Properties and Printability of the Synthesized Hydrogel Based on GelMA.

Gelatin methacryloyl (GelMA) has recently attracted increasing attention. Unlike other hydrogels, it allows for the adjustment of the mechanical properties using such factors as degree of functionalization, concentration, and photocrosslinking parameters. In this study, GelMA with a high degree of substitution (82.75 ± 7.09%) was synthesized, and its suitability for extrusion printing, cytocompatibility, and biocompatibility was studied. Satisfactory printing quality was demonstrated with the 15% concentration hydrogel. The high degree of functionalization led to a decrease in the ability of human adipose-derived stem cells (ADSCs) to adhere to the GelMA surface. During the first 3 days after sowing, proliferation was observed. Degradation in animals after subcutaneous implantation was slowed down.

Cartilage Formation In Vivo Using High Concentration Collagen-Based Bioink with MSC and Decellularized ECM Granules.

The aim of this study was to verify the applicability of high-concentration collagen-based bioink with MSC (ADSC) and decellularized ECM granules for the formation of cartilage tissue de novo after subcutaneous implantation of the scaffolds in rats. The printability of the bioink (4% collagen, 2.5% decellularized ECM granules, derived via 280 µm sieve) was shown. Three collagen-based compositions were studied: (1) with ECM; (2) with MSC; (3) with ECM and MSC. It has been established that decellularized ECM granules are able to stimulate chondrogenesis both in cell-free and MSC-laden scaffolds. Undesirable effects have been identified: bone formation as well as cartilage formation outside of the scaffold area. The key perspectives and limitations of ECM granules (powder) application have been discussed.

Bioprinting of Cartilage with Bioink Based on High-Concentration Collagen and Chondrocytes.

The study was aimed at the applicability of a bioink based on 4% collagen and chondrocytes for de novo cartilage formation. Extrusion-based bioprinting was used for the biofabrication. The printing parameters were tuned to obtain stable material flow. In vivo data proved the ability of the tested bioink to form a cartilage within five to six weeks after the subcutaneous scaffold implantation. Certain areas of cartilage formation were detected as early as in one week. The resulting cartilage tissue had a distinctive structure with groups of isogenic cells as well as a high content of glycosaminoglycans and type II collagen.

Mitochondrial redox state and Ca2+ sparks in permeabilized mammalian skeletal muscle.

Intact skeletal muscle fibres from adult mammals exhibit neither spontaneous nor stimulated Ca(2+) sparks. Mechanical or chemical skinning procedures have been reported to unmask sparks. The present study investigates the mechanisms that determine the development of Ca(2+) spark activity in permeabilized fibres dissected from muscles with different metabolic capacity. Spontaneous Ca(2+) sparks were detected with fluo-3 and single photon confocal microscopy; mitochondrial redox potential was evaluated from mitochondrial NADH signals recorded with two-photon confocal microscopy, and Ca(2+) load of the sarcoplasmic reticulum (SR) was estimated from the amplitude of caffeine-induced Ca(2+) transients recorded with fura-2 and digital photometry. In three fibre types studied, there was a time lag between permeabilization and spark development. Under all experimental conditions, the delay was the longest in slow-twitch oxidative fibres, intermediate in fast-twitch glycolytic-oxidative fibres, and the shortest in fast-twitch glycolytic cells. The temporal evolution of Ca(2+) spark frequencies was bell-shaped, and the maximal spark frequency was reached slowly in mitochondria-rich oxidative cells but quickly in mitochondria-poor glycolytic fibres. The development of spontaneous Ca(2+) sparks did not correlate with the SR Ca(2+) content of the fibre, but did correlate with the redox potential of their mitochondria. Treatment of fibres with scavengers of reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase, dramatically and reversibly reduced the spark frequency and also delayed their appearance. In contrast, incubation of fibres with 50 microm H(2)O(2) sped up the development of Ca(2+) sparks and increased their frequency. These results indicate that the appearance of Ca(2+) sparks in permeabilized skeletal muscle cells depends on the fibre's oxidative strength and that misbalance between mitochondrial ROS production and the fibre's ability to fight oxidative stress is likely to be responsible for unmasking Ca(2+) sparks in skinned preparations. They also suggest that under physiological and pathophysiological conditions the appearance of Ca(2+) sparks may be, at least in part, limited by the fine-tuned equilibrium between mitochondrial ROS production and cellular ROS scavenging mechanisms.

Metabolic regulation of Ca2+ release in permeabilized mammalian skeletal muscle fibres.

In the present study, the link between cellular metabolism and Ca2+ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca2+ release from the sarcoplasmic reticulum were detected with fluo-3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential-sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca2+] (with the Ca2+ indicator mag-rhod-2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca2+ release events were readily observed. The addition of L-glutamate or pyruvate reversibly decreased the frequency of Ca2+ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca2+] and promoted spontaneous Ca2+ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca2+ release events was not accompanied by a rise in global [Ca2+]i. Our results suggest that mitochondria exert a negative control over Ca2+ signalling in skeletal muscle by buffering Ca2+ near Ca2+ release channels.