Guide cells support muscle regeneration and affect neuro-muscular junction organization

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers

Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear.In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-100 microm in depth, which we termed primary; and (ii) smaller microcavities of 10-20 microm in diameter and 3-10 microm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold.In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future

Gene expression modulation in stretched muscle cells

Aim of this work was to evaluate the effects of mechanical stretching on the growth and differentiation of skeletal muscle cells. For this reason, we used a mechanical bioreactor that deformed the bottom of a seeded Petri dish. To investigate the effects of stretching on muscle gene expression, samples were analyzed in comparison to static ones without bioreactor stimulation. Cells morphology was analyzed with Wright’s staining. RT-PCR analysis revealed the influence of bioreactor’s action on the expression of specific muscle genes. Mechanical stress treatment resulted in improved cell proliferation and differentiation, due to the up-regulation of both Myf5 and MyoD transcription factors. Our results showed that the mechanic treatment improved the cell proliferation and differentiation due to the up-regulation of key muscle differentiation molecules, such as MyoD and Myf5

Video evaluation of the kinematics and dynamics of the beating cardiac syncytium: an alternative to the Langendorff method.

Many important observations and discoveries in heart physiology have been made possible using the isolated heart method of Langendorff. Nevertheless, the Langendorff method has some limitations and disadvantages such as the vulnerability of the excised heart to contusions and injuries, the probability of preconditioning during instrumentation, the possibility of inducing tissue edema, and high oxidative stress, leading to the deterioration of the contractile function. To avoid these drawbacks associated with the use of a whole heart, we alternatively used beating mouse cardiac syncytia cultured in vitro in order to assess possible ergotropic, chronotropic, and inotropic effects of drugs. To achieve this aim, we developed a method based on image processing analysis to evaluate the kinematics and the dynamics of the drug-stimulated beating syncytia starting from the video recording of their contraction movement. In this manner, in comparison with the physiological no-drug condition, we observed progressive positive ergotropic, positive chronotropic, and positive inotropic effects of 10 µM isoproterenol (ß-adrenergic agonist) and early positive ergotropic, negative chronotropic, and positive inotropic effects of 10 µM phenylephrine (alpha-adrenergic agonist), followed by a late phase with negative ergotropic, positive chronotropic, and negative inotropic trends. Our method permitted a systematic study of in vitro beating syncytia, producing results consistent with previous works. Consequently, it could be used in in vitro studies of beating cardiac patches, as an alternative to Langendorff's heart in biochemical and pharmacological studies, and especially when the Langendorff technique is inapplicable (e.g., in studies about human cardiac syncytium in physiological and pathological conditions, patient-tailored therapeutics, and syncytium models derived from induced pluripotent/embryonic stem cells with genetic mutations). Furthermore, the method could be helpful in heart tissue engineering and bioartificial heart research to "engineer the heart piece by piece." In particular, the proposed method could be useful in the identification of a suitable cell source, in the development and testing of "smart" biomaterials, and in the design and use of novel bioreactors and microperfusion systems

Cyclic mechanical cells stimulation of myoblasts in skeletal muscle tissue engineering: a preliminary study

The aim of this study is to evaluate how cyclic mechanical stretching influences skeletal muscle differentiation. To this purpose we realized a mechanical bioreactor that reproduces in vitro a cyclic mechanical stretching of cells seeded on to a polymeric scaffold. The bioreactor consists of three fundamental parts: a Petri dish (in polycarbonate Lexan®), the stretching scaffold device and a controlled DC motor. Firstly we performed tests of cellular adhesion on different membranes to identify the best cell culture surface using the murine muscular cell line C2C12, and after the identification of the culture surface we examined the role of cyclic mechanical stimulation on differentiation of C2C12 cells. We analysed the expression of genes involved in muscle differentiation by RT-PCR. Our results demonstrate that although the dynamic culture expressed a noticeable quantity of myosin (MLC3F) the cells did not significantly differentiate

Enhancement of a culture of human osteoblasts inside hydroxyapatite scaffolds via [2 mT; 75 Hz]-electromagnetic bioreactor

Biomaterials have been widely used in reconstructive bone surgery to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. In particular, porous hydroxyapatite is commonly employed owing to its biocompatibility; in addition , the in vitro modification of hydroxyapatite with osteogenic signals enhances the tissue regeneration in vivo, suggesting that the biomaterial modification could play an important role in tissue engineering . In this study we have followed a biomimetic strategy where electromagnetically stimulated SAOS -2 human osteoblasts proliferated and built their extracellular matrix inside porous hydroxyapatite. In comparison with control conditions , the electromagnetic stimulus (magnetic field , 2 mT; frequency , 75 Hz) increased , in vitro, the cell proliferation and the coating of hydroxyapatite with bone proteins (decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen). The physical stimulus aimed at obtaining a better in vitro modification of porous hydroxyapatite in terms of cell colonization and coating with osteogenic signals , like bone matrix proteins . The modified biomaterial could be used, in clinical applications, as an implant for bone repair