Fabrication of water-dispersible and cell-stimulating calcium phosphate nanoparticles immobilizing basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a therapeutic protein that can enhance angiogenesis, wound healing, and tissue regeneration; however, it is extremely unstable even under a normal physiological environment. Biocompatible calcium phosphate (CaP) nanoparticles (NPs) co-immobilizing bFGF, heparin, and ferucarbotran would be useful as a multifunctional delivery carrier of bFGF. In this study, such NPs were successfully fabricated by a coprecipitation process, using a labile supersaturated CaP solution containing bFGF, heparin, and ferucarbotran. The NPs showed relatively high negative zeta potential (-12 mV) because of the negatively charged heparin, which enabled their stable dispersion in water. The hydrodynamic diameter of the NPs was around 200 nm. Immunoreactive bFGF was released from the NPs in an acellular medium dose-dependently. The NPs promoted proliferation of baby hamster kidney fibroblasts (BHK-21 cells) and mouse osteoblastic MC3T3-E1 cells at a certain dose range, although they inhibited proliferation of rat pheochromocytoma (PC-12) cells. These results demonstrated that the effect of the NPs on cell proliferation was dependent on the cell type and dose, the details of which should be investigated in a future study.

Storable bFGF-Releasing Membrane Allowing Continuous Human iPSC Culture.

Basic fibroblast growth factor (bFGF) is a crucial supplement for culture media of human pluripotent stem cells. However, bFGF is extremely unstable under cell culture conditions, which makes frequent (generally every day) medium refreshment requisite. We recently developed a water-floatable, bFGF-releasing membrane via a simple bFGF adsorption process following oxygen plasma treatment by utilizing a polyethylene nonwoven fabric as an adsorbent. This membrane allowed sustained release of bFGF while floating on medium, thereby keeping the bFGF concentration in the medium sufficient for maintaining human-induced pluripotent stem cells (iPSCs) in a proliferative and pluripotent state for as long as 3 days. In this study, lyophilization was applied to the membrane to stabilize bFGF. The sustained bFGF-releasing function of the membrane was kept unchanged even after lyophilization and subsequent cryopreservation at -30 °C for 3 months. The cryopreserved membrane supported proliferation and colony formation of human iPSCs while retaining their viability and pluripotency in a medium-change-free continuous culture for 3 days. The present bFGF-releasing membrane is ready-to-use, storable for at least 3 months, and obviates daily medium refreshment. Therefore, it is a new and more practical bFGF supplement for culture media of human stem cells.

Controlled release of basic fibroblast growth factor from a water-floatable polyethylene nonwoven fabric sheet for maintenance culture of iPSCs.

Basic fibroblast growth factor (bFGF) is an essential supplement for culture media to support the proliferation of human pluripotent stem cells, while preserving their pluripotency. However, it is extremely unstable under cell culture conditions at 37 °C. Therefore, a culture medium supplemented with bFGF needs to be changed every day to maintain an effective concentration of bFGF. This study aimed to create a bFGF-releasing material via simple bFGF adsorption following oxygen plasma treatment by using a water-floatable polyethylene (PE) nonwoven fabric sheet as a bFGF-adsorbent material. Preliminary oxygen plasma treatment enhanced bFGF adsorption onto the sheet by increasing its surface water wettability. Based on the bFGF concentration in the adsorption solution, the resulting bFGF-adsorbed sheet showed different bFGF-release profiles in the culture medium. The bFGF-adsorbed sheet prepared under optimum conditions released bFGF in a sustained manner, maintaining the bFGF concentration in the culture medium of human induced pluripotent stem cells (iPSCs) at ≥10 ng mL-1 even without medium change for as long as 3 d. The bFGF released from the sheet retained its biological activity to support colony formation of iPSCs while preserving their pluripotency. This type of bFGF-releasing sheet can be used as a new form of bFGF supplement for the culture media of stem cells and would make a significant contribution to stem cell-based research and development.

Calcium phosphate nanoparticles prepared from infusion fluids for stem cell transfection: process optimization and cytotoxicity analysis.

This is the first study to report the use of infusion fluids for particle-mediated gene delivery with DNA-immobilized calcium phosphate (CaP) nanoparticles (NPs). In conventional CaP systems, CaP NPs are fabricated in labile supersaturated CaP solutions which are prepared from chemical reagents. In the present study, we fabricated CaP NPs via coprecipitation in labile supersaturated CaP solutions that were prepared from infusion fluids (even the water used was of injectable quality) instead of chemical reagents and demonstrated their gene delivery capabilities for the hard to transfect pluripotent stem cell (C3H10T1/2) along with the easy to transfect CHO-K1 cell. To achieve a high gene delivery capability by keeping the high safety level of our system intact, we varied the process parameters: coprecipitation temperature and time, along with the Ca and P concentrations of the CaP solution, without using additive agents (e.g. surfactants) other than infusion fluids and plasmids. The optimization of these process parameters led to a higher gene delivery capability compared with that of a commercial CaP system for both types of cells. MTT and protein assays showed that both our system and the commercial CaP system were not cytotoxic to both types of cells. Our CaP system has the advantages of high biological safety (due to injectable source materials), high serum-resistance, and relatively high and controllable gene delivery capability, depending on the process parameters. Thus, the present system warrants consideration for gene delivery applications.

