Regenerative medicine and the regeneration process
Regenerative medicine is an emerging field of biomedicine that deals with tissue engineering and molecular biology. It is defined as the process of creating living, functional tissues to repair or replace tissue or organ function lost due to age, disease, damage, or congenital defects . Such procedure differs from tissue reparation on the fact that the repair involves the healing of dead tissue by fibrous patching, while regeneration is the re-growth of parenchymal and stromal cells. Consequently, reparation induces tissue replacement with scar lacking of the functional capacity, while regeneration restores damaged tissues both structurally and functionally (restitutio ad integrum) . Hence, regenerative medicine is focused on human cells (somatic, adult stem cells and embryo-derived stem cells) and their mobilization, recruitment and integration into functional tissues. Therefore, the key issue in regeneration relates with the implementation of an appropriate environment (‘niche’) for cells recruitment and complete functional integration . Two alternative regenerative routes are available with the use of these tailored biomaterials. In “Tissue engineering” progenitor cells are seeded onto modified scaffolds. The cells grow outside the body, become differentiated and mime naturally occurring tissues. These tissue-engineered constructs are then implanted into the patients to replace diseased or damaged tissues. Clinical applications of these biomaterials include repair of articular cartilage, skin and vascular system. Differently, the “In situ tissue regeneration” approach involves the use of biomaterials in the form of powders, solutions, or doped microparticles to stimulate local tissue repair. Bioactive materials release chemicals by diffusion or network breakdown and activate the cells in contact with these stimuli. Clinical applications of this technique include surgery, surgical implants and hospital procedures [1, 4]. Fig. 1 schematizes the regeneration process.
The critical prerequisite for successful tissue regeneration is an appropriate extracellular matrix (ECM) remodeling, as ECM provides physical scaffolding for the cellular constituents and initiates crucial biochemical and biomechanical cues required for tissues morphogenesis, differentiation and homeostasis. Moreover, the ability of ECM to produce a “bridge” for normal tissue edges to regenerate counteracts the tissue’s natural response of fibroblast deposition and scar formation that leads to reparation instead of regeneration . Biomaterials scaffolds mime the actions of ECM. In fact, these constructs provide an environment that supports stem cells differentiation in the presence of other co-factors, such as serum-containing cell culture medium or biochemical supplements. Moreover, the synergic action of material inherent properties and released factors determines cells shift in phenotype .
Development of regenerative biomaterials
The development of novel regenerative biomaterials is an iterative process that involves the creation of increasingly safer, more reliable and more inexpensive replacements for damaged or diseased human tissues. In the last sixty years, four generations of products were developed with increased advantages. The first group, the “Bio-inert material”, is engineered to provide appropriate mechanical properties for surgical applications, corrosion resistance, and absence of injurious effects such as carcinogenicity, toxicity, allergy and inflammation. Examples are the hydroxyapatites, bioactive ceramics, metals and alloys .
The second class of biomaterials is the “Resorbable polymers”. These composites have natural or synthetic origin and the most used are polylactides, polyglycolides, polycaprolactones and trimethylcarbonates. The most prominent use of resorbable polymers regards the synthesis of drug-eluting stents that are used to maintain patency of the coronary arteries. Such devices contain cytostatic, cytotoxic, antithrombotic and/or anti-inflammatory agents . The third class of biomaterials is “Biocompatible nanocomposites” created to promote or inhibit specific cell activities. The nanoscale dimension enables nutrient transport and supports cell proliferation. Consequently, this morphology mimes the natural extracellular environment . Finally, the fourth generation of biomaterials is the “Biomimetic composites”, characterized by the ability to release bioactive molecules and to interact with stem cells. The stem cells/materials interfaces are complex and dynamic microenvironment in which cells and materials cooperate, leading to the remodeling of cells surroundings. In particular, the inherent properties of materials (e.g. adhesivity, stiffness, nanotopography, molecular flexibility or degradability) induce lineage-specific stem cells differentiation .
Enhancing regenerative approaches with nanoparticles
Since the 1970s, when Nobel Prize winner Christian de Duve described the structure and properties of lysosomes in biological tissues, drug administration protocols have significantly evolved due to the introduction of nanosized drug delivery systems . These latter can be defined as ultra-dispersed solid organic or inorganic structures displaying a sub-micrometer size, typically comprised between 10 and 100 nm. The upper limit is dictated by the vector’s ability to pass cellular interstices, while the lower limit is fixed by the threshold for first-pass elimination by kidneys. Moreover, such dimensions permit a good biodistribution of long-circulating nanocarriers . Then, the use of nanotechnology to improve current approaches in tissue and organ regeneration has received increased attention over the years thanks to the great versatility that they offer in terms of size and surface chemistry, allowing the utilization as carriers for the delivery of drugs, genetic material or growth factors (GFs). Indeed, a variety of nanoparticles has been developed for therapy; among them dendrimers, liposomes, polymer-based nanoparticles, micelles, carbon nanotubes and many more (Fig. 2).  In regeneration, nanoparticles improve the regenerative capabilities of biomaterials offering ways to control surface and mechanical properties. Moreover, incorporation of nanoparticles within biomaterials increases cellular adhesion, differentiation and integration of stem cells into the surrounding environment. Finally, the drug delivery capabilities of nanoparticles offer additional possibilities to increase the biological performance of biomaterials. 
