壳聚糖-Ⅰ型胶原复合膜的理化性能、抗菌性能及生物相容性的实验研究

发布时间：2018-05-19 17:16

本文选题：壳聚糖 + Ⅰ型胶原　；参考：《南方医科大学》2012年硕士论文

【摘要】：研究背景引导性骨再生(guided bone regeneration, GBR)膜具有屏障的作用,通过将骨引导性的基质保持在缺损区,屏蔽成纤维细胞等非成骨性细胞长入缺损区,允许骨组织在膜下空间内再生,从而减少移植骨骨量的丢失。胶原膜具有良好的生物相容性、可降解性、能够促进细胞的粘附和增殖的特点,作为引导性骨再生膜在临床上已被广泛应用。但纯胶原蛋白膜存在机械性能差、降解快、抗水性能差、不能与组织生长速度很好地匹配等问题。壳聚糖是一种可降解的天然聚阳离子生物多糖,降解产物为氨基葡萄糖等小分子,对人体无毒、无刺激,有较好的组织相容性。同时壳聚糖分子带正电荷,通过和细菌细胞膜上负电荷的相互作用,干扰细菌细胞膜功能,达到抗菌作用。本实验将Ⅰ型胶原与壳聚糖交联复合,制备具有良好力学强度、良好生物相容性及抗菌性能的引导骨再生膜修复材料,以改善胶原作为引导骨再生膜存在的不足。同时针对GBR术后存在软组织瓣裂开、膜暴露、感染等问题,通过在胶原中加入壳聚糖,使屏障膜具有抗菌性能,以减少膜暴露引起的感染,在促进成骨的同时,促进软组织的愈合,提高GBR手术的成功率。本实验使用提纯的牛肌腱Ⅰ型胶原(collagen, COL)和脱乙酰度85%的壳聚糖(chitosan, CS),按质量比为(COL:CS=3:1)制作壳聚糖-Ⅰ型胶原复合膜。将其与纯牛肌腱Ⅰ型胶原膜对比,检测其理化性能,并在两组膜上接种成纤维细胞和MG63成骨细胞,观察两组膜上所接种的细胞在膜表面的增殖情况,对壳聚糖-Ⅰ型胶原复合膜的理化性能和生物相容性进行初步研究。同时对其进行抗菌性能检测,评估壳聚糖-Ⅰ型胶原复合膜的抗菌作用,为其应用于引导性骨再生膜提供参考。目的制备壳聚糖-Ⅰ型胶原复合膜并研究其理化性能,检测其与成纤维细胞和MG63成骨细胞的生物相容性,以初步探讨壳聚糖-Ⅰ型胶原复合膜应用作为引导性骨再生屏障膜的可行性,同时对其进行抗菌性能检测,评估壳聚糖-Ⅰ型胶原复合膜的抗菌能力,从而为其应用于引导性骨再生提供参考。材料和方法 1.采用提纯的牛肌腱Ⅰ型胶原和壳聚糖,通过冷冻干燥法,使用热交联、化学交联技术制备壳聚糖-Ⅰ型胶原复合膜(实验组)和Ⅰ型胶原膜(对照组)。采用氨基酸含量分析、差示量热扫描法分析对牛肌腱Ⅰ型胶原性质和表征进行分析。通过红外光谱分析、扫描电镜分析、吸水率测试以及材料力学性能测试,检测实验组和对照组的理化性能。 2.0.5cm×0.5cm大小的实验组和对照组膜各20张,分别置于48孔板内,接种人牙龈成纤维细胞于孔板内培养,接种密度每孔5×104个,分别于1d、3d、5d、7d每组各取出5张膜,置于新48孔板中,每孔加入培养液200ml和MTS试剂40ml,孵育3.5h,测吸光度值(OD),比较两组膜上粘附的人牙龈成纤维细胞增殖水平。 3.1.0cm×1.0cm大小的实验组和对照组膜各12张,分别置于24孔板内,接种成骨肉瘤细胞系MG63于孔板内培养,接种密度每孔5×104个,分别于1d、3d、5d、7d每组各取出3张膜,置于新24孔板中,每孔加入培养液500ml和MTS试剂100ml,孵育3.5h,测吸光度值(OD),比较两组膜上粘附的MG63成骨细胞增殖水平。 4.人牙龈成纤维细胞接种于两组膜表面,接种后8h、1d、3d行生物材料扫描前处理之后,在扫描电镜(SEM)下观测细胞粘附、伸展及分化的形态变化。 5.成骨肉瘤细胞系MG63接种于两组膜表面,接种后4h及1d行生物材料扫描前处理之后,在SEM下观测细胞粘附、伸展及分化的形态变化。 6.实验组膜和对照组膜(10cm×1.0cm),每菌种每组各取3张膜,采用振荡法对两组膜行牙龈卟啉单胞菌、伴放线放线杆菌、金黄色葡萄球菌、大肠杆菌杆菌抗菌性能检测,平板计数法计数菌落回收数。 7.实验组膜和对照组膜(1.0cm×1.0cm)分别在3×108CFU/mL的牙龈卟啉单胞菌菌悬液和伴放线放线杆菌菌悬液培养24h,SEM观察细菌粘附膜表面情况。 8.以上统计学方法采用SPSS13.0统计分析软件,析因方差分析处理组和时间有无交互效应,多个样本均数比较采用单因素方差分析(One-way ANOVA),两个样本均数比较采用独立样本t检验比较,Levene'test检验方差是否齐性,方差齐则采用LSD样本均数间的两两比较,方差不齐则采用近似F检验的Melch法或Brown-Forsythe法进行方差分析后,Dunnett's T3多重比较。假设检验为双侧检验,检验标准0.05,P0.05时差异有统计学意义。实验结果 1.胶原氨基酸组成分析、差示量热扫描法分析结果表明牛肌腱Ⅰ型胶原的胶原3股螺旋结构是保持较完整,成分比例及性质表征符合Ⅰ型胶原的要求。红外光谱结果表明实验组具备了胶原和壳聚糖特征峰表现,并且两种材料之间形成大量氢键,分子间结合良好。扫描电镜分析发现实验组和对照组膜疏松面表面和横切面为多孔结构,实验组膜孔直径较大,为10～100μm。吸水率测试实验组膜吸水性为8.40±0.29,含水量为89.34±0.33%；对照组膜吸水性为9.25±1.13,含水量为89.09±2.12%；两组膜吸水性和含水量均无差异(P0.05)。