The Effect Of RhBMP-2Slow Release Formulation On Osteolysis Induced By Chromium Particles

BackgroundCurrently, artificial joint replacement is the effective method for treatment of bone and joint late diseases, which can effectively relieve joint pain, recover the function of joint and improve quality of life in patients. At present, more than2million people underwent artificial joint replacement in the whole world every year, and about20%of them required revision surgery. There are many common causes about joint revision surgery, such as infection, prosthesis aseptic loosening, periprosthetic fractures, joint instability and limited joint mobility, etc. The aseptic loosening caused by periprosthetic osteolysis is one of the main reason of Shortening the service life of artificial joint. With widely application of joint arthroplasty and growing numbers of patients who have undergone joint revision surgery, more and more attention has been focused on it. In order to relieve the pain and improve the quality of life, the advanced patients must have joint revision surgery, which has big difficulty in operation, high risk and much more cost.Therefore, how to find a kind of non-operation method for the prevention and treatment of periprosthetic osteolysis has become a very urgent and realistic problem.Maintenance of normal structure and function in bone tissue is a dynamic balance process and this balance is consist of bone formation and bone resorption metabolism. A certain amount of bone resorption will have equal amounts of bone formation. RANKL-RANK-OPG system is an important signal conditioning system in the process of osteoclast differentiation, activation and apoptosis. This system is not only involved in the regulation of physiological bone reconstruction, but also closely related to the occurrence of multiple bone diseases under pathological conditions. Recetor activatorof nuclear factonквligand (RANKL) combined with recetor activatorof nuclear factonкв(RANK) to induce osteoclast differentiation and osteolysis. Osteoprotegerin (OPG) can inhibit the effects of RANKL and the activity of osteoclasts to promote osteogenesis. Osteoblasts and bone marrow stromal cells express a certain amount of RANKL, which promote osteoclast differentiation and bone resorption and secretion of OPG to prevent the excessive bone resorption in physiological conditions. The coordination in RANKL/OPG rate is a key to maintain metabolism balance of bone. The rate of RANKL/OPG or RANK signal imbalance has become pathologic basis of many bone diseases, which may manifest as excessive bone loss and formation or disorders of bone remodeling. Thus it can be seen that RANKL and OPG is equally important to bone resorption, RANKL and OPG imbalance is the critical reason of osteoclast differentiation, massive osteolysis and bone loss. So the balance of RANKL/OPG in the periprosthetic tissue can reflect the condition of periprosthetic bone metabolism.Artificial joint interface can produce polyethylene, bone cement and metal particles because of the long-term friction. These wear particles can release a lot of osteolytic factors, such as:IL-1β,IL-6,TNF-a,etc. These osteolytic factors can stimulate RANKL gene expression in osteoclast precursor cells and inhibit the OPG production, which can cause the RANKL-RANK-OPG system imbalance in periprosthetic bone tissue. It also can promote osteoclast differentiation, activation and proliferation, then decrease osteoblast proliferation and extracellular matrix secretion by increasing the RANKL/OPG rate, result in increased periprosthetic osteolysis and decreased osteoblast, eventually lead to aseptic loosening of prosthesis. Therefore, elimination and reversal of the RANKL/OPG rate imbalance in RANKL/RANK/OPG system may be therapeutic targets for the treatment of aseptic loosening targets.Bone morphogenetic protein(BMP) is the strongest material for promoting osteogenesis at present, which can induce cells transformation from bone to cartilag. BMP belongs to the transforming growth factor-β(TGF-P) superfamily members, more than40kinds of BMP has been found, among which, the BMP-2is a important promoting factor in osteogenesis with the highest osteogenic activity. It can induce proliferation and differentiation of undifferentiated human mesenchymal stem cells to osteoblasts and promote osteogenesis. BMP-2regulate osteoblast and osteoclast differentiation, proliferation and functional activity in bone metabolism. It also can transmit information between cells and stroma by the way of autocrine, paracrine and cell adhesion and regulate differentiation and proliferation of bone cells. BMP-2plays an important role in bone reconstruction. The major roles of BMP-2pathway are as follows:BMP-2up-regulate the expression of inhibition of Smad protein through signal transduction in the extracellular and combination of Smad in the interior of the cell. These Smad proteins are transferred into the nucleus after phosphorylation, in order to regulate target gene and other transcription factors (including PEBP2alpha/Cbfal) and Smad protein stimulated by BMP-2singal coordinately induce expression of osteoblastic phenotype-related genes, then promote the differentiation and proliferation of osteoblasts. BMP-2is a important bone formation transcription factor in the process of bone metabolism because it involved in all stages of bone metabolism. BMP-2induce periosteum and bone marrow stromal cells to differentiate into osteoblasts and enhance bone regeneration, moreover, it activate or induce the perivascular mesenchymal cells to differentiate into chondrocytes and osteoblasts. In recent years,there are more and more studies about BMP-2and more and more attentions have been paid to the regulating effect on bone metabolism. Therefore, BMP-2may be the effective target of