Study On Biological Response To Wear Particles Of Ultra High Molecular Weight Polyethylene Loaded With Alendronate Sodium

Ultra high molecular weight polyethylene(UHMWPE)has been successfully used as one of the acetabular components of joint prosthesis due to its good wear resistance and excellent biocompatibility.Nevertheless,wear particles of UHMWPE are inevitable during service,and particles ≤ 10 μm could cause the strongest biological response.That is believed to be the principal factor leading to osteolysis and aseptic loosening of joint prosthesis.Therefore,it is necessary to find a way to reduce the damage caused by UHMWPE wear particles,thereby prevent wear particle-induced osteolysis and delaying prosthesis failure.The thesis developed UHMWPE loaded with alendronate(ALN),a drug used to treat osteolysis,and prepared a large number of clinically relevant micro-sized(≤ 10 μm)UHMWPE-ALN wear particles in vitro.The release behavior of ALN from UHMWPE-ALN wear particles was investigated.Inverted culture technique was adopted to evaluate the biological response of macrophages to the prepared wear particles.Then,alginate sodium(SA)beads were used to encapsulate cells and wear particles,serving as cell reactors to simulate the three-dimensional environment in vivo while ensuring that the wear particles could effective contact with cells.The novel long-term co-culture system could evaluate the cell response to clinically relevant micro-sized UHMWPE-ALN wear particles,and explore the mechanism of ALN-cell intactions.An air pouch model was adopted to evaluate the effect of clinically relevant micro-sized UHMWPE-ALN wear particles on the inflammatory response of mice,and reveal the mechanism of effect of released ALN on inflammatory tissue at histological and molecular biology levels.The main contents and results of this thesis are summarized as follows:1.The distribution of ALN in UHMWPE-ALN plates prepared by solvent evaporation and compression molding was studied by alternative indicator and random sampling method.Results showed that ALN distributed evenly in the plate and no obvious agglomeration was observed.A large number of clinically relevant micro-sized UHMWPE-ALN wear particles were obtained by vacuum gradient filtration after preparing initial wear particles using a rough rubbing pair.In vitro release found that,ALN from clinically relevant micro-sized UHMWPE-ALN wear particles was divided into two stages:the burst release(the first 24 h)and the slow(2-90 d).The smaller sized of clinically relevant micro-sized UHMWPE-ALN wear particles accelerated the ALN release.2.Clinically relevant micro-sized UHMWPE-ALN wear particles were co-cultured with macrophages by an inverted culture technique.It was found that wear particles had a chemotaxis effect on macrophages,and the cells could gradually engulf the wear particles.The ALN released from UHMWPE-ALN wear particles induced the death and inhibited the proliferation of macrophages.Furthermore,the released ALN reduced the secretions of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)by macrophages.These results suggested that ALN released from UHMWPE-ALN wear particles may inhibit wear particle-induced osteolysis by interfering with the cytokine pathway.3.SA beads of different diameters were prepared separately by a simple extrusion method and a high-voltage electrostatic spraying method.The morphology,permeability and stability of SA beads were studied.Results showed that SA beads maintained a long-term(>14 d)stability in vitro,and were permeable to proteins of different molecular weight,with smaller proteins being more permeable than larger ones.It was found that macrophages and UHMWPE-ALN wear particles effectively contacted each other and both were evenly distributed in SA beads.In addition,macrophages grew and proliferated normally in SA beads.It was shown that SA bead could be used as a cell reactor for the co-culture of cells and UHMWPE-ALN wear particles,and for evaluation of the long-term biological response of cells to wear particles.The method overcame the difficulties of cells-wear particles contact in other co-culture methods,and avoided the disadvantage associated with the long-term evaluation of cells to wear particles by regular inverted culture techniques.4.The long-term biological response of osteolysis-related cells(macrophages and osteoblasts)to clinically relevant micro-sized UHMWPE-ALN wear particles were evaluated by the above cell reactor method,in which the effect mechanism of ALN released from UHMWPE-ALN wear particles on macrophages and osteoblasts were revealed at the molecular level.Results showed that released ALN inhibited the proliferation and activity of macrophages,suppressed the secretion and expression of TNF-α,IL-6,IL-1β and nuclear factor κB receptor activator ligand(RANK)of macrophages,indicating inhibited the activation of osteoclasts and reduced osteolysis by interfering with the cytokine pathway.On the other hand,ALN acted on osteoblasts,promoted the proliferation and osteogenic differentiation,and increased the gene expression of osteoprotegrin(OPG),while inhibited the expression of RANK ligand(RANKL),in which way to inhibit the function of osteoclasts and reduce osteolysis.Thus,ALN released from clinically relevant micro-sized UHMWPE-ALN wear particles may act synergistically on macrophages and osteoblasts,inhibit osteolysis by interfering with the cytokine and RANKL/RANK signaling pathway.5.An air pouch model was established by injecting sterile air into the back of mouse,and subquently introducing clinically relevant micro-sized UHMWPE-ALN wear particles into the pouch to simulate the pathological process around joint prosthesis.The biological response of UHMWPE-ALN wear particles and the action mechanism of wear particles to inflammatory tissue at histological and molecular biological levels were studied in vivo.Hematoxylin and eosin(H&E)staining and histomorphometry showed that wear particles significantly stimulated the thickness of air pouch membrane and infiltration of inflammatory cells.In contrast,ALN released from UHMWPE-ALN wear particles significantly inhibited this phenomenon to alleviate the inflammatory response.Immunohistochemical and molecular biological analysis results showed that the injection of wear particles stimulated the protein and gene expressions of inflammatory cytokines(TNF-a and IL-6)in the air pouch membrane.ALN released from UHMWPE-ALN wear particles reduced the above expressions and inhibited the activation of osteoclasts.These effects may effectively inhibit osteolysis and prosthesis loosening by interfering with the cytokine pathway.In this thesis,SA bead was first used as a cell reactor to encapsulate cells and wear particles for the evaluation of the biological response and mechanisms of macrophages and osteoblasts to clinically relevant micro-sized UHMWPE-ALN wear particles.The cell reactor provided a long-term stable three-dimensional environment for co-culture of wear particles and cells,and ensured sufficient contact between wear particles and cells.This novel co-culture method effectively overcame the difficulties of cells-wear particles contact in other co-culture methods,and avoided the disadvantages associated with the long-term evaluation of cells to wear particles by inverted culture technique.The results of this study provide a scientific basis for the clinical application of UHMWPE-ALN,but also open up a new way to evaluate the biological response to micro-nano particles.