Time-dependent changes in adhesive force between chondrocytes and silk fibroin substrate.

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.

Evaluation of chondrocytes expression embedded in thermoresponsive poly(amino acid)s with sol-gel transition.

Poly(N-substituted alpha/beta-asparagine) was evaluated as a thermoresponsible and an injectable scaffold for cartilage regeneration. Solutions of this polymer are liquid state below 25 degrees C and nonfluid hydrogel above 35 degrees 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.

Frictional properties of regenerated cartilage in vitro.

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.

The anti-oxidative properties of alpha-tocopherol in gamma-irradiated UHMWPE with respect to fatigue and oxidation resistance.

Although addition of an antioxidant (alpha-tocopherol) is reported to prevent delamination in ultrahigh molecular weight polyethylene (UHMWPE) knee components, contribution of alpha-tocopherol as an antioxidant to the improvement of long-term fatigue performance of UHMWPE is an unknown mechanism. To solve this problem, bi-directional sliding fatigue tests were performed for gamma-irradiated (25 kGy), gamma-irradiated (25 kGy) with 0.3 wt% alpha-tocopherol added, and gamma-irradiated (25 kGy) with 0.3 wt% tocopheryl acetate added UHMWPE specimens. Internal defect initiation was quantified with scanning acoustic tomography (SAT). Also, oxidation index and crystallinity were obtained from infrared absorption spectra measured using Fourier transform infrared (FT-IR) microscopy. Only gamma-irradiated UHMWPE specimens resulted in severe fatigue fractures. alpha-Tocopherol-added UHMWPE specimens showed significantly lower projected area ratio of defects (1.80+/-0.82) than did gamma-irradiated (7.0+/-2.29) and tocopheryl acetate-added ones (8.50+/-2.01). The oxidation index of gamma-irradiated UHMWPE specimens (0.111+/-0.0052) was extremely higher compared to those of doped ones; 0.0179+/-0.0026 and 0.0144+/-0.0069 for alpha-tocopherol-added and tocopheryl acetate-added ones, respectively. The crystallinity of gamma-irradiated UHMWPE specimens (57.5+/-1.16) was lower compared to those of doped ones; 60.3+/-0.72 and 60.4+/-1.38 for alpha-tocopherol-added and tocopheryl acetate-added ones, respectively. The incorporation of alpha-tocopherol significantly improves the long-term fatigue performance of gamma-irradiated UHMWPE with oxidation stability. Also, the addition of alpha-tocopherol controls macromolecular structures resulting in the improvement of fatigue performance of UHMWPE.

Handling characteristics of poly(L-lactide-co-epsilon-caprolactone) monofilament suture.

A monofilament suture made of poly(L-lactide-co-epsilon-caprolactone) was examined by several mechanical tests to evaluate handling characteristics for tight tying. Six types of other monofilament sutures were also examined for comparisons. Two of these were nonabsorbable, while the others were absorbable sutures. Sutures consisting of glicolide were strongest among all the sutures examined. On the other hand, PROLENE and P(LA/CL) sutures showed high knot-pull strength despite low straight pull strength. Untying performance was evaluated by viscoelasticity, bending plasticity and tying test. A good correlation between tan delta and bending plasticity index was observed and the poly(L-lactide-co-epsilon-caprolactone) suture exhibited high tan delta, high bending plasticity and good resistance against untying.

Bio-artificial periosteum for severe open fracture--an experimental study of osteogenic cell/collagen sponge composite as a bio-artificial periosteum.

In an attempt to reduce complications in cases of severe open fracture, we developed a bio-artificial periosteum composed of osteogenic cells and collagen sponge. In the present study, we evaluated the osteogenic potential of the bio-artificial periosteum in vivo and in vitro. After 4-week incubation in vitro, the bio-artificial periosteum had high alkaline phosphatase activity and osteocalcin content. Moreover, energy dispersive X-ray analysis revealed numerous crystal structures consisting of P and Ca on the surface of the bio-artificial periosteum. Using a rat model for severe bone injury, we examined the bone formation process in defect sites covered with the bio-artificial periosteum. New bone formation occurred in the central part of the bone defect as well as at the bone edge. We conclude that by using the bio-artificial periosteum, the fracture site benefited from an improved osteogenic environment. These results indicate that a clinical trial to further evaluate this technique should be conducted.

Numerical simulations on fatigue destruction of ultra-high molecular weight polyethylene using discrete element analyses.

