Tissue-engineered cartilage using thermoresponsive gelatin as an in situ forming and moldable scaffold with chondrocytes: in vitro and in vivo performances

We devised a tissue-engineered cartilage using thermoresponsive gelatin, poly(N-isopropylacrylamide)-grafted gelatin (PNIPAAm-gelatin).

An aqueous solution of the PNIPAAm-gelatin can spontaneously gel above 34°C. This character of the PNIPAAm-gelatin might fit a gel to a given shaped chondral defect.

For in vitro study, chondrocytes isolated from the articular cartilage of Japanese White Rabbits were three-dimensionally cultured in PNIPAAm-gelatin gel for up to 12 weeks. The chondrocyte–PNIPAAm-gelatin constructs were evaluated at cell and tissue levels. At the cell level, cellular viability and the degree of cellular differentiation were assessed. At the tissue level, the appearance of the tissue, the amount of extracellular matrices (ECMs), and the mechanical properties were assessed by comparison with those of native hyaline cartilage. During 3 weeks of culture, the cellular viability was more than 95%. Confocal laser scanning microscopic images demonstrated that round-shaped cells, which are found in hyaline cartilage, were predominant in the construct. With an increase in culture time, the population of the cells arrested in the G₁/G₀ phase of the cell cycle in a three-dimensional condition was significantly higher than that in a monolayer condition. As type II collagen and sulfated-glycosaminoglycan specific to hyaline cartilage were detected in sections of the construct, little type I collagen, which is a marker of dedifferentiated chondrocytes, was detected. These results indicate that the inoculated cells could express their differentiated phenotype. Moreover, the amounts of ECMs increased and closed to that of native hyaline cartilage with time. Mechanical properties of the constructs tended to close towards those of native cartilage with culture time. These results indicate that cartilaginous tissue using PNIPAAm-gelatin can be reconstructed in in vitro conditions.

In animal studies, the combination of chondrocyte–PNIPAAm-gelatin constructs precultured for 2 weeks and the cell-incorporated PNIPAAm-gelatin solution were used as implants for chondral defects in the patellae of rabbits. Macroscopic evaluation of the implant harvested at 24 weeks postoperatively showed that, at the rough surface of the implants, tissue continuity to adjacent cartilage with minimal concave deformation was acquired. Tissue sections showed a homogeneous distribution of ECMs in the implanted tissue and no inflammatory cells. Moreover, mechanical properties of the constructs became closer to those of native cartilage.

These results indicate that PNIPAAm-gelatin should serve as an adequate scaffold for articular cartilage regeneration.