【作者】 包崇云； 王买全； R. Trukenmuller； 张强； 欧国敏； 宫苹； C.A. van Blitterswijk；

【Author】 CY Bao~1,MQ Wang~1,R Trukenmuller~2,Q Zhang~(1,3),GM Ou~1,P Gong~1,CA van Blitterswijk~2 (1.West China School of Stomatology,Sichuan University,Chengdu,China,610041; 2.Department of Tissue Regeneration,Institute of Biomedical Techonology, Twente University,Enschede,The Netherland,7500AE; 3.The Affiliated Stomatological Hospital of Nanchang University,Nanchang,China,330006)

【机构】 四川大学华西口腔医学院； Department of Tissue Regeneration,Institute of Biomedical Techonology,Twente University,Enschede,The Netherland,7500AE；

【摘要】 目的:通过制备精确控制微结构的材料样品,筛选影响C2C12细胞及S.a和P.g细菌粘附、增殖的关键微结构,探索优化实现软组织密封的经皮/穿龈部位材料形貌特征方法:①微米结构芯片样品制备:采用纳米HA浆料,制备表面亚微米粗糙度HA片。用激光消融法于HA片上23个大小为400μm×400μm区域(其中一个区域为对照),分别制作尺度为1μm,3μm, 5μm,10μm;深度与制备尺度相当;间距10μm,20μm;形状为线条、交叉线条、圆孔和三角形的微结构,形成HA微结构芯片。再以HA微结构芯片为模版,翻制形成与HA微结构一致的PDMS微结构芯片样品。②光镜、激光共聚焦显微镜、扫描电镜研究C2C12细胞、S.a和P.g细菌在微米结构芯片粘附、增殖行为结果:①SEM示在PDMS与HA制备成一样微结构模式的微结构芯片。②C2C12细胞在不同微米结构区域粘附、增殖数量不同,细胞更容易间距大,微结构尺度小的区域粘附与增殖,特别在亚微米凸凹晶粒及纳米界孔区域。两种细菌均主要粘附于微米结构区,沟裂越多,粘附细菌越多,沟裂宽度越大,粘附细菌越多,与微米结构的形状没有太大关系。而亚微米结构对照区域粘附较少。细胞、细菌在HA、PDMS相同的微结构区均表现出相似的粘附、增殖特性。讨论:生物材料的微结构和局部微环境的微形貌对细胞迁移、粘附、增殖、分化等行为均产生着影响,同时对微生物的粘附、增殖也产生影响。结论:①微米结构芯片可望为实现高通量筛选分析微结构对细胞及细菌行为的影响研究。2.细胞易于在亚微米结构粘附、增殖,细菌易于在微米结构而不易粘附于亚微米结构粘附、增殖,为进一步探索优化器件经皮/穿龈部位材料形貌制备提供初步理论依据。更多还原

【Abstract】 Purpose:Percutaneous -transmucosal devices refer to artifical devices remaining long - term in vivo and exercising specific function; this retention could be obtained by the means of through skin/gingiva or skin/ mucosa.It has been widely applied to dental implant、maxillofacial prosthesis、percutaneous implanted prosthetics and etc.But this kind of devices exist certain failures in practical application. The major reasons are peri - implantitis and deficiency of satisfactory combination between soft tissue and implanted materials.The chemical composition and physical morphology of percutaneous - transmucosal position determine the adhesion of bacterial and soft tissue,and influence the hermetization between soft tissue and implanted materials.Referring to physical morphology,most scholars suggest the surface of the position should be relative smooth.However,it still remains unclear that which kind of morphological characteristics is helpful to biological seal for soft tissue.In order to explore and optimize the physical patterns which can achieve the goal of soft tissue biological seal for percutaneous-transmucosal,micrometer structure specimen was made under precise control.Then screening the structure which can influence the adhesion and proliferation of C2C12、S.a and P.g.Method:A.The making of micrometer structure chips:Synthesis nanoHA slurries by wet heating treatment,and the HA disc with submicron range surface was made.The microstructure were divided into 23(400um×400um) areas,one of which was used as control and the others were fabricated with laser ablation as below:the sizes are 1um,3um,5um,10um respectively;the depths correspond with the size;the space are 10um,20um respectively;the shape are line, crosslock,round hole and triangle.The surface of HA microstructure chip were analyzed by XRD、SEM and Raman spectroscopy.Then we replicate with PDMS to get the same microstructure of nanoHA.The topography of the chips was observed by SEM.B.The adhesion and proliferation of C2C12 on microstructure chips:after defrostation and amplification,the cells were seeded on the HA specimen in 24 well culture plate at a density of 4×103 cells per well.For PDMS,the density is 4×104 cells per well.After culturing for 6h,1d,3d, 5d respectively,the cells were stained with DAPI,and then observed under invert phase contrast microscope and laser scanning confocal microscope.C.The adhesion and proliferation of bacterial on microstructure chips:two kinds of bacterial were inoculated into PDMS in 24 well culture plate,For S.a,the inoculative numbers are 1×106/well,and for P.g,the numbers are 1×108/well.Bacterial were cultured for 24hours and 48hours respectively,The influence of microstructure chip on the adhesion of S.a and P.g was analyzed by SEM and the bacteria were quantitative counted according to SEM.Result:Z A.SEM showed the particulate size of calcium phosphate ceramics slurries was 60nm×250nm.XRD showed their chemical composition was pure HA.Submicron HA disc could be fabricated by nanoHA slurries.Various shapes of microstructure could be fabricated by laser ablation.XRD and Raman spectroscopy showed the chemical composition of ceramics was still pure HA.Chips with the same micro pattern of HA disc could be replicated by PDMS.B.C2C12 cells were seeded on specimen.The numbers of the cell adhered to the specimen became higher as the prolongation of cultivate time.But the adhesive and proliferation numbers varied in different micrometer structure domains,which indicated that the cells were easier to adhere to the microstructure domains in those with bigger space and smaller size,especially in the submicron regions with original scraggly crystal but not be artifically fabricated.Although cellular behavior on specimen(HA、PDMS) was different,they all manifested similar property on the same microstructure.C.The two kinds of bacterial could mainly adhere to the microstructure,and the adhesive bacterial number was directly proportional to the number and the width of the grooves but had no significant relationship with microstructure shape.For those materials with submicron regions,less adhesion took place.Conclusion:A.NanoHA slurries were firstly adopted to fabricate ceramics composed of submicron and micrometer structure was made on ceramics by laser ablation.Then the same microstructure was replicated on PDMS.These structures worked as a foundation for the next high throughput screening which can analyze the influence of the microstructure on the behaviors of cells and bacteria B.Cells were inclined to be adhered and proliferate in submicron regions,whereas bacteria were inclined to be adhered and proliferate in microstructure regions,but not submicron regions,This result could provide preliminary theory and basis for exploring and optimizing physical pattern in percutaneous - transmucosal devices.更多还原