Abstract
Introduction: Fibroin sponge is purified silk protein from which high-strength gel sponge can be produced. The purified fibroin sponge causes no immune response. This study evaluates unique performances of the fibroin sponge for articular-cartilage regeneration, and mechanical properties of regenerated cartilage were also measured.
Methods: Refined silk yarn was dissolved in 9M lithium bromide aqueous solutions, and was frozen in −20& #8451 freezer for 12 hours. Hydrogel sponge was formed under the room temperature. Articular cartilage slices were taken from the proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits. The cartilage slices cut into small pieces and were digested with 0.25% trypsin in DMEM containing antibiotics for 30 min at 37& #8451. After rinsing with Tyrode’s balanced solution and centrifuging at 180 G for 5 min, the chondrocytes were isolated with 0.25% collagenase for 8 h at 37& #8451. These cells were harvested and inoculated into the fibroin sponge. The constructs of the chondrocytes and the fibroin sponge were cultured in DMEM containing 10& #65285 FCS and 50mL L-ascorbate for 4weeks. Indentation test and dynamic visco-elastic measurement were carried out for these constructs.
Results and discussion: Cell density of the inoculated chondrocytes was increased to about five times as much as initial volume. This regenerated tissue was intensely stained with safranin-O fast green and showed a meta-chromatic reaction. This also stained positively with immunostain for type & #8545 collagen, but negatively with immunostain for type & #8544 collagen. Mechanical tests showed that time constants of compressive creep and E’ values were increased with cultivation days, and the peak value and frequency of tan& #948 shifted to a lower amount. The change in dynamic visco-elastic properties of the regenerated cartilage is caused by synthesis of extracellular matrix.
The abstracts were prepared by Nico Verdonschot. Correspondence should be addressed to him at Orthopaedic Research Laboratory, University Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
