Cell death induced by mechanical compression on engineered muscle results from a gradual physiological mechanism

\u3cp\u3eDeep tissue injury (DTI), a type of pressure ulcer, arises in the muscle layers adjacent to bony prominences due to sustained mechanical loading. DTI presents a serious problem in the clinic, as it is often not visible until reaching an advanced stage. One of the causes can be direct mechanical deformation of the muscle tissue and cell. The mechanism of cell death induced by mechanical compression was studied using bio-artificial skeletal muscle tissues. Compression was applied by placing weights on top of the constructs. The morphological changes of the cytoskeleton and the phosphorylation of mitogen-activated protein kinases (MAPK) under compression were investigated. Moreover, inhibitors for each of the three major MAPK groups, p38, ERK, and JNK, were applied separately to look at their roles in the compression caused apoptosis and necrosis. The present study for the first time showed that direct mechanical compression activates MAPK phosphorylation. Compression also leads to a gradual destruction of the cytoskeleton. The percentage apoptosis is strongly reduced by p38 and JNK inhibitors down to the level of the unloaded group. This phenomenon could be observed up to 24. h after initiation of compression. Therefore, cell death in bio-artificial muscle tissue caused by mechanical compression is primarily caused by a physiological mechanism, rather than through a physical mechanism which kills the cell directly. These findings reveal insight of muscle cell death under mechanical compression. Moreover, the result indicates a potential clinical solution to prevent DTI by pre-treating with p38 or/and JNK inhibitors.\u3c/p\u3

Effect of culture conditions on endothelial cell growth and responsiveness

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions

Dissecting healthy and diseased hearts

Poster no abstract available

MicroRNA-1 and microRNA-206 improve differentiation potential of human satellite cells: A novel approach for tissue engineering of skeletal muscle

Innovative strategies based on regenerative medicine, in particular tissue engineering of skeletal muscle, are promising for treatment of patients with skeletal muscle damage. However, the efficiency of satellite cell differentiation in vitro is suboptimal. MicroRNAs are involved in the regulation of cell proliferation and differentiation. We hypothesized that transient overexpression of microRNA-1 or microRNA-206 enhances the differentiation potential of human satellite cells by downregulation quiescent satellite cell regulators, thereby increasing myogenic regulator factors. To investigate this, we isolated and cultured human satellite cells from muscle biopsies. First, through immunofluorescent analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we showed that in satellite cell cultures, low Pax7 expression is related to high MyoD expression on differentiation, and, subsequently, more extensive sarcomere formation, that is, muscle differentiation, was detected. Second, using qRT-PCR, we showed that microRNA-1 and microRNA-206 are robustly induced in differentiating satellite cells. Finally, a gain-of-function approach was used to investigate microRNA-1 and microRNA-206 potential in human satellite cells to improve differentiation potential. As a proof of concept, this was also investigated in a three-dimensional bioartificial muscle construct. After transfection with microRNA-1, the number of Pax7 expressing cells decreased compared with the microRNA-scrambled control. In differentiated satellite cell cultures transfected with either microRNA-1 or microRNA-206, the number of MyoD expressing cells increased, and a-sarcomeric actin and myosin expression increased compared with microRNA-scrambled control cultures. In addition, in a three-dimensional bioartificial muscle construct, an increase in MyoD expression occurred. Therefore, we conclude that microRNA-1 and microRNA-206 can improve human satellite cell differentiation. It represents a potential novel approach for tissue engineering of human skeletal muscle for the benefit of patients with facial paralysis

Essential environmental cues from the satellite cell niche: Optimizing proliferation and differentiation

The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-D-lysine and laminin coatings. Copyrigh