Aseptic loosening is a major cause of revision of total joint arthroplasty (TJA). Although crosslinked Ultra-high molecular weight polyethylene (UHMWPE) have improved wear resistance, residual radicals remaining in the material have a possibility to increase bio-reactivity of particles [2]. In this study, we attempt to evaluate the effects of irradiation and residual radicals on bio-reactivity of the material with a new method called the inverse culture method [1]. 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.Introduction
Material and methods
Loosening is concerned to be the major cause of revision in the artificial prosthesis. Wear debris of UHMWPE dispersed into the implant-bone interface are phagocytosed by macrophages releasing inflammatory cytokines such as TNF-α which leads to osteolysis and loosening eventually. It is known that the size and structure [1] as well as attached substances on particle surface such as endotoxin could affect the amount of cytokines released [2]. An 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.INTRODUCTION
MATERIALS AND METHODS
Ultra-High Molecular Weight Polyethylene (UHMWPE) wear debris is thought to be a main factor in the development of osteolysis (1). However, the method for the evaluation of the biological response to UHMWPE particles has not yet been standardized. In this study, four different types of UHMWPE particles were generated using a mechanized pulverizing method and the biological responses of macrophages to the particles were investigated using an inverted cell culturing process (2). 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).INTRODUCTION
MATERIALS & METHODS
It is essential to investigate the tribological maturation of tissue-engineered cartilage that is to be used in medical applications. The frictional performances of tissue engineered cartilage have been measured using flat counter surfaces such as stainless steel, glass or ceramics. However, the measured friction performances were significantly inferior to those of natural cartilage, likely because of cartilage adhesion to the counter surface. Tamura et al. reported that a poly (2- methacryloyloxyethyl phosphoryl-choline (MPC)) grafted surface shows low friction coefficient against cartilage without the adhesion to be equivalent to those for natural cartilage-on-cartilage friction. [1] On the other hand, Yamamoto et al. reported that applying a relative sliding movement had a potential to alter the expression of tribological function of regenerated cartilage of chondrocytes. [2] In this paper, the effects of the relative sliding movement on the expression of bone marrow stromal cells (BMSC)s were investigated using the poly(MPC) grafted surface as a counter surface. 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.Introduction
Material and methods
Dl-α-Tocopherol (VE)-blended non-crosslinked UHMWPE has been developed as a bearing surface material for knee prostheses due to the radical scavenging capabilities of vitamin E and has demonstrated a low wear rate in knee simulator testing [1,2]. In previous our study, VE-blended, crosslinked UHMWPE has demonstrated a low wear rate in hip simulator testing [3, 4]. As the radical scavenging capabilities also reduce the crosslinking degree of the material, multiple dose crosslinking has been investigated. However, these crosslinked UHMWPE materials may have different mechanical properties, as each crosslinking process, especially the annealing condition, is different. Additionally, there is little information about VE-blended, crosslinked UHMWPE with different annealing conditions. In this study, the effect of annealing temperature was investigated with regard to tensile strength, crosslink density, and crystallinity of VE blended, crosslinked UHMWPE. 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.Introduction
Method
UHMWPE resin powder (GUR 1050, Ticona, USA) was mixed with Background
Materials & Methods
Vitamin-E (VE)-blended UHMWPE has been developed as a bearing-surface material due to the antioxidant ability of VE and has demonstrated a low wear rate in knee simulator [1]. Additionally, in vitro biological response testing has revealed that wear particles from VE blended UHMWPE induce the secretion of inflammatory cytokines at significantly lower levels compared to conventional UHMWPE [2]. However, as the joint kinematics are different between the knee and the hip, it is not guaranteed that these improvements will be repeated in the hip. In this study, the wear resistance of VE-blended UHMWPE was evaluated in knee and hip simulator tests and the effects of VE concentration and electron-beam irradiation were investigated. 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.Introduction
Materials and Methods
Tamura et al. proposed a new friction test to measure the maturity of surface gel-hydration-like lubrication using MPC-polymer (2-Methacryloyloxyethyl phos -phorylcholine polymer) grafted surface as aãζζcounter surface. They suggested that the MPC-polymer grafted surface makes it possible to mimic in-vivo-like condition. Therefore, we can evaluate a lubricating ability of cartilage surface except for the possible effects of deformation resistance. By the way, reduction of lubricating ability of articular cartilage surface has much to do with pathogenesis of primary osteoarthritis. On the other hand, intraarticular injections of hyaluronic acid (HA) has been reported to have some clinical effect, however, it has not been clearly supported that HA restores a lubricating ability of injured cartilage surface. In the present study, the short-term effect of HA on injured cartilage surface's frictional performance was examined by the friction test using MPC-polymer grafted surface. 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.INTRODUCTION
METHODS
Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis1). Recently it has been suggested that LIPUS may be transmitted via Integrin: a protein which mediates cellular attachment between cells and extracellular matrix2). In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chndrocyte response to LIPUS was evaluated. 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 INTRODUCTION
EXPERIMENTAL METHODS
The tribological performance of grooved surfaces has been thoroughly analyzed, and such surfaces are thought to have great potential for hard-on-hard joint prosthesis. In related research, femtosecond laser-induced periodic surface structures (FLIPSSs) have been well developed to achieve grooved structures with submicron spacing (700nm) and amplitude (200nm). In this study, submicron-scale periodic grooved structures were made on SUS440C using a femtosecond laser, and its tribological performance was evaluated by both a pin-on-plate reciprocating sliding test and a ring-on-disk test. 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.Introduction
Method
Electron-beam-irradiated 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.INTRODUCTION
MATERIALS & METHODS
It have been reported that the wear volume of vitamin E-containing UHMWPE tested with a knee joint simulator was approximately 30% lower than that of virgin UHMWPE at 5 million cycles. However, the wear resistance mechanism of vitamin E-containing UHMWPE has not yet been clarified. The present study examines the effects of the addition of vitamin E on the frictional properties of ultra-high molecular weight polyethylene (UHMWPE) under several different load and serum conditions. Friction tests were carried out using a computer-controlled pin-on-disk friction test apparatus. The UHMWPE pin was mounted vertically at the tip of the leaf spring and linear reciprocating sliding motion for 2,000 cycles with an amplitude of 1 mm and a frequency of 1 Hz, was applied under 3 MPa or 30 MPa loading against Co-28Cr-6Mo alloy disk. The lubricant bath was filled with 5 ml of ultrapure water, fresh serum, post-friction (PF) serum or diluted-PF (DPF) which were kept at a temperature of 37°C. The friction force between the UHMWPE pin and the Co-28Cr-6Mo alloy disk was calculated from the displacement of the leaf spring during the sliding motion. Vitamin E-containing UHMWPE showed a significantly higher friction force than that of virgin UHMWPE in fresh serum lubricant at 30 MPa loading, while there were little differences in either ultrapure water or PF serum or DPF serum. And vitamin E-containing UHMWPE tends to exhibit a lower dynamic friction force within the first few hundred cycles in the case of all serum lubricants at 30 MPa loading. These results suggest that some interaction between the UHMWPE surface and the native conformation proteins was specifically affected by the addition of vitamin E and that some weeping of vitamin E might occur at early stage of sliding. Our results also suggest the importance of the conformational changes of serum proteins for the wear testing.
Discussion: The biological response to wear particles is strongly influenced by particle size and volume [
Physical environments play important roles for maturation of mechanical functions of tissue. In this study, effects of relative tribological movement on the expression of tribological function of regenerated synovial membrane were investigated. Fibroin sponge derived from silk was used as a three-dimension scaffold for the synovial membrane regeneration. Synovial cells were isolated from human synovial membrane, and were seeded onto the fibroin sponge. Magnetic stirring system (named Stirring Chamber) was used for culturing with relative slip motion where the cell-seeded side of the scaffold had been rubbed by a glass culture dish for 24 hours/day. Histological view of regenerated tissue of the dynamically cultured group (D group) showed extracellular-matrix-like eosinophilic meshwork structure formed continuously on the meshwork structure of the fibroin sponge. The newly formed tissue showed expression of collagen type I, especially on the surface of fibroin sponge. These structures were not seen in the statically incubated group (S group). Each group didn’t show expression of collagen type II. Frictional force was measured by using leaf spring method under the conditions of the sliding velocity: 0.8 mm/s, the loading time prior to sliding: 1 minute, and the applied load during the experiment: 0.029 N. The counterface for regenerated synovium was a flat stainless steel of which roughness was 0.06 μm Ra. All frictional experiments were performed in the saline solution and at room temperature (25°C). The friction coefficient of tissues cultured statically was 0.6–0.8, and that of tissues cultured with sliding motion was 0.2–0.4 at one week culturing, 0.3–0.5 at two weeks culturing. Our previous experiment showed that combination of fibroin-sponge scaffold and Stirring-chamber culturing system improved the tribological performance of regenerated cartilage tissue. The present study suggests that this combination have also a possibility for synovial cells to form functional lubricious membrane which can be used as anti-adhesion membrane for knee, ligament, and/or other surgical procedures. However, the deterioration of lubrication properties in the 2 weeks dynamically cultured group would indicate that the too long continuous tribological movement does not provide an optimal condition. More fine tribological loading history should be designed.
These results suggest that mechanism for the prevention of delamination by vitamin-E-addition is caused by increased elongation at break by increasing toughness at grain boundary. The addition of vitamin E is a simple and extremely effective method to prevent destruction of UHMWPE joint component.
