| The most ideal biological material is the personal bone tissue from oneself. As the evolution of natural biological materials for billions of years, biological materials are formed by a structure-complicated and sophisticated, wonderful principles and mechanism. As a complex, fresh autogenous bone is ingeniously combined with the inorganic and organic materials together. Hydroxyapatite mostly in inorganic materials are of highly osteoinductive, non-immunological rejection response. But autogenuous bone graft will increase the surgical trauma, and lead to local pain, paresthesia, or allergies and other complications in patients, and the source of bone is very limited. In recent years, the use of tissue-engineering bone to repair bone defects, not only has provided a method of repairing bone defects, but also has provided a new idea. New hope has been brought for the treatment of bone defects because of bone tissue engineering research. At present, artificial bones constructed by tissue engineering in vitro still remain in the laboratory and animal-experimental segments, but with the development of the related subjects, such as cell biology, engineering, immunology, material science, the research of bone tissue engineering will make a breakthrough progress and there is an extremely broad application prospects in industrial production and clinical applications. It will become a great potential for the development of high-tech industries in the 21st century, and offer enormous economic and social benefits. As the main inorganic component of human bone tissue, hydroxyapatite bioceramic is cheap, and has good biocompatibility, biodegradability, bone conductivity, and bone fusion. Particularly the porous coral hydroxyapatite can provide a lot of surface area/volume ratio, can ensure cell proliferation, differentiation, and high metabolic activity. Then it is particularly suitable for bone tissue engineering. With the increased mechanization of modern industrialization, a large number of significantly infectious bone defects increase caused by trauma, infection, cancer, accidents, wars, earthquakes. The traditional methods of treatment is that there is a debridement at first until the wound closed, and no signs of infection for 3 to 6 months, then there is a bone repairment secondly. The higher infective rate of bone graft in one-stage often occurs, and it has been regarded as taboos. And the clinical bacterial resistance to antibiotics is a growing problem, the role of fighting against microbial bone in antibiotics-loaded graft material is taken more and more limit, and microbial agent has been put forward higher requirements. To solve the traditional treatment methods to bring a range of issues, such as increasing treatment time, the difficulty, the patient's suffering and economic burden, traditional attitudes have been changed in recent years. Domestic and foreign scholars has made some progress for the anti-infective bone graft materials in basic experiments and clinical practice. To the end, we are cooperating with the Department of Materials Science and Engineering in Tsinghua University to develop an anti-infective bone scaffold materials, and a series of experimental research have shown that:The antibacterial bone repairment material has good biocompatibility, and can release slowly silver ions to effectively inhibit local residual bacteria in infective bone defect after debridement, as follow.This experiment divides into four parts: Section one:Development and Characterization of Silver-loaded coralline hydroxyapatite (Ag+-CHA) artificial bone.Objective:To develop Ag+-CHA artificial bone and study the physical and chemical characterization.Methods:The experiment was accomplished in Tsinghua University, Laboratory of Materials Science and Engineering from May to August in 2008. Experimental material:Ag+-CHA was prepared by immersing coralline hydroxyapatite (Coral Hydroxyapatite, CHA) particles into the some concentration of silver nitrate (Silver nitrate, AgNO3) solution, and frozen in freezed dryer after a certain time. X-ray diffraction method is applied on the Ag+-CHA, CHA and pure coral to investigate their crystal phase, structure and phase analysis; FT-IR spectroscopy is applied to analyze the composition of the sample material; SEM micrograph is applied to materials morphology and the aperture; Compression tests and three-point bending test are used to evaluate mechanical properties of bone repair scaffold. The experimental data is analyzed with One-Way ANOVA and LSD on mechanical properties by the software SPSS13.0.Results:①X-ray diffraction results revealed the characteristic peaks of Ag+-CHA, CHA, and pure coral, and there was no secondary phases, indicating that there was no other derivatives generated in the reaction between silver ions and the CHA, remaining the original crystalline structure; the same crystal plane phase at (211), (300) peak were lower than the CHA's. It showed that silver ions might react with the location of the HA crystal surface.②FT-IR spectroscopy are shown that they have a certain similarity,1456 cm-1 and 1413cm-1 are the material characteristic peaks of CO32-group, the same as pure coral. It showed that the most component of Ag+-CHA and CHA was CaCO3. The characteristic peaks of HA are 1031 cm-1,604 cm-1 and 563cm-1, shows that HA exists in the part of the Ag+-CHA and CHA already. The broadened HA features peaks on the FT-IR spectrum may be related to the transformation between silver ion of Ag+-CHA and the calcium ion of CHA, because of Ag+ ionic radius is larger than the Ca2+.