| Background:In recent years, as the rate of high-energy trauma injury caused by serious traffic accidents injuries, work and high-falling is increasing dramatically, the rate of serious open fracture dislocation and soft tissue is increasing significantly. Infection caused by wound contamination and bacteria residue is a big issue in clinical orthopedics. It is urgent for us to develop a kind of biological material which can not only fill bone defect effectively, but also can resist infection for a certain area. This kind of biological material should have good biocompatibility and can be degraded naturally in the body. At the early stage, it should be beneficial to bone growth. It should have the mechanical strength better than cancellous bone. This dissertation aims to develop a material with the above characteristics, which is expected to solve the problem.Purpose and significance:This dissertation aims to provide experimental evidence to develop a new biological material, which not only can be filled into bone, but also has anti-infective function. It studies the physical characteristics of a new artificial bone named norvancomycin-fibrin glue calcium phosphate bone substitute, acute cellular toxicitiy, anti-collapsibility and anti-rinse ability and the extraneous releasing law of novrancaomycin in the new material.Experimental Methods: 1, Research on physical characteristics of the new bone substitute of Norvancomycin-fibrin glue-calcium phosphateThe new composite bone substitute was obtained by the following steps. Add certain amount of norvancomycin (hereinafter referred as NV) into the artificial bone solid phase powder, which was made by a ratio of 3 to 1 between P-tricalcium phosphate calcium and monocalcium phosphate. Then fully grind these elements. By a solid-liquid ratio of 1 g:0.4ml, added 500 mmol/L citric acid solution into the mixture and fully mixed it. At the same time, by double cavity pushing accumulators, pushed both fibrinogen solution and thrombin solution into the artificial bone paste, which were of the same volume. (In contrast, the control groups of materials were made by mixing only P-calcium phosphate and monocalcium phosphateo at a ratio of 1g:0.4ml and then again mixing the mixtrue with citric acid solution.)Took appropriate amount of the above two kinds of artificial bone paste separately and fully mixed them. Then put the mixture into a tooling of 6mm internal diameter and 12mm height. Tested the setting time of them separately by using cement consistency condensation tester. The initial setting time was tested when the single-needle entered the position at 1/2 height of the sample material and the final setting time was tested when the single-needle entered into the material≦0.5mm. Still took another certain amount of the above two kinds of artificial bone paste and put them separately into a standard steel tooling of 6mm internal diameter and 18mm height. After them took shape, removed the tooling and put them into a constant temperature humidity chamber for 4 weeks for the purpose of fully hydration. After polishing test on the two ends of the sample materials, did the comprehensive strength test by electronic universal material testing machine. Next, did the curing time and curing strength tests on each group of materials for six times. In addition, took a few sample materials to do X-ray diffraction test and scanning electron microscope test. At the same time, tested the porosity of the two materials by drainage method. All the data from tests were analyzed by SPSS 13.0 to find out whether there were differences between the above two groups of materails by doing t-test.2, Research on the acute cellular toxicity, anti-collapsibility and anti-rinse of the materials①acute cellular toxicity test:Took appropriate amount of the two kinds of artificial bone samples made by the above methods. After sterilization with 60Co-y rays, based on the 30g/L, put certain amount of DMEM/F12 basal culture medium into the samples and extracted the materials for 72hours. Removed the samples from the solution and filtered the dregs. Added certain amount of fetal bovine serum according to the predetermined ratio into the extraction solution and made full culture medium containing 10% fetal bovine serum ready for use. For the purpose of comparison, made another full culture medium containing 10% fetal bovine serum by using normal DMEM/F12. Each sample set three repeated test groups.Got the bilateral femoral and tibial bone marrow fluid of the SD rat under sterile condition, which was killed by cervical dislocation. Then washed the marrow cavity separately by using three groups of full cellula culture medium which were made by the above methods. Next, cultured adhere cells (mainly were Mesenchymal stem cells) by full marrow method. When the cells were fused to approximately 85%-90%, counted the cells number rate in each culture medium based on cells counting plate and observed morphology of the cells at different growth stages with optical microscope. The outcomes were considered as the preliminary evaluation of acute toxicity effect of materials on cells.②Research on the anti-collapsibility and anti-rinse of the material:Tested the anti-collapsibility and anti-rinse of the material by using the traditional method of immersion and the method of simulating the operation situation, which cancellous bone was continuously bleeding. The later was an improved testing method to test the anti-rinse characteristics of materials, which was to give a certain dynamic pressure when the bone cement paste was injected. It measured the anti-rinse by a ratio between remaining weight (ml) after final coagulation and initial weight (m2). Each group of materials was tested three times and meanwhile the surface morphology and roughness were observed.3. Research on the extraneous releasing characteristics of antibiotics in vitro of the new norvancomycin-fibrin glue-calcium phosphate bone substituteMade the standard NV solution into a series concentration gradient of 200mg/L, 100mg/L,50mg/L,25mg/L,12.5mg/L,6.25mg/L.Under the room temperature, scanned the waves ranging from 200nm to 800nm by ultraviolet spectrophotometer and took the wavelength of the highest peak