Material and methods

UHMWPE particles (10µm diameter in average, Mitsui chemicals Co., LTD) along with irradiated particles (RAD, 300kGy electron irradiation) and particles annealed after the irradiation (RAD+ANN, 100°C 72 hours) are co-incubated with mouse macrophage cell line RAW264 using the inverse culture method. The amount of TNF-α was measured with ELISA.

MATERIALS AND METHODS

We cultured mouse macrophage cell line RAW 264 with spherical UHMWPE particles (8.7µm and 23µm diameter in average, Mitsui chemicals Co., LTD.) and LDPE particles (3.6µm and 5.8µm diameter in average, Sumitomo Seika Chemicals Co., LTD.) using the Inverse Culture Method for 24 hours before estimating the TNF-α generation by TNF- ALPHA QUANTIKINE ELISA KIT (R&D). Spherical UHMWPE particles (10µm diameter in average, Mitsui chemicals Co., LTD.) with E.coli original LPS (Enzo Life Sciences) attached to them were incubated with cells to see the effects of LPS on the bio-reactivity tests.

MATERIALS & METHODS

Virgin samples were manufactured via Direct Compression Molding (DCM) technique from UHMWPE GUR1050 resin powder (Ticona, USA). For vitamin E (VE)-blended sample, the resin was mixed with VE at 0.3 wt% and the mixture was then molded using DCM. The crosslinked virgin samples were made by gamma ray irradiation to UHMWPE GUR1020 resin sheet (Meditech, USA) with doses of 95kGy ±10% and annealed. The VE-blended crosslinked samples were made by electron beam irradiation to VE-blended samples with doses of 300kGy and annealed. The material conditions were summarized in Figure 1. To pulverize the samples, the Multi-Beads Shocker (Yasui Kikai, Japan) was used.

After pulverization, samples were dispersed in an ethanol solution and sequentially filtered through polycarbonate filters. Over 100 sections of the filter were selected randomly and images of the particles were analyzed using scanning electron microscope (SEM).

To analyze the macrophage biological response, an inverted cell culturing process was used (2). The mouse macrophage-like cells were seeded at densities of 4×105cells per well in a 96-well culture plate and incubated for 1h. UHMWPE particles suspended in the culture medium were then added to each well in the appropriate amount. After that, fresh medium was added to fill the wells, and a sealing film was used to cover the culture plate. The culture plate was then inverted to cause the UHMWPE particles interact with the adhered macrophages. The inverted culture plate was incubated for 8h. The amount of TNF-α was measured by enzyme-linked immunosorbent assay (ELISA).

Material and methods

BMSCs seeded onto fibroin sponge scaffolds were cultured by using the stirring chamber system (Figure 1), which can apply a relative tribological movement to the surface of the specimens. Three culture conditions were applied (dynamic in stirring chamber as frequency as 40 min [D1], as 40 sec [D2] and static in stirring chamber group [S]). The specimens were set into stirrer on a poly(MPC) grafted surface (MPC polymer coated surface, SANSYO).

As a counter surface in friction tests, the poly(MPC) grafted surface was prepared by atom transfer radical polymerization, and the regenerated cartilage was prepared by seeding 5×10⁵ cells (BMSCs from rat bone marrow) onto fibroin sponge scaffolds (8 mm diameter and 1 mm thickness) and by 14 days culture.

Method

VE blended samples were manufactured via direct compression molding following the blending of UHMWPE resin powder (GUR1050, Ticona Inc.) with VE (dl-α-tocopherol, Eisai Co. Ltd.) at 0.3wt%. The virgin samples were derived similarly, but without the addition of VE. Both materials underwent crosslinking by irradiation via a 10MeV electron beam at 300kGy and were then heat treated at several temperatures (25, 80, 110, 130 and 150 °C) for 24 hours.

Gel content, which can be interpreted as cross-link density, was determined by measuring the weight of the samples before and after soaking in decahydronaphthalene at 150 °C for twelve days.

Tensile tests were carried out following JIS K 7113, with the cross head speed set at 50 mm/min.

Crystallinity was determined by using DSC and integrating over the enthalpy curve from 80 to 150 °C and normalizing with the enthalpy of melting for 100% crystalline polyethylene.

Materials & Methods

UHMWPE resin powder (GUR 1050, Ticona, USA) was mixed with dl-a-Tocopherol (vitamin E) at 0.3 wt% and molded under direct compression at 25 MPa and 220°C. Virgin samples were produced by the same process, but without the addition of vitamin E (VE). Cylindrical pins (length: 40 mm, diameter: 3.5 mm) were then machined from these samples, packaged in a vacuum, and irradiated by electron-beam at 300 kGy. Samples were subsequently doped with either ascorbic acid 6-palmitate (Sigma, Japan) or ethanol (Ethanol 99.5%, Kishida, Japan) and subjected to a hydrostatic pressure of 100 MPa for 7, 14, and 21 days at room temperature. Radical measurements were made using ESR at 9.44 GHz and room temperature. All ESR spectra were recorded at 0.1 mW microwave power and 0.1 mT modulation amplitude.

