The Application Of Simvastatin For Repair Of Bone Defects And Promotion Of Osseointegration In Total Hip Arthroplasty

[Background]Bone defects and loosening of the prosthesis after total hip arthroplasty are two major challenges for today's orthopedic surgeons and research workers.Bone defects can be caused by infections, tumors, trauma, and a variety of congenital diseases. Autogenous bone, allograft bone and bone substitute materials were often used to repair bone defects in clinic. Limited sources, more postoperative complications and longer operative time limit the application of autograft bone graft for repair bone defect. Allograft bone graft has shortcomings of immunogenicity and pathogenicity. Bone substitute materials have shortcomings of slower absorption and poor Osteogenic potential. Funding of an ideal method for repair bone defect is imperative.Although total hip arthroplasty is a standard surgical technique, there are several complications associated with this procedure, including dislocation, infection, and loosening. Loosening, in which osteolysis attributed to particulate wear debris plays a key role, has emerged as one of the most frequent long-term complications of total hip arthroplasty, and is the most common indication for revision. In order to decrease the occurrence of loosening, many methods have been employed to enhance osseointegration, including surface coatings of implants and the utilization of growth factors.In clinical practice, recombinant human bone morphogenetic protein-2(rhBMP-2) has proved to be the most effective growth factor in this procedure; however, it has the disadvantages of a short shelf life, inefficient delivery to target cells, and high price. Finding effective and economic substitutes is imperative.The liposoluble statin, simvastatin (SIM), is an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, which is one of the rate-limiting enzymes of the mevalonate pathway, and has been widely used for lowering cholesterol and reducing heart attacks, thus providing an effective approach to the treatment of hyperlipidemia and arteriosclerosis. It has been reported that simvastatin can increase the expression of BMP-2mRNA in osteoblasts, promote bone formation. In addition, research has shown that simvastatin has a stimulatory effect on bone formation via osteoblastic differentiation of bone marrow stromal cells. Takenaka et al demonstrateed that simvastatin could stimulate bone formation via express of vascular endothelial growth factor (VEGF). Studies have confirmed that simvastation could stimulate bone formation in vivo.The aim of this study was to discuss the effects of simvastatin on repair of bone defect and promotion of osseointegration in total hip arthroplasty. This research is comprised of three sections. In section one and section two, macroporous CPCs were fabricated using SDS as a porogenic agent; Futher, SIM was introduced to enhance the osteoinductive properties of macroporous CPCs. The physical and mechanical characteristics of the test samples were investigated. Biological properties of the new CPCs were examined after intramuscular and endosteal implantation in rabbits. In section three, experiment was designed to evaluate whether simvastatin administered by injection could promote osseointegration in a canine total hip arthroplasty model.Section one:Simvastatin-loaded macroporous calcium phosphate cement:preparation, in vitro characterization[Objective]For preparation of SIM-containing CPCs, sodium dodecyl sulfate (SDS) as an air-entraining agent was added to the liquid phase and simvastatin (SIM) was homogenized with the solid phase. The initial setting time, macroporosity, Compressive strength, transformation of phase of the test samples were investigated.[Method]An aqueous solution of Na2HPO4with different amount of SDS as used as the liquid phase. Different amount of SIM was homogenized with the solid phase. The cement paste was made by mixing the powder and liquid phases at a powder-to-liquid ratio of2.5g/ml. The initial setting time was determined using a Gillmore needle according to the ASTM-C266-89standard. The macroporosity of the cement was determined using the density method. For the x-ray diffraction (XRD) analyses, the test specimens were removed from the physiological solution after three or seven days. Compressive test specimens were aged in the molds, held in air (37℃and100%relative humidity) for24hours. The morphology of the fracture surfaces of specimens used in the compressive test were examined using a scanning electron microscope.[Results]SDS had no significant effect on the cement's initial setting time, however could decrease the compressive strength significantly. On the fracture surfaces, there was evidence of spherical macroporous structures (50μm-120μm) homogeneously distributed in the case of CPCs that contained SDS. The macroporosity could reach to26.7%, when the concentration of SDS was300mM; The size of crystals was smaller as the reduction of concentration of SDS. SIM had no significant effect on the cement's initial setting time and macroporosity and did not lead to a significant decrease in the cement's compressive strength for a SIM content below10%; The SEM results showed that needle-like OHAp crystals were formed around and on the surface of the SIM rod. The XRD results showed that apatite was the predominant cement phase after seven days of soaking.[Conclusion]Introduction of SDS led to the incorporation of interconnected macrospores into the CPCs without significantly interfering with initial setting time, transformation of solid phase to hydroxyapatite, and biocompatibility. Regardless of the amount of SIM (1-10%wt) added to the CPC, it was compatible with the basic CPC. A large amount of SIM (10%wt) decreased the compressive strength of the cement. Section two:Simvastatin-loaded macroporous calcium phosphate cement: evaluation of in vivo performance[Objective]The short-term biocompatibility of porous/nonporous CPCs with or without SIM was examined using an intramuscular implantation model in rabbits. According the results of intramuscular implantation, specimens which have excellent biocompatibility and internal connection were selected for repair femoral condyle bone defect in rabbits.