Tissue engineering (TE) is a quite new and very promising approach to obtain the repair and regeneration of tissues and organs lost, damaged or compromised due to trauma, injury, disease or aging [20]. A key component of TE approach to bone regeneration is represented by natural or man-made scaffold that are used as template for the interactions between different types of cells, and the formation of bone extracellular matrix providing structural support to the newly formed tissue. An ideal scaffold should have the following features (1) a three-dimensional (3-D) and highly porous structure with interconnected pores to allow cells migration, flow transport of nutrients, and removal of metabolic waste; (2) biocompatibility and resorbability, with a rate of resorption similar to that of the forming new bone tissues; (3) a surface chemistry that favors cells attachment, proliferation and differentiation; (4) mechanical properties comparable to bone and soft tissues at the implantation site; (5) a possibility to be commercially produced and safely sterilized without any alteration of its properties [21–25]. Several approaches have been used in bone regeneration procedures, and calcium phosphate ceramics are, probably, extremely effective as scaffolds. There is a necessity of further studies of the best ways in which materials, cells and biologically active molecules could interact. Different types of cells and growth factors are two pivotal elements in bone biology/healing, and their interaction is extremely important towards an effective regeneration process. The best combination of materials, cells and growth factors seems to be a must for a very effective bone TE strategy [26]. A system to be used for bone repair and regeneration would ideally require osteoconductive and osteoinductive properties, so that new bone formation can be improved through an adequately shaped three-dimensional (3D) scaffold (osteoconduction ) and by a biological stimulus (osteoinduction ) [27]. Ceramic materials , e.g. hydroxyapatite, tricalcium phosphate and coralline-derived calcium phosphate, due to their inorganic nature and ionic composition, are extremely useful in several applications. These materials are known for their ability to bond to bone and stimulate new bone formation. 3D systems have been produced with the use of particulates or blocks having a porous interconnected structure [28]. The formation of 3D scaffolds in particulate or block shape creates a potential for their use either without cells (placing of the scaffold in the tissues, and its colonization by surrounding cells) or combining them in vitro with cells, creating a hybrid cell–material construct. These 3D scaffolds can be used also as a delivery system, releasing bioactive agents and enhancing the regenerative potentialities of the system [29]. The ability of micro-CT to evaluate 3-D structures in a non-destructive way has made its use and application extremely wide in several fields, such as physics, materials science, medicine, mineral processing and powder technology. Furthermore, the possibility to use synchrotron radiation X-ray sources has further improved the application of micro-CT due to its numerous advantages compared to conventional X-ray sources, including a higher beam intensity, a higher spatial coherence and the monochromaticity [30]. The monochromaticity property of synchrotron radiation reduces significantly the beam hardening effects, thus allowing to simplify the segmentation process of the whole image analysis. Synchrotron radiation X-ray micro-CT has been used to evaluate the 3-D porous architecture and microstructure of several different calcium phosphate scaffolds after a long-term healing period in humans [31]. In the last decade, bone substitute biomaterials have been used in combination with cells for the fabrication of artificial bone grafts. The use of multipotent mesenchymal stem cells (MSCs) has opened up new therapeutic perspectives for the in situ or in vitro TE of bone. The success of tissue regeneration is related to the structure of the scaffold and its ability to allow invasion by cells and tissues. This construct can then be placed in living tissues to act as replacement tissue after the in-vitro colonization of MSC. Blood vessels [32] begin to grow around and into the construct, and as the scaffold undergoes resorption, the newly formed bone tissue starts blending with the surrounding tissues and finally replaces the scaffold. The scaffolds can be reproducibly manufactured with a specific, desired structure obtained according to stochastic, fractal, or periodic principles. In recent years, the efforts in TE have been focused mainly on the characterization of the regenerative properties of different sources of stem cells (dental pulp, periodontal ligament, amniotic fluid) [33–36]. Amniotic derived stem cells (ADSCs) are an intermediate stage between embryonic stem cells and lineage-restricted adult progenitor cells. Their high proliferation rate together with their differentiation potential into cells of all three embryonic germ layers (ectoderm, endoderm and mesoderm) are important advantages over most of the known adult stem cell sources. In vitro studies and tests are needed to evaluate the attitude of the constructs to support cellular events such as adhesion, proliferation and osteogenic cells differentiation [37]. All the results obtained from in vitro and animal experimentations will give essential informations to try to transfer and apply these novel therapeutic strategies to the field of tissue regeneration.

