**3. Bio-nanocomposites**

a tissue with its functional properties [30]. The microscopic structure must allow nutrient diffusion as well as the efflux of metabolites no longer needed to the cell through the scaffold. Finally, the scaffold must have good mechanical properties, enabling handling during culture

One of the biggest health issues worldwide is organ failure derived from disease or a traumatic event; this has been resolved by transplantation of organs from living or deceased patient donors. The list of donors and recipients has increased in the last years and there are many patients on waiting lists for organ donation [31]. According to Gilpin and Yang [31], tissue engineering consists of three important aspects: the participating cells, the signaling molecules used and the scaffold. Scaffolds can be natural or synthetic. Natural scaffolds are derived from decellularization processes using chemical, enzymatic or physical methods. The resulting decellularized scaffold has to be recellularized either with one or different type of cells, in other cases

For more than 20 years, scientists started developing nano-bio-materials and it is thought that nano bio-composites will be more important than non-nanometric materials at the physiological level. The advancement in biomedical research due to the incorporation in biomaterials to

Decellularized scaffolds have been improved by combining them with biomaterials, not only to provide the extracellular matrix required for the cells to proliferate and differentiate but also to provide structural, biochemical and biomechanical support to the regenerated organ. Cheng et al. [35], developed silk-based scaffolds for bone regeneration, but their therapeutic efficacy was not optimal, therefore they developed a composite material of mesoporous bioactive glass/silk scaffold to improve mesenchymal stem cell regeneration activity in a rodent model for postmenopausal osteoporosis. They proved that the composite material provided the optimal environment for mesenchymal stem cell differentiation, attachment, and proliferation as treatment of osteoporotic defects [35]. Sterling and Guelcher [36] proposed another example of scaffolds to heal fractures derive for osteoporosis. In this research, the authors have argued that bone autografts (bone sample from the same patient), that have been used to improve fractured bone healing, have some pitfalls due to the limited amounts of bone that can be harvested, instead, hybrid scaffolds have been fabricated made with silk and calcium phosphate to stimulate bone formation and to reverse bone loss. The same group has shown that local delivery of recombinant bone morphogenic protein from microspheres made with polylactic glycolic acid has improved the mechanical properties of vertebrae in animal models [36]. We have been addressing some examples of the use of nanomaterials in conjunction with biological models or cells, but we are also going to show how these systems have to be visualized

Regarding tissue engineering, once the bio-engineered tissue is developed, it has to be evaluated in its structure and function. Histological and histochemical techniques have been used. For example, it is important not only to evaluate the 3D structure of scaffolds and its possible interaction with cells prior deciding on a biological or clinical application, but also the functionality of the cells contained the manufactured tissues. Different imaging techniques can be used to assure the efficiency of the biocomposites, such as ultrasound, microscopy, magnetic

resonance imaging (MRI), and other optical imaging techniques [20].

induced pluripotent stem cells are used to recapitulate organ functionality [31–33].

biological models has had a great impact in health sciences [34].

for further biomedical characterization.

in bioreactors and transplantation into the host [30].

30 Tissue Regeneration

Biomaterials research has been concerned with the use of nanomaterials to enhance the tissue regeneration process. In this regard, nanomaterials can be classified into organic and inorganic systems. Diversity in organic materials derived mainly from polymers, such as polysaccharides, collagen, and chitosan have been recently used with different morphologies into the biomedical application and stem cell differentiation [19]. In particular, the use of polymer NPs as carriers or drug delivery systems is promising materials used as neuroprotectors to avoid acute ischemic stroke, which is actually considered one of the most common causes of death worldwide [40]. Nanostructured drug delivery systems offer many advantages, such as the avoidance of drug degradation, the possibility to improve the pharmacokinetic profile and the specificity at nano scale.

NPs from different materials have been functionalized with bioactive molecules in order to describe their effects in cells and tissues. Bio-composites of silica NPs with fluorescent compounds from the tree *Eysenhardtia polystachya* were internalized into MCF-7 breast cancer cells and observed with confocal microscopy to analyze their possible anti-tumor effect [41].

Cells interact with each other through their own synthesized ECM, which provides support and allows proliferation and differentiation processes. In consequence, ECM produces high membrane adherence with specific ligands associated with signaling pathways and possible migration, which can regulate the cell growth [42, 43]. Our body possesses natural ECM, mainly conformed by fibrous proteins and proteoglycans, ranging in size from 50 to 500 nm [44]. In this regard, collagen is an important source of ECM, present in the majority of connective tissues, such as bone, skin, and tendons. It is confirmed by a three-dimensional protein network by nano-sized fibers, with high resistance and adherence [29, 45], and recently, many studies have been focused to mimic this behavior and replace it with functional materials.

