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Large osteochondral defects have been difficult to repair via tissue engineering treatments due to the lack of a sufficient number of source cells for repairing the defect and to the severe mechanical stresses affecting the replacement tissue. In the present study, whole-area osteochondral defects of rabbit patella were covered and wrapped with a fibroin sponge containing chondrocytes, with or without Green Fluorescent Protein (GFP) transgenic marking, on the surface facing the osteochondral defect. Five of eight osteochondral defects that were covered with the chondrocyte-seeded fibroin sponges showed hyaline cartilage-like repair containing no fibroin fragments at 6 weeks after surgery. The repaired tissue showed a layer formation, which showed intensive safranin-O and toluidine blue staining, and which showed positive type II collagen immunostaining. The average surface coverage of the repaired cartilage was 53%. On average, 48% of the cells in the repaired tissue were derived from GFP transgenic chondrocytes, which had been seeded in the fibroin sponge. The fibroin-sponge covering had the potential to allow the early repair of large osteochondral defects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1474-1482, 2016., (© 2016 Wiley Periodicals, Inc.)

Transgenic silkworm technology has enabled the biological properties of silk fibroin protein to be altered by fusion to recombinant bioactive proteins. However, few studies have reported the fabrication of genetically modified fibroin proteins into three-dimensional spongy structures to serve as scaffolds for tissue engineering. We generated a transgenic silkworm strain that produces fibroin fused to basic fibroblast growth factor (bFGF) and processed the fibroin into a spongy structure using a simple freeze/thaw method. NIH3T3 mouse embryonic fibroblasts grown on bFGF-fused fibroin sponges proliferated and spread out well, showing half the population doubling time of cells cultured on wild-type fibroin sponges. Furthermore, the number of primary rabbit articular chondrocytes growing on bFGF-fused fibroin sponges was around five-times higher than that of the wild-type control at 3-days post cell-seeding. As the physical properties of wild-type and bFGF-fused fibroin sponges were almost identical, it is suggested that bFGF fused to fibroin retained its biological activity, even after the bFGF-fused fibroin was fabricated into the spongy structure. The bFGF-fused fibroin sponge has the potential for widespread application in the field of tissue engineering, and the method of fabricating this structure could be applicable to other recombinant bioactive fibroin proteins., (© 2015 Wiley Periodicals, Inc.)

A quantitative analytical method was proposed for measuring cell co-migration, which was defined as two or more cells migrating together. To accurately identify and quantify this behavior, cell migration on fibroin substrates was analyzed with respect to intercellular distance. Specifically, cell size was characterized by major diameter, and then, based on these measurements and cell center data, a specific threshold distance for defining co-migration was determined after analyzing cell motion using the Voronoi diagram method. The results confirmed that co-migration occurrences of rounded cells were significantly more stable on fibroin than on ProNectin substrates under the present experimental conditions. The cell co-migration analysis method in this article was shown to be successful in evaluating the stability of cell co-migration and also suggested the presence of "critical distance" where two cells interact on fibroin substrates. With further research, the cell co-migration analysis method and "critical distance" may prove to be capable of identifying the aggregation behavior of other cells on different materials, making it a valuable tool that can be used in tissue engineering design.

Cell migration is one of the fundamental processes in histogenesis, and it is necessary to investigate such multicellular behavior quantitatively in cell regeneration studies. In this study, Voronoi diagram analysis was first confirmed in simulation testing, and then used to evaluate the multicellular behavior of chondrocytes on three different substrates: (1) wild-type fibroin (FIB); (2) L-RGDSx2 transgenic fibroin; (3) and collagen. The indices for the round factor average, round factor homogeneity, and area disorder (AD), calculated from Voronoi diagram analysis, were used to characterize the difference in spatiotemporal changes for the different chondrocyte populations, and a regression analysis of the AD index was used to measure the speed of cell aggregation. The results suggested that the arginine-glycine-aspartic acid-serine sequence affects aggregate formation of chondrocytes cultured on FIB. The Voronoi diagram analysis represents one of the promising quantitative analyses for cell regeneration studies.

