| BackgroundSince arthroplasty used clinically, it has become the main method of reconstructing joint function and achieved good results. However, with expansion of application, increasing number of operation and extension of service life,-the postoperation complications also increased. Among them, prosthesis aseptic loosening is the most common complication on the later period of artificial joint replacement. The factors which cause prosthetic loosening are mainly divided into two categories:one is mechanical factors, that is stress shielding around prosthesis, early loosening, prosthesis micromove, joint hydraulic pressure, loosed fixation of prosthesis, etc. Another is biological factors, which refers to that wear particles are generated in utilization and osteolysis is caused by a series of related biochemical reactions. At present, in the mainstream view was proposed the concept of "particles disease". It proposed that artificial joints produce a lot of wear particles, the wear particles stimulate monocytes-macrophages, osteoblasts, fibroblasts to produce osteolytic factors. Thereby it activates periprosthetic osteoclasts, produces bone resorption and bone dissolution. Then the gap appears between the prosthesis and the surrounding bone, so that the prosthesis loses support, and eventually lead to artificial joints aseptic loosening. The loosening artificial joints make further exacerbated wear. Thus formed a vicious cycle from wear to loosen.Wear particles include polyethylene particles, cement particles, ceramic particles and metal particles. There are many researches about polyethylene particles and bone cement particles. Titanium is common material for artificial joint prostheses and fracture internal fixation. Fretting between femoral stem and cement and bone interface, friction between femoral stem and bone, fretting between micro-porous coating, fretting between screws and acetabular cup, are the main source of titanium particles. So titanium particles is one of common metal particles after artificial joint replacement surgery. With the spread application of the new generation of metal-on-metal prosthesis, the periprosthetic osteolysis role of metal particles has gradually attracted people’s attention.When it refers to the effect of titanium particles on bone metabolism, the present study focused on osteoclasts. Studies suggest that the titanium particles can cause osteolysis by activation of osteoclasts. However, bone metabolism balance is a result of osteoclasts and osteoblasts joint action. As the main functional cells with osteogenic potential, osteoblasts are capable of synthesizing and secreting a variety of type I collagen and non-collagen protein, secreting mineralized substance as osteoid mineralization substances and alkaline phosphatase, thus promoting new bone formation. At the same time wear particles activate osteoclasts activity, inevitably closely contact with large amount of osteoblasts surrounding around. There is no in-depth study about whether it will effect the biological behavior of osteoblasts.Wear particles stimulate cells of membrane arround the prosthesis to release large amounts of osteolytic factors, which constitute a complex network inbody, and are involved in the bone resolved metabolic process around prosthesis alone or together. IL-6is mainly synthesized by osteoblasts, its primary role in the pathogenesis of osteoporosis is stimulates the original osteoclasts to differentiate into mature osteoclasts, increases the number of osteoclasts in trabecular bone, promote osteoclast differentiation, maturation, and by increasing the release of collagenase to promote bone matrix degradation, so that to increase bone resorptionand decrease BMD. IL-6as an important inflammatory mediator produced by stimulating wear particles, it involves in the pathological process of dissolution of bone around the prosthesis. In the synovial; fluid of the the patients who occurred prosthesis loosening after total hip arthroplasty, the levels of IL-6elevated, and therefore the increasing level of IL-6can be considered as the biomarker of bone dissolved around atificial prosthesis.Receptor activator of nuclear factor-KB belongs to type I transmembrane protein, it is one of the TNF family members. RANK is highly expressed on the surface of the osteoclast precursor cells. Receptor activator of nuclear factor-KB factor ligand binds to RANK expressed on the surface of osteoclast precursor cells and mature osteoclasts, promote the differentiation of osteoclasts and inhibit its apoptosis. Osteoprotegerin as a decoy receptor, competitively binding with RANKL, and thereby blocking the binding of RANKL to RANK on osteoclasts surface, inhibitting osteoclast maturation. The change of RANKL/OPG ratio is essential for oosteoclasts production. In general, when the RANKL/OPG ratio upregulates, the number of osteoclasts and their activity will increase, when the RANKL/OPG ratio downregulates, the number of osteoclasts and their activity will decrease. Therefore, the bony remodeling and the stability of bone mass around prothesis depended upon the balance between OPG and RANKL.At present, the treatment for joint prosthesis aseptic loosening is inhibitting osteolysis and bone resorption, including the control of osteolytic factors and osteoclasts. However, osteolysis is resulted from the co-effects of various factors through various methods. It is difficult to take complete control of prosthesis loosening by medicine which inhibits solo osteolytic factor. Some medicine which inhibit osteoclasts, such as calcitonin, would be