In this investigation we adopted a protocol of transient supplementation of serum-free media with TGF-β3 and applied a regimen of dynamic deformational loading whose timing was adjusted towards achieving the most robust mechanical properties. Studies by our group [25
] and by others [26
] have previously demonstrated that mechanical stimulus (physiologic deformational loading) can act synergistically with chemical stimuli (growth factors) to amplify the benefits conferred by either stimulus alone. Furthermore, it has been previously shown that the timing of the application of the growth factor can be critical; free-swelling cultures supplemented transiently with TGF-β3 consistently yielded cartilage-like tissue with higher mechanical properties than those derived from cultures with continuous (or no) growth factor supplementation [14
]. Similarly, work has been done demonstrating the utility of sequential growth factor protocols (e.g., TGF-β
1/FGF-2 followed by IGF-1) [26
The findings of this study indicate that coordination of the timing (introduction and duration) of the application of an appropriate chemical stimulus as well as the timing of the introduction of mechanical stimuli represents a strategy to optimize engineered tissue growth (i.e. a sequential
loading protocol). In Study 1, we have confirmed the earlier results of Byers et al. [14
] who found that discontinuation of TGF-β3 supplementation after two weeks in culture yields much better material properties than under continuous supplementation (). In Study 2, we have found that dynamic loading initiated at the same time as TGF-β3 supplementation yields significantly poorer properties than the free-swelling control group, after discontinuation of supplementation (). However, the application of deformational loading initiated after culturing with growth factor TGF-β3 for 2 weeks (Study 3) yields significantly stiffer chondrocyte-seeded agarose constructs than free-swelling controls. Using this sequential loading protocol, engineered constructs continued to display the dramatic improvement in properties associated with the removal of the growth factor (Studies 1 and 2) while benefiting from the deformational loading protocol. These constructs achieved the most favorable values for tissue-engineered cartilage constructs reported in the literature to date for the culture period prescribed. Young’s modulus and GAG levels achieved values similar to those of native cartilage after as little as 28 days in culture (). Dynamic modulus values, which are more representative of the functional tissue properties, however, remain at 32% of those manifested by native cartilage, after 42 days in culture (). As has been shown both theoretically [27
], and in vivo
], dynamic modulus values are largely influenced by collagen content and organization as well as construct permeability whereas the equilibrium modulus is influenced to a greater degree by GAG content.
Related to this observation, collagen levels for constructs in all the studies presented here remained relatively low (, , ) and were not different from levels achieved previously with optimal conditions using serum-supplemented media [25
]. This suggests that application of dynamic loading as well as the temporary supplementation of TGF-β3 has a much greater effect on GAG production compared to collagen production. In fact, the increase in the equilibrium compressive modulus over time of developing constructs can be attributed almost entirely to the increase in GAG levels.
While the average content of GAG and collagen were not statistically different between DDL versus FS constructs in Study 3, the compressive moduli were significantly stiffer (~15%) for DDL constructs (). We have previously reported that loaded and free-swelling constructs possess differences in levels of other extracellular matrix molecules (such as cartilage oligomeric matrix protein-COMP [31
], type IX collagen [24
]) and structural organization [17
] that may contribute to the disparate material properties observed, and ultimately influence chondrocyte mechanotransduction and the level of cartilage repair after implantation.
