钛表面定植壳聚糖衍生物的抑菌性能研究

发布时间：2018-05-12 17:06

本文选题：壳聚糖 + 钛　；参考：《第二军医大学》2013年博士论文

【摘要】：背景内植物材料已在骨科得到广泛应用，内植物周围感染问题也日益突出。骨科内植物表面宿主免疫低下、磨损颗粒损害周围组织、细菌耐药、生物膜形成等导致这类感染极难治疗，也导致住院时间延长，增加患者痛苦，增加并发症和死亡风险，也造成医疗资源的巨大支出。许多研究致力于内植物表面改性，但往往存在抑菌能力欠佳、导致细菌耐药、妨碍骨整合等缺陷。如能研制出新型抗菌内植物表面，使其具备良好的抗菌能力与生物相容性，且可有效预防细菌粘附与生物膜形成，甚至可促进骨整合，将有一定的研究与应用前景。第一部分目的合成不同取代度的氯乙酰硫脲羧甲基壳聚糖衍生物（CCMC），研究其抑菌性能、细胞毒性以及对骨髓间充质干细胞（MSCs）增殖与分化的影响。方法 1、以抑菌圈试验及抑菌率试验测试其对金黄色葡萄球菌、表皮葡萄球菌的体外抗菌性能； 2、以MTT比色法研究其对MC3T3-E1细胞的细胞毒性； 3、以MTT比色法测定其对MSCs增殖的影响； 4、以碱性磷酸酶活性和骨钙素含量测定其对MSCs分化的影响。结果 1、随着CCMC取代度的增高，其抑菌活性也逐渐增强； 2、0.88取代度的CCMC具有体外细胞毒性，另两种取代度的CCMC没有体外细胞毒性； 3、0.24与0.53取代度的CCMC对MSCs的增殖和分化均有一定促进作用，0.88取代度的CCMC对MSCs的增殖和分化均有一定抑制作用。第二部分目的测定不同取代度的CCMC的最小抑菌浓度及最小生物膜清除浓度，以及特定浓度下对金黄色葡萄球菌以及表皮葡萄球菌生物膜抑制率及对生物膜活性的影响。方法 1、以细菌定量法测定不同取代度的CCMC的最小抑菌浓度，以比色法测定各取代度CCMC的最小生物膜清除浓度； 2、以比色法测定各取代度CCMC在特定浓度下对三种生物膜的抑制率； 3、以荧光染色比较各取代度CCMC对表皮葡萄球菌生物膜活性的影响。结果 1、随着CCMC取代度的增高，最小抑菌浓度及最小生物膜清除浓度也逐渐减小； 2、相同浓度下，取代度越高，CCMC对三种生物膜的抑制率越高，0.88与0.53取代度的CCMC在较小浓度下即可有效抑制生物膜生长； 3、相同浓度下，取代度越高，CCMC对表皮葡萄球菌生物膜活性的抑制能力越强；随着浓度的增高，各取代度CCMC对表皮葡萄球菌生物膜活性的抑制能力越强。第三部分目的钛合金表面共价结合0.53取代度的CCMC（Ti-CCMC），通过与钛合金表面（Ti）的比较，研究这种钛表面抑制生物膜的能力、对成骨细胞粘附与增殖的影响，，建立新西兰大白兔感染模型，研究Ti-CCMC对感染局部骨组织的影响与全身性影响。方法 1、以荧光染色法比较Ti-CCMC与Ti在体外对生物膜活性的影响； 2、以细菌定量法比较Ti-CCMC与Ti在体外对表皮葡萄球菌粘附的影响； 3、比较Ti-CCMC与Ti对MC3T3-E1细胞粘附与增殖的影响； 4、以细菌培养定量检测法测定钛棒周围骨组织内细菌含量； 5、比较两组兔的体重变化，行血细菌培养、X线检查，研究两组兔全身性情况的差异； 6、对两组兔股骨中段髓腔行革兰氏染色，比较两组细菌局部迁移的差异。结果 1、Ti-CCMC体外对生物膜活性有明显抑制作用； 2、Ti-CCMCC由一定的抑制表皮葡萄球菌粘附的能力； 3、Ti-CCMC可促进MC3T3-E1细胞粘附与增殖； 4、Ti组骨组织内见细菌生长，Ti-CCMC组未见细菌生长； 5、两组兔体重比较无明显差异，Ti组术后X线检查发现股骨骨质破坏，Ti-CCMC组未见明显异常，未见骨溶解与透亮带； 6、Ti组兔股骨中段髓腔行革兰氏染色，可见革兰氏蓝染的细菌散在，Ti组未见细菌。结论壳聚糖酰基硫脲衍生物具有良好的抗菌能力、生物相容性与骨整合能力，可作为骨科内植物表面抗菌的新型材料，具有一定的研究与应用前景。
[Abstract]:background
Internal plant materials have been widely used in the Department of orthopedics, and the problems of the infection around plants are also becoming increasingly prominent. The low immunity of the plant surface host in the Department of orthopedics, the damage to the surrounding tissues, the resistance of the bacteria, the formation of biofilm, etc., lead to the extremely difficult treatment of this type of infection, and the prolonged hospitalization, the pain of the patients, the increase of the risk of complications and death. Many studies have contributed to the enormous expenditure of medical resources. Many studies have been devoted to the surface modification of plants, but they often have poor bacteriostasis, causing resistance to bacteria and impeding bone integration, such as developing a new type of antibacterial internal plant surface, making it good for antibacterial and biocompatibility, and effectively preventing bacterial adhesion and biofilm. It can even promote bone integration. It will have some research and application prospects.
Part one
objective
The synthesis of chloracetyl thiourea carboxymethyl chitosan derivative (CCMC) with different degrees of substitution was synthesized, and its antibacterial properties, cytotoxicity and the effect on the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs) were studied.
Method
1, the antibacterial activity of Staphylococcus aureus and Staphylococcus epidermidis was tested by inhibition zone test and inhibition rate test.
2, the cytotoxicity of MC3T3-E1 cells was studied by MTT colorimetry.
3, the effect of MTT on the proliferation of MSCs was determined by the colorimetric method.
4, the effects of alkaline phosphatase activity and osteocalcin content on MSCs differentiation were measured.
Result
1, with the increase of CCMC substitution degree, its antibacterial activity also increased.
CCMC with 2,0.88 substitution degree had cytotoxicity in vitro, and the other two CCMC of substitution degree had no cytotoxicity in vitro.
3,0.24 and 0.53 degree of substitution CCMC promoted the proliferation and differentiation of MSCs, and the 0.88 degree of substitution CCMC inhibited the proliferation and differentiation of MSCs.
The second part
objective