Several studies have been directed to design and understanding the composition and structure of new hybrid polymers. These hybrid materials are made of Au and Ag NPs supported on a polymer grill; the matrix prevents NPs aggregation, provides mechanical backing and keeps biocompatibility. In this area, CTS appears as a unique material with polycationic, chelating, and film forming properties. Additionally, through NPs incorporation its antibacterial

Trends in Tissue Regeneration: Bio-Nanomaterials http://dx.doi.org/10.5772/intechopen.75401 33

The AgNPs synthesis allows the production of stable metal NPs. When these NPs are incorporated in CTS electrospun fibers, it is possible to obtain high antimicrobial nanomaterials [62]. This behavior is generated due to the polycationic characteristics of CTS matrix and their interaction with the embedded AgNPs, linked by electrostatic attractions [63]. It has been reported that amine/hydroxyl groups presented in CTS matrix can interact with metal ions, in order to form stable complexes, and it is possible to in situ synthesize metal NPs in CTS solution, with high morphology control [64]. Moreover, AuNPs are also used due to their excellent biocompatibility and especially because it was found that CTS-AuNPs nanocomposites enhance the proliferation of human fibroblasts. This significant enhancement of biocompatibility may be due to the altered surface morphology. The size of the nanometric surface domains could have

The mechanical properties of CTS (e.g. swelling), are not good enough for medical applications; to solve this it was inserted into the structure, a natural synthetic polymer CTS-based, grafted with glycidyl methacrylate (CTS-g-GMA) [66]. This arrangement of polymers pro-

Ag and Au NPs show a collective oscillation of their electrons from the conduction band when they interact with a specific electromagnetic field; this property is called surface plasmon resonance (SPR). After all the evidence collected from the interaction between noble metals and natural polymers, the results are the evident success in the aggregation of AuNPs and AgNPs. This behavior was confirmed by UV-vis analyses, where the SPR bands were used to identify the metallic elements. As result, characteristic SPR for the AgNPs was located at 427 nm, while

Fibers obtained by electrospinning have been synthesized modifying the film method, the viscosity did not allow the correct stretched from the solutions, then it was necessary to add to the mixture polyethylene oxide and a surfactant to enhance the viscosity in order to obtain

These results are promising, the combined UV-vis spectra from the materials show SPR in 432 nm for AgNPs and 532 nm for AuNPs. Transmission electron microscopy (TEM) allows to observe fibers with several particles inserted in the surface, as presented in **Figure 2**. In this case, is not possible to determine if observed NPs are Ag or Au using only TEM imaging (elemental analyses show the presence of both elements), but the presence of them indicates that the NPs synthesis was successful and the electrospinning method is an option to perform

The major contribution of this research is that normally both metals, Au and Ag, are reduced chemically by separated and joined after these chemical reductions, nevertheless both nanostructured materials shown above as, films and fibers, underwent the chemical reduction *in situ*.

the SPR peak of the AuNPs was located around 530 nm [58], as shown in **Figure 1**.

materials with the characteristics to be used in biomedical applications.

effect increases and it can either stimulate or inhibit human cells activity [61].

an impact on cellular responses [65].

nanometric fibers of the polymer with NPs.

vided a new material with better biomedical applications.

The challenge in the research of materials able to replace the ECM is the recreation of a functional nanostructured network which allows cellular growth and differentiation. In fact, there are a lot of techniques for this task but there is one in particular that has been used more frequently in the recent years by researchers because it actually generates a fibrous structure like the ECM [46]. Electro spinning technique can produce nano-sized fibers from different sources, such as polymers, biocompatible systems, sol-gel, and nanocomposite materials. This technique generates three-dimensional porous fibers with high electrostatic attraction, associated with their high surface area/aspect ratio [47]. In this regard, this technique works from a solution (polymer, nanocomposite, and others) passed through a syringe, ending from a Taylor cone to control the efflux. A voltage source creates a drop and is collected at different distances to create variable morphology fibers. The surface tension produced between the collector and the needle is created by the electrostatic forces of the fibers [47].

Chitosan (CTS) has been defined as one of the most common biopolymers and chemically is a linear polymer derived from the deacetylated process of chitin, which is obtained from crustaceans [48]. The main characteristics of CTS are their biocompatibility and degradability [49], and can be easily processed in many different structures such as films, scaffolds, and fibers. CTS has been studied as antibacterial, biocompatible material, as a carrier for specific drug delivery and wound healing dressings [50, 51]. Some of its chemical properties are its solubility in organic acids [52] and low solubility in water. In order to improve the biological behavior of CTS material, different authors propose the addition of nano-sized structures to increase the physic-chemical and antibacterial properties, such as silver nanoparticles (AgNPs) [53] and gold nanoparticles (AuNPs) [54].

