| Background:CPC and PMMA have been widely studied respectively. The combination of CPC and PMMA is a mixture of CPC/PMMA, but we have no idea about whether the new mixture would still maintain its traditional advantages or not.Yet, through the study on reports of composite PMMA and other materials e.g. PG and PCL, preparation and characterization of drug-loaded PMMA/PG composites have been confirmed in 2002 . And partially biodegradable composites have been prepared with polymethyl methacrylate/poly (e-caprolactone) (PMMA/PCL) as an alternative to the drug delivery systems which can be polymerized in vivo and also, it can provide some structural support before degradation.So we believe that PMMA and CPC composite are feasible. New or modified PMMA formulations are being used in many clinical and experimental researches. Modifications to these fillers may vary from physicians and procedures. To date, no standardized formulations, biomechanical standards, or safety guidelines exist when preparing or modifying PMMA or any other bone void filler to be used in the spine. Polymethylmethacrylate is an effective vertebral augmen-tation filler material. It is inert, biomechanically sound, adaptable to different techniques and cost-effective.The choose of filler will depend on the eventual development of a material with good biomechanical and biological properties as well as good radiopacity and costeffectiveness.Although there lacks background about the preparation of composite bone cement, many scholars have obtained successful experience on PMMA, PG, PMMA and PCL composite materials. Hence, why not mixing them into new compounds, and then study whether the complexes are with good biomechanical and biological characteristics or not? Therefore, we have conducted experiments, the results of the study show that CPC and PMMA can be composited indeed, and bone cement composites have good biological characteristics and biomechanical characteristics.These advantages would offset the deficiencies of PMMA, which is neither degradable nor conducive to bone or new bone ingrowth. Therefore, if the composite can be made successfully, a new composite bone cement will be obtained with the advantages of both independent components (e.g., CPC and PMMA); in addition to the advantages of CPC listed above, PMMA will enhance the mechanical support of the material to meet the needs of new bone cement as the implantation material in the future.Objective:The study aims to research biological safety, biomechanics and tissue compatibility of calcium phosphate cement and Polymethyl Methacrylate composite bone cement mixed in different ratios.Methods and MethodsAccording to different mixing ratios of composite bone cement specimens, injection-type PMMA bone cement (Heraeus, Germany) was mixed with injection-type CPC bone cement (Ruibang company, China) to create a bone cement solid series with mass ratios of 3:1.2:1,1:1,1:2,1:5,1:10,1:15,1:20, corresponding to PMMA to CPC and vice versa according to the biological material experimental detection and safety standards in detecting the related experiments. CPC and PMMA were measured in accordance with the best ratio of liquid to solid (1ml:2g), and the concentrations of both CPC and PMMA were in accordance with the desired bone cement (3:1,2:1,1:1,1:2,1:5,1:10,1:15,1:20) mixing ratios. The bone cement mixing ratios(3:1,2:1,1:1,1:2,1:5,1:10,1:15,1:20) are in line with group75%, group 67%, group 50%, group 33%, group 16.7%, group 9.1%, group 6.25% and group 4.8%.A pasty mixture was created and then filled in a sterile mold for 60 s of compaction at 37℃ in a 100% humidity environment. Samples were removed and cured at 37℃ at 100% humidity for 23 hours.Cell toxicity testCPC group, PMMA group, CPC+PMMA composite bone cement group materials were arranged separately in mouse source medium (3 cm2/ml) at 37℃ for 120 h to prepare a medium extract of CPC, PMMA and composite bone cement group. Then, MC3T3-E1 osteoblastic progenitor cells were inoculated in a 10 cm2 dish and were cultured at 37 ℃,5%CO2,100% humidity for 2 days, during which time the cells grew logarithmically. The culture medium was then discarded and washed by PBS solution twice.0.25% trypsin was added into the culture dish (2 ml/10 cm2) until the cells became round in the medium after termination of digestion. Centrifugation in began at 1000 rpm for 5 min. The supernatant was then discarded, lml medium was added (i.e.,10μl), and the cells were plated at a density of 2.5×103-5×103/hole into a 96-well plate (5 holes/group). Cells were then