The effects of substrate material on the spatio-temporal behavior of cells is an important issue. Although cell aggregation has been observed on various fibroin substrates, the mechanisms of this aggregation have yet to be fully clarified. In this study, cell aggregation behavior on fibroin substrates were evaluated, focusing on the distance between each cell and the direction of individual cell migration. Our results showed that on fibroin substrates cells did not attract each other. However cells stayed close to adjacent cells over 24 hours of cultivation.

Chondrocytes distribution and cartilage formation in three types of fibroin sponges with different average pore sizes (40-80, 80-120 and 100-140 μm) was measured. The image processing was performed combining two methods to identify cells automatically: extraction of local maximum luminance and multi-threshold analysis. The results showed that initial accumulation of chondrocytes localized at surface area at 3 h in the small and medium-pore groups, however, the difference in the cell distributions become equivalent until 24 h after seeding. Cartilaginous tissue was well formed in each group at 21 days, and that in the smaller pore group tend to distribute at the surface area. Spherical tissues were located at the subsurface (200-600 μm below the surface) of the sponge in the medium- and large-pore groups at 21 days. Local cell aggregation was observed at 24 h at the same depth of the fibroin sponge as the spherical tissues observed at 21 days. These results suggest that the initial cell condensation process till 24 h after seeding play an important role in cartilage tissue formation.

Initial chondrocyte-silk fibroin interactions are implicated in chondrogenesis when using fibroin as a scaffold for chondrocytes. Here, we focused on integrin-mediated cell-scaffold adhesion and prepared cell adhesive fibroin in which a tandem repeat of the Arg-Gly-Asp-Ser (RGDS) sequence was genetically interfused in the fibroin light chain (L-chain) (L-RGDSx2 fibroin). We investigated the effects of the sequence on chondrocyte adhesion and cartilage synthesis, in comparison to the effects of fibronectin. As the physicochemical surface properties (e.g., wettability and zeta potential) of the fibroin substrate were not affected by the modification, specific cell adhesion to the RGDS predominately changed the chondrocyte adhesive state. This suggestion was also supported by the competitive inhibition of chondrocyte attachment to the L-RGDSx2 fibroin substrate with soluble RGD peptides in the medium. Unlike fibronectin, the expression of RGDS in the fibroin L-chain had no effect on chondrocyte spreading area but enhanced mRNA expression levels of integrins alpha5 and beta1, and aggrecan at 12 h after seeding. Although both the sequence and fibronectin increased cell adhesive force, chondrocytes grown on the fibroin substrate exhibited a peak in the force with time in culture. These results suggested that moderate chondrocyte adhesion to fibroin induced by the RGDS sequence was able to maintain the chondrogenic phenotype and, from the histology findings, the sequence could facilitate chondrogenesis., (2010 Elsevier Ltd. All rights reserved.)

Initial chondrocyte-material interactions are important for cell behaviors such as proliferation, phenotypic expression and matrix synthesis. Previously, we showed that chondrocytes cultured in/on silk fibroin scaffolds proliferate without dedifferentiating into fibroblast-like cells and that RGDS sequences genetically interfused in the fibroin light chain protein enhance cartilage tissue formation. In the present study, the adhesive force of chondrocytes was measured on fibroin substrates containing RGDS-expressing fibroin molecules produced by transgenic silkworms at the different densities of 0, 0.6, 1.5 and 3.0 mol%. The degree of chondrocyte attachment to fibroin substrates increased with the number of RGDS-expressing fibroin molecules. Moreover, the adhesive force per unit spreading area of a single cultured chondrocyte exhibited a peak that was higher with increased RGDS concentrations. The results of this study indicate that the RGDS sequences genetically interfused in the fibroin light chain protein exert effects on chondrocytes' adhesive behavior and can enhance cartilage tissue organization.