wave as detection wavelength and did experiments. Tested OD value in Norvancomycin solution of a series concentration gradient. All the data were analyzed by SPSS 13.0 to calculate its standard regression equation. Took appropriate amount of the two groups of materials to extract continuously in 0.9% normal saline. Then extracted the full extraction solution by clean syringes for OD value test. The initial lOd was sent to OD value test every 24 hours during extraction. then 5d every time was sent to OD value test. Removed the material sample and put it into a clean tube. Re-injected it with normal saline of the same amount for extraction again. Repeated the process several times until the antibiotic concentration of the extraction solution was approximate to zero. then the experiment was over.According to the standard regression equation derived from above tests, analyzed the relationship between antibiotic releasing quantity and time of the two groups of materials. As time (d) changes, measured the OD value of the two groups of materials, concentration x (mg/L) of vancomycin liquid, and the changes of the percentage P(%) of the accumulated released antibiotic quantity on the total dose. Analyzed the daily OD value of extraction solution with population pyramid by SPSS 13.0.Results:1, physical characteristics of the new composite materials:The initial and the final setting time of the new NV-FG-CPC material are much less than the control group, with 1.08 min less in initial setting time and 3.28 min less in final setting time than the control group respectively. As FG and NV are introduced into it, compressive strength of the new artificial bone decrease significantly. After 4 weeks of hydration, compressive strength of the NV-FG-CPC group are 6.43±1.09 Mpa, while CPC group 14.72±3.38Mpa (statistical test t=5.710, P<0.01). The porosity of the former one is 4 percent point higher than the latter. The main product of the new material after setting is still calcium phosphate. By electron microscopy, it is observed that FG connected the CPC crystals together. In addition, antibiotic particles, about 3-6μm in size, can be seen scattered on the background and they are quite different from the background particles2, Outcome of acute cellular toxicity and anti-rinse of the new materialPrimary culture cells grew well in each bottle of culture. After 14 days, the cells in the three bottles of culture came to be of 85-90% fusion rate. Cells in each bottle were of similar shape, which were mostly elongated spindle-shaped or short spindle-shaped. All the cells were in good condition and the orders of magnitude of all the three groups reached 106/L. After 24 hours, the purely CPC group in saline solution was surrounded by fog-like things, while NV-FG-CPC group didn't have this fogy phenomenon but there were only small amount of cement particles fell off. Repeated the experiments three times and it is seen that the glass tube of the NV-FG-CPC group still remained a large number of bone cement blocks, of which the average initial weight and the average final setting weight were 0.903g and 0.75g respectively and residue ml/m2 was 83.1%. The rinsed surface of bone cement was smooth and there was not any cement particles around it. In contrast, the bone cement remained in the glass tube of CPC group decreased significantly. The rinsed surface of the bone cement was rough and there were bone cement particles which were rinsed to spread in the container. By the same calculating method, outcomes were m20.840g, ml 0.567g and ml/m2 67.5%. The improved anti-rinse experiment shows that the new material has a more anti-rinse ability in a dynamic condition. The outcome is the same with the outcome of anti-collapsibility obtained by traditional testing method.3, The extraneous releasing raw of norvancomycin in vitro of the new materials:Under room condition of 25℃, standard NV solution has a maximum absorption peak at a wavelength of 226nm. The relationship between OD value and concentration x (mg/L) of the standard NV solution can be described as:OD= 0.013x+0.173 (R2=0.971, adjusting R2=0.964, F=133.58, P=0.00).The characteristics of norvancomycin in the new material are similar to traditional material. Both have an outbreak of releasing process at first, which last for 4 day for the new material and 6 days for the traditional material respectively. Then the NV-FG-CPC system, after an interval of 2 days, has a peak of sustained-releasing antibiotics, which lasts for about 8days. By comparison, the liquid concentration of CPC group decreased continuously. New material group Norvancomycin release time can be maintained for at least 6 weeks, which is much longer than the control group. At the 4th week, liquid concentration of the control group dropped sharply to be much below the lowest concentration requirement of antibiotic. The total releasing amount of norvancomycin in the new material reaches 90% over the total amount at the 30th days while the control group at the 15th dayMajor conclusions:①The new norvancomycin-fibrin glue-calcium phosphate bone substitute has good physical characteristics and it is highly operational for its self-setting time and compressive strength meet the clinical requirements. After curing, the main products of the new material remain calcium phosphate. As fibrin glue is added into the new material, porosity rate increases. By electron microscope, particles of antibiotics can be seen in this new composite material, which indicates that norvancomycin had successfully been introduced into the material②After norvancomycin and FG are introduced into the traditional calcium phosphate bone substitute at the same time, no new toxic substances is produced. The acute cellular toxicity experiment of the new material shows that it not only has good biological safety but also good anti-rinse ability.③The sustained-releasing time of the newly developed calcium phosphate-release system is prolonged significantly with fibrin glue being added to it. Its releasing time can be at least 6 weeks, which is much longer than traditional CPC group. In addition, norvancomycin in the extraction solution can maintain an adequate inhibitory concentration...
|
PDF Full Text Request