The formation of cartilaginous tissue in vitro is a promising alternative to repair damaged articular cartilage. However, recent attempts to tissue-engineer articular cartilage that has similar properties to the native tissue have proven to be difficult. The in vitroformed cartilaginous tissue typically has a similar proteoglycan content to native cartilage, but has a reduced collagen content and only a fraction of the mechanical properties. In this study, we investigated whether the intermittent application of cyclic shearing forces during tissue formation would improve the tissue quality. Chondrocyte cultures were stimulated a t a 2'%1 shear strain amplitude at a frequency of 1 Hz for 400 cycles every 2nd day. At one week, both collagen and proteoglycan synthesis increased (23 f 6% and 20 f 6'%1, respectively) over the unstimulated, static controls. At four weeks, an increased amount of tissue formed . Tissues that formed in the presence of shearing forces also displayed a 3-fold increase in compressive load-bearing capacity (stirnulatrri: 16 f 5, unstitnuluted: 5 f I kPa max. equilibrium stress) and a 6-fold increase in stiffness (stirnuluted 112 f 36, unstirnulutc'cl: 20 f 6 kPa max. equilibrium modulus) compared to the static controls. These results demonstrate that intermittent application of dynamic shearing forces over a four-week period improves the quality of cartilaginous tissue formed in vitro. Interestingly, low amplitudes of shear stimulation for short periods of time (6 min of stimulation applied every 2nd day) produced these changes.

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Tissue engineering of articular cartilage is a promising alternative for cartilage repair. However, it has been difficult to develop tissue in vitro that mimicks native cartilage. Cartilaginous tissue formed in vitro does not accumulate enough extracellular matrix, is deficient in collagen, and possesses only a fraction of the mechanical properties of native cartilage. In this study, we investigated whether long-term intermittent compressive stimulation would improve the quality of the generated tissue. Chondrocyte cultures were established on the surface of porous calcium polyphosphate substrates and allowed to form cartilaginous tissue. In vitro-formed tissues were subjected to different stimulation protocols for 1 week. The optimal mechanical stimulation parameters identified in this short-term study were then applied to the cultures for up to 4 weeks. Mechanical stimulation applied at a 5% compressive amplitude at a frequency of 1 Hz for 400 cycles every second day resulted in the greatest increase in collagen synthesis (37 +/- 9% over control) while not significantly affecting proteoglycan synthesis (2 +/- 8% over control). This condition, applied to the chondrocyte cultures for 4 weeks, resulted in a significant increase in the amount of tissue that formed (stimulated, 2.4 +/- 0.2 mg dry wt; unstimulated, 1.61 +/- 0.08 mg dry wt). Stimulated tissues contained approximately 40% more collagen (stimulated, 590 +/- 58 microg; unstimulated, 420 +/- 42 microg), and 30% more proteoglycans (stimulated, 393 +/- 34 microg; unstimulated, 302 +/- 32 microg) as well as displaying a 2- to 3-fold increase in compressive mechanical properties (maximal equilibrium stress: stimulated, 10 +/- 1 kPa; unstimulated, 5 +/- 1 kPa; maximal equilibrium modulus: stimulated, 80 +/- 23 kPa; unstimulated, 24 +/- 6 kPa). The results of this study demonstrate that intermittent mechanical stimulation can increase collagen synthesis and, when applied over a 4-week period, can accelerate extracellular matrix accumulation as well as improve the material properties of the developed tissue. Interestingly, only short periods of mechanical stimulation (6 min every second day) were needed to affect the quality of cartilaginous tissue formed in vitro.