cultured for 24 h at 37 ℃,5%CO2, 100% humidity. The original culture medium was then discarded, and the bone cement soaking liquid prepared was added at 100 μl/hole. These specimens were cultured for 24 h, after which CCK8 detection reagent (Sigma, America) was added; cultivation continued for an additional 3h-4 h (37℃,5%CO2,100% humidity). OD values were then measured in each group using the enzyme mark instrument volume at a wavelength of 450 nm. The relative cell growth rate was calculated in each group.Sensitization test Eleven bone cement samples were extracted under aseptic conditions. SD rats were reared in a quiet state following the principle of animal protection. A 1-ml aliquot of each leaching liquor was drawn into a disposable syringe for later use. The injection site on the medial thigh was disinfected, and the materials were injected via local intradermal injection at 0.1 ml per injection point. The control group was injected with physiological saline. The SD rats injection sites were observed for erythema, edema, induration and eschar formation after 15 min,30 min,1 h. and 24 h from injection and compared with the control group. The scale used for these observations based under the Magnusson and Kligman classification standard.Compressive strength test and tensile strength testThe bone cements were mixed uniformly and then injected into stainless steel molds at room temperature. The resulting specimens were cylindrical with diameters of 5 mm and heights of 10 mm. The bone cement specimens were placed on an Instron universal testing machine (n=10 times) to determine their compressive strength and tensile strength.Three-point bending test After storing in a water bath at 37℃ for 48±2 h, the flexural strength of the composite bone cement specimens was measured by a three-point bending test. Applying a universal testing machine (EZ20, Lloyd Instruments Ltd., UK), flexural strength tests were carried out with a supporting span of 50 mm with a crosshead speed of 5 mm/min until failure(n=10 times). Solidification time measurementA mold with a diameter of 10 mm and a height of 5 mm was prepared and then filled in with the different bone cement blends. These molds were placed into an environment at 37 ℃ and 100% humidity. A vertical pressure head on the bone cement surfaces was applied for 5 seconds to measure the degree of deformation into the bone cement using a Vicat apparatus every 30 seconds until the indentation could no longer be seen. The solidification time was measured from the end of filling the mold until no additional indentation was observed. This test was repeated three times and averaged for each bone cement sample. Scanning electron microscopy morphologyThe bone cement samples were immersed in ethanol to stop the hydration reaction and was allowed to dry naturally. A JSM-5600LV type low vacuum scanning electron microscope was then adopted to observe and detect structural changes in the bone cements’internal micro holes.500 cycles were completed for accuracy. Phase composition analysisThe tested samples were dried naturally at room temperature. Their structure and phase were analyzed with a X ’Pertpro type X-ray from the Holland PANalytical company. X-ray diffraction with a Cu rake, a tube voltage of 40keV, a tube current of 30 mA, a continuous scan range of 20 from 10°to 90°, and a scan rate of 15.24/min was applied. The phase of the samples was determined with the X-ray software Jade 5.0.Animal model of the bone defect To establish an animal model of the bone cement samples after implantation in a bone defect , muscle pouch tissues were investigated. SD rats were placed supine with fixed limbs and trunk. The inside of their skin on their lower limbs were disinfected using iodophor and alcohol after anesthesia. After a straight incision along the medial tibial surface, a gap in the muscle was produced to expose the periost. A hole was then drilled into the bilateral medial tibial bone using a hand drill with a borehole diameter of 5 mm, resulting in a borehole area of 19 mm2 and a medial tibial unicortical critical bone defect. The borehole area was greater than previously reported bone defect areas . The skin was sutured immediately after the bone defect was created to create the control group. For the experimental group, a muscle cover was sutured, the bone defect was created, and bone cement with a diameter of 5 mm and a thickness of 2 mm was implanted in bone defect; the skin was then sutured. The skin of the right lower limb was then cut, muscle tissues were separated to create