The minimum inhibitory concentration and minimum biofilm clearance concentration of CCMC with different degree of substitution, as well as the inhibitory rate of Staphylococcus aureus and Staphylococcus epidermidis in specific concentration and the effect on biofilm activity under specific concentration were measured.
Method
1, the minimum inhibitory concentration of CCMC with different degree of substitution was determined by bacterial quantitative method, and the minimum biofilm clearance concentration of each degree of substitution CCMC was determined by colorimetry.
2, the inhibition rates of three CCMC on specific biomembrane at different concentrations were determined by colorimetry.
3, the effects of substitution degree CCMC on the activity of Staphylococcus epidermidis biofilm were compared by fluorescence staining.
Result
1, with the increase of CCMC substitution degree, the minimum inhibitory concentration and minimum biofilm clearance concentration also decreased.
2, under the same concentration, the higher the degree of substitution, the higher the inhibition rate of CCMC to the three biofilms, and the inhibition of the biofilm growth under the smaller concentration of 0.88 and the 0.53 substitution degree.
3, under the same concentration, the higher the degree of substitution, the stronger the inhibition ability of CCMC to the activity of Staphylococcus epidermidis biofilm, and with the increase of concentration, the stronger the inhibition ability of each substitution degree CCMC to the activity of Staphylococcus epidermidis biofilm.
The third part
objective
The titanium alloy surface covalently combined with the 0.53 degree of substitution of CCMC (Ti-CCMC). By comparing with the titanium alloy surface (Ti), the effect of the titanium surface on the inhibition of the biofilm was studied. The effect on the adhesion and proliferation of osteoblasts was studied. The infection model of New Zealand white rabbits was established, and the effect of Ti-CCMC on the local bone tissue was studied and the effect of the whole body was studied.
Method
1, the effects of Ti-CCMC and Ti on biofilm activity in vitro were compared by fluorescence staining.
2, the influence of Ti-CCMC and Ti on the adhesion of Staphylococcus epidermidis in vitro was compared by bacterial quantitative method.
3, the effects of Ti-CCMC and Ti on adhesion and proliferation of MC3T3-E1 cells were compared.
4, bacterial culture was used to determine the bacterial content in bone tissue around titanium bar.
5, the weight changes of two groups of rabbits were compared, and blood bacterial culture and X-ray examination were performed to study the difference of the whole body condition between the two groups.
6, two groups of rabbits were treated with Gram staining in the middle femoral medullary cavity, and the difference of the local migration between the two groups was compared.
Result
1, Ti-CCMC significantly inhibited biofilm activity in vitro.
2, Ti-CCMCC inhibited the adhesion of Staphylococcus epidermidis to some extent.
3, Ti-CCMC can promote the adhesion and proliferation of MC3T3-E1 cells.
4, bacterial growth was observed in bone tissue of group Ti, and no bacterial growth was found in group Ti-CCMC.
5, there was no significant difference in body weight between the two groups. In group Ti, X-ray examination revealed femoral bone destruction, Ti-CCMC group showed no obvious abnormalities, no osteolysis and translucent zone.
6, in group Ti, the middle femoral medullary cavity of rabbits was stained with Gram stain. The bacteria of gram blue stain were scattered and no bacteria were found in group Ti.
conclusion
Chitosan acyl thiourea derivatives have good antibacterial ability, biocompatibility and bone integration ability, which can be used as new materials for antibacterial surface of plants in the Department of orthopedics, and has a certain research and application prospects.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

【共引文献】