Controlled superficial assembly of DNA-amorphous calcium phosphate nanocomposite spheres for surface-mediated gene delivery.

Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA-amorphous calcium phosphate (DNA-ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly's high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA-ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly's gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA-ACP nanocomposite spheres.

[The Interaction of Low-dose Droperidol, Propofol, and Sevoflurane on QTc Prolongation].

BACKGROUND: Droperidol is an effective antiemetic, but its use is limited because of the warning of drug-induced QT prolongation. Some reports showed that low-dose droperidol does not significantly probing QT interval. This study was aimed to determine the effect of low-dose droperidol (1.25 and 2.5 mg) on QTc interval, and the interaction among droperidol, propofol and sevoflurane. METHODS: Patients received either 1.25 mg (group L : n = 25) or 2.5 mg (group H : n = 25) droperidol, and fentanyl (3 µg x kg(-1)) was administered 2.5 min later. One minute after fentanyl administration, anesthesia was induced using propofol (1.5 mg x kg(-1)) and vecuronium. One minute after propofol administration, sevoflurane (3%) was started. Tracheal intubation was performed 3 min after propofol administration, and then sevoflurane was reduced to 1%. RESULTS: Compared to baseline, the QTc interval in group L was unchanged by droperidol. In group H, the QTc interval was significantly prolonged after droperidol injection, but recovered after propofol injection. After tracheal intubation, QTc interval was significantly prolonged in both groups. CONCLUSIONS: Droperidol's effect on QTc prolongation was shown at the dose of 2.5 mg but not 1.25 mg. This prolongation effect was offset by propofol, and was unchanged by sevoflurane.

Comparative efficacy of levobupivacaine and ropivacaine for epidural block in outpatients with degenerative spinal disease.

BACKGROUND: Levobupivacaine has less toxic potential on both the cardiovascular and central nervous system and has been widely used for postoperative epidural analgesia in surgical patients. However, there are few reports on the efficacy of epidural levobupivacaine in outpatients with lumbosacral radiculopathy. This study was carried out to evaluate the comparative efficacy of levobupivacaine and ropivacaine for epidural block in outpatients with degenerative spinal disease and sciatica. OBJECTIVE: We studied 32 patients (19 men and 13 women) with degenerative spinal disease and sciatica. STUDY DESIGN: The study was performed in a prospective, randomized, double blind, and crossover fashion. SETTING: Treatment room for outpatients. METHODS: The epidural block was produced with a caudal approach (0.125% levobupivacaine or 0.2% ropivacaine, 15 mL). The upper level of analgesia, lumbosacral pain, motor blockade, and hemodynamic changes were evaluated by pin prick, visual analogue scale (VAS), Bromage scale, and arterial blood pressure and heart rate at 15, 30, 60, and 90 minutes after epidural block, respectively. The recovery time to mobilization, ambulation, and spontaneous micturition were measured. RESULTS: There were no significant differences (P < 0.05) in the upper level of analgesia, VAS, and Bromage scale between 0.125% levobupivacaine and 0.2% ropivacaine throughout the time course. There were no significant differences in the recovery times to mobilization, ambulation, and spontaneous micturition between 0.125% levobupivacaine and 0.2% ropivacaine. There were no significant differences in arterial blood pressure and heart rate between the 2 trials throughout the time course. CONCLUSION: The results showed that 0.125% levobupivacaine and 0.2% ropivacaine for epidural block by a caudal approach provide similar lumbosacral pain relief, hemodynamic effects, and the degree and the recovery of motor blockade in outpatients with degenerative spinal disease and sciatica.

Improved gene transfer efficiency of a DNA-lipid-apatite composite layer by controlling the layer molecular composition.

Surface-mediated nonviral gene transfer systems using biocompatible apatite-based composite layers have potential use in tissue engineering applications. Herein, we investigated a relatively efficient system based on a DNA-lipid-apatite composite layer (DLp-Ap layer): an apatite (Ap) layer with immobilized DNA and lipid (Lp) complexes (DLp complexes). DLp-Ap layers were fabricated on substrates using supersaturated calcium phosphate coprecipitation solutions supplemented with DLp complexes, and the molecular compositions of the DLp-Ap layers were controlled by varying the net DNA concentrations and Lp/DNA ratios in the coprecipitation solutions. Increases in both the DNA concentration and Lp/DNA ratio in the coprecipitation solution increased the DLp complex content of the resulting DLp-Ap layer. However, a higher DLp complex content did not always provide increased gene transfer efficiency to the CHO-K1 cells, because there was a threshold content of approximately 10µg/cm(2). In addition, DLp-Ap layers with similar DLp complex contents exhibited different gene transfer efficiencies, most likely due to the different Lp/DNA ratios in the layers. Notably, the optimized Lp/DNA ratios in the coprecipitation solutions for maximizing the gene transfer efficiency were lower than those of the conventional particle-mediated lipofection systems. These findings will serve as a useful design guide for the preparation of DLp-Ap layers with high gene transfer efficiency.

