Effect of epigallocatechin-3-o-gallate and quercetin on the cryopreservation of cartilage tissue.

The effects of epigallocatechin-3-o-gallate (EGCG) and quercetin on the contents of extracellular matrix (ECM) in porcine cartilage at 4 °C were investigated. The addition of quercetin at 0.01 mM for the incubation of porcine cartilage disks at 4 °C for 2 week could suppress the decrease in ECM and the compliance of the disks, markedly greater than those of EGCG (1.0 mM).

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. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1474-1482, 2016.

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.

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.

The antioxidant and non-antioxidant contributions of vitamin E in vitamin E blended ultra-high molecular weight polyethylene for total knee replacement.

Vitamin E (VE) blended ultra-high molecular weight polyethylene (UHMWPE) has been developed in Japan as a material for use in total knee replacement (TKR). Various results have demonstrated that VE blended UHMWPE reduces the incidence of delamination failure and lowers the amount of wear produced during knee simulator testing. It was also found that wear particles from VE blended UHMWPE elicited a reduced biological response compared to conventional UHMWPE. A great deal of research concerning vitamin E (VE) stabilized ultrahigh molecular weight polyethylene (UHMWPE) has focused on VE's effects as an antioxidant and its ability to prevent the oxidative degradation of UHMWPE chains. However, other chemical and mechanical changes have been observed in VE blended UHMWPE that are unrelated to the oxidative protection that VE provides. This paper provides a general review of VE blended UHMWPE, with a particular focus on the non-antioxidant effects of VE. The potential application of VE blended UHMWPE in total hip replacement (THR), along with the differences in loading conditions between the knee and the hip are also discussed.

Quantification of cell co-migration occurrences during cell aggregation on fibroin substrates.

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.

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.

Evaluation of the clinical performance of ultrahigh molecular weight polyethylene fiber cable using a dog osteosynthesis model.

The purpose of this study was to assess the removability and biological reactivity of an ultrahigh molecular weight polyethylene (UHMWPE) fiber cable as a new biomaterial for osteosynthesis. We used a pull-out test and an implantation test to analyze the performance of the UHMWPE fiber cable using a dog model, and compared its characteristics with those of a wire cable and a soft wire. In the pull-out test, the UHMWPE fiber cable was as easy to remove as the soft wire, and both the UHMWPE fiber cable and the soft wire were significantly easier to remove than the wire cable. The fixation capability and the biological reactivity of the UHMWPE fiber cable were examined in an osteosynthesis model of the dog greater trochanter, and were compared with those of the soft wire. The bone union rate, assessed radiographically, was very similar when using the UHMWPE fiber cable and the soft wire. However, in the soft wire group, both the surface of the greater trochanter under the fixation material and the penetration area around the fixation material showed an increased tendency toward a biological reaction, including inflammation and granulation tissue formation, as compared to the UHMWPE fiber cable group. The UHMWPE fiber cable was as easily removed from the bone tissue as the soft wire, and was easier to remove than the wire cable. Additionally, the UHMWPE fiber cable caused reduced biological reactivity with the surrounding tissue, as compared with the soft wire. In conclusion, the UHMWPE fiber cable appeared to be a suitable fixation material for osteosynthesis.

Helical conformation endows poly-l-lactic acid fibers with a piezoelectric charge under tensile stress.

Poly-l-lactic acid (PLLA) has been clinically used as a bioabsorbable material and attains a piezoelectric charge upon molecular orientation by the application of a shear force to the C-axis of the crystal line region. Previous studies showed that implanted drawn PLLA films or rods accelerate the ossification due to piezoelectric effect. In this study, we originally designed helically-twisted PLLA fiber to produce piezoelectricity in bioabsorbable suture upon tensile stress. The piezoelectricity of the helical PLLA fibers was evaluated using a lock-in amplifier system in vitro. The ossification induced by helical PLLA fibers was examined by implanting them in the rat patellar ligament supporting a physiological tensile load. We observed that 57° and 45° twisted PLLA fibers generated a higher piezoelectric potential than did 27° twisted fibers. The animal experiment showed that the formation of osseous tissue around helical PLLA fibers was more significant than around non-helical control fibers at 4 weeks after their implantation. These results suggest that helical PLLA fiber may be useful for the surgical suture or artificial ligament, which connects to the bone.

Quantitative evaluation of fibroblast migration on a silk fibroin surface and TGFBI gene expression.

Cell migration plays important roles in natural processes involving embryonic development, inflammation, wound healing, cancer metastasis and angiogenesis. Cell migration on various biomaterials is also believed to improve the rate of wound healing and implant therapies in the tissue-engineering field. This study measured the distance traversed, or mileage, of mouse fibroblasts on a silk fibroin surface. Fibroblasts on the fibroin surface moved with better progress during 24 h than cells on collagen or fibronectin surfaces. Results obtained by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) revealed that fibroblasts on the fibroin surface expressed transforming growth factor ß-induced protein (TGFBI), which is an extracellular matrix (ECM) protein, stronger than on other surfaces in the early cell-culture stages. These results demonstrate that the fibroin surface shows higher potential to enhance cell migration and the production of ECM than a collagen or fibronectin surface.

Observation and quantification of chondrocyte aggregation behavior on fibroin surfaces using Voronoi partition.

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.

Static magnetic field effects on impaired peripheral vasomotion in conscious rats.

