| Part I The effects of Ti particles on the MC3T3-E1 cellsObjective: To test the cytotoxicity of Ti particles on the osteoblast-like cell and make sure the proper concentration of Ti particles suspension for the study.Methods: MC3T3-E1 cell was cultured with different concentrations of Ti particles(0,10,50,100 and 1000 particles numbers/cell number and named Ti-0, Ti-10, Ti-50, Ti-100, Ti-1000 group respectively). Observed phagocytosis phenomenon of the MC3TE-E1 cells and assessed cell viability by CCK8 48 hours after Ti particles treatment. The Col1α-I and IL-6 expression were compared by Real-Time PCR 5 days after treatment. On day 7 and day 14 assessed ALP activity by the ALP kit. On day 14 assessed matrix mineralization by Alizarin Red stain.Results: The phase contrast microscopy merged with ?uorescence micrograph showing the DAPI nuclear staining and the endocytosed particles by MC3T3-E1 cell in Ti-100 group, which confirmed the particles use in our study can be effectively endocytosed by MC3T3-E1. The viability of MC3T3-E1 cell were significantly inhibited by the Ti particles in a concentration dependent manner at 48 h, especially in the Ti-1000 group, the cell viability is only 28.3% of that in the Ti-0 group. The expression of Col1α-I was significantly inhibited in the Ti-100 group and Ti-1000 group compared with Ti-0 group, however the expression of IL-6 also appeared the down regulate trend in the Ti-1000 group. The ALP activity was significantly inhibited in the Ti-100 group on day 7 and day 14 compared with the Ti-0 group. Alizarin Red staining and semi-quantitative analysis showed that the mineralization level was not reduced but significantly increased in a concentration dependent manner except for Ti-1000 group.Conclusions: The Ti particles used in this study can significantly inhibit the cell viability and function of osteoblast-like cells. 100 particles/cell number reproducibly affected MC3T3-E1 functions without severely compromising cell viability. Part II The effects of LDI on the MC3T3-E1 co-cultured with Ti particlesObjective: To test LDI effect on the proliferation, differentiation and inflammatory cytokine IL-6 expression of MC3T3-E1 cells co-cultured with different concentration of Ti particles.Methods: MC3T3-E1 cell was cultured with different concentrations of Ti particles(0,10,50,100 and 1000 particles numbers/cell number and named Ti-0, Ti-10, Ti-50, Ti-100, Ti-1000 group respectively), after 24 h, the cells were irradiated with 0 and 0.5 Gy X-rays. Cell viability was determined with the CCK-8 assay 24 h,48 h and 72 h post irradiation. The PICP concentration in the supernatant was determined by ELISA kit at day 3 and day 7 post irradiation. On day 7 and day 14 assessed ALP activity by the ALP kit. The IL-6 expression were compared by Real-Time PCR on day 5 post irradiation. On day 14 assessed matrix mineralization by Alizarin Red stain.Results: The viability of MC3T3-E1 cell in the 0Gy group and 0.5Gy group all were significantly inhibited by the Ti particles at 24 h,48 h and 72 h time point. After 48 h and 72 h, the cell viability was significantly inhibited in the 0.5Gy groups compare to 0Gy groups. The PICP concentrations in the Ti-100 group were significantly lower than in the Ti-0 group(p<0.01);The concentration of PICP concentration in the 0 Gy-Ti-100 group was still lower than that of the 0 Gy-Ti-0 group(p<0.05), and X-ray irradiation significantly elevated the PICP concentration at 7 d post-irradiation(p<0.05). The ALP activity in the Ti-100 group were significantly lower than that in the Ti-0 group(p<0.05); The Ti particles still inhibited the ALP activity of MC3T3-E1 mildly, and X-ray irradiation significantly elevated the ALP activity of each groups at 14 d post-irradiation. The expression of IL-6 was significantly inhibited by X-ray irradiation. The Alizarin Red staining and semi-quantitative analysis showed that in the presence of Ti particles, X-ray irradiation can still promote calcification of MC3T3-E1 cells.Conclusions: Although LDI inhibited the cell proliferation mildly with or without Ti particles, LDI significantly promote the functions of MC3T3-E1 including increased the ALP activity and de novo synthesis of type I collagen in the presence of Ti particles. Furthermore, LDI can significantly inhibit the IL-6 expression in the presence of Ti particles. Part III The effects of LDI and Ti particles on the bone ingrowths into interface of the prosthesis in the Rabbit modelPart III The effects of LDI and Ti particles on the bone ingrowths into interface of the prosthesis in the Rabbit modelObjective: To make sure the effects of LDI on the bone ingrowths into interface of the prosthesis in the rabbit model with or without Ti particles.Methods: Twenty male New Zealand rabbits with an average weight of 2.1±0.2 kg(range: 1.8–2.3 kg) were randomly divided into the SHAM and 0.5 Gy groups. Sterile saline(0.2 m L) was injected into each the left femur(NS), while the right femur was injected with equal volumes of suspended Ti particles(Ti),then intramedullarly nailed with a titanium rod implant coated with hydroxyapatite. The animals in the 0.5 Gy group received the 0.5 Gy X-ray irradiation 2 days after the operation. All animals were euthanized 8 weeks after the operation. The PINP concentration were determined at day 0 2 weeks, 4 weeks and 8 weeks after operation. Assessed trabecular morphology around the implants 8 weeks after operation by Micro-CT, then the maximum push out force of simples were assessed by biomechanics test. Three samples of each group were chosen for bone histomorphology without decalcification 8 weeks after operation.Results: The serum PINP concentrations in both groups increased after irradiation and peaked 2 weeks later. These levels then decreased to approximately their original values. At week 2, the PINP concentration in the 0.5 Gy group was higher than that in the 0 Gy group. At 8 weeks postoperative, Ti particles only reduced the DA of trabecular around the implant and X-ray irradiation can reversed it. X-ray irradiation significantly enhanced the morphology of trabecular bone around the prosthesis, rate of new bone formation, the maximum push out force of the implants, however in the presence of Ti particles; the X-ray irradiation promotion effects were significantly counteracted. The bone histomorphology results showed that the interface membrane formed in 0Gy-Ti and a large number of Ti particles were observed around the implant. Meanwhile, mineralized fibrous tissue was observed around the Ti particles. In the 0.5Gy-Ti group, the interface membrane was thinner compared to the 0Gy-Ti.Conclusions: LDI significantly promotes osseointegration at and the biomechanical properties of the bone-implant interface in a rabbit model. Although the in the presence of Ti particles, the promotion effects of LDI on osseointegration were counteracted, the LDI significantly inhibited the inflammatory reaction induced by Ti particles. |
PDF Full Text Request