Preparation And Biological Study Of Digital Silver Coralline Hydroxyapatite Artifical Bone

BackgroundIn the worldwide, due to trauma, tumor and infection causes bone defects each year afflicts many patients, the need for bone graft transplantation. However, clinical treatment for bone defects of large-scale pollution using conventional bone graft material may caused to bone transplantation failure due to the secondary infection. And the growing problem of bacterial resistance to antibiotics in the clinical, graft material loaded antibiotics become increasingly localized, then put forward higher requirements for antimicrobial. In recent years, inorganic antibacterial materials for its effective antibacterial, higher security and stability has aroused widespread concern, compared with the organic and natural antibacterial agent, Inorganic antimicrobial agent has the advantages of broad spectrum antimicrobial, long lasting, does not produce drug-resistant, non-toxic side effects and so on. The antibacterial properties of Ag+is the strongest in the metal ion and also the earliest and most mature application of inorganic antibacterial agent.Over the years, the development of biological materials with antibacterial function instead of autologous bone repair of bone defects has been an important issue in medicine and materials science.. Bone tissue engineering for bone defects, especially of complex structures has brought new hope to repair bone defects. Bone tissue engineering consists of three key factors:signaling molecules (bone growth factors, bone inducing factor), scaffolds and target cells. The choice of scaffold materials is the core issue of the bone tissue engineering. At present,the scaffolds include the natural scaffolds, synthetic scaffolds and composite materials.Coral hydroxyapatite (CHA) is a natural coral by "hydrothermal reaction", the transition from the hydroxyapatite to retain the porous structure of the natural coral, good biocompatibility, with a larger aperture, higherporosity and pore transport rate. Large body of literature show that coral artificial bone is an ideal bone substitute materials, and because of its good compatibility and three-dimensional porous structure, is also considered the future of tissue engineering scaffolds and carriers.However, the CHA artificial bone is brittle, and can not be customized to the shape of the bone defect preparation of artificial stents, there is a certain shortage of its.L-polylactic acid (L-polylactic acid, of L-PLA) is an organic polymer material of one kind have been widely used in medicine, As the body of the implant has been certified by the U.S. Food and Drug Administration, and has been widely used in fixtures such as bone plates, bone screws, surgical suture lines. The advantage of it is the biodegradability, good biocompatibility, and easy shaping.However, a single type of material (natural scaffold or synthetic stent) is generally difficult to meet the requirements of scaffolds for bone tissue engineering for cell. Natural materials has good biocompatibility, cell recognition signal, which will help the proliferation of cell adhesion; artificial materials, compare to natural materials, Artificial material, although lacking in cell signaling, can be large-scale production,design and control structure, mechanical properties and degradation time, etc The composite material is a combination of several different materials through a certain method will be formed, or a compound formed by the biological material and growth factor,the materials can complement each other in performance, have already achieved good results in practical applications. Natural bio-derived materials, polymers, ceramic materials, either as a composite substrate, but also as a reinforcement and filler, with each other and combined to form the diverse nature of composite materialsRapid prototyping technology (Rapid Prototyping Technology, RPT) is a new forming technology based on the forming ideas of discrete accumulation, it and complete manufacturing rapid prototyping of biological model with high fast and accuracy from the CT data. It has been a rapid development since the1980s. It is a major breakthrough in the field of manufacturing technology and its impact on the manufacturing sector can be comparable with numerical control technology technology. In recent years, it has been widely used in the medical field, three-dimensional solid model can be made arbitrarily complex shapes, provides the possibility for bone tissue engineering scaffolds personalized manufacturing.If we can take advantage of rapid prototyping technology, to retain the natural coralline hydroxyapatite porous-like structure on the basis of the load silver ions, according to the morphology of bone defects, personalized preparation with anti-bacterial artificial bone scaffold repair material, whichwill greatly contribute to the development of antibacterial bone tissue engineering scaffolds.In this study. We use coral hydroxyapatite (CHA) by solution ion exchange method successfully loaded silver ions, to prepare different antibacterial silver content of silver hydroxyapatite