Yusuke Kambe

Effects of RGDS sequence genetically interfused in the silk fibroin light chain protein on chondrocyte adhesion and cartilage synthesis.

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.

Recombinant production and film properties of full-length hornet silk proteins

Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications.

Adhesive force behavior of single ATDC5 cells in chondrogenic culture

Cellular mechanical properties are implicated in numerous cell behaviors, but their involvement in cell differentiation process has remained unclear. Since mechanical interactions between chondrogenic cells and their surrounding environment heavily affect the maintenance of their differentiation phenotype, here, using a chondrogenic cell strain ATDC5, we evaluated cell mechanical properties (e.g., adhesive force and spring constant) and gene expression levels in differentiation culture. The adhesive force appeared to be affected by both cellular cytoskeletal and adhesive constructions. Treatment with Y27632, which accordingly inhibits actin polymerization, decreased the adhesive force while increased chondrogenic gene expressions, suggesting the both of them are interrelated via the mediation of actin cytoskeleton. However, the mechanical property did not represent chondrogenic differentiative stages as obviously as the biochemical characteristics. Meanwhile, interestingly, changes in cell distribution maps of the force in the differentiation process indicated that the cells have different levels of mechanical properties in the undifferentiated state, whereas they tend to converge when the differentiative stage is in a lull. These results reaffirm the cellular diversity during differentiation from a mechanical perspective and provide important information to the fields of generation and scaffold-based tissue regeneration, where cell-substrate adhesion plays a role.

Silk fibroin sponges with cell growth-promoting activity induced by genetically fused basic fibroblast growth factor.

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.

Vascular induction and cell infiltration into peptide-modified bioactive silk fibroin hydrogels

In hydrogel-based soft tissue engineering, vascular induction into a hydrogel as well as long-term volume retention is essential to maintain tissue shape and function without causing necrosis in the deeper part of the hydrogel. A silk fibroin (SF) hydrogel shows a sufficiently high mechanical strength to maintain its shape during implantation for a month, but it has not been well evaluated whether it has vascular-inducing bioactivity to achieve its replacement by vascularized tissues. Here, we produced a vascular-inducing peptide (VIP) containing an endothelial cell (EC)-adhesive REDV and vascular endothelial growth factor-mimicking QK peptides to modify the SF hydrogel. In vitro experiments showed that the modification of the SF hydrogel with VIP changed only biological properties of the hydrogel due to the bioactivity of VIP. Subcutaneous implantation of SF hydrogels in rats revealed isotropic EC migration into the hydrogels, which was followed by infiltration of macrophages and fibroblasts. Since these macrophages and fibroblasts appeared to degrade the SF network and to produce collagen, respectively, SF hydrogels were replaced gradually by regenerated tissues. VIP accelerated cell infiltration and doubled the formation of blood vessels in the regenerated tissue. These results suggest the potential of the VIP-modified SF hydrogel as a material for soft tissue engineering applications.

Early tissue formation on whole-area osteochondral defect of rabbit patella by covering with fibroin sponge.

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.

Cardiac differentiation of induced pluripotent stem cells on elastin-like protein-based hydrogels presenting a single-cell adhesion sequence

AbstractSubstrate-dependent cardiac differentiation of induced pluripotent stem cells (iPSCs) has been studied on various extracellular matrix (ECM)-derived substrates, such as collagen type I (Col-I). However, ECM-derived substrates have multiple cell-adhesive amino acid sequences and stimulate various signaling pathways in cells, making it difficult to clarify the mechanism of substrate-dependent stem cell differentiation. A substrate presenting one of these sequences is a powerful tool for elucidating the mechanism. We designed elastin-like proteins (ELPs) composed of repetitive VPGIG sequences with or without the RGD cell adhesion motif (ELP-RGD/ELP-Ctrl) and used a chemical crosslinker to generate hydrogels. By adjusting the ELP and crosslinker concentrations, we obtained ELP-Ctrl and ELP-RGD hydrogels with a Young’s modulus of 0.3 kPa. The ELP-Ctrl and ELP-RGD gels were used as a substrate for the cardiac differentiation of cultured murine iPSCs. Cells on the ELP-RGD gel showed four times higher gene expression of the contractile protein troponin T type 2 than those on a Col-I gel, which is an effective substrate for iPSC cardiac differentiation. The ELP-RGD gel might stimulate integrin-derived signaling pathways in the cells to promote cardiac differentiation. This study showed the potential of ELP hydrogels for studying substrate-dependent iPSC cardiac differentiation by enabling the control of cell-adhesive sequence presentation.We designed elastin-like proteins (ELPs) composed of repetitive VPGIG sequences with or without the RGD cell adhesion motif (ELP-RGD/ELP-Ctrl) and used tetrakis(hydroxymethyl)phosphonium chloride (THPC) as a chemical crosslinker to generate hydrogels. The ELP-RGD and ELP-Ctrl gels were used as substrates for cardiac differentiation culture of murine induced pluripotent stem cells (iPSC). Cells on the ELP-RGD gel showed four times higher expression of the contractile protein gene, troponin T type 2 (TnT2), than those on a collagen type I gel, which is an effective substrate for iPSC cardiac differentiation.

Development of a FRET-based recombinant tension sensor to visualize cell–material interactions

A Förster/fluorescence resonance energy transfer (FRET)-based molecular tension sensor was originally reported by the fusion of intracellular molecules, which has contributed to the elucidation of cell mechanotransduction. However, it is still unclear whether recombinant tension sensors can detect forces in the extracellular environment. Here, we developed a recombinant FRET-based tension sensor (rFRET-TS) and immobilized it to a glass surface. Fibroblasts seeded onto the surface likely bound to an RGDS peptide fused to one terminal of the rFRET-TS, and intra-molecular FRET was dominantly observed on the sensor-immobilized surface compared to inter-molecular FRET. Time-lapse FRET imaging showed that the rFRET-TS enabled the real-time visualization of forces between cells and material surfaces that stemmed from focal adhesion formation and actin cytoskeletal reorganization. This sensor is expected to be useful for clarifying cell-scaffold mechanical interactions by its insertion between protein molecules of the scaffold, which will provide clues for the control of cell behavior in/on scaffolds.

How do chondrocytes aggregate on fibroin substrate

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.