Investigation On Special Application Of Bisphosphonates And Sonodynamic Effect In Jicrylic Cement Implementation

Particle-induced osteolysis is a major cause of aseptic loosening after total joint arthroplasty. Bisphosphonates are commonly used to treat conditions associated with bone loss, such as osteoporosis, Paget's disease and skeletal carcinomatosis. Recently, several studies have shown that bisphosphonates can inhibit wear debris-induced osteolysis and increase peri-implant bone mineral density (BMD). However, side effects like fever, throat and stomach ulcers, and low bioavailability were observed after systemic treatment. Thus, bisphosphonate-loaded acrylic bone cement was proposed as local delivery system to reduce the risk of osteolysis and resulting prosthetic loosening. This proposal is similar to the addition of antibiotic to bone cement against infection. However, studies showed that liquid form of bisphosphonate led to incomplete polymerization of bone cement and weaken its mechanical strength. Powdered bisphosphonates proved to be a superior choice. Lewis et al. found that given the addition of0.21g of alendronate powder into50g of cement powder, there was no significant reduction in the fatigue life.Unfortunately, previous studies never focused on the effect of alendronate cement on wear debris-induced osteolysis in vivo. Also, it should be noted that bisphosphonates added to bone cement might reduce the mechanical properties of bone cement and led to the failure of the total joint replacement. Several studies have shown that liquid form of a bisphosphonate led to incomplete polymerization of acrylic bone cement and significantly reduced acrylic bone cement mechanical properties. Therefore, the aim of the present study is to comprehensively investigate the effect of drug load and fluid environment on the static mechanical properties, fatigue life, and micro structure of purified-alendronate-impregnated acrylic bone cement (PAIBC) in vitro and the related mechanism. In addition, the anti-resorptive efficacy of alendronate cement would be compared with systemic alendronate.Implant-related infection has been a devastating disaster for patients undergoing joint arthroplasty. Antibiotic-loaded bone cement is proven a keystone against the infection since it can provide high local delivery of antibiotic. Two categories are included based on the purposes:the prophylactic cement refers to prophylaxis against the infection with a low antibiotic load during a primary arthroplasty or a revision arthroplasty, whereas the therapeutic cement means curing the infection by using beads or spacers with a high antibiotic load between implant removal and reimplantation. High susceptibility to staphylococci, rare resistant strains, and weak toxicity render vancomycin typically the last resort or the drug of choice to be combined with aminoglycosides for loading into the therapeutic cement. Unfortunately, the therapeutic cement has met with mixed results. The relapse rate of the infection was reported to9%～18%. Such problem was mainly attributed to the poor antibiotic release from cement, of which vancomycin was one of the poorest. The length of time when local drug level exceeded the MIC (T>MIC), a key indicator for the antimicrobial activity of vancomycin-loaded bone cement (VLBC), was far from the required time for effective killing of bacteria. Furthermore, the long-term subtherapeutic release of antibiotic from cement will definitely select resistant strains. Anagnostakos et al. have shown that after prolonged time periods bacteria can even colonize gentamicin-vancomycin-loaded bone cement.Ultrasound, a popular medical device for sonoporation and bone healing, has been found to enhance antibiotic release from acrylic bone cement. However, the enhancement by previous milliwatt-level ultrasonication was confined in the early period of drug release. Then the burst release waned quickly to a sustained slow release, which did not contribute to adequately enhancing the drug release, extending the T>MIC, and avoiding drug-resistant strains. Furthermore, the mechanisms of improved drug release from cement are poorly understood. The nonthermal effect of ultrasound, mainly the stable cavitation and the radiation pressure, generates multidirectional acoustic microstreams which produces a high shear stress at drug-cement interfaces, allowing detachment of drug grains from the surface and pushing solution into acrylic matrix via craters and channels. Although it was reported that low-frequency pulsed wave ultrasound was found to be safe in vivo experiments, there were few reports on the effects of low-frequency pulsed wave ultrasound on the shear strength on at the cement-stem interface. Studies have suggested that the loosening failure of cemented stems was initiated by debonding