Approaches to tooth regeneration . The approaches to tooth regeneration are still in their infancy and face many obstacles. The different types of approaches that have been tried include: (a) remineralization of carious dentin by inorganic polyphosphates; (b) calcium phosphate coatings; (c) engineering of bone and tooth root using bioactive materials; (d) regeneration of different dental tissues with the use of different substances, i.e. amelogenins for the regeneration of the periodontal tissues, and calcium-phosphate ceramics and collagen for the reconstruction of the bone [38]. The deep and complete understanding of the principles that support the formation of teeth and periodontium represents the basic foundation to design innovative biomaterials to be used in the possible regeneration of these structures. When enamel undergoes demineralization , the residual mineral crystals can serve as templates for the new formation of apatite crystals [39]. The same process can be used in demineralized dentin, as for example can occur in a carious tooth, where dentin apatite crystals tend to remain and can be used as templates. It is also possible to attempt to remineralize dentin with the use of agents likes polyacrylic or polyaspartic acid [40]. These acids bind to collagen and serve to bind calcium and promoting apatite nucleation. The biomineralization processes, such as the formation of tooth enamel, is under the influence of various proteins such as the amelogenins. Mimicking nature, it has been tried to restore enamel by inducing remineralization of hydroxyapatite on the surface of the tooth. The regeneration of different parts of a tooth by implementation of biomimetic mineralization processes will represent a significant step for the future development of scaffolds for dental regeneration [41]. This will lead to the possible formation of teeth, and this fact could have an enormous benefit to human health with a huge socio-economic impact. Different hybrid composites will be evaluated to mimic the different regions of the tooth and of the periodontal tissues. These composites could have a structure as follows:

The different structure characteristics of the different tooth tissues could be obtaining by different degree of cross-linking. To regenerate the periodontal tissues could be used constructs composed of highly mineralized portions, i.e periodontal bone and cementum, connected by fibrous layers, mimicking the unmineralized periodontal ligament. Tooth regeneration could be a difficult but very important part of regenerative medicine in the future and could have a relevant importance in healthcare.

Graphene is a relatively new allotrope of carbon composed of a single layer of monocrystalline graphite with hybridized carbon atoms. Due to its structure, a one-atom-thick two-dimensional (2D) carbon material, graphene has attracted increasing attention in the past several years due to its high surface area, remarkable thermal conductivity, excellent charge mobility, and mechanical properties [42]. The unique structure and outstanding properties render graphene highly promising for a wide range of applications in the fields of electronics, sensor, and energy storage/conversion. Some of the other interesting aspects of graphene include high transparency toward visible light, high values of elasticity, unusual magnetic properties, and charge transfer interaction with molecules, that allowed it to gain a lot of interest in the biomedical field as a new component for biosensors, tissue engineering, and drug delivery. Graphene can be obtained following different approaches [43]. Interestingly, the majority of studies on chemistry of graphene do not involve “pristine” graphene, but rather carbon materials produced upon reduction of graphene oxide (GO) . Graphene-based materials show unique interactions with DNA and RNA, which make them attractive in DNA or RNA sensing and delivery. GO shows preferential adsorption of single stranded DNA over double stranded DNA and protects the adsorbed nucleotides from attack by nuclease enzymes opening up a wide range of application opportunities [44]. As opposed to interaction with DNA and RNA, there are only a few data on the interaction of graphene with proteins and lipids. It will be, hoewer, very important to understand interaction of graphene with lipid bilayer in the cell membrane. Protein adsorption on nanomaterials surface has received increasing attention for the past several years. This phenomenon affects in a significant way the behaviour of these materials in biological systems (e.g. cellular uptake and toxic responses). Nanomaterial surfaces are immediately covered by proteins, lipids, enzymes when put in a biological medium. These coated surfaces give new features to the nanosystem, i.e. hydrophilicity/hydrophobicity, surface charges and energy, topography [45, 46]. These new characteristics will produce the responses at the cell/tissue level. Due of their high specific surface area, the carbon family of nanomaterials including graphene has a potentially larger protein adsorption capacity than other nanostructures. After interaction with cells, tissues, or organs, graphene sheets surfaces change and have completely different biological properties. Hydrocarbons, organic molecules, and elements was reported to change the surface composition and surface