Biology subjects

Chirico, G.

Publications and source records attributed to Chirico, G..

3 recordsLinked to original sources

bioRxiv2025

Engineering macrophage responses through 3D scaffold microarchitecture

Biomaterial implantation in living organisms triggers a physiological response known as foreign body reaction, leading to the recruitment of macrophages, that can polarize either into a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Currently, there is growing interest in tailoring the physical properties of tissues and biomaterials to promote efficient tissue regeneration. Tridimensionality can profoundly influence macrophage behaviour; however, there is no clear consensus on the underlying mechanisms. 3D microstructures may play a crucial role in modulating immune cells, promoting anti-inflammatory responses, and supporting effective tissue repair and regeneration. In this study, we used two-photon polymerization to fabricate 3D scaffolds with large pores, measuring 50x50x20 m3, and small pores, measuring 15x15x15 m3. Both microstructures effectively influenced macrophage cytoskeletal organization and cellular metabolic activity. Notably, they were not sufficient to induce spontaneous macrophage polarization, indicating that they are intrinsically immunologically inert. When combined with chemical stimulation, as typically occurs in vivo, they elicited distinct responses. Specifically, as evidenced by the slight upregulation of the Arg1 marker, large pore sizes promoted an anti-inflammatory phenotype. Conversely, iNOS expression measurements indicated that small pores, which impose spatial constraints on macrophages, favoured a massive pro-inflammatory state. Our results demonstrate that 3D microstructures are versatile tools for multiple applications. Their precisely tunable architecture enables fine control over macrophage behaviour and immunomodulation, opening new avenues both for tissue engineering, by preventing fibrosis and promoting anti-inflammatory and pro-regenerative responses in vivo, and for the development of in vitro platforms to model inflamed tissues for screening anti-inflammatory drugs.

bioengineering↗

bioRxiv2023

Evidence for noisy oscillations of cAMP under nutritional stress condition in budding yeast

The Ras/cAMP/PKA pathway is one of the best studied signalling pathway in the budding yeast that regulates cellular responses to nutrients availability and stress. The cAMP levels and the PKA activity are also subjected to a strong negative feedback that operates either through the activity of the phosphodiesterase Pde1 and also on the amount of Ras2-GTP. We have previously made and simulated a dynamic model of the whole pathway and our results suggest the existence of stable oscillatory states that depend on the activity of the RasGEF (Cdc25) and RasGAP (Ira proteins) (Pescini et al. Biotechnol Adv 30, 99-107, 2012). Stochastic oscillations related to activity of the pathway were reported by looking at the nuclear localization of the trascription factors Msn2 and Msn4 (Gamedia-Torres et al. Curr Biol 17, 1044-9, 2007). In particular Medvedik et al. (PloS Biol 5, 2330-41, 2007) reported stable oscillations of the nuclear accumulation of Msn2 in condition of limited glucose availabiliy. We were able to reproduce the periodic accumulation of Msn2-GFP protein in yeast cells under condition of limiting glucose, and we tried to detect also in the same condition oscillations of cAMP levels in single yeast cells. We used a specific Fluorescence Resonance Energy Transfer (FRET) sensor based on a fusion protein between CFP-EPAC-YFP expressed in yeast cells. The FRET between CFP and YFP is controlled by cAMP concentration. This sensor allows us to monitor changes in cAMP concentrations in single yeast cell for a relative long time and a peak of cAMP was normally detected after addition of glucose to derepressed cells (Colombo et al. Biochem Biophys Res Commun 487, 594-99, 2017). Using this method we were able to detect noisy oscillations of cAMP levels in single yeast cells under condition of nutritional stress caused by limiting glucose availability (0.1%). We used Spectral analysis to discriminate between true oscillations and random noise. The oscillations were characterized by period of about 4-5 min, close to that observed for Msn2-GFP oscillations.

biophysics↗

bioRxiv2022

Microlenses fabricated by two-photon laser polymerisation for intravital cell imaging with non-linear excitation microscopy

Non-linear excitation microscopy offers several advantages for in-vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in-vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, we develop and test on fibroblast cell culture plano-convex microlenses to be used for non-linear imaging of biological tissue. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. We could retrieve magnified fluorescence images through the microlenses coupled to commercial confocal and two-photon excitation scanning microscopes. The signal-to-noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro-optics for optical in-vivo inspection of biological processes.

bioengineering↗