**2. Bone tissue engineering**

#### **2.1. The basic concepts**

Today great hope is set on Regenerative Medicine in all medical fields and, of course, it has developed to be of interest in orthopedics, being bone defects one of the main focus. In the last two decades, Regenerative Medicine approaches have been extensively studied to improve bone healing, or even generate functional bone tissue to substitute lost bone in orthopedics, neurosurgery, and dentistry. These types of studies include two different strategies of cellbased therapy: in the first approach, called Cell Therapy, cells are applied to substitute damaged cells within a tissue to reconstitute its integrity and function. The second approach, called Tissue Engineering, is more complex and encompasses three approaches: bioactive molecules (growth factors (GFs), cytokines, ECM compounds and hormones) that encourage tissue induction; cells and cell substitutes that will respond to the signals; the seeding of cells into three dimensional matrices, with specific adhesion properties and degradation rates, to compose a tissue-like construct to substitute lost parts of the tissue, or even organs; and a good nutritious support (angiogenesis) (Fig. 3).

Stem cells (SCs) are of particular interest in Regenerative Medicine, since they inhere several unique characteristics that distinguish them from other cell types. SCs represent unspecialized cells, which have the ability to differentiate into cells of all three germ layers, different adult cell types, and represent the only cell type which has the ability to renew itself indefinitely. It is important to distinguish embryonic stem cells (ESCs), which are truly pluripotent from multipotent adult stem cells and only found in early developmental stages of the organism. The successful dedifferentiation of somatic cells into a pluripotent ESC-like status by trans‐

of the plasma membrane that surrounds the ruffled border and serves as the point of attach‐ ment of the osteoclast to the underlying bone matrix. Active osteoclasts exhibit a characteristic polarity. Nuclei are typically located in the part of the cell most removed from the bone surface and are interconnected by cytoskeletal proteins (Watanabe et al., 1995). Osteoclasts contain multiple circumnuclear Golgi stacks, a high density of mitochondria, and abundant lysosomal

When a fracture occurs, a set of signals is triggered. These are both local signals and systemic ones; some of these signals are mediated by neuronal impulses (Nordsletten et al., 1994), by the haematoma at the site of the fracture and by the trauma caused to the tissues surrounding the fracture (Einhorn, 1998). These signals can be divided into two interactive and inter‐ changeable categories: inflammatory signals and bone building signals. These factors mitigate the migration of phagocytotic cells to the area of the fracture, removing the necrotic tissue and propagating the in-growth of new blood vessels to the site of the fracture, thus providing nutrients and cells to the fracture site and starting the healing cascade. If at the end of the healing process osteo-integration (of the new bone together with the native bone) is not achieved, even with the best type of scaffolds, the chances of long-term success are dismal

Today great hope is set on Regenerative Medicine in all medical fields and, of course, it has developed to be of interest in orthopedics, being bone defects one of the main focus. In the last two decades, Regenerative Medicine approaches have been extensively studied to improve bone healing, or even generate functional bone tissue to substitute lost bone in orthopedics, neurosurgery, and dentistry. These types of studies include two different strategies of cellbased therapy: in the first approach, called Cell Therapy, cells are applied to substitute damaged cells within a tissue to reconstitute its integrity and function. The second approach, called Tissue Engineering, is more complex and encompasses three approaches: bioactive molecules (growth factors (GFs), cytokines, ECM compounds and hormones) that encourage tissue induction; cells and cell substitutes that will respond to the signals; the seeding of cells into three dimensional matrices, with specific adhesion properties and degradation rates, to compose a tissue-like construct to substitute lost parts of the tissue, or even organs; and a good

Stem cells (SCs) are of particular interest in Regenerative Medicine, since they inhere several unique characteristics that distinguish them from other cell types. SCs represent unspecialized cells, which have the ability to differentiate into cells of all three germ layers, different adult cell types, and represent the only cell type which has the ability to renew itself indefinitely. It is important to distinguish embryonic stem cells (ESCs), which are truly pluripotent from multipotent adult stem cells and only found in early developmental stages of the organism. The successful dedifferentiation of somatic cells into a pluripotent ESC-like status by trans‐

vesicles that arise from the Golgi and cluster near the ruffled border.

(Avila et al., 2009).

**2.1. The basic concepts**

**2. Bone tissue engineering**

618 Regenerative Medicine and Tissue Engineering

nutritious support (angiogenesis) (Fig. 3).

**Figure 3.** The two strategies of stem cell application in Regenerative Medicine. Stem cells are either isolated from the patient (autologous transplantation) or from other donors (allogenous transplantation). The cells are expanden in vi‐ *tro* and either applied directly to the patient to substitute lost cells (Cell Therapy), or seeded into 3 dimensional scaf‐ folds (Tissue Engineering) and differentiated into the demanded cell type. The composed artificial tissue construct is subsequently implanted into patients' tissue defect*. Taken from A. Schmitt et al. 2012, with permission*

fection with four embryonic transcription factors, the so-called induced pluripotent stem cells (iPS cells), provide the possibility of autologous therapy with pluripotent and easily accessible cells in the future. Beside the great potential this technique undoubtedly represents, it bears some essential safety problems which are currently far from being solved. In contrast, a variety of multipotent adult SCs exists in assumedly all tissues of the organism. They are responsible for maintaining the integrity of the tissue they reside in. Usually, these adult SCs show limited differentiation potential to tissues of one germ layer.

