Nature-Inspired Processes and Structures: New Paradigms to Develop Highly Bioactive Devices for Hard Tissue Regeneration

Material scientists are increasingly looking to natural structures as inspiration for new-generation functional devices. Particularly in the medical field, the need to regenerate tissue defects claims, since decades, biomaterials with the ability to instruct cells toward formation and organization of new tissue. It is today increasingly accepted that biomimetics is a leading concept for biomaterials development. In fact, there is increasing evidence that the use of biomedical devices showing substantial mimicry of the composition and multi-scale structure of target native tissues have enhanced regenerative ability. As a relevant example, biomimetic materials have high potential to solve degenerative diseases affecting the musculoskeletal system, namely, bone, cartilage and articular tissues, which is of pivotal importance for most of human abilities, such as walking, running, manipulating, and chewing. In this respect, the adoption of nature-inspired processes and structures is an emerging fabrication concept, uniquely able to provide biomaterials with superior biological performance. The chapter will give an overview of the most recent results obtained in the field of hard tissue regeneration by using 3D biomaterials obtained by nature-inspired approaches. The main focus is given to porous hydroxyapatite-based ceramic or hybrid scaffolds for regeneration of bone and osteochondral tissues in neurosurgery and orthopedics

Multifunctional graphene oxide/biopolymer composite aerogels for microcontaminants removal from drinking water

Due to water depletion and increasing level of pollution from standard and emerging contaminants, the development of more efficient purification materials and technology for drinking water treatment is a crucial challenge to be addressed in the near future. Graphene oxide (GO) has been pointed as one of the most promising materials to build structure and devices for new adsorbents and filtration systems. Here, we analyzed two types of GO doped 3D chitosan-gelatin aerogels with GO sheets embedded in the bulk or deposited on the surface. Through combined structural characterization and adsorption tests on selected proxies of drinking water micropollutants, we compared both GO-embedded and GO-coated materials and established the best architecture for achieving enhanced removal efficiency toward con- taminants in water. To evaluate the best configuration, we studied the adsorption capacity of both systems on two organic molecules (i.e., fluoroquinolonic antibiotics ofloxacin and ciprofloxacin) and a heavy metal (lead Pb2\ufe) of great environmental relevance and with already proved high affinity for GO. The Pb monolayer maximum adsorption capacity qmax was 11.1 mg/g for embedded GO aerogels and 1.5 mg/g in coated GO-ones. Only minor differences were found for organic contaminants between coating and embedding approaches with an adsorption capacity of 5e8 mg/g and no adsorption was found for chitosan-gelatin control aerogels without GO. Finally, potential antimicrobial effects were found particularly for the GO-coated aerogels materials, thus corroborating the multifunctionality of the newly developed porous structures

Blending Gelatin and Cellulose Nanofibrils: Biocomposites with Tunable Degradability and Mechanical Behavior

Many studies show how biomaterial properties like stiffness, mechanical stimulation and surface topography can influence cellular functions and direct stem cell differentiation. In this work, two different natural materials, gelatin (Gel) and cellulose nanofibrils (CNFs), were combined to design suitable 3D porous biocomposites for soft-tissue engineering. Gel was selected for its well-assessed high biomimicry that it shares with collagen, from which it derives, while the CNFs were chosen as structural reinforcement because of their exceptional mechanical properties and biocompatibility. Three different compositions of Gel and CNFs, i.e., with weight ratios of 75:25, 50:50 and 25:75, were studied. The biocomposites were morphologically characterized and their total- and macro- porosity assessed, proving their suitability for cell colonization. In general, the pores were larger and more isotropic in the biocomposites compared to the pure materials. The influence of freeze-casting and dehydrothermal treatment (DHT) on mechanical properties, the absorption ability and the shape retention were evaluated. Higher content of CNFs gave higher swelling, and this was attributed to the pore structure. Cross-linking between CNFs and Gel using DHT was confirmed. The Young’s modulus increased significantly by adding the CNFs to Gel with a linear relationship with respect to the CNF amounts. Finally, the biocomposites were characterized in vitro by testing cell colonization and growth through a quantitative cell viability analysis performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the cell viability analysis was performed by the means of a Live/Dead test with Human mesenchymal stem cells (hMSCs). All the biocomposites had higher cytocompatibility compared to the pure materials, Gel and CNFs

