SILK FIBROIN AS A COMPONENT OF SCAFFOLDS FOR TISSUE ENGINEERING
Creators: name: MAROTTA, LAURA AMELIA
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: Since the scaffolds are implantable systems designed to promote the growth of new tissue, they have to maintain their characteristics in the physiologic environment for a relative long period, until the moment the new tissue is completely reconstituted and the scaffold can start to degrade. The scaffold features totally depend from the scaffold material, which must confer to the structure the suitable morphologic and mechanical properties. Natural fibroin is a proteic polymer that possesses excellent properties of robustness due to its crystalline highly organized conformation, but when it?s regenerated to be used for the scaffold preparation it?s obtained in the amorphous form. An important aim in this work was to find a strategy to induce the fibroin conformational change towards the crystalline stable form. As reported in Chapter 1 the first considered strategy was blending the fibroin solution with other hydrophilic polymers, in order to form a new ordered structure constituted by the interaction of fibroin and the polymers chains. PEGs with different molecular weight were selected since several works reported the feasibility of the realization of stable 2D scaffold and the modification of the surface characteristics of fibroin films by blending PEG. The results showed how PEGs with a molecular weight lower than 1500 in a percentage comprised between 5 and 10 % w/w on the fibroin weight, were able to induce the conformational change in the fibroin structure when the casting method was used. This part of the study demonstrated the importance of a low drying rate in permitting the conversion to the stable crystalline form induced by PEG addition, since fibroin and PEG chains need sufficient time to organize themselves in an ordered structure. The freeze-drying process, instead, didn?t allow PEG to induce of fibroin organization in the right conformation in 3D scaffolds. In order to obtain the fibroin conversion inside the 3D scaffold, another strategy, reported in Chapter 2, was tried to induce the conformational change after the scaffold production. The sterilization with steam under pressure was selected, firstly because sterility is a fundamental requirement that the scaffold have to accomplish and then because literature data report how high temperatures and vapour content are able to promote the transition from the protein amorphous form to the crystalline one. An autoclave treatment on fibroin scaffolds, indeed, allowed the fibroin conversion to the ?-sheet form and permitted to obtain scaffolds with suitable characteristics. Furthermore, an in-depth study was carried out on the formulation and process variables of the freeze drying method used for the 3D scaffold production. Interesting results were obtained with the addition of small amounts of DMSO to the fibroin solution before the lyophilisation process that, despite not having any effect in the increase of the amount of crystalline fibroin in the scaffold, was able to improve the scaffold mechanical properties. Regarding the study of the freezing phase of the freeze-drying process, the freeze thawing effect, induced by an increase in the sample temperature at the end of the freezing process, led to an increase in the mechanical properties of the scaffolds, even if the ?-sheet content of the protein resulted lower. This was due to the fact that fibroin fibres were highly oriented perpendicular to the horizontal surface, instead of randomly oriented within the scaffold structures obtained with the traditional freeze drying process. In Chapter 3, composite 3D scaffolds made of fibroin and PEG600 were characterized in order to compare their features with the ones of the pure fibroin. The structural analysis on the scaffolds was conducted both before and after an incubation period of 4 weeks in growth medium. The main result was that the PEG addition increased the scaffold porosity and pore size and was able to keep the scaffold architecture during all the incubation period, stabilizing the fibres organization inside the scaffold. The stabilization effect influenced also the scaffolds mechanical properties, which were tested evaluating the scaffold resistance to compression with a texture analysis. The composite scaffolds realized with the addition of PEG presented a lower compression resistance but the value remained constant for all the 4 weeks of incubation, demonstrating that the polymer stabilizing effect on the scaffold structure have repercussions on the scaffold mechanics as well. The positive effect of the PEG addition was extensively demonstrated also in the study of the diffusivity through the scaffolds of oxygen and some model molecules miming the structure of nutrients like glucose. The oxygen diffusivity, indeed, was constantly diminishing during the incubation time for the pure fibroin scaffolds, whilst it remained rather constant for the fibroin/PEG600 scaffolds, showing also in this case that PEG600 is able to maintain the scaffold architecture in a physiologic environment during a prolonged period of time. In conclusion, the stability during incubation of scaffolds made of fibroin can be improved by adding low molecular PEGs and modulating the drying conditions of the regenerated fibroin solutions. In particular, PEGs are able to promote the conversion of fibroin to the water resistant and mechanical robust ?-sheet form in 2D scaffolds, thanks to the slow water evaporation process typical of the casting drying method. In the case of 3D scaffolds obtained by freeze drying, the most relevant result concerned to the PEG addition is its significant capacity in stabilizing the fibroin scaffold structure in terms of morphology, mechanics and permeability during incubation for a relative long period of time. Moreover, freeze thawing allowed the realization of constructs with improved mechanical properties and a differentiated internal organization which can be relevant for hepatocytes differentiation and meniscus reconstruction. In the field of tissue engineering, fibroin can be considered as a versatile material for the realization of scaffolds with properties that can be modulated depending on the particular needs and that are able to accomplish their functions for the time required to the new tissue to regenerate.