02803nas a2200181 4500008004100000245012600041210006900167520217400236653001902410100001702429700002002446700001502466700001902481700001502500700001502515700001802530856007302548 2016 eng d00aClarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue0 aClarifying CLARITY Quantitative Optimization of the Diffusion Ba3 a

Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the “goodness” of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the approach, illustrating an example of how it can be used to determine the optimum clearing time for 1 mm-thick cerebellar slice from transgenic L7GFP mice, in which Purkinje neurons express the GFP (green fluorescent protein) tag. To validate the method, we evaluated confocal stacks of our samples using standard image processing indices (i.e., the mean pixel intensity of neurons and the contrast-to-noise ratio) as figures of merit for image quality. The results show that detergent-based delipidation for more than 5 days does not increase tissue clarity but the fraction of GFP in the tissue continues to diminish. The optimum clearing time for 1 mm-thick slices was thus identified as 5 days, which is the best compromise between the increase in light penetration depth due to removal of lipids and a decrease in fluorescent signal as a consequence of protein loss: further clearing does not improve tissue transparency, but only leads to more protein removal or degradation. The rigorous quantitative approach described can be generalized to any clarification method to identify the moment when the clearing process should be terminated to avoid useless protein loss.

10aBioengineering1 aMagliaro, C.1 aCallara, A., L.1 aMattei, G.1 aMorcinelli, M.1 aViaggi, C.1 aVaglini, F1 aAhluwalia, A. uhttp://journal.frontiersin.org/article/10.3389/fnins.2016.00179/full01564nas a2200133 4500008004100000022001300041245009100054210006900145520109700214653001901311100001501330700001401345856007101359 2015 eng d a0098135400aCFD modelling of a mixing chamber for the realisation of functionally graded scaffolds0 aCFD modelling of a mixing chamber for the realisation of functio3 a

Biological tissues are characterised by spatially distributed gradients, intricately linked with functions. It widely accepted that ideal tissue engineered scaffolds should exhibit similar functional gradients to promote successful tissue regeneration. Focusing on bone, in previous work we proposed simple methods to obtain osteochondral functionally graded scaffolds (FGSs), starting from homogeneous suspensions of hydroxyapatite (HA) particles in gelatin solutions. With the main aim of developing an automated device to fabricate FGSs, this work is focused on designing a stirred tank to obtain homogeneous HA-gelatin suspensions. The HA particles transport within the gelatin solution was investigated through computational fluid dynamics (CFD) modelling. First, the steady-state flow field was solved for the continuous phase only. Then, it was used as a starting point for solving the multi-phase transient simulation. CFD results showed that the proposed tank geometry and setup allow for obtaining a homogeneous suspension of HA micro-particles within the gelatin solution.

10aBioengineering1 aMattei, G.1 aVozzi, G. uhttp://www.sciencedirect.com/science/article/pii/S009813541500282300556nas a2200145 4500008004100000245008700041210006900128260001600197300001400213653001900227100001400246700001400260700001500274856012100289 2012 eng d00aCharacterization and modeling of mechanical behavior a discrete gradient structure0 aCharacterization and modeling of mechanical behavior a discrete  aRoma, Italy a130 - 13110aBioengineering1 aCaputo, M1 aVozzi, G.1 aMattei, G. uhttp://95.110.227.16/publications/characterization-and-modeling-mechanical-behavior-discrete-gradient-structure.html02057nas a2200169 4500008003900000245006000039210005800099260002000157520150900177653001901686100001501705700001801720700001601738700002501754700001501779856009301794 2012 d00aContinuous functionally graded materials (cFGMs) for TE0 aContinuous functionally graded materials cFGMs for TE aVienna, Austria3 a

Biological structures are not uniform but possess spatially distributed functions and properties, or functional gradients. To ensure functional, mechanical and structural integration, a tissue engineered (TE) scaffold has to reproduce these functional gradients. However the fabrication of functionally graded materials is challenging and usually an experimental trial-and-error approach is used. In this work we present a controlled method for the fabrication of cFGMs using the gravitational sedimentation of discrete solid particles within a primary fluid phase. To have an overall control over particle distribution, a time-varying dynamic viscosity solution (i.e. thermo-sensitive) was used as fluid phase. Computational fluid dynamic models were developed to have a fine control over particle distribution. Biomimetic osteochondral cFGMs scaffolds were fabricated using hydroxyapatite (HA) and gelatin. Glutaraldehyde was used to covalently bind gelatin-HA graded scaffolds. Mechanical properties were measured and correlated as a function of HA volume fraction. SEM-EDX analysis was used to further characterise HA content and its distribution within gelatin-HA cFGMs. Finally gelatin-HA cFGMs scaffold were seeded using periosteum derived progenitor cells, to investigate how the HA gradient modulates cell response. This approach represents an innovative yet simple tool for the fabrication of tailored cFGMs with biologically and physiologically relevant gradients for TE applications.

10aBioengineering1 aMattei, G.1 aAhluwalia, A.1 aFerretti, C1 aMattioli-Belmonte, M1 aTirella, A uhttp://95.110.227.16/publications/continuous-functionally-graded-materials-cfgms-te.html