powder, and mixed with polylactic acid (PLA) rapid prototyping and prepared the special shape of the digital silver bactericide artificial scaffolds by selective laser sintering. Through a series of experiments, study the Physicochemical properties, antimicrobial, biocompatibility of the artificial bone scaffolds. Through the repair Animal paragraph pollution bone defect model to investigate the feasibility applied to infected bone defects.Objections:1. To study the preparation methods of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds, and analyze their structural characteristics, physical and chemical properties.2. To study the Cell compatibility of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.3. To study the antimicrobial activity in vitro of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.4. To explore silver content and in vitro release rate of digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.5. To explore the histocompatibility and repair a large section of pollution bone defects in animal experiments of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.Methods and materials1. Coralline hydroxyapatite was mill into CHA particles after ball milling,take the particle size of140-200μm by sieved, soaked in solution of containing1O-2,10-310-4,10-5mmol/L AgN03from light, prepared silver hydroxyapatite powder with different quality of silver, and then with the L-PLA3:1quality mixed, With Simpleware3.1software, a digital cylindrical model was built by computer-aided design (CAD). Through the digital conversion, the model was transformed into STL file format, and then was imported into rapid prototyping machine. The digital silver coralline hydroxyapatite artificial bone scafflold (CHA/PLA-Ag) was prepared by selective laser sintering rapid prototyping (SLS RP) process. Use scanning electron microscopy, infrared spectroscopy, X-ray diffraction to detect the physical and chemical character and analyze the structure of the digital silver anti-bacterial artificial.2. Antibacterial properties of experiments in virto2.1Zone of inhibition experimentsRecover the Staphylococcus aureus ATCC25923and E. coli ATCC25922, Inoculated in a test tube containing LB liquid medium at37℃shaking culture Id. Take five kinds different cylindrical digital silver coralline hydroxyapatite artificial bone scafflold (CHA/PLA-Ag) prepared from soaking10-2,10-3,10-4,10-5mmol/L AgN03, the placed them in the inoculation of Staphylococcus aureus (inoculum of1.5×107CFU) and Escherichia coli (inoculum1.5×107CFU)90mm LB agar medium respectively, penicillin droplets as a positive control group. Cultured at37°C incubator for24hours, measuring the inhibition zone, and record the size of the zone of inhibition.Each strain was repeated six times.2.2Colony count experiments:prepare the Staphylococcus aureus and Escherichia to a final concentration of3×108CFU/mL bacterial suspension,the take100μL bacterial suspension drops containing silver and does not contain silver coral hydroxyapatite artificial bone surface and cultured for24h at37℃incubator. Material placed in5mL PBS for5min oscillation in the vortex instrument, eluting bacteria after24h.Take100μL eluate diluted with PBS to the appropriate concentration, to push the tablet at37℃incubator for3d,then count the colony of of plate culture, and calculate the antibacterial rate. The experiment was repeated six times for each species.3.Refer to the standard of GB/T16886.1-2001idt ISO10993-1:1997and GB/T 16175-2008, Cell toxicity test, hemolytic reaction, and biological evaluation of acute toxicity test, delayed hypersensitivity experiment were performed for biological evaluation of the contained silver hydroxyapatite artificial bone scaffolds.3.1Cytotoxicity:Take In vitro cell culture techniques to observe the changes of the silver hydroxyapatite artificial bone scaffolds extract on L-929cell morphology. MTT assay was used on the L-929cell proliferation, and to evaluate its cytotoxicity.3.2Hemolytic reaction:Detect the hemolytic rate after silver hydroxyapatite artificial bone scaffolds extract on New Zealand white rabbits, and to evaluate the hemolytic reaction.3.3Acute toxicity test:Mice by intraperitoneal injection of the extract of silver hydroxyapatite artificial bone scaffolds, and observed toxicity in mice, then to evaluate the toxicity of the materials.3.4Delayed hypersensitivity experiment:Guinea pig intradermal injection of Silver hydroxyapatite artificial bone scaffolds extracts to observe the allergic reactions of the skin of guinea pigs, then to evaluate the sensitization of the materials.4. use atomic absorption spectrometer to analyze Ag+content of the silver coral hydroxyapatite artificial bone scaffolds containing different quality of silver.10-3group digital silver hydroxyapatite artificial bone scaffolds were placed in the initial pH of7.451mL simulated body fluid (SBF).degradation in37℃incubator,1d,4d,7d,14d,21d,28d,49d to detect the release of silver ions in simulated body fluid and speed respectively.5.5mm x15mm pollution lacunar bone defect built in New Zealand white rabbit tibial metaphysis.The experimental group was implanted10-3mmol/L