NPs of noble metals are some of the most promising materials, owing to their high surface area and their facility of functionalization or coordination with organic molecules. For example, AuNPs are easily prepared in colloidal solutions. Novel research has been done exploring the potential use of AuNPs as phototherapeutic agents, in the detection and treatment of cancer, in gene therapy and in the transport and selective vectorization of drugs and macromolecules [17, 54]. Otherwise, the AgNPs are widely applied to produce artificial skin, sterilized materials, functional contraceptive devices, antibacterial surgical instruments, bone prostheses, bone coating, surface cleaners, antimicrobial paints, automotive upholstery, food storage, and others [55, 56].

Many synthesis methods have been designed to create blends with metallic NPs and enable the combination and/or synergism of their catalytic, electronic, and optical qualities. Therefore, synthesis of supported gold and silver NPs has attracted lots of attention, in view of their remarkable properties, which depend on the NP size and the amount of each material [57]; they have been used in oxidation reactions, tumor cell targeting and detection, H<sup>2</sup> O2 production and catalytic applications [58–60].

Several studies have been directed to design and understanding the composition and structure of new hybrid polymers. These hybrid materials are made of Au and Ag NPs supported on a polymer grill; the matrix prevents NPs aggregation, provides mechanical backing and keeps biocompatibility. In this area, CTS appears as a unique material with polycationic, chelating, and film forming properties. Additionally, through NPs incorporation its antibacterial effect increases and it can either stimulate or inhibit human cells activity [61].

conformed by fibrous proteins and proteoglycans, ranging in size from 50 to 500 nm [44]. In this regard, collagen is an important source of ECM, present in the majority of connective tissues, such as bone, skin, and tendons. It is confirmed by a three-dimensional protein network by nano-sized fibers, with high resistance and adherence [29, 45], and recently, many studies have

The challenge in the research of materials able to replace the ECM is the recreation of a functional nanostructured network which allows cellular growth and differentiation. In fact, there are a lot of techniques for this task but there is one in particular that has been used more frequently in the recent years by researchers because it actually generates a fibrous structure like the ECM [46]. Electro spinning technique can produce nano-sized fibers from different sources, such as polymers, biocompatible systems, sol-gel, and nanocomposite materials. This technique generates three-dimensional porous fibers with high electrostatic attraction, associated with their high surface area/aspect ratio [47]. In this regard, this technique works from a solution (polymer, nanocomposite, and others) passed through a syringe, ending from a Taylor cone to control the efflux. A voltage source creates a drop and is collected at different distances to create variable morphology fibers. The surface tension produced between the col-

Chitosan (CTS) has been defined as one of the most common biopolymers and chemically is a linear polymer derived from the deacetylated process of chitin, which is obtained from crustaceans [48]. The main characteristics of CTS are their biocompatibility and degradability [49], and can be easily processed in many different structures such as films, scaffolds, and fibers. CTS has been studied as antibacterial, biocompatible material, as a carrier for specific drug delivery and wound healing dressings [50, 51]. Some of its chemical properties are its solubility in organic acids [52] and low solubility in water. In order to improve the biological behavior of CTS material, different authors propose the addition of nano-sized structures to increase the physic-chemical and antibacterial properties, such as silver nanoparticles

NPs of noble metals are some of the most promising materials, owing to their high surface area and their facility of functionalization or coordination with organic molecules. For example, AuNPs are easily prepared in colloidal solutions. Novel research has been done exploring the potential use of AuNPs as phototherapeutic agents, in the detection and treatment of cancer, in gene therapy and in the transport and selective vectorization of drugs and macromolecules [17, 54]. Otherwise, the AgNPs are widely applied to produce artificial skin, sterilized materials, functional contraceptive devices, antibacterial surgical instruments, bone prostheses, bone coating, surface cleaners, antimicrobial paints, automotive upholstery, food storage,

Many synthesis methods have been designed to create blends with metallic NPs and enable the combination and/or synergism of their catalytic, electronic, and optical qualities. Therefore, synthesis of supported gold and silver NPs has attracted lots of attention, in view of their remarkable properties, which depend on the NP size and the amount of each material [57];

O2

produc-

they have been used in oxidation reactions, tumor cell targeting and detection, H<sup>2</sup>

been focused to mimic this behavior and replace it with functional materials.

lector and the needle is created by the electrostatic forces of the fibers [47].

(AgNPs) [53] and gold nanoparticles (AuNPs) [54].

and others [55, 56].

32 Tissue Regeneration

tion and catalytic applications [58–60].

