Il gel di fibrin come materiale per il tissue-mimicking: sintesi e caratterizzazione

In recent years, researches on biomaterials for biomedical applications have experienced significant growth, particularly to develop materials able to mimic properties of biological tissues. Natural biomaterials are usually preferred to synthetic ones due to advantages in terms of bioactivity, biocompatibility, non-toxic byproducts of biodegradation and intrinsic structural resemblance. The main disadvantages are risk of microbial contamination, immunogenic reactions, lack of reproducibility and poor and unstable mechanical properties. Among all natural biomaterials, fibrin gel has promising features: good porosity, deformability, visco-elasticity and biodegradability. Contrarily, rapid degradation and unstable mechanical properties were recognized as the main drawbacks to be overcome. Fibrin gel is a natural protein-based polymer naturally synthesized in human body during coagulation processes and its mechanism of formation inside human body is well known. This thesis work has tried to replicate fibrin gel in vitro together with physical and biological characterization. The protocol for the synthesis of fibrin gel has been accurately developed starting from intensive bibliographic research, passing through numerous attempts in laboratory to find the best concentration for each reagent and ending with the development of a protocol that permits the synthesis of repeatable, reproducible and long-lasting fibrin gel through a sol-gel reaction. Fibrinogen, dissolved in 0.9% sodium chloride solution and cell culture medium (1:1) and thrombin and calcium chloride dissolved in MilliQ-water are needed for fibrin gel production. The choice of using cell culture medium was taken thinking at the possible future ap- plications of fibrin gel, in which cells embedding is a crucial aspect. Indeed, the use of cell culture medium allows cells to survive for at least one day without the addition of nutrients and consequently the transition from sol to gel can take place without any interference, potentially modifying the material. Morphological characterization has been performed with SEM in high vacuum, low vacuum and environmental modes: the acquired images revealed a porous and fibrous structure perfectly comparable to what observed in the literature. Density measurements have been performed with pycnometer revealing that the density of fibrin gel is comparable with human soft tissues. Transient grating measurements have been used to assess speed of sound within fibrin gel and its storage longitudinal modulus: the analysis highlights fibrin gel compatibility with human soft tissues also for these two parameters. Biological characterization relies on the assessment of fibrin gel biocompatibility. It has been evaluated in terms of degradation time and cytocompatibility: results on degradation time highlight the ability to produce long-lasting fibrin gel using the developed protocol, particularly at least 60 days without cells and a 21-day experiment showed that it does not degrade in that time with cells embedded in it. Cytocompatibility has been assessed through viability tests based on resazurin assay revealing that cells are able to survive and proliferate inside fibrin gel. The results highlight the ability of fibrin gel to mimic a biological environment and pave the way to designing novel studies using an in vitro 3D scaffold. Potential applications include cancer research, side effects of drugs and pollutants and tissue engineering.