实验组膜湿态拉伸强度3.70±0.16MPa,明显高于对照组膜湿态拉伸强度0.94±0.06MPa(P0.001)。 2.MTS结果：两组膜上分别接种人牙龈成纤维细胞和MG63成骨样细胞,其吸光度值均随时间增大,呈明显的增殖趋势,即具有较好的细胞相容性；两组膜的细胞增殖差异没有统计学意义。 3.8h时人牙龈成纤维细胞在两组膜上粘附,细胞数目多,细胞呈长梭形,大小不一,胞浆丰富,大部分细胞伪足数目少。对照组膜上部分细胞已成铺平状,细胞之间有相互交联,实验组膜上接种的细胞之间有相互交联,但较对照组上的少。24h时人牙龈成纤维细胞在对照组膜和实验组膜上粘附,细胞完全伸展,细胞伸出突起拉伸细胞向多角形方向发展,细胞之间有相互交联,与膜的表面融为一体,细胞表面有明显的基质分泌。72h时人牙龈成纤维细胞覆层生长,完全覆盖了膜表面,并可见新增殖的细胞覆盖于平铺粘附于膜表面的细胞上,细胞形态多为圆形和梭形,增殖的细胞之间相互交通。 4.4h时MG63细胞在对照组膜上粘附,呈圆梭形,伪足粘附在膜表面,呈触须状,数量少；MG63细胞在实验组膜上开始伸展,呈长梭形,许多丝足从片足中伸出粘附在膜的表面。24h时MG63细胞在对照组膜上,细胞伸出突起拉伸细胞向多角形方向发展,细胞之间有相互交联,部分细胞已成铺平状,与膜的表面融为一体,其表面见大量细胞基质分泌物；MG63细胞在实验组膜上,呈铺平状,伸出大量伪足,伪足伸展充分,并往材料孔隙处生长并穿梭其中。 5.抗菌性能检测结果：壳聚糖-Ⅰ型胶原复合膜对牙龈卟啉单胞菌抗菌率达99.55%,对伴放线放线杆菌抗菌率达99.97%,对金黄色葡萄球菌抗菌率达99.80%,对大肠杆菌抗菌率达99.93%。单纯Ⅰ型胶原复合膜不具备抗菌性能。 6.实验组和对照组膜分别浸泡于牙龈卟啉单胞菌和伴放线放线杆菌菌悬液24h后,SEM观察可见Ⅰ型胶原膜(对照组膜)表面有大量的细菌黏附,细菌呈团块状分布；而壳聚糖-Ⅰ型胶原复合膜(实验组膜)表面黏附的细菌明显少于对照组,呈散在分布,同时实验组表面有的细菌皱缩,甚至出现溶解、破碎.。结论壳聚糖-Ⅰ型胶原复合膜对成纤维细胞和MG63成骨细胞的生长无抑制作用,相具有优良的结构及生物学性能,作为引导性骨再生膜材料有良好的应用前景。壳聚糖-Ⅰ型胶原复合膜具有较好的理化性能和表面形态,无细胞毒性,具有良好的表面相容性、良好的结构相容性,可望作为一种性能优良的引导性骨再生屏障膜。壳聚糖-Ⅰ型胶原复合膜对需氧菌和厌氧菌有较强的抗菌性能,对预防GBR术后感染,有一定的积极意义。
[Abstract]:Research background
The guided bone regeneration (GBR) membrane has a barrier function by keeping the bone guided matrix in the defect area, shielding non osteoblastic cells like fibroblasts and other non osteoblastic cells into the defect area, allowing the bone tissue to regenerate within the space of the membrane, thereby reducing the loss of bone mass of the transplanted bone. The collagen membrane has good biocompatibility. Biodegradable, can promote the characteristics of cell adhesion and proliferation. As a guided bone regeneration membrane, it has been widely used clinically. But the pure collagen membrane has poor mechanical properties, fast degradation, poor water resistance and can not match the growth rate of tissue well. Chitosan is a biodegradable natural polycationic polysaccharide, The degradation products are small molecules such as glucosamine, which are nontoxic to the human body and have no stimulation and have good histocompatibility. At the same time, the chitosan molecule has positive charge and interferes with the membrane function of bacterial cells through the interaction of negative charge on the membrane of bacterial cells to achieve antibacterial effect.