Design e sviluppo di materiali bio-ibridi multifunzionali per la medicina rigenerativa

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

Sintesi e caratterizzazione di nanosistemi biocompatibili per drug delivery nel trattamento dell'Alzheimer

Le nanoparticelle polimeriche offrono grandi vantaggi nella nanomedicina in quanto fungono da carrier per il Drug Delivery e possono essere molto utili per le malattie ancor oggi difficili da trattare quali le neurodegenerative come l’Alzheimer. In questo progetto di tesi sono stati creati nanocarrier polimerici utilizzando come polimero un copolimero a blocchi anfifilico noto come PLGA-b-PEG: con varie tecniche si sono ottenute micelle polimeriche nelle quali sono stati intrappolati come principi attivi sia un farmaco, il liraglutide, sia nanoparticelle di magnesio; il primo può ridurre le placche β-amiloidee, tipiche cause dell’Alzheimer, mentre le seconde possono aumentare la plasticità sinaptica anch’essa legata all’Alzheimer. Inoltre è stato sintetizzato e intrappolato anche un promettente agente diagnostico, ovvero nanoparticelle di ferro, utile per aumentare la sensibilità di tecniche di imaging quali MRI e per la rivelazione precoce di malattie. Tutti i sistemi sono stati caratterizzati con tecniche specifiche per valutare i parametri chiave quali dimensione, polidispersità, carica superficiale e concentrazione dei componenti e successivamente sono state utilizzate per studi biologici effettuati da collaboratori esterni. Tutto questo ha come obiettivo futuro la creazione di un carrier teranostico che racchiuderà al suo interno l’agente terapeutico e l’agente diagnostico per combinare i due effetti principali in un unico carrier

Nature-Inspired Unconventional Approaches to Develop 3D Bioceramic Scaffolds with Enhanced Regenerative Ability

Material science is a relevant discipline in support of regenerative medicine. Indeed, tissue regeneration requires the use of scaffolds able to guide and sustain the natural cell metabolism towards tissue regrowth. This need is particularly important in musculoskeletal regeneration, such as in the case of diseased bone or osteocartilaginous regions for which calcium phosphate-based scaffolds are considered as the golden solution. However, various technological barriers related to conventional ceramic processing have thus far hampered the achievement of biomimetic and bioactive scaffolds as effective solutions for still unmet clinical needs in orthopaedics. Driven by such highly impacting socioeconomic needs, new nature-inspired approaches promise to make a technological leap forward in the development of advanced biomaterials. The present review illustrates ion-doped apatites as biomimetic materials whose bioactivity resides in their unstable chemical composition and nanocrystallinity, both of which are, however, destroyed by the classical sintering treatment. In the following, recent nature-inspired methods preventing the use of high-temperature treatments, based on (i) chemically hardening bioceramics, (ii) biomineralisation process, and (iii) biomorphic transformations, are illustrated. These methods can generate products with advanced biofunctional properties, particularly biomorphic transformations represent an emerging approach that could pave the way to a technological leap forward in medicine and also in various other application fields

