Metal Prosthesis Wear Particles On Osteoclast Differentiation In Vitro

ObjectiveTo study a method of in vitro preparation and seperation for metallic wear particles around joint prosthesis, and evaluate its feasibility in medical experiments of prosthetic loosening. To observe the biologic characteristics of RAW264.7 cells,and to validate the feasibility of RANKL(Receptor Activator of Nuclear FactorκB Ligand) mediated differentiation of RAW264.7 cells into bone-resorptive Osteoclasts (OCs). To observe the action of metallic wear particles in the differentiation of RAW264.7 cells into bone-resorptive Osteoclasts.MethodPreparation of Metallic Wear Particles Around Joint Prosthesis by Vacuum Ball Mill in Vitro .Particle size,size distribution,chemical composition,and shape were characterized by element trace analysis, laser countersizer,scanning electron microscopy. Metal particles co-culture with RAW264.7,then detect cytotoxicity . RAW264.7 cells were treated with RANKL. RANKL-induced formation into OCs were observed after tartrate resistant acid phosphatase(TRAP) staining. Bone resorption pits were detected by scanning electron microscope. Particles and RAW264.7 cells were co-cultured, RT-PCR(Reverse Transcription-Polymerase Chain Reaction) test mRNA expression of RANK, TRAP and observation of particles on the RAW264.7 cells to osteoclast differentiation.ResultOur metal particles that prepare by vacuum ball mill that closely resembled particles found around solid and loose human prostheses(<5μm in diameter).The addition of metal particles(<10 mg/ ml in concentration) to RAW264.7 co-culture did not cause cell cytotoxicity,and we can use it in relative research of Cytology. RANKL-differentiated RAW264.7 cell cultures develop both TRAP+ mononuclear and multinucleated cells. Compared with sham control,statistical difference existed in the expression level(P<0.05).It has the ability of generating bone resorptive pits. Results of RT-PCR suggest that: metal wear particles increases the mRNA expression of RANK and TRAP in macrophage.Within a certain concentration, the number of osteoclast formation is proportional to the concentration of metal wear particles.ConclusionThis method is good enough to produce metallic wear particles that closely resembled particles in vivo, which has convenient ,reproducibility. and can be used in vitro studies of Cytology. RANKL can induce RAW264.7 cells to mature OCs. RAW264.7 cells are easier to obtain and culture.So RAW264.7 cells can be used as a preferable preosteoclast model.This methods has good repeatability and feasibility.It represent a relatively homogeneous population of pre-OCs. Modulation of the expression of RANK and TRAP on mononuclear phagocytic cells by metal wear particles occurred in the genomic level. Augmentation of RANK and TRAP expression play a positive role in cell transformation of RAW264.7 cells to osteoclasts with bone resorption function. Wear debris can enhance the formation of osteoclast from RAW264.7 induced by RANKL.