The Biological Activity Difference Between Mental Ion And Mental Particle

BackgroundJoint replacements as an effective strategy in treating bone and joint diseases, provides pleasing results in releasing pain and increase the motion range of joints. However, popularize joint replacements are somehow limited by relatively short lifespan of prosthesis. According to those long-term follow-up data, aseptic loosening plays a key role in this process. Aseptic loosening of the components are induced by biological factors and mechanical factors including material design compatibility and installation of the prosthesis. Fragments of wear debris may cause an inflammatory reaction involving IL1β(interleukin-1β) IL-6(interleukin-6) and TNF-α (TNF-α, tumor necrosis factor-a) with bone absorption which can cause loosening. This phenomenon is known as osteolysis. It is generally believed that the wear particles generated by the mechanical wear of the prosthesis can induce periprosthetic tissue cells to produce a series of biological reactions activating the osteoclasts causing periprosthetic osteolysis which is the main reason for long-term loosening of the artificial joints. With the technology development of material selection and process design wear and tear caused by mechanical loosening has been well controlled.The femoral prosthesis is commonly made of metal which is widely used in total hip arthroplasty because of its appropeiate stiffness and rigidity. However, osteolysis surrounding the host bone following metal prosthesis implantation made its application controversial. Osteolcast (OC) plays a significant role in osteolysis surrounding prosthesis. Numerous researches have evidenced the metal ion, Mainaly cobalt and chrome, located surrounding the femoral prosthesis.Bone is an active metabolic organ. Bone remodeling is important for regulating and maintaining skeletal integrity. In this process, OC absorbed the old bones meanwhile osteoblasts (OB, osteoblast) form the new bone. Therefore, bone reconstruction need these two processes precisely coordinate with each other. In the bone marrow microenvironment osteoclast differentiation of progenitor cells into mature OC, and bone resorption.Number of OB and OC under normal conditions and by a variety of factors and signaling to maintain stable levels, so that bone formation and bone resorption mediated by them in a State of balance.This balance is broken in pathological conditions, resulting in abnormal structure and function of bones, thus the corresponding clinical manifestations such as periprosthetic bone loss.Redevelopment is a complex process, subject to various cytokines, hormones and signaling pathways of regulation, mutual regulation between the OB and OC help bone resorption and bone formation in the bone remodeling process coupling between the two processes.RANK (RANK, Nuclear factor-B receptor activation factor) is type I transmembrane protein which belongs to TNF family. RANK highly expresse on the surface of osteoclast precursor. Nuclear factor Kb ligand receptor activation factor (RANKL, receptor activator of nuclear factor-K b ligand) binding to RANK not only promoting the differentiation of OC but also inhibiting the apoptosis of OC. OPG (OPG, osteoprotegerin) secreted by osteoblast can competitive binding to RANK against RANKL. RANKL/OPG rate is essential for OC. Ehance in the proportion of RANKL/OPG lead to the increase in the number of OC and its activity. Decrease in the proportion of RANKL/OPG will reduce the number of OC and its activity. In short, the normal bone remodeling and bone mass stability depends on the rate of RANKL/OPG.TNF-a is a kind of important inflammatory mediators which can can stimulate the inflammatory cells secrete IL-land IL-6lead to the macrophages and osteoclasts gathering at the place where inflammatory reaction take place. TNF-a can also stimulate osteoclast release PGE (PGE, prostaglandinE) which can induce osteoclastic bone resorption. In recent years, research has shown that TNF-α can promote human osteoblasts of osteoprotegerin ligand (OPGL) expression and osteoprotegerin physical distribution activity of osteoclast differentiation and maturation and increase its functionality, which further lead to implant loosening.Apoptosis is the process of programmed cell death controlled by genes. Stimulunts affect cells by initiating gene transcription and protein synthesis causing intracellular protease and activating endogenous DNA endonuclease. In recent years, studies have shown that around the aseptic loosening prosthesis exists the phenomenon of mononuclear macrophage apoptosis. Apoptosis of mononuclear macrophage will lead to more gathering of mononuclear macrophage causing a cascade reaction resulted in periprosthetic bone loss.Both metal particles and metal ions can induce Periprosthetic osteolysis leading to the loosening of prosthesis. But there is no relevant research comparing the effect between metal particles and metal ions on aseptic loosening and osteolysis. This study aim at compare the effect between metal particles and metal ions on aseptic loosening and osteolysis through vitro experiment providing reference for the choice of prosthesis materials.Purpose1. Prepare metal debris and metal ions which meet the requirements of the experiment. 