The Primary Study Of Particulate And Ion Forms Of Co-Cr Alloy Induced Aseptic Loosening

With the aging population growth, osteoarthritis incidence increased year by year. But the effective treatment of end-stage osteoarthritis is artificial joint replacement. Though artificial joint materials and design has been in the improved, aseptic loosening is still the most commen long-term complications. At present, the prosthesis revision is the main treatment for aseptic loosening, but the problem of prosthesis loosening is still not solved fundamentally.Along with the progress of science and technology, the material and design of artificial prosthesis is constantly updated. After the implantation in human body, the wear particles are produced along with the application of daily life, which contain Ti-6Al-4V, PMMA, UHMWPE and Co-Cr. At present a large number of experimental studies have shown that wear particles play a key role in the aseptic loosening of prosthesis, wear particles were swallowed by the periprosthetic cells, which include bone marrow mesenchymal stem cells, macrophages, bone cells, lymphocytes and fibroblasts, resulting in the release of cytokines, which will cause different inflammatory and immune response in the body, leading to the downregulation of bone cell function, function of osteoclast activation, eventually leading to osteolysis and aseptic loosening. Among these particles, the Co-Cr alloy is widely used in the manufacture of prosthesis as its high wear resistance, corrosion resistance and high temperature resistance. Therefore, we bring Co-Cr alloy in this research. As we know, once the prosthesis is implanted in the body, various ionic forms of Co-Cr alloy will be released around prosthesis as body fluid, furthermore the ionic forms of Co-Cr alloy are mostly Co2+ and Cr3+. So we study the Co-Cr particles and their ionic forms, and observe their similarities and differences between them.Bone quality is the dynamic equilibrium between bone loss and bone formation. The osteoclast from mononuclear macrophage can promote bone resorption to cause bone loss and osteoblast from bone marrow mesenchymal stem cells cause bone deposition to increase bone mass. Furthermore some inflammatory cytokines can lead to osteoclast activation and promote osteolysis. So that, osteoclast activation is an important factor to aseptic loosening. There are two important factors RANK and RANKL to adjust osteoclastogenesis, RANK is the membrane receptor of RANKL, belonging to the TNF receptor superfamily, which can be expressed in preosteoclasts, mature osteoclasts, chondrocytes, monocytes and tissue macrophages. The RANKL-RANK chain can initiate osteoclastogenesis and activation of mature osteoclasts. Furthermore, OPG/RANKL is also widely recognized as an important condition for regulating the differentiation and maturation of osteoclasts.Experimental models have been established to study the mechanism of osteolysis by wear particles and its corresponding ions, and verify molecular basis of differentiation and activation of osteoblasts and osteoclasts. At present, experimental models include experimental model for cells in vitro, animal model of prosthesis implantation, however, vitro model just explain the body process simply, and as to animal models, large animals such as rabbits, dogs, sheep and horses cost a lot, not easy to be managed, and sample size is limited, especially not suitable for the study of new treatment strategies, which in turn limits the widespread use of these animal models. Mouse has the advantages of gene homology with human, which is beneficial to biological research. By use of mouse, Dr.Wooley laboratory initiated mouse air pouch model to study cell responses and osteolysis effect, but its research time is short, which is not close to the body environment. So that we need to design an ideal model, which can be established to study the mechanism of molecular biology research around the prosthesis loosening osteolysis.In this research, we studied different effects to preosteoblasts challenged by different concentrations of particle and ion form of Co-Cr alloy in vitro, these effects included preosteoblast growth, differentiation, maturation, and participation in the inflammatory reaction, regulation of osteoclastogenesis. Finally, we constructed aseptic loosening animal model by implanting prosthesis and Co-Cr particle, through the model to further validate the role of preosteoblast in the regulation of osteoclast differentiation and maturation. MC3T3-E1 preosteoblasts (CRL-2594, ATCC, Manassas, VA) were cultured in an Alpha Minimum Essential Medium (α-MEM, Life Technologies, Grand Island, NY) containing 2 mM L-glutamine,1 mM sodium pyruvate,5% fetal bovine serum (FBS, Invitrogen, Grand Island, NY),100 U/mL penicillin (Invitrogen), and 100 mg/mL streptomycin (Invitrogen) at 37 ℃ and 5%CO2 atmosphere. For inducing osteoblast differentiation, MC3T3-E1 cells were cultured for three days in a-MEM supplemented with 5% FBS,10 mM β-glycerolphosphate (Sigma-Aldrich, St. Louis, MO),50μg/ml L-ascorbicacid (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich),100 U/mL penicillin and 100 mg/mL streptomycin. The differentiation of osteoblasts was determined by alkaline phosphatase staining. Induced cells were dispensed into each well of plates in the absence or presence of Co-Cr particles at 0.15,0.3, 0.625,1.25,2.5, or 5 mg/mL and the ion form Co(II), Cr(III) (respectively or together) at 