③The SEM and BSE images showed that the morphology of the SLCHAs depended on the content of Ag+, and the silver ions were uniformly distributed on the surface of SLCHAs. EDS results demonstrated that the silver content of the SLCHAs decreased along with the decrease of the concentration of silver nitrate.;④compression test method was used to analyze the compressive strength among the three kinds of materials, and there was no significant difference (F=3.147, P=0.196), the P values were 0.323,0.695,0.403 compared among them; The compression modulus among three kinds of material was also no significant difference (F=0.827, P=0.482), the P values were 0.491,0.604,0.247 compared among them; and the broken power in the three-point bending test was no significant difference (F=2.543, P=0.159), the P values were 0.080,0.731,0.131 compared.Conclusion:The physical and chemical properties of coralline hydroxyapatite still was maintained after loading silver ion and a three-dimensional porous framework has not been changed; The principle of Preparation is mainly the replacement reaction between silver ions and calcium, followed by the dissolution of silver ions-deposition. And with the increase of the concentration of silver nitrate solution, the content of silver in Ag+-CHA increased.Section two:Study on the content of silver in Ag+-CHA and releasing character in vitro.Objective:To develop antimicrobial Ag+-CHA artificial bone scaffold material, and the knowledge of Ag+-CHA is taken by silver content and releasing experiment in vitro.Methods:The total silver content in the SLCHAs and the average concentration in the releasing test were determined by inductively coupled plasma emission spectroscopy (ICP-OES), calculated the content of silver loading refer to the standard curve of realationship between the content of Ag and the concentration of AgN03. Simulated body fluid (SBF, pH=7.25) was used in the releasing test in vitro, and we try to reveal the relationship between the materials' strucure and the silver releasing. The experimental data is analyzed with Linear Regression and Curve Fit by the SPSS13.0.Results:The equation of Linear Regression:y=3E+007 x2+126863.3x+ 12.845 (R Square Change=0.997). According to Linear regression test, F=959.625, P=0.000, there is statistical significance. The Ag+-CHA has a well silver delivery capability, and the samples each are soaked into the liqiud of SBF, the titer of silver releasing after the 24 hours is 885.27±122.49μg/L at its peak, lower than that reported in the literature minimum inhibitory concentration of silver ions (MIC) 1.25μg/mL. Then gradually reduced,7 to 14 days to release the maintenance of a plateau, followed by a slow releasing in the following days a lower concentration. After 28 days, there are 79.80±6.69μg/L of silver ion release.Conclusion:The Ag+-CHA has been able to remain a long time the release of silver ions closely related to the 3D porous structure itself. The reversible ion exchange process shows more visiblely releasing performance and more effectively avoid the sudden release of silver ions to maintain the sustained release for a long time, effectively reducing the use of metallic silver to achieve its effect.Section three:Study on cytocompatibility of Silver-loaded coralline hydroxya-patite artificial boneObjective:To develop antimicrobial Ag+-CHA artificial bone scaffold material and study on the cytocompatibility of mouse embryonic osteoblasts line (MC3T3-E1) in vitro. Methods:The experiment was accomplished in Department of Orthopaedics, Guangzhou General Hospital of Guangzhou Military Command from June to September in 2009. Using cell culture technique in vitro, MC3T3-E1 cells were seeded on composites 10-3,8×10-5,10-5 mol/L Ag+-CHA, CHA and pure coral scaffold materials. Cell growth and reciprocity were monitored using MTT assay, ALP activity measurement, Inverted phase contrast microscope, Confocal laser scanning microscopes (CLSM), and SEM. The experimental data is analyzed with Univariate ANOVA on proliferation of osteoblasts by the SPSS 13.0.Results:①The results of MTT assay can be seen that significant differences-exist among the different groups (F=76.457, P=0.000). Proliferation of MC3T3-E1 cells is not significantly different from that on CHA, pure coral, and the lower silver concentrations (P=0.177,0.428) at days 1,3,5, but is significantly higher on the control group and 170μg/ml Ag+/CHA group (P=0.000,0.000,0.000,0.000,0.000). Compared to blank control group, the proliferation of the cells cultured in each scaffold increased with the increasing of culture time besides the CHA soaked with 170μg/ml Ag+ ions;②The results of ALP can be seen significant differences exist among the different groups(F=15.582, P=0.000). The group 10-3mol/L has more significant difference than other groups(P=0.000,0.000,0.000,0.000,0.004), indicating that cells have been growing slowest in this group, and ALP has been secreting at least; 8×10-5mol/L group,10-5mol/L group have no significant difference compared with the control group (P=0.340,0.068); CHA group showed no significant difference compared with pure coral group(P=0.292), and no significant difference exist in 8×10-5mol/L group and 10-5mol/L group(P=0.366,0.292,0.880), but there was significant difference between the control group (P=0.049,0.006,0.004, respectively), indicating that 8×10-5mol/L group,10-5mol/L group had no significant toxicity on cells, suitable for cell growth and adhesion on its surface. Meanwhile, the spatial structure of 3D porous material expands the surface area of cell adhesion, and is bebefit for the rapid proliferation of cell. The results are consistent with the MTT test.