a muscle cavity, and different bone cements were implanted into this muscle cavity. The muscle and skin were then sutured, the skin was disinfected with alcohol again, and each mouse was fed feeding after being numbered. X-rays of the implanted bone cements were recorded after 4 weeks and 15 weeks. The SD rats’limbs were fixed in the animal plate with a rubber band and the metabolism of the bone cement implantation in the lower limbs of SD rats was measured and recorded. Histological observationSD rats were sacrificed after the composite bone cements had been implanted for 15 weeks. The bone cements in the muscle cavity were removed, and the bone cements in the bone defects and surrounding bone were fixed with 10% formalin before observation with dyeing. Specimens were embedded in paraffin, decalcified and stained to observing the internal void structure and the degree of new bone formation on the bone cement materials.The weight loss rate calculationTransplantation of bone cement specimens weighing (Wo), Wo represents the initial weight, Bone cement specimens were implanted into the medial tibial muscle bag of SD rats before removing it after 15 weeks, and then the weight of (W1), W1 represent the weight of the specimen in vivo after degradation. The weight loss rate= (W0-W1)/W0×100% so as to evaluate the degradation of bone cements after implantation in vivo. Statistical analysisAll data are parametric after our statistical analysis using SPSS 19.0. They all follow a normal distribution after data exploring analysis and were expressed as mean ± SD (n= 5). The data were analyzed by one-way ANOVA. Should the data satisfied variance homogeneity, the regular F value and P value would be obtained, otherwise, Welch or Brown-Forsythe would be used to compare the population means.ResultsCell toxicity testCell toxicity grading standards are divided into six levels:level 0,≥100%; level 1, 75-99%; level 2,50-74%; level 3,24-49%; level 4,1-25%; level 5,0%. Level 0 is considered non-toxic, while level 5 is highly toxic. Compared with the control group, differences were found among the groups (PMMA group.4.8% group.6.25% group, 9.1% group and 16.7% group (P<0.05). Other groups of bone cement extracts had no effect on the relative MC3T3-el cell growth rate, and the toxic reaction was level 1 (i.e., non-toxic). Thus, the subjects of the bone cement material extracts (33% group,50% group,67% group,75% group and CPC group) did not experience cell toxicity.Sensitization test results The PMMA injection locations showed erythema on the skin, while the PMMA monomer group showed significant punctate erythema and edema on the skin with a Magnusson and Kligman score of 1 for prompt sensitization. There were no significant findings in other groups, as they shown a Magnusson and Kligman score of 0.Testing results of compressive strength and tensile strengthBoth the compressive strength and the tensile strength were found to enhance gradually with increasing PMMA concentration and decreasing CPC concentration in each test group. As shown, there were significant differences between the compressive strength of each test group. (P<0.05).Testing results of three-point bending testThe compressive strength was found to enhance gradually with increasing CPC concentration and decreasing PMMA concentration in each test group. As demonstrated, there were significant differences of the flexural strength in each test group (P<0.05).Results of curing timeThe curing time was longer in the CPC group (more than 11 min) but was shorter in the PMMA group (less than 2 min), and that of the composite cement groups were near 2-6 min. There were significant differences among the CPC group, the PMMA group and the groups of composite bone cements (P<0.05). Scanning electron microscopy morphologyTo reconcile each group bone cements by PMMA monomer, there were no significant differences found among CPC, PMMA and the composite bone cement in term of surfaces and structures, nor ded the interface between the CPC and PMMA composite bone cements; thus, good compatibility was found between these two materials. PPMA, type of microstructure, was also shown to distribute into the CPC with many benefits for the material’s mechanical properties, composite air permeability and electrical properties. Spherulitic grains of the CPC group were found as spherulites stacked close together. This did not rule out the adhesive effects of the PMMA monomer solvent, but with increasing PMMA concentration, the crystalline region loosened marginally.Even though this was not