| Research backgroudTo treat aggressive bone tumors such as bone giant cell tumor, curettage and bone graft is often used. Namely, fill bone defects with a variety of biological graft material after tumor curettage. There are two main drawbacks of this method, high recurrence rate and non-ideal filling materials.In order to reduce local recurrence, surgery combined with neo-adjuvant chemotherapy and postoperative chemotherapy is used. However, most chemotherapy drugs can cause systemic toxicity such as the liver, kidney, bone marrow and other organ damage. And it is difficult to reach an effective concentration in the bone.Besides, to fill the bone defects, various types of biocompatible graft materials have been developed. However, all these materials have shortcomings. For example, the source of autogenous bone graft is restricted, and the harvest may result in broad invasion. And allografts have drawbacks such as unforeseen immune response and disease transmission.In contrast, calcium phosphate cement (CPC) shows advantages such as moldability, biocompatibility, osteotransductivity and osteoconductivity, which make CPC very promising materials for clinical applications. Besides, CPC has additional advantages such as setting at ambient temperature, near neutral pHs, suitable porosity and a large surface area, these properties make it to be an attractive candidates as carriers of drug delivery system.Antibiotic-loaded calcium phosphate bone cement has been widely used for the treatment and prevention of osteomyelitis. This application suggests that CPC could serve as an excellent material for filling defects and for preparing effective antitumor drug delivery systems to deliver chemotherapy drug to peri-tissues progressively with time, control the local recurrence of bone tumors and reduce chemotherapy-related systemic toxicities.The laboratory studies on drugs incorporation into the cements cover different aspects. First, it is necessary to verify that addition of a drug does not influence the setting reaction not only in terms of the setting and hardening mechanisms but also basic crystal structure. Second, it is necessary to determine the drug-delivery properties of the cement in vitro. Third, the kinetics of drug release must be studied in vivo. Forth, the resorption of MTX-CPC and its effect on osteogenesis must be evaluated. Finally, but still importantly, the clinical performance of the drug-delivery system must be evaluated as well.We chose Methotrexate (MTX), a common antimitotic agent with mechanism of inhibiting dihydrofolate reductase (DHFR), as the incorporated chemotherapy drug. MTX has been confirmed to be effective against invasive bone tumor as giant cell tumor.To verify the first and second points, we added MTX into CPC, and preset MTX-CPC samples. Then a series of research were designed and performed. This previous study on MTX-CPC in vitro had shown that addition of 1%(weight/weight) MTX did not significantly alter the setting times (including initial and final setting time) of CPC. Although the mechanical properties (including the compressive strength and tensile strength) of the MTX-CPC significantly decreased when 1%MTX was added, the MTX-CPC remained compliant with the minimum requirements for clinical application. In vitro experiments on the release kinetics of MTX confirmed that MTX-CPC was a monolithic matrix system, with a burst effect of MTX release in the initial period after implantation and a sudden drop thereafter. MTX release at a level higher than the minimum effective concentration (1×10-8 mol/L) continued for 2-4months after implantation.To verify the third and forth points, we design and perform this research, and state the results in this article.PART I Release Kinetics Study of Methotrexate-Ioaded Calcium Phosphate Cement in VivoPurposeWe incorporated MTX in CPC and implanted them into rabbits to observe the in vivo release kinetics, and in vivo systemic toxicity and side-effects of the CPC-MTX system.Methods 1%(weight/weight) cylindrical MTX-CPC specimens (height 10mm, diameter 3mm) and control CPC specimens were preset. Each MTX-CPC specimen contained 1.00±0.02 mg MTX.36 New Zealand Rabbits were randomly divided into 2 groups.(1) Experiment group:this group contained 24 rabbits. We implanted MTX-CPC specimens into their femoral muscles.(2) Control group:this group contained 12 rabbits. We implanted control CPC specimens into their femoral muscles. Then 1 ml 0.1%MTX was injected intravenously from the rabbits'ear.Then following steps were taken.(1) Experiment group:venous blood samples and local muscle samples were obtained from 3 animals at various times (1,2,5,10,15,20,25,30 days post-implantation). Then the MTX concentrations of these samples were determined by high performance liquid chromatography (HPLC)(2) Control group:venous blood samples and local muscle samples were obtained from 3 animals in each group at various times (1,2,5,10 days post-implantation). Then the MTX concentrations of these samples were determined by HPLC method.(3) The release curve was determined according the MTX concerntration obtained at various times of experiment group.(4) The residual percentage of MTX in the MTX-CPC implants of experimental group was determined at designed time intervals (1,2,5,10,15,20,25,30 days post-implantation).(5) To determine the systemic toxicity, gastrointestinal side-effects and incision healing were observed. Rabbits in the 1 day and 10 day were sacrificed, and their kidneys and livers were obtained for histological examination to observe the cytotoxicity of MTX.(6) Statistical analysis:Data collected from local muscle concentration measurements and blood concentration measurements were expressed as mean±standard deviation (SD) and statistically analyzed using SPSS 13.0. Comparative studies of means were performed using two-sample t-test, with p< 0.05 indicating statistically significant difference.Results(1) During the experiment, no abnormalities were observed with regards to diet, urine, or stool. All incisions healed without complications. Histological examination of the liver and kidneys revealed no necrocytosis in the specimens at 1 day or 10 day.(2) Local release:The MTX release kinetics in vivo confirmed that MTX-CPC was a monolithic matrix system, with a burst effect in the initial stage and a sudden drop thereafter. The local concentration at day 5 was 3.24±0.81μg/ml, while the blood concentration was only 0.01±0.00μg/ml. The difference was significant (P |
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