62,125,250,500,1,000 μM, supernatant liquor was collected. MTT assay was performed to determine the mitochondrial activity by colorimetric reaction at 72 hours after incubation at 37℃ and 5% CO2 atmosphere. The live/total cell ratio was measured by CytoTox96(?) Non-Ratio Cytotoxicity Assay (Promega, Madison, WI). LDH activity in lysed cells and supernatant were assayed by colorimetric reaction with substrate mix in the kit. The cell viability and cytotoxicity were calculated by the relative medium LDH level over the cell lysis LDH. The differentiation of osteoblasts was determined by alkaline phosphatase staining and concentration of ALP protein. Real-time polymerase chain reaction (RT-PCR) was performed on samples of different groups to examine the gene expression profiles of monocyte chemo-attractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), runt-related transcription factor 2 (Runx2), osterix (Osx), and Low-density lipoprotein receptor-related protein 5 (Lrp5). In vivo, Titanium nails were implanted in the proximal tibia to imitate the knee joint replacement. In order to imitate aseptic loosening, we injected Co-Cr particles around the prosthesis. The Balb/c mice with the Ti-pin implantation and Co-Cr particle injected into tibial cavity were divided into 3 groups post-op:(1) Co-Cr group:mice were given an intra-articular injection of 50μl medium containing 5×105 MC3T3-E1 cells challenged by Co-Cr particles (n=12); (2) Co(Ⅱ) group:mice were given an intra-articular injection of 50μl medium containing 5×105 MC3T3-E1 cells challenged by ion Co(Ⅱ) (n=12); (3) loosening group:mice were given an intra-articular injection of 50μl medium containing 5×105 naive MC3T3-E1 cells (n=6); The fourth group was stable control group:mice only received the pin implantation without particle injection and cells infusion (n=6). After Ti-pin implantation and just before mice sacrifice(5 weeks later), μCT scanning was performed for bone densitometry. After sacrifice, the mouse limbs with the implant intact were immediately removed by disarticulating the knee joint, exposed the cap of pins and proximal tibia by removing all soft tissue and partial cartilage around the prosthetic joint for pull-out test. The peri-implant proximal tibiaes were fixed in formalin, decalcified the surrounding bone in the solution of 12% EDTA, lastly embedded in paraffin. Hematoxylin and eosin (HE) was operated to investigate the evidence of inflammation, bone formation or resorption around pin and the thickness of the soft-tissue membrane at the interface, The TRAP staining were proceeded to determine the number of osteoclast and its differentiation and maturity. Immunohistochemical (IHC) stains were proceeded to localize TNF-a, RANKL and Runx2 expression in the xenograft.In vitro, the naive cells and Cr(Ⅲ)-challenged cells exhibited polygonal morphology, whereas Co(Ⅱ) ion-challenged cells stretched more tentacles, some with bright areas. In the particle group, most cells appeared to have spindle morphology where phagocytosis was obviously present. The MTT assay suggested that all experimental groups suppressed proliferation of cells to a certain degree. LDH assay confirmed significant cytotoxic effects in cells challenged with particles (2.5 and 5 mg/mL) and ions (1,000 μM) compared to other tested groups. All types of particles and ions (with the exception of CrIII groups) exhibited significant negative influence on ALP activity in a dose-dependent manner (p< 0.01). PCR data have suggested that cells with Co-Cr particles dramatically promoted over-expression of MCP-1, TNF-α, and IL-6, whereas Co(Ⅱ) ions treatment predominately up-regulated expressions of RANKL, NFATc1, and down-regulated expression of OPG and Osx. In vivo, we have successfully established a mouse model of aseptic loosening. Intra-articular introduction of the MC3T3-E1 cells challenged by Co-Cr particles and ion Co(Ⅱ) to the SCID mouse pin-implant failure model resulted in reduced implant interfacial shear strength, thicker peri-implant soft-tissue formation, more TRAP+ cells and lower BMD compared to naive cells.In a word, MC3T3-E1 cells challenged by particle or ion form of Co-Cr alloy manifested different biological characteristics, which included cell morphology, proliferation, cytotoxicity, osteogenesis and inflammatory reaction. In the research, not only Co-Cr alloy particles but also metal ions interfere with preosteoblastic cells in osteoblast differentiation to a certain degree, furthermore, Co-Cr particle and Co(II) ion can promote MC3T3-E1 cells to express several inflammatory factor genes, then involved in inflammatory reaction and bone resorption around prosthesis, eventually resulted in aseptic loosening. What is more, ion Co(II) promoted preosteoblast to express RANKL gene and inhibited OPG gene expression, so that the ratio RANKL/OPG was higher, which is widely considered as important factor adjusting osteoclast differentiation and maturity. It is also confirmed by counting TRAP+cells around prosthesis in vivo. In other ways, Co-Cr particle promoted preosteoblast to express inflammatory genes such as MCP-1, TNF-a and IL-6, which involved in inflammatory reaction, at last resulted in aseptic loosening. In conclusion, not only Co-Cr particle but also Co(Ⅱ) ion interfered with the growth, maturation and functions of MC3T3-E1 cells, by which they can adjust osteoclast differentiation and maturity and eventually resulted in aseptic loosening.