AgNO3silver hydroxyapatite artificial bone scaffold materials for repair of bone defects, the control group bone defects implanted coral hydroxyapatite did not contain silver,while the blank control group bone defect was not implanted in any material.Results:1.ral hydroxyapatite (CHA) powder soaked in different concentrations of silver nitrate solution, by solution ion exchange method, prepared silver hydroxyapatite containing different silver content, then mixed the different containing silver CHA powder with PLLA according to the quality of3:1,and through the CAD and selective laser sintering rapid prototyping technology, can be successfully prepared digital silver bactericide and has a special shape of the artificial bone scaffolds. The color of digital CHA-Ag artificial bone is gray, but containing silver coral hydroxyapatite artificial bone soaked in10-2mol/L of AgN03is brownish black,its powder prior to molding is still gray, we think it becaude of its contained silver content is relatively high, during the preparation of molding laser beam glareing, Silver ions icombine oxygen to form silver oxide. Surface of artificial stent is roughness, regular cylindrical in shape, thickness uniformity, visible porous structure. The compressive strength of every materials are greater than1Mpa.2. In vitro antibacterial experiments.Zone of inhibition experiments:During the whole experiment of the digital CHA/PLA-Ag artificial bone material soaked10-4,10-5mmol/L of AgNO3concentration of silver nitrate, Staphylococcus aureus and Escherichia coli had no zone of inhibition,but the digital CHA/PLA-Ag artificial bone material for24hours on two kinds of bacteria Soaked10-2,10-3mmol/L of AgN03concentration of silver nitrate produced inhibition zone. The average inhibition zone diameter of silver artificial bone material soaked10-2mmol/L of AgN03to Staphylococcus aureus and Escherichia coli was13.00±0.71mm and12.30±0.71mm.respectively. while that of10-3CHA/PLA-Ag was11.51±0.09mm and11.00±0.15mm,respectively. To Staphylococcus aureus,various materials as well as with penicillin in the control group, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=5.149, P=0.020), the difference between the groups was significant statistical significance (F95370.727, P=0.000),pairwise comparisons were significant statistically significant (P=0.000). To E. coli, various materials as well as with penicillin in the control group, One-way ANOVA analysis of variance statistics showed that the homogeneity of variance (F=1.585, P=0.237),the difference between the groups was significant statistical significance (F=2692.000, P=0.000), pairwise comparisons were significant statisticallysignificance (P=0.000).Colony count experiments:There is no growth and reproduction of Staphylococcus aureus, Escherichia coli after exposure to silver CHA-Ag artificial bone material soaked10-2mmol/L of AgNO3, the antibacterial rate was100%; but the growth and reproduction of Staphylococcus aureus, Escherichia coli was reduced after exposure to silver CHA-Ag artificial bone material soaked10-3,10-4,10-5mmol/L of AgNO3, the antibacterial rate of Staphylococcus aureus was99.99%±0.00%,49.42%±0.28%and22.18%±0.26%respectively.,while that of Escherichia coli was99.99%±0.00%,44.66%±0.44%and21.09%±0.32%respectively. To Staphylococcus aureus, Comparison between various materials, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=6.154, P=0.004), the difference between the groups was significant statistical significance (F=2E+007, P=0.000),pairwise comparisons were significant statistically significant (P=0.000). To E. coli. Comparison between various materials, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=15.792, P=0.000),the difference between the groups was significant statistical significance (F=1E+007, P=0.000), pairwise comparisons were significant statistically significance (P-0.000).3. The majority of cell death, rounded growth and poor after extracts of100% soaked10-2mmol/L AgNO3CHA/PLA-Ag artificial bone cultured with L929cells2,4,7d.the cell was toxic and the toxicity grade was3. While there was strong growth of cells, observed under inverted microscope, cell membrane integrity, cells showed the typical shape of the spindle, adherent, after extracts of100%soaked10-3,10-4,10-5mmol/L AgNO3CHA/PLA-Ag artificial bone cultured with L929cells2,4,7d, the toxicity degree were1,0,0respectively. The The experimental results show that the cytotoxicity degree of10-3mmol/L AgNO3CHA/PLA-Ag artificial bone material extracts was0; the hemolysis rate was2.22%, less than5%, in line with the requirements of biomedical; The degree of acute toxicity grading was0; the sensitization scoring was0points,it showed that10-3mmol/L AgNO3CHA/PLA-Ag artificial bone material meet the medical standards for biological materials.4. The Ag+content of10-2,10-3,10-4,10-5group is2310.520±33.418,318.692±1.763,67.535±0.191and6.050±0.028μg/g respectively,.One-way ANOVA analysis of statistics showed that each group heterogeneity of variance (F=9.005, P=0.001),the difference between the groups was significant statistical significance (F=25847.567, P=0.000), pairwise