Marine-Inspired Approaches as a Smart Tool to Face Osteochondral Regeneration

The degeneration of osteochondral tissue represents one of the major causes of disability in modern society and it is expected to fuel the demand for new solutions to repair and regenerate the damaged articular joints. In particular, osteoarthritis (OA) is the most common complication in articular diseases and a leading cause of chronic disability affecting a steady increasing number of people. The regeneration of osteochondral (OC) defects is one of the most challenging tasks in orthopedics since this anatomical region is composed of different tissues, characterized by antithetic features and functionalities, in tight connection to work together as a joint. The altered structural and mechanical joint environment impairs the natural tissue metabolism, thus making OC regeneration even more challenging. In this scenario, marine-derived ingredients elicit ever-increased interest for biomedical applications as a result of their outstanding mechanical and multiple biologic properties. The review highlights the possibility to exploit such unique features using a combination of bio-inspired synthesis process and 3D manufacturing technologies, relevant to generate compositionally and structurally graded hybrid constructs reproducing the smart architecture and biomechanical functions of natural OC regions

Medicated Hydroxyapatite/Collagen Hybrid Scaffolds for Bone Regeneration and Local Antimicrobial Therapy to Prevent Bone Infections

Microbial infections occurring during bone surgical treatment, the cause of osteomyelitis and implant failures, are still an open challenge in orthopedics. Conventional therapies are often ineffective and associated with serious side effects due to the amount of drugs administered by systemic routes. In this study, a medicated osteoinductive and bioresorbable bone graft was designed and investigated for its ability to control antibiotic drug release in situ. This represents an ideal solution for the eradication or prevention of infection, while simultaneously repairing bone defects. Vancomycin hydrochloride and gentamicin sulfate, here considered for testing, were loaded into a previously developed and largely investigated hybrid bone-mimetic scaffold made of collagen fibers biomineralized with magnesium doped-hydroxyapatite (MgHA/Coll), which in the last ten years has widely demonstrated its effective potential in bone tissue regeneration. Here, we have explored whether it can be used as a controlled local delivery system for antibiotic drugs. An easy loading method was selected in order to be reproducible, quickly, in the operating room. The maintenance of the antibacterial efficiency of the released drugs and the biosafety of medicated scaffolds were assessed with microbiological and in vitro tests, which demonstrated that the MgHA/Coll scaffolds were safe and effective as a local delivery system for an extended duration therapy—promising results for the prevention of bone defect-related infections in orthopedic surgeries

Additive-Free Gelatine-Based Devices for Chondral Tissue Regeneration: Shaping Process Comparison among Mould Casting and Three-Dimensional Printing

Gelatine is a well-known and extensively studied biopolymer, widely used in recent decades to create biomaterials in many different ways, exploiting its molecular resemblance with collagen, the main constituent of the extra-cellular matrix, from which it is derived. Many have employed this biopolymer in tissue engineering and chemically modified (e.g., gelatin methacryloyl) or blended it with other polymers (e.g., alginate) to modulate or increase its performances and printability. Nevertheless, little is reported about its use as a stand-alone material. Moreover, despite the fact that multiple works have been reported on the realization of mould-casted and three-dimensional printed scaffolds in tissue engineering, a clear comparison among these two shaping processes, towards a comparable workflow starting from the same material, has never been published. Herein, we report the use of gelatine as stand-alone material, not modified, blended, or admixed to be processed or crosslinked, for the realization of suitable scaffolds for tissue engineering, towards the two previously mentioned shaping processes. To make the comparison reliable, the same pre-process (e.g., the gelatin solution preparation) and post-process (e.g., freeze-drying and crosslinking) steps were applied. In this study, gelatine solution was firstly rheologically characterized to find a formulation suitable for being processed with both the shaping processes selected. The realized scaffolds were then morphologically, phisico-chemically, mechanically, and biologically characterized to determine and compare their performances. Despite the fact that the same starting material was employed, as well as the same pre- and post-process steps, the two groups resulted, for most aspects, in diametrically opposed characteristics. The mould-casted scaffolds that resulted were characterized by small, little-interconnected, and random porosity, high resistance to compression and slow cell colonization, while the three-dimensional printed scaffolds displayed big, well-interconnected, and geometrically defined porosity, high elasticity and recover ability after compression, as well as fast and deep cell colonization