The beneficial effect of delayed compressive loading on tissue-engineered cartilage constructs cultured with TGF-beta3

Abstract

Objective: To determine whether the functional properties of tissue-engineered constructs cultured in a chemically-defined medium supplemented briefly with TGF-beta3 can be enhanced with the application of dynamic deformational loading.

Methods: Primary immature bovine cells (2-3 months old) were encapsulated in agarose hydrogel (2%, 30 x 10(6)cells/ml) and cultured in chemically-defined medium supplemented for the first 2 weeks with transforming growth factor beta 3 (TGF-beta3) (10 microg/ml). Physiologic deformational loading (1 Hz, 3 h/day, 10% unconfined deformation initially and tapering to 2% peak-to-peak deformation by day 42) was applied either concurrent with or after the period of TGF-beta3 supplementation. Mechanical and biochemical properties were evaluated up to day 56.

Results: Dynamic deformational loading applied concurrently with TGF-beta3 supplementation yielded significantly lower (-90%) overall mechanical properties when compared to free-swelling controls. In contrast, the same loading protocol applied after the discontinuation of the growth factor resulted in significantly increased (+10%) overall mechanical properties relative to free-swelling controls. Equilibrium modulus values reach 1306+/-79 kPa and glycosaminoglycan levels reach 8.7+/-1.6% w.w. during this 8-week period and are similar to host cartilage properties (994+/-280 kPa, 6.3+/-0.9% w.w.).

Conclusions: An optimal strategy 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.

Figure 1

Schematic of TGF-β3 time-course and loading time-course for each study. Loading was initiated at days indicated by arrow; TGF- β3 was supplemented during periods indicated by hatch marks.

Figure 2

a) Loading profile adjusted for system compliance delivered by bioreactor over time in culture. Blue line shows increasing tare strain as a result of increasing tissue thickness with time. Purple line shows decreasing applied dynamic strain as a result of tissue stiffening over time. b) Representative load vs. time curve of tissue-engineered constructs on day 42. A load of zero would have indicated platen lift-off. Inset represents full curve.

Figure 3

(Study 1) The effect of temporal application of TGF-β3 to a chemically defined medium: a) E_Y, b) G* at 1HZ, c) GAG, and d) collagen. *=p<0.005 for TGF continued vs. TGF discontinued (n=4).

Figure 4

(Study 2) The effect of dynamic deformational loading applied concurrently with exposure to TGF-β3: a) E_Y, b) G* at 1HZ, c) GAG, and d) collagen. *=p<0.005 for FS vs. CDL (n=5).

Figure 5

(Study 3) The effect of dynamic deformational loading initiated after the discontinuation of TGF-β3: a) E_Y, b) G* at 1HZ, c) GAG, and d) collagen. *=p<0.005 for FS vs. DDL (n=8).

Figure 6

(1) Safranin O staining for GAG, (2) Picrosirius Red staining for collagen, (3) hematoxylin and eosin staining for visualization of local multiplication of cell nuclei (Mag. 40x), and (4) Immunohistochemical staining for type II collagen. All slides taken from study 3 on either day 0 or day 42 with either free-swelling (FS) or dynamically-loaded (DL) groups.