Bone regeneration is a physiological process which can be observed in healing fracture and continuous remodeling along by adult life. Bone holds the most regenerative ability of human tissues contained on the mayor source of osteogenic cells capable for forming bones, the bone marrow (BM). However, a loss considerable amount of bone due to any anomalies such as severe trauma, skeletal deformations, bone tumor resection or periprosthetic osteolysis can obstruct this capacity. Nevertheless, the capacity of proliferation and differentiation of BMSCs, as well as cells concentration, are reduced with the increase of the age of the patient. Many *in vitro* studies were performed to investigate applicability of different SC types for bone regeneration. Here, promising capacity for differentiating towards bone cells, enhancing bone healing and vascularization could be proven for ESCs and different adult mesenchymal stem cells (MSCs). However, due to the ethical and safety concerns, which currently forbid appli‐ cations of ESCs or iPS cells in patients, we will focus on adult stem cells for therapeutic applications. Therefore, MSCs presently seem to be the most promising candidates for bone regeneration, due to their excellent osteogenic differentiation capacity. They can be isolated from a number of adult mesenchymal tissues, among others, umbilical cord blood, peripheral blood, placenta, synovial fluid, adipose tissue, skeletal muscle or BM, as mentioned, where they contribute to normal tissue turnover and repair. Recently, the multitude of cell surface markers used in various studies has been limited to a marker panel representing, in addition to plastic adherence and differentiation capacity, the minimal criteria for the identification of MSCs. The molecular mechanisms of human MSCs regulation and the importance of specific GFs during the different stages of osteogenic differentiation, as well as the secreted signaling proteins known as *Wnts,* implicated in various differentiation programs including osteogen‐ esis, are subjects of intensive research right now. Several studies have demonstrated improved results of MSCs therapy with genetically modified cells which produce osteogenic and angiogenic GFs in a local delivery of therapy strategy for bone healing. Also, there is recent information about the use of endothelial progenitor cells (EPCs) that improves the treatment of fracture and bone regeneration.

Besides their unique ability to differentiate into different cell types, MSCs secrete a variety of cytokines, showing anti-inflammatory activity and create an anabolic microenvironment. Furthermore, direct cell-cell contact immunomodulation has also been shown. On one hand, they indirectly influence tissue regeneration by secretion of soluble factors. On the other hand, they are able to modulate the inflammatory response. The differentiation potential of MSCs in bone engineering has been extensively studied *in vitro* and *in vivo.* By first time, Urist (1965), and Reddi and Huggins (1972) showed the capacity of a molecule, called bone morphogenetic protein (BMP), with potent osteoinductive properties in healing fractures and bone regenera‐ tion. Their experiments demonstrated the presence of osteoinductive cytokines in bone matrix that have abilities to induce MSCs differentiation into osteoblasts. The GFs, also including transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) or insulin-like growth factor (IGF), among others, are delivered of paracrine or autocrine manner, generating a chemotaxis process toward MSCs by recruitment, which induce their differentiation.

Extracellular matrix (ECM) is the native scaffold in most tissues. Besides the direct injection in the surrounding tissue, biomaterials are frequently used as carriers for cells, bioactive mole‐ cules and drugs. These materials have to be immune-compatible and nontoxic, whereas the bio-degradation process must neither release toxic substances nor tissue-toxic concentrations of degradation products. Scaffolds must be of three-dimensional structures, with great influence on cell growth and differentiation, and must be highly porous with interconnected pores of a diameter of at least 100 μm to allow ingrowth of cells and vessels. Despite the tissue engineering of bone, for which various inorganic materials, such as HA, calcium phosphate, calcium carbonate (due to their similarity to bone mineral, as well as their inherent biocom‐ patibility and osteoconductivity), or glasses was tested, mainly organic biomaterials have been investigated for scaffold production. These are either naturally derived, for example, collagen, fibrin, agarose, alginate, gelatin, silk or hyaluronic acid; or produced synthetically, such as polyhydroxyacids. Since natural bone consists of an ECM with nanosized apatitic minerals and collagen fibers that support bone cell functions, it is of interest to manufacture a synthetic biomimetic scaffold to i) contain nano-apatite crystals, together with fibers to form a matrix that supports cell attachment; ii) have mechanical properties similar to those of bone; and iii) encapsulate and support cells for osteogenic differentiation. Different methods have been employed in the fabrication of nanomaterials for bone engineering, such as the principle of electrospinning, that produce a variety of synthetic biomaterials, or the novel thermally induced phase separation (TIPS) technique to fabricate nanofibers to mimic natural collagen fibers. Rapid developments in this field of nanotechnology will be a key for many clinical benefits in the field of bone tissue engineering. The main advantage is that several novel biomaterials can be fabricated into nanostructures that closely mimic the bone in structure and composition. The optimization in the surface features of scaffolds has strongly improved cell behavior in terms of adhesion, proliferation, differentiation and tissue formation in three dimensions.