Condensation/aggregation process of rabbit-derived chondrocytes on a fibroin-coated patterned substrate was observed to estimate initial aggregation process in fibroin sponge. Chondrocytes were seeded on array of 160 microm diameter pits in three densities: 5 cells/pit (2.5 x 10(4) cells/cm(2), LOW), 15 cells/pit (7.5 x 10(4) cells/cm(2), MID) and 25 cells/pit (12.5 x 10(4) cells/cm(2), HIGH). In the MID and HIGH groups, cells tended to form aggregates after 24 h after cell seeding. In the LOW group, cell aggregate were not seen in a majority of the pits. Observation of aggregates using confocal laser scanning microscope showed that the chondrocytes at the interface of the fibroin surface tended to extend to the surface, developing an extensive network of stress fibers throughout the cytoplasm. On the other hand, chondrocytes in the other part of the aggregates maintained spherical shape, and most of the actin was localized in the cell cortex as opposed to in stress fibers. These results suggest two functional structures in the aggregates, which may explain the good balance between the maintenance of their differentiated phenotype and proliferation rate in the fibroin sponge.

In tissue engineering for cartilage repair using scaffold, initial chondrocyte-material interactions are significantly important for the following cell behaviors such as phenotypic expression and matrix synthesis. Silk fibroin scaffold is considered to be one of the useful materials in/on which chondrocytes can proliferate without dedifferentiating into fibroblast-like cells and can organize a hyaline-like tissue. For the purpose of seeking some useful aspects for designing scaffold, initial adhesive force of chondrocytes to the surface of fibroin substrate was measured by using a lab-made apparatus applying the cantilever beam method. It was found that the adhesive force per unit spreading area of chondrocytes on fibroin substrate had a clear peak between 6 and 12h after seeding. From the results of immunofluorescence staining for actin and vinculin during this period, it could be thought that an immature formation of actin fibers which was uniquely observed at the periphery of cells attaching to fibroin substrate did not contribute to the increase of adhesive force. Results in this study suggested that surface of the fibroin substrate was gradually covered with some substances which inhibit the adhesion during this period. These cell-material interactions have a possibility to be useful information for designing the adhesive performance of scaffold surface in cartilage regeneration.

Although tribological function is the most important mechanical property of articular cartilage, few studies have examined this function in tissue-engineered cartilage. We investigated changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into fibroin sponge. A reciprocating friction-testing apparatus was used to measure the friction coefficient of the regenerated cartilage under a small load. The specimen was slid against a stainless steel plate in a water vessel filled with physiological saline. The applied load was 0.03 N, the stroke length was 20 mm, and the mean sliding velocity was 0.8 mm/s. The friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the fibroin sponge surface. The friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer.

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.

The dynamic visco-elastic properties of regenerated cartilage tissue were measured to evaluate its mechanical function during cultivation. Harvested chondrocytes from 4-week-old Japanese white rabbits were inoculated into fibroin sponge at a cell concentration of about 5 x 10(7) cells/ml. Dynamic visco-elasticity measurements were performed under compressive loading to evaluate the load bearing function of the articular cartilage. The dynamic modulus and the dynamic loss of the regenerated cartilage increased and the peak value of tandelta, as well as the frequency at the peak, decreased with increasing cultivation time. The pores of the fibroin sponge became filled with newly formed tissue as cultivation time increased. These changes in the visco-elastic properties of the regenerated cartilage were compared with those of a model system, ethylene propylene diene monomer sponge with interstitial fluid, and appear to be a result of increased fluid flow resistance and internal loss. We conclude that the changes in the dynamic visco-elastic properties of the regenerated cartilage were caused because of narrowing of the fluid path by synthesized extracellular matrix.

The mechanical properties of regenerated cartilage tissue were measured to evaluate changes in their visco-elastic properties during cultivation. An indentation test and dynamic visco-elasticity measurements were carried out on cartilage tissue cultured with rabbit chondrocytes that had been inoculated into the fibroin sponge. A 1.5-mm-diameter porous indentor was used for the indentation test, in which time-dependent strain curves were derived from measurements taken under several loading conditions. Dynamic visco-elasticity measurements were performed under compressive loading to evaluate the load-bearing function of the articular cartilage. Although the amount of permanent deformation was not influenced by the duration of cartilage regeneration, the amount of creep deformation increased with longer cultivation. The E' value of the regenerated cartilage increased and the peak value of tan delta and the frequency at the peak became lower with longer cultivation. It is suggested that the changes in the time-dependent strain curves and dynamic visco-elastic properties of the regenerated cartilage were caused by maturation of the cultured cartilage tissue.