Aging is the most important risk factor for both the initiation and progression of degenerative cartilage diseases. Osteoarthritic cartilage degeneration may be due to the loss of viable chondrocytes due to apoptosis or physical stress. This degeneration is likely to be closely related to age-related changes, since aging chondrocytes and articular cartilage matrix undergo senescence-like changes, which increase the susceptibility of cells to degenerative processes and environmental or physiological stresses [27
]. As a result, chondrocytes from osteoarthritic patients might progress toward senescence more rapidly than those from normal individuals.
Aging chondrocytes also have important therapeutic ramifications. Recently, the treatment of articular cartilage defects have improved with the introduction of advanced tissue engineering techniques for autologous chondrocyte implantation (ACI) [28
]. ACI requires cell expansion in culture to provide sufficient amounts of chondrocytes for reimplantation. However, like all mammalian cells, normal adult chondrocytes have a limited mitotic potential and eventually enter a state of senescence [29
]. Moreover, the replicative life span of primary cells in culture are affected by the age of the donor, such that cells from older donors have a shorter life spans than those from younger donors [30
]. For example, in monolayer cultures of aged human chondrocytes, serial passages rapidly results in loss of phenotypic stability and proliferative capacity [7
]. Thus, to facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their replicative life span.
One possible method is transfection of hTERT
, which can immortalize or prolong the life span of various human cells, such as muscle satellite cells [31
], myoblasts [33
], fibroblasts [6
], and chondrocytes [7
]. Since most immortalized cells maintain their phenotype and state of differentiation, hTERT
-transfected cells are considered potential therapies for small-cell lung cancer [35
] and for postnatal neovascularization in severe ischemic disease [36
]. However, since chondrocytes uniquely maintain their phenotypes in 3-dimensional cultures [37
], it is not known whether hTERT
-immortalized chondrocytes maintain their state of differentiation.
Due to the aforementioned issues, in most cartilage tissue engineering studies, donor chondrocytes are usually from young animals. Our study is the first report that transfection of hTERT
can increase the replicative life span and therapeutic potential of tissue-engineered cartilage that is produced from ORA chondrocytes, which have a limited regenerative capacity. In addition, we used an ACHMS scaffold to maintain the phenotype of transfected chondrocytes, as indicated by the production of GAGs and type II collagen (Figures and ). These results are consistent with the findings of Piera-Velazquez et al. [8
In this study, we overcame the limited lifespan of ORA chondrocytes by transfection with hTERT and increased their growth rate up to 3-fold by cotransfection with GRP78 (Figure ). Specifically, hTERT/GRP78-transfected ORA chondrocytes grew at a constant rate for more than 20 PDL, whereas nontransfected chondrocytes stopped dividing after much fewer PDL. However, we were not able to completely immortalize chondrocytes, even those from young rabbits (PDL < 50). Although the additional transfection of SV40-TAg or mutant Ras could immortalize these cells, we did not choose this option because the transfected cells may have become cancerous. As a result, we focused on the phenotypic stability of GRP78 and hTERT.
is a candidate gene for gene therapy of muscular dystrophy [31
]. In contrast, GRP78
may have therapeutic applications for neuropathological conditions, such as Alzheimer's disease, because it protects cells from ER stress [11
]. ER stress can alter protein synthesis in cells [41
]. One mechanism by which ER stress promotes apoptosis in cells is by driving the accumulation of structurally abnormal proteins [42
], which are ordinarily repaired by ER chaperones to prevent age-related cell death. GRP78 is an example of a chaperone protein that regulates protein folding in the ER and thus contributes to cell survival [43
]. Since the increase in the expression of GRP78 during cell culture may help protect cells from ER stress, overexpression of GRP78 also may protect cultured chondrocytes independent of hTERT.
Due to a lack of cages for mutant rabbits, we were not able to perform animal transplantation experiments with chondrocytes that were transfected with hTERT or GRP78 alone. However, we believe that our in vitro and in vivo results from chondrocytes that were transfected with both hTERT and GRP78 are sufficient to support our conclusions. In the future, we plan to perform more animal experiments to elucidate the effects of GRP78.
In conclusion, our results showed that tissue-engineered cartilage that was grown from implanted in vivo with hTERT- and GRP78-transfected ORA chondrocytes in ACHMS scaffolds can repair articular cartilage defects in vivo (Figure ). The hTERT and GRP78-transfected ORA exhibited proliferative and differentiation activity in articular cartilage defects, resulting in the formation of hyaline cartilage. This study also shows that ORA chondrocytes potentially produce hyaline cartilage after genetic treatment, similar to chondrocytes from young animals. However, the mechanical strength of regenerated articular cartilage in large animals (i.e., sheep or pigs) needs to be investigated.