**3. Osteoclast differentiation and activation**

#### **3.1. Introduction**

Rheumatoid arthritis (RA) is a chronic inflammatory disease perpetuated by a dysregulated immune system. The cause remains grossly unknown, however both environmental and genetic factors are believed to contribute to RA development (Kochi 2010; Boissier 2011). The disease is characterized by joint inflammation, initially resulting in pain and swelling, and in a majority of the patients, erosion of bone and cartilage takes place (Lories 2011). The goal of current treatment regimens is to control inflammation, as well as to halt or retard the progres‐ sion of damage of the bone structure of the affected joint(s), as measured by X-ray analysis (Tan and Conaghan 2011). The RA joint, synovial fluid, and synovium contain a large variety of hematopoietic cells, which are situated in direct proximity to the articular cartilage and its underlying bone structures (Carvalheiro, da Silva et al. 2012; Shegarfi, Naddafi et al. 2012). Hence, the synovial fluid with its contents of immune cells and cytokines secreted from the cells in question, undoubtedly are responsible for the joint-destructive process (Iwamoto, Okamoto et al. 2008; Yamada, Nakashima et al. 2008).

#### **3.2. Osteoclast differentiation**

Physiological and pathological bone resorption is achieved by the osteoclast. Osteoclasts are large 20 to 100 μm multinucleated cells containing from three to 100 nuclei with many mitochondria, lysosomes, dense granules, vesicles, and an extensive Golgi network required for the synthesis and secretion of factors required to degrade bone matrix and subsequently phagocytose the resorbed products (Holtrop and King 1977). Tartrate-resistant acid phospha‐ tase (TRAP) (Minkin 1982), cathepsin K (Drake, Dodds et al. 1996), the calcitonin (CT) receptor (Hattersley and Chambers 1989), as well as αvβ3 integrin (Davies, Warwick et al. 1989) are marker gene products of the mature osteoclast (Teitelbaum 2000). The initial event in bone resorption is the attachment of the mature osteoclast to the bone matrix. Cell surface αvβ3 integrins bind to a plethora of extracellular matrix proteins synthesized and secreted by osteoblastic cells, including vitronectin, osteopontin, and bone sialoprotein. Arg-Gly-Aspcontaining peptides, Arg-Gly-Asp mimetics, and blocking antibodies to αvβ3 integrins have been shown to inhibit bone resorption, both *in vitro* and *in vivo*, indicating that this integrin plays a pivotal role in osteoclast functioning (Horton, Taylor et al. 1991). Once attached to bone, the osteoclast generates an isolated extracellular, acidic microenvironment between itself and the bone surface by creating a sealing zone structure unique to the osteoclast.

#### **3.3. Osteoclast activation**

teochondral signature. Variables analysed were much like those for the experiment with chondrocytes. However, here we measured mineralizing surface (Alizarin red S coloration) induced by the osteoblasts in a 2D culture.**C**. Co-culture of chondrocytes in alginate beads (or osteoblasts in 2D culture) in standard medium (Gordeladze 2011). The above mentioned experimental setting was used with chondrocytes differentiated from hMSCs in alginate beads in the ab‐ sence or presence of cytokines (IL-1, IL-6, IL-17, and TNFα), pre-microRNAs of all microRNA species of the osteochon‐ dral signature, or the set of cytokines along with the pre-microRNAs. Parameters analysed were: Q-PCR of transcription factors (TFs) and marker genes expressed by the chondrocytes, clinical score of alginate beads containing chondrocytes embedded in newly synthesized proteoglycan matrix, the number of Trap-positive osteoclasts, and the

*Termination*: Sclerostin expression then fades or ceases, and the process of bone formation comes to a halt. The newly deposited osteoid is subsequently mineralized, the bone surfaces return to their resting state, This is when bone-lining cells intercalate with osteomacs, and