The first way to improve the tissue regenerating capabilities of biomaterials is to combine them with nanomaterials to create nanocomposites. This new class of materials showed improved mechanical and/or biological performance, compared to analogous composites without nanoparticles, due to the changes in the classic laws of physics consequent to the manipulation at scales of around 100 nm.  In particular, the nanostructural topographical properties (nanotopography) of the materials is able to mime natural tissue, that can be defined as a nanostructured material consisting of collagen fibrils and proteins with dimensions in the 100 nm size or less, or bone tissue, which is a nanostructured composite composed of a polymer matrix (mainly collagen) reinforced with nanomater-sized ceramic particles (mainly carbonated HA) in order to stimulates the cells to grow.  Nanocomposites are produced using a wide range of nanostructured materials (e.g. ceramics, polymers and hydrogels). For example, hydroxyapatite (HA) the native mineral structure of bone, in its microscale dimension is a poor material for bone reconstruction due to its brittleness and slow degradation rate. However, incorporation of HA nanoparticles into polymeric materials has created promising scaffolds for bone tissue engineering. In fact, HA nanoparticles coated on polymeric poly lactide-co-glycolide (PLGA), scaffolds facilitated bone formation in a concentration-dependent manner . Similarly, polylactide (PLA) scaffolds coated with HA nanoparticles stimulated the expression of osteogenic proteins (e.g. BMP-2, osteopontin) on scaffold-attached bone marrow-derived mesenchymal stem cells and facilitated bone regeneration . Incorporation of metallic nanoparticles prepared from iron oxide or titanium into polymeric scaffolds, increased collagen and calcium deposition by osteoblasts, leading to enhanced tensile strength compared to non-metallic incorporated materials [16-17]. A study by Khang et al. showed that bone cells respond differently on submicrometer and nanometer scale titanium surfaces, and that small changes in nanomater surface features can have larger consequences towards bone regeneration . Nanoparticles of biphasic calcium phosphate have been shown to increase the tensile strength of a biomaterial composed of polyvinyl alcohol/gelatin nanomater . Table 1 recaps the synthesized nanocomposite materials.
Controlled release of biomolecules from biomaterials
Another strategy to build bioactive nanomaterials for tissue regeneration is by incorporating biomolecules directly into the materials, in order to promote stem cell attachment in situ. Thus, such materials not only act as a scaffold but also as a delivery vehicle for controlled release of bioactive molecules. The use of nanoparticles gives advantages because of their drug delivery capabilities, inherent mechanical and biological properties and ease of functionalization. For example, plasmids coated PLGA nanoparticles within a fibrin hydrogel complex were found to be capable of enhance bone regeneration . Similarly, PLGA nanospheres encapsulating plasmid and enclosed within a nanofibrous PLA scaffold showed controlled release of BMP-7 (plasmid) followed by ectopic bone formation . In another approach, block copolymer nanolithography was used to tune the size of gold nanoparticles on a polymeric surface. The selective immobilization of the plasmid BMP-2 on the gold nanoparticles offered the possibility of exactly controlling the release . Also proteins can be incorporated into nanosized biomaterials in order to stimulate bone tissue regeneration. For example, the extracellular matrix molecule osteopontin, a protein that plays an important role in bone remodeling, was incorporated in HA nanoparticles enclosed into a degradable matrix. The release was analyzed for its osteoinductive potential in a dog bone defect model . Biomaterials with chitosan nanocomplexes delivering angiogenesis and osteogenesis, stimulating factors demonstrated great bone tissue regeneration potential .
Biomaterials with PLGA nanoparticles and alginate microcapsules encapsulating, respectively, the factors BMP-2 and VEGF showed a positive effect on the formation of vascularized bone . Last developments of research regarded the development of ‘smart’ biomaterials with the ability to spatio-temporally control the dose, sequence and profile of release of several regeneration factors. Types of carriers are nanogels, cross-linked gelatin–polymer composites or gelatin-based coatings . In these systems, the biomaterials were produced by incorporating different layers that serve as matrices enabling internal architecture with controlled release properties. Temporal controlled release of nanoparticles from biomaterials can be achieved via responsive linkers. For example, in a study by Tokatlian et al. nanoparticles were immobilized to a biomaterial through the use of matrix metalloproteinases (MMPs) sensitive linkers.
In this way, cell-secreted MMPs are able to release the nanoparticles from the biomaterial in a temporally controlled manner . Another method to remotely control biomolecule release is with external stimuli such as light. For example, Shas S. et al. produced Photo-triggerable hydrogel-nanoparticle hybrid scaffolds for remotely controlled drug delivery . Table 2 recaps the examples of controlled release of biomolecules from biomaterials.
In the past few decades, nanoparticles have revolutionized the field of drug delivery due to their unique physical characteristics. Then, nanostructures were designed to fit multiple purposes. Specifically, their application in the field of regenerative medicine regarded the incorporation within biomaterials to increase cellular adhesion, differentiation and integration of stem cells into the surrounding environment. Such research is still in its beginning phase but is already making important contributions.
CONFLICT OF INTEREST
All authors declare no conflicts of interest in this paper.