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Results and Discussion
- Tissue engineering of cartilage in space

Cellular Viability Postflight.

Mir-grown constructs assessed 30 hr postflight were comparable to Earth-grown constructs with respect to cell viability and biosynthetic activity. Specifically, constructs from both groups consisted of 95-99% viable cells, as judged by trypan blue exclusion and intracellular esterase activity (Fig. 2), and incorporated radiolabeled tracers into macromolecules at comparable rates (Table 1). The latter may represent a rebound in chondrocyte metabolism in the Mir-grown constructs, which were assayed after return from space, in a manner analogous to the prolonged and time-dependent stimulation of chondrocyte metabolism that was observed in cartilage explants after the release of a compressive force (16). 

Construct Shape.

Constructs grown on Mir tended to become more spherical, whereas those grown on Earth maintained their initial discoid shape, as assessed histologically (Fig. 3 A and B) and from their respective aspect ratios (i.e., height to width) of 0.72 ± 0.08 and 0.62 ± 0.03 (P  in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1 g. In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion (22), increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. 

Construct Structure.

Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules (0.15-0.45 mm thick), as assessed by using safranin-O stain for GAG (Fig. 3 A-D) and immunohistochemical staining for collagen type II (data not shown). Constructs grown on Earth appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (Fig. 4 A and B), but the average collagen fiber diameter was similar in the two groups (22 ± 2 nm) and comparable to that previously reported for developing articular cartilage (23). Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis (24, 25). 

Construct Composition.

Constructs at the time of launch and after additional cultivation on Mir and on Earth contained 13 ± 1, 14 ± 0.8 and 19 ± 0.2 million cells, respectively. On Earth, construct wet weights increased 1.7-fold between 3 and 7 months, which could be attributed to increasing amounts of cartilage-specific tissue components (i.e., collagen type II and GAG) (Table 1). In contrast, on Mir construct wet weights increased 1.3-fold over the same time interval, because of deposition of collagen and unspecified components that were not GAG (Table 1). The PGA scaffold represented less than 0.3% of the final construct wet weight, based on previous degradation studies (8). The fraction of the total collagen that was type II decreased, but not significantly, from 92 ± 19% at launch to 78 ± 4% at landing, demonstrating relatively good maintenance of the chondrocytic phenotype. Constructs grown for 7 months on Mir and/or Earth had type II collagen fractions that were comparable to each other and to those previously reported for 8-month cultures of chondrocytes in alginate beads (26).

Construct Function.

Construct mechanical properties improved both on Mir and on Earth resulting in an increase in aggregate modulus, HA, and a decrease in hydraulic permeability, k (Table 1). Dynamic stiffness also increased with culture time and showed the characteristic frequency dependence of natural cartilage (12) (data not shown). Mechanical properties of Mir-grown constructs were inferior to those of Earth-grown constructs. In particular, the aggregate modulus of Earth-grown constructs was indistinguishable from natural calf cartilage and was 3-fold higher than that of Mir-grown constructs (Table 1). These data support the hypothesis that, in radially confined compression, HA is determined mainly by GAG content (12), whereas k and dynamic stiffness are likely to depend on a variety of factors, including collagen content and organization, GAG immobilization, and overall tissue architecture (12, 27).

The finding that constructs in the Earth group had markedly higher wet weights, GAG fractions, and aggregate moduli than constructs in the Mir group (Table 1) may be attributed to the effects of physical forces at unit gravity (i.e., settling-induced hydrodynamic and contact forces) on the growth and development of the engineered tissue. This finding is consistent with previous studies of cartilage explants in which dynamic compression (amplitudes of 1-5% and frequencies of 0.01-1 Hz) increased GAG and protein synthesis rates (16), and cyclic hydrostatic pressure (5 MPa, 0.25 Hz) increased GAG synthesis (28). The envisioned mechanism of mechanotransduction is analogous to that previously described for bone (29), involving four steps: mechanocoupling, biochemical coupling, signal transmission, and effector cell response.

Cells maintained viability and differentiated phenotype over 7 months both on Mir and on Earth. The observed differences between constructs in the two groups might be attributed to differences in construct cultivation conditions (i.e., free floating vs. gravity settling). In particular, spherical shape, relatively low GAG fraction and inferior mechanical properties of Mir-grown constructs might be attributed to the space environment. However, we cannot distinguish between the relative contributions of launch, exposure to microgravity and local environmental factors (e.g., cosmic radiation), and landing, emphasizing the need for the use of an in-flight, 1-g centrifuge for control groups in future flight experiments (30).

The present study is the longest cell culture experiment ever carried out in space and demonstrates the feasibility of microgravity tissue engineering. Final tissue constructs consisted of viable, metabolically active cells and were structurally and functionally cartilaginous, but constructs grown on Mir were smaller and mechanically inferior to those grown on Earth. Our results are consistent with previous reports that musculoskeletal tissues remodel in response to physical forces (31, 32) and are adversely affected by spaceflight (1, 2). The same cell-polymer-bioreactor system could be used for a variety of controlled microgravity studies aimed at improving our fundamental understanding of how gravity affects cell function and tissue development. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.

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