[Method]Sixty New Zealand White rabbits (mass:2.0-2.4kg) were randomly allocated into the twelve study groups. For each rabbit, an implant (nominal diameter and height of5mm and10mm, respectively) was inserted into the back muscles under general anesthesia. The implants inserted into the muscle, together with the surrounding tissues, were carefully taken out4weeks after surgery, and then decalcified, cut, and stained with hematoxylin and eosin. In sections made from intramuscular implants, special attention was given to the soft-tissue response and soft-tissue ingrowth into macropores. A histological grading scale for soft tissue implants was used to evaluate the response of the tissue surrounding the implant and at the implant surface.Twenty four New Zealand rabbits with an average weight of2.2kg were randomly divided into four groups (S0-M1, S0-M10, S300-M1, S300-M10; n=6rabbits in each group). For each rabbit, an implant (cylinder shape,5mm in diameter and10mm in height) was inserted into the back muscles in a manner similar to that described in Section2.3. The aim was to determine the concentration of SIM in blood with time, and the amounts remaining in the implants after rabbits were sacrificed at1day or30days.Twenty-four New Zealand White rabbits (average mass:3.0kg) were used. The animals were randomly divided into three groups of eight rabbits each (untreated group, S300-M0, and S300-M1). The cement paste was delivered into the trabecular bone of the femoral condyles under general anesthesia. The harvested cement cylinders in the bone defects were stained with modified ponceau trichroism bone stain and V-G stain. [Results]With the same amount of SIM, the reduction rate of total cholesterol concentration increased as macroporosity increased. With the same macroporosity, the reduction rate of total cholesterol concentration increased as the content of SIM increased. The tissue response to pure and porous CPCs without SIM or with1%SIM was similar. A connective tissue layer was observed to have formed around the implants and no inflammatory reaction was found. A thicker fibrous encapsulation and mild interaction were seen in cements containing5%SIM with or without SDS. When the content of SIM reached10%, a necrotic muscle layer could be detected around the cement and infiltrating inflammatory cells were observed between the necrotic muscle layer and normal muscle layer.The circulating blood concentrations of SIM in groups S0-M1and S300-M1were undetectable in the serum after5days. Significant differences were observed in the release values between the high dosage groups (S0-M10, S300-M10) and the low dosage groups (S0-M1, S300-M1). The release values of SIM from the microporous specimens were significantly lower than those of the porous samples. It can be seen that a large quantity of SIM remained after1day, of the order of71.6%,75.6%,82.2%and87.5%for S300-M1, S0-M1, S300-M10and S0-M10specimens, respectively. At the end of the experiment, the residual amounts of SIM in microporous specimens were higher than those in porous samples that contained the same amount of SIM. The residual percentage of SIM in the high dosage groups (S0-M10, S300-M10) were significantly higher than those in the low dosage groups (S0-M1, S300-M1).Bone ingrowth from the edge of the bone defect into the macropores of the cement was present in all implants (S300-M0, S300-M1). Remarkably, S300-M1implants were surrounded by a layer of primary bone outside the cement. The defects that had received no implant (untreated group) showed minimal bone formation at the defect borders. Histomorphometrical evaluation showed that the newly formed bone area of S300-M1(7.4±3.3%) was significantly higher than that of S300-M0(3.6±1.4%; p <0.05). The BIC was significantly higher in S300-M1(78.4±23.5%) than in S300-M0(54.3±14.6%; p<0.01). [Conclusion]Macroporous CPCs based on the use of SDS as an air-entraining agent are biocompatible in vivo. A large amount of SIM (10%wt) induced severe muscle necrosis, and produced an inflammatory reaction. Small amounts of SIM (1%wt) did not significantly affect biocompatibility of the CPCs, and was sufficient to enhance the osteoinductive potential of macroporous CPCs. Section three:Effects of simvastatin on osseointegration in a canine total hip arthroplasty model:An experimental study[Objective]The present study was designed to evaluate whether simvastatin administered by injection could promote osseointegration in a canine total hip arthroplasty model.[Method]Fifteen mongrel dogs were randomly divided into three groups of five dogs each (high dosage group, low dosage group and control group). High dosage group dogs received6.0mg/kg/day subcutaneous injections of simvastatin for30days. Low dosage group dogs received3.0mg/kg/day ubcutaneous injections of simvastatin for30days. Dogs in the control group received3.0mg/kg/day of isotonic saline. Blood samples were obtained to measure total cholesterol level before and after simvastatin administration. After12weeks, all dogs were sacrificed. Their femurs were removed and soft tissues were dissected. Femurs of10mm thickness were cut through the transverse plane5mm below the entotrochanter for histomorphometric analysis and mechanical testing. After being sputter-coated with a thin layer of carbon, the surface of the detached femoral component was examined using a scanning electron microscope. The atomic composition of the material covering the implant was determined by using an energy-dispersive spectrometer. [Results]There was a significant decrease in total cholesterol level in high dosage group (32%, P<0.05) and low dosage group (19%, P<0.05). The results of the push-out test showed that the shear strength of high dosage group (3.1±0.3MPa) was significantly higher than that of low dosage group (2.2±0.1MPa, P<0.05) and the control group(1.69±0.1MPa, P<0.01)at12weeks postoperatively. SEM showed that there was high density material deposited on the surface of the femoral component in all three groups.The area of femoral component covered by high density material was greater in high dosage group than in both low dosage group and the control group.The EDS results showed that the deposited material consisted mainly of the following elements:nitrogen, sulfur, calcium, phosphorus, and oxygen. The BIC was significantly higher in high dosage group (55.7%±4.0%) than in low dosage group (37.3%±4.2%, P<0.05)and the control group (30.5%±4.2%, P<0.01).[Conclusion]It seems reasonable to assume that systemic application of simvastatin by injection administration could stimulate osseointegration around implants in a dosage-dependent pattern without serious adverse reactions.Beyond that, clinical trials are needed to determine the effectiveness and optimum dosage schedule of simvastatin for enhancing osseointegration in humans.