We investigated the SMF effects on hemodynamics in the caudal artery-ligated rat as an in vivo ischemia model using noninvasive near-infrared spectroscopy (NIRS) combined with power spectral analysis by fast Fourier transform. Male Wistar rats in the growth stage (10 weeks old) were randomly assigned into four groups: (i) intact and nonoperated cage control (n = 20); (ii) ligated alone (n = 20); (iii) ligated and implanted with a nonmagnetized rod (sham magnet; n = 22); and (vi) ligated and implanted with a magnetized rod (n = 22). After caudal artery ligation, a magnetized or unmagnetized rod (maximum magnetic flux density of 160 mT) was implanted transcortically into the middle diaphysis of the fifth caudal vertebra. During the experimental period of 7 weeks, NIRS measurements were performed in 3- , 5- , and 7-week sessions and the vasomotion amplitude and frequency were analyzed by fast Fourier transform. Exposure for 3-7 weeks to the SMF significantly contracted the increased vasomotion amplitude in the ischemic area. These results suggest that SMF may have a regulatory effect on rhythmic vasomotion in the ischemic area by smoothing the vasomotion amplitude in the early stage of the wound healing process.

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.

A feasibility study for evaluation of mechanical properties of articular cartilage with a two-electrode electrical impedance method.

BACKGROUND: Since articular cartilage has important mechanical properties such as load-bearing, shock absorption and lubrication for activities in daily life, it is important to evaluate the mechanical properties of repaired cartilage in terms of whether those properties are the same as those of natural cartilage. The purpose of this study was to investigate the effectiveness of an electrical impedance method for quantitatively measuring the mechanical properties of cartilage. METHODS: Cartilage specimens were harvested from porcine knee joint, and two kinds of enzyme-treated cartilages were prepared to investigate the correlation between mechanical and electrical properties resulting from changes in the structure of the extracellular matrix. Collagenase solution and hyaluronidase solution were used to digest the collagen fibril and proteoglycan, respectively. Electrical impedance measurement, indentation test and biochemical analysis were carried out for the enzyme-treated cartilages. RESULTS: The water content increased with enzyme treatment time, and the permeability of the treated cartilages increased with decreasing glycosaminoglycan content for both types of enzyme-treated cartilages. The aggregate modulus and the electrical resistivity for both types of enzyme-treated cartilages decreased with increasing permeability after 12-h treatment. The aggregate modulus and the electrical resistivity for both types of treated cartilages decreased with increasing water content and permeability after 24-h treatment. The electrical resistivity and the aggregate modulus of articular cartilage depended not only on the water content, but also on the permeability, and the electrical resistivity for both types of enzyme-treated cartilages was found to be significantly linearly correlated with the aggregate modulus. CONCLUSIONS: These results showed that the aggregate modulus of articular cartilage can be estimated by measuring its electrical impedance.

Chondrocyte distribution and cartilage regeneration in silk fibroin sponge.

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.

Recovery Effects of a 180 mT Static Magnetic Field on Bone Mineral Density of Osteoporotic Lumbar Vertebrae in Ovariectomized Rats.

The effects of a moderate-intensity static magnetic field (SMF) on osteoporosis of the lumbar vertebrae were studied in ovariectomized rats. A small disc magnet (maximum magnetic flux density 180 mT) was implanted to the right side of spinous process of the third lumbar vertebra. Female rats in the growth stage (10 weeks old) were randomly divided into 4 groups: (i) ovariectomized and implanted with a disc magnet (SMF); (ii) ovariectomized and implanted with a nonmagnetized disc (sham); (iii) ovariectomized alone (OVX) and (vi) intact, nonoperated cage control (CTL). The blood serum 17-ß-estradiol (E(2)) concentrations were measured by radioimmunoassay, and the bone mineral density (BMD) values of the femurs and the lumbar vertebrae were assessed by dual energy X-ray absorptiometry. The E(2) concentrations were statistically significantly lower for all three operated groups than those of the CTL group at the 6th week. Although there was no statistical significant difference in the E(2) concentrations between the SMF-exposed and sham-exposed groups, the BMD values of the lumbar vertebrae proximal to the SMF-exposed area statistically significantly increased in the SMF-exposed group than in the sham-exposed group. These results suggest that the SMF increased the BMD values of osteoporotic lumbar vertebrae in the ovariectomized rats.

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.

Observation of chondrocyte aggregate formation and internal structure on micropatterned fibroin-coated surface.

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.

Effect of RGDS-expressing fibroin dose on initial adhesive force of a single chondrocyte.

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.

Strain-induced crystallization and orientation of vitamin E-blended ultrahigh molecular weight polyethylene.

The effects of vitamin E addition on the strain-induced crystallization and molecular chain orientation of ultrahigh molecular weight polyethylene (UHMWPE) were examined in order to clarify the wear mechanism of vitamin E-blended UHMWPE in knee prostheses. The structure changes of vitamin E-blended UHMWPE before and after tensile strain were analyzed by X-ray diffraction, Raman spectroscopy and image correlation method. The vitamin E-blended UHMWPE exhibited lower strain-induced crystallization than virgin UHMWPE but a higher Ic value in Raman spectroscopic analysis. The vitamin E-blended UHMWPE also exhibited a larger percentage of negative areal dilatation under tensile strain. These results suggest that the addition of vitamin E to UHMWPE decreases the strain-induced crystallization and increases the strain-induced orientation of the molecular chains present in the amorphous phase.