Design and development of bio-hybrid multifunctional materials for regenerative medicine

Biomimetics is a relatively recent multidisciplinary study embracing the use of nature as a model for innovative materials, structures and strategies. Biologically inspired approaches have been particularly attractive in several fields; in over 3.8 billion years of evolution, in fact, nature has introduced solutions maximizing functionality with reduced energy and materials and with no impact on environment, exactly the targets faced by the actual technological challenges. For these reasons researchers have been interested for years in trying to copy biological useful characteristics, including self-assembling and structural hierarchical organization, multifunctionality and environmental adaptability. In particular the last decade developments concerning nature-based materials and nature-inspired processes with potential biomedical applicability are achieving particular prominence thanks to their low impact on environment and exclusive high bio-compatibility. Bio-inspired materials have been proposed as excellent candidates in several fields such as cosmetic field, medical devices and tissue engineering; in particular, tissue engineering emerged as a viable therapeutic solution to regenerate diseased tissues. Associated to the concept of tissue engineering, the material science was involved and aimed at designing new custom-made scaffolds offering ideal environment for cell adhesion and migration, and regulating cellular proliferation and function by providing proper biochemical signals. Scaffolds are porous, degradable structures fabricated from either natural or synthetic polymers and other many kinds of inorganic materials. The optimization of their properties, so as to support cells, and the ability to degrade in response to matrix remodeling enzymes released by the cells is the key to the uniform regeneration of tissue. This PhD thesis had the aim to study natural processes and materials and mimic them to develop new bio-inspired and multifunctional materials capable to answer some need regarding tissue engineering, nanomedicine and cosmetic. The first material developed during PhD project was a hybrid composite for dentin regeneration. In this study by following a biomimetic approach we synthetized an hybrid composite in which the mineral phase, magnesium-doped hydroxyapatite nanoparticles (MgHA) are nucleated on a biopolymeric matrix (gelatin) resembling the chemico-physical features of the natural mineralized tissue. Chemic-physical evaluations demonstrated that biomineralization process took successfully place and the interaction between the two phases promoted the formation of a 60 wt% quasi-amorphous MgHA phase reproducing the chemical and physical features of the natural apatite. The hybrid composite (GelMgHA) was than blended with an hybrid polymeric matrix Gel/Chit (3/1 ratio) to obtain a 3D stable porous structure where the hybrid MgHA/Gel particles were homogeneously distributed, as SEM analysis confirms. By means of a controlled freeze-drying process was created microscopic channels whose structure is comparable to dentin tubules and suitable for cell penetration and matrix deposition. Furthermore, stability tests showed that the DHT cross-linking treatment performed on the dried scaffolds assure a low degradation rate and preservation of the 3D structure in physiological condition allowing the cell adhesion and proliferation before the structure destruction. 3D cell culture with mesenchymal stem cells highlight the promising properties of the new scaffolds for dentine regeneration. In detail, the chemical-physical features of the scaffolds, mimicking those of natural tissue, was suitable to stimulate cell adhesion with good cell/material interactions and the channel like porosity demonstrate to be suitable for long term cell colonization. MTT test established an increase in cell proliferation from day 1 to day 7 highlighting the absence of cytotoxicity confirming its high biomimicry and biocompatibility and its suitability as promising tool for 3D cell culture in dental regeneration. The second material developed during PhD project was a polymeric blend conveniently cross-linked for soft and hard tissue regeneration. In this study, blending processes are designed to combine the best properties of two bio-polymers and to obtain hybrid materials with improved mechanical performances without losing biocompatibility, chemical stability and flexibility. By means of cross-linking reaction and freeze-casting process, porous, stable and safe scaffolds for tissue regeneration based on gelatin and cellulose nano-fibers (CNF) were obtained. Several blend compositions and effective and safe chemical cross-linkers involving active groups of both gelatin and CNF were studied to stabilize the polymeric network and to control its degradation rate in simulated body conditions. In the first part the role of cross-linking was studied comparing genipin, hexamethylenediamine (HMDA) and dehydrothermal cross-linking treatment (DHT). SEM analysis, stability and viscoelasticity tests proved that by blending gelatine with suitable amounts of CNF and cross-linking with genipin, HMDA and DHT was possible to achieve well interconnected porous structures suitable for cell colonization with good performances in