Microstructure and Solute Segregation around the Melt-Pool Boundary of Orientation-Controlled 316L Austenitic Stainless Steel Produced by Laser Powder Bed Fusion.

For this article, we studied the microstructure and solute segregation seen around the melt pool boundary of orientation-controlled 316L austenitic stainless steel produced by laser powder bed fusion, using transmission electron microscopy and energy-dispersive x-ray spectroscopy. We found that the solidification cellular microstructures could be visualized with the aid of solute segregation (Cr and Mo) during solidification. Mn-Si-O inclusions (10-15 nm in diameter) were distributed along the lamellar boundaries, as well as in the dislocation cell walls. It is believed that the grain growth of the inclusions can be effectively suppressed by rapid quenching during the laser powder-bed fusion process. A thin region without cellular microstructures was observed at the melt-pool boundary. The cellular spacing widened near the bottom of the melt-pool boundary, owing to the decrease in the cooling rate. Atomic-structure analysis at the lamellar boundary by high-resolution transmission electron microscopy revealed a local interfacial structure, which is complementary to the results of electron back-scatter diffraction.

Hypoxia transactivates cholecystokinin gene expression in 3D-engineered muscle.

At high altitudes, the hypoxic atmosphere decreases the oxygen partial pressure in the body, inducing several metabolic changes in tissues and cells. Furthermore, it exerts potent anorectic effects, thus causing an energy deficit. Two decades ago, a marked increase in the resting level of plasma cholecystokinin (CCK) was observed in humans at the Mt. Kanchenjunga basecamp, located at 5100 m above the sea level, compared to sea-level control values. Interestingly, acute exercise also raises plasma CCK and exerts potent anorectic effects under normoxic conditions. However, the transcriptional regulations of Cck gene underlying these effects have not yet been established. Here, we employed acute electrical pulse stimulation (EPS) followed by microarray analysis to discover novel myokines in 3D-engineered muscle. Acute EPS affects the contractile function, inducing a decline in the contractile force. Surprisingly, microarray analysis revealed an EPS-induced activation of cholecystokinin receptor (CCKR)-mediated signaling. Furthermore, Cck was constitutively upregulated in 3D-engineered muscle, and its expression increased under hypoxic conditions. Notably, a hypoxia-responsive element was detected in the Cck promoters of mice and humans. Our results suggested that hypoxia transactivated Cck expression in 3D-engineered muscle. Furthermore, the elevation in plasma CCK levels following acute exercise or at high altitude might be partly attributed to myogenic cells.

Effect of heat stress on contractility of tissue-engineered artificial skeletal muscle.

The effects of heat stress on tissue like skeletal muscle have been widely studied. However, the mechanism responsible for the effect of heat stress is still unclear. A useful experimental tissue model is necessary because muscle function in cell culture may differ from native muscle and measuring its contractility is difficult. We previously reported three-dimensional tissue-engineered artificial skeletal muscle (TEM) that can be easily set in a measurement apparatus for quantitative evaluation of contractility. We have now applied TEM to the investigation of heat stress. We analyzed contractility immediately after thermal exposure at 39 °C for 24 or 48 h to evaluate the acute effects and after thermal exposure followed by normal culture to evaluate the aftereffects. Peak twitch contractile force and time-to-peak twitch were used as contractile parameters. Heat stress increased the TCF in the early stage (1 week) after normal culture; the TCF decreased temporarily in the middle to late stages (2-3 weeks). These results suggest that heat stress may affect both myoblast fusion and myotube differentiation in the early stage of TEM culture, but not myotube maturation in the late stage. The TCF increase rate with thermal exposure was significantly higher than that without thermal exposure. Although detailed analysis at the molecular level is necessary for further investigation, our artificial skeletal muscle may be a promising tool for heat stress investigation.

Development and evaluation of a removable tissue-engineered muscle with artificial tendons.

Tissue-engineered skeletal muscles were potentially useful as physiological and biochemical in vitro models. Currently, most of the similar models were constructed without tendons. In this study, we aimed to develop a simple, highly versatile tissue-engineered muscle with artificial tendons, and to evaluate the contractile, histological and molecular dynamics during differentiation. C2C12 cells were embedded in a cold type-І collagen gel and placed between two artificial tendons on a silicone sheet. The construct shrank and tightly attached to the artificial tendons with differentiation, finally detaching from the silicone sheet within 1 week of culture onset. We successfully developed a tissue-engineered skeletal muscle with two artificial tendons from C2C12 myoblasts embedded in type-І collagen gel. The isometric twitch contractile force (TCF) significantly increased during differentiation. Time to Peak Tension (TPT) and Half-Relaxation Time (1/2RT) were significantly shortened during differentiation. Myogenic regulatory factors were maximally expressed at 2 weeks, and subsequently decreased at 3 weeks of culture. Histological analysis indicated that myotube formation increased markedly from 2 weeks and well-ordered sarcomere structures were observed on the surface of the 3D engineered muscle at 3 weeks of culture. These results suggested that robust muscle structure occurred by 3 weeks in the tissue-engineered skeletal muscle. Moreover, during the developmental process, the artificial tendons might contribute to well-ordered sarcomere formation. Our results indicated that this simple culture system could be used to evaluate the effects of various pharmacological and mechanical cues on muscle contractility in a variety of research areas.