Objective To observe the expressions of extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase 9 (MMP-9) around the prosthesis, and to study the relationship between the expressions of EMMPRIN and MMP-9 and osteolysis around prosthesis. Methods Interface tissues were obtained at three Delee-Charnley acetabular sections and seven Gruen femur sections from 8 cases (8 hips) undergoing revision after total hip arthroplasty between February 2010 and January 2012, and were divided into osteolysis group and non-osteolysis group based on preoperative X-ray film and intraoperative observation; the tissues from another 8 patients with osteoarthritis undergoing total hip arthroplasty as the control group. The immunohistochemical staining and RT-PCR assays were used to determine the expressions of EMMPRIN and MMP- 9. The correlation between the positive cells and the severity of osteolysis were analyzed and compared. Results Histological examination showed that many macrophages, multinucleated giant cells assembled in the membrane of osteolysis zone, but many fibroblasts and synovial cells in non-osteolysis zones. EMMPRIN and MMP- 9 positive cells and gene expressions were observed in every group. The percentage of positive cells and gene expression of EMMPRIN and MMP-9 in osteolysis group were significantly higher than those in non-osteolysis and control groups (P lt; 0.05), but no significant difference was found between non-osteolysis group and control group (P gt; 0.05). The percentage of positive cells of EMMPRIN in zone III of acetabular was higher than that in zone I and zone II of revision hip (P lt; 0.05), but no significant difference between zone I and zone II (P gt; 0.05). The percentage of positive cells of MMP-9 in zone I and zone III was significantly higher than that in zone II of revision hip (P lt; 0.05), but no significant difference between zone I and zone III (P gt; 0.05). The expression of EMMPRIN from high to low in order was zones 1, 7, 4, 2, 3, 5, and 6 at femur; the values of zones 1, 7, and 4 were significantly higher than those of zones 2, 3, 5, and 6 (P lt; 0.05), but no significant difference among zones 1, 7, and 4, and among zones 2, 3, 5, and 6 (P gt; 0.05). The expression of MMP-9 from high to low in order was zones 1, 7, 4, 2, 3, 6, and 5 at femur; the values of zones 1 and 7 were significantly higher than those of zones 4, 2, 3, 6, and 5 (P lt; 0.05), and the values of zones 4 and 2 were significantly higher than those of zones 3, 6, and 5 (P lt; 0.05), but no significant difference between zone 1 and zone 7, between zone 4 and zone 2, and among zones 3, 5, and 6 (P gt; 0.05). Conclusion The expressions of EMMPRIN and MMP-9 have certain coherence. The over-expressions of EMMPRIN and MMP-9 may be one of the key points of inhibiting bone reconstruction and bone resorption at bone-implant interface under the stimulation of wear debris．
Objective To observe the effect of local injection of vascular endothel ial growth factor (VEGF) and VEGF antibody on the wear particle-induced osteolysis in the mouse air pouch model and to investigate the role of VEGF in the process of aseptic loosening of prosthesis. Methods The stem of metal hip prosthesis was obtained from the revision surgery.Metallic wear particles were made by vacuum ball mill ing. Wear particles suspension was prepared into the concentration of 10 mg/mL with PBS. Fifty female Kunming mice (aged 8-10 weeks, weighing about 25 g) were selected. Of 50 mice, 10 were used as the donors of bone graft, the other 40 were equally divided into control group (group A), particle group (group B), VEGF group (group C), and VEGF inhibited group (group D). Air pouches were made on the back of 40 mice by injecting sterile air subcutaneously. At 8th day, a graft of calvaria from the donor mice was implanted in air pouch. In groups B, C, and D, 0.5 mL wear particles suspension was injected into the air pouches, and in group A, 0.5 mL PBS was injected. Once a day at 6th and 7th days during the air pouch preparation and one time every two days after bone implantation, 0.2 mL recombinant human VEGF (rhVEGF) and VEGF antibody (Bevacizumab) were injected into the air pouches in groups C and D, respectively. In group A and group B, 0.2 mL sal ine was injected. Pouch tissues and bone were harvested at 2 weeks after bone implantation for HE staining, real-time fluorescent quantitative PCR and ELISA analyses. Results All mice survived to the end of experiment. The gross observation showed that there were mild redness, swell ing, and less neovascularization in air pouches in group A. There were obvious redness, swell ing, and more exudative and neovascularization in groups B, C, and D, most obvious in group C, the next in group B, then in group D. The histological and molecular biological analysis showed that inflammatory responses and osteolysis were obvious in group B and the pouch membrane thickness, the cell density, transforming growth factor α, interleukin 1β, and VEGF were significantly higher than those in group A (P lt; 0.05). The inflammatory responses and osteolysis were mostobvious in group C and the above-mentioned indexes were significantly higher than those in group B (P lt; 0.05). There were some inflammatory responses and osteolysis in group D, but the indexes were significantly lower than those in group B (P lt; 0.05) and were significantly higher than those in group A (P lt; 0.05). Conclusion VEGF can promote inflammatory responses and osteolysis in aseptic loosening of prosthesis. VEGF antibody can effectively inhibit wear particle-induced osteolysis.
