A Study Of A Novel Injectable Calcium Phosphate Bone Cement With Fibrin Glue

BackgroundsOBJECTIVE: Study the compound ofβ-TCP / MCPM bone cement and fibrin glue complex physics performance, to evaluation the fiber plastic eggsβ-TCP / MCPM properties of bone cement. Through the experimental biological safety study ofβ-TCP / MCPM calcium phosphate bone cement with fibrin glue in vivo for Bone defect reparing, feasibility as a guidance of clinical use for bone defect reparing.Methods: (1) physical properties of materials testing: 1. Hardening Time Measurements : Use Gilmore needle technique, which two quality are 113 and 445 g, respectively tip diameter 2.1 and 1.1mm thickness of needle to be tested. Not to be left obvious marks by the weight and leight neddle are the initial setting time and final setting time.2. Washout Resistance Test: The washout resistance was tested by shaping theβ—TCP/MCPM andβ-TCP/MCPM/FG sample into a small sphere by hand, and then placed it immediately in physiologiclike solution or distilled water (37°) for 24 h, and slightly shock its. The sample was considered to pass the washout resistance test if it did not visibly disintegrate in PLS.3. Materials development and Mechanical testing: Theβ—TCP/MCPM/FG samples were prepared by mixingβ—TCP/MCPM powder with the citric acid (or sodium citrate) at powder-to-liquid mass ratios (P/L) of 1g/0.3ml, blending after added fiber fibrin glue by a volume ratio of 2.5:1 , filled in the standard mold (with a diameter of 8 mm, 16 mm height), and conserve the sample in 37℃, and nearly 100% humidity conditions 72 h , marking preservation;andβ—TCP/MCPM by solid powder liquid ratio 1g/0.5ml made as the control group, the samples were used electronic universal testing machine to test the compressive strength, and calculate the compressive strength of the material.4. Examination of Surface Morphology: theβ-TCP/MCPM andβ-TCP/MCPM/FG group samples were maded as natural fracture, samples fracture surface by vacuum deposition of the instrument of spray, SEM magnification observed the samples structural characteristics under different SEM magnification , measurement of pore size, and wether there have different between the two materials . and take the fracture of theβ—TCP/MCPM/FG materials in high-temperature furnace 800℃calcined 8 h, in vacuum coating surface coated with gold, scanning electron microscopy observe the samples structural characteristics and size of the aperture, and the porosity of materials.5. X-ray diffraction (XRD) analysis: Powder X-ray diffraction (XRD) analysis was used to estimate the extent ofβ—TCP/MCPM andβ—TCP/MCPM/FG conversion to HA.(2). Biocompatibility and Safety of Experiments: 1. acute toxicity test: The mice were divided into groups of extract liquid and saline groups. Each injection of 1 ml. After injected 24 h, 72 h and 48h , weighing the mouse body weight, and observed whether they have any adverse reactions.2. hemolysis test: The fresh human blood anticoagulant diluted with saline to 2% human blood suspension. In different materials respectively in vitro extracts liquid saline group, normal saline (negative control) and double-distilled water (positive control) 2 mL. the further each tube were joined 2 mL of blood suspension, 37℃water bath for 1 h, observ whether there have hemolysis. 1000 r / min for 5 min. Supernatant from 1 mL. add 4 mL of 0.1% Na₂CO₃ Solution, 752 spectrophotometer at 540 nmoptical, test the density of the sample and calculate the rate of hemolysis.3. micronucleus test: The health Kunming mice which half male and half female were randomly divided into five groups, including three groups respectively intraperitoneal injection of different doses of theβ-TCP/MCPM saline extracts (0.1, 0.2, 0.4 ml) and the other two groups as a negative control group (NS) and the positive control group (cyclophosphamide 40 mg/kg). each 24-h intervalof the administration, After the fourth of 6 h administration of the animals were killed. Used bone marrow made into slide, Rett's (Wright) staining. In the oil microscope count, calculate the each animal of 1000 polychromatic erythrocytes containing micro-containing cells in the nucleus PCE cells.4. Cell culture experiments: take the logarithmic phase of MC3T3 osteoblast cell by trypsin digestion density of 5×10⁴ ml of cell suspension were inoculated into 96-hole culture plate, 100 ul of each hole, each 5 hole. After 24 h when cells adherent, disposable medium, by adding different concentrations of DMEM/F-12 extracts (undiluted group, 50% Solution Group, 10% Solution), pure culture fluid for the negative control group, with 0.64% Phenol medium as a positive control group. 