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The authors studied tissue engineering in space by using cartilage as a …

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Materials and Methods
- Tissue engineering of cartilage in space


Rotating bioreactors used for the first 3 months of cultivation on Earth were from Synthecon (RCCV-110, Houston, TX). The Biotechnology System (BTS) used for the subsequent 4 months of cultivation on either Mir or on Earth (see below) was custom-made according to the specifications of the Biotechnology Center at the National Aeronautics and Space Administration-Johnson Space Center. All other materials were obtained from previously specified sources (9).

Cell-Polymer Construct Preparation on Earth.

Chondrocytes were obtained by enzymatic digestion of full-thickness articular cartilage harvested from the femoropatellar grooves of 2- to 3-week-old bovine calves within 8 hr of slaughter (12). Scaffolds were 5-mm diameter × 2-mm thick discs made of PGA (the same material used to make Dexon absorbable surgical sutures) formed as a 97% porous mesh of 13-µm diameter fibers (8). Culture medium consisted of DMEM with 4.5 g/liter glucose, 10% fetal bovine serum, 10 mM N-2-hydroxy-ethyl piperazine N'-2-ethane sulfonic acid, 0.1 mM nonessential amino acids, 0.4 mM proline, 50 mg/liter ascorbic acid, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.5 µg/ml fungizone. Powdered medium components were sterilized by gamma irradiation (2.5-3.8 MRad) and rehydrated before use. Chondrocytes were seeded onto scaffolds (5 million cells per PGA disc) in spinner flasks stirred at 80 rpm in a 37°C humidified 10% CO2 incubator (13). After 6 days, cell-PGA constructs were transferred into rotating bioreactors (11 constructs per 110-ml vessel) configured as the annular space between a 5.75-cm diameter polycarbonate outer cylinder and a 2-cm diameter hollow inner cylinder covered with a 175-µm thick silicone membrane (14). The entire vessel was rotated as a solid body around its central axis while gas exchange was provided by pumping incubator air through the inner cylinder at 0.7-1.2 liters/min. Culture medium was replaced at a rate of 50% every 3-4 days, and the vessel rotation speed gradually was increased from 15 to 28 rpm over 3 months, to induce mixing by gravitational construct settling (7).

Construct Cultivation on Earth and on Mir.

After 3 months, constructs were transferred into each of two flight-qualified rotating, perfused bioreactors (BTS) for an additional 4 months of cultivation on either the Mir Space Station or on Earth (Fig. 1). Specifically, one BTS containing 10 constructs was transferred to Mir via the U.S. space shuttle STS-79 (9/16/96 launch) and brought back to Earth via STS-81 (1/22/97 landing). A second BTS with 10 constructs served as an otherwise identical study conducted on Earth, at the Johnson Space Center. In the BTS, the bioreactor was configured as the 125-ml annular space between a 5-cm diameter polycarbonate outer cylinder and a 1.5-cm diameter hollow inner cylinder covered with a 50-µm cut-off, 36% open area nylon mesh (Tetko, Kansas City, MO). A flat, thin 3.8-cm diameter disc attached near one end of the inner cylinder served as a viscous pump (15). Fluid entered the bioreactor behind the disc and exited via the mesh on the inner cylinder. In particular, the bioreactor was connected to a recirculation loop that included tubing (C-flex 15, Cole Parmer, Niles, IL), a peristaltic pump (Randolph model 250, Manchaca, TX), and a silicone membrane gas exchanger (Avecor model 0400-2A, Plymouth, MN). A mixture of 10% CO2 and 90% air flowed continuously through the gas exchanger at 0.6 ml/min. The temperature within the BTS was maintained at 37°C.

Medium was recirculated between the bioreactor and the gas exchanger at 4 ml/min for 20 min four times per day, and 50-100 ml of fresh medium was infused into the system approximately once per day. As a result, medium metabolic parameters were maintained within previously established target ranges [e.g., pH between 6.9 and 7.4; partial pressure of oxygen (pO2) between 71 and 127 mmHg] in both groups for the duration of the study, as assessed by using portable cartridges (G3+, I-Stat, Princeton, NJ). Concentration gradients within the bioreactor were minimized by differential rotation of the inner and outer vessel walls in the same direction at 10 and 1 rpm, respectively, in microgravity (ref. 15, and S. Kleis, personal communication), and by the convection associated with gravitational construct settling during solid body rotation of the bioreactor at 28 rpm in unit gravity (7). In the Mir group, gas bubbles were observed in the bioreactor between flight days 40 and 130. The amount of gas stabilized at approximately 20% of the total bioreactor volume, and the bubbles did not come into direct contact with the constructs, as assessed by videography. An equal amount of gas was introduced into the bioreactor in the Earth group, to match the conditions on Mir as closely as possible.

Construct Analysis.

Constructs were assessed at the time of launch (i.e., after 3 months of culture) and after 4 additional months on either Mir or Earth (i.e., after 7 months of culture), and compared with full-thickness natural calf articular cartilage (Table 1). Cell viability was assessed by using trypan blue exclusion and an intracellular esterase assay (Molecular Probes). Samples for light microscopy were fixed in 10% neutral buffered formalin, cross-sectioned, embedded, sectioned (5 µm thick), and stained with safranin-O for glycosaminoglycan (GAG). Samples for transmission electron micrography were fixed in Karnovsky's reagent (0.1 M sodium cacodylate with 2% paraformaldehyde and 2.5% glutaraldehyde), postfixed in 1% osmium tetroxide with 0.2 M collidine, embedded, and sectioned (70 nm thick). Samples for biochemical analyses were radiolabeled in mixed dishes for 18 hr in medium containing 35SO4 and 3H-proline, frozen, lyophilized, and enzymatically digested (16). Previously reported methods were used to quantitate cellularity (DNA) (17), macromolecular incorporation of radiolabeled tracers (16), GAG (18), type II collagen (19), and total collagen (20). Samples for mechanical studies were cored into 3-mm diameter × 2-mm thick discs, which excluded the upper and lower surfaces (approximately 0.5 mm thick) of both engineered and natural cartilage, placed in a cylindrical chamber, and subjected to confined compression and stress relaxation at increments of 10% strain up to a maximum of 40% strain as previously described (12, 21). At a static offset strain of 30%, sinusoidal strains of 0.5% amplitude were superimposed at frequencies of 0.025-1.0 Hz. Aggregate modulus (i.e., the ratio of incremental stress and incremental strain at equilibrium), dynamic stiffness (i.e., the ratio of the amplitude of the dynamic stress to the amplitude of the dynamic strain), and hydraulic permeability were calculated as described (12, 21).

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