本文選題：殼聚糖 + Ⅰ型膠原�。� 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景 引導(dǎo)性骨再生(guided bone regeneration, GBR)膜具有屏障的作用,通過將骨引導(dǎo)性的基質(zhì)保持在缺損區(qū),屏蔽成纖維細(xì)胞等非成骨性細(xì)胞長(zhǎng)入缺損區(qū),允許骨組織在膜下空間內(nèi)再生,從而減少移植骨骨量的丟失。膠原膜具有良好的生物相容性、可降解性、能夠促進(jìn)細(xì)胞的粘附和增殖的特點(diǎn),作為引導(dǎo)性骨再生膜在臨床上已被廣泛應(yīng)用。但純膠原蛋白膜存在機(jī)械性能差、降解快、抗水性能差、不能與組織生長(zhǎng)速度很好地匹配等問題。殼聚糖是一種可降解的天然聚陽(yáng)離子生物多糖,降解產(chǎn)物為氨基葡萄糖等小分子,對(duì)人體無毒、無刺激,有較好的組織相容性。同時(shí)殼聚糖分子帶正電荷,通過和細(xì)菌細(xì)胞膜上負(fù)電荷的相互作用,干擾細(xì)菌細(xì)胞膜功能,達(dá)到抗菌作用。 本實(shí)驗(yàn)將Ⅰ型膠原與殼聚糖交聯(lián)復(fù)合,制備具有良好力學(xué)強(qiáng)度、良好生物相容性及抗菌性能的引導(dǎo)骨再生膜修復(fù)材料,以改善膠原作為引導(dǎo)骨再生膜存在的不足。同時(shí)針對(duì)GBR術(shù)后存在軟組織瓣裂開、膜暴露、感染等問題,通過在膠原中加入殼聚糖,使屏障膜具有抗菌性能,以減少膜暴露引起的感染,在促進(jìn)成骨的同時(shí),促進(jìn)軟組織的愈合,提高GBR手術(shù)的成功率。 本實(shí)驗(yàn)使用提純的牛肌腱Ⅰ型膠原(collagen, COL)和脫乙酰度85%的殼聚糖(chitosan, CS),按質(zhì)量比為(COL:CS=3:1)制作殼聚糖-Ⅰ型膠原復(fù)合膜。將其與純牛肌腱Ⅰ型膠原膜對(duì)比,檢測(cè)其理化性能,并在兩組膜上接種成纖維細(xì)胞和MG63成骨細(xì)胞,觀察兩組膜上所接種的細(xì)胞在膜表面的增殖情況,對(duì)殼聚糖-Ⅰ型膠原復(fù)合膜的理化性能和生物相容性進(jìn)行初步研究。同時(shí)對(duì)其進(jìn)行抗菌性能檢測(cè),評(píng)估殼聚糖-Ⅰ型膠原復(fù)合膜的抗菌作用,為其應(yīng)用于引導(dǎo)性骨再生膜提供參考。 目的 制備殼聚糖-Ⅰ型膠原復(fù)合膜并研究其理化性能,檢測(cè)其與成纖維細(xì)胞和MG63成骨細(xì)胞的生物相容性,以初步探討殼聚糖-Ⅰ型膠原復(fù)合膜應(yīng)用作為引導(dǎo)性骨再生屏障膜的可行性,同時(shí)對(duì)其進(jìn)行抗菌性能檢測(cè),評(píng)估殼聚糖-Ⅰ型膠原復(fù)合膜的抗菌能力,從而為其應(yīng)用于引導(dǎo)性骨再生提供參考。 材料和方法 1.采用提純的牛肌腱Ⅰ型膠原和殼聚糖,通過冷凍干燥法,使用熱交聯(lián)、化學(xué)交聯(lián)技術(shù)制備殼聚糖-Ⅰ型膠原復(fù)合膜(實(shí)驗(yàn)組)和Ⅰ型膠原膜(對(duì)照組)。采用氨基酸含量分析、差示量熱掃描法分析對(duì)牛肌腱Ⅰ型膠原性質(zhì)和表征進(jìn)行分析。通過紅外光譜分析、掃描電鏡分析、吸水率測(cè)試以及材料力學(xué)性能測(cè)試,檢測(cè)實(shí)驗(yàn)組和對(duì)照組的理化性能。 2.0.5cm×0.5cm大小的實(shí)驗(yàn)組和對(duì)照組膜各20張,分別置于48孔板內(nèi),接種人牙齦成纖維細(xì)胞于孔板內(nèi)培養(yǎng),接種密度每孔5×104個(gè),分別于1d、3d、5d、7d每組各取出5張膜,置于新48孔板中,每孔加入培養(yǎng)液200ml和MTS試劑40ml,孵育3.5h,測(cè)吸光度值(OD),比較兩組膜上粘附的人牙齦成纖維細(xì)胞增殖水平。 