Objective To evaluate the effectiveness of prosthetic revision using custom-made long stem prosthesis and allograft-prosthesis composite (APC) for aseptic loosening after bone tumor resection. Methods Between January 2002 and June 2008, 14 patients with aseptic loosening after bone tumor resection were treated. There were 8 males and 6 females,aged 21-70 years (mean, 43.9 years). The locations were distal femur (8 cases), proximal femur (2 cases), and proximal tibia (4 cases). Pain of the affected l imb occurred after 6-31 years of prosthesis replacement and worsened when bearing and walking; 6 patients had shortened l imb. The functional results were assessed quantitatively according to the functional rating system of the Musculoskeletal Tumor Society (MSTS). The MSTS score was 16.36 ± 1.50 before revision. The X-ray films showed obvious prosthetic loosening and subsidence. The average time of symptom was 4.5 years (range, 3-9 years). In 7 patients having severe bone loss (the decrease of the thickness of cortical bone was more than 50%) and the prosthetic subsidence was more than 2 cm, the revision operation with the APC was performed; in 7 patients having less bone loss (the decrease of the thickness of cortical bone was less than 50%), the custom-made long stem prosthesis was performed. Results All wound healed by first intention. Two patients had temporary peroneal nerve paralysis and recovered after 3 months. All the patients were followed up 3.6 years on average (range, 2 years and 2 months-7 years) after revision. After revision, pain was rel ieved and the range of joint was improved. The MSTS score was 23.43 ± 2.56 at 12 months after revision showing significant difference when compared with the preoperative score (t=8.910, P=0.024). The X-ray films showed that lucency space l ine around stem cement in 2 patients at 12 months, and no prosthesis loosening and infection occurred. Conclusion The prosthetic revision after l imb salvage surgery with prosthesis for bone tumors was acceptable. The good functional results can be achieved by the revision with the APC or the custom-made long stem prosthesis according to the bone loss.
Objective Metal wear products cause the aseptic loosening of joint prosthesis. To investigate the effect of metal ions Co2+ and Cr3+ on the osteoblast apoptosis, cell cycle distribution, and secretion of alkal ine phosphatase (ALP), and to search a method to prevent and treat aseptic loosening. Methods The mouse calvarial osteoblasts (MC3T3-E1) were cultured in vitro to 3-5 generations (5 × 105 cells/ mL) and divided into 2 groups: the experimental group and the controlgroup. The osteoblasts were cultured in α-MEM medium containing 10%FBS (the control group), and the mixed solution ofCoCl2 and CrCl3 was added after the osteoblasts cultured in α-MEM medium containing 10%FBS attached completely (the experimental group). At 12, 24, and 48 hours after culture, the osteoblast apoptosis and the cell cycle distribution were assessed by flow cytometry; and ELISA method was appl ied to detect ALP content in serum supernatant. Results At 12, 24, and 48 hours after culture, the apoptosis rates in the experimental group (13.90% ± 0.52%, 14.80% ± 0.41%, and 13.40% ± 0.26%) were significantly higher than those in the control group (8.56% ± 0.31%, 8.19% ± 0.24%, and 2.15% ± 0.11%), (P lt; 0.05); G2M (dividing phase) distribution ratio significantly decreased and G0G1 (dormancy stage) distribution ratio significantly increased when compared with those in the control group (P lt; 0.05); and the absorbency (A) values of ALP were 0.955 ± 0.052, 0.624 ± 0.041, and 0.498 ± 0.026 in the exprimental group, and were 1.664 ± 0.041, 1.986 ± 0.024, and 2.192 ± 0.041 in the control group, showing significant differences between 2 groups (P lt; 0.05). Conclusion Metal ions Co2+ and Cr3+ have a marked effect on osteoblasts cell cycle distribution, which can make most of the cells to be in dormancy stage (G0G1), up-regulate the apoptosis rate and inhibit the releasion of ALP from osteoblasts.