Materials and Methods

VE blended samples (GUR_VE xx%) were manufactured via direct compression molding following the blending of UHMWPE resin powder with VE at several concentrations (0, 0.1, 0.3, 1.0%). Cross-linking for the VE samples was achieved by 10 MeV electron beam at several irradiance doses (30, 90, 300 kGy) and annealed below the melting point of UHMWPE.

Knee and hip simulator testing were carried out according to ISO 14243 and ISO 14242, respectively, and the volumetric wear was calculated. The gel fraction was determined by measuring the weight of the samples before and after soaking in decahydronaphthalene at 150°C. The oxidative resistance of the material was determined by measuring the Oxidation Index (OI) following ASTM F2102 before and after compulsory aging (ASTM2003). Radical measurements were made using high-sensitivity X-band ESR.

METHODS

Articular cartilage specimens were taken from porcine femoral condyle and cut into 5 mm diameter plugs. Their surfaces were wiped with particular papers soaked in saline solution. Thereafter, these specimens were preserved with 1 mL volume of HA and saline solution for 0, 3, 6, 9 hours. The concentration of HA was 1% (w/v) in saline solution (MW=9×10⁵ Daltons; Seikagaku corp., Tokyo, Japan). Friction test was carried out in saline solution under a constant pressure of 1.5 Mpa and a relative sliding velocity of 0.8 mm/s, with MPC-polymer grafted glass as counter surface. Besides, superficial layer of cartilage tissue was histologically observed by two kinds of staining method: Toluidine blue (pH7.0) staining and Toluidine blue (pH2.5) staining Then, the Toluidine blue (pH7.0) staining intensity on superficial tissue was quantitatively analyzed. As follows, images of the stained cartilage specimens were analyzed by ImageJ. Measure RGB program was used to average out luminance values of blue in 2.7 μm square area of superficial layer and middle layer. The ration of the mean value in superficial layer and it in middle layer was defined as Toluidine blue (pH7.0) Index.

EXPERIMENTAL METHODS

Three kinds of culture dishes coated with three diffrent fibroin aqueous solutions were prepared: 1 wild-type, 2 transgenic and 3 mixed. The wild-type aqueous solution was prepared from Bombyx mori silkworm cocoons. The transgenic aqueous solution was prepared from Bombyx mori silkworm cocoons in which RGD was interfused in the fibroin light chain³⁾. The mixed aqueous solution was prepared simply by blending RGD peptides with the wild-type fibroin aqueous solution. Chondrocytes were asepically harvested from the joints of 4-week-old Japanese white rabbits and then subcultured on T-flasks and seeded at 2.0 × 10⁵ cells/dish. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm² and a frequency of 1.71 MHz with a 200 ms tone burst repeated at 1.0 kHz, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. GAG production and the number of chondrocytes were measured by the Dimethylmethylene blue (DMMB) method⁴⁾ and the LDH method⁵⁾, respectively. Extracted mRNA from the chondrocytes was analyzed by using the Syber Green method, where the primers were designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the house-keeping gene, aggrecan and Sox 9. This data was analyzed using the two-sided Student's t-test.

Method

The pin-on-plate reciprocating test was performed using PAO6 (30.51cP at 37°C) as the lubricant. The pin and plate specimens were made of SUS440C and were polished to a surface roughness of 0.02μm Ra. The pin specimens were columnar in shape, and radial periodic grooved structures (700nm spacing x 200nm amplitude) were formed on the pin's outer periphery (from 4mm to 5mm in diameter).

The ring-on-disk test was performed using lubricants with different viscosity: PAO6 and PAO2 (4.60cP at 37°C). The ring-on-disk specimens were made of SUS440C and were polished to a surface roughness of 0.03μm Ra. Along the surface of the ring specimens, material was removed to create 4 elevated sections at 0°, 90°, 180° and 270°. These 4 sections were then polished and concentric grooved structures (700nm spacing x 200nm amplitude) were created along a 1.4mm circumferential path within each of these areas.

MATERIALS & METHODS

Both pure UHMWPE and Vitamin E added (0.3% w/w) resin was used to produce bulk specimens via vacuum direct compression molding at 220°C under 25 MPa for 30 min. Cylindrical pins (3.5 mm diameter, 40 mm length) for ESR measurement were then machined and placed in vacuum packaging. The pins were irradiated at 300 kGy, with half of each test group annealed at 80°C for 24 hours. Free radical measurements were made using a high-sensitive X-band ESR operating at 9.44 GHz. Detection of Vitamin E radicals was performed by comparing the characteristic symmetrical spectrum of oxidized Vitamin E to the spectra observed for the pins using both g-value and linewidth as references. Crosslink density was measured via gel fraction analysis and was performed in accordance with ASTM D2765. Thin sections (20 × 40 mm², 200 μm) were machined from the bulk specimens, which were then placed in vacuum packaging, irradiated and annealed at the same conditions as those for the ESR measurements. Two of these thin sections were then placed in a stainless-steel cage (200 µm pore diameter) and were immersed in decahydronaphtalene at 200°C for 24 hours. These specimens were then extracted using soxhlet extractor at 100°C for 24 hours and dried in vacuum at 150°C for 12 hours.