The AgNPs synthesis allows the production of stable metal NPs. When these NPs are incorporated in CTS electrospun fibers, it is possible to obtain high antimicrobial nanomaterials [62]. This behavior is generated due to the polycationic characteristics of CTS matrix and their interaction with the embedded AgNPs, linked by electrostatic attractions [63]. It has been reported that amine/hydroxyl groups presented in CTS matrix can interact with metal ions, in order to form stable complexes, and it is possible to in situ synthesize metal NPs in CTS solution, with high morphology control [64]. Moreover, AuNPs are also used due to their excellent biocompatibility and especially because it was found that CTS-AuNPs nanocomposites enhance the proliferation of human fibroblasts. This significant enhancement of biocompatibility may be due to the altered surface morphology. The size of the nanometric surface domains could have an impact on cellular responses [65].

The mechanical properties of CTS (e.g. swelling), are not good enough for medical applications; to solve this it was inserted into the structure, a natural synthetic polymer CTS-based, grafted with glycidyl methacrylate (CTS-g-GMA) [66]. This arrangement of polymers provided a new material with better biomedical applications.

Ag and Au NPs show a collective oscillation of their electrons from the conduction band when they interact with a specific electromagnetic field; this property is called surface plasmon resonance (SPR). After all the evidence collected from the interaction between noble metals and natural polymers, the results are the evident success in the aggregation of AuNPs and AgNPs. This behavior was confirmed by UV-vis analyses, where the SPR bands were used to identify the metallic elements. As result, characteristic SPR for the AgNPs was located at 427 nm, while the SPR peak of the AuNPs was located around 530 nm [58], as shown in **Figure 1**.

Fibers obtained by electrospinning have been synthesized modifying the film method, the viscosity did not allow the correct stretched from the solutions, then it was necessary to add to the mixture polyethylene oxide and a surfactant to enhance the viscosity in order to obtain nanometric fibers of the polymer with NPs.

These results are promising, the combined UV-vis spectra from the materials show SPR in 432 nm for AgNPs and 532 nm for AuNPs. Transmission electron microscopy (TEM) allows to observe fibers with several particles inserted in the surface, as presented in **Figure 2**. In this case, is not possible to determine if observed NPs are Ag or Au using only TEM imaging (elemental analyses show the presence of both elements), but the presence of them indicates that the NPs synthesis was successful and the electrospinning method is an option to perform materials with the characteristics to be used in biomedical applications.

The major contribution of this research is that normally both metals, Au and Ag, are reduced chemically by separated and joined after these chemical reductions, nevertheless both nanostructured materials shown above as, films and fibers, underwent the chemical reduction *in situ*.

**4. Clinical applications**

thermal burns.

spherical AgNPs around 7 nm, as presented in **Figure 3**.

**Figure 3.** AgNPs synthesized by *in situ* chemical reduction in CTS matrix.

The novel approach to use nanomaterials in regenerative medicine has established the design of functional tools to simulate, diagnose and stimulate cell growth of tissue or organs [16].

Trends in Tissue Regeneration: Bio-Nanomaterials http://dx.doi.org/10.5772/intechopen.75401 35

Burn wounds are a critical issue due to the widespread deaths due to the constant bacterial resistance to conventional antibiotics. In this regard, novel nanomaterials such as topic antimicrobial systems have been obtained to produce combined antibacterial/tissue regeneration responses in thermal burns. Luna-Hernández et al. [68] report the use of nanocomposites based on CTS/AgNPs synthesized by *in situ* chemical reduction method, obtaining embedded

In this research, controlled thermal burns produced in rats were treated with nanocomposites with different NPs concentration deposited at wound areas. These results showed the combined antibacterial responses to *S. aureus* and *P. aeruginosa*, depending on NPs concentration and the mesh formation of hydrated chitosan, which allowed bacterial penetration. As a result, significant tissue regeneration was shown in the thermal burns treated with CTS/AgNPs nanocomposites in comparison with untreated one, as presented in **Figure 4**. Also, histological assays showed important tissue regeneration responses in contact with nanocomposites, suggesting the myofibroblasts regeneration and accelerated healing processes compared to uncovered

Chemotherapy and radiation exert their effects by inhibiting tumor cell growth and by blocking tumor reformation. However, some cancer patients present tumor relapse due to cancer

**Figure 1.** UV-vis absorption spectra of silver nanoparticles (AgNPs), gold nanoparticles (AuNPs), and their corresponding nanomaterials formulated by AgNPs/CTS and AuNPs/CTS-GMA.

Is clear that there are more possibilities for NPs and natural polymers, here we have offered a slight landscape of that, additionally to Au and Ag different metals such as copper (Cu) also could be used in biomedical applications, but the noble metals are a field with an extensive list of contributions elsewhere [67].

**Figure 2.** Fibers of CTS with AuNPs and AgNP on the surface of the polymer.
