**3. Scaffolds in bone engineering**

#### **3.1. Scaffold properties**

physiologic concentrations [83]. Organic phosphate released after enzymatic hydrolysis of beta glycerol phosphate plays an important role in matrix mineralization. This free phosphate is usually applied in 5–10 mM concentrations for MSC bone differentiation. Ascorbic acid is a cofactor in the hydroxylation of prolines and lysine moiety of collagen molecules and is an abundant protein in the ECM. This reagent is used in 50–500 μM concentrations [84]. When MSCs are cultured in osteogenic media, they express the same markers as bone-forming osteoblasts that are responsible for laying down the matrix and mineral during new bone formation *in vivo*. The osteogenic differentiation of MSCs *in vitro* has been divided into three stages. The first stage (days 1–4) is the proliferation stage where a peak in the number of cells is seen. This is followed by early cell differentiation (from days 5 to 14), which is characterized by the transcription and protein expression of alkaline phosphatase (ALP). After this initial peak of ALP, its level starts to decline. Also found at an early stage is the expression of a collagen type I matrix onto which the mineral is deposited. The final stage (from days 14 to 28) results in a high expression of osteocalcin and osteopontin, followed by calcium and

**Figure 1.** Monolayer of mesenchymal stem cells derived from human bone marrow before (A) and after 3 weeks of (B)

In addition to osteogenic supplements, there are other substances that act as biochemical signals capable of triggering cellular processes like growth, proliferation or differentiation.

*Bone morphogenetic proteins* (*BMPs*) are a family of cytokines that stimulates the proliferation of chondrocytes and osteoblasts and increases extracellular matrix production. BMPs induce the differentiation of MSCs into osteoblasts. BMPs allow not only skeletal tissue formation during embryogenesis, growth, and adulthood but also bone healing process. In newborns' skeletons, BMPs can be found in the collagen fibers of the bone matrix and also in cells located in the periosteum and the bone marrow. After a fracture, BMPs' growth factors diffuse from bone matrix and activate osteoprogenitor cells which, in turn, produce more BMPs [86].

*Fibroblast growth factors* (*FGFs*) stimulate the proliferation of mesenchymal cells, osteoblasts and chondrocytes. FGFs enhance the growth of different tissues due to their angiogenic

The most common growth factors in bone tissue engineering are listed below.

phosphate deposition [4, 85].

230 Advanced Techniques in Bone Regeneration

differentiation. Arrows mark bone-forming nodules.

The evolution of bone implant devices has resulted in an increase in knowledge about the microenvironment where the replacement will occur, which results in changes in requirements and properties of the biomaterials used. This evolution can be measured by defining three different generations. However, these generations are not chronological but technological since there is currently active research and development for each. First-generation bone graft substitutes require the biomaterial to match the physical properties of the tissue to be replaced, while maintaining inertness with the tissue microenvironment. These include metals such as stainless steel, titanium and alloys; ceramics such as alumina and zirconia; and polymers such as silicone rubber, polypropylene and polymethylmethacrylate. Second-generation bone graft substitutes are made biodegradable with the aim that the rate of degradation matches the healing rate of the injured bone tissue. These biomaterials are based on the use of synthetic or natural polymers that can provide a controlled chemical breakdown under physiological conditions into inert products that can be resorbed by the body. Examples of the synthetic polymers include polylactide, poly(ε-caprolactone) and polyglycolide; and collagen, chitosan and hyaluronic acid for natural ones. The mechanical and osteoconductive properties of these polymers can be improved by forming composites with bioactive ceramics. Third-generation bone graft substitutes try to get closer to the autograft standard by using biomaterials capable of inducing specific cellular responses at the molecular level, by integrating the bioactivity and biodegradability of second-generation devices. This type of bone graft is based on the concept of bone tissue engineering, which focused on creating a device that enhances bone repair and regeneration by incorporating bone progenitor cells or/and bioactive signals (e.g., growth factors, small molecules) to stimulate cells into a scaffold made of various natural or synthetic biomaterials or their combination and with sufficient vascularization to allow access to nutrients to support this process. Nowadays, many groups worldwide seek to develop scaffolds with osteoinductive properties that would enhance bone healing. These scaffolds have to accomplish certain requirements and have to be:

*Biocompatible*—cells must populate the scaffold, adhere and proliferate. They should be able to migrate as well as differentiate. Overall, cell function should not be compromised. The scaffolds should enable unobstructed transport of nutrients, gases, signaling molecules, proteins and waste products in, out and within the scaffold.

