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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 μm diameter pits in three densities: 5 cells/pit (2.5×104 cells/cm2, LOW), 15 cells/pit (7.5×104 cells/cm2, MID) and 25 cells/pit (12.5×104 cells/cm2, 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. [ABSTRACT FROM AUTHOR]

Poly(N-substituted α/β-asparagine) was evaluated as a thermoresponsible and an injectable scaffold for cartilage regeneration. Solutions of this polymer are liquid state below 25°C and nonfluid hydrogel above 35°C, allowing an aqueous solution containing cells at room temperature to form a hydrogel with encapsulated cells at physiological body temperature. Chondrocytes were isolated from joint of 4-week-old Japanese white rabbits, dispersed within the thermoresponsive polymer solution and maintained for up to 72 hours in vitro. The polymer solutions demonstrated concentration-dependent inhibitory effect on chondrocytes multiplication. After the three-day cultivation, the survival rate of the chondrocytes fell into a 70~90% ranges among all the tested polymer concentrations. The morphology studies showed that there were some physical and/or chemical stress leading cells to necrosis and some extent apoptosis. Some physical and/or chemical stress may be applied, and over 70% of the chondrocytes could survive through the stress, suggesting that some phenotype could have been selected from the heterogeneous mixture of chondrocytes. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Cartilage regeneration using a fibroin sponge and a stirring chamber was investigated to improve the potential of articular cartilage tissue engineering. Chondrocytes seeded on the fibroin-sponge scaffolds were cultured in the stirring chamber (a bioreactor facilitating mechanical stimulation) for up to 3 weeks. Changes in DNA content, glycosaminoglycan (GAG) amount, integrin subunits α5 and β1 fluorescence intensity, and morphologic appearance, were studied to evaluate tissue maturity. Seeded scaffolds subjected to the stirring chamber demonstrated significant increases in both DNA content (38.9) and GAG content (54.3) at day 21 compared to the control group. In addition, the stirring chamber system facilitated a maturation of cartilage tissue showed by histologic examination, after a staining of proteoglycan and type II collagen. Clinical feasibility of the fibroin and stirring chamber system was evaluated using rabbit models with cartilage defect. Large defects on rabbit knee joints were repaired with regenerated cartilage, which resembles hyaline cartilage at 12 weeks after operation. These studies demonstrated the potential of such mechanically stimulated scaffoldcell constructs to support chondrogenesis in vivo. [ABSTRACT FROM AUTHOR]

Abstract: 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 0.03N, the stroke length was 20mm, and the mean sliding velocity was 0.8mm/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. [Copyright &y& Elsevier]

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°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. [ABSTRACT FROM AUTHOR]

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 5×10[sup 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 tanδ, 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. [ABSTRACT FROM AUTHOR]

Abstract: 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-RGDS×2 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 ζ 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-RGDS×2 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 α5 and β1, 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. [ABSTRACT FROM AUTHOR]