2. Observe the effect of metal debris on the differentiation of osteoclast in vitro.3. Observe the effect of metal ions on the expression RANKL and OPG secreted by osteoblast.4. Compare the biological activity difference between metal ions and metal debris on mononuclear macrophage.Method1. Prepare metal debris through the vacuum ball milling method. Analyse the elements of the metal debris. Observe its character through electron microscope. Measure its diameter by laser particle size analyzer. Detect its cytotoxicity by coculture with RAW264.7cell line.2. Treating RAW264.7cells which induced by RANKL with high concentration (10mg/ml) medium concentration (1.0mg/ml) low concentration (0.1mg/ml) metal debris respectively. Take count of TRAP (TRAP, tartrate-resistant acid phosphatase) stain positive multinucleate cells after7days culturing.3. In vitro culture of MC3T3-E1cell line. Divide it into two groups according to its medium. The experimental group including10mg/L C0Cl2and150mg/L CrC13in the culture medium defined as group A. The control group with normal culture medium defined as group B. Detect RANKL and OPG mRNA by RT-PCR. Detect RANKL and OPG protein by ELASIA.4. Mononuclear cells obtained from peripheral blood of30healthy volunteers. Coculture mononuclear cells with iron particles cobalt particles chromium particles titanium particles Iron ions chromium ions cobalt ions titanium ions and physiological saline respectively. Assess activity of the cells by MTT at6h12h24h36h48h respectively. Assess the expression of TNF-α protein by ELASIA at6h12h24h36h48h respectively. Results1. Median diameter volume mean diameter and surface mean diameter of metal particles obtained by vacuum ball milling is2.193μm3.018μm and2.119μm respectively. Diameter of98%particles is less than6.167μm. The diameter and biological activity in our experiment is in common with particles extracted from artificial joint interface tissue.In the testing concentration, typically less than1mg/ml, showed no cytotoxicity.2. The results show that as the wear particles suspension concentration increased RANKL induces formation of osteoclast cells increased. A number of osteoclast in the high concentration medium concentration low concentration group was higher than that in control group (P<0.05). A number of osteoclast in the high concentration medium concentration group was higher than that in low concentration group (P<0.05). A number of osteoclast in the high concentration group was higher than that in medium concentration group (P<0.05).3. The expression of RANKL OPG mRNA was increased with time in the experimental group. The expression of RANKL OPG mRNA was higher than that in the control group at2448h. Rate of RANKL/OPG mRNA is0.8601.232at2448h in the experimental group higher than that0.6950.688in the control group(P<0.05). Rate of RANKL/OPG protein at2448h in the experimental group was higher than that in the control group (P<0.05)4. The expression of TNF-a was higher in iron particles cobalt particles chromium particles titanium particles group at2436h than that in the control group (P<0.05). The expression of TNF-a was higher in cobalt particles chromium particles group than that in the iron particles titanium particles group (P<0.05). The expression of TNF-a was higher in cobalt ions group than that in the iron ions chromium ions titanium ions group. Iron particles cobalt particles chromium particles titanium particles can induce mononuclear cells to apoptpsis. Iron ions chromium ions cobalt ions titanium ions can induce mononuclear cells to apoptpsis. The ability of inducing mononuclear cells to apoptosis for cobalt particles chromium particles was greater than iron particles titanium particles. The ability of inducing mononuclear cells to apoptosis for iron ions cobalt ions was greater than chromium ions titanium ions.Conclusion1. Metal debris prepared by vacuum ball milling method not only simple and convenient but also has good reproducibility and productive thus can be used for vitro experiment.2. RAW264.7is a good osteoclast precursor cells model. RANKL induced RAW264.7to form osteoclasts is simple also has good repeatability. The number of osteoclase has increased after coculture with metal debris.3. Co2+, Cr3+interaction can stimulate osteogenesis cells increase expression of RANKL and OPGmRNA as well as the increase in the ratio of RANKL/OPG mRNA and greatly promote the secretion of the protein RANKL thus affecting the osteoclast precursor cells differentiate into osteoclasts and enhance the ability of bone absorption.4. Iron particles cobalt particles chromium particles titanium particles Iron ions chromium ions cobalt ions titanium ions can induce human mononuclear cells to apoptosis and its rate gradually increased over time.Metal particles and metal ions through stimulating mononuclear cells causing inflammatory reaction around the prosthesis and by inducing apoptosis of mononuclear cells lead to implant loosening. The biological activity of titanium was significantly lower than iron cobalt and chromium.