Coprecipitation of DNA-lipid complexes with apatite and comparison with superficial adsorption for gene transfer applications.

Apatite can mediate gene transfer into cells by serving as a safe and biocompatible immobilization matrix for DNA and transfection reagents. Recently, an apatite layer that immobilized DNA-lipid complexes was prepared by a coprecipitation process in a supersaturated calcium phosphate solution. This composite layer (DNA-lipid-apatite layer) showed a higher gene transfer capability than an apatite layer with superficially adsorbed DNA-lipid complexes (DNA-lipid-adsorbed apatite layer). In this study, the DNA-lipid-apatite layer and the DNA-lipid-adsorbed apatite layer were compared for their physicochemical properties and gene transfer capabilities. The higher gene transfer capability of the DNA-lipid-apatite layer compared with that of the DNA-lipid-adsorbed apatite layer was reconfirmed by a luciferase assay using epithelial-like CHO-K1 cells. Physicochemical structure analyses showed that the DNA-lipid-apatite layer possessed a larger capacity for DNA-lipid complexes than the DNA-lipid-adsorbed apatite layer. The DNA-lipid-apatite layer released DNA-lipid complexes in a slow and sustained manner, whereas the DNA-lipid-adsorbed apatite layer released them in short bursts. Consequently, the release of DNA-lipid complexes from the DNA-lipid-apatite layer was larger in amount and longer in duration than release from the DNA-lipid-adsorbed apatite layer. This difference in release profiles may be responsible for the higher gene transfer capability of the DNA-lipid-apatite layer compared with that of the DNA-lipid-adsorbed apatite layer. The coprecipitation process and the resulting DNA-lipid-apatite layer have many applications in tissue engineering.

Fabrication of a DNA-lipid-apatite composite layer for efficient and area-specific gene transfer.

A surface-mediated gene transfer system using biocompatible apatite-based composite layers has great potential for tissue engineering. Among the apatite-based composite layers developed to date, we focused on a DNA-lipid-apatite composite layer (DLp-Ap layer), which has the advantage of relatively high efficiency as a non-viral system. In this study, various lipid transfection reagents, including a newly developed reagent, polyamidoamine dendron-bearing lipid (PD), were employed to prepare the DLp-Ap layer, and the preparation condition was optimized in terms of efficiency of gene transfer to epithelial-like CHO-K1 cells in the presence of serum. The optimized DLp-Ap layer derived from PD had the highest gene transfer efficiency among all the apatite-based composite layers prepared in this study. In addition, the optimized DLp-Ap layer demonstrated higher gene transfer efficiency in the presence of serum than the conventional particle-mediated systems using commercially available lipid transfection reagents. It was also shown that the optimized DLp-Ap layer mediated the area-specific gene transfer on its surface, i.e., DNA was preferentially transferred to the cells adhering to the surface of the layer. The present gene transfer system using the PD-derived DLp-Ap layer, with the advantages of high efficiency in the presence of serum and area-specificity, would be useful in tissue engineering.

Fabrication of DNA-antibody-apatite composite layers for cell-targeted gene transfer.

Surface-mediated gene transfer systems using apatite (Ap)-based composite layers have received increased attention in tissue engineering applications owing to their safety, biocompatibility and relatively high efficiency. In this study, DNA-antibody-apatite composite layers (DA-Ap layers), in which DNA and antibody molecules are immobilized within a matrix of apatite nanocrystals, were fabricated using a biomimetic coating process. They were then assayed for their gene transfer capability for application in a specific cell-targeted gene transfer. A DA-Ap layer that was fabricated with an anti-CD49f antibody showed a higher gene transfer capability to the CD49f-positive CHO-K1 cells than a DNA-apatite composite layer (D-Ap layer). The antibody facilitated the gene transfer capability of the DA-Ap layer only to the specific cells that were expressing corresponding antigens. When the DA-Ap layer was fabricated with an anti-N-cadherin antibody, a higher gene transfer capability compared with the D-Ap layer was found in the N-cadherin-positive P19CL6 cells, but not in the N-cadherin-negative UV♀2 cells or in the P19CL6 cells that were pre-blocked with anti-N-cadherin. Therefore, the antigen-antibody binding that takes place at the cell-layer interface should be responsible for the higher gene transfer capability of the DA-Ap than D-Ap layer. These results suggest that the DA-Ap layer works as a mediator in a specific cell-targeted gene transfer system.