*Biodegradable*—the scaffold should be replaced with host/donor cells (tissues). Therefore, scaffolds must be biodegradable and byproducts must not be toxic. Ideal scaffold degradation should mirror the rate of new tissue formation.

*Biofunctional*—the scaffold should meet as many as possible functional requirements of the replaced tissue. Good scaffold should have specific mechanical properties and architecture, similar to properties of the replaced bone tissue. Properties like elasticity, permeability, compressibility, viscoelastic behavior, tensile strength and failure strain [91] should be similar and should give shape to the tissue that is regenerated on it [92]. It is very important to have strong, but at the same time, porous bone grafts. The pores should be big enough to allow smooth cell migration and proliferation besides vascularization and small enough to enable cell-to-cell communication and critical cell repopulation of the pores. Pores are crucial in a process of degradation. Their size should allow and promote scaffold degradation.

The main disadvantage of scaffolds is the lack of vascularization. Inspired by the nature of bone, different scaffolds have been studied extensively, and the main challenge is to precisely balance a desired structural strength and porosity. To design bone scaffolds, materials should have the desired biological properties for a specific application and should not be immuno‐ genic causing inflammatory response. The long-term goal is the development of the scaffold that can be applied in a clinical setting. Manufacturing technology should follow good manufacturing practice (GMP) procedures. Ultimately, the main goal is to develop scaffold that fulfills all previously mentioned requirements and has slow-release properties of bioactive molecule. Multiple factors (signaling peptides, adhesion peptides, growth factors, plasmid DNA, antibodies, microRNAs, etc.) can be incorporated into scaffolds to promote osteoblast migration, to manipulate tissue formation and to effectively enhance bone regeneration [93]. For instance, bone morphogenic protein 2 (BMP-2) was photo-crosslinked into biodegradable diblock copolymers PEG-PLA and was slowly released as the polymer degrades [94]. Another approach is to covalently bind the adhesion peptide like well-known arginine-glycineaspartate ligand or chemotactic factor like platelet-derived growth factors (PDGFs), to attract osteoblast and promote osteogenesis [95, 96] or incorporate angiogenic (FGFs [97]) and antiangiogenic factors to control scaffold vascularization [98]. MicroRNAs can post-transcription‐ ally regulate gene expression and alter bone regeneration [99]. There are many problems to that approach, and the major one is controlled release of bioactive substance together with its rapid dilution. To reduce the risk of BMP dilution following release from the scaffold, monoclonal anti-BMP antibodies are encapsulated within the scaffold [100].

