Ratiometric pH and O2 nanosensors were fabricated independently using a fluorophore that produces a signal proportional to the concentration of the analyte of interest, and a second fluorophore that produces a reference signal, insensitive to the analyte of interest. These fluorophores emit at different wavelengths and were either entrapped or covalently bound, within an inert optically transparent polymeric matrix. In this work uniform sized 3D micro-scaled alginate hydrogel constructs of approximately 100-300 $μ$m were fabricated. Through dual incorporation of HepG2 cells and nanosensors within these alginate constructs we aim to have a real-time, non-invasive method to measure microenvironmental pH and O2 content. Ratiometric fluorescent output from the microenvironment is used to monitor O2 concentrations and pH during cell culture. Measurements show that 3D micro-scaled constructs are suitable for cell growth and proliferation. Moreover O2 and pH values within the hydrogel cellularised microspheres are shown to have physiological values that enable the maintenance of the hepatic phenotype
The identification of the ideal cell source to generate cardiac tissue able to integrate into the host myocardium and with the contractile system is crucial for cardiac engineering. Amongst different cell sources so far proposed, human adult Cardiac Progenitor Cells (hCPCs) show the ability to proliferate and differentiate toward cardiac lineages when grown in appropriate microenvironmental conditions. It is widely accepted that conventional 2D cultures may provide a physiological environment for growing cells. For this reason the need to have an engineered microenvironment, matching physiological requirements, is crucial. A 3D context with spatial and time varying distribution of regulatory factors using mechanically matched scaffolds and bioreactors could represent an in vitro cell culture model being able to more closely reflects the in vivo conditions. In the present study, the possibility of using biocompatible and biodegradable scaffolds of collagen based or derivatives hydrogels in combination with Linneg/Sca-1pos hCPCs gathered from human heart biopsies was investigated. Bio-constructs were placed in the low shear, high flow MCmB (MultiCompartment modular Bioreactor) and the combined effects of dynamic culture conditions and 3D scaffolds on cell morphology and differentiation were studied in order to investigate the possibility of fabricating stem cell-derived cardiac patches to replace infarcted tissue.
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.
Monitoring and controlling the microenvironment of cell cultures is an ongoing challenge for many researchers. Much research has been conducted characterising individual aspects such as 3D architecture, mechanical properties, biochemicals, etc. The biggest deficits in existing models for monitoring analytes within the cellular environment is the lack of appropriate means for non invasive, real-time and integrated monitoring of the cellular responses. Nanosensors can overcome these issues: they are porous polymeric nanoparticles that are sensitive to a range of analytes including pH and O2. Microfabrication techniques are innovative tools to obtain controlled microstructures with a defined 3D architecture. In this work uniform sized 3D micro-scaled hydrogel constructs of approximately 300-400 um diameter were fabricated. Through dual incorporation of cells and nanosensors within these constructs we aim to have a real-time, non invasive method to measure microenvironmental pH value and O2 content. Ratiometric fluorescent output from the microenvironment is used to monitor O2 and pH during cell culture. Measurements show that 3D micro-scaled constructs are suitable for cell growth and proliferation. Moreover O2 content and pH within hydrogel cellularised microspheres are shown to have physiological values which enable the maintenance of the hepatic phenotype.
The role of materials mechanical properties is a new frontier for the evaluation of cell/material interaction, as well as for the determination of healthy/pathological state of a tissue. In these sense there is the need to have a unique method to measure materials viscoelastic properties. However, concerning with soft and highly hydrated constructs, the experimental set-up to precisely measure these properties is challenging because of the difficulty in defining zero stress or strain. To overcome these problems, we propose a novel and unique testing and data analysis technique (ERM) to derive materials viscoelastic properties. Results derived with this method can be compared to the ones obtained with standard testing techniques for viscoelastic materials. Pre-conditioning problems of testing soft and floppy materials are thus overcome, giving rise to have an accurate measure of viscous and elastic moduli of both hydrated materials and soft biological tissues. Small variations of measured properties can be also monitored with high precision, allowing a deeper investigation on the role of the scaffolding material or of tissue’s extracellular matrix (respectively in cell culture systems or in biomechanics measurements for the characterisation of soft tissues).
