RESUMO
Devices for in vitro culture of three-dimensional (3D) skeletal muscle tissues have multiple applications, including tissue engineering and muscle-powered biorobotics. In both cases, it is crucial to recreate a biomimetic environment by using tailored scaffolds at multiple length scales and to administer prodifferentiative biophysical stimuli (e.g., mechanical loading). On the contrary, there is an increasing need to develop flexible biohybrid robotic devices capable of maintaining their functionality beyond laboratory settings. In this study, we describe a stretchable and perfusable device to sustain cell culture and maintenance in a 3D scaffold. The device mimics the structure of a muscle connected to two tendons: Tendon-Muscle-Tendon (TMT). The TMT device is composed of a soft (E â¼ 6 kPa) porous (pore diameter: â¼650 µm) polyurethane scaffold, encased within a compliant silicone membrane to prevent medium evaporation. Two tendon-like hollow channels interface the scaffold with a fluidic circuit and a stretching device. We report an optimized protocol to sustain C2C12 adhesion by coating the scaffold with polydopamine and fibronectin. Then, we show the procedure for the soft scaffold inclusion in the TMT device, demonstrating the device's ability to bear multiple cycles of elongations, simulating a protocol for cell mechanical stimulation. By using computational fluid dynamic simulations, we show that a flow rate of 0.62 mL/min ensures a wall shear stress value safe for cells (<2 Pa) and 50% of scaffold coverage by an optimal fluid velocity. Finally, we demonstrate the effectiveness of the TMT device to sustain cell viability under perfusion for 24 h outside of the CO2 incubator. We believe that the proposed TMT device can be considered an interesting platform to combine several biophysical stimuli, aimed at boosting skeletal muscle tissue differentiation in vitro, opening chances for the development of muscle-powered biohybrid soft robots with long-term operability in real-world environments.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Músculo Esquelético , Diferenciação CelularRESUMO
Objective. In this study, we aimed to verify the beneficial effects of low-intensity pulsed ultrasound (LIPUS) stimulation on two cell types: H2O2-treated RSC96 Schwann cells and THP-1 macrophages, used to model neuropathic inflammation.Approach. Using a set-up guaranteeing a fine control of the ultrasound dose at the target, different frequencies (38 kHz, 1 MHz, 5 MHz) and different intensities (20, 100, 500 mW cm-2) were screened to find the most effective experimental conditions for triggering beneficial effects on metabolic activity and release of neurotrophic cytokines (ß-nerve growth factor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor) of RSC96 cells. The combination of parameters resulting the optimal one was applied to evaluate anti-inflammatory effects in terms of reactive oxygen species (ROS) and tumor necrosis factor-α(TNF-α) production, also investigating a possible anti-oxidant activity and mechanotransduction pathway for the anti-inflammatory process. The same optimal combination of parameters was then applied to THP-1 cells, differentiated into M1 and M2 phenotypes, to assess the effect on the expression and release of pro-inflammatory markers (TNF-α, interleukin (IL)-1ß, IL-6, IL-8) and anti-inflammatory ones (IL-10 and CD206).Main results.5 MHz and 500 mW cm-2were found as the optimal stimulation parameters on RSC96 cells. Such parameters were also found to suppress ROS and TNF-αin the same cell line, thus highlighting a possible anti-inflammatory effect, involving the NF-kB pathway. An anti-oxidant effect induced by LIPUS was also observed. Finally, the same LIPUS parameters did not induce any differentiation towards the M1 phenotype of THP-1 cells, whereas they decreased TNF-αand IL-8 gene expression, reduced IL-8 cytokine release and increased IL-10 cytokine release in M1-polarized THP-1 cells.Significance.This study represents the first step towards the use of precisely controlled LIPUS for the treatment of peripheral neuropathies.
Assuntos
Interleucina-8 , Doenças do Sistema Nervoso Periférico , Humanos , Interleucina-10 , Fator de Necrose Tumoral alfa , Doenças do Sistema Nervoso Periférico/terapia , Espécies Reativas de Oxigênio , Peróxido de Hidrogênio , Mecanotransdução Celular , Inflamação/terapia , Citocinas , Anti-Inflamatórios , Ondas UltrassônicasRESUMO
In this paper, we stimulated M1-like macrophages (obtained from U937 cells) with low-intensity pulsed ultrasound (LIPUS) to lower pro-inflammatory cytokine production. A systematic screening of different frequencies, intensities, duty cycles, and exposure times was performed. The optimal stimulation conditions leading to a marked decrease in the release of inflammatory cytokines were determined to be 38 kHz, 250 mW/cm2, 20%, and 90 min, respectively. Using these parameters, we verified that up to 72 h LIPUS did not affect cell viability, resulting in an increase in metabolic activity and in a reduction of reactive oxygen species (ROS) production. Moreover, we found that two mechanosensitive ion channels (PIEZO1 and TRPV1) were involved in the LIPUS-mediated cytokine release modulation. We also assessed the role of the nuclear factor κB (NF-κB) signaling pathway and observed an enhancement of actin polymerization. Finally, transcriptomic data suggested that the bioeffects of LIPUS treatment occur through the modulation of p38 MAPK signaling pathway.