Design of a custom-made device for real-time optical measurement of differential mineral concentrations in three-dimensional scaffolds for bone tissue engineering.

Monitoring bone tissue engineered (TEed) constructs during their maturation is important to ensure the quality of applied protocols. Several destructive, mainly histochemical, methods are conventionally used to this aim, requiring the sacrifice of the investigated samples. This implies (i) to plan several scaffold replicates, (ii) expensive and time consuming procedures and (iii) to infer the maturity level of a given tissue construct from a cognate replica. To solve these issues, non-destructive techniques such as light spectroscopy-based methods have been reported to be useful. Here, a miniaturized and inexpensive custom-made spectrometer device is proposed to enable the non-destructive analysis of hydrogel scaffolds. Testing involved samples with a differential amount of calcium salt. When compared to a reference standard device, this custom-made spectrometer demonstrates the ability to perform measurements without requiring elaborate sample preparation and/or a complex instrumentation. This preliminary study shows the feasibility of light spectroscopy-based methods as useful for the non-destructive analysis of TEed constructs. Based on these results, this custom-made spectrometer device appears as a useful option to perform real-time/in-line analysis. Finally, this device can be considered as a component that can be easily integrated on board of recently prototyped bioreactor systems, for the monitoring of TEed constructs during their conditioning.

Cell-specific Dynamic Clamp analysis of the role of funny If current in cardiac pacemaking.

We used the Dynamic Clamp technique for i) comparative validation of conflicting computational models of the hyperpolarization-activated funny current, If, and ii) quantification of the role of If in mediating autonomic modulation of heart rate. Experimental protocols based on the injection of a real-time recalculated synthetic If current in sinoatrial rabbit cells were developed. Preliminary results of experiments mimicking the autonomic modulation of If demonstrated the need for a customization procedure to compensate for cellular heterogeneity. For this reason, we used a cell-specific approach, scaling the maximal conductance of the injected current based on the cell's spontaneous firing rate. The pacemaking rate, which was significantly reduced after application of Ivabradine, was restored by the injection of synthetic current based on the Severi-DiFrancesco formulation, while the injection of synthetic current based on the Maltsev-Lakatta formulation did not produce any significant variation. A positive virtual shift of the If activation curve, mimicking the Isoprenaline effects, led to a significant increase in pacemaking rate (+17.3 ± 6.7%, p < 0.01), although of lower magnitude than that induced by real Isoprenaline (+45.0 ± 26.1%). Similarly, a negative virtual shift of the activation curve significantly lowered the pacemaking rate (-11.8 ± 1.9%, p < 0.001), as did the application of real Acetylcholine (-20.5 ± 5.1%). The Dynamic Clamp approach, applied to the If study in cardiomyocytes for the first time and rate-adapted to manage intercellular variability, indicated that: i) the quantitative description of the If current in the Severi-DiFrancesco model accurately reproduces the effects of the real current on rabbit sinoatrial cell pacemaking rate and ii) a significant portion (50-60%) of the physiological autonomic rate modulation is due to the shift of the If activation curve.