Objective Aseptic loosening of prosthesis is associated with peri prosthetical osteolysis caused by osteoclast activation. Receptor activator of nuclear factor kappa B (NF-κB) l igand (RANKL)/receptor activator of NF-κB (RANK) signalpathway is fundamental in osteoclast activation. To determine whether RANKL antibody can inhibit inflammatory osteolysis in a osteolysis model of mouse. Methods Sixty female BALB/c mice (aged 8-10 weeks, weighing 18-20 g) were selected. The skull bone piece was harvested from 20 mice as the donor of bone graft; the subcutaneous air pouches (2 cm × 2 cm) models were established on the back of the other 40 mice and the skull bone piece was inserted into the air pouches. The 40 mice were equally divided into groups A (negative control group), B (positive control group), C (low-dose RANKL antibody group), and D (high-dose RANKL antibody group). At 1 day after bone graft, 0.5 mL PBS was injected into the pouch of group A, 0.5 mL PBS containing titanium particle into groups B, C, and D. At 2 days before the titanium particle was injected, RANKL antibody (0.1 mL) were injected into the pouch of group C (50 μg/mL) and group D (500 μg/mL), respectively every day for 2 days, and 0.1 mL PBS into groups A and B. At 14 days after bone implantation, the pouchmembranes containing implanted bone were harvested for gross observation and histological analyse. Results All mice survived to the end of experiment, and incisions healed well. The gross observation showed that inflammatory responses, exudation, and vascular proliferation were obvious in group B, and were inconspicuous in groups A, C, and D. The histological analysis showed that significantly more infiltration of inflammatory cells, more obvious bone resorption, more bone collagen loss, and more positive staining area were observed in group B than in groups A, C, and D. There were significant differences in inflammatory cell number, pouch membrane thickness, bone collagen loss, and osteoclast content between group B and groups A, C, and D (P lt; 0.05). Conclusion RANKL antibody can directly blockRANKL/RANK signal pathway, which is an efficient therapy to inhibit bone absorption associated with implant wearing particles.
Objective Lots of metal ions accumulation and over-expression of receptor activator of NF-κB l igand (RANKL) around the prosthesis could be found in revision of total hip arthroplasty. To investigate the relationship between metal ions and aseptic loosening by observing the effects of Co2+ and Cr3+ ions on the expression of RANKL and osteoprotegerin(OPG) from osteoblast. Methods Osteoblasts were cultured in vitro at the density of 1 × 105 cells/mL, and were divided into 2 groups according to different culture solutions. In control group, osteoblasts were cultured with normal medium without CoCl2 and CrCl3. In experimental group, osteoblasts were cultured with the medium including CoCl2 (10 mg/ L) and CrCl3 (150 mg/L) solutions. The RT-PCR and ELISA methods were appl ied to detect the mRNA expression of RANKL and OPG and protein level at 24 and 48 hours after co-cultured, respectively. Results RT-PCR revealed that the mRNA expression of RANKL and OPG could be found in two groups at 24 and 48 hours after co-cultured, the expression was higher in the experimental group than in control group, especially the expression of RANKL, showing significant difference (P lt; 0.05). At 24 and 48 hours after co- cultured, the ratios of RANKL mRNA to OPG mRNA in the experimental group were 0.860 and 1.232, respectively, which were significantly higher than those in the control group (0.695 and 0.688,P lt; 0.05). ELISA revealed that the protein level of RANKL and OPG in experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion Co2+ and Cr3+ can stimulate the mRNA expressions of RANKL, OPG and secretion of those protein from osteoblasts, especially increase of the RANKL, which promotes the formation and activation of osteoblasts and the generation of aseptic loosening.
