| Objective: To Investigate the influence of bone cement particles on the biological behaviorof human nucleus pulposus cells. Methods: Human nucleus pulposus cells (HNPC)were treated withbone cement particles including calcium phosphate cement (CPC) and polymethylmethacrylate (PMMA)particles (PMMA1and PMMA2) of different concentrations. The effect of bone cement particles on thecell viability, cell cycle, apoptosis and senescence of HNPCs was observed at different time points.Results: CPC particles had no significant effect on the biological behavior of HNPCs. PMMA particlesat the concentration of0.001,0.01%v/v did not have a significant impact on the HNPCs cell viability,when the concentration was above of0.1%v/v, PMMA particles could significantly inhibit theproliferation activity of the nucleus pulposus cells (P<0.05). After3.0or6.0days of treatment with thePMMA1particles and6.0days of treatment with the PMMA2particles, at the same time point, theinhibition effect on HNPCs cell proliferation gradually was increased with increasing concentration ofthe particles; when the PMMA1particles was at the concentration of0.1,0.5and1.0%v/v, the PMMA2particles was at the concentration of1.0%v/v, the inhibition effect on the HNPCs cell proliferation wasstrengthened depending on the length of the particles treatment(P<0.01). When the concentrations were0.5and1.0%v/v, the PMMA1particles had greater inhibition than the PMMA2particles on the HNPCscell viability(P<0.05).When the concentration of the PMMA particles was above0.01%v/v, the G0/G1phase cells of HNPCs were increased significantly (P<0.05). After1.0,3.0or6.0days of treatment with the PMMA1particles and6.0days of treatment with the PMMA2particle, at the same time point, thecells proportion of the HNPCs in the G0/G1phase was gradually increased with increasingconcentration of the particles; when the concentration of the PMMA particles was0.5or1.0%v/v, theblocking rate of the HNPCs in the G0/G1phase was increased with time(P<0.01).Compared with thecontrol group, after6.0days of treatment with the PMMA1particles at the concentration of0.5and1.0%v/v, the proportion of necrosis and apoptosis of the HNPCs was significantly higher(P<0.01),thecells number of the positive senescence β-Galactosidase staining was increased significantly (P <0.01),p16INK4Aexpression was significantly higher in the HNPC cells. Conclusion: CPC particles don't havesignificant effect on the cell viability and the cell cycle of the nucleus pulposus cells. The treatment withPMMA particles at a certain concentration within a certain time period will have a significant impact onthe cell viability and the cell cycle of the nucleus pulposus cells, resulting in the decrease number ofliving cells, the blocking of the cells at the G0/G1phase. It works in a time-dependent anddose-dependent manner. The PMMA1particles have a greater inhibition than the PMMA2particles onthe HNPCs cell viability. Further studies showed that the PMMA1particles can contribute to necrosisand apoptosis of the nucleus pulposus cells, induce senescence of the nucleus pulposus cells. Objective: To Investigate the changes in gene expression of human nucleus pulposus cellstreated by bone cement particles. Methods: Human nucleus pulposus cells (HNPC)were treated withcalcium phosphate cement (CPC) and polymethylmethacrylate (PMMA) particles at the concentrationof1%v/v. Gene expression profile of selected genes including structure-related genes of extracellularmatrix (ECM), anabolic genes of ECM and catabolic genes of ECM was determined with fluorogenicquantitative real-time PCR at different time points. Results: Expression of aggrecan (ACAN), type Icollagen(COL1A1), type II collagen(COL2A1) was up-regulated by the CPC particles (3.2-fold, 2.05-fold and2.56-fold increase respectively compared to untreated cultures).The PMMA particlesincreased the expression of COL1A1and COL2A1(3.15-fold and3.71-fold increase respectivelycompared to untreated cultures), but the effect of PMMA on the expression of ACAN was notsignificant. CPC increased the expression of genes encoding bone morphogenetic protein (BMP2andBMP7)(3.17-fold and2.39-fold increase respectively), also increase the expression of the transcriptionfactor SOX9gene (2.81-fold) and tissue inhibitor of metalloproteinase (TIMP1and TIMP2)(2.47-foldand3.38-fold respectively).After PMMA treatment, the expression of BMP2, BMP7and SOX9genewas not increased significantly, but the expression of TIMP1and TIMP2was up-regulated (3.33-foldand3.36-fold respectively). Genes encoding matrix metallopeptidase (MMP3, MMP7, MMP9andMMP13) were overexpressed after CPC or PMMA treatment (3.8-fold,2.68-fold,3.39-fold and3.15-foldincrease respectively after CPC treatment;2.9-fold,3.81-fold,2.97-fold and3.81-fold increaserespectively after PMMA treatment).The CPC particles increased the expression ofIL1A, IL1B, andTNF gene (2.97-fold,4.44-fold and2.8-fold increase respectively), the PMMA particles increased theexpression of IL1B and TNF gene (2.18-fold