression was found on day 5 after radiation exposure. Irradiation induced aggressive invasive potential of the cells as determined in cell migration assays, particularly in cell lines with the highest c-Met expression. CONCLUSIONS: These results provide novel insights into both intracellular and extracellular dynamics of c-Met expression profiles upon irradiation of OTSCC cells in vitro. It might also suggest that radiation enhances cell migration, indicative of invasiveness, through c-Met up-regulation, at least for certain types of OTSCC cells.

Fungal Infected Adipose Stem Cells: The Effects of Novel Lipo-Niosome Nanoparticles Loaded with Amphotericin B and Thymus Essential Oil.

OBJECTIVE: In this study, we aimed to develop new Lipo-niosomes based nanoparticles loaded with Amphotericin B (AmB) and Thymus Essential Oil (TEO) and test their effectiveness in the treatment of fungal-infected human adipose stem cells (hASCs). MATERIALS AND METHODS: In this experimental study, optimal formulation of AmB and TEO loaded lipo-niosome (based on lipid-surfactant thin-film hydration method) was chemically, and biologically characterized. Therefore, encapsulation capacity, drug release, size, and the survival rate of cells with different concentrations of free and encapsulated AmB/ TEO were evaluated using the MTT method, and its antifungal activity was compared with conventional AmB. RESULTS: Lipo-Niosome containing Tween 60 surfactant: cholesterol: Dipalmitoyl phosphatidylcholine (DPPC): Polyethylene glycol (PEG) with a ratio of 20:40:60:3 were chosen as optimal formulation. Lipo-Niosomes entrapment efficiency was 94.15%. The drug release rate after 24 hours was 52%, 54%, and 48% for Lipo-AmB, Lipo-TEO, and Lipo-AmB/TEO, respectively. Physical and chemical characteristics of the Lipo-Niosomes particles indicated size of 200 nm and a dispersion index of 0.32 with a Zeta potential of -24.56 mv. Furthermore, no chemical interaction between drugs and nano-carriers was observed. The cell viability of adipose mesenchymal stem cells exposed to 50 µg/ml of free AmB, free TEO, and free AmB/TEO was 13.4, 58, and 36.9%, respectively. Whereas the toxicity of the encapsulated formulas of these drugs was 48.9, 70.8, and 58.3% respectively. The toxicity of nanoparticles was very low (8.5%) at this concentration. Fluorescence microscopic images showed that the antifungal activity of Lipo-AmB/ TEO was significantly higher than free formulas of AmB, TEO, and AmB/TEO. CONCLUSION: In this study, we investigated the efficacy of the TEO/AmB combination, in both free and encapsulatedniosomal form, on the growth of fungal infected-hASCs. The results showed that the AmB/TEO-loaded Lipo-Niosomes can be suggested as a new efficient anti-fungal nano-system for patients treated with hASCs.

Multifunctional PEGylated Niosomal Nanoparticle-Loaded Herbal Drugs as a Novel Nano-Radiosensitizer and Stimuli-Sensitive Nanocarrier for Synergistic Cancer Therapy.

Nowadays, radiotherapy is one of the most effective treatments for breast cancer. In order to overcome the radioresistance of cancer cells, radio-sensitizing agents can be used combined with irradiation to increase the therapeutic efficiency. Curcumin can enhance the radiosensitivity of cancer cells and decrease their viability by the accumulation of these cells in the G2 phase. The encapsulation of curcumin in a nanoniosomal delivery system increases aqueous solubility and bioavailability, resulting in increased radio sensitivity. The present study aimed to enhance the radio-sensitizing effect of the curcumin-containing nanoniosome (Cur-Nio) when combined with irradiation. Thus, curcumin (0.5 mg ml-1) was loaded on a PEGylated nanoniosome containing Tween 60, cholesterol, DOTAP, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) (at ratios of 70:30:10:5, respectively) by the thin-film hydration method. The particle size, zeta potential, entrapment efficiency, and drug-release rate of formulated nanoniosomes were determined. In order to assess cytotoxicity and apoptosis, different doses of irradiation along with various concentrations of free curcumin and Cur-Nio (single or in combination with irradiation) were treated with breast cancer cells. The particle size and zeta potential of Cur-Nio were reported to be 117.5 nm and -15.1 mV, respectively. The entrapment efficiency (EE%) and loading capacities were 72.3% and 6.68%, respectively. The drug-release rate during 6 h was 65.9%. Cell survival in the presence of curcumin at doses of 1 and 3 Gy showed a significant reduction compared with cells irradiated at 48 h and 72 h (p < 0.000). Also, the rate of cytotoxicity and apoptosis was significantly higher in cells treated with the combination of curcumin-containing nanoniosomes and irradiation in comparison with those treated with free curcumin. These findings indicate that the efficacy of pre-treatment with Cur-Nio as a radiosensitizer during radiotherapy enhances irradiation-induced breast cancer cell apoptosis and is a useful strategy to increase the effectiveness of breast cancer therapy.

Developmental expression of solute carrier family 26A member 4 (SLC26A4/pendrin) during amelogenesis in developing rodent teeth.

Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.

To B (Bone Morphogenic Protein-2) or Not to B (Bone Morphogenic Protein-2): Mesenchymal Stem Cells May Explain the Protein's Role in Osteosarcomagenesis.

Osteosarcoma (OS), a primary malignant bone tumor, stems from bone marrow-derived mesenchymal stem cells (BMSCs) and/or committed osteoblast precursors. Distant metastases, in particular pulmonary and skeletal metastases, are common in patients with OS. Moreover, extensive resection of the primary tumor and bone metastases usually leads to bone defects in these patients. Bone morphogenic protein-2 (BMP-2) has been widely applied in bone regeneration with the rationale that BMP-2 promotes osteoblastic differentiation of BMSCs. Thus, BMP-2 might be useful after OS resection to repair bone defects. However, the potential tumorigenicity of BMP-2 remains a concern that has impeded the administration of BMP-2 in patients with OS and in populations susceptible to OS with severe bone deficiency (e.g., in patients with genetic mutation diseases and aberrant activities of bone metabolism). In fact, some studies have drawn the opposite conclusion about the effect of BMP-2 on OS progression. Given the roles of BMSCs in the origination of OS and osteogenesis, we hypothesized that the responses of BMSCs to BMP-2 in the tumor milieu may be responsible for OS development. This review focuses on the relationship among BMSCs, BMP-2, and OS cells; a better understanding of this relationship may elucidate the accurate mechanisms of actions of BMP-2 in osteosarcomagenesis and thereby pave the way for clinically safer and broader administration of BMP-2 in the future. For example, a low dosage of and a slow-release delivery strategy for BMP-2 are potential topics for exploration to treat OS.