of the stem-cement interface because of a lack of chemical bonding, and the debonding at the interface is was primarily dominated by shear failure as proven with Finite Element Analysis. In the present study the kinetics of vancomycin release and the surface microstructure of VLBC under the watt-level ultrasonication with and without the pause insertion were contrasted. The shear strength, porosity and the extent of penetration of fluid into the cement-stem interface were determined and contrasted among the control group and ultrasound groups. Finally, the involved mechanisms were discussed. Part1Effects of local and systemic alendronate delivery on wear debris-induced osteolysis in vivoObjective:To investigate the effects of locally and systemically administered alendronate on wear-debris induced osteolysis in vivo.Methods:Endotoxin-free titanium particles were injected into rabbit femurs, prior to insertion of a non-weight-bearing polymethylmethacrylate plug into the distal femur canal. Then the particles were repeatedly injected into the knee2,4and6weeks after the implantation. Alendronate was incorporated at three different concentrations (0.1wt%,0.5wt%and1.0wt%) into bone cement for local delivery. For systemic delivery, alendronate was subcutaneously injected (1.0mg/kg/week) one week after the implantation and then once a week until sacrifice.Results:Eight weeks postoperatively, there was significant evidence of osteolysis surrounding the plug in the control group compared with markedly-blocked osteolysis in the local0.5wt%, local1.0wt%, and the systemic group. There was a concentration-dependent effect of alendronate on the improvement of periprosthetic bone mineral density. Notably, no significant difference was found between local0.5wt%alendronate and systemic alendronate in bone mineral density and implant fixation.Conclusions:Alendronate-loaded bone cement (0.5wt%) may be as effective as the systemic alendronate in inhibiting titanium particle-induced osteolysis. Part2Mechanical properties and micro-architecture of an acrylic cement impregnated with purified alendronate powderObjective:To investigate the static and fatigue properties of purified-alendronate-impregnated acrylic bone cement (PAIBC) in vitro, the effect of powder load and PBS immersion on biomechanics of PAIBC, and its related mechanism.Methods:PAIBC specimens were successfully manufactured for in-vitro assay, and randomly assigned to eight groups based upon the aging condition and the mass ratio between drug and cement (n=31-37):the control air group, the control PBS group, the0.1%PAIBC air group, the0.1%PAIBC PBS group, the0.5%PAIBC air group, the0.5%PAIBC PBS group, the1%PAIBC air group, and the1%PAIBC PBS group. According to the corresponding ASTM/ISO standards, their ultimate compressive. strength, compressive elastic modulus, ultimate tensile strength, ultimate flexuous strength, and fatigue life were tested systematically. The particle size distribution of purified alendronate powder was analyzed with the laser light scattering method. The fatigue test results, given as number of cycles-to-failure, were analyzed using the linearized format of the two-parameter Weibull function. The parameters of Weibull slope, Weibull characteristic fatigue life, Weibull mean number of fatigue stress cycles, and fatigue performance index were all calculated.Results:With drug load increased, there was an increase in ultimate compressive strength and ultimate tensile strength, and a decrease in fatigue life with statistical significance. When immersed in PBS for thirty days before the tests, the PAIBC specimens presented an overall significant decrease of ultimate compressive strength, compressive elastic modulus, ultimate tensile strength, and fatigue life. No effect of drug load or PBS immersion was noted on ultimate flexuous strength. The Weibull mean of fatigue life dropped by22.1%-26.1%after atmospheric aging and by 16.7%-18.9%after wet aging. The profile of particle size distribution presented a normal distribution. The diameter of drug particle ranged from4.67μm to44.21μm, with a mean diameter of20.77μm.Conclusions:Purified alendronate was a more homogeneous powder than tablet-ground powder, which contained particles with a lower range of sizes. The impregnation of purified bisphosphonate exerted less negative effect on the static and fatigue strength of acrylic bone cement, compared with liquid bisphosphonate and the tablet-ground one in literatures. The static strength of PAIBC was maintained high above the ASTM/ISO standards. Our study laid a biomechanical foundation for the potential clinical use of PAIBC. Part3Enhancement of intermittent insonation with low-frequency watt-level ultrasound on pharmacokinetics of vancomycin-loaded acrylic bone cement in vitroObjective:To investigate the effect