Rheumatoid arthritis (RA) is a chronic inflammatory disease perpetuated by a dysregulated immune system. The cause remains grossly unknown, however both environmental and genetic factors are believed to contribute to RA development (Kochi 2010; Boissier 2011). The disease is characterized by joint inflammation, initially resulting in pain and swelling, and in a majority of the patients, erosion of bone and cartilage takes place (Lories 2011). The goal of current treatment regimens is to control inflammation, as well as to halt or retard the progres‐ sion of damage of the bone structure of the affected joint(s), as measured by X-ray analysis (Tan and Conaghan 2011). The RA joint, synovial fluid, and synovium contain a large variety of hematopoietic cells, which are situated in direct proximity to the articular cartilage and its underlying bone structures (Carvalheiro, da Silva et al. 2012; Shegarfi, Naddafi et al. 2012). Hence, the synovial fluid with its contents of immune cells and cytokines secreted from the cells in question, undoubtedly are responsible for the joint-destructive process (Iwamoto,

Physiological and pathological bone resorption is achieved by the osteoclast. Osteoclasts are large 20 to 100 μm multinucleated cells containing from three to 100 nuclei with many mitochondria, lysosomes, dense granules, vesicles, and an extensive Golgi network required for the synthesis and secretion of factors required to degrade bone matrix and subsequently phagocytose the resorbed products (Holtrop and King 1977). Tartrate-resistant acid phospha‐ tase (TRAP) (Minkin 1982), cathepsin K (Drake, Dodds et al. 1996), the calcitonin (CT) receptor (Hattersley and Chambers 1989), as well as αvβ3 integrin (Davies, Warwick et al. 1989) are marker gene products of the mature osteoclast (Teitelbaum 2000). The initial event in bone resorption is the attachment of the mature osteoclast to the bone matrix. Cell surface αvβ3 integrins bind to a plethora of extracellular matrix proteins synthesized and secreted by

the remodelling cycle is concluded (Raggatt and Partridge 2010).

**3. Osteoclast differentiation and activation**

Okamoto et al. 2008; Yamada, Nakashima et al. 2008).

**3.2. Osteoclast differentiation**

eroded dentine surface (%).

484 Regenerative Medicine and Tissue Engineering

**3.1. Introduction**

As described in a previous paragraph, macrophage CSF (M-CSF) and receptor activator of NFκB ligand (RANK-L) are the most important factors known to date to drive osteoclast formation and activity (Lacey, Timms et al. 1998; Quinn, Elliott et al. 1998). M-CSF serves as a survival factor for osteoclast precursors due to up-regulation of Bcl-XL, inhibition of caspase-9 activation (Woo, Kim et al. 2002), and the support of mature osteoclast survival by preventing apoptosis (Fuller, Owens et al. 1993). Exposure to M-CSF also stimulates receptor activator of NFκB (RANK) expression in osteoclast precursor cells, thereby allowing RANK-L to drive formation of mature osteoclasts (Arai, Miyamoto et al. 1999). RANK-L is a trans-membrane protein expressed by activated osteoblasts, synovial fibroblasts, as well as T-cells. It can also be proteolytically cleaved by TNF convertase (TACE) to generate a soluble molecule that has osteoclastic activity at distal sites (Hofbauer, Khosla et al. 2000; Li, Toraldo et al. 2007). RANK-L induced osteoclastogenesis is inhibited by OPG (osteoprotegerin), a soluble decoy receptor for RANKL, which is also produced by a variety of cells including osteoblasts, synovial fibroblasts, B-cells, and T-cells (Simonet, Lacey et al. 1997; Hofbauer, Khosla et al. 2000; Takayanagi, Iizuka et al. 2000; Gillespie 2007; Li, Toraldo et al. 2007). In the arthritic joint, there is a significant macrophage infiltrate, and the extent of the subject infiltration correlates heavily with the extent of joint erosion in both RA patients and animal models with arthritis (Yanni, Whelan et al. 1994). Synovial macrophages isolated from different types of arthritis may differentiate *in vitro* to fully functional osteoclasts subsequent to RANK-L stimulation as well as independently of the RANK/RANK-L signalling pathway, i.e. after short term exposure to TNFα and IL-1α (Adamopoulos, Sabokbar et al. 2006). Stimulatory, co-stimulatory, and/or inhibitory signals may be provided by adjacent T-cell species present in the inflammatory infiltrate.