significant, it showed the formation of a continuous system after mixing and that the materials were compatible.The minimum aperture that bone cells needed for growth into should be 70 u m,200 u m-400 u m apertures of the general materials, making the most suitable sizes of range for bone cells to grow into as the percentage of above 70 u m pore aperture is very important for the bone cements’pore structure. This experiment above 70 u m aperture will be called the effective aperture of cell growth. By scanning electron microscopy observation, bone cement particles were spherical, and group CPC particles’aperture were loose; spherical particles of the group PMMA are arranged in dense between 100 u m-400 u m, pore aperture between 50um-100um, pore aperture of the group CPC/PMMA (75%), group CPC/PMMA (67%) and group CPC/PMMA (50%) between the group CPC and the group PMMA between 100 u m-200 u m. Therefore, the pore structure of the considered composite bone cements is suitable for the growth of bone cells. Analysis of X-ray diffractionTo compare the card of the control group with the powder diffraction standard of the International Diffraction Data Centre, PMMA bone cements are amorphous substances with no crystalline diffraction peaks; thus, it only showed the crystalline diffraction peaks of barium sulfate as a developer. The diffraction peaks which are shown by CPC crystalline are based on hydroxyapatite. The original crystallization properties, which included low CPC crystallinity, decreased significantly with increasing PMMA after the composite bone cements were mixed together. Sharp diffraction peaks in the XRD map were found to weaken, but the crystalline region with barium sulfate and hydroxyapatite with composite bone cement material still exist. The mixture of CPC and PMMA had no significant effect on the final products of the diffraction peaks of hydroxyapatite. Thus, there was no new crystalline phase appeared in the reactant (i.e., PMMA bone cements did not participate in the curing reaction of CPC bone cements).X-ray and Histological gross observationX-ray examinations of the bone cements were performed after 4 weeks. There were no significant degradations in the groups.15 weeks later, we had the X-ray examinations of the bone cements performed again. Except for the PMMA group, significant degradations appeared in both the CPC/PMMA group (50%) and CPC group. SD rat tibia was with a length of approximately 4.5 cm-5.0 cm after composite bone cement transplantation in vivo after 15 weeks. CPC/PMMA (50%) group and CPC group bone cements were degraded. And bone cells growth was found to have integrated with the surrounding bone tissues closely. PMMA group were found to not be degraded transplantation in vivo after 15 week. A portion of the composite bone cements (group 50%) was found to be degraded. The weight loss rate calculationPMMA group weight loss rate almost no change, CPC/PMMA (33%) group weight loss rate without significant difference, P> 0.05. Compared with the PMMA group, CPC/PMMA (50%,67%,75%) group and CPC group were significant differences in weight loss rate, P< 0.05. The most obvious change in CPC/PMMA (75%) group, the average weight loss rate is 6.78%.Discussion1. This study uses CPC and PMMA bone cement, in accordance with the mixture of different quality ratio, a composite CPC/PMMA bone cement are prepared, the preparation process is simple and reliable, the intervention of PMMA does not affect the properties of the CPC, with the gradual increasing of PMMA content, the mechanical strength is increasing, and the degradation rate in vivo gradually slow down, PMMA content of more than 50% can hardly degradable.2. Composite bone cement CPC/PMMA belongs to no cytotoxicity of biological material, has good cell compatibility.3.Composite bone cement CPC/PMMA non hematologic toxicity, does not cause delayed hypersensitivity, does not have systemic subacute toxicity, and has good biological safety, a composite bone cement CPC/PMMA meet the basic conditions for bone substitute material.4.To repairingthe critical bone defect of SDrat tibial bone by the composite cement CPC/PMMA, can think of composite bone cement is an alternative material and has good biological activity bone guided bone tissue.In summary, the better composite bone cement concentration are selected from them, group CPC/PMMA (50%), group CPC/PMMA (67%), group CPC/PMMA (75%) iprovide more variability and selectivity for the composite bone cement to obtain a better application. |
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