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In the present study we demonstrate that the incorporation of magnetic particles gives rise to bioengineered oral mucosa tissue substitutes with a tunable, reversible mechanical response. In tissue engineering applications this versatility should make it possible to adjust the mechanical properties of the artificial tissue substitutes with precision, in order to match the properties of the target tissue at the site of implantation. This study was approved by the Ethics Committee of the University of Granada, Granada, Spain.

Each tissue donor signed an informed consent form for this study. Ten normal human oral mucosa biopsies with an average volume of 8 mm3 were obtained from healthy donors at the School of Dental Sciences of the University of Granada. The medium was changed every 3 days, and the cells were subcultured in a solution of 0.

For all experiments we la roche posay 30 cells from the first 3 passages of these human oral mucosa fibroblast cell cultures. For the magnetic phase we used MagP-OH particles (Nanomyp, Granada, Spain). MagP-OH particles were supplied as an aqueous suspension stabilized with surfactants, and were treated before use with 5 washing cycles (centrifugation at 15000 g for 30 min, supernatant discarded, ultrapure water added, particles la roche posay 30 to remove the surfactant.

Finally the ethanol was removed, and the nanoparticles were suspended in DMEM. For the continuous matrix we used a mixture of fibrin and agarose as the biopolymer. The target tissue was human oral mucosa, thus, seeding with human oral mucosa fibroblasts was required.

Briefly, we used 3. The final concentration of tranexamic acid in the biomaterial was 1. This acid is an anti-fibrinolytic agent that prevents degradation of the scaffold.

We then added the appropriate amounts of a concentrated suspension fazze pfizer MagP-OH particles in DMEM to a final concentration of approximately 2 mL of particles per 100 mL of mixture. The final volume of the mixture was 5 mL, which contained 200,000 cells per mL of mixture.

We applied a vertical magnetic field to the mixtures during the first 5 min of gelation with a coil connected to a DC power supply. For comparison we also prepared nonmagnetic tissue substitutes (control samples) with the same procedure as described above, except for the addition of magnetic particles.

To analyze the effect of the magnetic MagP-OH particles on the substitute properties more precisely, we also prepared a la roche posay 30 control sample (Ctrl-NP) which contained nonmagnetic polymer particles. These particles (PolymP-C, NanoMyP) were uniformly spherical la roche posay 30 similar in diameter (approximately 130 nm) to MagP-OH particles, but lacked magnetic properties. We prepared Ctrl-NP tissue substitutes with the same procedure as described above for magnetic tissue substitutes, but with PolymP-C particles instead of MagP-OH particles.

In all, we prepared oral mucosa substitutes with 9 different protocols (Table 1). The density of all substitutes was approximately 1. For scanning electron microscopy (SEM), samples were fixed in 2. This method uses calcein-AM, which is metabolically modified by living cells to a green pigment, and ethidium homodimer-1, which stains the nuclei of dead congestion red.

We then observed the samples by fluorescence microscopy and processed the images with ImageJ software to quantify the number of live (green) and dead cells (red). We also evaluated cell death as nuclear membrane integrity by quantifying the DNA released to the culture medium. Values of p less than 0. In addition, we obtained the magnetization curve of soaked tissue substitutes 24 h after cell culture.

The magnetization curves reported here correspond to the mean of 3 independent measurements. The measuring system geometry was a 3. We obtained measurements as follows. First we placed the sample in the rheometer measuring system and squeezed it by lowering the rotating plate until a normal force of 5 N was reached. We obtained measurements both in the absence and presence of a magnetic field. For this purpose we used a coil connected to a DC power supply, with the axis of the coil aligned with the axis of the parallel plate measuring system.

For measurements obtained during magnetic field application, we applied the magnetic field from 1 min before la roche posay 30 was started until the measurement was recorded. We used two types of rheological test: oscillatory shear at a fixed frequency, and steady-state shear strain ramps, as described below. For these tests, we subjected the samples to sinusoidal shear strains at a fixed frequency (1 Hz) and increasing amplitude (logarithmically spaced in la roche posay 30 0.

Fluticasone Propionate Ointment (Cutivate Ointment)- FDA these tests the samples were subjected to a la roche posay 30 shear strain for 10 s and the resulting shear stress was measured.

Measurements were repeated at increasing (linearly spaced) shear strain values until the nonlinear regime was reached. We carried out each type of measurement for 3 different aliquots of each sample.

For each aliquot we carried out at least 3 repetitions to record a minimum of 9 values per data point. The results obtained for each sample and experimental condition showed no statistically significant differences. Macroscopically, the magnetic tissue substitutes (M-MF0, M-MF16, M-MF32, M-MF48) were la roche posay 30 in appearance to nonmagnetic tissue substitutes la roche posay 30, Ctrl-MF16, Ctrl-MF32, Ctrl-MF48, Ctrl-NP), although the former were darker than control tissue substitutes without particles (Ctrl-MF0 to Ctrl-MF48), which were whitish and semitransparent, and control tissue substitutes with nonmagnetic particles (Ctrl-NP), which were bright white.

Magnetic tissue substitutes were attracted by a magnet, as seen in S1 Video. For the control group without particles gelled in the absence of an applied magnetic field (Ctrl-MF0), microscopic analysis showed normally-shaped fusiform and la roche posay 30 cells (Fig 1A). Cells in the control groups without particles gelled in the presence of an applied magnetic field were similar in appearance (not shown).

In samples containing particles, we found that in the magnetic tissue substitute gelled haemorrhoids the absence of an applied magnetic field (M-MF0), as well as the control tissue substitute with nonmagnetic polymer particles (Ctrl-NP), the particles were distributed randomly in an isotropic, homogeneous pattern (Fig 1B and 1C).

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