ineffective after taking for a period of time, while the long-term utilization of bisphosphonate will lead to various serious toxic and side effects and the expensive price limits its application. Therefore, it is necessary to conduct further research.Prosthesis aseptic loosening is caused by the imbalance between bone formation and bone resorption. The reduction of bone formation also has important influence on prosthesis aseptic loosening. It may be another effective method to conduct treatment of joint prosthesis aseptic loosening by applying medicines to prompt bone formation around loosening prosthesis and make prosthesis biologically fixed. However, few related researches are at present. Icariin is a kind of "bone reinforcement" Chinese medicine, which can induced bone marrow mesenchymal stem cells to differentiate into osteoblasts, improve ALP activity, increase calcium salt content, prompt osteocalcin secretion, increase the number of calcified nodules and upregulate the expression of factors related to bone-formation, such as Runx-2, bFGF-1and Osterix etc. Icariin can prompt the recovery of bone fracture and increase bone mass of patients of osteoporosis. And it has a wide range of source, low cost, easy preparation, small side effects, high safety and incomparable advantages of traditional medicines, therefore it has been widely used for the treatment of osteoporosis. The pathological process of joint prosthesis aseptic loosening is similar to osteoporosis, both of them have decreased bone formation. For those osteoblasts around the loose prosthesis, whether icariin can promote bone formation, provide protective effect? In this study we observe the effects of icariin on osteoblasts stimulated by titanium particles in vitro, so as to provide experimental foundations for further animal experiment. Ojectives1、Study the effect of titanium particles on proliferation, phenotype and morphology of osteoblasts.2、Study the effect of icarrin on proliferation, differenciation of osteoblasts stimulated by titanium particles.3、Study the effect of icarrin on osteolytic factor IL-6secretion of osteoblasts stimulated by titanium particles.4、Study the effect of icarrin on OPG, RANKL mRNA expression of osteoblasts stimulated by titanium particles.Methods1、Calvarial osteoblasts of newborn rats were seperated in vitro by repeated enzyme digestion, and received primary culture. Alkaline phosphatase staining was carried out to identify them. Titanium culture medium was prepared and limulus assay was performed to exclude excessive endotoxin level. Titanium particles with concentrations of0.01,0.05,0.1,0.5as well as1mg/ml were respectively added to the third generation osteoblasts well grown(n=6). After cultured for seven days, OD was detected by CCK-8method to compare the influence of different titanium particles concentrations on osteoblasts proliferation. ALP activity and type I collagen expression were detected by ELISA to observe the effect of titanium particles on differentiation and phenotype of osteoblasts(n=6). The cells received double fluorescence staining with FITC-phalloidin and PI to observe cellular morphology variation of osteoblasts which swallowed titanium particles under laser scanning confocal microscope.2、Titanium particles were used to treat rats osteoblasts, and icariin with concentrations of10-5,10-6，10-7,10-8,10-9,10-10mol/L were added for interference respectively(n=6). The effect of icariin on proliferation of osteoblasts stimulated with titanium particles was observed after cultured for seven days, and the optimum drug concentration was screened out. Icariin with optimum drug concentration was added to intervene osteoblasts stimulated with titanium particles(n=6), ELISA method was carried out to detected ALP activity and type I collagen expression on the3rd7th、14th day after cultur, calcified nodules were stained by alizarin S red stain method on the21th day.3、The rats osteoblasts cultured in vitro were divided into five groups:the first group, the control group, osteoblasts; the second group, the tatinium particlesgroup, osteoblasts plus titanium particles; the third group, osteoblasts plus titanium particles plus10-10mol/L icariin; the fourth group, osteoblasts plus titanium particles plus10-9mol/L icariin; the fifth group osteoblasts plus titanium particles plus10-8mol/L icariin(n=6). They received differern intervene measures. After cultured for seven days, supernatant was extracted, and ELISA method was performed to detect IL-6concentration in the supernatant of each group.4、The rats osteoblasts cultured in vitro were divided into four groups:the first group, the control group,osteoblasts; the second group, titanium particles group, osteoblasts plus titanium particles; the third group, osteoblasts plus icariin; the fourth group, osteoblasts plus titanium particles plus icariin(n=6). Each group received differern intervene measures. After cultured for seven days, RT-PCR was carried out to detect relative expression of OPG and RANKL mRNA and RANKL/OPG ratio variation of each group.Results1、The differences of osteoblasts proliferation activity within each group was statistically significant (F=517.480, P=0.000). Different concentrations of titanium particles could inhibit the proliferation of osteoblasts, which had a significant difference (P<0.05) compared with the control group. The inhibition effect of titanium particles with concentration of lmg/ml on osteoblast proliferation activity was greater than those of titanium particles with concentration of0.01,0.05,0.1,0.5,1mg/ml (P<0.05). The effect of of titanium