**4. Effect of growth factor on glycosaminoglycan production in articular chondrocytes**

Growth factors, such as transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2), have demonstrated a great potential as cartilage anabolic factors because of their ability to induce matrix synthesis and promote repair in cartilage. TGF-β is one of the anabolic factors involved in cartilage maintenance and appears to be a good candidate for cartilage repair. TGF-β is a stimulator of extracellular matrix production, like collagen type II and proteoglycan (PG), in chondrocytes and it downregulates matrix-degrading enzymes (Edwards, et al. 1987). High amounts of TGF-β are stored in healthy cartilage (Pedrozo, et al. 1998, Redini F, et al. 1997, Morales, et al. 1991a,b, Burton-Wurster & Lust,1990), whereas in OA cartilage the expression of TGF-β is reduced (Verdier et al. 2003). FGF-2 belongs to the family of heparin-binding growth factors (Gospodarowicz, et al. 1987).This proteins are known to induce chemotactic, angiogenic, and mitogenic activity, and play an important role in early differentiation and development (Powers et al., 2000, Burgess & Maciag, 1989). Cell expansion in the presence of FGF-2 was shown to promote differentiation of stromal cells into cartilagi‐ nous tissues (Martin, et al. 1998). Similary, FGF-2 was shown to retain the chondrogenic differentiation potential of extensively expanded stromal cells (Tsutsumi, et al. 2001). Like wise, pellet cultures established from FGF-2 treated human mesenchymal stem cells were larger in size and contained a higher level of proteoglycans than untreated cells (Solchaga, et al. 2005). Martin et al. (1999a, 2001) showed that the FGF-2 treatment increased cell number during monolayer expansion and the differentiation capacity of expanded chondrocytes in subsequent 3-dimensional culture.

The most important problems in cartilage regeneration medicine are to supply nutrients to cells activated by grafting or growth factors and to maintain a healthy extracellular environ‐ ment. However, it has been shown that the extracellular osmotic pressure decreases with cartilage degeneration in candidates for treatment. In this study, cells were isolated from the cartilage from metacarpal phalangeal joints of 18-24 month bovine. Alginate beads containing cartilage cells collected from adult bovine (4 million cells/ml) were prepared. Three-dimen‐ sional culture was done for 5 days under a low osmotic condition (270 mOsm) as seen osteoarthritis or a normal osmotic condition like that of healthy cartilage (370 mOsm). Transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2) were added every day. GAG production was assessed by the dimethylmethylene blue (DMB) assay after 5 days. Fig. 8 shows the effect of osmorality and growth factor on the amount of GAG accumulated with time in culture. Significantly more GAG was accumulated by cells cultured at 370 Osm than low osmoralities (270 mOsm). The cells cultured with the growth factors (TGFβ or FGF-2) were more active and accumulated significantly more GAG accumulated than cells cultured without the growth factors. However, GAG accumulated was significantly lower for cells cultured at 270 mOsm than those at 370 mOsm. Thus, the clinical application of cartilage regeneration medicine needs to be advanced by providing appropriate physiological condi‐ tions with consideration of age-related cartilaginous changes.

The interrelationships between cell density, cell viability and activity, and diffusion distance resulting from nutrient supply constraints, limit the rate at which GAG can be accumulated in three-dimensional constructs. GAG accumulation depends on GAG production per cell and on cell density. GAG accumulation thus appears necessarily slow, and the general finding that cultures of >7 months are required to achieve concentrations of GAG similar to those seen in vivo may not be easily overcome (Kellner et al. 2002, Roughley, 2004). An increase in GAG Importance of Extracellular Environment for Regenerative Medicine and Tissue Engineering of Cartilagious Tissue http://dx.doi.org/10.5772/55566 555