of intermittent insonation on pharmacokinetics and microstructure of vancomycin-loaded acrylic bone cement (VLBC) in vitro under low-frequency elevated-intensity ultrasound, and its related mechanism.Methods:Twenty-one VLBC cylindrical specimens were successfully manufactured for in-vitro assay, and randomly assigned to three groups (n=7):the control group, the continuous-insonation group and the intermittent-insonation group. All specimens, from which both insonation groups were insonated for14d, were immersed in40-ml PBS. During28-d observation PBS from four specimens per group was extracted and analyzed with fluorescence polarization immunoassay at designated time intervals, respectively. Four pharmacokinetic parameters, the duration of time for which antimicrobial concentration exceed the minimum inhibitory concentration (T>MIC), the drug release during the first day, the drug release between14d and28d, the cumulative release during14d, were all calculated from plots of drug-release-versus-time curve. Exponential-rise-to-maximum model was adopted to fit the plots of drug cumulative release. After immersion of14d, three specimens were dehydrated, desiccated and coated with a thin layer of gold. Their surface microstructures were analyzed with scanning electron microscopy.Results:T>LMIC of the intermittent-insonation group increased by6.56d and7.09d compared with the control group (P<0.05) and the continuous-insonation group (P<0.05), respectively. However, T>MIC of the continuous-insonation group was lower than the control group by0.53d (P<0.05). The total release of intermittent-insonation group at sub-inhibitory drug level decreased by0.16mg and0.19mg compared with the control group (P<0.01) and the continuous-insonation group (P<0.01), respectively. The cumulative release of intermittent-insonation group during14d increased by2.88mg and2.81mg compared with the control group (P<0.01) and the continuous-insonation group (P<0.01), respectively. The goodness of fit with exponential-rise-to-maximum model was satisfying for three groups (P<0.01). M0, Mmax-Mo and K in intermittent-insonation group significantly increased compared with the control group and the continuous-insonation group. K and N in intermittent-insonation group appeared lower compared with the control group despite increase in Mo.Conclusions:Under the intermittent insonation with elevated-intensity low-frequency ultrasound, both the total drug release and T>MIC of VLBC will increase; in addition, the long-term release of sub-inhibitory drug release will almost cease. The exponential-rise-to-maximum model is suitable for predicting the drug release of VLBC with insonation. Our pharmacokinetic data and fitting curve confirmed the significant enhancement of intermittent insonation with low-frequency elevated-intensity ultrasound on surface liberation and internal diffusion of drug from VLBC. Part4Effect of Low-Frequency Pulsed Wave Ultrasound on Micro-Architecture and Mechanics of Stem-Cement InterfaceObjectives: Several studies have shown that low-frequency pulsed wave ultrasound can effectively enhance and accelerate antibiotic release from bone cement. The mechanisms may be associated with the effects of detaching forces and pushing forces by acoustic microstream. We hypothesized that low-frequency pulsed wave ultrasound improved interfacial porosity and penetration of fluid through the cement-stem interface.Methods:The1%vancomycin-loaded acrylic bone cement-stem interface samples were successfully manufactured and randomly divided into three groups:the control group,450mW/cm2ultrasound group and1200mW/cm2ultrasound group. Two ultrasound groups were exposed to a local ultrasonic field for7days, then immersed in37℃PBS for23days, while the control group immersed in PBS for30days with no ultrasonication. After30-day immersion, the shear strength and the porosity of the stem-cement interface were determined. Two additional specimens from each group were investigated for the extent of penetration of fluid into the stem-cement surface.Results:The450mW/cm2ultrasonication had no significant effect on the cement-stem interface (p<0.05). The mean shear strength decreased by14%and the interfacial porosity increased by9%in the1200mW/cm2ultrasound group compared with the control group; also, there was a higher porosity and much more penetration of fluid at the interface close to the ultrasound transducer in the1200mW/cm2group compared to the control group.Conclusions:The1200mW/cm2low-frequency pulsed wave ultrasound significantly enhanced porosity and penetration of fluid at the interface which resulted in the lower interfacial initial stability. But the effect of low-frequency pulsed wave ultrasound on the fatigue strength of the stem-cement deserved further investigation with the cyclic loading tests in vivo.