In this experiment, collagen I was crosslinked with chitosan to prepare a guided bone regeneration membrane with good mechanical strength, good biocompatibility and antibacterial properties, in order to improve the deficiency of collagen as a guiding bone regeneration membrane. At the same time, the problems of soft tissue flap opening, membrane exposure, infection and so on after GBR were added to the collagen. Chitosan, which makes the barrier membrane have antibacterial properties, reduces infection caused by membrane exposure, promotes bone formation, promotes the healing of soft tissue, and improves the success rate of GBR operation.
In this experiment, the purified collagen (COL) and 85% deacetylation of chitosan (chitosan, CS) were used to produce chitosan - type I collagen composite membrane according to the mass ratio (COL:CS=3:1). Compared with the pure bovine collagen type I collagen membrane, the physical and chemical properties were detected, and the two groups of membranes were inoculated with fibroblasts and MG63 osteoblasts. A preliminary study on the physical and chemical properties and biocompatibility of the chitosan - type I collagen composite membrane was carried out by investigating the proliferation of the cells inoculated on the membrane on the membrane surface. The antibacterial properties of the chitosan - type I collagen composite membrane were detected and the antibacterial effect of the chitosan - I collagen composite membrane was evaluated to provide reference for its application to the guided bone regeneration membrane.
objective
The chitosan - type I collagen composite membrane was prepared and its physical and chemical properties were studied. The biocompatibility with fibroblasts and MG63 osteoblasts was detected. The feasibility of chitosan - I collagen composite membrane as a guiding bone regeneration barrier membrane was preliminarily discussed. At the same time, the antibacterial properties of the composite membrane were detected and the chitosan - type I collagen composite membrane was evaluated. Therefore, it can provide reference for guiding bone regeneration.
