The particles around the artificial joint prosthesis can cause the recruitment and phagocytosis of macrophages, and the activation of macrophages after phagocytosis of particles occurs, releasing a series of cytokines and forming inflammatory granulomas, which eventually lead to osteolysis and loosening. A variety of particles exist around the prosthesis, including UHMWPE particles, titanium alloy particles and bone cement particles, etc. Clinical histological observations have revealed that the strength of osteolysis induced by particles of different materials varies, with the strongest effect of UHMWPE particles. The exact mechanism of this difference in local biological response due to different particle materials is still unknown and there is a lack of relevant experimental studies. Macrophage phagocytosis is the initial step in the periprosthetic osteolysis response, and the stronger the phagocytosis, the higher the activation, and the stronger the inflammatory and osteolysis response. Accordingly, the authors hypothesized that the difference in the degree of particle-induced osteolysis was related to the difference in the degree of macrophage phagocytosis induced by particles. In this experiment, we hypothesized that different particles induced different macrophage phagocytosis responses, and compared the differences in macrophage phagocytosis responses induced by two types of particles, UHMWPE and Ti-6AL-4V, to explore the reasons for the differences in biological responses induced by particles of different materials at the cellular level. Particles and cells 1. Particles: UHMWPE particles were kindly given by Professor Wooley H. of John D. Dingle VA Medical Center, USA. The particles are irregular in morphology with an average diameter of 2.6 μm (diameter range 0.8-23 μm,). Ti-6AL-4V particles were made by the orthopedic laboratory of the Ninth People’s Hospital of Shanghai Second Medical University, Patent No. 03142073.7. The process of making Ti-6AL-4V material was made by loading a mill block made of Ti-6AL-4V material into a mill jar of the same material, adding 70 mL of DMEM, placed on a shaker and shaken for 21 days, and the particles within the DMEM were extracted by gradient centrifugation. The produced Ti-6AL-4V pellets were morphologically variable with an average diameter of 3.9 μm (diameter range 0.6-31 μm,). To remove the endotoxin, the pellets were suspended in 75% ethanol, shaken and washed four times at room temperature for 1 h each, and soaked in 100% ethanol overnight. The endotoxin content was determined to be less than 0.25 EU/mL. The treated pellets were suspended and immersed in culture medium. 2. RAW 264.7 cells: RAW 264.7 cells (purchased from the Institute of Cell Research, Chinese Academy of Sciences, Shanghai, China) belong to the mouse monocyte/macrophage line, which has the dual characteristics of macrophage and osteoclast precursor cells, and were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃ and 5% CO2. The experiments were divided into 2 groups according to the different pellets, and UHMWPE pellets and Ti-6AL-4V pellets were added to the culture medium and the concentration was adjusted to 0.1 mg/mL, so that RAW 264.7 cells were cultured under pellet-stimulated conditions. Comparative observation of phagocytosis of RAW 264.7 cells on two kinds of particles 1. Determination of optimal cellular response density: This experiment used the MTT method to find the optimal cellular response density to particles. Macrophages (RAW 264.7 cells) were co-cultured with UHMWPE particles and Ti-6AL-4V particles at densities of 1×103/mL, 1×104/mL, 1×105/mL and 1×106/mL, respectively, and MTT cell viability assay was performed after 72h. The results showed that RAW264.7 cells at a density of 1×105/mL were most sensitive to stimulation with 0.1 mg/mL concentration of particles. 2. Observation of particle phagocytosis: RAW 264.7 cells with a density of 1×105/mL were co-cultured with 0.1mg/mL of UHMWPE particles and Ti-6AL-4V particles, respectively, and cell phagocytosis of particles was observed under light microscope at the 1st, 4th, 12th, 48th and 72h after culture. And the ratio of cells containing phagocytosed granules to the total number of cells in each field of view was counted under 100x light microscope field of view at the 1st, 4th, 48th and 72th h. The statistical analysis was also repeated three times. Statistical treatment The chi-square test was performed using SPSS10.0 statistical software, and р < 0.05 was considered statistically significant. Results Macrophages phagocytosed UHMWPE particles UHMWPE particles stimulated macrophages to produce a rapid and strong phagocytic response. only 1 h after the contact of RAW 264.7 cells with UHMWPE particles, some cells started to phagocytose the particles. As the incubation time increased, more and more cells phagocytosed the particles, and by 48 h, 96.4% of the cells contained phagocytosed particles. 