and3.08-fold increase respectively)Most gene expressionpeak appeared at the1.0-day and3.0-day time point after treatment of bone cement particles, but theexpression peak ofSOX9, TIMP1, and TNF gene appeared at the6.0-day time point after treatment ofCPC particles. Conclusion: Gene expression of structure-related genes of ECM and metabolism-relatedgenes of ECM is affected significantly by the treatment with bone cement particles. The PMMAparticles may be more effective than the CPC particles in promoting catabolic metabolism of ECM.After treatment of bone cement particles, asynchronous changes in ECM-related gene expression maylead to difficult to maintain the dynamic balance of ECM metabolism. Objective: To Investigate the speed, the degree and the immunohistochemical changes ofrabbits lumbar intervertebral disc degeneration induced by bone cement particles injection. Methods:60New Zealand white rabbits were randomly divided into the blank control group, the degenerationcontrol group, the sham operation group, the PMMA group and the CPC group. An annular puncturemodel was established using16G needles in the degeneration control group. The26G needles werechosen in the sham operation group. In PMMA group and the CPC group, a suspension of PMMA orCPC particles at the concentration of1.0%v/v was injected into lumbar discs of rabbits using26Gneedles. The samples were examined with imaging modalities, histological examination,immunohistochemical analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) at different time points. Results: The X-ray examination showed significant disc spacenarrowing was observed in the PMMA group compared to those before stabbing at3-week time point(P<0.01),the disc heights were decreased progressively until12weeks after surgery(P<0.01), thedecrease in the disc heights was significantly higher than that of the sham operation group(P<0.01), butwas less pronounced compared to the degeneration control group (P<0.01). There was no significantdifference between the CPC group and the sham operation group (P>0.05).The MRI findings showedthere was a significant difference of the disc signal intensity between different groups (P<0.01). TheThompson scores of the PMMA group were higher than those of the sham operation group at the6-week and12-week time points (P<0.005). There was no significant difference between the CPC groupand the sham operation group, and also between the PMMA group and the degeneration control group.Histologic studies showed there was a significant difference of the histologic scores between differentgroups (P<0.01). The histologic scores were significantly higher in the degeneration control group thanthose in the blank control group at all three time points (P<0.005).The histologic scores weresignificantly higher in the PMMA group than those in the sham operation group and the CPC group atall three time points(P<0.005).The histologic scores were significantly lower in the CPC group thanthose in the degeneration control group. There was no significant difference between the CPC group andthe sham operation group, and also between the PMMA group and the degeneration control group.Immunohistochemical staining of type II collagen showed strong positive staining in extracellularmatrix of the blank control group, the sham operation group and the CPC group. Mean optical density of these groups was significantly higher than those of the degeneration control group and the PMMAgroup at different time points (P<0.05). Immunohistochemical staining of matrix metallopeptidase7showed the number of positive cells was lower in the blank control group, the sham operation group andthe CPC group. Positive cells percentage of these groups were significantly lower than those of thedegeneration control group and the PMMA group at different time points (P<0.05).Progressive increaseof positive cells percentage was observed in the degeneration control group and the PMMA group at the3-week and6-week time points, but the positive cells percentage was decreased at the12-week timepoint (P<0.05).TUNEL test showed the apoptotic index of the degeneration control group and thePMMA group was significantly higher than those of the blank control group, the sham operation groupand the CPC group(P<0.01). The apoptotic index of the degeneration control group and the PMMAgroup reached the summit at the6-week time point, then decreased at the12-week timepoint(P<0.05).Conclusion: Injections of the PMMA particles decreased the synthesis of type II collagen,increased the expression of matrix metallopeptidase7, and induced apoptosis of the nucleus pulposuscells, resulting in degeneration of intervertebral discs. Imaging findings showed decreased disc heightsand weakened disc signal intensity. Degenerative disc changes caused by the PMMA particles wereslower and milder than disc degeneration of classic annular puncture model. The CPC particles injectionhad minimal impact on the intervertebral disc. |
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