Materials and methods
1. using the purified bovine tendon type I collagen and chitosan, the chitosan - type I collagen composite membrane (experimental group) and type I collagen membrane (control group) were prepared by thermal crosslinking and chemical crosslinking by freeze-drying and drying. The properties and characterization of bovine collagen type I were analyzed by amino acid content analysis and differential calorimetry. The physical and chemical properties of the experimental group and the control group were detected by FTIR, SEM, water absorption and mechanical properties.
The 2.0.5cm * 0.5cm experimental group and the control group were 20 sheets, respectively placed in the 48 hole plate, inoculated with human gingival fibroblasts in the orifice plate, and inoculated with 5 x 104 per pore, respectively, in 1D, 3D, 5D, 7d, each of the 5 membranes, and placed in a new 48 hole plate. Each hole was added to the cultured liquid 200ml and MTS reagent 40ml, incubated 3.5H, and measured the absorbance value (OD), and measured the absorbance value (OD). The proliferation of human gingival fibroblasts adhered to the membrane was compared with that of the two groups.
The 3.1.0cm * 1.0cm experimental group and the control group were 12 sheets each, respectively placed in the 24 orifice plate, inoculated into the osteosarcoma cell line MG63 in the orifice plate, and inoculated with 5 x 104 per pore, respectively, in 1D, 3D, 5D, 7d, respectively, in each group of 3 membranes, and placed in a new 24 hole plate, each hole was added to the cultured liquid 500ml and MTS reagent 100ml, incubated 3.5H, and measured the absorbance value (OD), The proliferation of MG63 osteoblasts in the two groups was compared.
4. gingival fibroblasts were inoculated on the two groups of membrane surfaces. After inoculation, 8h, 1D, and 3D were treated with biological materials before scanning, and the morphological changes of cell adhesion, extension and differentiation were observed under scanning electron microscope (SEM).
5. adult osteosarcoma cell line MG63 was inoculated on the surface of two groups. After inoculation, 4H and 1D were treated with biological materials before scanning. The morphological changes of cell adhesion, extension and differentiation were observed under SEM.
6. the membrane of the experimental group and the control group (10cm x 1.0cm), 3 films were taken in each group. The two groups of porphyrinomonas gingivalis, Actinobacillus Actinobacillus, Staphylococcus aureus and Escherichia coli were detected by oscillating method, and the number of colony recovery was counted by plate counting method.
7. the membrane of experimental group and the control group (1.0cm x 1.0cm) were cultured in 3 x 108CFU/mL of porphyromoninomonas gingivalis suspension and Actinobacillus actinomycetes suspension for 24h, and the surface of the bacterial adhesion membrane was observed by SEM.
More than 8. statistical methods used SPSS13.0 statistical analysis software, analysis of variance analysis of variance analysis of the processing group and time have no interaction effect, the number of samples are compared to single factor analysis of variance (One-way ANOVA), two samples are compared by independent sample t test comparison, Levene'test test variance is homogeneous, variance homogeneous use LSD samples The 22 comparison between the average number and the variance of the variance, the Melch or Brown-Forsythe method of the approximate F test was used for the analysis of the variance and the multiple comparison of Dunnett's T3. The hypothesis test was bilateral test, the test standard 0.05, and the difference of P0.05 were statistically significant.