Objective To introduce the occurrence mechanisms, prevention, and treatment measures of prosthetic aseptic loosening. Methods The recent original articles about prosthetic aseptic loosening were extensively reviewed and analyzed. Results Prosthetic aseptic loosening was a very complex process involving many mechanical and biological aspects. The main mechanical factors included prosthetic materials, shapes and sizes, implant fixation methods (including surfacetreatments), cl inical installation, interface micromotion, stress shielding, implant wear, interface integrity, and peri prosthetic high hydraulic pressure, etc.; the main biological factors included the types and sizes of wear particles, cell-activated responses, cytokine release, enzyme activation and allergic reactions to wear particles, etc.. Many measures should be adopted to effectively prevent and treat it, including improving materials and designs of prostheses, fixation techniques, surgical techniques, and drug treatments. Conclusion Prosthetic aseptic loosening is still a troublesome compl ication after joint replacements in orthopaedics, and more attention should be paid for its effective prevention and treatment.
Objective To evaluate the mechanisms of p42/p44 kinase phosphorylation in cell models and to investigate the effect of simvastatin on the prevention and treatment of aseptic loosening of prosthesis by observing the influence of simvastatin on the levels of tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) of human peri pheral blood mononuclear cell (PBMC) challenged with titanium particles. Methods PBMC from 45 mL peripheral blood of healthy adult voluntary donators, were separated and cultured, and divided into 5 groups according to different culturemedium: group A, PBMC and titanium particles; group B, PBMC and titanium particles with 1 × 10-5 mol/L simvastatin; group C, PBMC and titanium particles with 1 × 10-6 mol/L simvastatin; group D, PBMC and titanium particles with 1 × 10-7 mol/L simvastatin; and group E, PBMC and titanium particles with the extracellular signal-regulated kinase (ERK1/2) inhibitor U0126. The contents of TNF-α and MCP-1 were tested by ELISA after 24 hours of culture. PBMC were pretreated with different medium grouping as groups A, B, C, D, and E for 60 minutes, and were challenged with titanium particles for 30 minutes and 60 minutes, then the level of ERK1/2 expression was tested by Western blot. Results In groups A, B, C, D, and E, the absorbance (A) values of TNF-α were 1.115 5 ± 0.243 6, 0.693 6 ± 0.354 3, 0.695 7 ± 0.387 3, 0.716 4 ± 0.478 9, and 0.263 5 ± 0.101 6, respectively; and the A values of MCP-1 were 1.421 0 ± 0.105 3, 0.915 1 ± 0.411 3, 1.003 5 ± 0.464 2, 1.102 0 ± 0.353 9, and 0.271 3 ± 0.145 1, respectively. The levels of TNF-α and MCP-1 in group A were significantly higher than others, showing significant differences (P lt; 0.05). There were significant differences between group E and groups B, C, and D (P lt; 0.05), between group B and groups C, D (P lt; 0.05); no significant difference between group C and group D (P gt; 0.05). Western blot results showed the expression of ERK1/2 in all groups at 30 minutes and 60 minutes of culture. The levels of ERK1/2 expression were 1.612 1 ± 0.068 2, 1.078 1 ± 0.072 8, 1.268 7 ± 0.223 1, 1.439 7 ± 0.180 1, and 0.732 0 ± 0.110 4 in groups A, B, C, D, and E, respectively; showing significant differences between groups (P lt; 0.05). Conclusion ERK1/2 is a phosphorylated protein after stimulated by wear particles; it is also one of the most important cell signal ing activation of macrophage. Simvastatin can inhibit the expression of bone absorptive factors induced by wear particles and may be used in the prevention and treatment of aseptic loosening of prosthesis.