ability to induce matrix synthesis and promote repair in cartilage. TGF-β is one of the anabolic factors involved in cartilage maintenance and appears to be a good candidate for cartilage repair. TGF-β is a stimulator of extracellular matrix production, like collagen type II and proteoglycan (PG), in chondrocytes and it downregulates matrix-degrading enzymes (Edwards, et al. 1987). High amounts of TGF-β are stored in healthy cartilage (Pedrozo, et al. 1998, Redini F, et al. 1997, Morales, et al. 1991a,b, Burton-Wurster & Lust,1990), whereas in OA cartilage the expression of TGF-β is reduced (Verdier et al. 2003). FGF-2 belongs to the family of heparin-binding growth factors (Gospodarowicz, et al. 1987).This proteins are known to induce chemotactic, angiogenic, and mitogenic activity, and play an important role in early differentiation and development (Powers et al., 2000, Burgess & Maciag, 1989). Cell expansion in the presence of FGF-2 was shown to promote differentiation of stromal cells into cartilagi‐ nous tissues (Martin, et al. 1998). Similary, FGF-2 was shown to retain the chondrogenic differentiation potential of extensively expanded stromal cells (Tsutsumi, et al. 2001). Like wise, pellet cultures established from FGF-2 treated human mesenchymal stem cells were larger in size and contained a higher level of proteoglycans than untreated cells (Solchaga, et al. 2005). Martin et al. (1999a, 2001) showed that the FGF-2 treatment increased cell number during monolayer expansion and the differentiation capacity of expanded chondrocytes in

The most important problems in cartilage regeneration medicine are to supply nutrients to cells activated by grafting or growth factors and to maintain a healthy extracellular environ‐ ment. However, it has been shown that the extracellular osmotic pressure decreases with cartilage degeneration in candidates for treatment. In this study, cells were isolated from the cartilage from metacarpal phalangeal joints of 18-24 month bovine. Alginate beads containing cartilage cells collected from adult bovine (4 million cells/ml) were prepared. Three-dimen‐ sional culture was done for 5 days under a low osmotic condition (270 mOsm) as seen osteoarthritis or a normal osmotic condition like that of healthy cartilage (370 mOsm). Transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2) were added every day. GAG production was assessed by the dimethylmethylene blue (DMB) assay after 5 days. Fig. 8 shows the effect of osmorality and growth factor on the amount of GAG accumulated with time in culture. Significantly more GAG was accumulated by cells cultured at 370 Osm than low osmoralities (270 mOsm). The cells cultured with the growth factors (TGFβ or FGF-2) were more active and accumulated significantly more GAG accumulated than cells cultured without the growth factors. However, GAG accumulated was significantly lower for cells cultured at 270 mOsm than those at 370 mOsm. Thus, the clinical application of cartilage regeneration medicine needs to be advanced by providing appropriate physiological condi‐

The interrelationships between cell density, cell viability and activity, and diffusion distance resulting from nutrient supply constraints, limit the rate at which GAG can be accumulated in three-dimensional constructs. GAG accumulation depends on GAG production per cell and on cell density. GAG accumulation thus appears necessarily slow, and the general finding that cultures of >7 months are required to achieve concentrations of GAG similar to those seen in vivo may not be easily overcome (Kellner et al. 2002, Roughley, 2004). An increase in GAG

subsequent 3-dimensional culture.

554 Regenerative Medicine and Tissue Engineering

tions with consideration of age-related cartilaginous changes.

**Figure 8.** Effect of extracelluler osmotic change and growth factors on GAG accumulation/tissue volume. This figure gives pooled data for 2 representative osmolality from 6 separate experiments for cells cultured by articular chondro‐ cytes. GAG production at 370 mOsm was about 42.1±9.1 μg/ml/day, while it was only about 26.3 ± 4.3 μg/ml/day at 270 mOsm. During culture at 370 mOsm, GAG production was increased about 2-3 times by addition of growth fac‐ tors, while there was a clear decrease in the response of GAG production to growth factors at 270 mOsm compared with that seen at 370 mOsm. Incubation with growth factors, enhances GAG production during culture at a normal osmotic pressure, but cell function is decreased in degenerated cartilage. Values are mean ± standard error. (\*: Scheffe between 270 mOsm and 370 mOsm medium, #: P<0.05, Scheffe between control and every growth factors).

production rate per cell can be induced by addition of growth factors, but the relative increase which can be achieved is limited (usually two–threefold under optimal conditions) and the consequent increase in metabolic demand can lead to a fall in pH in the construct center and thus severely limit growth factor efficacy. In this study, addition of TGF-β, and FGF-2 to constructs was found to have big effect on the concentration of accumulated GAG under low osmoralities. Thus, increasing cell metabolism potentially should increase GAG deposition, but leads to a more nutrients demands.