comparisons were significant statistically significance (P=0.000). Ag+content of10-3materials in vitro SBF showed the trend of slow release, fast release within7days of the initial soak, after the release rate gradually slowed down.5.Each group of experimental animals eat normally, and can free activity after2weeks after operation. The wound of10-3CHA/PLA-Ag group animals has no significant swelling, no crack, no exudate pus,it healed well; The wound of CHA/PLA group animals was poor, dehiscence, swelling and exudate pus, the secretions of bacterial culture was Staphylococcus aureus, two weeks after surgery soft tissue around wood swelling reduced.and gradually healed; While that of the blank control group was mild swelling, no significant exudate pus, healing after one week.During 3months,there was no death of animals and pathologic fracture. General observation showed the rabbit tibia defects of experimental group and CHA/PLA group healed completely,but that of blank control group almost no bone repair by fibrous tissue filling. The same time-point comparison of radiological examination score of experimental group, CHA/PLA group and blank control group,One-way ANOVA analysis of statistical results showed that each group homogeneity of variance (P>0.05),the difference between the groups was significant statistical significance (P<0.05), pairwise comparisons were significant statisticallysignificance (P=0.000). Among them, the difference of scores at each time point of experimental group and CHA/PLA group has no statistical significance (P>0.05). This showed the bone repair capacity was no significant difference between the experimental group and CHA/PLA group, while the experimental group, CHA/PLA group and control group at each time point differences in scores have significant statistical significance (P<0.05), the scores of the experimental group and CHA/PLA group were higher than the control group, demonstrating the experimental group and CHA/PLA group have a significant role in bone repair.Conclusion1. In this study, coral hydroxyapatite (CHA) powder soaked in different concentrations of silver nitrate solution, by solution ion exchange method, prepared silver hydroxyapatite containing different silver content, then mixed the different containing silver CHA powder with PLLA according to the quality of3:1,and through the CAD and selective laser sintering rapid prototyping technology, can successfully prepared digital silver bactericide and has a special shape of the artificial bone scaffolds,the process is simple and easy. The compressive strength of materials are greater than1Mpa.2. In vitro antibacterial tests of CHA/PLA-Ag artificial bone material showed that the it has an antibacterial effect against to Staphylococcus aureus and Escherichia coli.The zone of inhibition experiments of CHA/PLA-Ag artificial bone material soaked10-4,10-5mmol/L of AgN03against to the above two kinds of bacterial were negative in vitro, Colony number of experiments show that the antibacterial rate of less than90%,they do not meet the standards of medical antibacterial materials,While that of the materials soaked10-2,10-3mmol/L of AgNO3against to the above-mentioned two kinds of bacteria was Obvious. The colony number of experiments show its antibacterial effect is obvious, antibacterial rate of more than90%, has good antibacterial properties, in line with the standards of medical antibacterial materials.3. Contained silver artificial bone scaffolds in vitro biological tests showed that soaking10-2mmol/L of AgNO3material extract has cytotoxicity, does not meet the medical standards for biological materials. But the cell toxicity grading of CHA/PLA-Ag artificial extracts soaked10"3,10-4,10-5mmol/L AgNO3were1,0,0. It show that with the material as containing silver content increased, the greater the cytotoxicity. The CHA/PLA-Ag artificial bone material soaked10-3,10"4,10-5mmol/L AgNO3meet the medical standards for biological materials, Which CHA/PLA-Ag silver artificial bone scaffolds soaked10-3mmol/L has no hemolytic reaction, no systemic toxicity and allergenicity, has good biocompatibility.4. The containing silver and release experiments of contained silver CHA/PLA-Ag artificial materials show that the artificial bone material containing silver increased with increasing silver ion concentration in solution, Silver ions in the scaffolds can maintain prolonged release characteristics, so that it can effectively avoid the early massive release of silver ions, to maintain a long period of sustained slow release, and effectively reduce the toxic effects of silver ions on the tissue cells. 5. In the repair of rabbit tibial metaphysis pollution lacunar bone defect model, contained silver CHA/PLA-Ag artificial materials prepared by soaked10-3mmol/L AgNO3can significantly inhibited the bone infection and repair the bone defects, the results showed that hydroxyapatite artificial bone combined with PLLA after containing silver did not significantly affect the osteogenesis,and has antimicrobial, biocompatibility and bone conduction,.It is a good prospect of new antibacterial bone repair alternative materials.