Growth Factor Supplementation and Dynamic Hydrostatic Pressurization for Articular Cartilage Tissue Engineering

INTRODUCTION Articular cartilage transmits the stresses (5-12 MPa) that occur in joints with loading [1]. In vivo, a number of mechano-electrochemical signals arise with deformation of cartilage [2]. One of these signals, hydrostatic pressurization, occurs as a result of the high water content of the tissue and the small pore size. With deformation, fluid is constrained from rapidly leaving the tissue, and so pressurizes. This fluid pressurization has been shown both theoretically and experimentally to support upwards of 90% of the applied stress [3]. In vitro, chondrocytes respond to pressurization by altering their biosynthetic rates; dynamic pressurization generally leads to increases, while static pressure leads to decreases [4,5]. In monolayer culture of chondrocytes, dynamic pressurization (10 MPa, 1 Hz, 4 hours/day for 4 days) increased matrix gene expression [6]. Long-term growth of chondrocytes seeded in PGA felts demonstrated that intermittent hydrostatic pressure increased matrix deposition [7]. Based on these findings, we developed a custom bioreactor for applying dynamic hydrostatic pressurization (DHP) to chondrocyte-seeded agarose hydrogels [8]. With this device, we have recently reported a ~2 fold increase in the aggregate modulus compared to free swelling control, with similar increases in proteoglycan content after one month of DHP culture (3 MPa, 0.33 Hz) [8]. In other studies, using deformational loading, we had also previously shown that growth factors (IGF-1 and TGF-β1) interact synergistically with mechanical signals to increase tissue growth [9]. In the present study, we examined the growth of chondrocyte-seeded agarose hydrogels with growth factor supplementation for long term culture in free swelling conditions or with dynamic hydrostatic pressurization. MATERIALS AND METHODS Cell Culture: Chondrocyte-seeded agarose hydrogels were prepared as previously described [10]. Briefly, immature bovine chondrocytes were suspended in 2% agarose (Type VII, Sigma) at 60 million cells/ml. Disks (Ø 4.76 x 2.25 mm) were cored, and cultured in petri dishes (15 to 20 disks) with 30 ml of high glucose DMEM (supplemented with 10% fetal bovine serum, buffers, antibiotics, amino acids, and 50 μg/ml fresh ascorbic acid) at 37°C and 5% CO2. Media, supplemented with the growth factors TGF-β1 (10 ng/ml), IGF-1 (300 ng/ml), or their combination, were changed daily. Dynamic Hydrostatic Pressurizaton: Dynamic hydrostatic pressurization (DHP) was applied using a custom-feedback controlled pressure bioreactor [8]. For pressurization, constructs were placed in sealed sterile plastic bags with 7 ml of fully supplemented DMEM (with growth factors where appropriate). Control samples were similarly sealed, and placed inside the incubator adjacent to the pressure bioreactor. DHP was applied with a triangular waveform with a peak pressure of 3 MPa and a frequency of 0.33 Hz for four hours per day, five days per week for four weeks. After loading, constructs were returned to free swelling culture in 30 ml of DMEM supplemented as above. Every two weeks, 3-4 samples were removed from culture for analysis. Mechanical Testing: Mechanical testing was carried out on constructs and native tissue (n=5) using stress relaxation tests in unconfined compression with a ramp compressive strain to 10% of the measured thickness. After equilibrium was reached, a sinusoidal displacement of 40 um was applied at