#### **3.2. Scaffold types**

With respect to source of biomaterials, scaffolds can be divided into two main groups: the ones made from natural and the ones made from synthetic materials. The natural biomaterials are obtained from natural sources and processed to make desired scaffolds. A few decades ago, researchers have discovered that decalcified bone matrix (DBM) possesses inherent osteoin‐ ductive properties (http://www.ncbi.nlm.nih.gov/pubmed/4870495), and DBM was used in the treatment of clinical orthopedic situations which has shown favorable results [101, 102]. Decellularized ECM (mammalian extracellular matrix) scaffolds recovered from allografts (tissue from individuals of same species) and xenografts (tissue from individuals of different species) have a desired three-dimensional porous structure and can be repopulated by host bone-forming cells. ECM is a complex of different glycosaminoglycans, glycoproteins and huge number of different small proteins. The cells can easily attach, grow and differentiate with excellent viability. Decellularization and treatments such as freeze-drying, irradiation and washing with acid minimize their immunogenicity, but some epitopes can still be recognized by the host. These treatments prevent any infection to be transferred from the tissue, but can affect their mechanical and biological properties [97]. Most commonly used biological materials for bone tissue engineering are chitosan, collagen, hyaluronic acid, alginate, elastin, cellulose, fibrin, gelatin, etc. Chitosan is a hydrophilic, linear polysaccharide (suh, matthew, application of chitosan-based) obtained by alkaline deacetylation of chitin from shrimp and other crustacean shells. It has many beneficial properties, such as biocompatibility (no inflammatory or allergic reaction, (chatelet, damour, influence of the degree), biodegrad‐ ability (it is naturally degraded by hydrolytic enzymes such as lysozyme) and no toxicity [103]. Since collagen is the most abundant protein in various tissues including bone, scaffolds made of collagen are very attractive for biomedical applications. Collagen is composed from two α1 chains and one α2 chain wrapped together by hydrogen and covalent bonds to form righthanded triple helix. These fibers spontaneously pack together to form long thin fibrils of similar structure. Collagen is an attractive material for a scaffold synthesis because its mechanical properties can be altered by crosslinking, either with different chemicals (glutaraldehyde, formaldehyde, etc.) or with physical treatments (UV irradiation, heating, etc.) [104–106]. Hyaluronic acid is a simple linear polysaccharide composed of a repeating disaccharide, and it is hydrophilic, nonimmunogenic, and easy to modify and produce. It is easily replaced by extracellular matrix produced by host cells due to hyalurodinase degradation. These materials have a huge biological activity; they promote cell adhesion as well as cell growth. They are biodegradable, allowing host cells to replace the scaffold with their own extracellular matrix. The main drawbacks are their poor mechanical properties limiting their use as bone grafts and the reproducibility of their synthesis. Immunogenicity, limited physical and mechanical stability as well as limited resource of biomaterials have encouraged researches to develop composites using synthetic materials.

scaffolds should enable unobstructed transport of nutrients, gases, signaling molecules,

*Biodegradable*—the scaffold should be replaced with host/donor cells (tissues). Therefore, scaffolds must be biodegradable and byproducts must not be toxic. Ideal scaffold degradation

*Biofunctional*—the scaffold should meet as many as possible functional requirements of the replaced tissue. Good scaffold should have specific mechanical properties and architecture, similar to properties of the replaced bone tissue. Properties like elasticity, permeability, compressibility, viscoelastic behavior, tensile strength and failure strain [91] should be similar and should give shape to the tissue that is regenerated on it [92]. It is very important to have strong, but at the same time, porous bone grafts. The pores should be big enough to allow smooth cell migration and proliferation besides vascularization and small enough to enable cell-to-cell communication and critical cell repopulation of the pores. Pores are crucial in a

The main disadvantage of scaffolds is the lack of vascularization. Inspired by the nature of bone, different scaffolds have been studied extensively, and the main challenge is to precisely balance a desired structural strength and porosity. To design bone scaffolds, materials should have the desired biological properties for a specific application and should not be immuno‐ genic causing inflammatory response. The long-term goal is the development of the scaffold that can be applied in a clinical setting. Manufacturing technology should follow good manufacturing practice (GMP) procedures. Ultimately, the main goal is to develop scaffold that fulfills all previously mentioned requirements and has slow-release properties of bioactive molecule. Multiple factors (signaling peptides, adhesion peptides, growth factors, plasmid DNA, antibodies, microRNAs, etc.) can be incorporated into scaffolds to promote osteoblast migration, to manipulate tissue formation and to effectively enhance bone regeneration [93]. For instance, bone morphogenic protein 2 (BMP-2) was photo-crosslinked into biodegradable diblock copolymers PEG-PLA and was slowly released as the polymer degrades [94]. Another approach is to covalently bind the adhesion peptide like well-known arginine-glycineaspartate ligand or chemotactic factor like platelet-derived growth factors (PDGFs), to attract osteoblast and promote osteogenesis [95, 96] or incorporate angiogenic (FGFs [97]) and antiangiogenic factors to control scaffold vascularization [98]. MicroRNAs can post-transcription‐ ally regulate gene expression and alter bone regeneration [99]. There are many problems to that approach, and the major one is controlled release of bioactive substance together with its rapid dilution. To reduce the risk of BMP dilution following release from the scaffold,

process of degradation. Their size should allow and promote scaffold degradation.

monoclonal anti-BMP antibodies are encapsulated within the scaffold [100].