To observe the effect of different dosage of curcumin on expression of MMP-2 and MMP-9 in the tissue of cystiform in air-pouch mouse models after the injection of polyethylene wear particles, and to investigate its mechanism of intervening inflammatory response induced by wear particles. Methods Seventy-two kunming strain mice were used to establ ish air-pouch animal models by referring to the method of Yang et al. and injecting 3 mL suspension of ultrahigh molecular weight polyethylene wear particles (concentration 1 × 108 cells/mL) into dorsal cyst cavity. Then the animals were randomized into 3 groups (n=24 per group): group A (control group), 0.6 mL/day normal sal ine by gavage; group B(low-dosage experimental group), 0.6 mL/day curcumin solution at a concentration of 1.6 mg/mL by gavage; group C (highdosage experimental group), 0.6 mL/day curcumin solution at a concentration of 3.2 mg/mL by gavage. General condition of the animals was observed after operation. The mice were killed 3, 7 and 14 days after operation (8 mice per group at a time), the tissue of cystiform was harvested to receive gross, histology and immunohistochemistry observation, as well as RT-PCR and Western blot detection. Results All mice survived till the end of experiment. White cystiform tissue was evident on the back of mice subcutaneously in each group. For diameter of the cyst cavity at each time point, group A was obviously greater than groups B and C, and group C was significantly less than group B. Microscope observation showed that inflammatory response in group A was ber than that of groups B and C, and group C was obviously less than group B at 7 and 14 days. There was a significant difference between groups B and C and group A in terms of MMP-2 and MMP-9 expression at 7 and 14 days after curcumin del ivery (P lt; 0.05), and no significant differences were evident at 3 days (P gt; 0.05). There was no significant difference between group B and group C in MMP-2 expression at 7 days after curcumin del ivery (P gt; 0.05), and significant difference was evident at 14 days (P lt; 0.05). There was significant difference bewteen group B and group C in MMP-9 expression at 7 and 14 days after curcumin del ivery (P lt; 0.05). Nuclear translocation of NF-κB P65 was inhibited remarkably after curcumin del ivery,and there were significant differences among three groups at 7 and 14 days (P lt; 0.05), and no significant differences were evident at 3 days (P gt; 0.05). Conclusion Ultra-high molecular weight polyethylene wear particles can stimulate expression of MMP-2 and MMP-9 in cystiform tissue. Curcumin can restrain expression of MMP-2 and MMP-9 in cystiform tissue of air-pouch animal models, and expression of MMP-2 and MMP-9 may be regulated by the activation of NF-κB.
ObjectiveTo investigate the expression changes of the receptor activator of nuclear factor-κB ligand (RANKL) in the peripheral blood of patients with aseptic loosening of the implant after total hip arthroplasty (THA) by comparing with that of patients with femoral neck fracture and to analyze the correlation between RANKL expression and aseptic loosening. MethodsBetween January 2008 and January 2013,the peripheral blood were harvested from 58 patients with aseptic loosening of the implant after THA (trial group) and 63 patients with femoral neck fracture (control group).The 2 groups were well matched,with no significant differences in age and gender (P>0.05).The expressions of the RANKL mRNA and RANKL protein were evaluated by quantitative real-time PCR and Western blot respectively.At the same time,the concentration of RANKL was also measured by ELISA. ResultsThe expression of the RANKL mRNA in the trial group was 18.30±1.09,which was significantly higher than that of control group (1.00±0.05)(t=125.390,P=0.000).The relative RANKL protein expression values in trial group and control group were 0.856±0.254 and 0.404±0.102 respectively,showing significant difference (t=13.032,P=0.000).The results of ELISA showed that the concentration of RANKL in trial group [(3.553 5±0.129 7) ng/mL] was significantly higher than that of control group [(1.912 3±0.126 2) ng/mL] (t=18.124,P=0.000). ConclusionThe high RANKL expression in peripheral blood is probably correlated with aseptic loosening of the implant in patients undergoing THA,which possibly is the prognostic factor of aseptic loosening of the implant.