3.1.0cm×1.0cm大小的實(shí)驗(yàn)組和對(duì)照組膜各12張,分別置于24孔板內(nèi),接種成骨肉瘤細(xì)胞系MG63于孔板內(nèi)培養(yǎng),接種密度每孔5×104個(gè),分別于1d、3d、5d、7d每組各取出3張膜,置于新24孔板中,每孔加入培養(yǎng)液500ml和MTS試劑100ml,孵育3.5h,測(cè)吸光度值(OD),比較兩組膜上粘附的MG63成骨細(xì)胞增殖水平。 4.人牙齦成纖維細(xì)胞接種于兩組膜表面,接種后8h、1d、3d行生物材料掃描前處理之后,在掃描電鏡(SEM)下觀測(cè)細(xì)胞粘附、伸展及分化的形態(tài)變化。 5.成骨肉瘤細(xì)胞系MG63接種于兩組膜表面,接種后4h及1d行生物材料掃描前處理之后,在SEM下觀測(cè)細(xì)胞粘附、伸展及分化的形態(tài)變化。 6.實(shí)驗(yàn)組膜和對(duì)照組膜(10cm×1.0cm),每菌種每組各取3張膜,采用振蕩法對(duì)兩組膜行牙齦卟啉單胞菌、伴放線放線桿菌、金黃色葡萄球菌、大腸桿菌桿菌抗菌性能檢測(cè),平板計(jì)數(shù)法計(jì)數(shù)菌落回收數(shù)。 7.實(shí)驗(yàn)組膜和對(duì)照組膜(1.0cm×1.0cm)分別在3×108CFU/mL的牙齦卟啉單胞菌菌懸液和伴放線放線桿菌菌懸液培養(yǎng)24h,SEM觀察細(xì)菌粘附膜表面情況。 8.以上統(tǒng)計(jì)學(xué)方法采用SPSS13.0統(tǒng)計(jì)分析軟件,析因方差分析處理組和時(shí)間有無交互效應(yīng),多個(gè)樣本均數(shù)比較采用單因素方差分析(One-way ANOVA),兩個(gè)樣本均數(shù)比較采用獨(dú)立樣本t檢驗(yàn)比較,Levene'test檢驗(yàn)方差是否齊性,方差齊則采用LSD樣本均數(shù)間的兩兩比較,方差不齊則采用近似F檢驗(yàn)的Melch法或Brown-Forsythe法進(jìn)行方差分析后,Dunnett's T3多重比較。假設(shè)檢驗(yàn)為雙側(cè)檢驗(yàn),檢驗(yàn)標(biāo)準(zhǔn)0.05,P0.05時(shí)差異有統(tǒng)計(jì)學(xué)意義。 實(shí)驗(yàn)結(jié)果 1.膠原氨基酸組成分析、差示量熱掃描法分析結(jié)果表明牛肌腱Ⅰ型膠原的膠原3股螺旋結(jié)構(gòu)是保持較完整,成分比例及性質(zhì)表征符合Ⅰ型膠原的要求。紅外光譜結(jié)果表明實(shí)驗(yàn)組具備了膠原和殼聚糖特征峰表現(xiàn),并且兩種材料之間形成大量氫鍵,分子間結(jié)合良好。掃描電鏡分析發(fā)現(xiàn)實(shí)驗(yàn)組和對(duì)照組膜疏松面表面和橫切面為多孔結(jié)構(gòu),實(shí)驗(yàn)組膜孔直徑較大,為10～100μm。吸水率測(cè)試實(shí)驗(yàn)組膜吸水性為8.40±0.29,含水量為89.34±0.33%；對(duì)照組膜吸水性為9.25±1.13,含水量為89.09±2.12%；兩組膜吸水性和含水量均無差異(P0.05)。實(shí)驗(yàn)組膜濕態(tài)拉伸強(qiáng)度3.70±0.16MPa,明顯高于對(duì)照組膜濕態(tài)拉伸強(qiáng)度0.94±0.06MPa(P0.001)。 