Ultra-high molecular weight polyethylene (UHMWPE) is a heterogeneous material composed of a networked substructure of grain boundary and grain aggregation. A new numerical model based on the discrete element method (DEM) was proposed to examine microscopic defect formation and propagation in UHMWPE. Numerical simulations were carried out using this model under two types of loading condition: unidirectional repetitive compression (simple loading) and bidirectional repetitive compression (switched loading). Subsurface defects were initiated and propagated in the vicinity of grain boundaries under both loading conditions. The defect propagation behavior was especially sensitive to grain boundary allocation under switched loading. An increase in defects was more rapid under switched loading than under simple loading. These numerical results showed qualitatively good agreement with experimental ones. It is suggested that the newly developed numerical method based on the DEM is a promising method to investigate fatigue behavior of a heterogeneous material such as UHMWPE under complicated loading conditions.

Defect initiation at subsurface grain boundary as a precursor of delamination in ultrahigh molecular weight polyethylene.

In order to examine the initiation mechanism of delamination in ultrahigh molecular weight polyethylene (UHMWPE) knee components, a bi-directional sliding fatigue test was performed for three types of UHMWPE specimens: nonirradiated, gamma-irradiated (25 kGy) and gamma-irradiated (25 kGy) with 0.1% vitamin E added. Sliding surfaces of post-tested UHMWPE specimens were observed using an optical microscope and a scanning electron microscope. Also, surface roughness was measured at the sliding surfaces of UHMWPE specimens. Delamination was observed only in gamma-irradiated specimens. A networked structure of surface asperity that resembled grain boundary was observed prior to delamination in gamma-irradiated specimens. Surface roughness in the gamma-irradiated specimens, higher than in any other specimen, showed a rapid increase prior to delamination. Detailed observation using an optical microscope and a scanning electron microscope showed microscopic crack initiation along subsurface grain boundaries in gamma-irradiated specimens. These results suggest that subsurface crack initiation is a precursor of delamination and is accelerated by oxidative degradation due to gamma irradiation. Of the three types of specimens, UHMWPE with vitamin E added showed the lowest surface roughness values at all measuring points. The addition of vitamin E is effective in improving wear resistance and fatigue performance of UHMWPE.

Gamma-irradiation aggravates stress concentration along subsurface grain boundary of ultra-high molecular weight polyethylene (UHMWPE) under sliding fatigue environment.

Numerical simulations were carried out using a DEM-based model under a sliding fatigue environment to investigate the influences of differences of mechanical properties between intra-granular portions and inter-granular portions on local stress-strain fields and to determine the effect of gamma-irradiative degradation of UHMWPE components on delamination. The predicted stress fields in the gamma-irradiated UHMWPE component were highly nonuniform and were quite different from those predicted in the non-irradiated UHMWPE component. The stress distributions were intensely affected by the structural inhomogeneousness of networked grain boundaries. Marked stress concentrations were observed along subsurface grain boundaries in the gamma-irradiated UHMWPE. The gamma-irradiated UHMWPE showed a steep increase in the maximum equivalent stresses for an increase in the number of sliding. As the number of sliding increased, moreover, the region where the maximum equivalent stress was observed, moved deep into the specimen. These findings suggest that fatigue damage with plastic deformation proceeds mainly at a subsurface region in the gamma-irradiated UHMWPE and at a superficial region in the non-irradiated one. Therefore, gamma-irradiation is an important factor in accelerating fatigue crack initiation resulting in delamination.

Microscopic destruction of ultra-high molecular weight polyethylene (UHMWPE) under uniaxial tension.

To examine the effects of a networked substructure of granular agglomerate on a style of destruction in prosthetic ultra-high molecular weight polyethylene (UHMWPE), uniaxial tensile simulations were carried out using the numerical model based on the discrete element method (DEM). The numerical simulations were performed taking the difference of mechanical characteristics between inter-granular and intra-granular portions of UHMWPE into consideration. A significant increase in stress and strain was observed along grain boundaries where micro cracks were initiated. This finding suggests that the large difference of mechanical properties between intra-granular and inter-granular portions causes significantly increased local stresses and strains in the vicinity of grain boundaries. The tensile simulation resulted in intra-granular destruction, which had good agreement with a result of previous experimental observation. This is presumably because the directions of principal shear stresses do not coincide with those at stress-concentrated grain boundaries. The dependence of the style of material destruction on forms of loading application could be explained by the relationship between directions of principal shear stress and reorientations of stress-concentrated grain boundaries.

Culture of chondrocytes in fibroin-hydrogel sponge.

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 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.

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.

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.

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.

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.

Reduction of wear volume from ultrahigh molecular weight polyethylene knee components by the addition of vitamin E.

Wear performance and debris-size distribution of vitamin E (DL-alpha tocopherol, VE)-added ultrahigh molecular weight polyethylene (UHMWPE) was evaluated using a knee-simulator test. VE was mixed with GUR 1050 UHMWPE powder at 0.3 wt%, and the tibial components of the knee joint were made by direct compression molding. The VE-added UHMWPE showed consistently lower wear volume throughout the test.