**1. Introduction**

Regenerative medicine (RM) is a therapeutic approach that aims to restore structure and function of damaged tissues and organs, in particular to find a solution for those that become perma‐ nently damaged and untreatable [1].

RM can be potentially applied to different tissues [2], and one of the most promising fields is that related to bone [3, 4].

Tissue regeneration is a complex task that encompasses completely restoring the lost struc‐ ture, including its micro-architecture and consequently its functionality. As for bone regener‐ ation, optimal healing is achieved when certain prerequisites are met, namely, osteoinduction, osteoconduction, osteogenesis, and mechanical stability [5].

Osteoinduction is the process that allows the recruitment and stimulation of immature preosteoblastic cells to mature osteoblasts and to produce new bone [6]. This phenomenon is regulated by a class of molecules known as inductive agents, mainly represented by bone morphogenetic proteins (BMPs) [3]. As a consequence of osteoinduction, osteogenesis can be achieved. Osteogenesis is carried out by osteoblasts, and consists in the formation of new bone. To improve the outcome of bone regeneration, biomaterials are often used to fill the gaps created by lost tissue. Such biomaterials must be osteoconductive, i.e., capable of supporting bone deposition on their surface [6]. Finally, mechanical stability of the healing site is the fourth factor to consider in order to reach regeneration of sound bone and avoid formation of fibrous tissue [5].

RM for bone tissue currently includes four approaches: molecular, cellular, use of bone substitutes, and tissue engineering (TE).

Progresses in molecular biology and a deeper knowledge of the mechanisms of fracture healing at a molecular level have allowed for the identification of a large number of key molecules that can be used locally or systematically to enhance bone repair [7]. Autologous cells can be an alternative or complementary choice for healing bone fracture. Mesenchymal stem cells (MSCs) have been proposed as a useful in regenerative interventions. MSCs can be collected from bone marrow [8], from peripheral blood [9], or from adipose tissue [10, 11]. Further possibilities to harvest MSCs in dental applications could be other types of stem cells direct‐ ly isolated from oral tissues such as the dental pulp (DPSCs) or the periodontal ligament (PDLSCs) [12–14]. As mentioned before, biomaterials have also been proposed as a tool to provide a substrate for new bone cells to deposit new bone, acting as gap fillers and osteo‐ conductive scaffold. A wide number of synthetic bone substitutes are now available includ‐ ing hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and calcium-phosphate cements, glass ceramics, and biocompatible metals [15, 16].

These different approaches are often combined and the investigation of the optimal condi‐ tions and tools to regenerate a tissue created a field called tissue engineering.

#### **1.1. Tissue engineering**

**1. Introduction**

330 Advanced Techniques in Bone Regeneration

nently damaged and untreatable [1].

substitutes, and tissue engineering (TE).

glass ceramics, and biocompatible metals [15, 16].

osteoconduction, osteogenesis, and mechanical stability [5].

that related to bone [3, 4].

tissue [5].

Regenerative medicine (RM) is a therapeutic approach that aims to restore structure and function of damaged tissues and organs, in particular to find a solution for those that become perma‐

RM can be potentially applied to different tissues [2], and one of the most promising fields is

Tissue regeneration is a complex task that encompasses completely restoring the lost struc‐ ture, including its micro-architecture and consequently its functionality. As for bone regener‐ ation, optimal healing is achieved when certain prerequisites are met, namely, osteoinduction,

Osteoinduction is the process that allows the recruitment and stimulation of immature preosteoblastic cells to mature osteoblasts and to produce new bone [6]. This phenomenon is regulated by a class of molecules known as inductive agents, mainly represented by bone morphogenetic proteins (BMPs) [3]. As a consequence of osteoinduction, osteogenesis can be achieved. Osteogenesis is carried out by osteoblasts, and consists in the formation of new bone. To improve the outcome of bone regeneration, biomaterials are often used to fill the gaps created by lost tissue. Such biomaterials must be osteoconductive, i.e., capable of supporting bone deposition on their surface [6]. Finally, mechanical stability of the healing site is the fourth factor to consider in order to reach regeneration of sound bone and avoid formation of fibrous

RM for bone tissue currently includes four approaches: molecular, cellular, use of bone

Progresses in molecular biology and a deeper knowledge of the mechanisms of fracture healing at a molecular level have allowed for the identification of a large number of key molecules that can be used locally or systematically to enhance bone repair [7]. Autologous cells can be an alternative or complementary choice for healing bone fracture. Mesenchymal stem cells (MSCs) have been proposed as a useful in regenerative interventions. MSCs can be collected from bone marrow [8], from peripheral blood [9], or from adipose tissue [10, 11]. Further possibilities to harvest MSCs in dental applications could be other types of stem cells direct‐ ly isolated from oral tissues such as the dental pulp (DPSCs) or the periodontal ligament (PDLSCs) [12–14]. As mentioned before, biomaterials have also been proposed as a tool to provide a substrate for new bone cells to deposit new bone, acting as gap fillers and osteo‐ conductive scaffold. A wide number of synthetic bone substitutes are now available includ‐ ing hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and calcium-phosphate cements,

These different approaches are often combined and the investigation of the optimal condi‐

tions and tools to regenerate a tissue created a field called tissue engineering.

Tissue engineering (TE) was first defined in 1988 at the first *TE symposium* in California, as "*an interdisciplinary field of research that applies the principles of engineering and the life sciences towards the development of biological substitutes that restore, maintain and improve tissue function*". It has been demonstrated that TE offers great potential in clinical applications [17, 18], and, in particular, bone tissue engineering seems to harbor a great potential. At present, bioabsorba‐ ble scaffolds combined with bone-marrow aspirate and osteoinductive factors (BMPs) have yielded promising results [16], and, more recently, the applicability of a β-TCP scaffold seeded with autogenous bone-marrow cells for bone reconstruction has been shown in a sheep model [19]. Moreover, TE has been used to improve fracture healing and to augment the boneprosthesis interface in arthroplasty, with promising results and safety [20, 21].

#### *1.1.1. Scaffold*

Scaffolds are a central concept in TE. They are 3D porous structures designed to promote cell adhesion, proliferation, and extracellular matrix deposition in order to allow for the restora‐ tion of damaged tissue [22].

Scaffolds can be divided into biological and synthetic materials. Biological scaffolds are derived from human and animal tissues, whereas synthetic ones are made of artificial biomaterials [23]. As materials of biological origin, although often possessing favorable characteristics, suffer from scarce availability, safety concerns and sometimes possibility of inflammatory or even immune responses, synthetic biomaterials have been the center of increasing attention. The state of art on scaffolds has evolved over the last years and in‐ volves the employment of natural or synthetic polymers. Collagen is the most abundant polymer in tissues and, as a consequence, among the most investigated material for the production of natural-derived scaffolds [24–26]. Together with collagen, chitosan, alginate, and cellulose are promising biomaterial for bone tissue engineering applications [27–30]. Among the synthetic polymers used for scaffold fabrication, polylactic-co-glycolic acid (PLGA) and polycaprolactone (PCL) are probably the most studied [31]. However, their characteris‐ tics for TE applications are still suboptimal compared with those of natural polymers [4]. Alternatively to the use of polymers, calcium phosphate, apatite forms, and bioglasses find wide application in bone engineering [32]. Regardless of their chemistry, the main feature scaffolds should possess is biocompatibility.
