**2. Biocompatibility**

The concept of biocompatibility is widely used within biomaterial science, but it is still uncertain what it really means. When it was first used in the early 1940s, a material was considered biocompatible if it could be placed in contact with tissues without altering them: a biocompatible material was conceived to be ideally inert. However, as research progressive‐ ly revealed that a true biological inertia is not possible, because any thing that enters in contact with a tissue induces a non-self response from the host immune system, the concept of

biocompatibility had to be necessarily reviewed. For years materials were considered biocompatible if they were non-toxic, non-immunogenic, non-carcinogenic, non-irritant, and so on against human body. During the 1980s, new evidences brought about another change of view and lead to a more modern definition of biocompatibility. First, it was clear that materials always react with tissues and that they are not inert. Second, it was shown that biological responses to biomaterial are different across tissues, and that the tissue itself affects material biocompatibility. Third, the scientific community realized that some clinical situation require that materials get degraded and removed from the host after accomplishing their function [33]. Accordingly to these concepts, a widely accepted definition of biocompatibility was outlined at the Consensus Conference in Boston in 1987 as follows: "Biocompatibility refers to the ability of a material to perform with an appropriate host response in a specific situation" [34].

In conclusion, focusing on this definition, a material is inserted into a tissue to perform a function, not simply lie inertly, and tissue responses to the material have to be adequate to the specific desired applications [35].

Biocompatibility as defined above is a pivotal concept for TE and scaffolds fabrication. A scaffold can be considered for *in vivo* application if it has been proven to be biocompatible *in vitro*, i.e., if it can support cell adhesion and proliferation. Cellular responses, in turn, heavi‐ ly depend on protein adsorption on the scaffold surface. Protein adsorption on materials is a spontaneous phenomenon that can be accompanied by protein denaturation, i.e., alteration of protein conformation and function [36]. Protein denaturation on to surfaces may occur for different reasons, mainly due to the chemical and physical characteristics of the material, and for that, a series of methods to enhance the biocompatibility of the surfaces have been developed.

#### **2.1. Modern approaches to enhance scaffold biocompatibility**

It has been solidly established that shortly after implantation biomaterials are covered with a thin layer of host proteins, and it is believed that the state of adsorbed proteins play a key role in scaffold colonization from cells [37]. Therefore, controlling the amount, composition and conformation of adsorbed proteins is a viable approach to obtain a highly biocompatible surface [38]. In recent years, several strategies have been developed to guide protein adsorp‐ tion and thus to improve cell adhesion, including immobilizing short fragment or proteins on scaffolds, or chemically and physically modifying scaffold surfaces.

#### *2.1.1. Chemical and physical treatments*

It has been demonstrated that some proteins bind preferentially certain chemical groups. For example it has been shown that fibrinogen binds methyl (–CH3) functionalized surfaces, but not carboxy (–COOH) ones, whereas the hydroxy (–OH) groups enhance the affinity for albumin over fibrinogen [39–41]. Therefore, the first strategy developed to control protein adsorption on scaffolds was enriching surfaces with functional groups, by combining chemical and physical treatments.

Chemical graft modification entails surface activation through different methods, such as chemical reactions or UV, plasma, and ozone exposure [42], followed by covalent grafting of the desired functional groups. Chemical grafting has been used to improve hemocompatibil‐ ity of vascular grafts by enriching them with heparin and polyethylene glycol (PEG or PEO). The drawbacks of this approach include the loss of protein mobility at the material surface, because they are covalently bound and the possible release of toxic monomers [38].

To overcome issues associated to chemical graft deposition, self-assembled monolayers (SAMs) were developed. SAMs was widely used to study *in vivo* responses of implanted biomaterials in the past, although nowadays is limited to gold- and silver-coated surfaces [38, 43, 44].

An increasingly popular method to graft surfaces with functional groups is plasma modifica‐ tion. Plasma is considered the fourth state of matter and it is obtained when gases are excited by specific electromagnetic frequencies. Plasma modification is cheap and seems to be very effective, but it is still being currently investigated for the development of biomedical devices, including metals, polymers, and ceramics [38, 45].

