Scaffold Requirements

2.1.1.1 Biocompatibility

This is the first criterion that every tissue - engineered scaffold must meet [6].

William defines the term biocompatibility as:

"the capability of a material to facilitate natural cellular and molecular activity within a scaffold in the absence of systemic toxicity" [8].

Biocompatible scaffolds allow cells to adhere, migrate and proliferate on their surface without the risk of triggering dangerous inflammatory responses [6], and/or potentially toxic effects, both locally and systemically [9].

Good biocompatibility also promotes osteoconductivity, osteoblast proliferation and osteoinductivity [8].

In order for the scaffold to be biocompatible it is necessary to carefully choose the material with which it is manufactured. Suitable materials for BTE scaffolds must bind firmly to the natural bone in situ and promote new bone growth [9].

It has been shown that scaffolds in HA are highly biocompatible and improve the tissue repair process: thanks to the release of calcium and phosphate ions, osteogenesis is greatly accelerated [8].

Methods for controlling the scaffold biocompatibility can be found in ISO 10993 – 1 [10].

2.1.1.2 Porosity and Pore Size

Pore size and porosity percentage of the scaffolds is an essential parameter to get a physiological tissue development [11]. The ideal scaffold for cancellous bone tissue should have an interconnected porous structure with porosity > 80% [2], where more than 60% of the pores should have a size between 100 - 400 μm and at least 20 % is expected to be smaller than 20 μm [3].

Porosity percentage and pore size directly affect the osteoinductive and osteoconductive capabilities of the scaffold [12].

A suitable porosity range confers to the confers to the scaffold adequate mass transport properties for cell migration, attachment and interaction with the biological environment [6], as well as the passage of nutrients and bioactive molecules. Moreover, it has been demonstrated that suitable porosity features could improve vascularization and spatial organization between cell growth and ECM production, thus leading to a considerable enhancement of the biomineralization process [8].

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Large pores (around 200-300 µm), in fact, lead to direct osteogenic pathways [2],[9]. The prevailing opinion in the literature is that in order to allow greater cell migration and proliferation, and the consequent formation of new tissue, the pore size must be between 200-400 µm [13].

Smaller pores, instead, (closer 100 μm) were found to be beneficial to chondrogenesis [14].

However, too small pores, lead to poor vascularisation [11] and limited cell migration, causing the formation of cell capsules around the edges of the scaffold [9].

It is, however, necessary for the pores not to be too large, as this would excessively decrease the mechanical resistance of the final product [9], [6].

The ideal degree of porosity must be found so as to allow sufficiently high permeability and interconnecivity for nutrient supply and waste removal and adequate stiffness and resistance to loads transmitted from the healthy bone adjacent to the scaffold [2].

Interconnectivity (Figure 2) is a key requirement to ensure the transport of nutrients and the elimination of waste products [15].

An in vivo study with HA scaffolds has shown that low pore interconnection only enables pore penetration and chondroidal tissue formation but does not guarantee proper bone tissue growth [15].

The shape of the pores can also influence the mechanical and osteogenic properties of the scaffold [6].

Gong et al. performed fatigue tests (cyclic stress - strain) on scaffolds with triangular and circular pores, with a total porosity of about 60%. The study found that scaffolds with circular pores are more resistant than those with triangular pores [6].

Figure 2.Pores and interconnection of pores showed in HA scaffold [16].

Optical microscopy (light or electron microscopy technique) or microcomputer tomography can be applied in the middle parts of the scaffold to investigate the macro and micropores [10].

2.1.1.3 Mechanical Properties

For the scaffold to be functional, it must have biomechanical properties comparable to those of the adjacent healthy bone [12],[8].

The scaffold must maintain a certain stiffness until the newly formed tissue has achieved sufficient structural integrity to physically support itself [8].

It must have a Young's module similar to that of natural bone in order to avoid stress shielding and promote early tissue regeneration [17].

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Good mechanical properties could be obtained by building anisotropic scaffolds with oriented pores [9]. Pore size and interconnection and architecture affect the mechanical properties of the scaffolds [15].

For example, studies on HA scaffolds have shown that decreasing the pore size results in higher compressive strength [15].

The compressive strength is the most common test done for ceramic scaffolds and it is used to calculate the stress – strain curve [18].

In accordance with ASTM F2883-11, for implantable ceramic scaffolds, compressive strength would be tested according to ASTM C1424 for advanced ceramics [18].