2.MTS結(jié)果：兩組膜上分別接種人牙齦成纖維細(xì)胞和MG63成骨樣細(xì)胞,其吸光度值均隨時(shí)間增大,呈明顯的增殖趨勢(shì),即具有較好的細(xì)胞相容性；兩組膜的細(xì)胞增殖差異沒有統(tǒng)計(jì)學(xué)意義。 3.8h時(shí)人牙齦成纖維細(xì)胞在兩組膜上粘附,細(xì)胞數(shù)目多,細(xì)胞呈長(zhǎng)梭形,大小不一,胞漿豐富,大部分細(xì)胞偽足數(shù)目少。對(duì)照組膜上部分細(xì)胞已成鋪平狀,細(xì)胞之間有相互交聯(lián),實(shí)驗(yàn)組膜上接種的細(xì)胞之間有相互交聯(lián),但較對(duì)照組上的少。24h時(shí)人牙齦成纖維細(xì)胞在對(duì)照組膜和實(shí)驗(yàn)組膜上粘附,細(xì)胞完全伸展,細(xì)胞伸出突起拉伸細(xì)胞向多角形方向發(fā)展,細(xì)胞之間有相互交聯(lián),與膜的表面融為一體,細(xì)胞表面有明顯的基質(zhì)分泌。72h時(shí)人牙齦成纖維細(xì)胞覆層生長(zhǎng),完全覆蓋了膜表面,并可見新增殖的細(xì)胞覆蓋于平鋪粘附于膜表面的細(xì)胞上,細(xì)胞形態(tài)多為圓形和梭形,增殖的細(xì)胞之間相互交通。 4.4h時(shí)MG63細(xì)胞在對(duì)照組膜上粘附,呈圓梭形,偽足粘附在膜表面,呈觸須狀,數(shù)量少；MG63細(xì)胞在實(shí)驗(yàn)組膜上開始伸展,呈長(zhǎng)梭形,許多絲足從片足中伸出粘附在膜的表面。24h時(shí)MG63細(xì)胞在對(duì)照組膜上,細(xì)胞伸出突起拉伸細(xì)胞向多角形方向發(fā)展,細(xì)胞之間有相互交聯(lián),部分細(xì)胞已成鋪平狀,與膜的表面融為一體,其表面見大量細(xì)胞基質(zhì)分泌物；MG63細(xì)胞在實(shí)驗(yàn)組膜上,呈鋪平狀,伸出大量偽足,偽足伸展充分,并往材料孔隙處生長(zhǎng)并穿梭其中。 5.抗菌性能檢測(cè)結(jié)果：殼聚糖-Ⅰ型膠原復(fù)合膜對(duì)牙齦卟啉單胞菌抗菌率達(dá)99.55%,對(duì)伴放線放線桿菌抗菌率達(dá)99.97%,對(duì)金黃色葡萄球菌抗菌率達(dá)99.80%,對(duì)大腸桿菌抗菌率達(dá)99.93%。單純Ⅰ型膠原復(fù)合膜不具備抗菌性能。 6.實(shí)驗(yàn)組和對(duì)照組膜分別浸泡于牙齦卟啉單胞菌和伴放線放線桿菌菌懸液24h后,SEM觀察可見Ⅰ型膠原膜(對(duì)照組膜)表面有大量的細(xì)菌黏附,細(xì)菌呈團(tuán)塊狀分布；而殼聚糖-Ⅰ型膠原復(fù)合膜(實(shí)驗(yàn)組膜)表面黏附的細(xì)菌明顯少于對(duì)照組,呈散在分布,同時(shí)實(shí)驗(yàn)組表面有的細(xì)菌皺縮,甚至出現(xiàn)溶解、破碎.。 結(jié)論 殼聚糖-Ⅰ型膠原復(fù)合膜對(duì)成纖維細(xì)胞和MG63成骨細(xì)胞的生長(zhǎng)無抑制作用,相具有優(yōu)良的結(jié)構(gòu)及生物學(xué)性能,作為引導(dǎo)性骨再生膜材料有良好的應(yīng)用前景。殼聚糖-Ⅰ型膠原復(fù)合膜具有較好的理化性能和表面形態(tài),無細(xì)胞毒性,具有良好的表面相容性、良好的結(jié)構(gòu)相容性,可望作為一種性能優(yōu)良的引導(dǎo)性骨再生屏障膜。殼聚糖-Ⅰ型膠原復(fù)合膜對(duì)需氧菌和厭氧菌有較強(qiáng)的抗菌性能,對(duì)預(yù)防GBR術(shù)后感染,有一定的積極意義。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R783.1

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