#### *2.1.2. Immobilization of RGD and other recognition sequences for integrins*

One of the most recent approaches developed to enhance scaffold biocompatibility is the surface immobilization of small peptides able to mimic proteins involved in cell adhesion, to enrich scaffolds with docking points for cells (Ruoslahti, 1996). The best investigated peptide is the arginine-glycine-aspartic acid (RGD) motif, an ubiquitous adhesive sequence found in many ECM proteins responsible for their interaction with cellular integrin receptors [46]. Several groups have studied the *in vitro* ability of RGD and related motifs to improve osteoblast adhesion, migration, and gene expression [47–49]. Moreover, coating titanium implants with the RGD peptide has been shown to induce a direct activation of macrophages, osteoblasts, and osteoclasts in rat tibia and femur and in dog femur [50–52].

However, Hennessy et al. enriched hyaluronic acid disks with RGD and observed poor cell adhesion and inhibitory effects of the RGD binding domain, probably due to the fast adsorp‐ tion of fibronectin, vitronectin and fibrinogen within 30 min, which competed with RGD motifs to bind integrins [53].

#### *2.1.3. Surface coatings*

biocompatibility had to be necessarily reviewed. For years materials were considered biocompatible if they were non-toxic, non-immunogenic, non-carcinogenic, non-irritant, and so on against human body. During the 1980s, new evidences brought about another change of view and lead to a more modern definition of biocompatibility. First, it was clear that materials always react with tissues and that they are not inert. Second, it was shown that biological responses to biomaterial are different across tissues, and that the tissue itself affects material biocompatibility. Third, the scientific community realized that some clinical situation require that materials get degraded and removed from the host after accomplishing their function [33]. Accordingly to these concepts, a widely accepted definition of biocompatibility was outlined at the Consensus Conference in Boston in 1987 as follows: "Biocompatibility refers to the ability of a material to perform with an appropriate host response in a specific situation" [34].

In conclusion, focusing on this definition, a material is inserted into a tissue to perform a function, not simply lie inertly, and tissue responses to the material have to be adequate to the

Biocompatibility as defined above is a pivotal concept for TE and scaffolds fabrication. A scaffold can be considered for *in vivo* application if it has been proven to be biocompatible *in vitro*, i.e., if it can support cell adhesion and proliferation. Cellular responses, in turn, heavi‐ ly depend on protein adsorption on the scaffold surface. Protein adsorption on materials is a spontaneous phenomenon that can be accompanied by protein denaturation, i.e., alteration of protein conformation and function [36]. Protein denaturation on to surfaces may occur for different reasons, mainly due to the chemical and physical characteristics of the material, and for that, a series of methods to enhance the biocompatibility of the surfaces have been

It has been solidly established that shortly after implantation biomaterials are covered with a thin layer of host proteins, and it is believed that the state of adsorbed proteins play a key role in scaffold colonization from cells [37]. Therefore, controlling the amount, composition and conformation of adsorbed proteins is a viable approach to obtain a highly biocompatible surface [38]. In recent years, several strategies have been developed to guide protein adsorp‐ tion and thus to improve cell adhesion, including immobilizing short fragment or proteins on

It has been demonstrated that some proteins bind preferentially certain chemical groups. For example it has been shown that fibrinogen binds methyl (–CH3) functionalized surfaces, but not carboxy (–COOH) ones, whereas the hydroxy (–OH) groups enhance the affinity for albumin over fibrinogen [39–41]. Therefore, the first strategy developed to control protein adsorption on scaffolds was enriching surfaces with functional groups, by combining chemical

**2.1. Modern approaches to enhance scaffold biocompatibility**

scaffolds, or chemically and physically modifying scaffold surfaces.

specific desired applications [35].

332 Advanced Techniques in Bone Regeneration

*2.1.1. Chemical and physical treatments*

and physical treatments.

developed.

The application of coatings that mimic the ECM could be an alternative method to improv‐ ing scaffold biocompatibility. In particular, coatings for bone biomaterials should promote the creation of a suitable environment for osteoblast, osteoclasts, and progenitor cells, that promote implant integration, by improving bone/implant contact (BIC) [46]. Coating titanium im‐ plants with collagen, which is the most abundant protein in bone tissue, supports *in vitro* adhesion, migration, and differentiation of osteoblasts [54, 55]. Similarly, coatings of hydrox‐ yapatite-based scaffold with chondroitin sulfate (CS), wide spread in cancellous and cortical bone, Hyaluronic acid (HA) or heparin have demonstrated to increase BMPs secretion and consequently osteoblasts differentiation [56, 57].

All the issues connected to the strategy described, prompted us to develop a new method to enhance scaffold biocompatibility by using a novel class of molecules, called aptamers, to improve protein adsorption and cell adhesion.