clinical treatment of aseptic loosening caused by metal wear particles.In this article, we evaluate the bone resorption of rat skull by use of the rat bone-implanted air pouch models of aseptic loosening induced by wear particles.The computer image analysis technique have been used for the determination of the ratio of osteoclasts was confirmed by TRAP staining in rat skull. We detect the RANKL and OPG expression by using the method of Western-blot and RT-PCR for assessing bone metabolism rate, we observe rhBMP-2on bone resorption and the expression of OPG and RANKL in the model and explore the rhBMP-2in the role of aseptic loosening in order to search for a new method in prevention and treatment of it.Objective1. To observe the effects of different concentrations of rhBMP-2on the bone resorption and the expression of RANKL、OPG and then evaluate the effects of rhBMP-2on anti-osteolysis in animal models by using the rat bone-implanted air pouch models and different concentrations of rhBMP-2intervention.2. To explore the meaning and feasibility of the rhBMP-2in the prevention and treatment of osteolysis.Method50SD rats were randomly divided into5groups (the blank group, chromium particles group,50μg/ml rhBMP-2slow-released system+CR particle group,100μ g/ml rhBMP-2slow-released system+CR particle group and200μg/ml rhBMP-2slow-released system+CR particle group), other10SD rats were used as a peripheral bone graft donor. The airbags were constructed on the back of rats by filtering air20ml, every2day1times and total3times. If there is no marked inflammatory response such as redness, pus, induration and seeping at the injection site after one week, it can Indicates that the animal airbag model was successfully constructed, other10rats were killed and taken the skull, repaired and maintained into the blade approximately10mm×10mm size, renovated of soft tissue and cleaned around to protect the periosteal bone surfaces. The rats with successful modeling were under intraperitoneal anesthesia and then dressing good bone implants in the airbag. The model successfully after24hours, the rats in blank group were injected with6ml saline in the airbag, the rats in chromaffin granules group were injected with2ml chromium particles suspension and4ml saline, the rats in50μg/ml rhBMP-2slow-released system+CR particle group,100μg/ml rhBMP-2slow-released system+CR particle group and200μg/ml rhBMP-2slow-released system+CR particle group were not only injected with2ml chromium particles suspension in airbag, but also injected with4ml rhBMP-2slow-released system fluid suspension after ultrasonic dispersion with the concentration of50μg/ml,100μg/ml, 200μg/ml. The rats were killed and taken the tissues from airbag after2weeks normal feeding. The image-pro-plus5.0have been used for the determination of the ratio of osteoclasts was confirmed by TRAP staining in rat skull. We detected the RANKL and OPG expression by using the method of Western-blot and tested the expression of RANKLmRNA、OPGmRNA by RT-PCR. Recording test results for each group.Data were statistically analyzed with the software package SPSS13.0, The mean value of each group was checked by one-way ANOVA and P<0.05was considered significant difference, evaluated the effect of rhBMP-2slow-released system on bone metabolism.Results1. The Western-blot results showed:There are expression of RANKL and OPG in each group of models. Expression of RANKL and OPG and the rate of RANKL/OPG in chromium particles group were the highest in the five groups (P<0.05). In the rhBMP-2slow-released system intervened groups, these were decreased with the concentration increased of rhBMP-2slow-released system, all the differences between groups have statistical significance (P<0.05); there were no obvious difference between the blank group and200μg/ml rhBMP-2slow-released system+chromium particles group (P>0.05).2. The resules of the ratio of TRAP stained osteoclasts by using image-pro-plus5.0showed:TRAP stained osteoclasts can be found in each group of models. The ratio of TRAP stained osteoclasts in chromium particles group were the highest in the five groups (P<0.05). In the rhBMP-2slow-released system intervened groups, these were decreased with the concentration increased of rhBMP-2slow-released system, all the differences between groups have statistical significance (P<0.05); there were no obvious difference between the blank group and200vg/ml rhBMP-2slow-released system+chromium particles group(P=0.844, P>0.05).3. The RT-PCR results showed:There are expression of RANKLmRNA and OPGmRNA in each group of models. Expression of RANKLmRNA and OPGmRNA and the rate of RANKLmRNA/OPGmRNA in chromium particles group were the highest in the five groups (P<0.05). In the rhBMP-2slow-released system intervened groups, these were decreased with the concentration increased of rhBMP-2slow-released system, all the differences between groups have statistical significance (P<0.05); there were no obvious difference between the blank group and200μg/ml rhBMP-2slow-released system+chromium particles group (P>0.05).Conclusion1. Chromium particles can cause increased the rate of RANKL/OPG in the tissue within the rat bone-implanted air pouch models and promote osteolysis. This model has simulated the biological and histological characteristics of joint prosthesis aseptic loosening.2. According to the experiment, there was a positive correlation between the ratio of TRAP stained osteoclasts and the RANKL/OPG rate. This is also further proof that the rate of RANKL/OPG in the RANKL/RANK/OPG system can reflect the bone metabolism.3. Within a certain concentration range, rhBMP-2slow-released system can efficiently promote bone formation and inhibit bone resorption by significantly decreasing the RANKL/OPG rate in the tissue within the rat bone-implanted air pouch models.