energy, which affected the protein absorption and cell attachment, proliferation, differentiation, and final integration in the tissues [47–51]. The high active surface area of graphene, over other nanomaterials, is one of the main advantages of graphene based materials, which allows a high-density drug loading. Due to the specific geometry of graphene (2D structure), both sides of a single layer graphene sheet can be used as a substrate for the controlled adsorption of molecules and functional groups for surface modification [52]. For instance, it has been shown that covalent attachment of chitosan, folic acid, and polyethylene glycol (PEG) to GO produces a potential platform for the delivery of anti-inflammatory and water insoluble anticancer drugs such as doxorubicin (Dox) and SN38, a camptothecin analogue [53]. The idea is to study drug-graphene interaction as to be able to deliver, in a controlled manner and exploiting graphene-drug interactions, drug in proper quantity directly to the desired target site. It is important to understand such interactions from two points of view, one for biomedical applications and other for their toxicity and biocompatibility. Like other employed materials in nanomedicine, toxicity of the graphene is strongly dependent to its physicochemical properties (e.g., size and its distribution, surface charge, particulate state, number of layers, surface functional groups and particularly shape). One of the most important issues for biomedical applications of graphene is its short- and long-term toxicity [54–56]. Carbon-based materials (carbon nanotubes or nanocrystalline diamonds) have been widely tested for both their potential toxicological risks and their possible use in biomedical applications. Moreover, the antibacterial activity of graphene-based materials can be used in wound healing applications to prevent infections or to potentiate and protect the integration process of diffeent types of biomaterials biomaterials. Graphene, when used as a delivery vehicle, could, probably, potentiate the effects of antibacterial drugs. Gene therapy to treat genetic disorders and cancer is another area where graphene could be usefully employed. Successful gene therapy requires efficient and safe gene vectors that protect DNA from nuclease degradation as well as facilitate DNA uptake with high transfection efficiency. Graphene has been explored for applications in gene delivery, gene–drug co-delivery and protein delivery [43, 55, 57]. Osteogenic differentiation of Mesenchymal Stem Cells was enhanced on titanium surfaces coated with GO carrying BMP-2, compared to titanium surfaces coated only with BMP-2 [58]. In vivo studies in mouse also showed a higher new bone formation when using titanium–GO–BMP2 implants compared to only titanium or titanium–GO or titanium–BMP2 implants; these new composites could be very effective carriers for delivery of drugs. Several studies have emphasized the potential of graphene-based materials as drug and gene delivery vehicles in vitro; however, there is a need to demonstrate their potential in vivo with particular focus on safety, biodistribution and efficacy [59]. Therefore, graphene is an ideal model material for experiments with adherent (anchorage-dependent) cells (e.g. osteoblasts, mesenchymal stromal cells (MSCs), etc.). Adhesion of osteoblasts is a crucial prerequisite to subsequent cell functions, such as proliferation, synthesis of proteins (e.g. proteins of extracellular matrix (ECM), morphogenic factors and osteoinductive molecules) and formation of mineral deposits [43, 60, 61]. Adhesion is generally dependent on time, adhesive forces at the cell/material interface, and surface topography. Cell adhesion is primarily mediated by integrins, a widely expressed family of transmembrane adhesion receptors. Upon ligand binding, integrins rapidly associate with the actin cytoskeleton and cluster together to form focal adhesions (FAs) , which are discrete complexes that contain structural (e.g. vinculin) and signalling molecules (e.g., focal adhesion kinase). FAs are central elements in the adhesion process because they function as structural links between the cytoskeleton and ECM to mediate stable adhesion and migration. Furthermore, in combination with growth factor receptors, FAs activate signalling pathways that regulate transcription factor activity and direct cell growth and differentiation [62]. Human MSCs are mononuclear cell population adherent to tissue culture plastic and have been isolated from adult bone marrow. They are capable of further proliferation as well as differentiation into multiple lineages involved with connective tissue (osteoblasts, adipocytes and chondrocytes) when exposed to various growth factor combinations or substrates with different topography and rigidity). Thus, these cells may serve as a good model for testing the possible increased/accelerated differentiation induced by adhesion onto graphene surfaces. Recently, the utilization of graphene foam, a 3D porous structure, showed to be succesfully used as a novel scaffold for Neural Stem Cells (NSCs) in vitro. It was found that three-dimensional graphene foams (3D-GFs) can not only support NSC growth, but also keep cell at an active proliferation state. These findings show that 3D-GFs could offer a powerful platform for NSC research and neural tissue engineering [63].