hydrogels) appear to be very promising for the development of a new strategy that could stimulate neural tissue and axonal regrowth, thus inducing the formation of new synapses for the restoring of the lost neural circuits.89,93 In order to act as a bridging implant, an ideal scaffold should possess specific characteristics: the ability to fit the cystic cavity, proper morphology, mechanical properties and degradation rate, the ability to deliver cells and growth factors and, preferably, electrical conductivity.6,94-97 In particular, regarding the morphology, an ideal scaffold should possess oriented/channel-like pores in order to guide the neuronal and axonal growth6,94,98 with pore dimension around 50-100 µm for scaffold colonization.94,99,100 The electrical conductivity can be achieved by the introduction of conductive polymers, such as polypirrole100 or carbon nanotubes (CNTs), as described in the following paragraphs.
1.3.2.1 Scaffolds design for neural tissue engineering
Among the wide range of possible materials that can be employed for the preparation of scaffolds for neural tissue engineering, biopolymer-based biomaterials are very promising candidates for their biocompatibility, gel forming properties, biodegradability and bioactivity.94 Several methods and processes can be used for the preparation of scaffolds and hydrogels with oriented pores, containing alginate or chitosan as structural components. In alginate hydrogels these structures can
INTRODUCTION
be obtained thanks to an oriented gelation101 or through mechanical stretching of the materials.102 Anisotropic porous structures can also be obtained by means of directional supercritical CO2
foaming,103 after the freeze-casting of hydrogels that have been frozen in an oriented way on a cooled plate98,104,105 or by the Ice Segregation Induced Self Assembly (ISISA) process in a liquid nitrogen bath106-110 or in dry ice.111
For example Francis et al. reported the preparation of a scaffold with aligned pores prepared with chitosan and alginate: the scaffold possesses mechanical properties similar to the neural tissue and was able to guide the neurite growth of the dorsal root ganglia.94 Alginate, chitosan and gelatin-based scaffolds have been successfully used, in combination with GFs or RGD peptides, to promote neural differentiation and nerve regeneration.112-114
Specific biologically relevant proteins and polysaccharides can be used as bioactive components within the scaffolds. For example, hyaluronic acid has proved to reduce astrocyte proliferation thus helping to attenuate the inflammatory response and gliosis in the surrounding tissue;115 moreover, it was demonstrated that the presence of hyaluronic acid supports angiogenesis and inhibits glial scar formation.116 Fibrin has been used for the preparation of scaffolds for the delivery of neurotrophin-3 (NT-3), resulting into enhanced neural fiber sprouting in rats;117 on the other hand, the aligned fibers of fibronectin mats can orient the axonal growth in rat damaged spinal cord.118 1.3.2.2 Sustained neurotrophine synthesis: co-cultures with engineered mesoangioblasts An interesting approach for the implementation of neural scaffolds and the improvement of their bioactivity is represented by the incorporation of cells that are able to synthesize and locally release neurotrophines (NTs) such as NT-3, Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF). This approach guarantees a sustained, selective and controlled release for long periods. Sasaki et al. showed an improved locomotor recovery for rat after the transplantation of gene-modified human mesenchymal stem cell (hMSCs) that overexpressed BDNF.119 These cells were also used in combination with agarose scaffolds and showed an improved tissue regeneration.98 Genetically engineered neural stem cells that overexpressed NT-3, seeded in poly(ɛ-caprolactone) (PCL) scaffolds, proved to increase behavioural and electrophysiological recovery in rats with hemisection surgery in the spinal cord.120
INTRODUCTION
A promising cell lineage that can be used in this approach is represented by the mesoangioblasts (MABs): these cells are self-renewal multipotent progenitors of mesodermal tissue that have already been utilized for tissue engineering121 and can be isolated from small biopsies of postnatal human skeletal muscle.122 Su et al. showed that it is possible to genetically engineer these cells to induce the production and release of NGF and BDNF.123 The positive effects of these cells were proved in terms of viability and electrophysiological activity of primary neuronal cells and adult organotypic hippocampal slices when cultured in the presence of MABs conditioned media.
Moreover, the effect of BDNF produced by the engineered MABs is higher than that obtained with the administration of recombinant BDNF.123
Altogether, these results show the great potential of the development of scaffolds enriched with bioactive components and genetically modified cells, to be employed in the design of bridging implant strategy, since the supplementation of NTs and of other bioactive compounds can sustain the survival and functional recovery of neurons by modulating the post-injury microenvironment.
1.4 BONE TISSUE REGENERATION
Bone tissue is a specialized form of connective tissue that plays key roles in several physiologic functions: to name a few, protection and support for organs, movement, blood production, storage and homeostasis of calcium and other minerals, blood pH regulation, mesenchymal and hemopoietic cell progenitors housing.5 It is mainly composed of inorganic mineral crystals, that accounts for the 60-70% of the dry mass; the principal mineral component is hydroxyapatite (HAp, Ca10(PO4)6(OH)2), but there are also small amounts of other inorganic salts.124 The remaining part of the dry mass is composed for the 10–20% by collagen fibers, with a prevalence of collagen type I, proteoglycans and non-collagenous proteins such as osteocalcin, osteopontin, osteonectin, fibronectin and thrombospondin.125-127 According to the structure, bone can be categorized in two types: the cortical (compact) and the trabecular (cancellous or spongy) bone. The 80% of the skeletal bone is composed of cortical bone; it possesses a high elastic modulus (~20 GPa) and a low porosity (5-10%) and its average ultimate compression strength is 105-131 MPa.128,129 In contrast, the porosity of trabecular bone is higher (approximately 50–95%), but it presents lower mechanical properties: the elastic modulus ranges from 25 to 240 MPa, while the ultimate compressive strength ranges from 0.2 to 10.4 MPa.75,130
INTRODUCTION
1.4.1 BONE TISSUE DAMAGES AND COMMON THERAPEUTIC APPROACHES