particles on osteoblast proliferation activity showed no significantly difference between concentration of0.01mg/ml and0.05mg/ml (P=0.240). The differences of osteoblasts ALP activity and type I collagen expression within each group was statistically significant (ALP:F=828.024, P=0.000; type I collagen:F=2258.835, P=0.000). Different concentrations of titanium particles could significantly inhibit ALP activity and type I collagen expression of osteoblasts, there was significant difference (P<0.05) compared with the control group. The inhibition effect of titanium particles with concentration of lmg/ml on osteoblast ALP activity and type I collagen expression was greater than those of titanium particles with concentration of0.01,0.05,0.1,0.5,1mg/ml (P<0.05). The effect of of titanium particles on osteoblast ALP activity and type I collagen expression showed no significantly difference between concentration of0.01mg/ml and0.05mg/ml (P=0.240). Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts swallowed with titanium particles.2、The differences of osteoblasts proliferation activity within each group was statistically significant (F=829.652, P=0.000). Icarrin with concentrations of10-10-10-6mol/L could inhibit decreased proliferation ability of osteoblasts stimulated by titanium particles, which showed significant difference compared with the titanium particles group (P<0.05).10-8mol/L was icarrin’s optimum drug concentration, which had significant differences compared with other various concentrations of icariin groups (P<0.05). When the concentration of icariin rise to10-5mol/L, it inhibitted the proliferation of osteoblasts (P<0.05). Factorial analysis showed that there was significant differences of ALP concentration within different groups (Fgroup=445.548, P=0.000), so were different time points (Ftime=194.242, P=0.000), and there existed interaction (Fgroup×time=81.439, P=0.000). One-way ANOVA analysis showed that, there were significant differences within different groups of the same time point (3d:F=18.699, P=0.000;7d:F=129.110, P=0.000;14d:F=459.210, P=0.000), so were different time point of the same group (control: F=130.968, P=0.000; Ti:F=23.070, P=0.000; ICA:F=232.660, P=0.000; Ti+ICA: F=43.024, P=0.000). There were significant differences of type I collagen expression within different groups (Fgroup=1843.514, P=0.000), so were different time points (Ftime=341.296, P=0.000), and there existed interaction (Fgroup×time=250.979, P=0.000). One-way ANOVA analysis showed that, there were significant differences within different groups of the same time point (3d:F=86.658, P=0.000;7d: F=923.149, P=0.000;14d:F=1293.625, P=0.000), so were different time point of the same group (control:F=268.229, P=0.000; Ti:F=206.053, P=0.000; ICA: F=519.974, P=0.000; Ti+ICA:F=120.072, P=0.000). Pairwise comparisons showed that, ALP activity and type I collagen expression of osteoblasts in titanium particles group on3d,7d,14d are lower than that of control group during the same period (P<0.05). while ALP activity and type I collagen expression of osteoblasts in titanium particles+icarrin groups on7d,14d.are higher than that of titanium particles groups during the same period (P<0.05). The role of icariin had a time-effect relationship,, ALP activity and type I collagen expression of titanium particles+icarrin reached the highest on7d,which had significant difference compared with that of3d and7d (P<0.05). Alizarin red stain showed that the amount of cacified nodules and distribution area of osteoblast were higher in icarrin intervene group than in titanium particles group.3The differences of IL-6expression of osteoblasts within each group was statistically significant (F=7202.778, P=0.000). Compared with the control group, titanium particles obviously promoted the osteoblasts’expression of IL-6(P<0.05). Different concentrations of icariin could significantly inhibit IL-6expression mediated by titanium particles, which was significant different compared with the titanium particles group(P<0.05). The inhibition effect of icarrin with concentration of108mol/L on IL-6expression was greater than those of icarrin with concentrations of10-10、10-9mol/L(P<0.05).4、The differences of OPG mRNA、RANKL mRNA expression of osteoblasts and RANKL/OPG ratio within each group were statistically significant (OPG:F=4277.834P=0.000; RANKL:F=5599.081P=0.000; RANKL/OPG: F=1334.919P=0.000). Compared with the control group, OPG mRNA expression decreased, RANKL mRNA expression increased, and RANKL/OPG ratio rised in titanium particles group, which showed statistic difference (P<0.05). After icarrin intervention, OPG mRNA expression rised, while RANKL mRNA expression decreased, and RANKL/OPG ratio declined, which showed statistic difference compared with the titanium particles group (P<0.05)Conclusions1-, Titanium particles can inhibit the proliferation and differenciation ability of cultured rat osteoblasts in vitro, decreased bone formation. Titanium particles can be phagocytosed by osteoblasts and make changes in cell morphology and cytoskeleton.2、Icariin can reverse the inhibition of effect of titanium particles on osteoblasts proliferation, differentiation, maturation and mineralization, maintain cell phenotype and promote bone formation.3、Icariin can inhibit titanium particles-mediated osteoblasts’secretion of osteolytic factor IL-6, thus inhibit osteolysis.4、Icariin can regulate osteoblasts OPG, RANKL mRNA expression mediated by titanium particles, thus inhibit differenciation and maturation of osteoclasts.. |
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