**7. Conclusions**

Transforming growth factor-β (TGF-β) (Morales, et al. 1988, 1991a,b, Luyten, et al. 1988, van Osch, et al. 1998, Diao, H. et al. 2009,), bone morphogenetic protein-2 (BMP-2) (van Beuningen, et al. 1998, Blaney Davidson, et al. 2007), bone morphogenetic protein-7 (BMP-7) (Chubinskaya, et al. 2007, Hayashi , et al. 2008), insulin growth factors-1 (IGF-1) (Luyten, et al. 1988, van Osch, et al. 1998, Fortier, et al. 2002, Goodrich, et al. 2007) and fibroblastic growth factor (FGF) (Martin, et al. 1999a, Maehara, et al. 2010), to constructs was found to have little effect on the concentration of accumulated GAG although it increased construct size. In addition, GAG production rates appear to fall with time in culture in many different systems also limiting GAG accumulation (Mercier et al., 2004). Increasing cell density potentially should increase GAG deposition, but leads to a lower activity per cell, and also, in general, has not been found to increase GAG deposition rates (Panossian et al., 2001). It should also be noted that tissue in vivo cannot support too high a cell density, so in vitro culture of constructs at high cell density

Culture conditions such as stirring or perfusion (Freyria et al., 2000, Seidel et al., 2004) appear able to overcome diffusive transport initially, but as GAG concentrations rise and the hydraulic permeability of the construct falls, convective transport also is reduced and rates of GAG deposition slow. GAG concentrations were reported to reach 5% by wet weight within 2 months but took a further 5 months to increase to 7% GAG. In view of the long culture times which appear necessary to achieve the required GAG composition in vitro, achievement of in vivo concentration before implantation of a construct may be an unrealistic and possibly unnecessary goal for tissue engineered disc. It has been suggested that about 2-3 fold amount of GAG can be produced using anabolic growth factors. Even if such growth factors are used, more than 100 days of culture is thought to be necessary. Furthermore, it has been reported that turnover of GAG in the cartilage tissue takes about 2 - 3 years (Maroudas, 1975). So, GAG is slowly synthesized in the biological condition, and a long time is necessary to construct articular cartilage with adequate mechanical strength even if cells are maintained in active status by three-dimensional culture. Anabolic growth factors were obvious tools to enhance cartilage repair. Recently, Carragee et al (2011). reported that the bone morphogenic protein-2 (BMP-2) has cancer risk associated with the use of BMP-2 in spinal fusion surgery. In the future, orthopaedic surgeons must exercise caution to use the anabolic growth factors in clinics.

At present, the target diseases of treatment utilizing bioengineering (tissue engineering) such as chondrocytes implantation are local lesions such as traumatic cartilage defect and osteo‐ chondritis dissecans (Bittberg, 1999, Minas, 2001, Ochi, et al. 2002, Robert, et al. 2003) (Fig. 16A). For regeneration of extensive degenerated cartilage in OA, it is necessary to secure a large amount of chondrocytes for implantation. If a large graft is implanted, a nutritional problem may occur as explained above. In addition, the subchondral bone needs to be healthy to obtain the normal function of articular cartilage such as dispersion of load. But, lesion of OA is not localized in the cartilage layer but involves the subchondral bone (e.g., osteosclerosis) (Fig.16B). The osteosclerosis which is the subchondral bone, covers the bone-cartilage junction with age and looks to close the nutritional route through these vascular system (Trueta, 1963, Havelka, et al. 1984). Therefore, even if regeneration of cartilage is achieved by means of hyaline cartilage, the regenerated cartilage may sustain overload and may be degenerated

could lead to cell death after implantation.

562 Regenerative Medicine and Tissue Engineering

There is increasing interest in the using biological methods to repair osteoarthritis. Biological repair depends on the articular cartilage maintaining a population of viable and active cells. Adequate nutrition of the cartilaginous tissue influences the outcome of such therapies and, hence, must be considered to be a crucial parameter. Therefore, it is very important to maintain an appropriate physicochemical environment to achieve successful cartilage repair by biological methods and tissue engineering procedures.