With respect to source of biomaterials, scaffolds can be divided into two main groups: the ones made from natural and the ones made from synthetic materials. The natural biomaterials are obtained from natural sources and processed to make desired scaffolds. A few decades ago, researchers have discovered that decalcified bone matrix (DBM) possesses inherent osteoin‐ ductive properties (http://www.ncbi.nlm.nih.gov/pubmed/4870495), and DBM was used in

**3.2. Scaffold types**

proteins and waste products in, out and within the scaffold.

should mirror the rate of new tissue formation.

232 Advanced Techniques in Bone Regeneration

Typically, two individual groups of synthetic biomaterials are used in the fabrication of bone grafts: ceramics and synthetic polymers. Ceramics polymers (inorganic oxides and salts), such are hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and biphasic calcium phosphate (BCP) are mechanically stiff and have very low elasticity, making them suitable only for bone tissue grafts. Ceramics perfectly imitate natural bone structure, and cell interaction with ceramics promotes proliferation as well as differentiation of osteoblasts [107].

Synthetic polymers, such as polystyrene, polyglycolic acid (PGA) and poly-l-lactic acid (PLLA) acid, have the rewarding and satisfying properties because their architecture can be adjusted and changed by the composition of the polymer as well as by altering the synthesis method. However, cell might have difficulties to attach and proliferate on their surface, so there is always the risk of rejection due to reduced bioactivity. Degradation of synthetic polymers becomes the major issue because most of them are degraded by hydrolysis, causing lower local pH and cell necrosis [108].

Since ceramics have excellent osteoinductive properties but low mechanical strength, and synthetic polymers exhibit poor osteoinductivity but better mechanical strength and degrad‐ ability, in the past decade researches have been trying to develop the scaffolds made of ceramic and polymer composites. Most commonly used 3D composites are made of synthetic polymers such as poly(lactic) acid (PLA), PGA, poly(ε-caprolactone) (PCL), poly(lactic-co-glycolide) (PLGA), poly(propylene fumarate) (PPF) and natural polymers such as collagen type I and chitosan. These composites have rigid sponge-like structures often containing HA (133-138 from three-dimensional alexander). Hydroxyapatite increases attachment of mesenchymal stem cells, differentiation to osteoprogenitors and promotes cell survival [109, 110].

#### **3.3. The ideal scaffold for bone tissue engineering**

The ideal scaffold is difficult to obtain and should be biocompatible, bioresorbable, osteocon‐ ductive (must allow bone cells to adhere, proliferate and secrete extracellular matrix), ostein‐ ductive (with the ability to induce new bone formation), osteogenic (should act as MSCs and osteoblasts reservoir), structurally similar to bone enabling formation of strong bonds with surrounding bone tissue, as well as it should be easy to use and cost-effective. New approach includes development of methods to isolate and transplant bone tissue-forming cells, bioactive matrix materials that act as tissue scaffolds mimicking what happens in nature, and delivery of bioactive molecules within scaffolds. In the past two decades, many 3D systems have been

**Figure 2.** Bone graft grown on scaffold made of chitosan and hyaluronic acid. Section of graft stained with hemalaun/ eosin staining shows equal cell distribution, formation of extracellular matrix and scaffold residues.

studied and some have been commercialized for clinical application. The main advantage of this system is that cells grown in 3D environment that is more representative to natural bone tissue. Final goal is to produce a "living" scaffold providing mechanical support, bioactive signal as well as cells with osteogenic potential. Researchers have developed bioreactors to mimic physiological conditions. The main advantage is that this system enables controlled manipulation of all variables. In contrast to classic static *in vitro* cultures, bioreactors allow to apply mechanical stimuli that are very important in osteogenic differentiation [111]. Many different bioreactors to promote good osteogenesis (Koller reactors [112]), spinner flask bioreactors, [113] have been investigated. Recently developed perfusion bioreactors have shown high efficiency in uniformed cell seeding on a scaffold, enhanced proliferation, great supply of oxygen and nutrients throughout the scaffold as well as enhanced osteogenic differentiation because the pump forces the medium to flow through the scaffold (**Figure 2**) [114–117].