72 h later the cells that phagocytosed the particles reached saturation, and the proportion of cells containing phagocytosed particles no longer increased. At the same time, it was observed that the cells did not proliferate after phagocytosis, but showed apoptotic phenomena such as cell body enlargement, fried egg-like shape and cell shedding. When the medium was changed to normal medium, the cells containing the phagocytosed particles continued to apoptosis and fell off, while the remaining small amount of cells without phagocytosed particles started to proliferate until the whole culture dish was covered. Macrophage phagocytosis of Ti-6AL-4V particles Compared with UHMWPE particles, Ti-6AL-4V particles caused macrophage phagocytosis to appear later and the proportion of cells that phagocytosed particles was relatively small, a phenomenon that was more pronounced early in culture. At 1h after co-culture of RAW 264.7 cells with Ti-6AL-4V particles, few cells phagocytosed the particles. At 4h after culture, there was a small amount of cellular phagocytosis of the pellet. As the culture time increased, the number of cells engulfing the pellet gradually increased, but the proportion was significantly less compared with the UHMWPE pellet group in the same time period. By 72h, more than 70% of the cells contained phagocytic granules. The morphological changes of cells that phagocytosed granules were small, and rarely showed shedding of apoptosis. Discussion UHMWPE is the main particle causing osteolysis A variety of wear particles exist around the artificial joint prosthesis, such as UHMWPE particles, titanium particles, bone cement particles, pure titanium particles and cobalt-chromium-molybdenum particles, which can cause a biological reaction around the prosthesis and undergo a series of pathological changes, eventually leading to osteolysis and loosening. Clinical histological observation shows that UHMWPE particles are the most common and numerous around the prosthesis, and are more closely related to the biological reaction around the prosthesis than other particles, and are the most important particles causing osteolysis and loosening: when the UHMWPE particles in the boundary membrane exceed 1×1010/g, osteolysis and loosening will almost certainly occur, and the service life of the prosthesis will be significantly shortened. The reason why UHMWPE particles are more likely to cause osteolysis than other particles has not been fully elucidated. The main mechanism by which particles cause osteolysis and loosening is by stimulating the biological response of the cells surrounding the prosthesis, resulting in an altered local biochemical environment and imbalance in bone metabolism. Macrophages are the main defense-responsive cells in the body and have the most direct and close relationship with granules. According to histological observation, only a small amount of granules and macrophages existed in the boundary membrane around the well-fixed prosthesis, while the boundary membrane of the loosened prosthesis with local thickening contained a large amount of granules, and at the same time a large number of macrophages were recruited to engulf the granules, and the number of macrophages was proportional to the number and volume of granules, indicating that granules can cause macrophages to recruit around the prosthesis and be engulfed by them. The results of this experiment also showed that macrophages began to phagocytose the UHMWPE particles after only 1 h of contact with them, and the phagocytosis increased significantly with time, which verified that macrophages do have the ability to phagocytose the particles. After phagocytosis of particles, macrophages were activated and underwent a series of reactions, which caused changes in the surrounding biochemical environment as well as changes in their own traits. First, after phagocytosis, macrophages secrete a variety of biochemical mediators and cytokines, including TNF-α, IL-1, IL-6, and MMP, which cause inflammatory granuloma and bone resorption around the prosthesis. Secondly, macrophages are the precursor cells of osteoclasts, and in the case of granule stimulation, the number of differentiated into osteoclasts increases and the bone resorption capacity is enhanced, causing local bone metabolic imbalance and periprosthetic osteolysis. Experiments have proved that the above biological changes of macrophages must occur only after contact with or phagocytosis of the particles, and if the contact between particles and cells is prevented, the above changes will not occur. Comparison of phagocytosis response between UHMWPE particles and titanium particles This experimental study showed that UHMWPE particles could cause rapid and strong phagocytosis response of macrophages compared with Ti-6AL-4V particles.UHMWPE particles were phagocytosed immediately after contact with macrophages, and the phagocytosis phenomenon became more and more obvious with the extension of time, and the proportion of cells containing particles increased rapidly, and by 72h it had reached the saturation state of phagocytosis. In contrast, in the Ti-6AL-4V particle group, the phagocytosis phenomenon appeared later, and the proportion of cells containing phagocytosed particles was significantly less than that in the UHMWPE particle group during the same time period. Previous studies have suggested that the main reason why UHMWPE particles are the main factor contributing to the occurrence of osteolysis is that they are numerous, far exceeding the sum of the number of other particles, and therefore cause a stronger biological response. And the results of the present experiment showed that UHMWPE particles stimulated phagocytosis of macrophages more strongly and rapidly than titanium alloy particles even at the same particle density. Since macrophages need to phagocytose the particles before the biological reaction occurs, it can be speculated that UHMWPE particles themselves also induce stronger phagocytosis and biological reaction of macrophages than other particles, producing more inflammatory mediators and cytokines, which explains from another perspective that UHMWPE particles are more likely to induce osteolysis than other particles. In conclusion, UHMWPE particles induce faster and more intense macrophage phagocytosis than Ti-6AL-4V particles, and this leads to a series of biochemical reactions, which ultimately lead to stronger periprosthetic osteolysis and loosening.