The delayed applied loading protocol found to be efficacious in the current studies is in direct contrast to that which we have previously reported to be optimal for constructs cultured with serum supplemented media, where the highest mechanical properties (E Y
=185 kPa on day 56 [32
]) were obtained when dynamic deformational loading was applied at the earliest possible time (i.e., day 0). One way to explain these results may be to consider the much greater contribution of the growth factor to the observed tissue growth relative to that induced by the application of dynamic loading. The mechanisms behind the drastic increases in growth associated with transient supplementation of TGF-β3 is not yet well understood, however these results suggest that the TGF-β3 preconditions cells toward high anabolic activity that is manifest once the growth factor is removed (Study 1). Continuous loading with or without growth factors in the presence of FBS does not have this same suppressive result [25
] and may be due to the presence of other growth factors such as IGF-I or other proteins that can regulate TGF-β growth factors in serum [33
The mechanism behind the detrimental effect of applied dynamic loading in the presence of TGF-β3 (Study 2) is a new finding that warrants further discussion. One clue may lie in the concentration of the growth factor within the construct. Theoretical models performed by our laboratory for molecules of similar size to TGF-β3 (~25 kDa, R&D Systems) indicate that the concentration of the molecule within the tissue construct under dynamic loading conditions can be elevated ~2–3 fold compared to free diffusion conditions [35
]. The presence of TGF-β3 binding proteins in the elaborated matrix [36
], such as reported for insulin-like growth factor I (IGF-I) in native cartilage [38
], can also result in a greater concentration of growth factor retained in the construct compared to the culture media. Therefore, dynamic loading in combination with binding protein and proteoglycan interactions may increase the concentration of TGF-β3 localized in the construct into the range where the growth factor can begin to elicit a negative response. This threshold concentration where catabolic effects have been observed has been reported to occur at culture media concentrations of approximately 20–50 ng/mL [40
]. To test this hypothesized mechanism, a study of the dose response to TGF-β3 with and without dynamic loading is planned for future research. This hypothesis would be supported if doses of TGF-β3 lower than used here were to combine with dynamic loading to yield better properties than free-swelling controls; and if doses of TGF-β3 above a certain threshold were to produce poorer properties than lower doses, under free-swelling conditions.
The results of this study address a number of important issues related to functional tissue engineering of articular cartilage. The most positive outcome is the finding that temporary supplementation of TGF-β3 followed by dynamic loading can produce an equilibrium modulus and GAG content which match those of native tissue over a culture period of 4 to 6 weeks only; the dynamic modulus and collagen content remain lower than in native tissue, but are as good as, or better than reported in previous studies. However, there are a number of practical issues that remain to be addressed. First, the modest improvement observed in the mechanical properties with dynamic loading in Study 3 (~11% for EY
and ~17% for G*) suggests that free-swelling culture may be a less costly alternative, precluding the need to load constructs on a daily basis. While this may be true for the culture conditions employed in this study, our previous studies demonstrate that dynamic loading can be far more beneficial than free swelling under other culture conditions[11
]. Since the production of higher levels of collagen remains a challenge, it may well be that the elusive culture conditions which can promote rapid protein synthesis might also benefit significantly from dynamic loading, possibly by increasing the expression of cell receptors to growth factors and signaling proteins.
Second, it may be argued than any beneficial outcomes observed with immature chondrocytes are of limited value for current clinical strategies, which rely on mature autologous cells. Indeed, although immature bovine cells respond favorably to supplementation of TGF-β3, preliminary work from our lab (not shown) suggests that, in fact, mature primary chondrocytes do not respond as robustly. This is likely due to known decreases in the expression of TGF-β receptor and signaling proteins that occurs with age [42
], and additional strategies are thus required to supplement the successful techniques achieved in this study when using mature cells. It may also be noted that the strategies employed in this study might be successful on alternative sources of immature cells, such as embryonic stem cells.
It appears that, as in bone [44
], the structure and function of cartilage reflects the physical demands to which it is subjected. Cartilage from weight-bearing and non-weight bearing regions (the source for autologous grafts) have been reported to be distinct in structural organization as well as cells, with chondrocytes from loaded regions exhibiting greater expression of intermediate filaments than their counterparts in less loading regions [45
]. This disparity in chondrocyte populations appears to be an adaptation to their physical environment. Our contention is that chondrocytes subjected to loading during pre-culture (i.e., preconditioning) may better acclimate to the physiologic loading environment that they are exposed to post-implantation. This concept of cell memory has been described in the bone remodeling literature, where it has been suggested that acquired long-term memory of a mechanical loading environment may influence the responsiveness of bone tissue to external stimuli (e.g.[46
]). Similarly, the presence (or absence) of extracellular matrix molecules such as type II collagen, the presence or absence of focal adhesions, and mechanical and morphological changes to the cell membrane in response to preconditioning with growth factors [47
] have all been shown to influence the response of chondrocytes to mechanical loading. Only with in vivo
studies, or possibly with the proper in vitro model of cartilage repair [51
], can the efficacy of applied loading bioreactors on functional cartilage repair be assessed. The findings of the current study suggest that an optimal strategy using well-characterized conditions for the functional tissue engineering of articular cartilage, particularly to accelerate construct development, may incorporate sequential application of different growth factors and applied deformational loading.