Study On Clinically Relevant Micro-Metre Sized Uhmwpe Wear Particles Loaded With Alendronate Sodium

Although total joint replacement is one of the most successful surgeries in clinic, wear particles of artifical joint still lead to the periprosthetic osteolysis and aseptic loosening, which are the major reasons for the late failure of artifical joint. Among the wear particels of artifical joint, ultra high molecular weight polyethylene (UHMWPE) wear particles account for 70%～90% of the total wear particles and result in the most significant biological response due to its lower wear-resistance in the prosthetic joint components, and it is the major cause of aseptic loosening resulting from osteolysis around artifical joint. Therefore, it is essential to study the effect of UHMWPE wear particles on aseptic loosening.In our previous studies, UHMWPE was loaded with alendronate sodium (ALN) which were anti-osteolysis drugs in clinic. The influence of UHMWP-ALN and UHMWPE wear particles on macrophages were evaluated by co-cultured. However, UHMWPE-ALN wear particles less than 40 μm were used in our previous cell studies because it was difficult to separate UHMWPE-ALN wear particles due to the electrostatic interaction and wear particles shape. Clinical studies showed that the majority size of wear particles is in 0.1-10 μm, which are the most biologically reactive. In this study, the preparation and characterization, in vitro ALN release and cell evaluation of clinically relevant micro-metre sized UHMWPE-ALN wear particles were studied. In addition, UHMWPE-ALN wear particles and macrophages were co-cultured by inverted and normal culture models by different researchers in our previous studies, which was difficult to judge the cell response by the different cell culture models. In the present study, the effects of the inverted and normal culture models to macrophages were studied simultaneously. Furthermore, the response of macrophage to the compound of UHMWPE-ALN or UHMWPE wear particles and Co-Cr particles were also studied.The UHMWPE-ALN and UHMWPE were prepared by the solvent evaporation and hot-pressing. The UHMWPE-ALN and UHMWPE wear particles were produced by the light-duty bench grinder and then were filtrated by membrane gradually under subnormal ambient. UHMWPE-ALN and UHMWPE wear particles were characterized by laser diffraction wear particle analyzer, scanning electron microscopy(SEM), fourier transform infrared spectroscopy (FT-IR), X ray diffraction (XRD), and differential scanning calorimetry (DSC) analysis. In vitro release of ALN from UHMWPE-ALN wear particles with the clinically relevant micro-metre sized were studied by immersed in phosphate buffer saline (PBS) and measured by ninhydrin colorimetric method. The influence of UHMWPE-ALN wear particles, UHMWPE wear particles, and the compound of UHMWPE-ALN or UHMWPE wear particles with Co-Cr particles on macrophages were evaluated by inverted and normal culture models, respectively. The morphology of macrophages was observed by optical microscope and SEM. The proliferation of macrophages were investigated by methyl thiazolyl tetrazolium(MTT) assay. The injury degree of macrophages was evaluated by the release of lactate dehydrogenase (LDH). The expressions of pro-osteolytic cytokines (interleukin-6, IL-6 and tumor necrosis factor-a, TNF-a) were investigated by enzyme-linked immunosorbnent assay (ELISA).The results of laser scattering particle size analyzer and SEM showed that the average particle size of UHMWPE-ALN and UHMWPE wear particles which were filtrated by membrane gradually under subnormal ambient were 6.98 and 5.65 μm. The particle size distribution was concentrated,90% of which range from 0.1 to 10 μm. The morphology of most wear particles were in block or flake. And they were similar with the size and morphology of UHMWPE wear particles in clinic. The above data indicated that clinically relevant micro-metre sized wear particles were obtained. The results of FTIR showed that the addition of ALN didn’t change the chemical structure of UHMWPE. Similarly, the preparation and separation process of wear particles didn’t change the chemical structure of UHMWPE and ALN. Oxidation reaction happened on UHMWPE and ALN were stable during the hot-pressing procedure. XRD and DSC results demonstrated that the hot-pressing procedure, the preparation and separation process of wear particles and the addition of ALN had no influence on crystal structure and crystallite dimension of UHMWPE, but the crystallinity of sheet groups was decreased than that of wear particles and powder groups.The profile of in vitro ALN release from UHMWPE-ALN wear showed two stages: the initial burst release (the first 24 h) and the latter slow release (1 to 90 d). The release of enriched ALN on the surface of wear particles played an important part during the burst release stage, and the particle size of wear particles had a great influence on the drug release rate. While the dispersion of aggregated wear particles and the diffusion of ALN in UHMWPE-ALN wear particles were the main causes of ALN release during the second stage, which conformed to the Higchi drug diffusion model. There was no oxidation phenomenon of UHMWPE-ALN wear particles after immersing in PBS at 37℃ for 90 d.Cell experiment results showed that macrophages had phagocytosis and chemotaxis effect to the clinically relevant micro-metre sized wear particles. The morphology of macrophages changed due to the addition of wear particles. The MTT values of macrophages co-cultured with UHMWPE-ALN wear particles were both lower than those of UHMWPE wear particles by inverted and normal culture models, the results indicated that ALN could inhibit the activity of macrophages. The MTT values of macrophages were the highest when they were co-cultured with UHMWPE wear particles in a concentration of 0.02 mg/mL. There was no significant difference in the activity of macrophages by inverted and normal culture models. However, ALN obviously inhibited the activity of macrophages by the inverted culture model. LDH results showed that the damage of the macrophages by the inverted culture model was bigger than that by the normal culture model. The results of ELISA indicated that clinically relevant micro-metre sized UHMWPE-ALN and UHMWPE wear particles could promote the TNF-α and IL-6 release of macrophages. The secretion of TNF-α and IL-6 were not simultaneous, and the expression of TNF-α were higher than that of IL-6. After co-culturing with the compound of UHMWPE-ALN or UHMWPE wear particles and Co-Cr particles, the proliferation of macrophages were promoted by UHMWPE wear particles, while inhibited by Co-Cr particles. The promotion effect of UHMWPE wear particles on the proliferation of macrophages was no influence because of the addition of Co-Cr particles.The present study provided a potential method for generating clinically relevant micro-metre sized UHMWPE-ALN wear particles, and provided a basis for applications in clinics.