For example, to replace cortical bone a good scaffold should have a compressive strength ~ 130 - 180 MPa and Young’s module ~ 12 -18 GPa, as it is described in Chapter 1).

In addition, it has been proven that the mechanical strength of the scaffold affects the mechanotransduction properties of the bone cells attached to the surface. There seems to be a correlation between mechanotransduction and the potential osteoinductive properties of the scaffold [12].

Not all anchorage - dependent cells respond similarly to scaffold stiffness[11]:

endothelial cells, for example, migrate and proliferate more easily on more rigid substrates [11].

2.1.1.4 Biodegradability

As the tissue regenerates, the scaffold must degrade in a controlled manner over time [8].

The biodegradability of the scaffold depends on the type of material and its application: TCP-based scaffolds show a rate of bioresorption similar to bone neoformation, whereas HA scaffolds are generally characterized by better chemical durability [8].

Usually the degradation of the biomaterial is influenced by the presence of organic ions in physiological fluids [7].

The rate of scaffold degradation may change depending on the different printing mode used. It will therefore be necessary to test different production methods to find the optimal one [8]. It can be calculated in accordance with ISO 10993-14 [18].

2.1.1.5 Surface properties and interaction with cells

The scaffold needs to be made in complex and even irregular shapes. L’osso umano ha una struttura molto complessa dal livello della nanoscale fino alla macroscale. The scaffold should serve as a model for natural bone growth and should therefore imitate the hierarchical structure of natural bone. With regard to cortical bone, for example, its internal architecture should be similar to the small vascular channels, Volkmann's channels and the gaps in the osteocytes and Hanversian channels [9].

As already mentioned, the scaffold needs careful design in order to achieve a high level of interconnected porosity [9].

Several studies have shown that the characteristics of the scaffold surface affect the amount, type and conformation of proteins and cells that will adhere to it [9].

A rough surface may improve cell adhesion, but excessive roughness must be avoided, or the cells may fail to develop focal adhesion plaques [4].

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The performance of a scaffold depends on the interaction between its surface and biological fluids and is often mediated by certain proteins that are absorbed by the biological fluid [19].

Chemistry, roughness and surface topography strongly influence the protein layer on which the formation of surface bonds directly aimed at binding only certain types of cells depends.

Proteins create a specific interface to which cells can respond to the macrostructure of the scaffold as well as the chemical properties of the surface determine how cells attach to the structure [19].

Surfaces with nanometric topography increase the availability of proteins and amino acids by promoting cell adhesion to a large extent [19].

Some examples in the literature suggest incorporating growth factors into scaffolds because they can improve and speed up the growth of new bone. in fact, morphogenic bone proteins (BMPs) help the development of both bone and cartilage and can trigger the differentiation and proliferation of osteoprogenitor cells. vascular endothelial growth factor (VEGF) has often been used because it can improve blood vessel formation [14].

Finally, the synthesis of the material and the production of the scaffold should allow the sterilisation process to be easily marketable [2].

Table 2. summarises the main features of the scaffold and their effects.

Table 2. Characteristics of the scaffold and their desirable effects [14].

Scaffold Requirements Desirable Features

Biocompatibility  Non-toxic breakdown products

 Non-inflammatory scaffold components, avoiding immune rejection Biodegradability  Biodegradability

 Controlled scaffold degradation which can complement tissue ingrowth whilst maintaining sufficient support

 Degradable by host enzymatic or biological processes

 Allows invading host cells to produce their own extracellular matrix Bioactivity  Scaffold materials that can interact with and bind to host tissue

 Osteoconductive and osteoinductive properties

 Inclusion of biological cues and growth factors to stimulate cell ingrowth, attachment and differentiation

Scaffold Architecture  Interconnected pores allowing diffusion and cell migration

 Microporosity to present a large surface area for cell-scaffold interactions

 Macroporosity to allow cell migration and invasion of vasculature

 Sufficient porosity to facilitate cell ingrowth without weakening mechanical properties

 Pore size tailored to target tissue and cells

 Inbuilt vascular channels to enhance angiogenesis in vivo

Mechanical Properties  Compressive, elastic and fatigue strength comparable to host tissue allowing cell mechanoregulation to occur and structural integrity to remain in vivo

 Scaffold material that can be readily manipulated in the clinical environment to treat individual patient bone defects

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