③Surface topography of MC3T3-E1 cells seeding for 1 days:The transparent and round cells seeding on 10-3mol/L Ag+-CHA were free, folded and unadhered, presenting shrinkage and low proliferation; The osteoblasts were proliferated much better on the 8×10-5mol/L group,10-5mol/L group than that on the surface 10-3mol/L. The confluent cells were shaped in fusiform and arranged tightly in fasciculation or finger print, with the narrow interval and gathering growth.④CLSM image shows the morphology of the 8×10-5mol/L group is the same as the other two groups.⑤SEM image shows material cells were spindle-shaped than the flat surface of the tiny villi and folds, the cell contact area with the material protruding pseudopods formed duck webbed adhesive and wrapping materials, the surface shows that their secretion of bone matrix.Conclusion:The MTT, ALP activity, inverted phase contrast microscopy, laser confocal and scanning electron microscopy observation of the full range of multi-angle cells in response to the natural framework of the material. The cell proliferation, growth and well, indicates that material has good cytocompatibility. The results of MTT and ALP activity quantitatively show that the low-silver content loaded in Ag+-CHA and non-loaded materials are basically the same in the biocompatibility.Section four:Study on the repairment with Ag+-CHA bone graft in infected radial bone defects in rabbitsObjective:To investigate the therapeutic effect of the Ag+-CHA artificial bone on a large number of bone defects.Methods:36 New Zealand white rabbits were selected, and randomly assigned to four groups with nine rabbits in each group. A rabbit model of infected and defect radial bone segment with 15mm. The animals all were followed by implant of Ag+-CHA in group A, CHA in group B, situ autogenous bone in group C and those in group D were left without anything grafting, as a control group. Respectively, specimens were harvestd after 2,6,10 weeks after the above procedures and were then subjected to gross observation, radiographic examination, histological observation to compare their therapeutic effect on bone tissue repairment. The experimental data is analyzed with Repeated-Measures ANOVA and LSD on the results of radiographic examination by the SPSS 13.0.Results:The different groups of materials for repairing_bone defects:2w general observations can be seen when the A, B groups the surface of granulation tissue graft and some translucent bone coverage, with the host bone callus stump some connection between the graft-free apparent shift; C group graft and host bone callus, some connected to graft inactivity; D group to muscle bone defect filled with fibrous tissue.6w, when A, B groups the surface of thin bone graft covered stump with the host bone callus between the large number of connections, a more solid; C group of cortical bone graft and host continuous, but uneven, no significant graft shift; D group in order to muscle defect was still filled with fibrous tissue. 10w, when A, B two consecutive graft bone covering the surface of a clear, stable graft; C group graft and host bone cortex into one row; D group in order to muscle defect was still filled with fibrous tissue. X-ray results can be seen among the groups at different time points, there was a significant difference (F=11.537, P=0.000); Autogenous bone was the best than the other groups compared with the significant difference (P=0.004,0.010, 0.000), indicating that autologous bone tissue in large segmental bone defect repair is still the best materials; Ag+-CHA group and the CHA group showed no significant difference(P=0.524), indicating that lower silver content of Ag+-CHA was the same as non-loaded silver; Control group had significant differences compared with other groups (P=0.000,0.000,0.000), and the role of bone repairment was obviously inhibited owing to the infection of large segment of rabbit radial bone the bone defect, and the animal model is successful.Conclusion:Based on the idea of bionics and previous study on scaffold material for bone repair, we prepared a new type of antimicrobial scaffold materials for infected bone defects. The composition and structure of bone material are similar to natural bone inorganic ingredients and porous structure of cancellous bone. The animal experiments in vivo proved that this material has good biocompatibility and bone conductivity, and is an effective bone repair-scaffold materials, can be achieved the same effect as non-silver materials for bone repairment, and there is hope to become the preferred antiinfected bone repair material and has broad market prospects.In summary, the preparation process of anti-bacterial Ag+-CHA bone substitute materials is simple, and antimicrobial properties of silver ions is achieved more desirablely by slow-release. There are good bone conductivity and osteogenic effect in the large infective bone defect, it is conceivable that the anti-bacterial artificial bone material may have a widespread application prospect in treating infective bone defect to prevent infection as a good-assisted treatment. |
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