experimental result
Analysis of the composition of 1. collagen amino acids, the result of differential calorimetry analysis showed that the 3 strand spiral structure of collagen type I collagen was kept more complete, the proportion and characterization of the composition conformed to the requirement of type I collagen. The infrared spectrum showed that the experimental group had the characteristic peaks of collagen and chitosan, and the formation of the two kinds of materials was large. The surface and transverse surface of the membrane of the experimental group and the control group are porous structure. The membrane pore diameter of the experimental group is larger. The membrane water absorbability of the experimental group is 8.40 + 0.29, the water content is 89.34 + 0.33%, the water absorbability of the film is 9.25 + 1.13 and the water content is 89.09 + 2., and the water content is 89.09 + 2.. 12%, there is no difference in water absorption and water content in the two groups (P0.05). The wet tensile strength of the membrane in the experimental group is 3.70 + 0.16MPa, which is significantly higher than that of the control group with the wet tensile strength of 0.94 + 0.06MPa (P0.001).
2.MTS results: the two groups were inoculated with human gingival fibroblasts and MG63 osteoblasts respectively on the membrane. The absorbance values of the two groups were all increased with time, showing a significant tendency to proliferate, that is, good cytocompatibility, and there was no significant difference in cell proliferation between the two groups.
At 3.8h, the human gingival fibroblasts were adhered to the two groups of membranes, the number of cells was large, the cells were long spindle shaped, the size was different, the cytoplasm was rich, and the number of most of the cells was small. The cells on the membrane of the control group had been paving and cross linking between the cells, and there were cross linking between the cells inoculated on the membrane in the experimental group, but compared with the less.24h on the control group. Human gingival fibroblasts adhere to the membrane of the control group and the membrane of the experimental group. The cells extend completely. The cells extend to the polygonal direction with protruding stretching cells. The cells are interlinked with each other, and the cells are fused with the surface of the membrane. When the surface of the cell surface has a clear matrix of.72h, the human gingival fibroblasts overlay and cover the surface of the membrane completely. Moreover, the newly proliferated cells were covered in the cells that lay on the surface of the membrane. The cells were mostly round and fusiform, and the proliferating cells interacted with each other.
At 4.4h, MG63 cells adhered to the membrane of the control group and showed a circular spindle shape. The pseudo foot adhered to the surface of the membrane and showed a tentacle shape. The number of MG63 cells began to stretch on the membrane of the experimental group and showed a long shuttle shape. Many of the silk feet extended from the foot to the surface of the membrane on the membrane, and the MG63 cells were on the control group, and the cells protruded out the elongated cells to the polygon direction. There are cross linking between the cells, some cells have become paving, and the surface of the membrane is integrated into one, and the surface of the cells is a large number of cell matrix secretions; MG63 cells are paving in the membrane of the experimental group, extending a large number of pseudo feet, extending the pseudo foot fully, and growing and shuttling into the pores of the material.
5. antibacterial performance test results: the antibacterial rate of chitosan - type I collagen composite membrane to Porphyromonas gingivalis was 99.55%, the antibacterial rate to Actinobacillus actinomycetes was 99.97%, the antibacterial rate of Staphylococcus aureus was 99.80%. The antibacterial rate of Escherichia coli was 99.93%. simple type I collagen composite membrane.
6. the membrane of the experimental group and the control group was soaked in Porphyromonas gingivalis and Actinobacillus actinomycetes 24h respectively. The SEM observation showed that the surface of type I collagen membrane (the control group membrane) had a large number of bacterial adhesion, and the bacteria were mass in the mass distribution, but the surface adhesion bacteria of the chitosan type I collagen composite membrane (experimental group membrane) were obviously less than the control group. Scattered and distributed on the surface of the experimental group, some bacteria collapsed and even dissolved and broken.
conclusion
Chitosan - type I collagen composite membrane has no inhibitory effect on the growth of fibroblasts and MG63 osteoblasts. It has excellent structural and biological properties. As a guided bone regeneration membrane material, it has good application prospects. Chitosan - type I collagen composite membrane has good physical and chemical energy and surface morphology, no cytotoxicity, and is good. The surface compatibility and good structural compatibility are expected to be a good leading bone regeneration barrier membrane. Chitosan - type I collagen composite membrane has strong antibacterial properties for aerobic and anaerobic bacteria. It has certain positive significance for preventing infection after GBR.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R783.1

【参考文献】