37°C at 5% CO 2 incubator of culture, respectively 24, 48, 72 h after removal, each hole by adding 20 ul 0.5% MTT to continue to foster a 4 h. Disposable medium, by adding DMSO 150 ul/ hole, 5 m in shock, Meibiaoyi 490 nm measured OD value of every hole. Calculated the relative cell proliferation rate.Results: The results showed that adding fibrin glue can significantly prolong the composite bone cement setting time of the initial and final time condensate, final setting time than initial setting time delay 2-4 seconds. Allβ—TCP/MCPM/FG showed excellent washout resistance;they remained stable and hardened while immersed in PLS, no obvious collapse phenomenon. But control group, there have obviously collapsing, materials incomplete. Theβ—TCP/MCPM andβ—TCP/MCPM/FG bone cement materials compressive strength are 19.63±1.98MPa and 14.72±1.81MPa.Both samples XRD spectra are the same as the standard of HAP XRD spectra consistent, XRD spectra peak near at 32°, the two samples have the same crystalline phase, adding FG Without a new phase made, mainly into HAP. Scanning electron microscope see the rules ofβ—TCP/MCPM from Corynebacterium parvum crystal and a flat-shaped crystal, crystal interconnected attachments, irregular crystals filled with porous, about the size microporous (2-3 um) about Voidage 46.84%.β—TCP/MCPM/FG composite materials within the crystals throughout theβ-TCP/MCPM,β-TCP/MCPM Crystal closely linked. Crystal is about the size of the pores (2-5 um) and about 48.06% porosity. After calcination temperature composite materials, see the aperture than the microporous calcined before increasing the diameter of microporous (2 - 7 um) about 57.58% porosity, and there will be an extended Fissureβ—TCP/MCPM microporous connected. The acute systemic toxicity test results shows that the average weight of the mice in 24,48,72 h after injection of the saline group and the change basically are the same, and there are no any of mice appear death or coma, respiratory depression, difficulty in breathing, limbs and other toxic reactions limited activities after injecting, which shows that the artificial bone material composite no acute systemic toxicity. The hemolysis test results shows that there no significant hemolysis in the experimental group and the negative control group, the positive control group have significant hemolysis, the rate of the experimental group hemolytic no more than 5%, meet standard requirements. The micronucleus test result shows that the rate test group and negative control group there were no significant difference in the micronucleus;-negative control group rate with the positive control group there have significant differences. The new composite materials shows no genetic toxicity. Cytotoxicity test showes that the experimental group and the negative control group of mice MC3T3 osteoblast growth and differentiation well, maintain spindle cells, adherent growth, higher cell density, multi-spindle and longer and see circular polygon cell division, the positive control group cells stop growth and differentiation , the cells changed round narrowed, with more floating dead cells ; The experimental group to MC3T3 osteoblast cells were cytotoxic I level, the control group wereâ…£-â…¤.Conclusion: 1.The results showed that the bone cement material with suitable setting time and better washout resistanc properties and can accelerate the degradation ofΒ—TCP/MCPM itself, by adding fibrin glue after the compressive strength of the composite material to the cancellous bone strength requirements to meet for the non-load-bearing bone defect filling ;β—TCP/MCPM/FG porosity markedly improved when after calcination, So with the fibrin glue degradation the void will be increasedgradually, Promote the degradation of materials, which will help the new bone growth,Promote the bone defect repair.2. The new compound fibrin glue calcium phosphate cement materials have goodbiocompatibility and biosafety, reach the biocompatibility and biological of securityrequirements, and as injectable calcium phosphate cement meet the basic securityneeds for clinical application.