physiological conditions. In the second part, several blend compositions (1:0, 1:1, 2:1, 1:2, and 0:1) were studied to obtain scaffolds with different mechanical properties and biodegradability in simulated body conditions, able to induce specific cell differentiation. The evaluation of blends with different CNF:Gel ratio demonstrates the blend powerful able to improve the single polymer properties. In particular, polymeric blend reveals some different characteristics in comparison to pure polymers; this is a proof of blend concept. Furthermore, all blends are capable to promote and support the cell colonization and proliferation confirming that blending different polymeric matrices it is possible to engineer new nano-composite materials with improved properties respect to the original raw materials. Finally, the biomineralization process occurred successfully, but only CNF/MgHA40 and CNF/MgHA@Gel maintained a 3D dried structure after freeze-drying. Analysis showed a composite similar to the bone highlighted with a Ca/P ratio not stoichiometric and a MgHA poorly crystalline. Anyway, the final shape is not a robust scaffold yet. The third material developed during PhD project was a drug delivery nanosystems that allows to improve the drug efficacy, increasing the drug’s concentration that goes to target site and lowering the collaterals effects. In particular, magnetic hybrid nanobeads (MHNs) were created composed by alginate and Fe-hydroxyapatite nanoparticles that are both biocompatible and bioresorbable. Alginate derives by a brown algae and it is very investigated for its biodegradability, biocompatibility, low cost and capability of gelation with multivalent cations. Fe-hydroxyapatite is used because it allows to absorb or link on its surface a lot of target as bioactive molecules, moreover, Fe(II) and Fe(III) ions confer on apatite magnetic properties without containing secondary phases like magnetite that accumulates in the body and could have side effect on long-term. In this way, it is possible to drive nanosystem to the desired site before releasing bioactive molecules with the use of external magnetic field. A bio-inspired mineralization approach was followed to synthesize a superparamagnetic hybrid composite consisting of Fe-doped apatite nanocrystals nucleated onto alginate polymeric matrices. An oil-in-water emulsification process following by cross-linking technique was settled to obtain egg-like hybrid composites featuring uniform size distribution and exposure of mineral phase at the nanobeads surface. Chemical-physical analyses highlighted that MHNs exhibited biomimetic composition, adequate swelling properties and stability in physiological-like environment and superparamagnetic properties. Finally, MHNs did not negatively affect the cell viability and the cell proliferation over the time resulting as a promising magnetic drug delivery systems suitable for smart applications in nanomedicine. Finally, the last material developed during PhD project was a hybrid composites used in sunscreen formulation composed of iron- and titanium-doped hydroxyapatite and gelatin (GelFeTiHA) or titanium-doped hydroxyapatite and gelatin (GelTiHA) capable together to protect by UVA and UVB ray avoiding the whitening effect and the photocatalytic effect which can damage the tissues provoking skin disease. The use of sunscreens as protective barriers against skin damage and cancer, by absorbing harmful UVA and UVB rays, is becoming an increasingly important issue; such products are usually based on TiO2 that is able to reflect, scatter, and absorb UV radiation, thus preventing sunlight-related skin disorders such as sunburn and skin photodamage. However, it is well known to generate reactive oxygen species (ROS) under photoexcitation, it has to be chemically modified when used in sunscreens. HA-based materials have been developed for tissue regeneration and for drug delivery system and they have high biocompatibility and biomimicry. However, unmodified hydroxyapatite (HA) does not absorb in the UV range, but an interesting feature of biomimetic HA is to incorporate some foreign ions in its lattice decreasing its crystallinity. The purpose of this project is to modify HA structure with iron and titanium ions to obtain a UV-absorbing material. In the biomineralization process, the mineral phase (HA) is nucleated on polymeric fibres (gelatin) obstructing the particles’ growth and promoting small nanoparticles suitable to be dispersed in sunscreen cream, without damaging skin for the penetration of nanoparticles. Several analyses were performed to evaluate their morphology (SEM), the properties of mineral phase (XRD, FTIR, TGA) and their application in sunscreen field (UV/visible test, photodegradation test). Results demonstrate that only the sample with gel-TiHA revealed a high reflectance in the UVA and UVB range, however, the sample with gel-FeTiHA showed a good absorption in the UVB range. Although the reduced properties of gel-FeTiHA in terms of reflectance index, its combination with gel-TiHA is important because the presence of Fe ions in different amounts provide for a brown colour range avoiding the whitening effect typical of highly protective sunscreen. Finally, both samples do not form radicals and/or reactive species under irradiation highlighting their possible use in sunscreen field.